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WO2012040579A2 - Criblage moléculaire à travers des matrices de rigidité définie - Google Patents

Criblage moléculaire à travers des matrices de rigidité définie Download PDF

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Publication number
WO2012040579A2
WO2012040579A2 PCT/US2011/052990 US2011052990W WO2012040579A2 WO 2012040579 A2 WO2012040579 A2 WO 2012040579A2 US 2011052990 W US2011052990 W US 2011052990W WO 2012040579 A2 WO2012040579 A2 WO 2012040579A2
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Prior art keywords
cell
cells
array
screening
populations
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WO2012040579A3 (fr
Inventor
Daniel Tschumperlin
Justin Mih
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Harvard University
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Harvard University
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    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity

Definitions

  • the present invention relates to systems, compositions and methods for screening of cells.
  • cells e.g., adherent cells
  • present disclosure provides methods for cell-based screening (e.g., using multiwell plates) featuring substrates of user-defined stiffness. The methods are used to assess the magnitude and character of cellular drug responses across a physiological stiffness range.
  • a stiffness-tunable polymer substrate e.g., polyacrylamide
  • methods of screening for candidate compounds e.g., small molecules, macromolecules, proteins, nucleic acids, pharmacological agents, pharmacological inhibitors, etc.
  • stiffness-dependent biology e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors.
  • a “candidate compound” is meant a chemical, protein, nucleic acid, or other agent, be it naturally-occurring or artificially-derived.
  • Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.
  • pharmaceutical agent or “pharmaceutical composition” is meant any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, (ed. A. R. Gennaro), Mack Publishing Co., Easton, Pa., 2000.
  • a method for screening for compounds that have cell proliferation modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell proliferation activity is affected by the presence of the test agents as compared to a control.
  • a shear modulus of about 10 to about 1,000,000 Pascals e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000
  • a method for screening for screening for compounds that have cell proliferation modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell proliferation activity of any one of the cell or cell populations
  • a method for screening for compounds that inhibits cell proliferation comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell proliferation.
  • a shear modulus of about 10 to about 1 ,000,000 Pascals
  • a method for screening for compounds that have cell adherence modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell adherence activity is affected by the presence of the test agents as compared to a control.
  • the methods are used to identify compounds that modulate adherence in a physiological setting based on their affect on cells cultured on a substrate characterized by an elastic modulus mimicking the in vivo microenvironment.
  • a method for screening for screening for compounds that have cell adherence modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell adherence activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.
  • a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa,
  • a method for screening for compounds that inhibits cell adherence comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell adherence.
  • a shear modulus of about 10 to about 1 ,000,000 Pascals
  • a method for screening for compounds that have cell spreading modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell spreading activity is affected by the presence of the test agents as compared to a control.
  • a shear modulus of about 10 to about 1,000,000 Pascals e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000
  • a method for screening for screening for compounds that have cell spreading modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell spreading activity of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.
  • a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about
  • a method for screening for compounds that inhibits cell spreading comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1 ,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that inhibits cell spreading.
  • a shear modulus of about 10 to about 1 ,000,000 Pascals
  • a method for screening for compounds that have apoptotic modulating activity comprising: contacting a cell or cell population with a test agent, wherein the cell or cell population is adherent to a hydrogel having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); and determining whether cell viability is affected by the presence of the test agents as compared to a control.
  • a shear modulus of about 10 to about 1,000,000 Pascals
  • the test agent is known to induce apoptosis in cells.
  • a method for screening for screening for compounds that have apoptotic modulating activity comprising: placing cells or cell populations in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cells or cell populations with a test agent; and determining whether cell viability of any one of the cell or cell populations of the array is affected by the presence of the test agents as compared to a control.
  • the test agent is known to induce apoptos
  • a method for screening for compounds that induces apoptosis comprising: placing a cell or cell population in assigned locations in an array of compliant substrates having a shear modulus of about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, from about 10 to about 900,000 Pa); contacting the cell or cell population with a test agent; and determining which cell or cell population populations are rendered sensitive to drug treatment, thereby identifying a drug that induces apoptosis.
  • the test agent is known to induce apoptosis in cells.
  • a shear modulus of about 10 to about 1,000,000 Pascals e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10
  • the substrates have a shear modulus of about 0.3 to about 55 kilo Pascals.
  • each location in the array of compliant substrates comprises a unique shear modulus.
  • the array of compliant substrates comprises a shear modulus that spans the entire stiffness range (e.g. , 0.3-55 kPa).
  • the cell or cell population stained or left unstained, and if stained, is fixed prior to staining the cell or cell population.
  • the cell or cell population is not fixed prior to staining the cell or cell population.
  • the cells are stained for stained for F- actin and/or nuclei using methods well known in the art.
  • the cell or cell population is imaged, thereby performing automated fluorescent imaging.
  • the cell or cell population is imaged using autofocusing to capture images of cells on all substrates.
  • the images are detected and processed, e.g. , using a computer or other processor.
  • the cell or cell population is imaged using a Pathway HTTM fluorescence imaging system with autofocusing set at 400 nm step stage positioning.
  • Imaging of the cell or cell population comprises imaging cellular morphology.
  • the array comprises a 6 well plate, a 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, a 384 well plate, a 1536 well plate, a 3456 well plate, or a 9600 well plate.
  • a shear modulus of about 10 to about 1,000,000 Pascals e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa,
  • the cells or cell populations are contacted with a nucleic acid composition, and it is determined whether cellular phenotype of any one of the cell or cell populations of the array is affected by the presence of the nucleic acid composition as compared to a control.
  • the nucleic acid composition is an inhibitor of ribonucleic acid (RNA) selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA).
