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WO2025212481A1 - Method for correlating cellular responses to therapy with their molecular properties - Google Patents

Method for correlating cellular responses to therapy with their molecular properties

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Publication number
WO2025212481A1
WO2025212481A1 PCT/US2025/022261 US2025022261W WO2025212481A1 WO 2025212481 A1 WO2025212481 A1 WO 2025212481A1 US 2025022261 W US2025022261 W US 2025022261W WO 2025212481 A1 WO2025212481 A1 WO 2025212481A1
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WO
WIPO (PCT)
Prior art keywords
cells
group
cell
response
therapy
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Pending
Application number
PCT/US2025/022261
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French (fr)
Inventor
Steven William METZGER
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Individual
Original Assignee
Individual
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Publication date
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Publication of WO2025212481A1 publication Critical patent/WO2025212481A1/en
Pending legal-status Critical Current
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the disclosure is directed to a method for correlating a group of cells' response to a therapy with their molecular properties.
  • the response of the group of cells to a therapy is measured.
  • the group of cells is embedded in a matrix material that can be sectioned while preserving the spatial distribution of the cells.
  • the matrix is sectioned into at least a first section and a second section, with the first containing at least a portion of the cells.
  • the group of cells in the first section is characterized to determine at least one molecular property, and the response of the group of cells to the therapy is correlated with these properties.
  • the methods are further directed to correlating the response of a second group of cells to one or more molecular properties.
  • the response of the second group of cells to the therapy is measured.
  • the second group of cells is embedded in the matrix material while maintaining the spatial distribution of the second group of cells in the matrix material.
  • the first section comprises at least a portion of the second group of cells.
  • the spatial distribution of cells may be relative to the general matrix material, or other groups of cells, such as the first group of cells.
  • the at least portion of the second group of cells embedded in first section is characterized to determine at least one molecular property of the at least a portion of the second group of cells.
  • the response of the second group of cells is correlated to the properties of the second group of cells.
  • the method is directed to additionally correlating the response of a plurality of cells to one or more molecular properties of each of the plurality of groups of cells.
  • a therapy is introduced to each group of cells.
  • the response to each group of cells to the therapy is measured.
  • Each group of cells is embedded into a matrix material capable of being sectioned while maintaining a spatial distribution of each group of cells in the matrix material.
  • the matrix material is sectioned to create at least a first section and a second section, wherein the first section includes at least a portion of each group of cells.
  • a portion of each group of cells embedded in first section is characterized to determine at least one molecular property of the at least a portion of the group of cells.
  • the response of the group of cells to the properties of the group of cells is correlated.
  • the disclosure aims to address the limitations of conventional approaches by developing methods that analyze group of cells without the need for extensive mass amplification through time-consuming culturing.
  • This approach can provide actionable information about the response of a group of cells, or different groups of cells, to a therapy within a clinically relevant timeframe, thereby facilitating more rapid and personalized treatment decisions.
  • the technique described requires a fewer number of doublings, the challenges associated with overgrowth of a subset of the group of cells (due to differing growth rates) are negligible.
  • the lack of sample availability is mitigated by expanding the type of starting sample from biopsy samples to other sample types, including but not limited to blood, effusions, and lymphatic fluid.
  • the disclosure offers a method for combining traditional molecular analysis with response of viable cells to a therapy.
  • This may include at least one of the following; measuring protein abundance or presence using antibodies, measuring messenger RNA levels, and identifying mutations in an individual group of cells.
  • This combined approach creates a unified picture by linking functional data from viable cells with molecular markers including known biomarkers.
  • the method integrates these analyses by incorporating biomarkers into the functional assay itself or by converting samples to a format compatible with the standard mounted tissue methodology. This ensures that functional data and biomarker information are aligned at the cellular level.
  • This integration has the potential to revolutionize functional precision medicine for patients. By providing a more comprehensive picture, it could lead to faster and more accurate identification of effective therapies, ultimately improving patient outcomes.
  • the disclosed methods can be used to identify biomarkers that predict responses to therapies.
  • traditional molecular analysis techniques such as antibody-based measurement of protein presence or abundance, messenger RNA levels, and nucleic acid mutations, with functional precision medicine readouts, researchers can link the functional response of an individual group of cells to specific molecular properties. This can lead to the development of more accurate predictive biomarkers that can guide patient selection in clinical trials and inform treatment decisions in clinical practice.
  • the disclosure can also be used to study the mechanisms of resistance to therapy. By exposing a group of cells to therapy and monitoring their response over time, researchers can identify the molecular pathways and genetic alterations (gene expression) that contribute to the development of resistance to therapy. This knowledge can inform the development of strategies to overcome resistance, such as the design of compounds that address specific mechanisms of resistance or the use of combination therapies that prevent treatment failure or the emergence of resistance.
  • Cancer research for pharmaceutical and diagnostic purposes needs tools that uncover complex fundamental oncogenic pathways to develop effective therapies for over 200 subtypes characterized by diverse etiologies.
  • the disclosure provides cancer diseases as a non-limiting example to demonstrate the applicability of the methods disclosed herein.
  • a therapy refers to any stimulus, whether a therapeutic compound or a heat, cold, radiation, or other stimulus, that when provided to a group of cells causes a measurable response from the group of cells.
  • the therapy can have an effect on a disease or disorder, symptom of a disease or disorder, severity of a disease or disorder, or severity of symptoms of a disease or disorder.
  • a therapeutic compound refers to a compound that, when provided to a group of cells, causes a measurable response from the group of cells.
  • the therapeutic compound can be a drug such as those listed in the Orange Book or Purple Book (FDA, both of which are incorporated herein by reference in their respective entirety)or putative drug.
  • the therapy which may include, without limitation, a small molecule, biologic (e.g., monoclonal antibody, peptide, or protein), nucleic acid-based therapeutic, or other pharmacologically active agent, can be prepared in a form suitable for the assay system.
  • the compound may be dissolved in an aqueous solvent and may include solvents to dissolve therapy in dimethyl sulfoxide (DMSO), wherein in the case of DMSO the final concentration in the assay can be less than 1% (v/v), such as less than 0.1% (v/v), to reduce cytotoxicity relative to higher solvent levels.
  • DMSO dimethyl sulfoxide
  • a physiologically compatible buffer such as phosphate-buffered saline (PBS) with a pH that can be neutral relative to acidic or basic conditions within a range including but not limited to 7.2-7.6, may be employed to maintain stability and activity.
  • PBS phosphate-buffered saline
  • a cellular or tissue model relevant to the therapeutic target can be selected, which may include established cell lines (e.g., MCF-7 for breast cancer, A549 for lung cancer), primary tissues derived from patient samples (e.g., tumor biopsies, pleural effusions), or combinations thereof.
  • the model may correspond to the candidate therapy’s mechanism of action, potentially targeting specific molecular alterations (e.g., EGFR mutations, HER2 overexpression) or biological pathways (e.g., apoptosis, immune activation).
  • Primary tissues can provide a heterogeneous cellular environment, including stromal and immune components, reflective of in vivo conditions relative to cell lines alone.
  • a range of concentrations of the candidate therapy can be prepared via serial dilution, wherein in some cases the dilution can be moderate relative to single-step adjustments within a range including but not limited to 3-fold or 10-fold dilutions spanning 1 nM to 100 pM, to establish a dose-response curve.
  • concentration range may be guided by prior in vitro or in vivo data, pharmacokinetic parameters (e.g., Cmax), or standard screening protocols as outlined by the National Cancer Institute (NCI) or similar guidelines. Dilutions can be conducted in the assay medium to potentially ensure compatibility with cellular or tissue conditions.
  • the assay can incorporate controls to potentially ensure data validity.
  • the solvent or buffer used for therapy formulation e.g., DMSO, PBS
  • DMSO, PBS may be added alone at concentrations equivalent to those in the test conditions to assess solvent- related effects on group of cells or tissue preparations (vehicle control).
  • a known therapeutic agent with established efficacy against the selected model e.g., trastuzumab for HER2-positive cells, nivolumab for immune checkpoint inhibition
  • Untreated group of cells or tissue preparations in growth medium alone may be included to establish baseline proliferation, viability, or immune activity relative to treated conditions (negative control).
  • the candidate therapy and controls can be introduced to the seeded group of cells in the assay format using a pipette or automated liquid handling system.
  • the volume added wherein in some cases the volume can be small relative to larger additions within a range including but not limited to 10-50 pL, may be consistent across wells to maintain uniform conditions relative to variable dosing.
  • allogeneic or autologous immune cells can be added to the assay system to evaluate immune-mediated effects of the candidate therapy.
  • Allogeneic immune cells may be sourced from healthy donors, while autologous immune cells can be derived from the same patient as the primary tissue sample.
  • the immune cells may be introduced at a ratio to target cells or tissue, wherein in some cases the ratio can be moderate relative to extreme proportions within a range including but not limited to 1 :1 to 10:1 effector-to-target ratio, and can be pre-activated (e.g., with IL-2 or anti-CD3 antibodies) to enhance reactivity.
  • This addition can allow assessment of immune cell infiltration, cytotoxicity, or cytokine production (e.g., IFN- y, TNF-a) in response to the therapy.
  • the method may further comprise incubating the group of cells, with or without added immune cells, with the candidate therapy for a defined period, wherein in some cases the period can be extended relative to shorter durations within a range including but not limited to 24-96 hours, followed by measurement of cellular or tissue response.
  • the method can be adaptable to high-throughput screening formats, wherein a library of candidate therapies may be tested concurrently across multiple cellular or tissue models, with or without immune cell co-cultures.
  • the assay conditions including model type, therapy concentration, immune cell inclusion, and detection methods, can be adjusted based on the specific therapeutic class (e.g., immunomodulatory vs. cytotoxic) or disease indication under investigation, potentially ensuring flexibility relative to fixed protocols.
  • the addition of the therapy occurs after a portion of time the group of cells are not in the presence of the compound.
  • the sample is split, enabling the contents to be tested in response to multiple therapies simultaneously (for example and not wishing to be limiting, therapies A, B, C, D in parallel) or one after another (for example and not wishing to be limiting in series therapies A, B, C, then D) or a mixture of simultaneously and in parallel (for example and not wishing to be limiting therapies A then B in parallel with therapies C then D).
  • the sample is split then incubated for a portion of time prior to challenge with therapies or therapy.
  • the sample can be split then incubated for a portion of time to allow for at least one of the following; growth, normalization, or induction of at least one of the following; a cell type, all cells, a mixture of cells prior to challenge with therapies or therapy.
  • the sample can be split and then incubated with a separate therapy prior to challenge to therapies with or without having the benefit of growth prior stimulation.
  • More than one therapy can be provided to a group of cells and at least one condition is absent of a therapy and serves as a control.
  • the therapy represents a potential or existing therapy.
  • the therapy uses a means for effectiveness that is at least one of the following: a chemical, biological, or electromagnetic radiosensitization.
  • the therapy selectively targets cancer cells, diseased cells, or foreign cells associated with a pathogen, thereby reducing damage to healthy tissues.
  • the therapy is a combination of therapies. The observation of the response to a plurality of therapy is performed simultaneously in a plurality of separate wells. The wells can be part of a microplate.
  • the response can be measured as compared to a reference or expected response.
  • the response can be measured as compared to a control measurement prior to adding the therapy.
  • the measurement is made a first time for the control, and a second time to determine the measured response.
  • cell generally refers to any cell, prokaryotic or eukaryotic, including plant, yeast, worm, insect and mammalian without limitation.
  • Mammalian cells include, without limitation; primate, human and a cell from any animal of interest, including without limitation; mouse, hamster, rabbit, dog, cat, domestic animals, such as equine, bovine, murine, ovine, canine, feline, etc.
  • the term includes cellular constituents and cellular products made by or associated with the group of cells (e.g. connective tissue like material, extracellular matrices, released extracellular cytokines, other signaling molecules, hormones, proteins, adenosine triphosphate, other cells such as blood cells, waste products, secretions).
  • the group of cells are physically or functionally associated with each other.
  • the group of cells behaves in a tissue like manner performing aspects of functions associated with at least one of the following; epithelial, connective bone, blood, cartilage, muscle, nervous tissue.
  • the group of cells behaves in an organ-like manner.
  • the organ-like manner replicates at least some of the in vivo normal or diseases functionality.
  • the method can be applied to greater than ten of the group of cells.
  • the number of the group of cells can depend on the experimental or therapeutic context. There is no strict maximum number of the group of cells, as the method’s applicability can be limited only by practical considerations such as sample availability, matrix capacity, and/or the ability to accurately measure and correlate responses across the plurality of the group of cells.
  • the association of the cells determines a response to the group of cells.
  • the group of cells may include cancerous and non- cancerous cells.
  • the group of cells exhibit properties of complex in vitro models of disease.
  • the group of cells that are adjacent to each other within a tissue form a distinct subpopulation or microenvironment.
  • the disclosure allows the characterization of heterogeneity within and across the group of cells to capture the heterogeneity and dynamics of the entire population of cells.
  • the disclosure describes methods for longitudinal tracking of the group of cells prior to and after the addition of a therapy.
  • a first step of measuring a response to a therapy is not performed.
  • measurements are performed at least one time point and in other cases across a plurality of timepoints.
  • the measurement is localized and can be tracked or monitored over time.
  • the measuring of the group of cells occurs prior to or during sample preparation. In some embodiments, measuring occurs after sample preparation. In some embodiments, measuring occurs after settling of the sample. In some embodiments, measuring occurs after cells are actively transported or settled. In some embodiments, the measuring occurs after entombment. In some embodiments, measuring occurs prior to, during, or after growth of the group of cells. In some embodiments, the group of cells are fixed and then measured. In some embodiments, the measuring of the group of cells occurs prior to exposure to a therapy. In some embodiments, measuring of the group of cells occurs after a duration of growth in the presence of therapy. In some embodiments, measuring the group of cells occurs after at least one of the following; cell permeabilization, clearing or clarification, dehydration, embedding, or epitope restoration. In some embodiments, measurement of the group of cells occurs after sectioning or mounting.
  • the volume is a droplet of Matrigel containing the group of cells.
  • the volume of a droplet is partitioned from another solution.
  • the volume is of aqueous nature that is partitioned from a non-aqueous volume.
  • the volume contains a unique barcode that is utilized for tracking each volume.
  • the group of cells are uniquely barcoded enabling tracking of a substantial portion of the group of cells in a volume.
  • a non-uniform electric field is utilized to move a substantial portion of the group of cells to a portion of the volume prior to imaging via dielectrophoretic forces.
  • an electro-osmotic force is applied to the volume to cause a portion of the volume to migrate for the purposes of imaging the volume.
  • the concentration locates the group of cells at a surface.
  • the cells are in proximity to each other.
  • the group of cells are in the same plane for detection.
  • the cells detected are at an air water interface.
  • the group of cells are on a surface enabling facile means of detection.
  • the group of cells are on a surface within a chamber having at least one inlet.
  • the chamber having at least one inlet is located on the top of a chamber forming a well that contains fluid by gravitational means.
  • the group of cells are physically separated from each other using physical wells confining the group of cells within boundaries.
  • the chamber has an inlet and an outlet. In some embodiments, there are a plurality of chambers.
  • the inlet comprises an opening for fluid introduction and the outlet represents a porous substrate.
  • the fluid containing the group of cells is actively passed through the porous substrate and captured on porous substrate such that the group of cells of interest are retained.
  • the plurality of chambers is comprised of a fluidic device that manages fluid flow to each chamber.
  • the fluidic device supports sequential fluid transfers to and through the chambers.
  • the fluidic transfers are accomplished with a one to many relationship of fluid source to the plurality of chambers.
  • the surface can be removed from the chamber components or plurality of chamber components.
  • the surface is removed and the group of cells are retained on the surface. In some embodiments, the group of cells retained on the surface are further interrogated.
  • the selection of materials utilized affords a means of performing steps that can support the use of at least one of the following conditions, the use of aqueous solutions, the use of organic solvents, temperatures extremes that result in phase transitions of materials or coatings of the materials such as melting, freezing, or vaporization occur.
  • the materials utilized may have dissimilar or similar properties to the embedding materials that include but are not limited to hydrophobicity, surface energy, thermal conductivity, thermal expansion properties.
  • the material properties are useful for the desired effect to cause adherence or non-adherence in at least one of the following, cells, entombment materials, solutions, or embedding materials.
  • the vessel can be treated with a modifier to alter the surface energy or chemical properties, potentially reducing the adhesion of the embedded material used in later steps of the method.
  • the modifier may include, without limitation, a chemical coating, a physical treatment, or a combination thereof, which can be selected to adjust the vessel’s surface characteristics relative to untreated surfaces.
  • the modifier may comprise a silane-based compound, such as a fluorinated silane, which can reduce surface energy by creating a hydrophobic layer, or a polyethylene glycol (PEG)-based coating, which may provide a non-stick surface, thereby potentially decreasing the adhesion of embedded materials like paraffin or PEG used for histological processing of a group of cells.
  • the treatment can involve applying a plasma treatment, wherein the surface energy can be adjusted by exposure to a plasma gas, such as oxygen or argon, for a duration that can be short relative to longer periods, such as within a range including but not limited to 1-10 minutes.
  • a plasma gas such as oxygen or argon
  • the reduction in adhesion can facilitate the release of embedded material, such as a paraffin block containing a group of cells, from the vessel, potentially improving the efficiency of histological analysis and preserving the integrity of the sample for downstream detection of molecular properties.
  • the material can be a surfactant, such as a non-ionic surfactant including but not limited to polysorbate 20 or Triton X-100, which may be applied to the vessel surface to create a hydrophilic layer, potentially reducing the adhesion of hydrophobic embedding materials relative to untreated surfaces.
  • a surfactant such as a non-ionic surfactant including but not limited to polysorbate 20 or Triton X-100, which may be applied to the vessel surface to create a hydrophilic layer, potentially reducing the adhesion of hydrophobic embedding materials relative to untreated surfaces.
  • embedding chemistries such as methacrylate or acrylamide or other free radical chemistries
  • the sacrificial layer may comprise molecules such as polysaccharides or polypeptides, which can form a temporary coating that adheres to the vessel and later detaches, potentially facilitating the removal of the embedded material without compromising the structural integrity of the group of cells within the sample.
  • the treatment may occur at any point in the methods described herein that can be advantageous to the process before removal of the embedded material, potentially ensuring compatibility with subsequent steps depending on the specific workflow requirements.
  • Embedding is for the purposes of immobilizing the group of cells in a matrix material.
  • the removal of the matrix material may be performed prior to sectioning the matrix material.
  • Entombment is a process in which a viable group of cells are added to an entombing material to limit the movement of a viable group of cells.
  • entombment provides a scaffold allowing for the differentiation of the group of cells.
  • the entombment material can also promote the ability to track cells. Entombment of the group of cells can be used to aid the measurement of a response on a surface or in a volume.
  • the entombment provides a scaffold that signals or promotes cellular behavior consistent with the development of normal or diseased tissues for the group of cells.
  • the entombment material is a hydrogel like material or of substantially increased viscosity to limit the motion of a group of cells during the imaging time period and effectively entomb the group of cells in the volume.
  • the entombment porosity limits diffusion of extracellular products from the group of cells.
  • the component is included in the initial volume. In some embodiments the initial volume is exchanged with a volume that contains the component. In some embodiments, entombment of a group of cells reduces the movement of the group of cells and enables a reduction in the imaging frequency without losing track of the group of cells over time. [0081] In some embodiments the component is of sufficient viscosity to enable tracking over time with the advantage of lowering the required detection frequency to monitor and track the group of cells. In some embodiments, a component utilizing intramolecular interactions provides a network effectively trapping the group of cells in place or slowing cellular velocity. In some embodiments, a component with intermolecular interactions provides a network effectively trapping the group of cells in place or slowing cellular velocity. In some embodiments the component contains both intra- and intermolecular interactions to support a network effectively trapping the group of cells in place or slowing cellular velocity.
  • TLM- Tracker Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28(17), 2276-2277. https://doi.Org/10.1093/bioinformatics/bts424
  • the group of cells can be measured following cell fixation and optionally permeabilization.
  • Cell fixation can be performed with or without permeabilizing the cells.
  • the clarifying solutions include at least one of the following; xylene, toluene, benzene, chloroform, methyl salicylate, methyl benzoate, butyl acetate, isopropyl acetate, amyl acetate, propylene oxide, and limonene.
  • the group of cells can be measured following a dehydration step.
  • Dehydration can be performed with or without fixation or permeabilizing the cells.
  • Dehydration can be performed with or without clearing or clarification.
  • removal of water is required. In some embodiments, the removal of water is required prior to embedding. In some embodiments, the water is evaporated from the sample. In some embodiments, the water is replaced with less hydrophilic solvent. In some embodiments, a sample is dehydrated using an agent that is miscible with water such that the sample can be rid of water prior to embedding of the sample in compatible water soluble or water insoluble wax for the histology purposes. In some embodiments, multiple rounds of dehydration are performed with increasing concentrations of dehydrating reagent. In some embodiments, dehydration is accomplished using mixtures of alcohol and water. In some embodiments, the alcohol percentage volume composition is less than 10%.
  • the alcohol percentage of the dehydration solution can be greater than 90%. In some embodiments, the alcohol percentage of the dehydration solution can be 100%. In some embodiments the mixture of alcohol percentage may increase with sequential dehydration steps.
  • ethyl alcohol is utilized as a dehydrant. In some embodiments at least one of the following; methanol, acetone, denatured alcohol, butanol, dioxane, tetrahydrofuran, ethylene glycol, propylene glycol, isopropyl alcohol is used as a dehydrant.
  • Epitope Restoration can be performed with or without the following; fixation or permeabilizing the cells, clearing or clarification, dehydration.
  • the material requires restoration of epitopes to enable measurement using probes that target specific molecules contained by a group of cells. In some embodiments, the restoration is required after fixation.
  • Protease- induced Epitope Retrieval is utilized.
  • enzymes including at least one of; Proteinase K, Trypsin, and Pepsin have been used successfully to restore the binding of an antibody to its epitope.
  • Heat-induced Epitope Retrieval is utilized to restore epitopes.
  • the heating is performed in at least one of the following; microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths. It is known to those well skilled in the art there are a wide range of epitope restoration techniques available. The methods disclosed herein are not meant to be limiting.
  • histological stains can be employed to detect molecular properties within a group of cells, potentially revealing structural and biochemical characteristics relevant to therapeutic response assessment.
  • Hematoxylin and Eosin (H&E) staining may be utilized, wherein hematoxylin can bind to acidic molecules, including but not limited to DNA and RNA in the nucleus, potentially staining them a color that can be dark relative to lighter shades, such as blue, while eosin may bind to basic proteins in the cytoplasm and extracellular matrix, potentially staining them a color that can be light relative to darker shades, such as pink, thereby revealing general cellular organization and some molecular distribution.
  • H&E stain staining may be utilized, wherein hematoxylin can bind to acidic molecules, including but not limited to DNA and RNA in the nucleus, potentially staining them a color that can be dark relative to lighter shades, such as blue, while eosin may bind to basic proteins in the cytoplasm and extra
  • Periodic Acid- Schiff (PAS) staining can target carbohydrates, including but not limited to glycogen and mucopolysaccharides, by reacting with sugar molecules, potentially indicating their presence in a group of cells or tissue preparations.
  • Oil Red O staining may be applied to detect lipids by binding to neutral fats, potentially highlighting molecular properties related to fat storage or metabolism.
  • Trichrome stains can differentiate collagen, which may be rich in specific amino acids, from muscle or cytoplasm, potentially reflecting protein composition and distribution within the sample. These staining methods may facilitate the visualization of molecular properties, supporting the characterization of a group of cells for diagnostic or pharmaceutical development purposes.
  • the term "probe,” “probe reagent” or “probe molecule” refers to an entity (e.g., but not limited to, a molecule, a particle, a composite entity, or a multi-molecular entity) that interacts with or binds to a target molecule or an analyte for the analysis of the target or the analyte.
  • an entity e.g., but not limited to, a molecule, a particle, a composite entity, or a multi-molecular entity
  • the nature of the interaction or binding is noncovalent, e.g., by hydrogen, electrostatic, or van der Waals interactions, however, binding can also be covalent.
  • Probe reagents can be entities (e.g., but not limited to, molecules, particles, composite entities, or multi-molecular entities) capable of undergoing binding or molecular recognition events with target molecules. Probe reagents can be naturally-occurring, recombinant or synthetic. Examples of the probe reagent can include, but are not limited to, a nucleic acid, an antibody or a portion thereof, an antibody-like molecule, an enzyme, a cell, an antigen, a small molecule, a protein, a peptide, a peptidomimetic, an aptamer, and any combinations thereof. By way of example only, in immunohistochemistry, the probe reagent can include an antibody specific to the target antigen to be analyzed.
  • Locked nucleic acid is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo conformation.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such LNA oligomers are generally synthesized chemically.
  • Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA.
  • nucleic acid can include a modified nucleic acid.
  • Modified nucleic acids are well known in the art.
  • a nucleic acid described herein can comprise one or more nucleic acid modifications known in the art.
  • the nucleic acid can comprise one or more nucleic acid modifications selected from the group consisting of internucleotide linkage modifications (intersugar linkage modifications), sugar modifications, nucleobase modifications, backbone modifications/replacements, and any combinations thereof.
  • Exemplary internucleotide linkage modifications include, but are not limited to, phosphorothioate, phosphorodithioate, phosphotriester (e.g.
  • alkyl phosphotriester alkyl phosphotriester
  • aminoalkylphosphotriester alkyl-phosphonate (e.g., methyl-phosphonate), selenophosphate, phosphoramidate (e.g., N- alkylphosphoramidate), boranophosphonate, and the like.
  • Exemplary sugar modifications include, but are not limited to, 2'- O-Me (2'- O-methyl), 2'- O-MOE (2- O-methoxyethyl), 2'-F, 2'- 0-[2-(methylamino)-2-oxoethyl] (2'- O-NMA), 2'-S-methyl, 2'-O-CH 2-(4'-C) (LNA), 2'-O-CH 2CH 2-(4'-C) (ENA), 2'-0-aminopropyl (2'-O-AP), 2'-0-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-O-DMAEOE), arabinose sugar, and the like.
  • nucleobase modifications include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 5-methylcytosine (5- me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other 6-alkyl derivatives of adenine and guanine; 2-propyl and other 2-alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-propynyl uracil; 5-propynyl cytosine; 6-azouracil; 6-azocytosine; 6-azothymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
  • Exemplary backbone modifications include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA ( aegPNA), backnone-extended pyrrolidine PNA ( bepPNA), and the like.
  • PNA peptide nucleic acid
  • aegPNA aminoethylglycyl PNA
  • bepPNA backnone-extended pyrrolidine PNA
  • the term "antigens” generally refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as, without limitation, an antibody, and additionally capable of being used in an animal to elicit the production of antibodies capable of binding to an epitope of that antigen.
  • An antigen may have one or more epitopes.
  • the term "antigen” can also refer to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by Major Histocompatibility Complex (MHC) molecules.
  • TCR T cell receptor
  • MHC Major Histocompatibility Complex
  • An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a T cell epitope and is given in adjuvant.
  • An antigen can have one or more epitopes (B- and T-epitopes). The specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens.
  • Antigens as used herein may also be mixtures of several individual antigens.
  • a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
  • peptidomimetic refers to a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide
  • the group of cells , the constituents of the group of cells , or the products of the group of cells are detected using probes.
  • the probe binds to a molecule having a nucleic acid, protein, carbohydrate, or lipid-like characteristics.
  • the probe binds to epigenetic targets.
  • Some examples, not meant to be limiting include at least one of the following; DNA methylation, histone modifications, non-coding RNAs, Chromatin remodeling complexes, Polycomb and Trithorax group proteins, Bromodomain-containing proteins, histone deacetylases (HDACs) and histone acetyltransferases (HATs), or DNA demethylases
  • the probe binds antigen or antigen like targets for the purposes of detection
  • the probes are a member of aptamer, antibody, and nucleic acid binding protein classes of reagents.
