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WO2025245123A1 - Méthodes et matériaux d'évaluation de traitements du cancer - Google Patents

Méthodes et matériaux d'évaluation de traitements du cancer

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Publication number
WO2025245123A1
WO2025245123A1 PCT/US2025/030210 US2025030210W WO2025245123A1 WO 2025245123 A1 WO2025245123 A1 WO 2025245123A1 US 2025030210 W US2025030210 W US 2025030210W WO 2025245123 A1 WO2025245123 A1 WO 2025245123A1
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WIPO (PCT)
Prior art keywords
cancer
microcancer
mammal
cancer treatment
models
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PCT/US2025/030210
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English (en)
Inventor
Panagiotis Z. Anastasiadis
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Publication of WO2025245123A1 publication Critical patent/WO2025245123A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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

Definitions

  • Diffuse gliomas are the most common malignant brain tumors in adults and include isocitrate dehydrogenase mutant (IDH-mut) astrocytomas, IDH-mut and chromosome lp/19q-codeleted oligodendrogliomas, and IDH-wildtype glioblastomas (Louis et al., Neuro Oncol, 23: 1231 (2021)).
  • IDH-mut isocitrate dehydrogenase mutant
  • chromosome lp/19q-codeleted oligodendrogliomas Despite multi-modal treatment, gliomas remain incurable.
  • Genomic and transcriptomic profiling have shown that high-grade gliomas (HGGs) are molecularly complex, with multiple cancer-driving events and oncogenic pathways concurrently deregulated in each tumor (N. Cancer Genome Atlas Research, Nature, 455: 1061 (2008)).
  • This document provides methods and materials for assessing cancer treatments and/or treating mammals (e.g., humans) having a brain cancer (e.g., a brain cancer such as glioma).
  • mammals e.g., humans
  • a brain cancer e.g., a brain cancer such as glioma
  • the methods and materials provided herein can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma).
  • one or more samples obtained from a mammal (e.g., a human) having a cancer (e g., a brain cancer such as glioma) can be used to generate a collection of ex vivo 3- dimensional (3D) microcancer culture models (sometimes referred to herein as microcancer models) representative of that mammal’s cancer that can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating that particular mammal’s cancer (e.g., that particular human’s glioma) based, at least in part, on a reduction of the cell viability of one or more of that mammal’s microcancer models following contact with a particular candidate anti-cancer treatment.
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • microcancer models representative of that mammal’s cancer that can be used to identify one or more candidate anti-cancer treatments that are likely to
  • one or more samples obtained from a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) can be used to generate a collection of microcancer models that can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating that particular mammal’s cancer (e.g., that particular human’s glioma) based, at least in part, on comparing the cell viability in one or more of that mammal’s microcancer models that were contacted with a particular candidate anti-cancer treatment to the cell viability in a one or more of that mammal’s microcancer models that were not contacted with the candidate anti-cancer treatment and/or any potential anti-cancer treatment to identify those that promote reduced cell viability.
  • a mammal e.g., a human having a cancer
  • a cancer e.g., a brain cancer such as glioma
  • microcancer models e.
  • a first potential anti-cancer treatment such as a combination of drug 1 and drug 2 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • a second potential anti-cancer treatment such as a combination of drug 3 and drug 4 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • a third potential anti-cancer treatment such as a combination of drug 5 and drug 6 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • a fourth potential anti-cancer treatment such as a combination of drug 1 and drug 3 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • a fifth potential anti-cancer treatment such as a combination of drug 1 and drug 4 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • a seventh potential anti-cancer treatment such as a combination of drug 1 and drug 6 can be exposed to one to ten microcancer models of a particular mammal (e.g., a human)
  • an eighth potential anti-cancer treatment such as a combination of drug 2 and drug 3 can be exposed to one to ten microcancer models of a particular mammal (e.g.. a human)
  • the cell viabilities of each can be compared to the cell viabilities of one or more controls (e.g., a subset of that mammal’s microcancer models that was not exposed to any potential anticancer treatment) to identify one or more candidate anti-cancer treatments as having the ability to promote reduced cell viability and thereby as being likely to be effective against that particular mammal’s cancer.