  • RNA ribonucleic acid
  • shRNA short hairpin RNA
  • the phenotype comprises cellular morphology, cellular adhesion, or cytoskeleton function.
  • Methods of determining an individualized course of treatment of a subject are carried out by providing a cell population from a subject or providing a sample of cells obtained from an individual, e.g., a human patient, to be treated. Subsequently, the cells or cell population are placed in assigned locations in an array of compliant substrates having a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about 400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa, from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, or from about 10 to about 900,000 Pa).
  • a shear modulus of between about 10 to about 1,000,000 Pascals (e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about 10 to about 200,000 Pa, from about 10 to about 300
  • the cells or cell population are contacted with a test agent, and cell viability, cell proliferation activity, adherence activity, or spreading activity of any one of the cells or cell populations of the array is detected, measured and analyzed, e.g., using a computer or other processor. Based on the collected and processed data, a determination is made as to whether any of the parameters are reduced or increased by the presence of the test agent compared to a control.
  • the individualized course of treatment is selected base on performance of the test compound in the in vitro assay that simulates the in vivo condition. Such a determination is particularly useful for selecting treatment for abnormal conditions, e.g. , cancer, hypertension, fibrosis.
  • the test agent is administered to the subject to treat the abnormal condition, i.e. , disease.
  • Polynucleotides, polypeptides, or other agents are purified and/or isolated.
  • an "isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purified compounds are at least 60% by weight (dry weight) the compound of interest.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest.
  • a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.
  • a purified or isolated polynucleotide ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Purified also defines a degree of sterility that is safe for administration to a human subject, e.g. , lacking infectious or toxic agents.
  • nucleotide or polypeptide that has been separated from the components that naturally accompany it.
  • nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
  • isolated nucleic acid is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes.
  • the term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybridgene, i.e., a gene encoding a fusion protein.
  • Isolated nucleic acid molecules according to the present invention further include molecules produced
  • a small molecule is a compound that is less than 2000 daltons in mass.
  • the molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
  • Figure 1 Integration of matrix stiffness into a multiwell plate for routine cell- based screening, (a) Hydrogel shear modulus (G) is specified by acrylamide:bisacrylamide content and measured by atomic force microscopy indentation (circles). The density of gel- bound collagen (lines) is tuned independently of stiffness, (b) Prototypical multiwell plate configuration, (c) Emergence of gradually increasing (blue), abrupt transition (green) and stiffness-insensitive (orange) cell accumulation following 72 hours of culture across a physiological stiffness range.
  • G Hydrogel shear modulus
  • A549 human lung adenocarcinoma cell line
  • NHDF normal human dermal fibroblast
  • NHLF normal human lung fibroblast
  • NIH 3T 3 mouse embryonic fibroblast cell line
  • MDCKII Madin-Darby canine kidney epithelial cell line
  • hMSC human mesenchymal stem cell
  • 16HBE14o- human bronchial epithelial cell line
  • RLE6TN rat lung epithelial cell line
  • L292 mouse fibroblast cell line
  • HEK29 human embryonic kidney cell line
  • Figure 4 Schematic of polyacrylamide hydrogel incorporation into a multiwell plate.
  • FIG. 7 Divergent responses to ROCK inhibitors. The effects of Y-27632, fasudil hydrochloride and H-l 152 on the accumulation of NHLFs on 400 Pa versus rigid substrates. The divergent responses to GSK 429286 treatment are recapitulated in hMSCs and NIH3T3 cells.
  • Figure 8 ROCK inhibition rescues the cell spreading defect observed on low stiffness substrates. NHLFs were fixed following 48 hours of culture under the indicated conditions and stained for f-actin (green) and nuclei (blue).
  • Figure 9 is a bar chart showing the effect of siRNA targeting non-muscle myosin heavy chain Ila (MYH9) on fibroblasts.
  • Figure 10 is a series of photomicrographs illustrating automated imaging of cell morphology in a 384 well plate. Seven cell types cultured across increasing substrate stiffness, stained for F-actin (red) and nuclei (blue). Images were obtained at 200X magnification.
  • Figure 11 is a schematic illustrating an autofocusing imaging system to detect, measure, and process data regarding morphology, proliferation, protein expression, and other parameters.
  • Cells are cultured in assigned locations in an array of compliant substrates. The cells are imaged utilizing an autofocusing microscope. Finally, a computer/processor is utilized to visualize, measure, and analyze physical parameters of cellular morphology.
  • the physical environment of a living cell influences its ability to proliferate, metabolize, differentiate and remodel.
  • Living cells specify lineage and express different phenotypic and physical states with extreme responsiveness to stiffness of their underlying matrix.
  • the stiffness of the extracellular matrix is a vital mechanical cue that regulates cellular fate and function (Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Cell 126, 677-689 (2006); Discher, D.E., Janmey, P. & Wang, Y. Science 310, 11 39-114 3 (2005)).
  • stiffening or softening of tissue that occurs in a number of diseases, such as sclerodoma, atherosclerosis, emphysema, and fibrosis of the lung, liver and kidney.
  • the compliant surface culture plate technology opens up the field to the assessment of stiffness-dependent cell behaviors at a level of detail that is not currently possible. For example, simulation of cell growth/behavior to contact with fat tissue is carried out by growing cells on a surface characterized by a shear modulus of fat (approximately 10 Pascal). Different tissue types are characterized by different stiffness, e.g., normal brain tissue has a shear modulus of approximately 200 Pascal.
  • Cell growth/behavior also differs relative to the disease state of a given tissue, e.g., the shear modulus of normal mammary tissue is approximately 100 Pascal, whereas that of breast tumor tissue is approximately 2000 Pascal.
  • normal liver tissue has a shear modulus of approximately 300 Pascal compared to fibrotic liver tissue, which is characterized by a shear modulus of approximately 800 Pascal.