  • the cellular products are generated by metabolic processes and detected.
  • the cellular products are cytokines, chemokines, or other cell signaling molecules.
  • the cellular products are detected extracellularly.
  • the group of cells are detected using probes with complementary nucleic acid sequences to targeted regions.
  • the complementary nature of the probes may be incomplete, having a single or plurality of bases mismatched or missing relative to the targeted sequence.
  • the hybridization assays are used to probe nucleic acid sequences for detection.
  • the nucleic acid target is messenger RNA.
  • Exemplary methods for nucleic acid detection using hybridization that are applicable to the methods are additionally described herein include, without limitation, the methods described in U.S. Pat. No. 7,473,767, US patent publication no. 2007/0166708, and US application number US 2010/0261026.
  • in situ PCR of tissue as described without limitation by (Janiszewska et al. 2015), the entire contents incorporated herein, is performed amplifying the region or regions of interest and facilitating robust detection through additional steps of targeting the amplified regions with probes.
  • the PCR probes are allelespecific, and will amplify the DNA if a specific SNV is present.
  • copy number alterations compare the detection intensity of the targeted genomic region to that of a reference region to determine whether there are copy number alterations (deletions or amplifications) in the group of cells.
  • a plurality of padlock probes is utilized for detection, the probe sequence is amplified then later detected.
  • the probes utilized are padlock probes as described, without limitation, by (Larsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods. 2004 Dec;1 (3):227-32. doi: 10.1038/nmeth723.) with the entire contents incorporated herein.
  • detection of RNA is accomplished using ROLL FISH as described by (Wu, C., Simonetti, M., Rossell, C. et al. RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin- embedded tumor tissue samples.
  • a single response is measured. In other variations, a plurality (i.e. , two or more) responses are measured.
  • a plurality of probes is utilized. In some embodiments, a plurality of probes and a plurality of tags are utilized.
  • an additional aspect of specificity is utilized to enhance the fidelity of the detection of targets. Exemplary methods described within United States Patent 9677131 , United States Patent 9777315, United States Patent 10731206, and United States Patent 9902993. The entire contents of these patent documents are incorporated herein by reference without limitation.
  • a plurality of probes is used for a target.
  • the plurality of probes contains complementary nucleic acid sequences.
  • the complementary sequences hybridize providing a means for additional specificity of the binding of the probes to the target effectively reducing non-specific probe binding sources of noise.
  • the described approach is particularly useful for detection in volumes.
  • the complementary probes direct subsequent amplification of the sequence.
  • the amplification incorporates reporter mechanisms.
  • the reporter mechanisms are incorporated after amplification.
  • the measuring of the targets is accomplished with probes that contain a reporter mechanism that enables detection of the probe and by extension the presence of the target and further by extension the detection of the group of cells , substantial constituents of the group of cells , or products of the cell or the group of cells.
  • the probes are directly tagged with fluorescent reporter molecules.
  • the tagging is accomplished through additional means of using an additional probe to detect the presence of the precursor probe.
  • secondary detection methods commonly employed in antibody detection assays are utilized.
  • the additional means of tagging is accomplished with the covalent attachment of nucleic acid oligomers to serve as bar codes for each specific probe.
  • the reporter molecules are utilized to enable multiplexing. In some embodiments, multiplexing is accomplished through temporal methods as described herein. In some embodiments, the reporter molecules selectively report enabling the simultaneous detection of a plurality of reporter molecules.
  • the reporter mechanism amplifies the signal generated through the use of techniques known to those skilled in the art. In some embodiments, amplification is achieved using rolling circle amplification.
  • the reporter mechanism generates a detectable fluorescent signal and is attached to the probe providing a means for localization of the target by detectable fluorescence. In some embodiments, the reporter mechanism is detectable using a fluorescence polarization. In some embodiments, the reporter mechanism is detectable using fluorescence resonance energy transfer. In some embodiments, the reporter is a radioactive emitter incorporated into the probe or probes. In some embodiments, the reporter mechanism generates a chemiluminescent signal. In some embodiments, the reporter mechanism generates an electrochemical signal. In some embodiments, the reporter mechanism generates a magnetization or magnetic flux signal. In some embodiments, the report is a colorimetric report.
  • the reporter amplifies the optical detection using at least one of the following; absorbance, scattering, refraction, raman, or brillouin.
  • the reporter is a precipitant.
  • the precipitant is detected by the production of color or absorbance, scatter, or other optical technique.
  • the presence of the reporter alters the impedance.
  • a combination of reporter mechanisms is utilized to detect any number of target-probes.
  • Detection is accomplished by any means available in the art that is capable of detecting the specific signals on a given reporter mechanism utilized.
  • Detection is accomplished using means readily implemented by those skilled in the art.
  • fluorescent detection is further described where in suitable consideration of appropriate excitation sources can be readily determined.
  • Possible sources can include but are not limited to arc lamp, xenon lamp, lasers, light emitting diodes or some combination thereof.
  • the appropriate excitation source is used in conjunction with an appropriate optical detection system, for example an inverted fluorescent microscope, an epi-fluorescent microscope or a confocal microscope.
  • a microscope is selected that detects with enough spatial resolution to separate distinct signals.
  • the reporter may be the molecular mass of a molecule measured from a group of cells.
  • the detection of the mass is detected using mass spectrometry, that may include matrix-assisted laser desorption/ionization (MALDI).
  • MALDI matrix-assisted laser desorption/ionization
  • the detection is of sufficient frequency in order to track the shifting of cells relative to their initial position such that all cells, and their corresponding group of cells, can be tracked over time.
  • the initial cells can be assigned to an initial group of cells.
  • the cells may move overtime and a stationary reference point is used to identify spatial location of area containing the moving cells during repeated detection.
  • a vessel is imaged repeatedly.
  • a vessel is imaged repeatedly and the features of the microplate are utilized as reference points that create a bounding area that contains the cells.
  • the microplate wells contain features wherein the purpose of features is to act as a reference point or plurality of reference points.
  • the entire volume or surface that contains the group of cells is detected over time.
  • areas or regions of interest are first identified such that the later amount of area or volume over time is reduced.
  • the regions of interest are determined by sampling all the volume or area for a period of time then identifying regions of interest within this area or volume that are useful based on useful detections.
  • sample preparation at least one of the following; sample preparation, entombment, permeabilization, clearing or clarification, dehydration, embedding, and fixation are performed prior to measuring the response.
  • at least one of the following measures is of utility.
  • the measurement of the group of cells can be utilized for the purposes of determining the cellular state of the group of cells.
  • probes utilized for detection of the group of cells can be utilized for the purposes of determining the cellular state of the group of cells.
  • the detection of the state of the group of cells includes molecules having substantial characteristics of one of the following proteins, carbohydrates, nucleic acids, or lipids.
  • the state of cells is characterized by epigenetic events detected.
  • detection of the presence and abundance (or lack thereof) of receptors or ligands is determined.
  • enzymes are detected.
  • the production of a substrate is detected by enzymes.
  • enzymes as used here generally refers to a protein molecule that catalyzes chemical reactions of other substances without it being destroyed or altered upon completion of the reactions.
  • the term can include without limitation naturally occurring enzymes and bioengineered enzymes or mixtures thereof.
  • Examples of enzyme families include kinases, dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyl transferases, decarboxylases, transaminases, racemases, methyl transferases, formyl transferases, and a- ketodecarboxylases.
  • the cellular pathway or cellular mechanism includes the interaction between cells or the same or distinct populations of cells. In some embodiments, the cellular pathway or cellular mechanism involves the immunologically and non-immunologically related group of cells.
  • the detection of the group of cells can be utilized for the purposes of determining the viability of a group of cells.
  • cell viability using MTT or CellTiter-G Io assays or apoptosis activity using caspase-3/7 are utilized.
  • intracellular constituents of the group of cells are measured to assess cell death as defined by the penetration of a viable cell membrane impermeable dye including but not limited to propidium iodide or YOPRO.
  • the group of cells is determined to be organized in such a way that the response to external therapy is coordinated by the collective response of the group of cells.
  • the group of cells may be of distinct types relative to one another as detected by some means.
  • the functional response detected is immunologically related.
  • the ligand detected is the programmed cell death ligand 1 on cancer cells.
  • immunosuppressive molecules expressed or their receptors are detected.
  • immune checkpoints such as programmed death-ligand 1/programmed death-1 (PD-L1/PD-1), galectin-9/TIM-3, IDO1 , LAG-3, and CTLA4 are detected .
  • the group of cells may transition between cellular states such as the Mesenchymal-to-epithelial transition and be detected.
  • the group of cells, including distinct cells that operate in an organized manner may be determined as the fundamental entity in which a population model of the group of cells can be constructed.
  • the measurement of the group of cells can be utilized for the purposes of measuring growth of a cell or the group of cells.
  • the group of cells are monitored over time to assess the group of cells prior to a therapy.
  • the number of cells is enumerated over time.
  • the mass or volume of cells is measured over time.
  • the number of division events is measured over time.
  • cellular constituents produced by the group of cells are assessed using a measure of cell state.
  • the measure of cell state correlates to growth of the group of cells.
  • the measure of cell state correlates to the function of the group of cells producing a cellular constituent of relevance to growth or the expected production of said cellular constituent.
  • insulin is produced and detected indicative of proliferation or stasis and of utility.
  • BrdU incorporation is utilized to measure growth.
  • the monitoring of the group of cells over time can be utilized to longitudinally track and measure over time providing a means for monitoring the growth kinetics.
  • a rate of change can be determined by detection over a period of time.
  • the measure for the rate of change can increase (i.e., growth) or decrease (i.e., growth arrest).
  • the rate of change can be instantaneous with monitoring over a short period of time relative to the cellular division rate.
  • an average rate of change over a longer period of time can be determined.
  • the intra cellular morphology, cellular morphology, proliferation rates, and organizational structure of the group of cells are utilized for the purposes of differentiating cell types.
  • centromere 17 CEP17
  • erythroblastic oncogene B-2 ERBB2
  • enumeration of CEP17 and ERBB2 signals detection and enumeration in the nuclei of the group of cells.
  • a control of non-tumor-containing tissue is compared.
  • the detected group of cells are isolated from one another, physically or in silico, providing a means for analysis of each group of cells separately.
  • the group of cells detected by any means previously described, may be determined as the fundamental entity in which characterization of the population of the group of cells can be generated to characterize the sample.
  • the measurements capture the heterogeneity of the differentiated cells of the same type or the group of cells containing different cell types. In some embodiments, measuring indicates the presence of a specific cell population.
  • a plurality of measures can be utilized to serve to indicate or increase the confidence in the indication that a cell is of a certain type or class or has a certain product produced as a function of the cell life cycle.
  • the detected group of cells is determined to have likely or has originated from an original cell. In some embodiments the determination of origination is determined by detecting growth of the group of cells. In some embodiments, the detected group of cells may be determined as a fundamental entity in which characterization of the population of all the group of cells can be generated.
  • the sum total of at least one or a plurality of detections correlates to the responsiveness to the therapy.
  • the embedding material is at least one of the following; Carbowax, polyethylene glycol (PEG), polyethylene glycol fatty acid esters, such as polyethylene glycol monostearate, stearic acid, polyethylene oxide, Poly(vinyl methyl ether), carboxymethyl cellulose salts or acid forms, agarose, agar, polypropylene glycol, and hexadecanol.
  • PEG polyethylene glycol
  • polyethylene glycol fatty acid esters such as polyethylene glycol monostearate, stearic acid, polyethylene oxide
  • Poly(vinyl methyl ether) polyoxymethyl cellulose salts or acid forms
  • agarose agarose
  • agar polypropylene glycol
  • polypropylene glycol polypropylene glycol
  • hexadecanol hexadecanol
  • the embedding material infiltrates the material that entombs a group of cells.
  • the embedding is accomplished using hydrogels comprising polymerizable monomers selected from the group of acrylic and methacrylic monomers, wherein said monomers are capable of forming hydrophilic, three- dimensional networks upon polymerization.
  • monomers include acrylic acid (AA), polyacrylic acid (PAA) hydrogels; methacrylic acid (MAA), polymethacrylic acid (PMAA) networks; and 2-hydroxyethyl methacrylate (HEMA).
  • Additional embodiments encompass 2- hydroxyethyl acrylate (HEA); N-isopropylacrylamide (NIPAAm), and N,N-dimethylacrylamide (DMA).
  • Further monomers in this category include acrylamide (AAm), widely utilized in polyacrylamide (PAAm); methacrylamide (MAM); 2-acrylamido-2-methylpropane sulfonic acid (AMPS); glycidyl methacrylate (GMA), polyethylene glycol diacrylate (PEGDA) and polyethylene glycol dimethacrylate (PEGDMA).
  • the embedding matrix material may comprise vinyl monomers.
  • monomers include vinyl pyrrolidone (VP), which forms poly(vinylpyrrolidone) (PVP); vinyl alcohol (VA), derived from hydrolyzed poly(vinyl acetate) to yield poly(vinyl alcohol) (PVA) hydrogels crosslinked physically or chemically; vinyl acetate (VAc), serving as a precursor to PVA-based gels; and N-vinylcaprolactam (NVCL).
  • VP vinyl pyrrolidone
  • VA vinyl alcohol
  • VAc vinyl acetate
  • NVCL N-vinylcaprolactam
  • the monomers are selected such that they infiltrate the material that entombs the group of cells.
  • the embedding is accomplished using hydrogel compositions derived from natural and bio-derived monomers.
  • monomers include chitosan, that may be modified with vinyl groups such as methacrylate to enable crosslinkable hydrogel formation; hyaluronic acid (HA), that may be functionalized (e.g., as HA-methacrylate, HA-MA); and alginate, that may be similarly modified with polymerizable groups, though often crosslinked ionically.
  • Additional embodiments comprise gelatin that may be functionalized to generate methacryloyl (GelMA), a crosslinkable derivative of gelatin, dextran, that may be functionalized (e.g., as dextran methacrylate); cellulose, that may be modified into derivatives such as hydroxyethyl cellulose methacrylate; and starch, that may be adapted with acrylate or other polymerizable groups.
  • GelMA methacryloyl
  • dextran a crosslinkable derivative of gelatin
  • dextran that may be functionalized (e.g., as dextran methacrylate)
  • cellulose that may be modified into derivatives such as hydroxyethyl cellulose methacrylate
  • starch that may be adapted with acrylate or other polymerizable groups.
  • embedding incorporates zwitterionic monomers, characterized by both positive and negative charges.
  • Such monomers include sulfobetaine methacrylate (SBMA), forming poly(SBMA) hydrogels; carboxybetaine methacrylate (CBMA); and phosphorylcholine methacrylate (MPC) in poly(MPC) hydrogels.
  • embedding encompasses hydrogel-forming compositions comprising synthetic monomers tailored for specialized properties. These monomers include ethylene glycol monomethacrylate (EGMA); diethylene glycol methacrylate (DEGMA), oligo(ethylene glycol) methacrylate (OEGMA); itaconic acid (IA) and maleic acid (MA); vinylsulfonic acid (VSA); and 4-vinylpyridine (4VP).
  • EGMA ethylene glycol monomethacrylate
  • DEGMA diethylene glycol methacrylate
  • OEGMA oligo(ethylene glycol) methacrylate
  • IA itaconic acid
  • MA maleic acid
  • VSA vinylsulfonic acid
  • 4VP 4-vinylpyridine
  • embedding compositions further comprise crosslinking monomers to facilitate the formation of a three-dimensional network essential for gelation.
  • crosslinking monomers include N,N'-methylenebis(acrylamide) (MBA); ethylene glycol diacrylate (EGDA); triethylene glycol dimethacrylate (TEGDMA); and 1 ,4-butanediol diacrylate (BDDA). These difunctional monomers may be copolymerized with the aforementioned monomers to achieve the desired degree of crosslinking and mechanical strength.
  • embedding utilizes polymerization conducted via free radical polymerization, including thermal, photo-, or redox-initiated methods, though alternative techniques such as enzymatic crosslinking or ionic gelation may be employed, particularly for natural monomers.
  • polymerization is achieved using methods of low cytotoxicity.
  • mixtures of the previously mentioned materials are used for embedding.
  • the embedding of the group of cells is accomplished by freezing.
  • embedding methods described herein are performed prior to freezing.
  • additives are utilized to reduce ice crystal formation during the freezing process.
  • solvents are utilized to select a freezing temperature advantageous for sectioning (e.g. dimethyl sulfoxide, having an elevated freezing temperature relative to water).
  • the freezing is performed consistent with cryostat methodologies.
  • At least one of the following steps; sample preparation, entombment, the group of cells adherence to a surface, permeabilization, clearing or clarification, dehydration, embedding, and fixation locks the position of the cells relative to each other such that the tracking of a group of cells can be assigned to locations.
  • the spatial movement of the group of cells can be less than 500 microns.
  • the spatial movement of cells can be determined to ensure that the measured response corresponds to the characterized multiplex properties - i.e., that the same cells or group of cells are compared.
  • the spatial movement of the group of cells can be less than 100 microns. In some embodiments, the spatial movement of the group of cells can be less than 50 microns. In some embodiments, the spatial movement of the group of cells can be less than 25 microns. In some embodiments, the spatial movement of the group of cells can be less than 10 microns. In some embodiments, the spatial movement of the group of cells can be less than 1 micron relative to the final position prior to fixation. In further variations, the spatial movement of the group of cells can be relative to the final position prior and/or after embedding the group of cells.
  • the location of the group of cells can be registered with respect to the position prior to fixation can be corrected or compensated for algorithmically during signal analysis using any art-recognized computer-implemented algorithms.
  • the group of cells are first measured or a portion of the measuring steps performed before removal from the vessel and sectioning Removal from Vessel or Chamber
  • the mounting adapts to specific requirements: for mass spectrometry, such as matrix-assisted laser desorption/ionization (MALDI), tissues may be affixed to conductive metal plates or glass slides coated with a thin layer of gold or silver, optimizing ionization and detection of molecular species. Alternatively, cryosections can be thaw-mounted directly onto these substrates to preserve biomolecular properties. For Raman spectroscopic analysis, samples may be deposited on low-background substrates like calcium fluoride (CaF 2 ) to reduce spectral interference or signal enhancing substrates such as mirrored stainless steel, with sections either air-dried or maintained in a hydrated state depending on the analyte of interest. Characterizing Molecular Properties
  • the responses of the group of cells to therapies are utilized to determine the response of a cell type to a therapy for diagnostic and/or pharmaceutical development.
  • the therapies are compared to the no therapy control.
  • the cells are assessed independently from other cells with respect to a parallel no therapy condition.
  • the interaction of cells associated with the host immune response is assessed.
  • immune cells are useful for diagnostic and/or pharmaceutical development.
  • immune cells and other cells are useful for diagnostic and/or pharmaceutical development.
  • the interaction between immune cells and non-immune cells is assessed.
  • the interaction between the group of cells is determined by the cellular constituents or cellular products.
  • the cellular constituents produced are cytokines, chemokines, or other cellular signaling elements.
  • the heterogeneity assessed correlates to the response and is useful for diagnostic and/or pharmaceutical development.
  • the correlated response predicts the efficacy of the therapy for diagnostic or pharmaceutical development purposes.
  • a training set of clinical outcome data is compared to the response observed and the information associated with the interrogation of the group of cells and their cellular products.
  • the response observed is further refined and broken down into sub groups prior to the comparison to the clinical information.
  • the observed response is utilized to predict the benefit of the therapy for a patient whose materials were used to generate the response.
  • the response is used to predict the progression free or overall survival expected for that therapy for that particular patient.
  • algorithms operate at the level of distinct entities comprised of separately detected the group of cells as described herein or by any means known to those skilled in the art.
  • population analysis means analysis of all entities measured for providing utility for diagnostic and/or pharmaceutical development purposes.
  • computational algorithms are employed to analyze the response and molecular properties, reconstructing a population model that captures the diversity of responses and molecular characteristics across the sample.
  • This model may incorporate machine learning techniques to identify predictive biomarkers, classify cell types, or infer therapeutic efficacy based on the combined functional and molecular signatures.
  • the heterogeneity of the group of cells including differences in cell state, differentiation, or spatial distribution, is quantified and correlated with therapy outcomes, offering a refined understanding of disease progression and treatment response.
  • algorithms are required for the identification of the individual group of cells.
  • the detected individual group of cells are the fundamental entity of affording the construction of a population model useful for characterizing the response for diagnostic and/or pharmaceutical development purposes.
  • features are utilized to characterize the detections described for each entity.
  • data analysis of the features or each entity alone or as a population are algorithmically correlated to predicted response for diagnostic and/or pharmaceutical development purposes.
  • the extraction of features may be accomplished using traditional image analysis techniques including the registration of images, segmentation of areas of interest using thresholds, and extraction of features such as the change in area over time for these areas can be employed.
  • Object detection frameworks such as R- CNN, Fast R-CNN, Faster R-CNN, YOLO, and SSD, can be applied to detect and localize the group of cells in the images.
  • Object detection frameworks such as R- CNN, Fast R-CNN, Faster R-CNN, YOLO, and SSD, can be applied to detect and localize the group of cells in the images.
  • FCNs fully convolutional networks
  • U-Net and DeepLab can be employed to accurately segment individual cells or the group of cells from the background and from each other.
  • Generative Models such as Variational Autoencoders (VAEs) and Generative Adversarial Networks (GANs), can be used for data augmentation and for modeling the distribution of the group of cells. By training these networks in an adversarial manner, GANs can generate realistic images of the group of cells that capture the diversity and variability of real samples.
  • VAEs Variational Autoencoders
  • GANs Generative Adversarial Networks
  • spatial coordinates or unique identifiers can be assigned to each group of cells, capturing their positions via imaging or barcoding.
  • the embedding and sectioning steps are conducted in a manner that maintains the original spatial arrangement within the matrix.
  • Molecular characterization on the sections thus provides spatially resolved data.
  • the method generates a wealth of data by extracting features from images of the individual group of cells and monitoring their response to therapy over time.
  • the tracked group of cells are differentiated by the expression properties of messenger RNA.
  • the presence and amount of expression of oncogenes is utilized for segmenting into classes of cells. The following section describes some of the methods that can be used to correlate the longitudinal data with patient outcomes, but these examples are not intended to be limiting.
  • Feature Extraction and Selection Relevant features capture the morphological, textural, and dynamic properties of the group of cells. These features may include the group of cells size, shape, intensity, texture descriptors, and growth rates, among others. Feature selection techniques, such as principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), or recursive feature elimination (RFE), can be applied to identify the most informative features that discriminate between the different group of cells populations or treatment responses.
  • PCA principal component analysis
  • t-SNE t-distributed stochastic neighbor embedding
  • RFE recursive feature elimination
  • Time Series Analysis The data from the group of cells tracked over time can be treated as time series.
  • Time series analysis techniques such as autoregressive integrated moving average (ARIMA) models, polynomial and hill curve fitting functions, hidden Markov models (HMMs), or long short-term memory (LSTM) networks, can be employed to model the temporal dynamics of the group of cells behavior and the responses to therapies.
  • ARIMA autoregressive integrated moving average
  • HMMs polynomial and hill curve fitting functions
  • HMMs hidden Markov models
  • LSTM long short-term memory
  • Clustering and Subpopulation Identification Clustering algorithms, such as k- means, hierarchical clustering, or density-based spatial clustering of applications with noise (DBSCAN), can be used to associate the group of cells with similar features or response patterns. Distinct subpopulations within the heterogeneous population can be identified. The disclosure can uncover specific cell types or states that may be associated with treatment sensitivity or resistance. These subpopulations can then be further characterized by their molecular properties or functional properties to gain insights into the mechanisms underlying their behavior.
  • DBSCAN density-based spatial clustering of applications with noise
  • Machine learning models can be trained to predict patient response based on the longitudinal group of cells data.
  • Supervised learning algorithms such as logistic regression, support vector machines (SVMs), random forests, or gradient boosting machines (GBMs), can be used to classify cells or patients into responder and non-responder groups. These models can learn the complex relationships between the extracted group of cells features, time-dependent behavior, and patient outcomes, enabling the prediction of treatment efficacy for new patients.
  • the provides a method of correlating a response of a group of cells to multiplex properties of the group of cells, comprising: introducing a therapy to a plurality of cells and/or group of cells cultured in a vessel under controlled conditions; measuring a response of the group of cells to the therapy using microscopy to generate longitudinal imaging data indicative of cellular dynamics; embedding the group of cells into a material capable of being sectioned while maintaining the spatial distribution of the physical group of cells on the matrix material; sectioning the matrix material to create at least a first section and a second section, wherein the first section comprises at least a portion of the group of cells; characterizing the at least a portion of the group of cells embedded in the first section to determine molecular properties, including nucleic acid and protein properties, using probes compatible with spatial omics analysis; and correlating the response of the physical group of cells to the molecular properties of the group of cells.
  • the method generates structured data, comprising imaging-derived features and molecular properties, configured for analysis by transformer-based artificial intelligence (Al) models to characterize cellular states, interactions, and responses at a tissue level, addressing limitations in prior art techniques that fail to preserve spatial and molecular integrity.
  • Al transformer-based artificial intelligence
  • Effective visualization techniques are essential for interpreting and communicating the complex patterns and relationships in the longitudinal group of cells data.
  • Tools such as t-SNE plots, principal component analysis (PCA) plots, or heatmaps can be used to visualize the high-dimensional group of cells feature space and identify clusters or trends.
  • Interactive dashboards can be developed to allow researchers and clinicians to explore the data, compare different treatment conditions, and assess individual patient responses. Such methods can be effectively repurposed for Pharmaceutical development purposes.
  • NLP natural language processing
  • LLM large language models
  • the individual group of cells can be considered as the "tokens" that collectively make up the overall cell population.
  • Each individual group of cells cell or cell group is characterized by a unique set of features, including at least one of the following; growth dynamics data from imaging-based methods and molecular properties profiling data. These features can be thought of as the "vocabulary" that describes the behavior and characteristics of each individual group of cells cell or cell group.
  • the disclosure enables the application of machine learning techniques, such as those used in LLMs, to analyze and model responses of the group of cells to therapy.
  • the training data for the LLM-like algorithm will be derived from a large cohort of patients, each with unique disease characteristics and treatment histories. To ensure the model's robustness and generalizability, it is essential to include a diverse range of patient samples, covering various disease stages, and prior treatment regimens. This heterogeneous dataset will enable the model to learn the complex relationships between group of cells behavior, responses to therapies, and patient outcomes across a wide spectrum of clinical scenarios.
  • the group of cells will be cultured under conditions that allow for growth and exposed to a panel of therapies, including both standard-of-care treatments and experimental compounds.
  • the tokenization process will be applied consistently across all patient samples.
  • Each individual group of cells will be treated as a unique "token,” characterized by a set of features derived from the imaging and molecular profiling data. This tokenization step ensures that the LLM-like algorithm can process the data in a uniform manner, regardless of the patient source.
  • the process of training an LLM-like algorithm on the tokenized individual group of cells cell or cell group data is implemented as follows:
  • [0267] 1 Data Acquisition: Obtain a sample comprising the group of cells from a patient and culture under conditions that allow for growth. Expose the group of cells to one or more therapy conditions and detect the response of an individual group of cells using imaging-based methods over time. Utilize a polynomial to fit the response of an individual group of cells to parametrize the response, probe the cells with molecular probes to detect biomarkers, genetic alterations, cell type, cellular constituents, or cellular components.
  • Tokenization Segment the parameterized response and molecular profiling data into an individual group of cells , treating each individual group of cells as a unique "token.” Extract relevant features from each token, such as parameters relating to response, morphology, and expression levels of specific biomarkers or genetic alterations. These features will serve as the input data for the LLM-like algorithm.