  • one or more controls e.g., a subset of that mammal’s microcancer models that was not exposed to any potential anticancer treatment
  • This document also provides methods and materials for treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) by administering an anti-cancer treatment that is selected based, at least in part, on whether or not the anti-cancer treatment was identified as being a candidate anti-cancer treatment likely to be effective against that particular mammal’s cancer using the microcancer models as described herein.
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • a mammal e.g., a human having a cancer (e.g., a brain cancer such as glioma) can be administered an anti-cancer treatment that is selected based, at least in part, on its ability to promote reduced cell viability in one or more microcancer models for that mammal as compared to one or more control microcancer models for that mammal that were not exposed to any potential anti-cancer treatments.
  • a cancer e.g., a brain cancer such as glioma
  • an anti-cancer treatment that is selected based, at least in part, on its ability to promote reduced cell viability in one or more microcancer models for that mammal as compared to one or more control microcancer models for that mammal that were not exposed to any potential anti-cancer treatments.
  • that candidate anti-cancer treatment can be classified as being likely to be effective in treating that particular mammal (e.g., that particular human).
  • that candidate anti-cancer treatment identified as being likely to be effective in that particular mammal can be administered to that particular mammal to treat that mammal’s cancer.
  • Having the ability to identify candidate anti-cancer treatments that are likely to be effective against a particular mammal's cancer as described herein (e.g., based, at least in part, on a reduction of the cell viability in one or more of that mammal’s microcancer models that were contacted with the candidate anti-cancer treatment) provides a unique and unrealized opportunity to provide an individualized approach for selecting cancer therapies based on the likelihood of success, thus providing cost-effective care with better outcomes.
  • multi-omic data about a mammal’s cancer e.g., whole genome sequencing data and/or RNAseq data
  • RNAseq data can be used to select possible candidate anti -cancer treatments to be assessed using that mammal’s microcancer models as described herein.
  • certain genetic mutations present is a mammal’s cancer can be used to select candidate anti-cancer treatments designed to exploit those genetic mutations of the cancer.
  • one aspect of this document features a method for identifying a candidate anti-cancer treatment likely to be effective in treating a mammal having cancer.
  • the method comprises (or consists essentially of. or consists of): (a) culturing a cell suspension of a sample of the cancer in each of a plurality of individual locations of a culture vessel in the presence of culture medium that does not contain hydrocortisone to generate a microcancer model of the cancer in each of the plurality of individual locations, (b) contacting one or more of the microcancer models with a potential anti-cancer treatment, and (c) identifying the potential anti-cancer treatment as having the ability to reduce cell viability of the microcancer model contacted with the potential anti-cancer treatment as compared to cell viability of one or more control microcancer models of the cancer not contacted with the potential anti-cancer treatment, thereby identifying the potential anti-cancer treatment as being the candidate anticancer treatment.
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the culture medium can be a medium that does not contain ROCK inhibitor.
  • the culture medium can contain ROCK inhibitor.
  • the culture medium can be removed from the culture vessel and replaced with a culture medium that does not contain ROCK inhibitor during the contacting step (b).
  • the potential anti-cancer treatment can comprise a single compound.
  • the single compound can be selected from the compounds set forth in Table 1.
  • the potential anti-cancer treatment can comprise a combination of two compounds, a combination of three compounds, a combination of four compounds, a combination of five compounds, or a combination of six compounds.
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • Each of the plurality of individual locations can contain 500 to 30,000 cells prior to the step (b).
  • the culture medium can be medium that does not contain hydrocortisone.
  • the culture medium can be medium that does not contain ROCK inhibitor.
  • the culture medium can contain ROCK inhibitor.
  • the culture medium can be removed from the culture vessel and replaced with a culture medium that does not contain ROCK inhibitor during the contacting step (b).
  • the potential anti-cancer treatment can comprise a single compound.
  • the single compound can be selected from the compounds set forth in Table 1.
  • the potential anti-cancer treatment can comprise a combination of two compounds, a combination of three compounds, a combination of four compounds, a combination of five compounds, or a combination of six compounds.
  • the combination can be selected from the
  • this document features a method for identify ing a candidate anticancer treatment likely to be effective in treating a mammal having cancer.