  • Growth, signal transduction, gene or protein expression/secretion, as well as other physiologic parameters are altered in response to contact with different substrate stiffness and evaluated in response to contact with substrates characterized by mechanical properties that simulate different tissue types or disease states.
  • the terms "soft substrate” and "stiff substrate” are relative terms depending upon the tissue type being tested.
  • exemplary wells of a multiwall plate comprise wells with substrates that are 10, 20, 50, 100, 200, 400, 800, 1000, 2000, 5000, 10000, or more Pa as well as intermediate values.
  • the present invention provides for methods of screening for compounds (e.g. , small molecules, macromolecules, etc.) that modulate stiffness-dependent biology (e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors).
  • compounds e.g. , small molecules, macromolecules, etc.
  • stiffness-dependent biology e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors.
  • devices and methods related to a stiffness-tunable polyacrylamide high-throughput screening system there is provided devices and methods related to a stiffness-tunable polyacrylamide high-throughput screening system.
  • methods of screening for compounds e.g., small molecules,
  • stiffness-dependent biology e.g., cell proliferation, cell adhesion, cell spreading, apoptosis, signaling events, and/or detection of soluble and insoluble factors.
  • methods for screening for compounds that modulate stiffness-dependent biology comprising contacting a cell adherent to a compliant substrate having a shear modulus (i.e. , stiffness) of about 10 to about
  • cells are transferred into a suitable tissue culture plate (e.g., multiwell plate) having a surface covered with a compliant substrate having a shear modulus of about 10 to about 1,000,000 (e.g., from about 10 to about 100,000, from about 10 to about 150,000 Pa, from about 10 to about 200,000, from about 10 to about 300,000, from about 10 to about 400,000, from about 10 to about 500,000, from about 10 to about 600,000, from about 10 to about 700,000, from about 10 to about 800,000, from about 10 to about 900,000).
  • tissue culture plate e.g., multiwell plate
  • a compliant substrate having a shear modulus of about 10 to about 1,000,000 e.g., from about 10 to about 100,000, from about 10 to about 150,000 Pa, from about 10 to about 200,000, from about 10 to about 300,000, from about 10 to about 400,000, from about 10 to about 500,000, from about 10 to about 600,000, from about 10 to about 700,000, from about 10 to about 800,000,
  • the cells are brought into contact with a test agent and after some incubation period, the effect of the test agent on a stiffness-dependent biology is assessed.
  • the effect of the test agent on a stiffness-dependent biology is assessed.
  • cells may be brought into contact with a test agent and after some incubation period (e.g., 72 hours) the impact of the test agent on the degree of proliferation of a cell or cell population is assessed.
  • the cells are allowed to adhere to the compliant substrate in the presence or absence of test agent.
  • test agent for example, where cell adherence is the stiffness- dependent biology, cells may be brought into contact with a test agent at the time of plating, and after some incubation period (e.g., 72 hours) the impact of the test agent on the degree of adherence of a cell or cell population is assessed.
  • the incubation period may be from 4 hours to 96 hours (e.g., about 4, 8, 12, 24, 36, 48, 72, 96 hours and points in-between).
  • the cells are cultures for several days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. days).
  • the effect of the test agent on a stiffness-dependent biology may be assessed in terms of a degree of impact such as a 10% to 1000% increase or decrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase or decrease in a specified activity).
  • a degree of impact such as a 10% to 1000% increase or decrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase or decrease in a specified activity).
  • a method for screening for compounds that have cell proliferation modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell proliferation activity is affected.
  • the cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments.
  • the detecting step comprises detecting whether cell proliferation pathways have been modulated (e.g. , activated or inhibited).
  • a method for screening for compounds that have cell adhesion modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell adhesion activity is affected.
  • the cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments.
  • the detecting step comprises detecting whether cell adhesion pathways have been modulated (e.g. , activated or inhibited).
  • a method for screening for compounds that have cell spreading modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether cell spreading activity is affected.
  • the cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments.
  • the detecting step comprises detecting whether cell spreading pathways have been modulated (e.g. , activated or inhibited).
  • a method for screening for compounds that have apoptotic modulating activity comprising contacting a cell or population of cells with a test agent and detecting whether apoptotic activity is affected.
  • the cell or cell population are adherent cultures on the user-defined matrices (e.g., compliant substrate such as a hydrogel) of the present embodiments.
  • the detecting step comprises detecting whether apoptotic pathways have been modulated (e.g. , activated or inhibited).
  • cells e.g., tumor cells
  • the assay dish may be a 6-well, 12-well, 24-well, 96-well, 384-well or 1536-well assay dish.
  • the cells are contacted with a selected drug to be screened and the cell response to selected drug is assessed (e.g., viability or sensitivity).
  • the arrays can be designed to take advantage of systems developed for current assay formats, such as detection systems and robotic systems and the like which are designed to handle 6-well, 12-well, 24-well, 96-well, 384-well, 1536-well plates, or even 9,600- microwell plates, for example.
  • the present invention is not limited to the presently used microtiter plate configurations but provides for any configuration necessary to take advantage of industry standards as well as provides the flexibility to design for novel configurations. Tissue Culture Substrate/Solid Support - Mutiwell Systems
  • the invention provides an apparatus including a tissue culture substrate or solid substrate suitable for tissue culture (e.g., multi- well plate) and a gel affixed to a surface of the plate.
  • tissue culture substrate may be any surface or solid support suitable for use in tissue culture.
  • the tissue culture substrate is in the form of multiwell, glass-bottom plates.
  • the thickness of the gel is less than 1 millimeter (e.g., less than 800 ⁇ , less than 600 ⁇ , less than 500 ⁇ , less than 400 ⁇ , less than 300 ⁇ , less than 200 ⁇ , less than 150 ⁇ , less than 100 ⁇ , less than 75 ⁇ , less than 50 ⁇ , etc.). In some embodiments, the thickness of the gel is between 50 ⁇ and 150 ⁇ (e.g., ⁇ or less than 100 ⁇ ).