  • Data Preprocessing Normalize and standardize the feature data across all tokens to ensure comparability. Split the tokenized data into training, validation, and test sets.
  • Model Architecture Design an LLM-like architecture that can process the tokenized cell data. This may involve adapting existing LLM architectures, such as the transformer model, to handle the specific input features and output targets relevant to cell behavior and responses to therapies.
  • Validation and Testing Evaluate the trained model's performance on the validation and test sets, assessing its ability to accurately predict the response of a patient’s individual group of cells to therapy. Fine-tune the model as needed to improve its performance. To evaluate the model's performance and ensure its clinical relevance, the trained algorithm will be applied to an independent validation dataset, also consisting of tokenized data and patient outcomes. This validation step will assess the model's ability to accurately predict patientspecific responses to therapies and outcomes based solely on the behavior and characteristics of their group of cells. The model's performance can be further refined through techniques such as cross-validation and hyperparameter tuning.
  • the plate was placed in an incubator for an additional 24 hrs
  • the plate was removed from the incubator and a 15 uL of a 10x stock concentration of each therapy including a “no therapy” control (Sacituzumab-govitecan, trastuzumab deruxtecan, emtansine, deruxtecan, docetaxel).
  • a “no therapy” control (Sacituzumab-govitecan, trastuzumab deruxtecan, emtansine, deruxtecan, docetaxel).
  • a total of 8, 3-fold serial dilutions from the cMax, the maximum concentration of the therapy achieved in serum after dosing, for each therapy tested was achieved in 40 test conditions performed including a "no therapy” control run in triplicate.
  • the plate was imaged immediately after dosing (dosing time 0) and then returned to the incubator.
  • the plate was imaged at 24, 48, 72, and 96 hrs post dosing
  • step M the plate was fixed by first removing 140 uL of fluid from all the wells and replacing with 100 uL of a 1x fixative solution (2% PFA, pH 7.4, Sigma). The wells were incubated for 2 hrs.
  • a 1x fixative solution 2% PFA, pH 7.4, Sigma.
  • the silos obtained from each well were placed in a standard embedding tray with the well surface facing down in a 3 x 3 array preserving the ordering to match the microplate layout.
  • a paraffin block of the silos was created by standard blocking procedures.
  • the signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
  • H&E cancerous cells
  • FISH substantial HER2 signal
  • probe free imaging growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
  • a line listing for 49 unique entities of individual detections from example 1 control (no therapy) is shown below for illustrative purposes. Such detections are used as independent orthogonal metrics useful for characterization of the group of cells leading to in aggregate an analysis of all the group of cells at a population level. In the control condition (no therapy) there is a mixture of responses for each measure. EPCAM+ signal intensities correlate with HER2+ signal as well as the H&E manual assessment of each group of cells. The combined information for Unique ID 44 and 18 suggests an incorrect categorization.
  • H&E results are suggestive of a cancer cell that is not recognized with high confidence using H&E alone but can putatively recategorized for the purposes of describing a response to therapy by creating an algorithm that weighs EPCAM+, Division Rate, and HER2+ in addition to H&E for classification of cancer cells in test conditions.
  • Low frequency detection and increased confidence in classification of cell type is critical for accurate characterization of population response to no therapy conditions.
  • the signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
  • H&E cancerous cells
  • FISH substantial HER2 signal
  • probe free imaging growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
  • ISBER Best Practices Recommendations for Repositories: Fifth Edition (ISBER Best Practices, 5th Edition)
  • TLM- Tracker Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28(17), 2276-2277. https://doi.org/10.1093/bioinformatics/bts424
  • VISION an open-source software for multi-dimensional image analysis of cellular biophysics. Journal of Cell Science, 137(5), jcs262166. https://doi.org/10.1242/jcs.262166

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Abstract

Methods of correlating a response of a group of cells to therapy to one or more molecular properties of the group of cells by embedding cells in media is provided.

Description

METHOD FOR CORRELATING CELLULAR RESPONSES TO THERAPY WITH THEIR MOLECULAR PROPERTIES
PRIORITY
[0001] This application claims the benefit under 35 U.S.C. §119(e) of the U.S. Provisional Patent Application No. 63/572,297, entitled Integrated Functional and Molecular Characterization of cells and Cell Groups to Guide Therapy, which was filed March 31 , 2024, the content of which is incorporated by reference herein in its entirety.
BACKGROUND
[0002] Delays in obtaining diagnostic results often impede timely treatment, particularly in time-sensitive diseases such as cancer, where early intervention is crucial. Many patients receive insufficient actionable guidance, resulting in a lack of clear, personalized treatment options. The practicality of diagnostic methods is further constrained by difficulties in obtaining or preserving specific sample types, which limits their broader application. Additionally, the inconsistent predictive value of biomarkers across different diseases and patient profiles makes it challenging to develop reliable and universally effective diagnostic tools.
[0003] The high failure rate in drug discovery efforts is primarily attributed to the poorly understood nature of normal and disease states. The inherent complexity and variability of diseases pose significant challenges in developing treatments that are effective across diverse patient populations. This lack of comprehensive understanding not only impedes the creation of effective therapies but also contributes to the substantial costs and extended timelines associated with bringing new drugs to market.
SUMMARY
[0004] In a first aspect, the disclosure is directed to a method for correlating a group of cells' response to a therapy with their molecular properties. The response of the group of cells to a therapy is measured. The group of cells is embedded in a matrix material that can be sectioned while preserving the spatial distribution of the cells. The matrix is sectioned into at least a first section and a second section, with the first containing at least a portion of the cells. The group of cells in the first section is characterized to determine at least one molecular property, and the response of the group of cells to the therapy is correlated with these properties.
[0005] In some variations, the methods are further directed to correlating the response of a second group of cells to one or more molecular properties. The response of the second group of cells to the therapy is measured. The second group of cells is embedded in the matrix material while maintaining the spatial distribution of the second group of cells in the matrix material. The first section comprises at least a portion of the second group of cells. The spatial distribution of cells may be relative to the general matrix material, or other groups of cells, such as the first group of cells. The at least portion of the second group of cells embedded in first section is characterized to determine at least one molecular property of the at least a portion of the second group of cells. The response of the second group of cells is correlated to the properties of the second group of cells.
[0006] In some variations, the method is directed to additionally correlating the response of a plurality of cells to one or more molecular properties of each of the plurality of groups of cells. A therapy is introduced to each group of cells. The response to each group of cells to the therapy is measured. Each group of cells is embedded into a matrix material capable of being sectioned while maintaining a spatial distribution of each group of cells in the matrix material. When the matrix material is sectioned to create at least a first section and a second section, wherein the first section includes at least a portion of each group of cells. A portion of each group of cells embedded in first section is characterized to determine at least one molecular property of the at least a portion of the group of cells. The response of the group of cells to the properties of the group of cells is correlated.
[0007] The disclosure aims to address the limitations of conventional approaches by developing methods that analyze group of cells without the need for extensive mass amplification through time-consuming culturing. This approach can provide actionable information about the response of a group of cells, or different groups of cells, to a therapy within a clinically relevant timeframe, thereby facilitating more rapid and personalized treatment decisions.
[0008] Because the technique described requires a fewer number of doublings, the challenges associated with overgrowth of a subset of the group of cells (due to differing growth rates) are negligible. In another aspect, the lack of sample availability is mitigated by expanding the type of starting sample from biopsy samples to other sample types, including but not limited to blood, effusions, and lymphatic fluid.
[0009] Additionally, the disclosure offers a method for combining traditional molecular analysis with response of viable cells to a therapy. This may include at least one of the following; measuring protein abundance or presence using antibodies, measuring messenger RNA levels, and identifying mutations in an individual group of cells. This combined approach creates a unified picture by linking functional data from viable cells with molecular markers including known biomarkers. The method integrates these analyses by incorporating biomarkers into the functional assay itself or by converting samples to a format compatible with the standard mounted tissue methodology. This ensures that functional data and biomarker information are aligned at the cellular level. This integration has the potential to revolutionize functional precision medicine for patients. By providing a more comprehensive picture, it could lead to faster and more accurate identification of effective therapies, ultimately improving patient outcomes.
[0010] In some variations, new candidate therapies can be identified. In other variations, combination therapies can be developed. By exposing cells derived from different patients to various combinations of therapy and monitoring their response at an individual group of cells level, researchers can determine which combinations are most effective for specific subtypes or genotypes. This can guide the development of new treatments, including personalized combination therapies that are tailored to individual patients, improving treatment outcomes and reducing the risk of resistance.
[0011] The disclosed methods can be used to identify biomarkers that predict responses to therapies. By integrating traditional molecular analysis techniques, such as antibody-based measurement of protein presence or abundance, messenger RNA levels, and nucleic acid mutations, with functional precision medicine readouts, researchers can link the functional response of an individual group of cells to specific molecular properties. This can lead to the development of more accurate predictive biomarkers that can guide patient selection in clinical trials and inform treatment decisions in clinical practice.
[0012] The disclosure can also be used to study the mechanisms of resistance to therapy. By exposing a group of cells to therapy and monitoring their response over time, researchers can identify the molecular pathways and genetic alterations (gene expression) that contribute to the development of resistance to therapy. This knowledge can inform the development of strategies to overcome resistance, such as the design of compounds that address specific mechanisms of resistance or the use of combination therapies that prevent treatment failure or the emergence of resistance.
[0013] Cancer research for pharmaceutical and diagnostic purposes needs tools that uncover complex fundamental oncogenic pathways to develop effective therapies for over 200 subtypes characterized by diverse etiologies. The disclosure provides cancer diseases as a non-limiting example to demonstrate the applicability of the methods disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawings, that are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure
[0015] Figure 1 illustrates a schematic comparison of data mapping strategies for associating a response of a group of cells with molecular properties, contrasting a conventional approach with the disclosed method, in accordance with an illustrative embodiment. The figure depicts the relationship between response measures and molecular properties, including but not limited to measured multiplexed mRNA properties, protein properties, and epigenetic properties.
DETAILED DESCRIPTION
[0016] The following description is presented to allow any person skilled in the art to make and use the embodiments, and is provided in the context of a particular application. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. It should be understood that the following descriptions are not intended to limit the embodiments to one preferred embodiment. To the contrary, it is intended to cover alternatives, modifications, and equivalents as can be included within the spirit and scope of the described embodiments as defined by the appended claims. Thus, the disclosure is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.
[0017] As described herein, the disclosure is directed to a method for correlating a group of cells' response to a therapy with their molecular properties. The response of the group of cells to a therapy is measured. The group of cells is embedded in a matrix material that can be sectioned while preserving the spatial distribution of the cells. The matrix is sectioned into at least a first section and a second section, with the first containing at least a portion of the cells. The group of cells in the first section is characterized to determine at least one molecular property, and the response of the group of cells to the therapy is correlated with these properties
[0018] The disclosure provides a number of advantages over conventional research methodologies. The methods can be applied to non-cancer disease states diagnostics and drug discovery.
[0019] The methods provide a facile way to analyze cells for various biomolecules such as, without limitation; carbohydrates, nucleic acids, lipids, proteins and specific targets such as, without limitation; genomic alterations, epigenetic changes, RNA levels, protein activity. This analysis can then be directly compared with the characterization of the response of a viable group of cells to a therapy condition. This information can be linked together at a single group of cells level providing an unprecedented level of characterization of the heterogeneity of all the group of cells for the purposes of determining effectiveness of the therapy for pharmaceutical development or diagnostic applications.
Obtaining a sample of Tissue or Bodily Fluid
[0020] In general, samples include a group of cells. In various embodiments, the samples may be at least one of biopsy specimens, bodily fluids, bone marrow aspirates, lymph node specimens, cytology specimens, blood, urine, nasal swabs, buccal swabs, formalin-fixed paraffin-embedded (FFPE) tissue blocks, fresh frozen tissue samples, tissue microarrays (TMAs), 2D cultures, liquid cultures, organoids, spheroids, stool samples, sputum samples, skin scrapings or swabs, and nail clippings or hair samples. Samples can be removed from a living organism, such as by surgery. In some embodiments, the sample material was obtained from a living person. In some cases, the material may have been obtained from a carcass, cadaver or organ obtained from a living organism. The samples can be sorted, or be freshly acquired (i.e., used within 24 hours of being acquired). [0021] Examples of sample collection can be found in ISBER Best Practices: Recommendations for Repositories: Fifth Edition (ISBER Best Practices, 5th Edition), which is incorporated herein by reference in its entirety.
[0022] In some embodiments, multiple samples are obtained. In some embodiments, the samples are preserved in a manner that maintains cellular integrity. In further embodiments, the samples are cryopreserved tissue, cells, cell, or cultured cells. In some embodiments, the samples utilize cell cultures.
[0023] In some variations, the cells are cancer cells. The term "cancer1 used herein refers to any cell derived from any type of cancer, including but not limited to carcinomas, sarcomas, leukemias, lymphomas, and myelomas originating from various tissues such as breast, prostate, lung, colon, skin, brain, bone, blood, and others. The cancer samples may encompass various stages of disease progression, including but not limited to early-stage, locally advanced, and metastatic cancers. Cancer samples can be derived from primary tumors, metastatic lesions, or circulating tumor cells found in bodily fluids. Furthermore, cancer samples may exhibit different levels of differentiation, ranging from well-differentiated to poorly differentiated or undifferentiated states. The disclosure is applicable to all types of cancer samples regardless of their tissue of origin, stage, or differentiation status.
[0024] In some embodiments a resected specimen is utilized. In some embodiments, the sample is obtained by invasive sampling. In some embodiments, cells circulating in body fluids are isolated for use. In some embodiments, skin punches or skin materials cut from patients are utilized. In some embodiments, a biopsy procedure is performed to obtain sample material. In some embodiments a protected specimen brush or bronchial lavage is utilized. In some embodiments, lymph fluid is collected. In some embodiments, a thoracic duct cannulation is utilized to collect lymph fluid. In some embodiments sample fluid is utilized and obtained by centesis. In some embodiments, sample fluid is obtained from amniocentesis, arthrocentesis, lumbar puncture, paracentesis, pericardiocentesis, and thoracentesis then utilized. In some embodiments, a bone biopsy is utilized. In some embodiments the samples were frozen or previously preserved prior to processing.
Sample Preparation
[0025] The sample can be prepared or modified.
[0026] For example, a portion of the sample can be processed. In some embodiments, the sample can be mechanically separated into portions, and a substantial portion utilized for the purposes of further characterization. In some embodiments, the sample is sufficiently fluid such that volumetric means can be utilized to separate portions of the sample.
[0027] Examples of sample processing techniques include, but are not limited to, placing the material on a substrate with or without the aid of an attachment mechanism, resuspending the material in suspension, or diluting the sample in sufficient media to support cellular growth. In some embodiments, mechanical processing of a sample can be performed (e.g. , without limitation, homogenizing, centrifuging, vortexing, sectioning and shearing), separation of a sample (e.g., without limitation, filtering, centrifuging, electrophoresis), and any combinations thereof. In some embodiments, the purpose is to obtain cells for subsequent study.
[0028] In some embodiments, sample preparation maintains the spatial organization of the group of cells as received prior to sample preparation. In some embodiments, sample preparation involves handling of the material to add fluid, manipulate physically, or transfer to another format.
[0029] In some embodiments, the sample preparation disperses the group of cells. In some embodiments, cell types can be purified or enriched. In some embodiments the enrichment step corresponds to growth of the sample using media that selectively cultures the cell type of interest. The enrichment can be achieved, for example, by selectively capturing the cell type of interest and separating from the remaining group of cells and materials in the sample. In some embodiments, the selective isolation utilizes a probe as described herein. In some embodiments, the Epithelial Cell Adhesion Molecule (EPCAM) target and antibody probe is used to selectively capture cells as described further in Ring A, Mineyev N, Zhu W, Park E, Lomas C, Punj V, Yu M, Barrak D, Forte V, Porras T, Tripathy D, Lang JE. EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low. Oncotarget. 2015 Dec 29;6(42):44623-34. doi: 10.18632/oncotarget.5977, which is incorporated herein by reference in its entirety.
[0030] In some embodiments, the target may be Human Epidermal growth factor Receptor 2 (HER2) recognized by an antibody probe. In some embodiments, the antibody probe for HER2 is trastuzumab monoclonal antibody. In some embodiments, the probe is attached to a magnetic bead that enables magnetic separation of the desired cell type from the sample. In some embodiments, the probe is attached to a density modifying component. In some embodiments the density modifier is different from the remaining materials in the sample such that separation through differential density techniques can be used.
[0031] In some embodiments, enzymatic or chemical methods of disruption of the tissue may be performed to separate cells from each other and other components of the sample. Enzymes or chemical methods can be selected to disrupt the Extracellular Matrix (ECM). Alternatively, enzymes can target various fibrous proteins like collagen, elastin, and fibronectin, as well as complex carbohydrates like glycosaminoglycans. Enzymes or chemical methods are selected to preferentially or completely disrupt Cell Adhesion Molecules (CAMs). In some embodiments, enzymes or chemical methods can be selected to degrade cadherins, selectins, and integrins. In some embodiments, enzymes or chemical methods can be selected to disrupt cell to cell interactions. Enzymes or chemical methods can be selected to disrupt epithelial tissues. Enzymes or chemical methods can be selected to disrupt specialized structures that link the cytoskeletons of adjacent cells, providing strong adhesion to resist mechanical stress. Enzymes or chemical methods can be selected to disrupt gap junctions. Enzymes or chemical methods can be selected to disrupt linkages of cells to the extracellular matrix, specifically to a type of ECM called the basement membrane. In some embodiments, enzymes or chemical methods can be selected to disrupt the junctions connecting the actin filament bundle in one cell with that in the next cell. Enzymes or chemical methods can be selected to disrupt cells from basement membranes.
[0032] In some embodiments, a combination of enzymes or chemicals can be utilized to prepare the sample. In some embodiments, a reduced number or type of enzymes and chemical methods can be utilized to accomplish separating cells into individual or small group of cells while preserving aspects of the cellular machinery so as to reduce the disruption of normal cellular processes. In some embodiments, the sample preparation techniques preserve immune cells.
[0033] In some embodiments, a substantial portion of the group of cells are selectively removed from the sample. In some embodiments, the selected group of cells are removed from the samples. In some embodiments, the removal of non-cell components, or cellular components interfere with the methods described. In some embodiments, selective lysis of cell populations can be performed to enrich the sample for a type of cell of interest or eliminate a type of cell from the sample. In some embodiments the red blood cells are lysed selectively using osmotic lysis. In some embodiments, osmotic lysis is achieved by exchanging the sample volume or diluting the sample in a hypotonic solution of ammonium chloride or sodium citrate and water to rupture the membrane of red blood cells. In some embodiments, a commercial kit can be utilized to selectively lyse red blood cells such as Lysis Buffer (eBioscience), Lysing Solution (BD), Red Blood Cell Lysis Buffer (Thermo Fisher Scientific). In some embodiments, lysis of the red blood cell membrane is accomplished with a dilute solution of a detergent wherein the detergent concentration is insufficient to lyse other cells but lytic for red blood cells. In some embodiments, mechanical force is applied to rupture red blood cells. In some embodiments, the mechanical force can be applied by sonication, centrifugation, or passage through a microporous filter.
[0034] In some embodiments, the sample is intact and the cellular constituents are not disrupted. The material is incubated in a fluid for a substantial period of time. The fluid is exchanged multiple times. The fluid contains nutrients required for growth of the sample. The sample is a section of material and in some cases a matrix material. In some embodiments the section of the material prepared for use and further characterization.
[0035] In some embodiments, a shipping device is utilized for the dual purposes of sample preparation and transportation from collection to the laboratory location where testing is performed. The sample shipping device controls temperature so as to increase the viability of the material prior to testing. The sample shipping device contains materials that are mixed with the sample to preserve, grow, or perform aspects of the sample preparation process. In some embodiments, portions of the methods described within this application are incorporated into the shipping device. The shipping device logs information for later analysis.
[0036] All methods can be performed as described in “Comprehensive Sampling and Sample Preparation: Analytical Techniques for Scientists” ISBN 978-0-12-381374-9, 2012, Academic Press, Janusz Pawliszyn, which is incorporated herein by reference in its entirety.
[0037] Techniques, including but not limited to sample preparation techniques known to those skilled in the art such are described in the following references, each of which is incorporated by reference herein in its entirety:
1. Chow, Sue, David Hedley, Patricia Grom, Robert Magari, James W. Jacobberger, and T. Vincent Shankey. 2005. “Whole Blood Fixation and Permeabilization Protocol with Red Blood Cell Lysis for Flow Cytometry of Intracellular Phosphorylated Epitopes in Leukocyte Subpopulations.” Cytometry. Part A: The Journal of the International Society for Analytical Cytology 67 (1): 4-17.
2. Eslami-S, Zahra, Luis Enrique Cortes-Hernandez, and Catherine Alix-Panabieres. 2020. “Epithelial Cell Adhesion Molecule: An Anchor to Isolate Clinically Relevant Circulating Tumor Cells.” Cells 9 (8). https://doi.Org/10.3390/cells9081836.
[0038] Techniques, including but not limited to additional details describing the culturing of primary tissues are provided in the following references, each of which is incorporated herein by reference in its entirety:
1. Clevers, H. (2016). Modeling development and disease with organoids. Cell, 165(7), 1586-1597. doi:10.1016/j.cell.2016.05.082.
2. Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., Van Es, J. H., Van den Brink, S Clevers, H. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology, 141 (5), 1762-1772. doi:10.1053/j.gastro.2011.07.050.
3. Lancaster, M. A., & Knoblich, J. A. (2014). Generation of cerebral organoids from human pluripotent stem cells. Nature Protocols, 9(10), 2329-2340.
4. Huch, M., Dorrell, C., Boj, S. F., van Es, J. H., Li, V. S., van de Wetering, M., ... Clevers, H. (2013). In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature, 494(7436), 247-250. doi:10.1038/nature11826.
5. Kretzschmar, K., & Clevers, H. (2016). Organoids: Modeling development and the stem cell niche in a dish. Developmental Cell, 38(6), 590-600. doi:10.1016/j.devcel.2016.08.014.
Introducing a Therapy
[0039] In some embodiments, drug sensitivity screening assays evaluate the efficacy of a candidate therapy against a group of cells obtained from samples including but not limited to primary tissues, cells harvested from blood or other patient fluids, establish cell lines, and cryopreserved materials. Such assays used to analyze or evaluate therapeutic potential, establishing dose-response relationships, and supporting pharmaceutical development or diagnostic applications, with particular consideration of immune reactivity and the potential integration of allogeneic or autologous immune cells.
[0040] The following disclosure outlines steps that can be employed to incorporate a candidate therapy into drug sensitivity screening assay against a group of cells, potentially ensuring compatibility with diverse cellular models and reliable measurement of responses, including immune-mediated effects. Without wishing to be bound by theory, the method of the disclosure may comprise the steps delineated under the subheadings below, which can be performed in the order presented or in any suitable sequence as determined by one skilled in the art.
Preparation of the Therapy
[0041] A therapy refers to any stimulus, whether a therapeutic compound or a heat, cold, radiation, or other stimulus, that when provided to a group of cells causes a measurable response from the group of cells. The therapy can have an effect on a disease or disorder, symptom of a disease or disorder, severity of a disease or disorder, or severity of symptoms of a disease or disorder. A therapeutic compound refers to a compound that, when provided to a group of cells, causes a measurable response from the group of cells. The therapeutic compound can be a drug such as those listed in the Orange Book or Purple Book (FDA, both of which are incorporated herein by reference in their respective entirety)or putative drug. The therapeutic compound can have an effect on a disease or disorder, symptom of a disease or disorder, severity of a disease or disorder, or severity of symptoms of a disease or disorder. The therapeutic compound can be selected to correspond to a specific cell type. Therapeutic compounds can include compound in a salt, solvate, ester form, or combination thereof.
[0042] In some embodiments, the therapy, which may include, without limitation, a small molecule, biologic (e.g., monoclonal antibody, peptide, or protein), nucleic acid-based therapeutic, or other pharmacologically active agent, can be prepared in a form suitable for the assay system. For small molecules, the compound may be dissolved in an aqueous solvent and may include solvents to dissolve therapy in dimethyl sulfoxide (DMSO), wherein in the case of DMSO the final concentration in the assay can be less than 1% (v/v), such as less than 0.1% (v/v), to reduce cytotoxicity relative to higher solvent levels. For biologies, a physiologically compatible buffer, such as phosphate-buffered saline (PBS) with a pH that can be neutral relative to acidic or basic conditions within a range including but not limited to 7.2-7.6, may be employed to maintain stability and activity.
[0043] In some embodiments, the stability and solubility of the candidate therapy can be confirmed under assay conditions, including but not limited to temperature, wherein in some cases the temperature can be elevated relative to room temperature within a range including but not limited to 35-37°C; pH, wherein in some cases the pH can be neutral relative to extreme conditions within a range including but not limited to 6.5-7.5; and incubation duration, wherein in some cases the duration can be extended relative to shorter periods within a range including but not limited to 24-96 hours. Stability may be evaluated using analytical techniques such as high-performance liquid chromatography (HPLC) or spectroscopy, potentially ensuring degradation can be less than a detectable threshold during the assay timeframe.
Selection of an Appropriate Cellular or Tissue Model
[0044] In some embodiments, a cellular or tissue model relevant to the therapeutic target can be selected, which may include established cell lines (e.g., MCF-7 for breast cancer, A549 for lung cancer), primary tissues derived from patient samples (e.g., tumor biopsies, pleural effusions), or combinations thereof. The model may correspond to the candidate therapy’s mechanism of action, potentially targeting specific molecular alterations (e.g., EGFR mutations, HER2 overexpression) or biological pathways (e.g., apoptosis, immune activation). Primary tissues can provide a heterogeneous cellular environment, including stromal and immune components, reflective of in vivo conditions relative to cell lines alone.
Culturing Conditions for Therapy Introduction
[0045] In some embodiments, the therapy can be introduced to group of cells from cell lines or primary tissues that can be cultured in an appropriate growth medium tailored to the model type, potentially supplemented with nutrients and growth factors (e.g., epidermal growth factor (EGF), interleukin-2 (IL-2) for immune cells) as needed. Culture conditions may be sustained at a temperature, wherein in some cases the temperature can be elevated relative to room temperature within a range including but not limited to 35-37°C, and CO2 concentration, wherein in some cases the concentration can be elevated relative to ambient air within a range including but not limited to 4-6%, conducive to cellular proliferation or tissue integrity relative to less optimal environments. Primary tissues may be maintained as explants, organoids, or dissociated cells, depending on the assay design.
[0046] In some embodiments, the therapy can be introduced to group of cells can be harvested, resuspended, and seeded into multiwell plates (e.g., 96-well, 384-well) or other assay formats (e.g., microfluidic devices) at a predetermined density or mass, wherein in some cases the density for cell lines can be moderate relative to sparse or overcrowded conditions within a range including but not limited to 1 ,000-10,000 cells per well, and the mass for primary samples can be small relative to larger fragments within a range including but not limited to 1-5 mg. The seeding density or mass may be chosen to permit measurable growth, functional response, or immune reactivity during the assay period, as verified by preliminary experiments or historical data, and can be less than a level that would lead to overcrowding or degradation.
Introduction of the Candidate Therapy and Optional Immune Cells
[0047] In some embodiments, a range of concentrations of the candidate therapy can be prepared via serial dilution, wherein in some cases the dilution can be moderate relative to single-step adjustments within a range including but not limited to 3-fold or 10-fold dilutions spanning 1 nM to 100 pM, to establish a dose-response curve. The concentration range may be guided by prior in vitro or in vivo data, pharmacokinetic parameters (e.g., Cmax), or standard screening protocols as outlined by the National Cancer Institute (NCI) or similar guidelines. Dilutions can be conducted in the assay medium to potentially ensure compatibility with cellular or tissue conditions.