  • the method comprises (or consists essentially of. or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality of locations comprises a microcancer model of the cancer, (b) identify ing a potential anti-cancer treatment that involves administering a single drug at a set dose to the mammal, (c) contacting one or more of the microcancer models with the single drug at a concentration within 5 percent of C max of the set dose in plasma or at the location of the cancer within the mammal, and (d) identifying the contacting of step (c) as having the ability to reduce cell viability of the one or more microcancer models contacted with the single drug as compared to cell viability of one or more control microcancer models of the cancer not contacted with the single drug, thereby identifying the potential anti-cancer treatment as being the candidate anti-cancer treatment
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the single compound can be selected from the compounds set forth in Table 1.
  • this document features a method for identifying a candidate anticancer combination treatment likely to be effective in treating a mammal having cancer.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality of locations comprises a microcancer model of the cancer, (b) identifying a potential anti-cancer treatment that involves administering a combination of two drugs, each at a set dose, to the mammal, (c) contacting one or more of the microcancer models with a first drug of the two drugs at a concentration within 5 percent of Cmax of the set dose of the first drug in plasma or at the location of the cancer within the mammal, (d) contacting one or more of the microcancer models with a second drug of the two drugs at a concentration within 5 percent of Cmax of the set dose of the second drug in plasma or at the location of the cancer within the mammal, (e) contacting one or more of the microcancer models with the
  • the identifying step (f) can comprise using a plot, where the x axis is the percent inhibition of cell viability of the first drug at the concentration of step (c) and the y axis is the percent inhibition of cell viability of the second drug at the concentration of step (d), wherein a first x,y data point is plotted based on the percent inhibition of cell viability of step (c) for (x) and the percent inhibition of cell viability- of step (d) for (y), and wherein a second X,Y data point is plotted based on the percent inhibition of cell viability- of step (e) for the combined exposition to the first drug for (X) and second drug for (Y) at the concentrations of step (e).
  • the mammal can be a human.
  • the cancer can be a brain cancer, or the cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the combination can be selected from the combinations of compounds
  • this document features a method for identifying a candidate anticancer treatment likely to be effective in treating a mammal having cancer.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality of locations comprises a microcancer model of the cancer, (b) contacting one or more of the microcancer models with a potential anti-cancer treatment, (c) conducting whole genome sequencing of a sample of the cancer or of the microcancer model to identify genetic mutations within the cancer, and optionally conducting whole genome sequencing of a germline sample of the mammal, and optionally conducting RNAseq of a sample of the cancer or of the microcancer model to identify expression profiles of the cancer, (d) identifying the potential anti-cancer treatment as having the ability to reduce cell viability of the one or more microcancer models contacted with the potential anti-cancer treatment as compared to cell viability of one or more control microcancer models of the cancer not contacted
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the potential anti-cancer treatment can comprise a single compound.
  • the single compound can be selected from the compounds set forth in Table 1.
  • the potential anti-cancer treatment can comprise a combination of two compounds, a combination of three compounds, a combination of four compounds, a combination of five compounds, or a combination of six compounds.
  • the combination can be selected from the combinations of compounds set forth in Table 2.
  • the potential anti-cancer treatment can be selected based on the genetic mutations identified within the cancer.
  • this document features a method for identifying a candidate anticancer treatment likely to be effective in treating a mammal having cancer.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality of locations comprises a microcancer model of the cancer, (b) contacting one or more of the microcancer models with a potential anti-cancer treatment in the presence of one or more dyes that stain cells for cell death or cell death by apoptosis, autophagic cell death, or necrosis, (c) conducting live imaging of the one or more microcancer models to observe cell death, (d) identifying the potential anti-cancer treatment as having the ability to reduce cell viability of the one or more microcancer models contacted with the potential anti-cancer treatment as compared to cell viability of one or more control microcancer models of the cancer not contacted with the potential anti-cancer treatment, thereby identifying the potential anti-cancer treatment as being the
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the potential anti-cancer treatment can comprise a single compound.
  • the single compound can be selected from the compounds set forth in Table 1.
  • the potential anti-cancer treatment can comprise a combination of two compounds, a combination of three compounds, a combination of four compounds, a combination of five compounds, or a combination of six compounds.