  • multiple shear modulus gels are casted and derivatized in a multi-well glass-bottom plate.
  • a collection of wells with varying shear modulus i.e., stiffness
  • a multi-well plate is fabricated with a range of compliant substrates and used to test cellular responses across a plate with well each of which contain a substrate with stiffness ranging from e.g., 50 to 150,000 Pascals.
  • lung fibroblast proliferation and apoptosis which is strongly dependent upon substrate shear modulus can be tested.
  • Cells can be grown in the plates, and can be manipulated and analyzed in a manner consistent with conventional multi-well plates.
  • Modern (circa 2008) microplates generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells.
  • shear modulus gels varying in orientation, can be casted and derivatized in a multi-well glass-bottom plate.
  • a collection of wells with varying shear modulus i.e., stiffness
  • an apparatus including a tissue culture substrate or solid substrate suitable for tissue culture (e.g., multi- well plate) and a gel affixed in at least a first well in the plate.
  • a multi- well plate can use synthetic matrix-coated hydrogels to span a physiological range of shear modulus values.
  • a 96-well plate which is a generally used format for biological assays, can be used.
  • the system can also be extended to other formats that are amendable to high- throughput screening (384- well plates or other multiple of 96) or cell cultivation (petri dish).
  • a polyacrylamide hydrogel is polymerized as a thin, optically transparent layer which is affixed to the bottom of each well.
  • the elasticity can be tuned over the range of typical soft tissues (heart, lung, kidney, liver, muscle, neural, etc.) from elastic modulus -20 Pascals (fat) to -100,000 Pascals (skeletal muscle) or more.
  • prior art cell culture dishes made from polystrene plastic have a stiffness of -3,000,000,000 Pascals.
  • the well can be a multi-well configuration such as a 96- well assembly comprising a 12 x 8 matrix of wells in a plate (e.g., a Matrical 96-well assembly).
  • the well can also be a 6-well, 24- well, 384-well configurations.
  • the well can include standard multi-well plates used to study various biological endpoints under different interventions.
  • the bottom of the well bottom can be glass to allow for observation of cells placed within the well.
  • the present invention provides for methods for fabricating user defined matrices (e.g., hydrogels) with elastic properties covering a broad, physiologically relevant range.
  • the hydrogel solution can be delivered unto a surface of the tissue culture substrate (e.g., well) via a dispensing system (e.g., pipette, automated liquid dispensing system).
  • a dispensing system e.g., pipette, automated liquid dispensing system.
  • Different concentrations of the hydrogel solution can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa.
  • Polymer hydrogels suitable for use with the present embodiments include, but are not limited to, the following: acrylamide/bis-acrylamide, polyalkylimide, poly(N- vinyl formamide), polyvinyl alcohol, poly(ethylene glycol), polydimethylsiloxane, silicone, glycosaminoglycans, hyaluronic acid, chondroitin sulfate, polysaccharide, self-assembling peptides, collagen, gelatin, fibrin, methylcellulose, and agarose.
  • the polymerization solution can be comprised of variable ratios of acrylamide:bis-acrylamide, and can be delivered into the well via a dispensing system (e.g., pipette, automated liquid dispensing system). Different concentrations of the acrylamide / bis-acrylamide mixture can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa.
  • a dispensing system e.g., pipette, automated liquid dispensing system.
  • Different concentrations of the acrylamide / bis-acrylamide mixture can be used to produce gels of different shear modulus, for example, ranging from 20 to 100,000 Pa.
  • hydrogels can be affixed (i.e., firmly attached) to the bottom of a well (e.g., wells in 24, 96, 384 well plates).
  • the firm attachment enables long-term cell cultures, as well as compatibility with some assay reagents that may cause the hydrogels to shrink and detach.
  • the plates can support the attachment and growth of different cell types and can be compatible with standard multi-well plate assays.
  • the mechanical properties of the hydrogels can be reproducible and stable to increase the shelf life of the substrate.
  • the hydrogel solution is delivered unto a surface of a well such that wells can be covered with gels of uniform thickness and static or various shear moduli.
  • a 96- well plate for example, can be loaded with plurality of polymerization solutions in different wells.
  • the wells can be covered with gels of uniform thickness and various shear moduli (e.g., 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, and 51200 Pa).
  • the well columns can be loaded with gels of increasing shear modulus in ascending order (i.e., 50, 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 Pa respectively).
  • the hydrogels are optically transparent, thin ( ⁇ 100 ⁇ ) and bound covalently to the entire surface of each well.
  • each well can typically receive 1-2 microliters of polymerization solution.
  • a 96- well assembly typically will receive about 5 microliters.
  • the amount of polymerization solution can change based on the desired thickness of resulting gel.
  • the delivery system can be a pipette or similar liquid dispensing system (e.g., BioTek Microplate Liquid dispensing system).
  • the glass plate can be hydrophobic glass. As an example, and not a limitation, a circular well, the diameter of the glass plate is less than the diameter of the well.
  • methods of fabricating hydrogels includes placing a first polymerizing solution into a well, covering the first polymerizing solution with a plate, such that the area of the plate is less than the area of the well, conjugating the first polymerizing solution with a ligand, placing a second polymerizing solution into the well, such that oxygen in the air substantially inhibits polymerization at the air-liquid interface; and detoxifying the well.
  • a second polymerizing solution can be distributed evenly by tapping the well.
  • the second polymerizing solution can substantially cover a ring shaped area defined by the edge of the first polymerizing solution and the well.
  • the second polymerizing solution can affix the first polymerizing solution to the well.