[0048] In some embodiments, the assay can incorporate controls to potentially ensure data validity. The solvent or buffer used for therapy formulation (e.g., DMSO, PBS) may be added alone at concentrations equivalent to those in the test conditions to assess solvent- related effects on group of cells or tissue preparations (vehicle control). A known therapeutic agent with established efficacy against the selected model (e.g., trastuzumab for HER2-positive cells, nivolumab for immune checkpoint inhibition) can be included to confirm assay sensitivity and reproducibility relative to untested conditions, potentially capturing immune reactivity where applicable (positive control). Untreated group of cells or tissue preparations in growth medium alone may be included to establish baseline proliferation, viability, or immune activity relative to treated conditions (negative control).
[0049] In some embodiments, the candidate therapy and controls can be introduced to the seeded group of cells in the assay format using a pipette or automated liquid handling system. The volume added, wherein in some cases the volume can be small relative to larger additions within a range including but not limited to 10-50 pL, may be consistent across wells to maintain uniform conditions relative to variable dosing.
[0050] In some embodiments, allogeneic or autologous immune cells (e.g., peripheral blood mononuclear cells (PBMCs), T cells, natural killer (NK) cells) can be added to the assay system to evaluate immune-mediated effects of the candidate therapy. Allogeneic immune cells may be sourced from healthy donors, while autologous immune cells can be derived from the same patient as the primary tissue sample. The immune cells may be introduced at a ratio to target cells or tissue, wherein in some cases the ratio can be moderate relative to extreme proportions within a range including but not limited to 1 :1 to 10:1 effector-to-target ratio, and can be pre-activated (e.g., with IL-2 or anti-CD3 antibodies) to enhance reactivity. This addition can allow assessment of immune cell infiltration, cytotoxicity, or cytokine production (e.g., IFN- y, TNF-a) in response to the therapy.
[0051] In some embodiments, the method may further comprise incubating the group of cells, with or without added immune cells, with the candidate therapy for a defined period, wherein in some cases the period can be extended relative to shorter durations within a range including but not limited to 24-96 hours, followed by measurement of cellular or tissue response.
[0052] In some embodiments, the method can be adaptable to high-throughput screening formats, wherein a library of candidate therapies may be tested concurrently across multiple cellular or tissue models, with or without immune cell co-cultures. The assay conditions, including model type, therapy concentration, immune cell inclusion, and detection methods, can be adjusted based on the specific therapeutic class (e.g., immunomodulatory vs. cytotoxic) or disease indication under investigation, potentially ensuring flexibility relative to fixed protocols.
[0053] In some embodiments the addition of the therapy occurs after a portion of time the group of cells are not in the presence of the compound.
Testing of Multiple Therapies
[0054] In some embodiments, the sample is split, enabling the contents to be tested in response to multiple therapies simultaneously (for example and not wishing to be limiting, therapies A, B, C, D in parallel) or one after another (for example and not wishing to be limiting in series therapies A, B, C, then D) or a mixture of simultaneously and in parallel (for example and not wishing to be limiting therapies A then B in parallel with therapies C then D). In some embodiments, the sample is split then incubated for a portion of time prior to challenge with therapies or therapy. The sample can be split then incubated for a portion of time to allow for at least one of the following; growth, normalization, or induction of at least one of the following; a cell type, all cells, a mixture of cells prior to challenge with therapies or therapy. The sample can be split and then incubated with a separate therapy prior to challenge to therapies with or without having the benefit of growth prior stimulation. More than one therapy can be provided to a group of cells and at least one condition is absent of a therapy and serves as a control. In some embodiments, the therapy represents a potential or existing therapy. In some embodiments, the therapy uses a means for effectiveness that is at least one of the following: a chemical, biological, or electromagnetic radiosensitization.
[0055] In some embodiments, the therapy selectively targets cancer cells, diseased cells, or foreign cells associated with a pathogen, thereby reducing damage to healthy tissues. [0056] In some embodiments, the therapy is a combination of therapies. The observation of the response to a plurality of therapy is performed simultaneously in a plurality of separate wells. The wells can be part of a microplate.
Measuring a Response to group of cells to the Therapy
[0057] The response can be measured as compared to a reference or expected response. Alternatively, the response can be measured as compared to a control measurement prior to adding the therapy. In this case, the measurement is made a first time for the control, and a second time to determine the measured response. In some embodiments, there are a plurality of control measurements made prior to adding the therapy and at least one additional measure after the therapy has been added.
[0058] The term “cell” or "cells" used herein generally refers to any cell, prokaryotic or eukaryotic, including plant, yeast, worm, insect and mammalian without limitation. Mammalian cells include, without limitation; primate, human and a cell from any animal of interest, including without limitation; mouse, hamster, rabbit, dog, cat, domestic animals, such as equine, bovine, murine, ovine, canine, feline, etc. The cells may be of synthetic or natural origin spanning a wide variety of tissue types without limitation such as; hematopoietic, neural, mesenchymal, cutaneous, mucosal, stromal, muscle spleen, reticuloendothelial, epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary, T-cells etc. Stem cells, embryonic stem (ES) cells, ES- derived cells and stem cell progenitors are also included, including without limitation, hematopoeitic, neural, stromal, muscle, cardiovascular, hepatic, pulmonary, gastrointestinal stem cells, etc. Yeast cells may also be used as cells in some embodiments of the methods described herein. In some embodiments, the cells can be ex vivo or cultured cells, e.g. in vitro. For example, for ex vivo cells, cells can be obtained from a subject, where the subject is healthy and/or affected with a disease. Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art [0059] As used herein, the term "group of cells ” refers to a single cell, a plurality of single cells, a plurality of cells of the same or different cell type or state as determined by any detectable means, a cell group or cluster of cells, a plurality of a cell group or cluster of cells, a cell group or cluster of cells of a different type or state as determined by any detectable means. The term includes cellular constituents and cellular products made by or associated with the group of cells (e.g. connective tissue like material, extracellular matrices, released extracellular cytokines, other signaling molecules, hormones, proteins, adenosine triphosphate, other cells such as blood cells, waste products, secretions). In some embodiments the group of cells are physically or functionally associated with each other. In some embodiments, the group of cells behaves in a tissue like manner performing aspects of functions associated with at least one of the following; epithelial, connective bone, blood, cartilage, muscle, nervous tissue. In some embodiments, the group of cells behaves in an organ-like manner. In some embodiments, the organ-like manner replicates at least some of the in vivo normal or diseases functionality.
[0060] As used throughout herein, the method can be applied to a plurality of groups of cells. This includes any number of the group of cells. In one variation, the method is applied to two of the group of cells. In another variation, the method is applied to three of the group of cells. In another variation, the method is applied to four of the group of cells. In another variation, the method is applied to five of the group of cells. In another variation, the method is applied to six of the group of cells. In another variation, the method is applied to seven of the group of cells. In another variation, the method is applied to eight of the group of cells. In another variation, the method is applied to nine of the group of cells. In another variation, the method is applied to ten of the group of cells. In some variations, the method can be applied to greater than ten of the group of cells. The number of the group of cells can depend on the experimental or therapeutic context. There is no strict maximum number of the group of cells, as the method’s applicability can be limited only by practical considerations such as sample availability, matrix capacity, and/or the ability to accurately measure and correlate responses across the plurality of the group of cells. [0061] In some embodiments, the association of the cells determines a response to the group of cells. In some embodiments, the group of cells may include cancerous and non- cancerous cells. In some embodiments, the group of cells exhibit properties of complex in vitro models of disease. In some embodiments, the group of cells that are adjacent to each other within a tissue, form a distinct subpopulation or microenvironment. The group of cells can also refer to clustered circulating tumor cells or cells that demonstrate coordinated behavior or communication or immune cells. The group of cells may proliferate two-dimensionally on a flat surface, three-dimensionally, or in a shape consistent with an in vivo organ or tissue.
[0062] Various methods, including culturing of the group of cells, are further described in the references listed below, each of which is incorporated herein by reference in its entirety:
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6. Acanda De La Rocha AM, Berlow NE, Fader M, Coats ER, Saghira C, Espinal PS, Galano J, Khatib Z, Abdella H, Maher OM, Vorontsova Y, Andrade-Feraud CM, Daccache A, Jacome A, Reis V, Holcomb B, Ghurani Y, Rimbias L, Guilarte TR, Hu N, Salyakina D, Azzam DJ. Feasibility of functional precision medicine for guiding treatment of relapsed or refractory pediatric cancers. Nat Med. 2024 Apr;30(4):990-1000
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15. Coussens NP, et al. Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery. Clin Transl Sci. 2018 Sep;11(5):461-470. doi: 10.1111/cts.12570
[0063] The disclosure allows the characterization of heterogeneity within and across the group of cells to capture the heterogeneity and dynamics of the entire population of cells. The disclosure describes methods for longitudinal tracking of the group of cells prior to and after the addition of a therapy.
When Measuring Occurs
[0064] In some embodiments, a first step of measuring a response to a therapy is not performed. In some embodiments, measurements are performed at least one time point and in other cases across a plurality of timepoints. In some embodiments, the measurement is localized and can be tracked or monitored over time.
[0065] In some embodiments, the measuring of the group of cells occurs prior to or during sample preparation. In some embodiments, measuring occurs after sample preparation. In some embodiments, measuring occurs after settling of the sample. In some embodiments, measuring occurs after cells are actively transported or settled. In some embodiments, the measuring occurs after entombment. In some embodiments, measuring occurs prior to, during, or after growth of the group of cells. In some embodiments, the group of cells are fixed and then measured. In some embodiments, the measuring of the group of cells occurs prior to exposure to a therapy. In some embodiments, measuring of the group of cells occurs after a duration of growth in the presence of therapy. In some embodiments, measuring the group of cells occurs after at least one of the following; cell permeabilization, clearing or clarification, dehydration, embedding, or epitope restoration. In some embodiments, measurement of the group of cells occurs after sectioning or mounting.
[0066] In some embodiments, the measurements of the group of cells include measurements of their group of cells. In some embodiments, measurements are made of products that may be at located intracellularly, extracellularly, cell surface bound, within the extracellular matrix, or in proximity to the cell surface [0067] In some embodiments the group of cells are detected in a volume. Alternatively, in some embodiments, the group of cells are detected on a surface or in proximity to a surface. Measurements in a Volume (Volume, Vessel) [0068] In some embodiments, the group of cells are in a volume. In some embodiments, the volume containing the group of cells is smaller than the entire volume. In some embodiments, the density of the group of cells is such that statistically it is unlikely that the group of cells occupy space such that one cell does not occlude another from detection. In some embodiments the detection is performed in near real time. In some embodiments the near real time detection is performed in a smaller portion of the entire volume.
[0069] In some embodiments the volume is partitioned into many smaller volumes that confines the group of cells enabling facile detection. In some embodiments, the detected group of cells tracked over a period of time. In some embodiments, a volume is utilized such that statistically it is probable the volume contains a single group of cells . In some embodiments, a volume is utilized such that statistically an average number of the group of cells is achieved in the volume. In some embodiments the volume is placed on a surface. In some embodiments, an array of volumes is placed on a surface. In some embodiments, the volume effectively confines the group of cells within the volume of the droplet. In some embodiments, the volume is submerged or contained within a liquid. In some embodiments the volume is a droplet of Matrigel containing the group of cells. In some embodiments, the volume of a droplet is partitioned from another solution. In some embodiments, the volume is of aqueous nature that is partitioned from a non-aqueous volume. In some embodiments, the volume contains a unique barcode that is utilized for tracking each volume. In some embodiments, the group of cells are uniquely barcoded enabling tracking of a substantial portion of the group of cells in a volume.
[0070] In some embodiments, the sample is first settled for a portion of time prior to imaging to the group of cells. In some embodiments, the group of cells are actively settled by applying an external force. In some embodiments, the settling time is reduced by accelerating the settling using centrifugal force. In some embodiments, a density of the fluid is selected to separate the group of cells from the remaining group of cells. In some embodiments, a density gradient is utilized to a separate group of cells of interest from the remaining group of cells. [0071] In some embodiments, an electrokinetic force on the charged group of cells is utilized to move a substantial portion of the group of cells to a portion of the volume prior to imaging. In some embodiments, a non-uniform electric field is utilized to move a substantial portion of the group of cells to a portion of the volume prior to imaging via dielectrophoretic forces. In some embodiments, an electro-osmotic force is applied to the volume to cause a portion of the volume to migrate for the purposes of imaging the volume. In some embodiments, the concentration locates the group of cells at a surface.
Measurements on Surfaces (Chamber, Fluidic Device)
[0072] In some embodiments, the cells are in proximity to each other. In some embodiments, the group of cells are in the same plane for detection. In some embodiments the cells detected are at an air water interface. In some embodiments, the group of cells are on a surface enabling facile means of detection. In some embodiments, the group of cells are on a surface within a chamber having at least one inlet. In some embodiments, the chamber having at least one inlet is located on the top of a chamber forming a well that contains fluid by gravitational means. In some embodiments, the group of cells are physically separated from each other using physical wells confining the group of cells within boundaries. In some embodiments, the chamber has an inlet and an outlet. In some embodiments, there are a plurality of chambers.
[0073] In some embodiments, the inlet comprises an opening for fluid introduction and the outlet represents a porous substrate. In some embodiments, the fluid containing the group of cells is actively passed through the porous substrate and captured on porous substrate such that the group of cells of interest are retained.
[0074] In some embodiments the plurality of chambers is comprised of a fluidic device that manages fluid flow to each chamber. In some embodiments, the fluidic device supports sequential fluid transfers to and through the chambers. In some embodiments, the fluidic transfers are accomplished with a one to many relationship of fluid source to the plurality of chambers. In some embodiments the surface can be removed from the chamber components or plurality of chamber components. In some embodiments, the surface is removed and the group of cells are retained on the surface. In some embodiments, the group of cells retained on the surface are further interrogated.
Vessel And Chambers
[0075] In some embodiments, the selection of materials utilized affords a means of performing steps that can support the use of at least one of the following conditions, the use of aqueous solutions, the use of organic solvents, temperatures extremes that result in phase transitions of materials or coatings of the materials such as melting, freezing, or vaporization occur. In some embodiments, the materials utilized may have dissimilar or similar properties to the embedding materials that include but are not limited to hydrophobicity, surface energy, thermal conductivity, thermal expansion properties. In some embodiments, the material properties are useful for the desired effect to cause adherence or non-adherence in at least one of the following, cells, entombment materials, solutions, or embedding materials. In some embodiments, the vessel can be made from materials including but not limited to glass, plastic, or a combination thereof, and may be configured as a microplate, a petri dish, or a flow chamber, depending on the requirements of the method for supporting measuring a response of a group of cells. The microplate can contain a number of wells that may vary relative to smaller or larger formats, such as within a range including but not limited to 6 to 1536 wells, potentially accommodating various throughput needs. Plates may comprise substrates such as glass-bottom or polystyrene, which can be compatible with detection methods including but not limited to brightfield or fluorescence imaging, colorimetric detection ensuring versatility across analytical techniques. In some embodiments, materials are selected for suiting the combination of conditions. In some embodiments, the vessel can be a flow chamber having an inlet and an outlet, wherein the inlet may allow introduction of fluids or reagents, and the outlet can facilitate removal of waste or excess materials, potentially supporting dynamic assays. An example of a flow chamber device can be found in the reference Yamagishi, M., Ohara, O., & Shirasaki, Y. (2020). Microfluidic immunoassays for time-resolved measurement of protein secretion from single cells. Annual Review of Analytical Chemistry, 13(1), 15-32, incorporated herein in its entirety. [0076] The flow chamber may have a single fluidic path that serves the fluidic needs of the method, such as delivering a therapy or washing solutions in a controlled manner, potentially enabling uniform exposure of a group of cells to conditions while maintaining sample integrity for downstream analysis.
[0077] In some embodiments, the vessel can be treated with a modifier to alter the surface energy or chemical properties, potentially reducing the adhesion of the embedded material used in later steps of the method. The modifier may include, without limitation, a chemical coating, a physical treatment, or a combination thereof, which can be selected to adjust the vessel’s surface characteristics relative to untreated surfaces. For example, the modifier may comprise a silane-based compound, such as a fluorinated silane, which can reduce surface energy by creating a hydrophobic layer, or a polyethylene glycol (PEG)-based coating, which may provide a non-stick surface, thereby potentially decreasing the adhesion of embedded materials like paraffin or PEG used for histological processing of a group of cells. In some embodiments, the treatment can involve applying a plasma treatment, wherein the surface energy can be adjusted by exposure to a plasma gas, such as oxygen or argon, for a duration that can be short relative to longer periods, such as within a range including but not limited to 1-10 minutes. In some embodiments, the reduction in adhesion can facilitate the release of embedded material, such as a paraffin block containing a group of cells, from the vessel, potentially improving the efficiency of histological analysis and preserving the integrity of the sample for downstream detection of molecular properties. In some embodiments, the material can be a surfactant, such as a non-ionic surfactant including but not limited to polysorbate 20 or Triton X-100, which may be applied to the vessel surface to create a hydrophilic layer, potentially reducing the adhesion of hydrophobic embedding materials relative to untreated surfaces. In some embodiments, there may be a sacrificial layer of molecules that can adsorb to the vessel wall and are designed to chemically crosslink to embedding chemistries, such as methacrylate or acrylamide or other free radical chemistries, and release from the wall at a later point in time, potentially triggered by a change in conditions, including but not limited to temperature, wherein in some cases the temperature can be elevated relative to room temperature within a range including but not limited to 50-70°C, or pH, wherein in some cases the pH can be acidic relative to neutral conditions within a range including but not limited to 4-6. In some embodiments, the sacrificial layer may comprise molecules such as polysaccharides or polypeptides, which can form a temporary coating that adheres to the vessel and later detaches, potentially facilitating the removal of the embedded material without compromising the structural integrity of the group of cells within the sample. In some embodiments, the treatment may occur at any point in the methods described herein that can be advantageous to the process before removal of the embedded material, potentially ensuring compatibility with subsequent steps depending on the specific workflow requirements.
Measuring Entombed Group of Cells
[0078] Embedding is for the purposes of immobilizing the group of cells in a matrix material. The removal of the matrix material may be performed prior to sectioning the matrix material.
[0079] Entombment is a process in which a viable group of cells are added to an entombing material to limit the movement of a viable group of cells. In some variations, entombment provides a scaffold allowing for the differentiation of the group of cells. The entombment material can also promote the ability to track cells. Entombment of the group of cells can be used to aid the measurement of a response on a surface or in a volume. In some embodiments, the entombment provides a scaffold that signals or promotes cellular behavior consistent with the development of normal or diseased tissues for the group of cells. In some embodiments, the entombment material is a hydrogel like material or of substantially increased viscosity to limit the motion of a group of cells during the imaging time period and effectively entomb the group of cells in the volume. In some embodiments, the entombment porosity limits diffusion of extracellular products from the group of cells.
[0080] In some embodiments, the component is included in the initial volume. In some embodiments the initial volume is exchanged with a volume that contains the component. In some embodiments, entombment of a group of cells reduces the movement of the group of cells and enables a reduction in the imaging frequency without losing track of the group of cells over time. [0081] In some embodiments the component is of sufficient viscosity to enable tracking over time with the advantage of lowering the required detection frequency to monitor and track the group of cells. In some embodiments, a component utilizing intramolecular interactions provides a network effectively trapping the group of cells in place or slowing cellular velocity. In some embodiments, a component with intermolecular interactions provides a network effectively trapping the group of cells in place or slowing cellular velocity. In some embodiments the component contains both intra- and intermolecular interactions to support a network effectively trapping the group of cells in place or slowing cellular velocity.
[0082] In some embodiments, the entombment material can be a gelling agent. In some embodiments, the gelling agent can be a hydrogel. In some embodiments the gelling agent is at least one of the following; agar-agar, agarose, alginate, chitosan, carrageenan, polyethylene glycol, hyaluronic acid, alginate, and chitosan, cellulose, dextran, agarose, pullulan, gellan gum, collagen, gelatin, fibrin, albumin, silk fibroin, polyacrylamide, polyacrylic, polyvinyl alcohol (PVA) and poly(N-vinylpyrrolidone) (PVP), poly(N-isopropylacrylamide) (PNIPAM), pluronic block copolymers, and poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the gel strength is provided by at least one of the following basement-membrane matrix extracted from Engelbreth-Holm-Swarm mouse sarcomas (Matrigel).
[0083] Various techniques and materials, including but not limited entombment materials in the alternate to Matrigel, are described in the following references, each of which is incorporated herein by reference in its entirety:
1. Kaur, Savneet, Impreet Kaur, Preety Rawal, Dinesh M. Tripathi, and Ashwini Vasudevan. 2021 . “Non-Matrigel Scaffolds for Organoid Cultures.” Cancer Letters 504 (April): 58-66.
2. Aisenbrey, Elizabeth A., and William L. Murphy. 2020. “Synthetic Alternatives to Matrigel.” Nature Reviews. Materials 5 (7): 539-51.
3. Marchini, Amanda, and Fabrizio Gelain. 2022. “Synthetic Scaffolds for 3D Cell Cultures and Organoids: Applications in Regenerative Medicine.” Critical Reviews in Biotechnology 42 (3): 468-86.
4. Nguyen, Eric H., William T. Daly, Ngoc Nhi T. Le, Mitra Farnoodian, David G. Belair, Michael P. Schwartz, Connie S. Lebakken, et al. 2017. “Versatile Synthetic Alternatives to Matrigel for Vascular Toxicity Screening and Stem Cell Expansion.” Nature Biomedical Engineering 1 (July), https://doi.orq/10.1038/s41551-017-0096.
5. Kozlowski, Mark T., Christiana J. Crook, and Hsun Teresa Ku. 2021 . “Towards Organoid Culture without Matrigel.” Communications Biology 4 (1): 1387.
[0084] In some embodiments, entombment is accomplished using hydrogels comprising polymerizable monomers selected from the group of acrylic and methacrylic monomers, wherein said monomers are capable of forming hydrophilic, three-dimensional networks upon polymerization. Such monomers include acrylic acid (AA), polyacrylic acid (PAA) hydrogels; methacrylic acid (MAA), polymethacrylic acid (P AA) networks; and 2- hydroxyethyl methacrylate (HEMA). Additional embodiments encompass 2-hydroxyethyl acrylate (HEA); N- isopropylacrylamide (NIPAAm), and N,N-dimethylacrylamide (DMA). Further monomers in this category include acrylamide (AAm), widely utilized in polyacrylamide (PAAm); methacrylamide (MAM); 2-acrylamido-2-methylpropane sulfonic acid (AMPS); glycidyl methacrylate (GMA), polyethylene glycol diacrylate (PEGDA) and polyethylene glycol dimethacrylate (PEGDMA). [0085] In some embodiments, the entombment may comprise vinyl monomers. Such monomers include vinyl pyrrolidone (VP), which forms poly(vinylpyrrolidone) (PVP); vinyl alcohol (VA), derived from hydrolyzed poly(vinyl acetate) to yield poly(vinyl alcohol) (P A) hydrogels crosslinked physically or chemically; vinyl acetate (VAc), serving as a precursor to PVA-based gels; and N-vinylcaprolactam (NVCL).
[0086] In some embodiments, the entombment is accomplished using hydrogel compositions derived from natural and bio-derived monomers. Such monomers include chitosan, that may be modified with vinyl groups such as methacrylate to enable crosslinkable hydrogel formation; hyaluronic acid (HA), that may be functionalized (e.g., as HA-methacrylate, HA-MA); and alginate, that may be similarly modified with polymerizable groups, though often crosslinked ionically. Additional embodiments comprise gelatin that may be functionalized to generate methacryloyl (GelMA), a crosslinkable derivative of gelatin, dextran, that may be functionalized (e.g., as dextran methacrylate); cellulose, that may be modified into derivatives such as hydroxyethyl cellulose methacrylate; and starch, that may be adapted with acrylate or other polymerizable groups.
[0087] In some embodiments, entombment incorporates zwitterionic monomers, characterized by both positive and negative charges. Such monomers include sulfobetaine methacrylate (SBMA), forming poly(SBMA) hydrogels; carboxybetaine methacrylate (CBMA); and phosphorylcholine methacrylate (MPC) in poly(MPC) hydrogels.
[0088] In some embodiments, entombment encompasses hydrogel-forming compositions comprising synthetic monomers tailored for specialized properties. These monomers include ethylene glycol monomethacrylate (EGMA); diethylene glycol methacrylate (DEGMA), oligo(ethylene glycol) methacrylate (OEGMA); itaconic acid (IA) and maleic acid (MA); vinylsulfonic acid (VSA); and 4-vinylpyridine (4VP).
[0089] In some embodiments, entombment compositions further comprise crosslinking monomers to facilitate the formation of a three-dimensional network essential for gelation. Such crosslinking monomers include N,N'-methylenebis(acrylamide) (MBA); ethylene glycol diacrylate (EGDA); triethylene glycol dimethacrylate (TEGDMA); and 1 ,4-butanediol diacrylate (BDDA). These difunctional monomers may be copolymerized with the aforementioned monomers to achieve the desired degree of crosslinking and mechanical strength. [0090] In some embodiments, entombment utilizes polymerization conducted via free radical polymerization, including thermal, photo-, or redox-initiated methods, though alternative techniques such as enzymatic crosslinking or ionic gelation may be employed, particularly for natural monomers. In some embodiments the polymerization is achieved using methods of low cytotoxicity.
[0091] In some embodiments, mixtures of the previously mentioned materials are used for entombment.
[0092] In some embodiments, the group of cells are first allowed to settle and/or actively transported to the surface prior to gelling. In some embodiments, the group of cells are gelled during the settling process. In some embodiments, the group of cells are settled and then gelled by triggering a reduction in effective viscosity by a means that many include, change in temperature, activating by exposure to light, adding a component that triggers a change in viscosity or increases gel strength. In some embodiments, the gelling is accomplished through the evaporation of fluid effectively concentrating the gelling agent and triggering gelation or increasing the viscosity effectively trapping the group of cells in place or slowing cellular velocity.
Mitigations to Distortion by Meniscus Effects and Evaporate Fluid Losses
[0093] In some embodiments, the group of cells are contained within a vessel. In some embodiments, the meniscus curvature at the air liquid interface of the vessel is reducing the optical artifacts associated with transmitted light illumination and optical interrogation of the group of cells. In some embodiments the walls of the vessel are designed to match the surface energy of the liquid containing the group of cells such that the meniscus curvature is reduced. In some embodiments, evaporation loss is reduced with the addition of a material over the culture media. In some embodiments, a liquid overlay is utilized to reduce the meniscus curvature and/or evaporative losses. In some embodiments, an oil overlay is utilized to match the surface energy of the vessel walls. In some embodiments, a viscous solution or gel-like substance is overlaid on the liquid containing the group of cells to reduce the meniscus curvature and/or evaporative losses.
Measuring a Response to the group of cells
[0094] At least one of the following aspects of the measurement are recorded including the time of acquisition, the spatial location, the quantitative results of the measurement.
[0095] In some embodiments, the measurement is acquired using an imaging device including but not limited to charge coupled devices (CCDs), complementary metal-oxide semiconductor (CMOS) devices, Indium Gallium Arsenide sensors, photomultiplier tubes, avalanche photodiodes, electron multiplying CCDs, scientific CMOS devices, or other similar devices.
[0096] In some embodiments, the elements of the imaging devices are utilized to determine the spatial and temporal measurements of the group of cells. In some embodiments the intensity and number of the elements, individually or in aggregate, are utilized to quantitatively assign a measure to the group of cells
[0097] Further descriptions, including but not limited to description of the methods utilized for the measurement of a response is further described in the references listed below, each of which is incorporated herein by reference in its entirety:
1. Larsen, B. M., Kannan, M., Langer, L. F., Khan, A. A., Igartua, C., & Salahudeen, A. A. (2021). A pan-cancer organoid platform for precision medicine. Cell Reports, 36(4), 109429. https://doi.Org/10.1016/j.celrep.2O21.109429
2. Larsen, B. M., Stein, M., Kartouny, M., Salahuadeen, A., Kannan, M., Khan, A., Sincher Freire, V., & Zhang, Y. (2021). Large scale organoid analysis (U.S. Patent Application No. US 2021/0172931 A1). U.S. Patent and Trademark Office.