  • the combination can be selected from the combinations of compounds set forth in Table 2.
  • the one or more dyes can be selected from the group consisting of annexin V fluorescent conjugates, caspase 3/7 specific fluorescent dyes.
  • this document features a method for treating a mammal having a brain cancer.
  • the method comprises (or consists essentially of, or consists of) administering a compound set forth in Table 1 to the mammal.
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • this document features a method for treating a mammal having a brain cancer.
  • the method comprises (or consists essentially of, or consists of) administering a combination of compounds set forth in Table 2 to the mammal.
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • this document features a method for treating a mammal having a brain cancer with a candidate anti -cancer combination treatment identified as being likely to be effective in treating the mammal.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality' of locations comprises a microcancer model of the cancer, (b) identifying a potential anti-cancer treatment that involves administering a single drug at a set dose, to the mammal, (c) contacting one or more of the microcancer models with the single drug at a concentration within 5 percent of Cmax of the set dose in plasma or at the location of the cancer within the mammal, (d) identifying the contacting of step (c) as having the ability to reduce cell viability of the one or more microcancer models contacted with the single drug as compared to cell viability of one or more control microcancer models of the cancer not contacted with the single drug, thereby identifying the potential anti-
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the single compound can be selected from the compounds set forth in Table 1.
  • this document features a method for treating a mammal having a brain cancer with a candidate anti -cancer combination treatment identified as being likely to be effective in treating the mammal.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, w herein each of the plurality of locations comprises a microcancer model of the cancer, (b) identifying a potential anti-cancer treatment that involves administering a combination of two drugs, each at a set dose, to the mammal, (c) contacting one or more of the microcancer models with a first drug of the two drugs at a concentration within 5 percent of Cmax of the set dose of the first drug in plasma or at the location of the cancer within the mammal, (d) contacting one or more of the microcancer models with a second drug of the two drugs at a concentration within 5 percent of Cmax of the set dose of the second drug in plasma or at the location of the cancer within the mammal, (e
  • the identify ing step (f) can comprise using a plot, where the x axis is the percent inhibition of cell viability of the first drug at the concentration of step (c) and the y axis is the percent inhibition of cell viability of the second drug at the concentration of step (d), wherein a first x,y data point is plotted based on the percent inhibition of cell viability of step (c) for (x) and the percent inhibition of cell viability of step (d) for (y).
  • the mammal can be a human.
  • the cancer can be a brain cancer, or the cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the combination can be selected from the combinations of compounds set forth in Table 2.
  • this document features a method for identifying a candidate anticancer treatment likely to be effective in treating a mammal having cancer and previously treated with a prior anti-cancer treatment.
  • the method comprises (or consists essentially of, or consists of): (a) obtaining a culture vessel comprising a plurality of locations, wherein each of the plurality of locations comprises a microcancer model of the cancer, wherein each of the microcancer models was exposed to the prior anti-cancer treatment while the mammal was receiving the prior anti-cancer treatment, (b) contacting one or more of the microcancer models wi th a potential anti-cancer treatment, and (c) identifying the potential anti-cancer treatment as having the ability to reduce cell viability of the one or more microcancer models contacted with the potential anti-cancer treatment as compared to cell viability of one or more control microcancer models of the cancer not contacted with the potential anti-cancer treatment, thereby identifying the potential anti-cancer treatment as being the candidate anticancer treatment.
  • the mammal can be a human.
  • the cancer can be a brain cancer.
  • the brain cancer can be a glioma.
  • the culture vessel can be an ultra-low attachment plate.
  • the culture vessel can be a hanging drop plate.
  • the potential anti -cancer treatment can comprise a single compound.
  • the single compound can be selected from the compounds set forth in Table 1.
  • the potential anti-cancer treatment can comprise a combination of tw o compounds, a combination of three compounds, a combination of four compounds, a combination of five compounds, or a combination of six compounds.
  • the combination can be selected from the combinations of compounds set forth in Table 2.
  • Figure 1 shows a schematic overview of an ex vivo glioma platform to identify individualized targeted treatments for HGG.
  • Figure 2 is a flow- chart indicating the number of patients enrolled and that proceeded with following applications for next generation sequencing (NGS) and microcancer model generation and drug testing.