  • Acrylamide, and other polymer solutions can be highly toxic to cells, so it is generally necessary to include a detoxification process.
  • glutathione can be added to detoxify free, unpolymerized acrylamide (e.g., a solution of glutathione can be dispensed into the well and incubated for several hours prior to seeding the well with cells).
  • Shear Moduli can be added to detoxify free, unpolymerized acrylamide (e.g., a solution of glutathione can be dispensed into the well and incubated for several hours prior to seeding the well with cells).
  • the shear modulus of the gels can cover a range or be kept static.
  • a multi-well tray can include gels with a standard shear modulus (e.g., 5000, 10,000, 17,000 Pa) in each well.
  • the configuration of wells encompassing the stiffness range of adipose tissue to skeletal muscle can be specified.
  • the shear modulus of the cell culture substrate of the device is in the range of the tissue type to be evaluated.
  • the shear modulus may range from about 10 to about 3,000,000,000 Pascals (Pa). In some embodiments, the shear modulus may range from about 10 to about 100,000 Pascals (Pa), including from aboutlO Pa to about 100,000 Pa, from about 20 Pa to about 100,000 Pa, from about 100 Pa to about 100,000 Pa, from about 400 Pa to about 100,000 Pa, from about 800 Pa to about 100,000 Pa, from about 1200 Pa to about 100,000 Pa, from about 1600 Pa to about 100,000 Pa, from about 3200 Pa to about 100,000 Pa, from about 6400 Pa to about 100,000 Pa, from about 12800 Pa to about 100,000 Pa, from about 25600 Pa to about 100,000 Pa, from about 50,000 Pa to about 100,000 Pa, from about 10 Pa to about 51200 Pa, from about 20 Pa to about 51200 Pa, from about 100 Pa to about 51200 Pa, from about 400 Pa to about 51200 Pa, from about 800 Pa to about 51200 Pa, from about 1200 Pa to about 51200 Pa, from about 1600 Pa to about 51200 Pa, from about 3200 Pa to about 51200 Pa, from about 6400 Pa to about 51
  • the hydrogels may be further modified or treated with a substance (e.g., matrix protein) to foster cell attachment.
  • a substance e.g., matrix protein
  • the otherwise inert gels may be functionalized with equivalent amounts monomeric collagen or other suitable attachment ligand or combinations of attachment ligands.
  • suitable attachment ligands include, but are not limited to, the following: collagen I, collagen IV, fibronectin, vitronectin, laminin, or RGD peptides. Attachment ligands can be coupled to the gel surface using any means known in the art. The uniformity of ligand binding can be assessed with anti-ligand and anti-IgG-coated fluorescent beads.
  • the attachment ligand (e.g., collagen) can be fixed at a density between 1 ⁇ g/ml and 100 ⁇ g/ml (e.g., between 1 ⁇ g/ml and 100 ⁇ g/ml, between 1 ⁇ g/ml and 80 ⁇ g/ml, between 1 ⁇ g/ml and 60 ⁇ g/ml, between 1 ⁇ g/ml and 50 ⁇ g/ml, between 1 ⁇ g/ml and 40 ⁇ g/ml, between 1 ⁇ g/ml and 20 ⁇ g/ml, between 1 ⁇ g/ml and 10 ⁇ g/ml, between 1 ⁇ g/ml and 5 ⁇ g/ml, between 2 ⁇ g/ml and 100 ⁇ g/ml, between 2 ⁇ g/ml and 80 ⁇ g/ml, between 2 ⁇ g/ml and 60 ⁇ g/ml, between 2 ⁇ g/ml and 50 ⁇ g/ml, between 2 ⁇ g/ml
  • the concentration of serum present in the culture media may be optimized between 0% and 15% for each test condition (e.g., between 1% and 10%, between 3% and 10%, between 5% and 10%, between 1 % and 15%, between 3% and 15%, between 5% and 15%, between 10% and 15%, between 1 % and 3%, between 1 % and 5%, between 3% and 5%, etc.)
  • Cells e.g., between 1% and 10%, between 3% and 10%, between 5% and 10%, between 1 % and 15%, between 3% and 15%, between 5% and 15%, between 10% and 15%, between 1 % and 3%, between 1 % and 5%, between 3% and 5%, etc.
  • the cells may be primary cells or of an established cell line.
  • Cells may be of a type selected from the group consisting of cardiovascular, gastrointestinal, kidney, genitourinary, musculoskeletal, nervous system, oral, breast, periodontal, or skin cell or progenitor thereof. Cells may be normal or transformed cells.
  • the cells are tumor cells of known genotype and/or representative of a particular form of cancer.
  • cells are seeded on a hydrogel (e.g., rapidly casted hydrogels) of specified stiffness over a tissue culture substrate.
  • cells are seeded at subconfluent density (e.g., less than 100 cells/mm 2 , less than 90 cells/mm 2 , less than 80 cells/mm 2 , less than 70 cells/mm 2 , less than 60 cells/mm 2 , less than 50 cells/mm 2 , less than 40 cells/mm 2 , less than 30 cells/mm 2 , less than 20 cells/mm 2 , less than 15 cells/mm 2 , less than 10 cells/mm 2 , less than 5 cells/mm 2 , and points in between).
  • subconfluent density e.g., less than 100 cells/mm 2 , less than 90 cells/mm 2 , less than 80 cells/mm 2 , less than 70 cells/mm 2 , less than 60 cells/mm 2 , less than 50 cells/mm 2 , less than 40 cells/mm 2 , less than 30 cells/mm 2 , less than 20
  • Cells grown in the multiple shear modulus plate can be fixed and immunologically stained, or isolated for gene expression and protein analysis. Attachment-dependent cell types can be studied, including fibroblasts, smooth muscle, endothelial, epithelial, tumor, osteoid, and neuronal. The plate can serve as a tool to direct the differentiation of adult or embryonic stem cells.