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T., & Theis, F. J. (2013). An automatic method for robust and fast cell detection in bright field images from high-throughput microscopy. BMC Bioinformatics, 14, 297. https://doi.Org/10.1186/1471-2105-14-297
4. Kannan, M., Larsen, B., Khan, A., & Salahudeen, A. (2023). Artificial immunohistochemical image systems and methods (U.S. Patent No. US 202301455084 A1).
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5. Yoshikawa, A. L., Susaki, E. A., Omura, T., Takahashi-Kanemitsu, A., & Taketo, M. M. (2024). Blueprints from the plane to space: Outlook of the next-generation three-dimensional histopathology. Cancer Science, 115(1), 1-10. https://doi.org/10.1111/cas.16095
6. Shannon, M. J., Eisman, S. E., Lowe, A. R., Sloan, T. F. W., & Mace, E. M. (2024). cellPLATO - an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data. Journal of Cell Science, 137(20), Article jcs261887. https://doi.org/10.1242/jcs.261887
7. Skylaki, S., Hilsenbeck, O., & Schroeder, T. (2016). Challenges in long-term imaging and quantification of single-cell dynamics. Nature Biotechnology, 34(11), 1137-1144. https://doi.org/10.1038/nbt.3713
8. Askary, A., Chen, W., Choi, J., Du, L. Y., Elowitz, M. B., Gagnon, J. A., Schier, A. F., Seid, S., Shendure, J., Stadler, T., & Tran, M. (2024).
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10. Eilken, H. M., Nishikawa, S.-l., & Schroeder, T. (2009). Continuous single-cell imaging of blood generation from haemogenic endothelium. Nature, 457(7231), 896-
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11 . Meijering, E., Dzyubachyk, O., Smal, I., & van Cappellen, W. A. (2009). Tracking in cell and developmental biology. Seminars in Cell and Developmental Biology, 20(8), 894-902. https://doi.Org/10.1016/j.semcdb.2009.07.004 12. Weber, F., Isakr, S., Ragaller, F., Schlegel, J., Plochtberger, B., Sezgin, E., & Andronico, L. A. (2024). VISION - an open-source software for multi-dimensional image analysis of cellular biophysics. Journal of Cell Science, 137(5), jcs262166. https://doi.org/10.1242/ics.262166
13. Bray, MA., Singh, S., Han, H. et al. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nat Protoc 11 , 1757-1774 (2016). https://doi.orq/10.1038/nprot.2016.105
14. Stossi F, Singh PK, Safari K, Marini M, Labate D, Mancini MA. High throughput microscopy and single cell phenotypic image-based analysis in toxicology and drug discovery. Biochem Pharmacol. 2023 Oct;216:115770. doi: 10.1016/j.bcp.2023.115770
15. Yu, Y., Xueyan, C., Jieying, P., Huiheng, N., Yaojun, Z., Zemeng, Z., Yufei, B., Xianwen, R., Jiesheng, L., Dongfang, W., & Min-Min, C. (2024). Pan-cancer single-cell dissection reveals phenotypically distinct B cell subtypes. Cell, 206(6), 638. https://doi.Org/10.1016/i.cell.2024.06.038
[0098] In some embodiments, the optical methods are utilized to measure the group of cells, without limitation, and by example a method such as darkfield, white light (absorbance), phase contrast, differential interference contrast (DIC), side scatter, forward of reverse scatter, or other scattered light method wherein the signal correlates to an increase in the group of cells volume or mass. In some embodiments, quantitative phase contrast approaches are utilized. In some embodiments, holographic imaging techniques are employed. Further description of the methods utilized is further described in the references listed below, each of which is incorporated herein by reference in its entirety:
1. Winter, P. W., & Shroff, H. (2014). Faster fluorescence microscopy: Advances in high speed biological imaging. Current Opinion in Chemical Biology, 20, 46-53. https://doi.Org/10.1016/j.cbpa.2014.04.008
2. El-Schich, Z., Molder, A. L, & Gjbrloff Wingren, A. (2018). Quantitative phase imaging for label-free analysis of cancer cells — Focus on digital holographic microscopy. Applied Sciences, 8(7), Article 1027. https://doi.org/10.3390/app8071027
3. Goldberg, D. A., Howson, D. C., Metzger, S. W., & Buttry, D. A. (2005). Sensitive and rapid biodetection (International Publication No. WO 2005/027714 A2). World Intellectual Property Organization.
4. Metzger, S. W., Howson, D. C., Goldberg, D. A., & Buttry, D. A. (2017). Rapid microbial detection and antimicrobial susceptibility testing (U.S. Patent No. US 9,657,327 B2). U.S. Patent and Trademark Office.
5. Shirasaki, Y., Yamagishi, M., Suzuki, N., Izawa, K., Nakahara, A., Mizuno, J., Shoji, S., Heike, T., Harada, Y., Nishikomori, R., & Ohara, O. (2014). Real-time single-cell imaging of protein secretion. Scientific Reports, 4, Article 4736. https://doi.org/10.1038/srep04736 6. Liu, T., Yamagichi, Y., Shirasaki, Y., Shikada, K., Yamagishi, M., Hoshino, K., Kaisho, T., Takemoto, K„ Suzuki, T., Kuranaga, E., Ohara, O., & Miura, M. (2014). Single-cell imaging of caspase-1 dynamics reveals an all-or-none inflammasome signaling response. Cell Reports, 8(1), 974-982. https://doi.Org/10.1016/j.celrep.2014.07.012
7. Klein, J., Leupold, S., Biegler, I., Biedendieck, R., Munch, R., & Jahn, D. (2012). TLM- Tracker: Software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies. Bioinformatics, 28(17), 2276-2277. https://doi.Org/10.1093/bioinformatics/bts424
8. Prisbey, L., Metzger, S. W., & Gusyatin, O. (2024). Lens-free holographic optical system for high sensitivity label-free cell and microbial growth detection and quantification for screening, identification, and susceptibility training (U.S. Patent Application No. US 2024/0385099 A1). U.S. Patent and Trademark Office.
9. Zhou, Y., Mao, S., & Fei, P. (2024). Light sheet fluorescence microscopy: Advancing biological discovery with more dimensions, higher speed, and lower phototoxicity. The Innovation, 5(5), Article 100692. https://doi.Org/10.1016/j.xinn.2024.100692
10. Ravian, L. van Ineveld, Kleinnijenhuis, M., Alieva, M., de Blank, S., Barrera Roman, M., van Vliet, E. J., Martinez Mir, C., Johnson, H. R., Bos, F., Heukers, R., Chuva de Sousa Lopes, S. M., Dekkers, J. F., Wehren, E., Rios, A. C., Drost, J., Maxima Center for Pediatric Oncology, & Utrecht, T. N. (2021). Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D. Nature Biotechnology, 39(10), 1239-1245. https://doi.Org/10.1038/S41587-021-00926-3
[0099] In some embodiments, the measurement of a group of cells is accomplished by an increase in variability of signal intensity or structure of the image within a region of interest detected and defined using an optical method. In some embodiments, variability of signal intensity or structure is obtained from a holographic image. In some embodiments, the image is obtained from an inline hologram.
[00100] In some embodiments, surface plasmon resonance is utilized. In some embodiments, the group of cells interact with surface plasmon resonance resulting in detection. In some embodiments, spectroscopic methods are used to monitor the group of cells.
[00101] In some embodiments, light sheet microscopy can be used to measure the group of cells selectively illuminating a single plane of the sample, allowing rapid, three- dimensional imaging of living cells and tissues with virtual or optical sectioning capability. [00102] In some embodiments, Fourier transform spectroscopy, infrared spectroscopy are utilized for detection. In some embodiments, inelastic scattering approaches such as Brillouin or Raman spectroscopy are utilized. In some embodiments, spectroscopic techniques are utilized to image a surface area or volume.
[00103] In some embodiments, the molecular mass and the measurement using mass spectrometry provides a means for measuring the group of cells. This approach leverages advanced mass spectrometry techniques, such as Matrix-Assisted Laser Desorption/lonization (MALDI), to ionize and analyze biomolecules with high sensitivity and precision, enabling detailed characterization of cellular components. Additionally, techniques like laser microdissection coupled with mass spectrometry allow for spatially resolved molecular profiling, isolating specific cellular regions or rare cell populations to enhance the accuracy and specificity of the measurements.
[00104] In some embodiments, the electrical properties of the surface area or volume change over time in response to the number of cells in the initial group of cells. In some embodiments, the local resistance of the surface is altered. In some embodiments, the resistance change is frequency dependent. In some embodiments, the probe free method measures a change in frequency of the resonant structure of a micro machined cantilever in the presence of the group of cells.
Additional Processing
[00105] In some embodiments, the group of cells can be measured following additional processing. Cell fixation and permeabilization, clearing or clarification, dehydration, and epitope restoration are described in Bancroft's Theory and Practice of Histological Techniques (Eighth Edition) ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Formatebook, PublisherElsevier Masson, Language:English, Authors. Kim Suvarna, Editors. Kim Suvarna incorporated by reference herein in its entirety.
Cell Fixation and Permeabilization
[00106] In some variations, the group of cells can be measured following cell fixation and optionally permeabilization. Cell fixation can be performed with or without permeabilizing the cells. In some embodiments, at least one of the following performed for the purposes of facilitating measuring and include; fixation, permeabilization, clearing or clarification, dehydration.
[00107] In some embodiments, the group of cells are fixed. In some embodiments, fixation is performed prior to measuring a response probing. In some embodiments, the cellular constituents or cellular products are intracellular, requiring permeabilization of the cell outer membrane to provide access to the intracellular constituents or products.
[00108] In some embodiments, a covalent chemical fixative is utilized. In some embodiments, the chemical fixative contains at least one of the following; an aldehyde reagent such as formalin, paraformaldehyde, glyoxal, or other equivalent chemical agent for the purposes of cell fixation.
[00109] In some embodiments, permeabilizing agents are used to provide access to targets for probes intracellularly, in some embodiments, permeabilization of the outer membrane can be achieved by exposure to alcohol reagents. In some embodiments at least one of the following; ethanol, methanol, or other water miscible alcohols can be used to permeabilize cell membranes enabling access to intracellular targets. In some embodiments, a mixture of water and alcohol is utilized. In some embodiments, a 70 percent mixture of alcohol and water is utilized.
[00110] In some embodiments, a hemolytic fixative (CytoRich Red Preservative) is added. In some embodiments, a detergent is used for its hemolytic properties. In some embodiments, a non-ionic detergent is used. In some embodiments, Triton X 100 is used. In some embodiments, an ionic detergent is used. In some embodiments, sodium dodecyl sulfate (SDS) is used (Chow et al. 2005).
Clearing or Clarification
[00111] In some variations, the group of cells can be measured following clearing or clarification. Clearing or clarification can be performed with or without fixation and permeabilizing the cells.
[00112] In some embodiments, the material is clarified. In some embodiments, the material is clarified after fixation. In some embodiments, an organic solvent or mixture of solvents is utilized. In some embodiments of at least one of the following, the solution is methanol with or without hexane or tetrahydrofurane (THF) alone. In some embodiments, the solution is at least one of the following; ethylsalicilate, benzyl alcohol, benzyl benzoate, dichloromethane and dibenzyl ether. In some embodiments, combinations of solutions are utilized. In some embodiments, ethylsalicilate, benzyl alcohol, benzyl benzoate, dichloromethane and dibenzyl ether are the final clarifying solutions as described by (Richardson, Douglas S., and Jeff W. Lichtman. 2015. “Clarifying Tissue Clearing.” Cell 162 (2): 246-57, incorporated herein by reference in its entirety) without limitation and the entire contents incorporated herein. In some embodiments, the clarifying solutions include at least one of the following; xylene, toluene, benzene, chloroform, methyl salicylate, methyl benzoate, butyl acetate, isopropyl acetate, amyl acetate, propylene oxide, and limonene.
Dehydration
[00113] In some variations, the group of cells can be measured following a dehydration step. Dehydration can be performed with or without fixation or permeabilizing the cells. Dehydration can be performed with or without clearing or clarification.
[0100] In some embodiments, removal of water is required. In some embodiments, the removal of water is required prior to embedding. In some embodiments, the water is evaporated from the sample. In some embodiments, the water is replaced with less hydrophilic solvent. In some embodiments, a sample is dehydrated using an agent that is miscible with water such that the sample can be rid of water prior to embedding of the sample in compatible water soluble or water insoluble wax for the histology purposes. In some embodiments, multiple rounds of dehydration are performed with increasing concentrations of dehydrating reagent. In some embodiments, dehydration is accomplished using mixtures of alcohol and water. In some embodiments, the alcohol percentage volume composition is less than 10%. In some embodiments, the alcohol percentage of the dehydration solution can be greater than 90%. In some embodiments, the alcohol percentage of the dehydration solution can be 100%. In some embodiments the mixture of alcohol percentage may increase with sequential dehydration steps. In some embodiments, ethyl alcohol is utilized as a dehydrant. In some embodiments at least one of the following; methanol, acetone, denatured alcohol, butanol, dioxane, tetrahydrofuran, ethylene glycol, propylene glycol, isopropyl alcohol is used as a dehydrant. Epitope Restoration [0101] Epitope restoration can be performed with or without the following; fixation or permeabilizing the cells, clearing or clarification, dehydration.
[0102] In some embodiments, the material requires restoration of epitopes to enable measurement using probes that target specific molecules contained by a group of cells. In some embodiments, the restoration is required after fixation. In some embodiments, Protease- induced Epitope Retrieval is utilized. In some embodiments, enzymes including at least one of; Proteinase K, Trypsin, and Pepsin have been used successfully to restore the binding of an antibody to its epitope. In some embodiments, Heat-induced Epitope Retrieval is utilized to restore epitopes. In some embodiments, the heating is performed in at least one of the following; microwave ovens, pressure cookers, vegetable steamers, autoclaves, or water baths. It is known to those well skilled in the art there are a wide range of epitope restoration techniques available. The methods disclosed herein are not meant to be limiting.
Histological Stains for Measurements of the group of cells
[0103] In some embodiments, histological stains can be employed to detect molecular properties within a group of cells, potentially revealing structural and biochemical characteristics relevant to therapeutic response assessment. Hematoxylin and Eosin (H&E) staining may be utilized, wherein hematoxylin can bind to acidic molecules, including but not limited to DNA and RNA in the nucleus, potentially staining them a color that can be dark relative to lighter shades, such as blue, while eosin may bind to basic proteins in the cytoplasm and extracellular matrix, potentially staining them a color that can be light relative to darker shades, such as pink, thereby revealing general cellular organization and some molecular distribution. Periodic Acid- Schiff (PAS) staining can target carbohydrates, including but not limited to glycogen and mucopolysaccharides, by reacting with sugar molecules, potentially indicating their presence in a group of cells or tissue preparations. Oil Red O staining may be applied to detect lipids by binding to neutral fats, potentially highlighting molecular properties related to fat storage or metabolism. Trichrome stains can differentiate collagen, which may be rich in specific amino acids, from muscle or cytoplasm, potentially reflecting protein composition and distribution within the sample. These staining methods may facilitate the visualization of molecular properties, supporting the characterization of a group of cells for diagnostic or pharmaceutical development purposes.
[0104] For those skilled in the art, a description of techniques for visualizing the group of cells using techniques such as immunohistochemical or histochemical staining methods are described in Bancroft's Theory and Practice of Histological Techniques (Eighth Edition) ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Format:ebook, PublishecElsevier Masson, Language:English, Authors. Kim Suvarna, Editors. Kim Suvarna incorporated by reference herein in its entirety.
Probes for Measurements of the group of cells
[0105] Probes can be used to detect target molecules without fixation or permeabilizing the cells, clearing or clarification, dehydration, or epitope restoration. In other variations, probes can be used to detect the presence of specific molecules in cells once permeabilized.
[0106] As used interchangeably herein, the term "probe," "probe reagent" or "probe molecule" refers to an entity (e.g., but not limited to, a molecule, a particle, a composite entity, or a multi-molecular entity) that interacts with or binds to a target molecule or an analyte for the analysis of the target or the analyte. Typically, the nature of the interaction or binding is noncovalent, e.g., by hydrogen, electrostatic, or van der Waals interactions, however, binding can also be covalent. Probe reagents can be entities (e.g., but not limited to, molecules, particles, composite entities, or multi-molecular entities) capable of undergoing binding or molecular recognition events with target molecules. Probe reagents can be naturally-occurring, recombinant or synthetic. Examples of the probe reagent can include, but are not limited to, a nucleic acid, an antibody or a portion thereof, an antibody-like molecule, an enzyme, a cell, an antigen, a small molecule, a protein, a peptide, a peptidomimetic, an aptamer, and any combinations thereof. By way of example only, in immunohistochemistry, the probe reagent can include an antibody specific to the target antigen to be analyzed. An ordinary artisan can readily identify appropriate probe reagents for the target molecules or analytes of interest to be detected in various bioassays. In some embodiments, the probe reagent can be multi- molecular. For example, in one embodiment, the probe reagent can comprise a particle, an antibody, biotin and/or streptavidin, or any combinations thereof.
[0107] In some embodiments, the probe reagents can be modified by any means known to one of ordinary skill in the art. Methods to modify each type of probe reagents are well recognized in the art. Depending on the types of probe reagents, an exemplary modification includes, but is not limited to genetic modification, biotinylation, labeling (for detection purposes), chemical modification (e g., to produce derivatives or fragments of the probe reagent), and any combinations thereof. In some embodiments, the probe reagent can be genetically modified. In some embodiments, the probe reagent can be biotinylated.
[0108] The term "nucleic acids" used herein generally refers, without limitation, to polymers (polynucleotides) or oligomers (oligonucleotides) of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar linkages. The term "nucleic acid" also includes polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Exemplary nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acids (PNA), and polymers thereof in either single- or double-stranded form. Locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo conformation. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such LNA oligomers are generally synthesized chemically. Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA. DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds. PNA is generally synthesized chemically. Unless specifically limited, the term "nucleic acids" encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues ( Batzer, et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka, et al., J.
Biol. Chem. 260:2605-2608 (1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). The term "nucleic acid" should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, single (sense or antisense) and double-stranded polynucleotides.
[0109] In some embodiments, the term "nucleic acid" described herein, without limitation, can include a modified nucleic acid. Modified nucleic acids are well known in the art. Thus, a nucleic acid described herein can comprise one or more nucleic acid modifications known in the art. For example, the nucleic acid can comprise one or more nucleic acid modifications selected from the group consisting of internucleotide linkage modifications (intersugar linkage modifications), sugar modifications, nucleobase modifications, backbone modifications/replacements, and any combinations thereof. Exemplary internucleotide linkage modifications include, but are not limited to, phosphorothioate, phosphorodithioate, phosphotriester (e.g. alkyl phosphotriester), aminoalkylphosphotriester, alkyl-phosphonate (e.g., methyl-phosphonate), selenophosphate, phosphoramidate (e.g., N- alkylphosphoramidate), boranophosphonate, and the like. Exemplary sugar modifications include, but are not limited to, 2'- O-Me (2'- O-methyl), 2'- O-MOE (2- O-methoxyethyl), 2'-F, 2'- 0-[2-(methylamino)-2-oxoethyl] (2'- O-NMA), 2'-S-methyl, 2'-O-CH 2-(4'-C) (LNA), 2'-O-CH 2CH 2-(4'-C) (ENA), 2'-0-aminopropyl (2'-O-AP), 2'-0-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-O-DMAEOE), arabinose sugar, and the like. Exemplary nucleobase modifications include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 5-methylcytosine (5- me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other 6-alkyl derivatives of adenine and guanine; 2-propyl and other 2-alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-propynyl uracil; 5-propynyl cytosine; 6-azouracil; 6-azocytosine; 6-azothymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methyl and other 7-alkyl derivatives of adenine and guanine; 8-azaguanine; 8-azaadenine; 7- deazaguanine; 7-deazaadenine; 3-deazaguanine; 3-deazaadenin; universal base; and any combinations thereof. Exemplary backbone modifications include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA ( aegPNA), backnone-extended pyrrolidine PNA ( bepPNA), and the like.
[0110] As used herein, the term "antigens" generally refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as, without limitation, an antibody, and additionally capable of being used in an animal to elicit the production of antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes. The term "antigen" can also refer to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by Major Histocompatibility Complex (MHC) molecules. The term "antigen", as used herein, also encompasses T-cell epitopes. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a T cell epitope and is given in adjuvant. An antigen can have one or more epitopes (B- and T-epitopes). The specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.
[0111] In some embodiments, the probe reagent can be an antibody or a portion thereof, or an antibody-like molecule. In such embodiments, the probe reagents can be used to, for example, detect and/or identify pathogen type or species, the presence of cell or disease markers, cellular protein expression levels, phosphorylation or other post-translation modification state, or any combinations thereof.
[0112] As used herein, the term "aptamers" generally means, without limitation, a single-stranded, partially single-stranded, partially double-stranded or double-stranded nucleotide sequence capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules. In some embodiments, the aptamer recognizes the non- oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers can include, without limitation, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branchpoints and nonnucleotide residues, groups or bridges. Methods for selecting aptamers for binding to a molecule are widely known in the art and easily accessible to one of ordinary skill in the art.
[0113] As used herein, the term "antibody" or "antibodies" refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. The term "antibodies" also includes "antibody-like molecules", such as fragments of the antibodies, e.g., antigen-binding fragments. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. "Antigen-binding fragments" include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Linear antibodies are also included for the purposes described herein. The terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard immunological meanings ( Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)). Antibodies or antigen-binding fragments specific for various antigens are available commercially from vendors such as R&D Systems, BD Biosciences, e- Biosciences and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art.
[0114] As used herein, the term "Complementarity Determining Regions" (CDRs; i.e. , CDR1 , CDR2, and CDR3) generally refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat ( i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. , Seguences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" ( i.e. about residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987), each of which is incorporated by reference herein in its entirety). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
[0115] The expression "linear antibodies" generally refers to the antibodies described in Zapata et al. , Protein Eng., 8(10):1057-1062 (1995), which is incorporated herein by reference in its entirety. Briefly, these antibodies comprise a pair of tandem Fd segments (VH -CH1-VH- CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
[0116] The expression "single-chain Fv" or "scFv" antibody fragments, as used herein, is intended to mean antibody fragments that comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. (Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994)).
[0117] The term "diabodies," as used herein, refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) Connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. ( EP 404,097; WO 93/11161 ; Hollinger et ah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993), each of which is incorporated herein by reference in its entirety).
[0118] As used herein, the term "peptidomimetic" refers to a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide
[0119] In some embodiments, the group of cells , the constituents of the group of cells , or the products of the group of cells are detected using probes.
[0120] In some embodiments the probe binds to a molecule having a nucleic acid, protein, carbohydrate, or lipid-like characteristics. In some embodiments, the probe binds to epigenetic targets. Some examples, not meant to be limiting include at least one of the following; DNA methylation, histone modifications, non-coding RNAs, Chromatin remodeling complexes, Polycomb and Trithorax group proteins, Bromodomain-containing proteins, histone deacetylases (HDACs) and histone acetyltransferases (HATs), or DNA demethylases [0121] In some embodiments, the probe binds antigen or antigen like targets for the purposes of detection
[0122] In some embodiments, the probes are a member of aptamer, antibody, and nucleic acid binding protein classes of reagents. In some embodiments, the cellular products are generated by metabolic processes and detected. In some embodiments, the cellular products are cytokines, chemokines, or other cell signaling molecules. In some embodiments, the cellular products are detected extracellularly. [0123] In some embodiments, the group of cells are detected using probes with complementary nucleic acid sequences to targeted regions. In some embodiments, the complementary nature of the probes may be incomplete, having a single or plurality of bases mismatched or missing relative to the targeted sequence. In some embodiments, the hybridization assays are used to probe nucleic acid sequences for detection. In some embodiments, hybridization assays are used to probe and detect ribosomal RNA, messenger RNA, transfer RNA, and DNA. In some embodiments, fragments of nucleic acids may be detected. In some embodiments the detection of one or more nucleic acid sequences is performed. In some embodiments, the detection of one or more nucleic acid sequences corresponds to an insertion or deletion or single nucleotide variation (SNV). In some embodiments, the detection of one or more nucleic acid sequences corresponds to a translocation relative to an expected sequence. In some embodiments, the nucleic acid target is a DNA sequence found in a chromosome or in the mitochondria. In some embodiments, the nucleic acid target is an RNA sequence. In some embodiments, the nucleic acid target is messenger RNA. Exemplary methods for nucleic acid detection using hybridization that are applicable to the methods are additionally described herein include, without limitation, the methods described in U.S. Pat. No. 7,473,767, US patent publication no. 2007/0166708, and US application number US 2010/0261026.
[0124] In some embodiments, in situ PCR of tissue as described without limitation by (Janiszewska et al. 2015), the entire contents incorporated herein, is performed amplifying the region or regions of interest and facilitating robust detection through additional steps of targeting the amplified regions with probes. In some embodiments, the PCR probes are allelespecific, and will amplify the DNA if a specific SNV is present. In some embodiments, copy number alterations compare the detection intensity of the targeted genomic region to that of a reference region to determine whether there are copy number alterations (deletions or amplifications) in the group of cells. In some embodiments, a plurality of padlock probes is utilized for detection, the probe sequence is amplified then later detected.
[0125] In some embodiments, the probes utilized are padlock probes as described, without limitation, by (Larsson C, Koch J, Nygren A, Janssen G, Raap AK, Landegren U, Nilsson M. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods. 2004 Dec;1 (3):227-32. doi: 10.1038/nmeth723.) with the entire contents incorporated herein. In some embodiments, detection of RNA is accomplished using ROLL FISH as described by (Wu, C., Simonetti, M., Rossell, C. et al. RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin- embedded tumor tissue samples. Commun Biol 1 , 209 (2018). https://doi.org/10.1038/s42003- 018-0218-0), or described by (Raj, A., van den Bogaard, P., Rifkin, S. et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5, 877-879 (2008). https://doi.org/10.1038/nmeth.1253 ), or smFISH as described (Crosetto, N., Bienko, M. & van Oudenaarden, A. Spatially resolved transcriptomics and beyond. Nat Rev Genet 16, 57-66 (2015). https://doi.org/10.1038/nrg3832), the entire contents of the each reference is incorporated herein by reference.
Plurality of Measurements
[0126] In some variations, a single response is measured. In other variations, a plurality (i.e. , two or more) responses are measured.
[0127] In some embodiments, a plurality of probes is utilized. In some embodiments, a plurality of probes and a plurality of tags are utilized.
[0128] In some embodiments, an additional aspect of specificity is utilized to enhance the fidelity of the detection of targets. Exemplary methods described within United States Patent 9677131 , United States Patent 9777315, United States Patent 10731206, and United States Patent 9902993. The entire contents of these patent documents are incorporated herein by reference without limitation. In some embodiments, a plurality of probes is used for a target. In some embodiments, the plurality of probes contains complementary nucleic acid sequences. In some embodiments, the complementary sequences hybridize providing a means for additional specificity of the binding of the probes to the target effectively reducing non-specific probe binding sources of noise. In some embodiments, the described approach is particularly useful for detection in volumes.
[0129] In some embodiments, the complementary probes direct subsequent amplification of the sequence. In some embodiments, the amplification incorporates reporter mechanisms. In some embodiments the reporter mechanisms are incorporated after amplification.