  • NGS next generation sequencing
  • Figure 3 is a schematic showing the trajectory of each case from patient enrollment, NGS. microcancer model generation and drug testing, to quality control.
  • FIG. 4 shows the clinical parameters and glioma relevant molecular alterations of patient samples.
  • RTK, MEK, and PI3K pathway components indicated.
  • TSGs tumor suppressor genes
  • LoF loss-of-function
  • GoF gain-of-function
  • Figures 5A-5B show the characterization of patient derived microcancer models with parental glioblastoma including staining for Ki-67 proliferation marker in PDX GBM8 orthotopic xenograft tissue and a GBM8 microcancer model (pCancer) (Figure 5A), and cell viability, measured by nM ATP over 12-day of culture, for microcancer models (PT425) ( Figure 5B).
  • Figure 6 is a genetic landscape comparing patient-derived microcancer models (pCancer) and parental glioblastoma and showing copy number alterations (CNAs) and mutation alterations (lollipops) in U plot (top), CNAs in horizontal view (middle top), heterozygosity status (middle bottom), and single base substitutions (SBS) mutational signature (bottom). Copy number gains, losses, diploid (grey) and DNA junctions (lines) indicated.
  • CNAs copy number alterations
  • lollipops mutation alterations
  • SBS single base substitutions
  • Figures 7A-7C show that glioma-relevant mutations identified in parental tumor are retained in microcancer models (pCancer) with a similar frequency (Figure 7A), the overlap in mutations captured by whole exome sequencing (WES) ( Figure 7B), and the correlation between allele frequency of all mutations identified by WES (Figure 7C) in microcancer models (pCancer) and parental tumor.
  • Figures 8A-8B are comparisons between microcancer models and parental glioblastoma illustrated by a Venn diagram showing the similarity and difference of DNA junction numbers captured by mate pair sequencing (MPseq) in microcancer models (pCancer) and corresponding parental tumor PT311 (MPseq) (Figure 8A) and pTERT mutation status in the parental tumor PT311 and microcancer models identified by clinical testing (Neuro-Oncology expanded gene panel (NONCP), and PCR ( Figure 8A).
  • MPseq mate pair sequencing
  • NONCP Neuro-Oncology expanded gene panel
  • Figure 8A PCR
  • Figure 9 shows the work for microcancer model (pCancer) generation and drug testing followed by biomarker identification.
  • Figures 10A-10D are dose-response curves of microcancer models (pCancer) (PT303) treated with pimasertib (Figure 10A), capivasertib ( Figure 10B), and navitoclax (Figure IOC) and a heatmap showing % inhibition at Cmax for each culture condition ( Figure 10D).
  • Figure 11 is a scatter plot depicting % viabi li ty of all replicates performed at different times of drug testing, each data point representing drug responses measured at first and second experiments, showing correlation between experiments. Pearson’s (r p ) and Spearman’s (r s ) correlation analyses performed.
  • Figure 12 is a schematic illustrating agents targeting key glioma-relevant pathways.
  • Figures 13A-13B show the normalized microcancer model dose-response curves for neratinib ( Figure 13 A) and paxalisib ( Figure 13B) across all samples.
  • Figures 14 is a three-tier drug response (Figure 14A) showing ⁇ 35% (minimal response, MR; light gray), 35-70% (partial response, PR; gray), and >70% (strong response, SR; dark gray) of growth inhibition at Cmax for indicated inhibitors. Stacked bar graphs depict the percentage of differential responses for each drug (right) and case (bottom).
  • Figures 15A-15C are normalized dose-response curves for osimertinib (Figure 15A), foretinib (Figure 15B), and sunitinib (Figure 15C) targeting indicated RTKs, with Cmax indicated.
  • Figure 16 is a stacked bar graph showing the percentage of differential responses, ⁇ 35% (minimal response, MR; light gray), 35-70% (partial response. PR; gray), and >70% (strong response, SR; dark gray), for birabresib, mivebresib, and trotabresib, and JQ1 plotted for comparison.