  • the cell is a stem cell or progenitor cell such as an embryonic stem cell or induced pluripotent stem cell.
  • Such a stem cell may be a pluripotent cell of mesodermal, ectodermal or endodermal origin.
  • a stem cell is of mesodermal origin.
  • a stem cell is a hematopoiteic progenitor cell.
  • Exemplary cells to be used in screening methods include endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, ASCs, preadipocytes, differentiated or de-differentiated adipocytes, keratinocytes, unipotent and multipotent progenitor and precursor cells, as well as lymphocytes and precursors thereof.
  • the cell is a cancer (tumor) cell, e.g., lung, breast, colon, prostate, pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries, urinary tract, rectal tract, or is a melanoma or leukemia.
  • the cell is a non-tumor (normal) tissue derived cell or lineage committed cell, e.g. a breast, liver, prostate, brain, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, muscle, thyroid, prostate, uterus, cervix, ovaries, urinary tract, rectal tract, pancreatic or bladder cell.
  • the cell is a patient-derived primary cells (e.g., tumor-derived, non- tumor (normal) tissue derived).
  • the selected drug or test agents may be any drug known to interact with cell proliferative pathways, cell adhesion pathways, cell spreading pathways, apoptotic pathways, etc.
  • the selected drug or test agent is a small molecule.
  • Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • peptides e.g., peptoids
  • amino acids amino acid analogs
  • polynucleotides polynucleotide analogs
  • a small molecule is a compound that is less than 2000 daltons in mass (e.g., a small molecule inhibitor).
  • the molecular mass of the small molecule compounds is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
  • RNA interference e.g. , small interfering RNA
  • exemplary genes to be target include those that are involved in cell adhesion and cytoskeleton function.
  • pharmacological agents are utilized to inhibit genes of interest.
  • antibodies e.g., neutralizing antibodies that bind to cell surface proteins such as integrins or growth factor receptors, are screened to identify those cell surface proteins or structures that play a role in cell stiffness dependency.
  • the expression of such genes/gene products are unique to substrate stiffness characteristics and can identify molecules that are differentially expressed on diseased cells compared to normal cells. For example, a cell surface protein is present on diseased cells but not normal cells or are expressed. Such proteins are then exploited for targeted drug delivery strategies.
  • the selected drugs or test agent is a known
  • chemotherapeutic agent is art recognized and is intended to include those chemical and biological agents, including small molecules and larger molecules, such as peptides, proteins, lymphokines, antibodies, tumor necrosis factor, conjugates of antibodies with toxins, and other chemical or biological molecules which have an anti-tumor effect (e.g., inhibits cancer cell growth such as by inducing cancer cell senescence or death).
  • Chemotherapeutic agents to be screened fall into the following exemplary categories: modulators of intracellular signaling proteins, alkylating agents, antimetabolites, antitumor antibiotics, plant alkaloids, terpenoids, topoisomerase inhibitors, taxanes, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof. See also Table 1-1 in Example 1 below.
  • Common chemotherapeutic agents include, but are not limited to, Sorafenib (4- [4- [[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl-pyridine-2- carboxamide), Gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4- ylpropoxy)quinazolin-4-amine) , Imatinib (4- [(4-methylpiperazin- 1 -yl)methyl] -N- [4-methyl- 3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide), PLX5568, Dasatinib (BMS- 354825), Staurosporine, midostaurin, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlor
  • Imatinib is used to treat chronic myelogenous leukemia (CML), gastrointestinal stromal tumors (GISTs) and other cancers.
  • CML chronic myelogenous leukemia
  • GISTs gastrointestinal stromal tumors
  • Chemotherapeutic agents include inhibitors or modulators of intracellular signaling proteins. Categories of such agents include: protein kinase inhibitors; Src family tyrosine kinases inhibitors; BCR/ABL inhibitors; MEK inhibitors; Raf inhibitors; SH2 55 inhibitors; PI3K inhibitors; and JNK inhibitors. The use of these inhibitors may be selected based on the target cell being screened. For example, MEKi in melanoma, PI3K inhibitors in lung cancer cell lines.
  • compositions to be screened include small molecules (e.g., less than 2,000 daltons molecular mass), antibodies, or polynucleotides (e.g., RNAi molecules).
  • small molecules e.g., less than 2,000 daltons molecular mass
  • antibodies e.g., antibodies
  • polynucleotides e.g., RNAi molecules.
  • the table below provides a list of protein kinase inhibitors.
  • Dasatinib is dual BCR/ABL and Src family of tyrosine kinases inhibitors may be used in chronic myelogenous leukemia (CML) and metastatic melanoma.
  • CML chronic myelogenous leukemia
  • MEK inhibitors MEKi
  • MEK is a key protein kinase in the RAS/RAF/MEK/ERK pathway, which signals for cancer cell proliferation and survival.
  • MEK is frequently activated in cancer, in particular in tumors that have mutations in the RAS and RAF oncogenes.
  • MEK also regulates the biosynthesis of the inflammatory cytokines TNF, IL-6 and IL-1, which can act as growth and survival factors in cancer.
  • Examples of MEK inhibitors include: 2-(2-amino-3-methoxyphenyl)-4-oxo-4H- [l]benzopyran; 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro- benzami- de; ARRY-162; ARRY-300; AZD6244 (ARRY-886); and AZD8330 (ARRY-704).
  • Categories of MEK inhibitors include: Substituted diarylamines (U.S. Patent No. 7001905); 4-arylamino, 4-aryloxy, and 4-arylthio diarylamines and derivatives (U.S. Patent No.