[0130] It is noted that to those skilled in the art, there are additional embodiments and combinations of methods described to achieve the goals of nucleic acid detection. A review article of useful approaches (Gaspar I, Ephrussi A. Strength in numbers: quantitative singlemolecule RNA detection assays. Wiley Interdiscip Rev Dev Biol. 2015 Mar-Apr;4(2):135-50. doi: 10.1002/wdev.170. Epub 2015 Jan 21. PMID: 25645249; PMCID: PMC5024021), the entire contents without limitation incorporated herein, describes additional techniques that are not discussed for brevity.
[0131] In some embodiments, the probe binds to targets that are accessible with having to access the cell membrane. In some embodiments, the probe binds to targets in cells that have their membrane compromised or sufficiently permeable to support detection of targets internal to the cell membrane. In some embodiments, the specific probes are present on the outer membrane or diffuse away from the cell and can be accessed without crossing the cellular wall and do not require permeabilization of the cell. In some embodiments, the detection of cellular products localizes the products enabling association of the cellular products with the group of cells. In some embodiments, the extracellular signal is detected and localized using a precipitation reaction or localized by capture agents. [0132] In some embodiments, the measuring of the targets is accomplished with probes that contain a reporter mechanism that enables detection of the probe and by extension the presence of the target and further by extension the detection of the group of cells , substantial constituents of the group of cells , or products of the cell or the group of cells. In some embodiments, the probes are directly tagged with fluorescent reporter molecules. In some embodiments, the tagging is accomplished through additional means of using an additional probe to detect the presence of the precursor probe. In some embodiments, secondary detection methods commonly employed in antibody detection assays are utilized. In some embodiments, the additional means of tagging is accomplished with the covalent attachment of nucleic acid oligomers to serve as bar codes for each specific probe. In some embodiments, the reporter molecules are utilized to enable multiplexing. In some embodiments, multiplexing is accomplished through temporal methods as described herein. In some embodiments, the reporter molecules selectively report enabling the simultaneous detection of a plurality of reporter molecules.
Reporter Mechanisms Used with Probes
[0133] In some embodiments, a reporter mechanism is utilized to provide a means for the detection of any number of probes. In some embodiments, a reporter mechanism is utilized to provide a means for the detection of any number of hybridization events. In some embodiments, a reporter mechanism is utilized to provide a means for the detection of any number of target-probe events. In some embodiments, the reporter mechanism is detectable using an exclusion of signal approach. In some embodiments, the reporter signal kinetics are utilized to enhance detection.
[0134] In some embodiments, the reporter mechanism amplifies the signal generated through the use of techniques known to those skilled in the art. In some embodiments, amplification is achieved using rolling circle amplification.
[0135] In some embodiments, the reporter mechanism generates a detectable fluorescent signal and is attached to the probe providing a means for localization of the target by detectable fluorescence. In some embodiments, the reporter mechanism is detectable using a fluorescence polarization. In some embodiments, the reporter mechanism is detectable using fluorescence resonance energy transfer. In some embodiments, the reporter is a radioactive emitter incorporated into the probe or probes. In some embodiments, the reporter mechanism generates a chemiluminescent signal. In some embodiments, the reporter mechanism generates an electrochemical signal. In some embodiments, the reporter mechanism generates a magnetization or magnetic flux signal. In some embodiments, the report is a colorimetric report.
[0136] In some embodiments, the reporter amplifies the optical detection using at least one of the following; absorbance, scattering, refraction, raman, or brillouin. In some embodiments, the reporter is a precipitant. In some embodiments, the precipitant is detected by the production of color or absorbance, scatter, or other optical technique. In some embodiments, the presence of the reporter alters the impedance. In some embodiments, a combination of reporter mechanisms is utilized to detect any number of target-probes.
Detection of Reporter Mechanism(s)
[0137] Detection is accomplished by any means available in the art that is capable of detecting the specific signals on a given reporter mechanism utilized.
[0138] Detection is accomplished using means readily implemented by those skilled in the art. For the purposes of illustration and not wishing to be limiting, fluorescent detection is further described where in suitable consideration of appropriate excitation sources can be readily determined. Possible sources can include but are not limited to arc lamp, xenon lamp, lasers, light emitting diodes or some combination thereof. The appropriate excitation source is used in conjunction with an appropriate optical detection system, for example an inverted fluorescent microscope, an epi-fluorescent microscope or a confocal microscope. Preferably, a microscope is selected that detects with enough spatial resolution to separate distinct signals. [0139] In some embodiments, the reporter may be the molecular mass of a molecule measured from a group of cells. In some embodiments the detection of the mass is detected using mass spectrometry, that may include matrix-assisted laser desorption/ionization (MALDI).
Measurement Frequency, and Longitudinal Tracking
[0140] In some embodiments, the detection is of sufficient frequency in order to track the shifting of cells relative to their initial position such that all cells, and their corresponding group of cells, can be tracked over time. In some embodiments, the initial cells can be assigned to an initial group of cells. In some embodiments, the cells may move overtime and a stationary reference point is used to identify spatial location of area containing the moving cells during repeated detection. In some embodiments, a vessel is imaged repeatedly. In some embodiments, a vessel is imaged repeatedly and the features of the microplate are utilized as reference points that create a bounding area that contains the cells. In some embodiments, the microplate wells contain features wherein the purpose of features is to act as a reference point or plurality of reference points.
[0141] In some embodiments, the entire volume or surface that contains the group of cells is detected over time. In some embodiments, areas or regions of interest are first identified such that the later amount of area or volume over time is reduced. In some embodiments, the regions of interest are determined by sampling all the volume or area for a period of time then identifying regions of interest within this area or volume that are useful based on useful detections.
Measures of the group of cells
[0142] In some embodiments, at least one of the following; sample preparation, entombment, permeabilization, clearing or clarification, dehydration, embedding, and fixation are performed prior to measuring the response. In some embodiments at least one of the following measures is of utility.
[0143] Measures known to those skilled in the art and the corresponding methods are described in the following:
1. Current Protocols in Molecular Biology (ISSN 1934-3639), published by John Wiley & Sons, Inc.,
2. Current Protocols in Chemical Biology, (n.d.). Wiley Online Library. Retrieved March 24, 2025, from https://currentprotocols.onlinelibrarv.wilev.com/iournal/19406509,
3. Current Protocols in Cytometry, (n.d.). Wiley Online Library. Retrieved March 24, 2025, fro m https://currentprotocols.onlinelibrarv.wiley.com/iournal/19349297,
4. Current Protocols in Immunology, (n.d.). Wiley Online Library. Retrieved March 24, 2025, from https://currentprotocols.onlinelibrarv.wiley.com/iournal/1934368X,
5. Current Protocols in Neuroscience, (n.d.). Wiley Online Library. Retrieved March 24, 2025, from https://currentprotocols.onlinelibrarv.wiley.com/iournal/19348517,
6. Current Protocols in Nucleic Acid Chemistry, (n.d.). Wiley Online Library. Retrieved March 24, 2025, from https://currentprotocols.onlinelibrarv.wiley.com/iournal/19349289 [0144] each of which is incorporated herein in its entirety by reference.
Cellular State
[0145] In some embodiments, the measurement of the group of cells can be utilized for the purposes of determining the cellular state of the group of cells.
[0146] In some embodiments, probes utilized for detection of the group of cells can be utilized for the purposes of determining the cellular state of the group of cells.
[0147] In some embodiments, the detection of the state of the group of cells includes molecules having substantial characteristics of one of the following proteins, carbohydrates, nucleic acids, or lipids. In some embodiments, the state of cells is characterized by epigenetic events detected. In some embodiments, detection of the presence and abundance (or lack thereof) of receptors or ligands is determined. In some embodiments, enzymes are detected. In some embodiments, the production of a substrate is detected by enzymes.
[0148] The term "enzymes" as used here generally refers to a protein molecule that catalyzes chemical reactions of other substances without it being destroyed or altered upon completion of the reactions. The term can include without limitation naturally occurring enzymes and bioengineered enzymes or mixtures thereof. Examples of enzyme families include kinases, dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyl transferases, decarboxylases, transaminases, racemases, methyl transferases, formyl transferases, and a- ketodecarboxylases.
[0149] In some embodiments, the cellular pathway or cellular mechanism includes the interaction between cells or the same or distinct populations of cells. In some embodiments, the cellular pathway or cellular mechanism involves the immunologically and non-immunologically related group of cells.
[0150] In some embodiments, the detection of the group of cells can be utilized for the purposes of determining the viability of a group of cells. In some embodiments, cell viability using MTT or CellTiter-G Io assays or apoptosis activity using caspase-3/7 are utilized. In some embodiments, intracellular constituents of the group of cells are measured to assess cell death as defined by the penetration of a viable cell membrane impermeable dye including but not limited to propidium iodide or YOPRO.
[0151] In some embodiments, the group of cells is determined to be organized in such a way that the response to external therapy is coordinated by the collective response of the group of cells. In some embodiments, the group of cells may be of distinct types relative to one another as detected by some means.
[0152] In some embodiments, the functional response detected is immunologically related. In some embodiments, the ligand detected is the programmed cell death ligand 1 on cancer cells. In some embodiments, immunosuppressive molecules expressed or their receptors are detected. In some embodiments, immune checkpoints such as programmed death-ligand 1/programmed death-1 (PD-L1/PD-1), galectin-9/TIM-3, IDO1 , LAG-3, and CTLA4 are detected . In some embodiments, the group of cells may transition between cellular states such as the Mesenchymal-to-epithelial transition and be detected. In some embodiments, the group of cells, including distinct cells that operate in an organized manner, may be determined as the fundamental entity in which a population model of the group of cells can be constructed.
[0153] In some embodiments, extracellular products in proximity to the group of cells are measured over time to assess cell state. In some embodiments, extracellular products are contained and measured over time to assess cell state. In some embodiments the diffusion of extracellular products is reduced by entombing the group of cells or using a viscous solution to limit diffusion. In some embodiments, the extracellular products are measured at a frequency that determines production of the extracellular products in a shorter time period such that the amount integrated over the entire period can be determined. In some embodiments, the rate of change is measured for a period of time. In some embodiments, the extracellular signal is detected and localized using a precipitation reaction or localized by capture agents.
Growth
[0154] In some embodiments, the measurement of the group of cells can be utilized for the purposes of measuring growth of a cell or the group of cells.
[0155] In some embodiments, the group of cells are monitored over time to assess the group of cells prior to a therapy. In some embodiments, the number of cells is enumerated over time. In some embodiments, the mass or volume of cells is measured over time. In some embodiments, the number of division events is measured over time. In some embodiments, cellular constituents produced by the group of cells are assessed using a measure of cell state. In some embodiments, the measure of cell state correlates to growth of the group of cells. In some embodiments, the measure of cell state correlates to the function of the group of cells producing a cellular constituent of relevance to growth or the expected production of said cellular constituent. In some embodiments, insulin is produced and detected indicative of proliferation or stasis and of utility. In some embodiments, BrdU incorporation is utilized to measure growth.
[0156] In some embodiments, the monitoring of the group of cells over time can be utilized to longitudinally track and measure over time providing a means for monitoring the growth kinetics. In some embodiments, a rate of change can be determined by detection over a period of time. In some embodiments, the measure for the rate of change can increase (i.e., growth) or decrease (i.e., growth arrest). In some embodiments, the rate of change can be instantaneous with monitoring over a short period of time relative to the cellular division rate. In some embodiments, an average rate of change over a longer period of time can be determined. Cell Interaction
[0157] In some embodiments the interaction of the cells is detected by means of associating the one with the other demonstrating a non-random or stochastically driven specific interaction. In some embodiments, the nature of the interaction is for one cell to impact the viability of the other. In some embodiments, the interaction negatively affects the other cell through a mechanism associated with at least one of the following; the attack of the cell, trigger of programmed cell death, nutritional competition, release of extracellular components, or other mechanism. In some embodiments the molecules responsible for the negative interaction are detected and associated with a cell, its cell constituents, its cell products, or its group of cells. Spatial Distribution of Cells
[0158] In some embodiments, the spatial presence of molecular targets is detected. In some embodiments, the sum total of targets is detected. In some embodiments, the heterogeneity of cell distribution is detected and characterized. In some embodiments, the target density is normalized by a cell type and characterized. In some embodiments, the induction of immunosuppressive cells accumulates and is detected. In some embodiments, the distribution of a known or previously unknown biomarker containing cells is detected. In some embodiments, the distribution of HER2 is detected.
Cell Differentiation
[0159] In some embodiments, the intra cellular morphology, cellular morphology, proliferation rates, and organizational structure of the group of cells are utilized for the purposes of differentiating cell types.
[0160] In some embodiments, cell differentiation from other types is achieved using measurements of the group of cells. [0161] In some embodiments, the cells are differentially probed using a probe to a nucleic acid sequence that is specific for a cell type. In some embodiments, cell specific probes, such as EPCAM (Nicolazzo C, Gradilone A, Loreni F, Raimondi C, Gazzaniga P. EpCAMIow Circulating Tumor Cells: Gold in the Waste. Dis Markers. 2019 Sep 17;2019: 1718920. doi: 10.1155/2019/1718920. PMID: 31636732; PMCID: PMC6766153.), can be used to monitor targets associated with tumor cells and in the presence of non-tumor cells found in tissue samples. In some embodiments, mRNA expression properties for a group of cells can be used to classify by cell type. In some embodiments, the use of a probe to detect a single nucleotide variant is utilized for cell differentiation. In some embodiments, a plurality of probes is used to detect a single nucleotide variant for the purposes of cell differentiation. In some embodiments, a plurality of probes is used to detect a plurality of single nucleotide variants for the purposes of cell differentiation. In some embodiments, a probe or probes are utilized to differentiate stem cells from other cells. In some embodiments, a probe or probes are utilized to differentiate mesenchymal to epithelial transition states of cells.
Cell Death Assessment
[0162] In some embodiments the probes are used to assess the death of the group of cells. In some embodiments, the criteria for death are determined by the integrity of the cell wall.
Biomarker Detection
[0163] In some embodiments, the group of cells are processed using a standard hematoxylin and eosin (H&E) histology stain. In some embodiments, the tracked cells of the group of cells are differentiated by the analysis of standard histology staining. In some embodiments, the analysis from standard H&E stains is recorded by computerized means to classify the tracked group of cells. In some embodiments, the biomarker has known or previously unknown clinical significance. In some embodiments, specific antibody probes are utilized to bind to specific target proteins for diagnostics purposes. In some embodiments, protein expression levels are detected using antibody recognition and the group of cells with varying levels of expression are compared. In some embodiments, the detected group of cells is accomplished using immunolabeling techniques using antibodies against such targets, including but not limited to cytokeratins and Homeobox protein cdx2 and detected using means familiar to those skilled in the art. In some embodiments, enumeration of the group of cells containing the following protein targets including but not limited to Anaplastic lymphoma kinase (ALK) translocation, Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3) translocation, and rearranged during transfection (RET) translocation, mismatch repair (MMR), and HER2 is achieved with detection.
[0164] In some embodiments, the target is a biomarker of known or potential clinical significance on the group of cells detected using probes as well known to those skilled in the art. In some embodiments, the target is a single nucleotide variant. In some embodiments, the single nucleotide variant is detected on a chromosome.
[0165] Nucleic acids (e.g., RNA or DNA) can be identified or measured. In some embodiments, the nucleic acid targets specific genes or portions of a specific gene. In some embodiments, the nucleic acid targets are for specific fusion genes. In some embodiments the fusion gene targets are for Anaplastic lymphoma kinase (ALK) translocation, Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3) translocation, and rearranged during transfection (RET) translocation. In some embodiments, the targets contain both a gene target and a marker for the specific portion of the chromosomal DNA. In some embodiments, the centromere 17 (CEP17) and erythroblastic oncogene B-2 (ERBB2) genes are targeted. In some embodiments, enumeration of CEP17 and ERBB2 signals detection and enumeration in the nuclei of the group of cells. In some embodiments, a control of non-tumor-containing tissue is compared.
Utility of Measures
[0166] In some embodiments, the detected group of cells are isolated from one another, physically or in silico, providing a means for analysis of each group of cells separately.
[0167] In some embodiments, the group of cells , detected by any means previously described, may be determined as the fundamental entity in which characterization of the population of the group of cells can be generated to characterize the sample.
[0168] In some embodiments, the measurements capture the heterogeneity of the differentiated cells of the same type or the group of cells containing different cell types. In some embodiments, measuring indicates the presence of a specific cell population.
[0169] In some embodiments, a plurality of measures can be utilized to serve to indicate or increase the confidence in the indication that a cell is of a certain type or class or has a certain product produced as a function of the cell life cycle.
[0170] In some embodiments, the detected group of cells is determined to have likely or has originated from an original cell. In some embodiments the determination of origination is determined by detecting growth of the group of cells. In some embodiments, the detected group of cells may be determined as a fundamental entity in which characterization of the population of all the group of cells can be generated.
[0171] In some embodiments, the detections determine the cells are of the same lineage.
[0172] In some embodiments, the detections of the types previously described represent orthogonal or complementary measures of each fundamental entity. In some embodiments, at least one of the detections and measures from the detections are utilized for in silico creation of a population model of the entities. In some embodiments, the effect is upon the cell growth or production of cellular constituents or cellular products. In some embodiments, the observation of the effect is measured over a time period. In some embodiments, the cellular constituents or cellular products are detected after substantial interaction time has elapsed. In some embodiments, the observation of the effect is at the endpoint after a portion of time. In some embodiments, the detection of cellular constituents or cellular products occurs after proliferation or cell death.
[0173] In some embodiments, the response is determined by at least one measure. In some embodiments, the response is determined by two or more measures.
[0174] The response can be determined by comparing measures of the group of cells tested in the presence of the therapy and compared to the measures of the group of cells tested in the absence of the therapy. In other embodiments, the sample treated with the compound is not compared to the portion of the sample that is not treated with the compound. [0175] In some embodiments, the response is determined by comparing the prior measurements of the group of cells to the measurements made after the therapy is present. [0176] In some embodiments, the response is determined for each group of cells. In some embodiments, the aggregate of the group of cells is utilized to determine the response of the population.
[0177] In some embodiments, the heterogeneity of cell death correlates to the response to the therapy. In some embodiments, the correlated response predicts the efficacy of the therapy for diagnostic or pharmaceutical development purposes.
[0178] In some embodiments, the heterogeneity of the measures of the group of cells detected correlates to the response to the therapy. In some embodiments, the correlated response predicts the efficacy of the therapy for diagnostic and/or pharmaceutical development purposes.
[0179] In some embodiments, the density of targets per unit area or unit volume correlates to the responsiveness to the therapy.
[0180] In some embodiments, the receptors or ligands are correlated with a response to the therapy. In some embodiments, the response to the therapy is related to altering, blocking, initiating, accelerating, or continuing unchanged a targeted cellular pathway or cellular mechanism.
[0181] In some embodiments, the monitoring of the group of cells over time can be utilized to longitudinal track the response over time providing a means for monitoring the kinetics of the response to a therapy. In some embodiments, a rate of change can be determined by detection over a period of time and correlated with a therapy. In some embodiments, the measure for the rate of change can increase (i.e., growth) or decrease (i.e., growth arrest) in response to the therapy.
[0182] In some embodiments, the heterogeneity of the cellular interaction correlates to the response to the therapy.
[0183] In some embodiments, the characterization of the sample can capture the heterogeneity of the response of differentiated cells of the same type or group of cells containing different types to a therapy. [0184] In some embodiments, measuring indicates the presence of a specific cell population. In some embodiments, a plurality of probes can be utilized to serve to indicate or increase the confidence in the indication that a cell is of a certain type or class or has a certain product produced as a function of the cell life cycle or in response to some therapy.
[0185] In some embodiments, protein expression levels are detected using antibody recognition and the group of cells with varying levels of expression are compared to their response to the therapy.
[0186] In some embodiments, the sum total of at least one or a plurality of detections correlates to the responsiveness to the therapy.
[0187] In some embodiments, the spatial distribution of detections of at least one or a plurality of detections correlates to the responsiveness to the therapy. In some embodiments, the distribution of HER2 detections is useful for correlating to the response of an Antibody Drug Conjugate (ADC) that targets HER2.
[0188] In some embodiments, the population model is utilized to determine if substantial heterogeneity in the response to therapy across one of the detection types or combinations of the detection types. In some embodiments, the population model characterizes the sample. In some embodiments, all measures derived from all detections are of utility for predicting the patient's response to a therapy. In some embodiments, at least one for the detections is of utility for predicting the patient’s response to the therapy. In some embodiments the response to a potential therapy is used for diagnostic and/or pharmaceutical development purposes. Embedding
[0189] Embedding uses an embedding matrix material to immobilize the group of cells in place relative to the other group of cells. Embedding can allow removal of the group of cells embedded in matrix material, and sectioning of the matrix material. In some embodiments, embedding is performed to prepare the matrix material along with the group of cells for later sectioning. In contrast to entombment, in which the group of cells are maintained as viable cells, the embedding process is independent of maintaining the viability of the group of cells. In some variations, the embedded group of cells are viable cells. In some variations, the embedded group of cells are not viable cells.
[0190] In some embodiments, the embedding matrix material is the same as entombment material. In these embodiments, entombment materials such as matrigel or synthetic analogs (as described in the following reference Aisenbrey, E.A., Murphy, W.L. Synthetic alternatives to Matrigel. Nat Rev Mater 5, 539-551 (2020). https://doi.org/10.1038/s41578-020-0199-8 the entire contents are incorporated herein) are utilized as embedding matrix materials. In some embodiments, it is advantageous to embed the entombed group of cells again using the same material as used for entombment. For example, 1 - 2 wt% can be used for entombment, and a higher concentration can be used for embedding. In some embodiments, the use of the entombed material physically reinforces or strengthens the material in preparation for sectioning.
[0191] In some embodiments, the material is embedded in a matrix material that acts to preserve or further preserve the group of cells. In some embodiments, the embedded matrix material also facilitates handling of the matrix material while preserving the delicate biological structures. Embedding methods those to those known and skilled in the art are described in Bancroft's Theory and Practice of Histological Techniques (Eighth Edition) ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Format:ebook, PublisherElsevier Masson, Language:English, Authors. Kim Suvarna, Editor: S. Kim Suvarna incorporated by reference herein in its entirety.
[0192] In some embodiments, the embedding matrix material is paraffin. In some embodiments, paraffin wax, or similar embedding matrix material, is diluted in a solvent and the solvent evaporates creating a thin section of paraffin, or similar embedding matrix material, that can be processed with standard histological processing techniques as with sectioned material.
[0193] In some embodiments, the sample is embedded in an alternative matrix material such as histogel as described by (La Fortune KA, Randolph ML, Wu HH, Cramer HM.
Improvements in cell block processing: The Cell-Gel method. Cancer Cytopathol. 2017 Apr;125(4):267-276. doi: 10.1002/cncy.21814.) the entire contents incorporated herein without limitation. In some embodiments, the wax like substance is a wax as described in the following references and their entire contents incorporated herein without limitation
[0194] Embedding in a matrix material may be accomplished using such as the methods described in the following each of which is incorporated herein in its entirety by reference:
1. Havnar, C., Holokai, L, Ichikawa, R., Chen, W., Scherf, A., & Shamir, E. R. (2024). Histogel-based techniques for embedding organoids in paraffin blocks enable high throughput downstream histopathological analyses. Journal of Histotechnology, 47(4), 1-10. https://doi.Org/10.1080/01478885.2024.2398381
2. Graham, E. T. 1982. “Improved Diethylene Glycol Distearate Embedding Wax.” Stain Technology 57 (1): 39-43.
3. Quarterly Journal of Microscopical Science. 1958.
4. Richardson, Douglas S., and Jeff W. Lichtman. 2015. “Clarifying Tissue Clearing.” Cell 162 (2): 246-57.
5. Sage, M. 1972. “Polyethylene Glycol Distearate 600 with 10 per Cent 1-Hexadecanol; a Superior Embedding Wax for Warm Climates.” Stain Technology 47 (6): 313-15.
6. Steedman, H. F. 1947. “Ester Wax; a New Embedding Medium.” The Quarterly Journal of Microscopical Science 88 (1): 123-33.
7. Steedman, H.F. 1949. “An Ester Wax for Use in the Tropics.” Nature 164 (4182): 1084. 8. Steedman, H.F. 1957. “Polyester Wax; a New Ribboning Embedding Medium for Histology.” Nature 179 (4574): 1345.
9. Steedman, Hugh Fletcher. 1960. Section Cutting in Microscopy.
10. Taleporos, P. 1974. “Diethylene Glycol Distearate as an Embedding Medium for High Resolution Light Microscopy.” The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society 22 (1): 29-34.
11. Ghosh, R. N., Thomas, J., R., V. B., N. G., D., Janardanan, A., Namboothiri, P. K., & Peter, M. (2023). An insight into synthesis, properties and applications of gelatin methacryloyl hydrogel for 3D bioprinting. Materials Advances, 4(20), 4657-4673. https://doi.orq/10.1039/d3ma00715d
[0195] In some embodiments, the embedding is performed using a water soluble wax matrix material as described in (Bancroft's Theory and Practice of Histological Techniques (Eighth Edition) ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Formatebook, PublisherElsevier Masson, Language:English, Authors. Kim Suvarna, Editors. Kim Suvarna incorporated by reference herein in its entirety and (Culling, C. F. A., R. T. Allison, and W. T. Barr. 2014. Cellular Pathology Technique. Elsevier.) the entire contents incorporated herein without limitation.
[0196] In some embodiments, the embedding material is at least one of the following; Carbowax, polyethylene glycol (PEG), polyethylene glycol fatty acid esters, such as polyethylene glycol monostearate, stearic acid, polyethylene oxide, Poly(vinyl methyl ether), carboxymethyl cellulose salts or acid forms, agarose, agar, polypropylene glycol, and hexadecanol.
[0197] In some embodiments, the embedding material infiltrates the material that entombs a group of cells. In some embodiments, the embedding is accomplished using hydrogels comprising polymerizable monomers selected from the group of acrylic and methacrylic monomers, wherein said monomers are capable of forming hydrophilic, three- dimensional networks upon polymerization. Such monomers include acrylic acid (AA), polyacrylic acid (PAA) hydrogels; methacrylic acid (MAA), polymethacrylic acid (PMAA) networks; and 2-hydroxyethyl methacrylate (HEMA). Additional embodiments encompass 2- hydroxyethyl acrylate (HEA); N-isopropylacrylamide (NIPAAm), and N,N-dimethylacrylamide (DMA). Further monomers in this category include acrylamide (AAm), widely utilized in polyacrylamide (PAAm); methacrylamide (MAM); 2-acrylamido-2-methylpropane sulfonic acid (AMPS); glycidyl methacrylate (GMA), polyethylene glycol diacrylate (PEGDA) and polyethylene glycol dimethacrylate (PEGDMA).
[0198] In some embodiments, the embedding matrix material may comprise vinyl monomers. Such monomers include vinyl pyrrolidone (VP), which forms poly(vinylpyrrolidone) (PVP); vinyl alcohol (VA), derived from hydrolyzed poly(vinyl acetate) to yield poly(vinyl alcohol) (PVA) hydrogels crosslinked physically or chemically; vinyl acetate (VAc), serving as a precursor to PVA-based gels; and N-vinylcaprolactam (NVCL).
[0199] In some embodiments the monomers are selected such that they infiltrate the material that entombs the group of cells.
[0200] In some embodiments, the embedding is accomplished using hydrogel compositions derived from natural and bio-derived monomers. Such monomers include chitosan, that may be modified with vinyl groups such as methacrylate to enable crosslinkable hydrogel formation; hyaluronic acid (HA), that may be functionalized (e.g., as HA-methacrylate, HA-MA); and alginate, that may be similarly modified with polymerizable groups, though often crosslinked ionically. Additional embodiments comprise gelatin that may be functionalized to generate methacryloyl (GelMA), a crosslinkable derivative of gelatin, dextran, that may be functionalized (e.g., as dextran methacrylate); cellulose, that may be modified into derivatives such as hydroxyethyl cellulose methacrylate; and starch, that may be adapted with acrylate or other polymerizable groups.
[0201] In some embodiments, embedding incorporates zwitterionic monomers, characterized by both positive and negative charges. Such monomers include sulfobetaine methacrylate (SBMA), forming poly(SBMA) hydrogels; carboxybetaine methacrylate (CBMA); and phosphorylcholine methacrylate (MPC) in poly(MPC) hydrogels.
[0202] In some embodiments, embedding encompasses hydrogel-forming compositions comprising synthetic monomers tailored for specialized properties. These monomers include ethylene glycol monomethacrylate (EGMA); diethylene glycol methacrylate (DEGMA), oligo(ethylene glycol) methacrylate (OEGMA); itaconic acid (IA) and maleic acid (MA); vinylsulfonic acid (VSA); and 4-vinylpyridine (4VP).
[0203] In some embodiments, embedding compositions further comprise crosslinking monomers to facilitate the formation of a three-dimensional network essential for gelation. Such crosslinking monomers include N,N'-methylenebis(acrylamide) (MBA); ethylene glycol diacrylate (EGDA); triethylene glycol dimethacrylate (TEGDMA); and 1 ,4-butanediol diacrylate (BDDA). These difunctional monomers may be copolymerized with the aforementioned monomers to achieve the desired degree of crosslinking and mechanical strength.
[0204] In some embodiments, embedding utilizes polymerization conducted via free radical polymerization, including thermal, photo-, or redox-initiated methods, though alternative techniques such as enzymatic crosslinking or ionic gelation may be employed, particularly for natural monomers. In some embodiments the polymerization is achieved using methods of low cytotoxicity.
[0205] In some embodiments, mixtures of the previously mentioned materials are used for embedding.
[0206] In some embodiments, the embedding of the group of cells is accomplished by freezing. In some embodiments embedding methods described herein are performed prior to freezing. In some embodiments, additives are utilized to reduce ice crystal formation during the freezing process. In some embodiments, solvents are utilized to select a freezing temperature advantageous for sectioning (e.g. dimethyl sulfoxide, having an elevated freezing temperature relative to water). In some embodiments, the freezing is performed consistent with cryostat methodologies.
[0207] Additional description of embedding - including freezing and the use of cryostat methodology - can be found in Bancroft's Theory and Practice of Histological Techniques (Eighth Edition). ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Formatebook, PublisherElsevier Masson, Language:English, Authors. Kim Suvarna, Editors. Kim Suvarna incorporated by reference herein in its entirety.
Positional Considerations of Fixation and Dehydration
[0208] In some embodiments, at least one of the following steps; sample preparation, entombment, the group of cells adherence to a surface, permeabilization, clearing or clarification, dehydration, embedding, and fixation locks the position of the cells relative to each other such that the tracking of a group of cells can be assigned to locations. In some embodiments, the spatial movement of the group of cells can be less than 500 microns.
[0209] When the response of the physical group of cells is correlated to the multiplex properties of the group of cells, the spatial movement of cells can be determined to ensure that the measured response corresponds to the characterized multiplex properties - i.e., that the same cells or group of cells are compared. In some embodiments, the spatial movement of the group of cells can be less than 100 microns. In some embodiments, the spatial movement of the group of cells can be less than 50 microns. In some embodiments, the spatial movement of the group of cells can be less than 25 microns. In some embodiments, the spatial movement of the group of cells can be less than 10 microns. In some embodiments, the spatial movement of the group of cells can be less than 1 micron relative to the final position prior to fixation. In further variations, the spatial movement of the group of cells can be relative to the final position prior and/or after embedding the group of cells.
[0210] In some embodiments where there is or expects to be a spatial movement of the group of cells relative to a fixation step due to shrinkage, the location of the group of cells can be registered with respect to the position prior to fixation can be corrected or compensated for algorithmically during signal analysis using any art-recognized computer-implemented algorithms.
Sectioning
[0211] In some embodiments, sectioning is performed. In some cases, the sectioning is performed at room temperature. In some embodiments, the embedded material is frozen and the sectioning is performed while the embedded material remains frozen. Further details for sectioning in general can be found in Bancroft's Theory and Practice of Histological Techniques (Eighth Edition) ISBN:9780702068874, 070206887X, Page count:536, Published:2019, Format:ebook, Publisher:Elsevier Masson, Language:English, Authors. Kim Suvarna, Editors. Kim Suvarna incorporated by reference herein in its entirety.
Virtual Sectioning or Optical Sectioning
[0212] Methods utilized to virtually section materials and known to those skilled in the art such are described in the following each of which is incorporated herein in its entirety by reference:
1. Xu, X., Su, J., Zhu, R., Li, K., Zhao, X., Fan, J., & Mao, F. (2025). From morphology to single-cell molecules: High-resolution 3D histology in biomedicine. Molecular Cancer, 24, 63. https://doi.orq/10.1186/s12943-025-02240-x
2. Rappaz, B., Breton, B., Shaffer, E., & Turcatti, G. (2014). Digital holographic microscopy: A quantitative label-free microscopy technique for phenotypic screening. Combinatorial Chemistry & High Throughput Screening, 17(1), 80-88.
10.2174/13862073113166660062
3. Prisbey, L., Metzger, S. W., & Gusyatin, O. (2024). Lens-free holographic optical system for high sensitivity label-free cell and microbial growth detection and quantification for screening, identification, and susceptibility training (U.S. Patent Application No. US 2024/0385099 A1). U.S. Patent and Trademark Office.
4. Zhou, Y., Mao, S., & Fei, P. (2024). Light sheet fluorescence microscopy: Advancing biological discovery with more dimensions, higher speed, and lower phototoxicity. The Innovation, 5(5), Article 100692. https://doi.Org/10.1016/j.xinn.2024.100692
Vessel/Fluidic Device Deconstruction
[0213] In some embodiments, a honeycomb well structure forming the walls of the vessel containing the volume containing the group of cells is removed leaving a matrix material containing the group of cells. In some embodiments, the substrate contains cells on the surface. In some embodiments, a volume containing the entombed group of cells remains on the substrate after the honeycomb well structure is removed.
[0214] In some embodiments, the vessel is constructed of a matrix material amenable to microtoming using a large microtome such as the 8000 Sledge Microtome (Bright Instruments) with a maximum section size of 250 mm x 210 mm. In some embodiments the vessel is a microplate. In some embodiments, the vessel is made from acetal, paraffin, or polyoxymethylene (Delrin). In some embodiments, the sectioned vessel slices containing the group of cells are mounted on glass substrates.
[0215] In some embodiments, the chamber walls are formed by a layer of matrix material containing the group of cells. In some embodiments, the layer is removed leaving a surface containing the group of cells. In some embodiments, a surface containing the entombed group of cells remains after the layer is removed. In some embodiments, the surface containing the group of cells are not sectioned. Partially Performed in Vessel or Chamber
[0216] In some embodiments, the group of cells are first measured or a portion of the measuring steps performed before removal from the vessel and sectioning Removal from Vessel or Chamber
[0217] In some embodiments the group of cells, their cellular constituents, or cellular products are transferred from a vessel or a chamber to another vessel or a chamber prior to measuring. In some embodiments, the position of the group of cells, their cells, their cellular constituents and their cellular components are preserved relative to one another when they are removed from the vessel. In some embodiments, the additional vessel is a glass substrate. In some embodiments, the glass substrate is the shape and size typical of tissue mounted slides for the purposes of histological staining. In some embodiments, the measuring performed in the additional vessel can be registered with detection of the group of cells. In some embodiments, a fiduciary marker or plurality of fiduciary markers are utilized to provide landmarks for registering the detections occurring in the two vessels or two chambers or between a vessel and a chamber. In some embodiments, the transfer can occur multiple times to multiple vessels or multiple chambers.
[0218] In some embodiments, the vessel is constructed of a material with different coefficient of thermal expansion relative to the matrix material used for embedding. In some embodiments, the vessel containing the matrix material is frozen to cause the release of the matrix material. In some embodiments the vessel is a microplate. In some cases, the removal of the matrix material generates a negative mold of the microplate structure. In some embodiments, the mold is further embedded in another material such that the original mold can be identified visually relative to the further embedding material.
[0219] In some embodiments, the negative mold is cooled such that the further embedding material does not melt the negative mold. In some embodiments, the melting temperature of the further embedding material is lower than the negative mold such that the negative mold is not melted by the further embedding material. In some embodiments, the embedding matrix material is paraffin and the microplate is made from polystyrene.
[0220] In some embodiments the matrix material from a chamber or a vessel is frozen and the thermal coefficient of expansion relative to the vessel material is dissimilar that the frozen volume is released. In some embodiments, the frozen material can be sectioned using a cryostat then transferred to a substrate.
[0221] In some embodiments, measuring is partially performed such that a combinatorial approach can be used to barcode the position and scan and capture this information before the group of cells and their cells, cellular constituents, and/or cellular components are removed from the vessel enabling a later reconciliation of measuring performed later with the initial location of the group of cells prior to removal. [0222] In some embodiments a fiduciary marker or plurality of fiduciary markers are utilized and can link the tracked group of cells and cellular product information with subsequently described further measuring of the group of cells and their cells, cellular constituents, or cellular products.
[0223] In some embodiments, the section thickness is determined by the method used to measure the molecular properties. In some embodiments, the section thickness can be less than 1 micron. In some embodiments, the section thickness can be less than 3 microns. In some embodiments, the section thickness can be less than 5 microns. In some embodiments, the section thickness can be less than 10 microns. In some embodiments, the section thickness can be less than 20 microns. In some embodiments, the section thickness can be less than 50 microns. In some embodiments, the section thickness can be less than 100 microns. In some embodiments, the section thickness can be less than 200 microns. In some embodiments, the section thickness can be less than 500 microns. In some embodiments, the section thickness can be less than 1000 microns. In some embodiments, the section thickness can be less than 2000 microns. In some embodiments, the section thickness can be less than 5000 microns.
Mounting of Sections for Analysis
[0224] In some embodiments, the sectioned material is affixed to a planar substrate to facilitate analysis. In some embodiments, the planar substrate is glass slides, quartz coverslips, or coated substrates having specialized conductive coatings like indium tin oxide (ITO) to support compatibility with diverse imaging and analytical modalities.
[0225] In some embodiments, samples are mounted onto glass slides with the assistance of adhesives. Additional mounting variations include the use of adhesive tapes for fragile samples, microfluidic chambers for dynamic analysis. In some embodiments, the adhesive is precoated on the planar substrate. In some embodiments, poly-L-lysine, amino silane, or photopolymerizable adhesive. In some embodiments, the substrates enable precise excision of targeted cellular regions while maintaining structural integrity under laser exposure to facilitate laser microdissection. In some embodiments, sections may be mounted on polymer- coated substrates or polyethylene naphthalate (PEN) membrane slides.
[0226] In some embodiments, the mounting adapts to specific requirements: for mass spectrometry, such as matrix-assisted laser desorption/ionization (MALDI), tissues may be affixed to conductive metal plates or glass slides coated with a thin layer of gold or silver, optimizing ionization and detection of molecular species. Alternatively, cryosections can be thaw-mounted directly onto these substrates to preserve biomolecular properties. For Raman spectroscopic analysis, samples may be deposited on low-background substrates like calcium fluoride (CaF2) to reduce spectral interference or signal enhancing substrates such as mirrored stainless steel, with sections either air-dried or maintained in a hydrated state depending on the analyte of interest. Characterizing Molecular Properties
[0227] In some embodiments, the sectioned material is measured using previously mentioned methods described herein. In some embodiments, the measurements are multiplexed, enabling the simultaneous or sequential assessment of a plurality molecular properties of the group of cells within a single sample. In some embodiments, the molecular properties include spatial-omics molecular data. In some embodiments, the molecular data relates to protein expression, nucleic acid sequences, epigenetic modifications, and metabolic products
[0228] In some embodiments, the plurality probes are applied serially such that further multiplexed measurements of molecular properties may be achieved.
[0229] In some embodiments, the initial probes are stripped prior to the next application of subsequent probes such that the signal can be removed and colors used for detection can be reused further multiplexing the number of probes. In some embodiments, measuring is performed in which a multitude of probes are applied to a sample, allowing the measurement of many groups of cells.
[0230] Various methods, including multiplexed molecular properties, can be measured using such as the methods described in the following references, each of which is incorporated herein by reference in its entirety:
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[0231] The multiplexed measurements may leverage a combination of detection techniques, such as optical imaging (e.g., fluorescence microscopy, holographic imaging, or light sheet microscopy), spectroscopic methods (e.g., Raman or Brillouin spectroscopy), and mass spectrometry (e.g., MALDI-based molecular profiling), applied to the sectioned material. These techniques enable the spatially resolved detection of multiple targets, including biomarkers, cellular constituents, and extracellular products, while preserving the positional context of the group of cells established during earlier processing steps, such as entombment, fixation, or embedding.
[0232] In some embodiments, the molecular properties are characterized by employing a plurality of probes — such as antibodies, nucleic acid sequences, or aptamers — each conjugated to distinct reporter mechanisms (e.g., fluorescent tags, barcodes, or colorimetric precipitants). These probes target specific molecular features, including protein expression levels (e.g., HER2, ALK, or PD-L1), nucleic acid alterations (e.g., single nucleotide variants, translocations, or copy number changes), and cellular states (e.g., viability, proliferation, or differentiation). The use of temporal-sequential detection or spectral separation allows for the simultaneous interrogation of dozens to hundreds of targets within the same sample, enhancing the resolution of heterogeneity across the group of cells.
[0233] In some embodiments, the probe contains a nucleic acid barcode that multiplexes detection. In some embodiments, the multitude of probes can then be identified in a temporally-sequential manner. Exemplary temporal-sequential methods using hybridization applicable to the methods described herein include, without limitation, those described in European Patent Application EP 2794928 B1 , European Patent Application EP 4108782 A1 , and United States Patent US 11 ,170,334 B2. The entire contents of these patent documents are incorporated herein by reference.
Correlating the Response and Molecular Properties
[0234] The resulting data are correlated with the spatial and temporal tracking of the group of cells performed prior to sectioning, enabling a direct linkage between responses (e.g., growth kinetics, cell death, or interaction dynamics) and molecular properties. For example, the response of an individual group of cells to a therapy — measured as changes in viability, proliferation rate, or extracellular product secretion — can be correlated or mapped to specific biomarker expression patterns or genetic mutations detected in the same cells.
[0235] The upper portion of Figure 1 represents a conventional approach for correlating response measures of a group of cells to molecular properties. The group of cells may be labeled as "1 ," "2," through "n," each associated with a response measure (and the group of cells are not embedded in a matrix capable of being sectioned). The molecular properties can include, without limitation, mRNA properties 1 through n, protein properties 1 through n, and epigenetic properties 1 through n, as indicated on the right side of the schematic. In the conventional approach depicted, a many-to-many mapping occurs because the conventional methods teach away from embedding by intentionally disrupting the spatial position of the group of cells to measure the molecular properties, wherein each response measured may be associated with multiple molecular properties, and each molecular property may correspond to multiple response measures. The limitations of the conventional approach thus teach away from preserving the spatial distribution of the group of cells, resulting in the inability to associate mRNA, protein, or epigenetic properties with a specific group of cells resulting in the aggregate data masking individual or rare responses.
[0236] The lower portion of Figure 1 illustrates an embodiment of the disclosed method for associating response measures of a group of cells with molecular properties. The group of cells may be labeled as "1 ," "2," through "n," each associated with a response measure. The disclosed method may employ a one-to-one mapping, as depicted in Figurel , wherein each response measure may be directly linked to a specific set of multiplexed molecular properties. These properties can include, without limitation, mRNA properties 1 through n, protein properties 1 through n, and epigenetic properties 1 through n, as shown on the right side of the schematic. The benefits of the disclosed method may be emphasized, including the direct association of response measures with molecular properties, enabling precise characterization of individual responses within a group of cells.
[0237] In some embodiments, a method is provided wherein a plurality of sections are utilized to correlate to the response. In some embodiments, the plurality of sections may be employed to reconstruct a three-dimensional (3D) volume. In some embodiments, the reconstruction can include aligning and stacking the sections, potentially with the assistance of image registration techniques or fiduciary markers, to generate a digital model representative of the original sample. In some embodiments, the 3D reconstruction may preserve the spatial distribution of the cells, thereby enabling the mapping of molecular properties — ascertained through analysis of each section — to their respective positions. In some embodiments, the method can integrate this 3D molecular profile with response, which may be tracked spatially and temporally, to correlate the effects of the therapy with molecular properties across the entire volume. In some embodiments, the utility of the described method reveals heterogeneity and region-specific variations in treatment efficacy. Alternatively, in some embodiments, a specific group of cells demonstrates a response of interest; a particular section containing said group may be identified and selected for analysis.
[0238] This integrated approach facilitates the identification of subpopulations with distinct therapeutic sensitivities or resistance mechanisms, providing actionable insights for both diagnostic and pharmaceutical applications.
Diagnostic and/or Pharmaceutical Development Applications
[0239] The following embodiments represent a portion of all possible applications and are not limiting in any way. In some embodiments, the responses of the group of cells to therapies are utilized to determine the response of a cell type to a therapy for diagnostic and/or pharmaceutical development. In some embodiments, the therapies are compared to the no therapy control. In some embodiments, the cells are assessed independently from other cells with respect to a parallel no therapy condition. In some embodiments, the interaction of cells associated with the host immune response is assessed. In some embodiments, immune cells are useful for diagnostic and/or pharmaceutical development. In some embodiments, immune cells and other cells are useful for diagnostic and/or pharmaceutical development. In some embodiments, the interaction between immune cells and non-immune cells is assessed. In some embodiments, the interaction between the group of cells is determined by the cellular constituents or cellular products. In some embodiments, the cellular constituents produced are cytokines, chemokines, or other cellular signaling elements. In some embodiments, the heterogeneity assessed correlates to the response and is useful for diagnostic and/or pharmaceutical development. In some embodiments, the correlated response predicts the efficacy of the therapy for diagnostic or pharmaceutical development purposes.
[0240] In some embodiments, a training set of clinical outcome data is compared to the response observed and the information associated with the interrogation of the group of cells and their cellular products. In some embodiments, the response observed is further refined and broken down into sub groups prior to the comparison to the clinical information. In some embodiments, the observed response is utilized to predict the benefit of the therapy for a patient whose materials were used to generate the response. In some embodiments, the response is used to predict the progression free or overall survival expected for that therapy for that particular patient.
Correlating Via Algorithms
[0241] In some embodiments, algorithms operate at the level of distinct entities comprised of separately detected the group of cells as described herein or by any means known to those skilled in the art. In some embodiments, population analysis means analysis of all entities measured for providing utility for diagnostic and/or pharmaceutical development purposes.
[0242] In some embodiments, computational algorithms are employed to analyze the response and molecular properties, reconstructing a population model that captures the diversity of responses and molecular characteristics across the sample.
[0243] This model may incorporate machine learning techniques to identify predictive biomarkers, classify cell types, or infer therapeutic efficacy based on the combined functional and molecular signatures. The heterogeneity of the group of cells , including differences in cell state, differentiation, or spatial distribution, is quantified and correlated with therapy outcomes, offering a refined understanding of disease progression and treatment response.
[0244] In some embodiments, algorithms are required for the identification of the individual group of cells. In some embodiments, the detected individual group of cells are the fundamental entity of affording the construction of a population model useful for characterizing the response for diagnostic and/or pharmaceutical development purposes. In some embodiments features are utilized to characterize the detections described for each entity. In some embodiments, data analysis of the features or each entity alone or as a population are algorithmically correlated to predicted response for diagnostic and/or pharmaceutical development purposes.
Feature Extraction from Images
[0245] In some embodiments, the extraction of features may be accomplished using traditional image analysis techniques including the registration of images, segmentation of areas of interest using thresholds, and extraction of features such as the change in area over time for these areas can be employed.
[0246] The methods can be further enhanced by incorporating various machine learning techniques to analyze the images of the group of cells and their response to therapy. These machine learning methods can be used to automatically detect, segment, and classify cells, as well as to predict sensitivity to therapy based on cellular features. The following examples illustrate some of the machine learning approaches that can be applied within the scope of this disclosure, but they are not intended to be limiting.
[0247] Convolutional Neural Networks (CNNs): CNN architectures, such as AlexNet, VGGNet, ResNet, and Inception, can be utilized to achieve state-of-the-art performance in tasks like image classification, object detection, and segmentation. In the context of this disclosure, CNNs can be trained to classify cell types, detect cellular structures, or predict responses to therapies based on the appearance of cells in the images.
[0248] Transfer Learning: By using the learned weights from a pre-trained CNN as a starting point, transfer learning can significantly reduce training time and improve performance on the target task of analyzing images. Fine-tuning, where the pre-trained model is further trained on the target dataset.
[0249] Object Detection and Segmentation: Object detection frameworks, such as R- CNN, Fast R-CNN, Faster R-CNN, YOLO, and SSD, can be applied to detect and localize the group of cells in the images. For segmentation, fully convolutional networks (FCNs) and their variants, like U-Net and DeepLab, can be employed to accurately segment individual cells or the group of cells from the background and from each other.
[0250] Generative Models: Generative models, such as Variational Autoencoders (VAEs) and Generative Adversarial Networks (GANs), can be used for data augmentation and for modeling the distribution of the group of cells. By training these networks in an adversarial manner, GANs can generate realistic images of the group of cells that capture the diversity and variability of real samples.
[0251] Few-Shot Learning: Few-shot learning learns from a limited number of labeled examples per class, which is particularly relevant when dealing with rare events or limited patient data. Approaches like Siamese Networks and Prototypical Networks use metric learning to compare query images with a few labeled examples and make predictions based on similarity. Meta-learning techniques, such as Model-Agnostic Meta-Learning (MAML), can also be applied to quickly adapt to new disease types or therapy with limited data.
[0252] It is important to note that the specific machine learning algorithms and architectures mentioned here are examples, not meant to be limiting, and the disclosure can be adapted to incorporate any suitable machine learning approach that can improve the analysis of the group of cells images and the prediction of responses to therapies for diagnostic and/or pharmaceutical development purposes. It is further noted that such methods are useful for characterizing the population of responses, providing a means for correlating or predicting patient response.
Data Analysis
[0253] To ensure accurate correlation between the response and molecular properties, the spatial location of the group of cells is preserved and tracked throughout the method.
During response measurement, spatial coordinates or unique identifiers can be assigned to each group of cells, capturing their positions via imaging or barcoding. The embedding and sectioning steps are conducted in a manner that maintains the original spatial arrangement within the matrix. Molecular characterization on the sections thus provides spatially resolved data. By aligning the spatial data from the response measurement with that from the molecular analysis — using techniques like image registration — the method enables precise, location- specific correlation of response and molecular properties. The resulting correlated measures of each group of cells generates a population distribution that can be useful for diagnostic and drug development purposes described herein.
[0254] The method generates a wealth of data by extracting features from images of the individual group of cells and monitoring their response to therapy over time. In some embodiments, the tracked group of cells are differentiated by the expression properties of messenger RNA. In some embodiments, the presence and amount of expression of oncogenes is utilized for segmenting into classes of cells. The following section describes some of the methods that can be used to correlate the longitudinal data with patient outcomes, but these examples are not intended to be limiting.
[0255] Feature Extraction and Selection: Relevant features capture the morphological, textural, and dynamic properties of the group of cells. These features may include the group of cells size, shape, intensity, texture descriptors, and growth rates, among others. Feature selection techniques, such as principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), or recursive feature elimination (RFE), can be applied to identify the most informative features that discriminate between the different group of cells populations or treatment responses.
[0256] Time Series Analysis: The data from the group of cells tracked over time can be treated as time series. Time series analysis techniques, such as autoregressive integrated moving average (ARIMA) models, polynomial and hill curve fitting functions, hidden Markov models (HMMs), or long short-term memory (LSTM) networks, can be employed to model the temporal dynamics of the group of cells behavior and the responses to therapies.
[0257] Clustering and Subpopulation Identification: Clustering algorithms, such as k- means, hierarchical clustering, or density-based spatial clustering of applications with noise (DBSCAN), can be used to associate the group of cells with similar features or response patterns. Distinct subpopulations within the heterogeneous population can be identified. The disclosure can uncover specific cell types or states that may be associated with treatment sensitivity or resistance. These subpopulations can then be further characterized by their molecular properties or functional properties to gain insights into the mechanisms underlying their behavior.
[0258] Machine Learning for Response Prediction: Machine learning models can be trained to predict patient response based on the longitudinal group of cells data. Supervised learning algorithms, such as logistic regression, support vector machines (SVMs), random forests, or gradient boosting machines (GBMs), can be used to classify cells or patients into responder and non-responder groups. These models can learn the complex relationships between the extracted group of cells features, time-dependent behavior, and patient outcomes, enabling the prediction of treatment efficacy for new patients. [0259] In some embodiments, the provides a method of correlating a response of a group of cells to multiplex properties of the group of cells, comprising: introducing a therapy to a plurality of cells and/or group of cells cultured in a vessel under controlled conditions; measuring a response of the group of cells to the therapy using microscopy to generate longitudinal imaging data indicative of cellular dynamics; embedding the group of cells into a material capable of being sectioned while maintaining the spatial distribution of the physical group of cells on the matrix material; sectioning the matrix material to create at least a first section and a second section, wherein the first section comprises at least a portion of the group of cells; characterizing the at least a portion of the group of cells embedded in the first section to determine molecular properties, including nucleic acid and protein properties, using probes compatible with spatial omics analysis; and correlating the response of the physical group of cells to the molecular properties of the group of cells. The method generates structured data, comprising imaging-derived features and molecular properties, configured for analysis by transformer-based artificial intelligence (Al) models to characterize cellular states, interactions, and responses at a tissue level, addressing limitations in prior art techniques that fail to preserve spatial and molecular integrity.
[0260] Integration with Clinical Data: To gain a more comprehensive understanding of patient response, it is crucial to integrate the longitudinal group of cells data with clinical information, such as patient demographics, tumor characteristics, images and information from radiology, and treatment history. Multivariate statistical methods, such as multiple regression, canonical correlation analysis (CCA), or partial least squares (PLS) regression, can be used to model the relationships between the group of cells derived features and clinical variables. By incorporating clinical data, the disclosure can identify patient-specific factors that modulate treatment response and develop more personalized predictive models.
[0261] Visualization and Interpretation: Effective visualization techniques are essential for interpreting and communicating the complex patterns and relationships in the longitudinal group of cells data. Tools such as t-SNE plots, principal component analysis (PCA) plots, or heatmaps can be used to visualize the high-dimensional group of cells feature space and identify clusters or trends. Interactive dashboards can be developed to allow researchers and clinicians to explore the data, compare different treatment conditions, and assess individual patient responses. Such methods can be effectively repurposed for Pharmaceutical development purposes.
Large Language Models
[0262] The detection and characterization of the individual group of cells , as described in the disclosure, can be conceptualized as a form of tokenization, similar to the process used in natural language processing (NLP) and large language models (LLMs). In NLP, tokenization involves breaking down a text corpus into smaller units called tokens, which can be individual words, subwords, or characters. These tokens serve as the input fortraining LLMs, enabling the models to learn patterns and relationships within the text data.
[0263] In the context of the disclosure, the individual group of cells can be considered as the "tokens" that collectively make up the overall cell population. Each individual group of cells cell or cell group is characterized by a unique set of features, including at least one of the following; growth dynamics data from imaging-based methods and molecular properties profiling data. These features can be thought of as the "vocabulary" that describes the behavior and characteristics of each individual group of cells cell or cell group.