  • PI3K inhibitors PI3Ki
  • PI3K inhibitors are Wortmannin (Calbiochem, La Jolla, Calif.; Cat. No. 681675), benzo[b]thiophene-2-carboxamide, 5,6-dimethoxy-3-phenoxy-N-lH-tetrazol-5- yl-.
  • any compound is a PI3K inhibitor if it is able to block the catalytic activity of PI3K.
  • Other PI3K inhibitors known in the art can also be utilized in the present invention.
  • JNK inhibitors JNKi
  • Examples include: Anthra[l,9-cd]pyrazol-6(2H)-one (selective c-Jun N-terminal protein kinase (JNK) inhibitor).
  • JNK selective c-Jun N-terminal protein kinase
  • Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.
  • An alkylating agent may include, but is not limited to, a nitrogen mustard, an ethylenimene, a
  • methylmelamine an alkyl sulfonate, a nitrosourea or triazines.
  • They include but are not limited to: busulfan, chlorambucil, cisplatin, carboplatin, chlorambucil, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), Melphalan (PAM), oxaliplatin, cis-Diammminedichloroplatinum (II) (CDDP), nitrosoureas such as N,N'-bis(II- chloroethyl)-N-nitrosourea (BCNU), nitrogen mustards, ethyleneimine compounds, alkyl sulphonates, cisplatin and dacarbazine.
  • busulfan chlorambucil, cisplatin, carboplatin, chlorambucil, cyclophosphamide (Cytoxan
  • Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, antimetabolites specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, methotrexate, folic acid, purine or pyrimidine antagonists, 6-Mercaptopurine, fluorodeoxyuridine, cytosine arabinoside, azathioprine, mercaptopurine and thioquinone.
  • 5-FU 5-fluorouracil
  • Ara-C cytarabine
  • fludarabine gemcitabine
  • methotrexate methotrexate
  • folic acid purine or pyrimidine antagonists
  • 6-Mercaptopurine fluorodeoxyuridine
  • Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogs and derivatives thereof may be isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.
  • Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
  • Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
  • Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include, but are not limited to, compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules.
  • Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity.
  • the vinca alkaloids include: Vincristine, Vinblastine, Vinorelbine, and Vindesine.
  • Podophyllotoxin is a plant-derived compound which is said to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide.
  • Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific and therefore work in all phases of the cell cycle. Examples of antitumor antibiotics include, but are not limited to, actinomycin, adriamycin, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.
  • RNAi drugs and other drugs that affect genes involved in tumorigenesis and metastasis may have different effects on cells depending on the matrix stiffness upon which they are tested (reflective of the in vivo environment).
  • nucleic acids e.g., RNAi and other nucleic acid-based therapies
  • RNAi is a naturally occurring mechanism that controls gene expression at the post-transcriptional level.
  • double-stranded interfering RNAs target complementary mRNAs for degradation, resulting in selective silencing of specific proteins.
  • This characteristic of RNAi makes it a valuable laboratory research tool, both in cells and in whole animal models.
  • RNAi libraries which are composed of reagents that systematically target every gene in the genome, has made it possible to conduct highthroughput screens that interrogate phenotypes associatedwith the loss-of-function of many genes simultaneously.
  • RNAi By suppressing gene expression and therefore protein function, RNAi, to a certain extent, models the pharmacological inhibition of a target protein and is therefore an effective tool for proof-of-principle experiments to identify and validate cancer drug targets.
  • the drug discovery process can be broadly summarized into five stages: target identification, target validation, high-throughput compound screening, lead optimization and clinical trials.
  • target identification target validation
  • high-throughput compound screening lead optimization
  • clinical trials The use of RNAi on cells cultured on user-defined substrate stiffnesss, and in particular high-throughput RNAi approaches, improves and enhances many of the stages of drug discovery, and in doing so streamline the process from the initial target identification to the development of a drug for use in the clinic.
  • the substrate assay systems and methods here provide optimal fidelity to the clinical situation and provides a means of target identification to distinguish key targets that are essential for the survival and behavior of tumor cells in a clinically relevant setting but are redundant in normal cells by testing proliferation, cell morphology, cell mobility, invasion and metastasis, sustained angiogenesis, limitless replicative potential, the ability to evade apoptosis, insensitivity to anti-growth signals, and self sufficiency in growth signals.
  • Some chemotherapy agents do not fall into the previous categories based on their activities. They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin. Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP).
  • An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer.
  • a substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essential thrombocytosis and malignant melanoma.
  • a methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease.
  • An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer.
  • a substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essential thrombocytosis and malignant melanoma.
  • a methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH
  • adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.
  • the otherwise inert gels are functionalized with equivalent amounts monomelic collagen, ensuring that substrate modulus is the lone variable in the system (Figure la and Figure 5). With this method, over 100 plates can be assembled and made ready for cell culture within a single day. [00120] As an initial assessment of the platform's capabilities, we surveyed the effect of substrate stiffness on the accumulation (net change in cell number from 4 to 72 hours) of 10 cell types ( Figure lc). Cells were seeded at a subconfluent density (15 cells/mm 2 ) in 10% serum and cultured across five shear moduli spanning a broad, physiological range.
  • Table 1-1 Molecular actions of exemplary tested compounds and classes of compounds to be screened.
  • blebbistatin is a potent inhibitor of non-muscle myosin II
  • compounds that target known regulators of myosin II activity Inhibition of Rho- associated kinase (ROCK) by GSK 429286 ( Figure 2b), Y-27632, Fasudil hydrochloride and H-l 152 ( Figure 7a), all succeeded in recapitulating the divergent, stiffness-specific response to blebbistatin.
  • ROCK inhibition also rescued the cell spreading defect observed on 100 and 400 Pa substrates ( Figure 8), consistent with its growth-promoting effects in these contexts.