[0264] By tokenizing the cell population into an individual group of cells , the disclosure enables the application of machine learning techniques, such as those used in LLMs, to analyze and model responses of the group of cells to therapy. The training data for the LLM-like algorithm will be derived from a large cohort of patients, each with unique disease characteristics and treatment histories. To ensure the model's robustness and generalizability, it is essential to include a diverse range of patient samples, covering various disease stages, and prior treatment regimens. This heterogeneous dataset will enable the model to learn the complex relationships between group of cells behavior, responses to therapies, and patient outcomes across a wide spectrum of clinical scenarios.
[0265] For each patient in the cohort, the group of cells will be cultured under conditions that allow for growth and exposed to a panel of therapies, including both standard-of-care treatments and experimental compounds. The tokenization process will be applied consistently across all patient samples. Each individual group of cells will be treated as a unique "token," characterized by a set of features derived from the imaging and molecular profiling data. This tokenization step ensures that the LLM-like algorithm can process the data in a uniform manner, regardless of the patient source.
A Large Language Model Implementation
[0266] In one embodiment, the process of training an LLM-like algorithm on the tokenized individual group of cells cell or cell group data is implemented as follows:
[0267] 1 . Data Acquisition: Obtain a sample comprising the group of cells from a patient and culture under conditions that allow for growth. Expose the group of cells to one or more therapy conditions and detect the response of an individual group of cells using imaging-based methods over time. Utilize a polynomial to fit the response of an individual group of cells to parametrize the response, probe the cells with molecular probes to detect biomarkers, genetic alterations, cell type, cellular constituents, or cellular components.
[0268] 2. Tokenization: Segment the parameterized response and molecular profiling data into an individual group of cells , treating each individual group of cells as a unique "token." Extract relevant features from each token, such as parameters relating to response, morphology, and expression levels of specific biomarkers or genetic alterations. These features will serve as the input data for the LLM-like algorithm. [0269] 3. Data Preprocessing: Normalize and standardize the feature data across all tokens to ensure comparability. Split the tokenized data into training, validation, and test sets. [0270] 4. Model Architecture: Design an LLM-like architecture that can process the tokenized cell data. This may involve adapting existing LLM architectures, such as the transformer model, to handle the specific input features and output targets relevant to cell behavior and responses to therapies.
[0271] 5. Training: Train the LLM-like model on the tokenized cell data, using techniques such as unsupervised pre-training and supervised fine-tuning. The model will learn to predict the response dynamics and molecular properties of an individual group of cells based on their input features and the test conditions they were exposed to. During the training process, the LLM-like algorithm will be exposed to the tokenized data and corresponding patient outcomes from the entire cohort. The model will learn to recognize the complex relationships between the input features (i.e., growth dynamics and molecular properties of the individual group of cells ) and the output targets (i.e., patient-specific responses to therapies and outcomes). By training on a large and diverse dataset, the model will be able to generalize these learned relationships to new, unseen patient samples.
[0272] 6. Validation and Testing: Evaluate the trained model's performance on the validation and test sets, assessing its ability to accurately predict the response of a patient’s individual group of cells to therapy. Fine-tune the model as needed to improve its performance. To evaluate the model's performance and ensure its clinical relevance, the trained algorithm will be applied to an independent validation dataset, also consisting of tokenized data and patient outcomes. This validation step will assess the model's ability to accurately predict patientspecific responses to therapies and outcomes based solely on the behavior and characteristics of their group of cells. The model's performance can be further refined through techniques such as cross-validation and hyperparameter tuning.
[0273] 7. Interpretation and Application: Use the trained model to generate predictive scores or probabilities for each individual group of cells , indicating their likelihood of responding to specific therapy. Integrate these scores across the entire population to predict patient-specific responses to therapies and identify effective therapies. Once validated, the LLM-like algorithm can be applied to new patient samples in a clinical setting. For each new patient, their group of cells will undergo the same tokenization process, generating a set of input features that capture the unique behavior and characteristics. The trained model will then process these input features and generate patient-specific predictions of responses to therapies and outcome.
[0274] Once the tokenization is complete, the data will be organized and analyzed on an individual patient basis. This involves grouping the tokenized data by patient and integrating it with relevant clinical information, such as the patient's diagnosis, stage, treatment history, and eventual outcome (e.g., responses to therapies, progression-free survival, overall survival). By structuring the data in this way, the LLM-like algorithm can learn to associate specific patterns of the group of cells behavior and responses to therapies with patient-level outcomes.
[0275] By applying LLM-like techniques to tokenized data, the disclosure enables a powerful new approach to modeling and predicting the heterogeneous responses of the group of cells to therapy. This approach combines the high-resolution analysis of the individual group of cells with the ability of LLMs to learn complex patterns and relationships from large datasets. The resulting models can provide rapid, patient-specific predictions of responses to therapies, facilitating the development of personalized treatment strategies. By training the LLM-like algorithm on a large, diverse cohort of patients and applying the tokenization process consistently across all samples, the disclosure enables a powerful new approach to predicting patient-specific responses to therapies and outcomes. This approach leverages the high- resolution analysis of the individual group of cells , the ability of LLMs to learn complex patterns from large datasets, and the integration of patient-level clinical data to provide actionable insights for personalized treatment.
LLM Guard Rails
[0276] In addition to learning from the tokenized data and patient outcomes, the LLM- like algorithm will incorporate certain guard rails based on established clinical knowledge and best practices. These guard rails will help ensure that the model's predictions are consistent with well-validated biomarkers and a priori understanding of responses to therapies. By integrating this domain-specific knowledge into the learning process, the LLM-like algorithm can provide more reliable and interpretable predictions that align with conventional clinical guidelines.
[0277] A guard rail will be the incorporation of known biomarker-outcome relationships. For many disease types, there are well-established biomarkers that have been validated as predictors of responses to therapy or patient prognosis. For example, in breast cancer, the presence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are routinely used to guide treatment decisions. The LLM-like algorithm will be trained to recognize and prioritize these validated biomarkers when making predictions, ensuring that its outputs are consistent with established clinical practice.
[0278] Another important guard rail will be the encoding of a priori knowledge about the group of cells behavior and a response to therapy. One such principle is the understanding that robust growth in the presence of a therapy is a strong indicator of resistance to that therapy. This knowledge will be incorporated into the LLM-like algorithm through a combination of data preprocessing and model architecture design.
[0279] During data preprocessing, the tokenized data will be annotated with additional features that capture the relationship between growth and the response to therapy. For example, the group of cells that exhibit robust growth in the presence of a specific therapy will be correlated as "resistant" to that therapy. This annotation step ensures that the model is explicitly trained to recognize the association between robust growth and resistance.
[0280] In terms of model architecture, the LLM-like algorithm can be designed to include specific modules or layers that encode the a priori knowledge about growth-resistance relationships. For instance, the model may include a decision rule that automatically classifies the group of cells as resistant if their growth rate exceeds a certain threshold in the presence of a therapy. Alternatively, the model may be trained with a loss function that heavily penalizes predictions that fail to associate robust growth with resistance.
[0281] By incorporating these guard rails into the LLM-like algorithm, the disclosure ensures that the model's predictions are grounded in established clinical knowledge and best practices. The integration of validated biomarker-outcome relationships and a priori understanding of the group of cells behavior helps to constrain the model's learning process, reducing the risk of spurious or clinically implausible predictions.
[0282] Furthermore, the inclusion of these guard rails enhances the interpretability of the model's outputs. By aligning the model's predictions with established clinical principles, healthcare professionals can more easily understand and trust the model's recommendations. This interpretability is crucial for the adoption and integration of the LLM-like algorithm into clinical decision-making processes.
[0283] In summary, the LLM-like algorithm described in the disclosure will incorporate guard rails based on established clinical knowledge and best practices, such as validated biomarker-outcome relationships and the a priori understanding that robust growth indicates resistance to therapy. These guard rails will be integrated through data preprocessing, model architecture design, and customized loss functions. By grounding the model's learning process in domain-specific knowledge, the disclosure ensures that its predictions are reliable, interpretable, and consistent with conventional clinical guidelines, facilitating the development of personalized treatment strategies that are both data-driven and clinically sound.
Additional Considerations
[0284] By leveraging these algorithm approaches, the disclosure can transform the vast amounts of image-derived data into actionable insights for predicting patient response and guiding personalized treatment. In some embodiments, the longitudinal nature of the individual group of cells provides a unique opportunity to capture the dynamic aspects of these fundamental entities and their response to therapy, enabling a more comprehensive understanding of the underlying biological processes for diagnostic and/or pharmaceutical development purposes. In some embodiments, static endpoint analysis correlates to the response to the therapy. In some embodiments, the correlated response predicts the efficacy of the therapy for diagnostic or pharmaceutical development purposes.
[0285] Finally, it is important to emphasize that the specific data analysis techniques mentioned here are not meant to be limiting, and the disclosure can incorporate any suitable computational or statistical approach that can extract meaningful patterns and correlations from the longitudinal individual group of cells. Examples
[0286] The following examples illustrate various aspects of the disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the disclosure.
Example 1: Paraffin Embedded
A. A 5 ml_ remnant sample of pleural fluid was collected from a patient during a palliative thoracentesis procedure from a biospecimen vendor. The patient had a prior diagnosis of endocrine refractory HER2 negative breast cancer. The sample was centrifuged at 500 relative centrifugal force (RCF) for 15 minutes and the supernatant was removed and 5 mL of growth media was added then the pellet was resuspended in solution by vortexing. The growth media used was DMEM/F12 with supplements including Epidermal Growth Factor, Noggin, R-spondin 1, N-acetylcysteine, B27 supplement, Nicotinamide , Y-27632.
B. A 5 uL T rypan Blue stained aliquot was placed on a haemocytometer and cell counts were enumerated (all cells) and an initial concentration of 1 x 107 cells/mL was determined.
C. 369 uL of the sample was added to 13.3 mL of growth media containing a 1 :200 dilution of EPCAM antibody probed with Cy5 (Abeam) in a 15 mL conical tube. 21.6 uL of a 4.5 x 106 particle per mL 1 urn 3 color fluorescent polystyrene latex bead (Polysciences) working solution was added to the conical tube. The tube was vortexed vigorously and placed on an ice bath for 20 minutes.
D. The diluted and 4 °C chilled sample was vortexed and 754 uL of Matrigel Matrix was added to the conical tube.
E. A 4 °C precooled pipette tip was placed on a p1000 pipette (set to 900 uL) and the contents of the conical tube were pipette mixed 10x while the conical tube was maintained on ice bath.
F. 145 uL of cooled solution was aliquoted into all wells of a 4 °C precooled 96 Glass Bottom 60180-P334 polystyrene microplate (Thermo Scientific) and centrifuged at 37 °C for 20 minutes at 500 RCG.
G. The plate was removed from the centrifuge and placed on a EVOS M5000 FL microscope. Bright field and EPCAM images were acquired at 10x Magnification across all wells with 10 mS exposure for both BF (white light) and FL channels (635 nM). A total of 10 slices space 20 microns in z direction using a 20x objective with 0.4 NA.
H. The plate was placed back into the incubator
I. After 24 hours of incubation, the plates were scanned 5 times every 60 minutes in BF and FL.
J. The plate was placed in an incubator for an additional 24 hrs K. The plate was removed from the incubator and a 15 uL of a 10x stock concentration of each therapy including a no therapy control (Sacituzumab-govitecan, trastuzumab deruxtecan, emtansine, deruxtecan, docetaxel). A total of 8, 3-fold serial dilutions from the cMax, the maximum concentration of the therapy achieved in serum after dosing, for each therapy was achieved in 40 test conditions performed including a "no therapy" control run in triplicate.
L. The plate was imaged immediately after dosing (dosing time 0) and then returned to the incubator.
M. The plate was imaged at 24, 48, 72, and 96 hrs post dosing
N. After completion of step M. the plate was fixed by first removing 140 uL of fluid from all the wells and replacing with 100 uL of a 1x fixative solution (2% PFA, pH 7.4, Sigma). The wells were incubated for 2 hrs.
O. Briefly, after fixation samples were washed with MilliQ-grade (16.2 MQ cm) water (4 x 15 min) and then subjected to a series of progressive (aqueous) isopropanol: 70% (1 x 30 min); 95% (3 x 15 min) and 100% (3 x 1 h) wash steps. Low-pressure vacuum was applied at each step to assure complete infiltration.
P. Afterwards, samples were immersed in molten paraffin wax (Paraplast grade, melting temperature 56-58 °C, Merck) and allowed to embed o/n. After that, samples were subjected to low-pressure vacuum for 5-10 min (with heat) and allowed to infiltrate for a further 24 h to optimize embedding. At least one extra paraffin change used to remove isopropanol.
Q. The plates were frozen at -20 °C and the paraffin silos of each well were removed from the plate.
R. The silos obtained from each well were placed in a standard embedding tray with the well surface facing down in a 3 x 3 array preserving the ordering to match the microplate layout. A paraffin block of the silos was created by standard blocking procedures.
S. Histological sections (5 pm thick) were obtained using a rotary microtome (Leica Microsystems). Deparaffinization and rehydration were done using isopropanol as well.
T. Slides were heated to 65°-70 °C (to melt paraffin without damaging tissue) before immersing in 100% isopropanol, which was repeated until complete removal of wax. Slides were then immersed in 95% v/v aqueous isopropyl alcohol (2 x 1 min), followed by 70% v/v aqueous isopropyl alcohol (30 s) and finally brought to MilliQ-grade water (6 min) prior to staining.
U. Traditional staining using Hematoxylin and Eosin (H&E) was performed on the sectioned material
V. FISH was performed with HER-2/neu immunostaining was performed using rabbit antihuman c-erbB-2 oncoprotein as primary antibody (Dako, Copenhagen, Denmark) at 1 OO dilution. Binding of the primary antibody was checked by Dako Quick-Staining, probed Streptavidin-Biotin System (LSAB; Dako, USA), followed by the addition of diaminobenzidine (DAB) as a chromogen. W. The slides were imaged EVOS M5000 FL
X. The analysis of the acquired images is described briefly for the purposes of illustration. Images obtained from growth, during exposure to therapies tested, H&E, and FISH for each well were registered and aligned relative to another using the visible boundaries of the wells or the stained features to obtain longitudinal information about the response of each group of cells in each well in the microplate. The images acquired during growth were utilized to determine areas that were changing. Standard image processing techniques applied a threshold for detection growth or response to the therapies tested obtaining integrated intensity values for areas defining each group of cells. The defined areas for each group of cells was registered to the H&E and FISH images. The signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
Y. Algorithmic determination predicted complete responses with Sacituzumab-govitecan, trastuzumab deruxtecan, deruxtecan. No response predicted with emtansine, deruxtecan, docetaxel
Example 2: Water Soluble Wax Embedded
A. An aliquot of the previously mentioned pleural effusion sample was processed as described above in Example 1 except for after completion of the fixation described in step N the following steps were performed.
B. The wells were washed with an increasing series of 40C prewarmed PEG (MW 1500) solutions (10%, 50%, and 100%) then incubated for 10 minutes at 40C then cooled rapidly at - 20 °C .
C. The plates were frozen at -20 °C and the PEG silos of each well were removed from the plate.
D. The silos obtained from each well were placed in a standard embedding tray with the well surface facing down in a 3 x 3 array preserving the ordering to match the microplate layout. A block of the silos was created by standard blocking procedures using 100% PEG solution.
E. Histological sections (5 pm thick) were obtained using a rotary microtome (Leica Microsystems).
F. Slides were heated to 65°-70° (to adhere tissue to slides) before immersing in 100% isopropanol, which was repeated until complete removal of PEG. Slides were then immersed in 95% v/v aqueous isopropyl alcohol (2 x 1 min), followed by 70% v/v aqueous isopropyl alcohol (30 s) and finally brought to MilliQ-grade water (6 min) prior to staining.
G. Traditional staining using Hematoxylin and Eosin (H&E) was performed on the sectioned material
H. FISH was performed with HER-2/neu immunostaining was performed using rabbit antihuman c-erbB-2 oncoprotein as primary antibody (Dako, Copenhagen, Denmark) at 1 OO dilution. Binding of the primary antibody was checked by Dako Quick-Staining, probed Streptavidin-Biotin System (LSAB; Dako, USA), followed by the addition of diaminobenzidine (DAB) as a chromogen.
I. The slides were imaged EVOS M5000 FL
J. The analysis of the acquired images is described briefly for the purposes of illustration. Images obtained from growth or response to the therapies tested, H&E, and FISH for each well were registered and aligned relative to another using the visible boundaries of the wells or the stained features to obtain longitudinal information about the response of each group of cells in each well in the microplate. The images acquired during growth were utilized to determine areas that were changing. Standard image processing techniques applied a threshold for probe-free measurement of growth or response to or response to the therapies tested obtaining integrated intensity values for areas defining each group of cells. The defined areas for each group of cells was registered to the H&E and FISH images. The signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
Example 3: Matrigel Domes
A. A 15 mL aliquot of the remnant sample processed up to and including step B of Example 1 was mixed with 3.75 mL of Matrigel Matrix (25% v/v) in a conical tube and vortexed.
B. A 4 °C precooled pipette tip was placed on a p1000 pipette (set to 900 uL) and the contents of the conical tube were pipette mixed 10x while the conical tube was maintained on ice bath.
C. A total of 5 droplets, each droplet was 20 uL in volume, were added to each well using a 4 °C precooled 96 Glass Bottom 60180-P334 polystyrene microplate (Thermo Scientific).
D. The plate was allowed to warm to room temperature and a 50 uL liquid overlay of growth media was added to each well then placed on a EVOS M5000 FL microscope. Bright field and EPCAM images were acquired at 10x Magnification across all wells with 10 mS exposure for both BF (white light) and FL channels (635 nM). A total of 10 slices space 20 microns in z direction using a 20x objective with 0.4 NA.
E. The plate was placed back into the incubator
F. After 24 hours of incubation, the plates were scanned 5 times every 60 minutes in BF and FL.
G. The plate was placed in an incubator for an additional 24 hrs
H. The plate was removed from the incubator and a 15 uL of a 10x stock concentration of each therapy including a “no therapy” control (Sacituzumab-govitecan, trastuzumab deruxtecan, emtansine, deruxtecan, docetaxel). A total of 8, 3-fold serial dilutions from the cMax, the maximum concentration of the therapy achieved in serum after dosing, for each therapy tested was achieved in 40 test conditions performed including a "no therapy" control run in triplicate.
I. The plate was imaged immediately after dosing (dosing time 0) and then returned to the incubator.
J. The plate was imaged at 24, 48, 72, and 96 hrs post dosing
K. After completion of step M. the plate was fixed by first removing 140 uL of fluid from all the wells and replacing with 100 uL of a 1x fixative solution (2% PFA, pH 7.4, Sigma). The wells were incubated for 2 hrs.
L. Briefly, after fixation samples were washed with M illiQ-grade (16.2 MQ cm) water (4 x 15 min) and then subjected to a series of progressive (aqueous) isopropanol: 70% (1 x 30 min); 95% (3 x 15 min) and 100% (3 x 1 h) wash steps. Low-pressure vacuum was applied at each step to assure complete infiltration.
M. Afterwards, samples were immersed in molten paraffin wax (Paraplast grade, melting temperature 56-58 °C, Merck) and allowed to embed o/n. After that, samples were subjected to low-pressure vacuum for 5-10 min (with heat) and allowed to infiltrate for a further 24 h to optimize embedding. At least one extra paraffin change used to remove isopropanol.
N. The plates were frozen at -20C and the paraffin silos of each well were removed from the plate.
O. The silos obtained from each well were placed in a standard embedding tray with the well surface facing down in a 3 x 3 array preserving the ordering to match the microplate layout. A paraffin block of the silos was created by standard blocking procedures.
P. Histological sections (5 pm thick) were obtained using a rotary microtome (Leica Microsystems). Deparaffinization and rehydration were done using isopropanol as well.
Q. Slides were heated to 65°-70° (to melt paraffin without damaging tissue) before immersing in 100% isopropanol, which was repeated until complete removal of wax. Slides were then immersed in 95% v/v aqueous isopropyl alcohol (2 x 1 min), followed by 70% v/v aqueous isopropyl alcohol (30 s) and finally brought to MilliQ-grade water (6 min) prior to staining.
R. Traditional staining using Hematoxylin and Eosin (H&E) was performed on the sectioned material
S. FISH was performed with HER-2/neu immunostaining was performed using rabbit antihuman c-erbB-2 oncoprotein as primary antibody (Dako, Copenhagen, Denmark) at 1 : 100 dilution. Binding of the primary antibody was checked by Dako Quick-Staining, probed Streptavidin-Biotin System (LSAB; Dako, USA), followed by the addition of diaminobenzidine (DAB) as a chromogen.
T. The slides were imaged EVOS M5000 FL
U. The analysis of the acquired images is described briefly for the purposes of illustration. Images obtained from growth or response to the therapies tested, H&E, and FISH for each well were registered and aligned relative to another using the visible boundaries of the wells or the stained features to obtain longitudinal information about the response of each group of cellsin each well in the microplate. The images acquired during growth were utilized to determine areas that were changing. Standard image processing techniques applied a threshold for probe-free measurement of growth or response to the therapies tested obtaining integrated intensity values for areas defining each group of cells. The defined areas for each group of cells was registered to the H&E and FISH images. The signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
V. Algorithmic determination predicted complete responses with Sacituzumab-govitecan, trastuzumab deruxtecan, deruxtecan. No response predicted with emtansine, deruxtecan, docetaxel
Example 4: Measures from Detections, Utility of Cell Differentiation
[0287] A line listing for 49 unique entities of individual detections from example 1 control (no therapy) is shown below for illustrative purposes. Such detections are used as independent orthogonal metrics useful for characterization of the group of cells leading to in aggregate an analysis of all the group of cells at a population level. In the control condition (no therapy) there is a mixture of responses for each measure. EPCAM+ signal intensities correlate with HER2+ signal as well as the H&E manual assessment of each group of cells. The combined information for Unique ID 44 and 18 suggests an incorrect categorization. Re-evaluation of these H&E results are suggestive of a cancer cell that is not recognized with high confidence using H&E alone but can putatively recategorized for the purposes of describing a response to therapy by creating an algorithm that weighs EPCAM+, Division Rate, and HER2+ in addition to H&E for classification of cancer cells in test conditions. Low frequency detection and increased confidence in classification of cell type is critical for accurate characterization of population response to no therapy conditions.
Example 5: Measures of Response
[0288] By applying an algorithm that utilizes EPCAM+, Division Rate, and HER2+ in addition to H&E a classification of cancer cells is obtained for each test condition. A corresponding population response is shown for the population and shown to increase with therapy concentration.
Example 6: Screening of Potential Therapy Application
A. An aliquot of the previously mentioned pleural effusion sample was processed as described above in Example 1 except the therapies included a library of 40 candidates was run in triplicate at a single concentration including a “no therapy” control. Additionally, after completion of the fixation described in step N the following steps were performed.
B. The wells were washed with an increasing series of 40C prewarmed PEG (MW 1500) solutions (10%, 50%, and 100%) then incubated for 10 minutes at 40C then cooled rapidly at - 20 °C .
C. The plates were frozen at -20 °C and the PEG silos of each well were removed from the plate.
D. The silos obtained from each well were placed in a standard embedding tray with the well surface facing down in a 3 x 3 array preserving the ordering to match the microplate layout. A block of the silos was created by standard blocking procedures using 100% PEG solution. E. Histological sections (5 pm thick) were obtained using a rotary microtome (Leica Microsystems).
F. Slides were heated to 65°-70° (to adhere tissue to slides) before immersing in 100% isopropanol, which was repeated until complete removal of PEG. Slides were then immersed in 95% v/v aqueous isopropyl alcohol (2 x 1 min), followed by 70% v/v aqueous isopropyl alcohol (30 s) and finally brought to MilliQ-grade water (6 min) prior to staining.
G. Traditional staining using Hematoxylin and Eosin (H&E) was performed on the sectioned material
H. FISH was performed with HER-2/neu immunostaining was performed using rabbit antihuman c-erbB-2 oncoprotein as primary antibody (Dako, Copenhagen, Denmark) at 1 OO dilution. Binding of the primary antibody was checked by Dako Quick-Staining, probed Streptavidin-Biotin System (LSAB; Dako, USA), followed by the addition of diaminobenzidine (DAB) as a chromogen.
I. The slides were imaged EVOS M5000 FL
J. The analysis of the acquired images is described briefly for the purposes of illustration. Images obtained from growth or response to the therapies tested, H&E, and FISH for each well were registered and aligned relative to another using the visible boundaries of the wells or the stained features to obtain longitudinal information about the response of each group of cells in each well in the microplate. The images acquired during growth were utilized to determine areas that were changing. Standard image processing techniques applied a threshold for probe-free measurement of growth or response to therapies tested obtaining integrated intensity values for areas defining each group of cells. The defined areas for each group of cells was registered to the H&E and FISH images. The signal contained within the areas was integrated and the collective signal was utilized to determine whether the areas contained cancerous cells (H&E), substantial HER2 signal (FISH), and growth in the presence or absence of a therapy (probe free imaging) on the group of cells basis for all areas identified changing during growth imaging.
K. The response profiles were assessed and the top 5 candidates were identified for further pharmaceutical development.
[00114] Various aspects of any and all method steps described herein may use processes, compositions, and any other material described in the following references, each of which is incorporated herein by reference in its entirety.
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Claims

1. A method of correlating a response of a group of cells to therapy to one or more molecular properties of the group of cells comprising: introducing a therapy to the group of cells; measuring a response of the group of cells to the therapy; embedding the group of cells into a matrix material capable of being sectioned while maintaining a spatial distribution of the group of cells in the matrix material; sectioning the matrix material to create at least a first section and a second section, wherein the first section comprises at least a portion of the group of cells; characterizing the at least a portion of the group of cells embedded in first section to determine at least one molecular property of the at least a portion of the group of cells; and correlating the response of the group of cells to the properties of the group of cells.
2. The method of claim 1 , further comprising correlating the response of a second group of cells to one or more molecular properties of the second group of cells, for the second group of cells, measuring a response of the second group of cells to the therapy; embedding the second group of cells into the matrix material capable of being sectioned while maintaining the spatial distribution of the second group of cells in the matrix material; wherein, in the sectioning step, the first section comprises at least a portion of the second group of cells; characterizing the at least portion of the second group of cells embedded in first section to determine at least one molecular property of the at least a portion of the second group of cells; and correlating the response of the second group of cells to the properties of the second group of cells.
3. The method of claim 1 , further comprising correlating the response of a plurality of groups of cells to one or more molecular properties of each of the plurality of groups of cells, for each group of cells introducing a therapy to each group of cells; measuring a response of each group of cells to the therapy; embedding each group of cells into a matrix material capable of being sectioned while maintaining a spatial distribution of each group of cells in the matrix material; wherein, in the sectioning step, the first section comprises at least a portion of each group of cells; characterizing a portion of each group of cells embedded in first section to determine at least one molecular property of the at least a portion of the group of cells; and correlating the response of the group of cells to the properties of the group of cells.
4. The method of claim 1 , comprising introducing multiple therapies to the group of cells.
5. The method of any one preceding claim, wherein the response in the measuring step is different from the at least one molecular property.
6. The method of any one preceding claim, wherein measuring comprises a single response.
7. The method of one of claims 1 - 5, wherein a plurality of responses is measured.
8. The method of any one preceding claim, wherein the at least one molecular property is a multiplex of molecular properties.
9. The method of any one preceding claim, wherein the at least one molecular property is a gene expression or protein or reflects an epigenetic modification.
10. The method of any one preceding claim, wherein the at least one molecular property is growth of the group of cells.
11 . The method of any one preceding claim, further comprising obtaining a sample of tissue or bodily fluid comprising the group of cells from a patient.
12. The method of claim 11 , wherein the sample is acquired within 24 hours.
13. The method of any one preceding claim, further comprising evaluating the efficacy of the therapy.
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