  • Multiwell plate fabrication Glass-bottom, black-walled, 96-well plates (Matrical Biosciences) were treated with an aqueous solution of g-methacryloxypropyltrimethoxysilane (Acros Organics) for 1 hour, rinsed in distilled water and allowed to dry. Solutions containing 0.10% ammonium persulfate, 0.15% tetramethylethylenediamine and variable ratios of acrylamide: bisacrylamide (Bio-Rad) were delivered into selected wells of the multiwell plate.
  • a custom built, 96-pin array with affixed borosilicate glass squares (Hausser Scientific) rendered hydrophobic by treatment with SurfaSil (Pierce) was used to sandwich the solutions between opposing glass surfaces and achieve a final gel thickness of ⁇ 100 ⁇ . After 10 minutes, the array was removed and the polymerized gels were rinsed with distilled water. To derivatize the gels, 50 ⁇ of sulfo-SANPAH (G-Biosciences) at 50 ⁇ g/ml in 50 mM HEPES buffer, pH 8.5 was delivered to each well and activated by UV exposure.
  • the solution was replaced with 100 ⁇ of collagen (PureCol) in PBS at 10 ⁇ g/ml (unless otherwise indicated) and incubated at room temperature for 4 hours.
  • the gels were rinsed with PBS and UV- sterilized prior to validation studies and cell seeding.
  • Chemiluminescence Substrate (Pierce) was added to each well. Images were captured using a CCD camera (Syngene) within a linear range of detection, and average pixel densities were evaluated in Adobe Photoshop 6.0.
  • Apoptosis was assessed using a fluorescence -based, ApoONE Caspase 3/7 Activity Assay (Promega).
  • Epithelial-Mesenchymal Transition induction Subconfluent A549 cells were cultured in media supplemented with 10 ng/ml of recombinant TGFj31 (R&D Systems) for 3 days, and withdrawn prior to cell seeding in multiwell plates.
  • Drug screening GF10920 3X, NSC2 3766, simvastatin, PP1 , FAK Inhibitor 14, SU9516, okadaic acid, cantharidin, taxol, IPA 3, cytochalasin D, calpeptin, Y-276 32, GSK429286 and fasudil hydrochloride were purchased from Tocris Bioscience;
  • NHLFs in multiwell plates were fixed in 10% formalin and blocked in 1 % goat serum. F-actin and nuclei were stained with Alexa Fluor 488- Phalloidin and Hoescht 33342 (both from Invitrogen), respectively. Fluorescence images were captured with a Nikon TE 300 fluorescent microscope.
  • fibroblasts were treated with siRNA targeting non-muscle myosin heavy chain Ila (MYH9) or a scrambled siRNA control for 72 hours, then seeded on 400 Pa and rigid substrates and cultured for an additional 72 h.
  • Figure 9 shows the fold change in cell number over the initial seeding density for each indicated concentration of siRNA. Results were normalized against the effect of negative control siRNA. MYH9 knockdown exerted divergent effects on growth promotion as matrix stiffness varied from rigid to 400 Pa gels, recapitulating the effect of blebbistatin.
  • Example 3 Automated fluorescent imaging in a 384 well plate
  • PA gels were fabricated spanning the entire stiffness range (0.3- 55 kPa) in a 384 well plate and seeded 7 cell types at various densities. After 24 hours in culture, the cells were fixed and stained to visualize f-actin and nuclei. Using autofocusing, we captured images of cells on all substrates, including glass ( Figure 10). Prominent morphological transitions in the 1-6 kPa range were evident in many of the primary and immortalized cell lines, with the exception of L929 cells, which were virtually indistinguishable under all stiffness conditions.

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La présente invention concerne des procédés de criblage basés sur des cellules qui recourent à des substrats d'une rigidité définie par l'utilisateur. Lesdits procédés peuvent être utilisés pour évaluer l'importance et la nature des réponses cellulaires à un médicament dans une gamme de rigidité physiologique.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013186408A1 (fr) * 2012-06-12 2013-12-19 Universida De Zaragoza Dispositif de culture cellulaire et procédé associé audit dispositif
WO2015020614A1 (fr) * 2013-08-07 2015-02-12 Agency For Science, Technology And Research Procédés, appareils et systèmes de culture de cellules et de tissus

Non-Patent Citations (5)

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Title
KOSTIC, A. ET AL.: 'Differential matrix rigidity response in breast cancer cell lines corelates with the tissue tropism' PLOS ONE. vol. 4, no. 7, 23 July 2009, page E6361 *
LAU, K. C. ET AL.: 'Automated imaging of circulating fluorocytes for the diagnosis of erythropoietic protoporphyria: A pilot study for population screening' JOURNAL OF MEDICAL SCREENING. vol. 15, no. 4, 2008, pages 199 - 203 *
MCDANIEL, D. P. ET AL.: 'The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype' BIOPHYSICAL JOURNAL. vol. 92, no. 5, 08 December 2006, pages 1759 - 1769 *
REHFELDT, F. ET AL.: 'Cell responses to the mechanochemical microenvironment - Implications for regenerative medicine and drug delivery' ADVANCED DRUG DELIVERY REVIEWS. vol. 59, 14 August 2007, pages 1329 - 1339 *
SEMLER, E. J.: 'Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance' BIOTECHNOLOGY AND BIOENGINEERING. vol. 89, no. 3, 22 December 2004, pages 296 - 307 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013186408A1 (fr) * 2012-06-12 2013-12-19 Universida De Zaragoza Dispositif de culture cellulaire et procédé associé audit dispositif
WO2015020614A1 (fr) * 2013-08-07 2015-02-12 Agency For Science, Technology And Research Procédés, appareils et systèmes de culture de cellules et de tissus
US9976113B2 (en) 2013-08-07 2018-05-22 Agency For Science, Technology And Research Methods, apparatuses, and systems for cell and tissue culture

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