  • Figures 17A-17D are w aterfall plots of % inhibition at Cmax for microcancer models treated with capivasertib (Figure 17A), CC-115 ( Figure 17B). navitoclax ( Figure 17C). and vorinostat ( Figure 17D) and the status of the genetic features related to the drug target for each case.
  • Figure 18 is a scatter plot with bars (mean ⁇ SEM) showing JQ1 efficacy in microcancers derived from tumors with wildtype or mutated pTERT or ATRX. P-value determined by unpaired t-test.
  • Figures 19A-19B show TCGA ( Figure 19A) and pathway -based ( Figure 19B) transcriptional subtypes (second row) with glioma subtype (first row) and normalized enrichment scores (NES) of each signature shown.
  • NES indicating the degree of activation of each signature from high (1, dark grey, dashed) to no (0, gray, no dash).
  • SPL representing the complexity of signatures involved from high (0, black) to low (1, white).
  • Figures 20A-20B show the association of assigned subtypes between the TCGA and pathway -based transcription classifiers in glioma ( Figure 20 A) and the subtype assignment using the TCGA and pathway-based transcription classifications for individual cases ( Figure 20B).
  • Figures 21A-21B are correlation plots showing the relationship between transcriptional subtype enrichment scores (ES, scale: 0-100) of TCGA ( Figure 21 A) and pathway based transcriptional (txn) subtype ( Figure 2 IB) (x-axes) and microcancer model response (% Inhibition (Inh) at Cmax; scale: 0-100) to JQ1, vorinostat, CC-115, capivasertib, and navitoclax (y-axes).
  • Figures 22A-22D are distribution plots showing % inhibition at Cmax from cases with low-to-high response from left to right to JQ1 (Figure 22A), vorinostat (Figure 22B), CC-115 (Figure 22C), and capivasertib (Figure 22D).
  • Responders (R) and non-responders (NR) are cases above the yellow dashed lines and below the grey dashed lines, respectively.
  • Figures 23A-23D are heatmaps depicting statistically significant differentially- expressed genes for indicated subtypes between responder (R) and non-responder (NR) microcancer groups to JQ1 ( Figure 23A), vorinostat (Figure 23B), CC-115 ( Figure 23C), and capivasertib (Figure 23D).
  • Figures 24A-24D are heatmaps showing differentially expressed genes for indicated gene sets between responder (R) and non-responder (NR) microcancer model groups following treatment with JQ1 ( Figure 24A), vorinostat (Figure 24B and Figure 24C), and CC- 115 ( Figure 24D).
  • Raw- Z-Score from low to high and % inhibition at Cmax (% Inh) are indicated.
  • Figure 25 is a schematic showing that Hippo kinases inactivate YAP/TAZ signaling and prevent DGC to G-STEM conversion.
  • Figures 26A-26C are correlation plots showing relationships between enrichment of indicated signatures (x-axes) and pCancer response to JQ1 (Figure 26A), CC-115 ( Figure 26B). and vorinostat ( Figure 26C). Linear correlation lines indicated. Signatures linked to Hippo inactivation (GOBP Positive Reg Hippo and DGC signature) and YAP/TAZ activation (G-STEM signature) separated by dashed vertical line. Grey, statistically significant p-values and corresponding r p .
  • Figure 27 is a heatmap depicting differentially expressed genes in the MT0RC1 signaling hallmark between CC-115 responder (R) and non-responder (NR) microcancer model groups.
  • Figure 28 shows the effect of combination strategies on microcancer model drug responses.
  • An example dose-response curve graph indicates % inhibition of single agents and combination treatment at each drug’s Cmax, and conversion to a heatmap or an X-Y scatter plot show combination treatment being more effective that corresponding single agents.
  • Figure 29 is a graph depicting the effect of 7 drug combinations compared to their corresponding single agent treatments.
  • Figures 30A-30B are heatmaps depicting differential responses of combination treatments compared to single agents at each Cmax and the companion stacked column graph ( Figure 30A-B) shows the relationship between drug combinations and single agent treatments and response category across cases.
  • Figures 31 A-3 IE show that JQ1 treatment resulted in alterations of transcriptional subtypes and enrichments of different gene sets, with corresponding enrichments in biological functions, protein complexes, and signaling pathways in non-responder (NR) PT431 and responder (R) PT440 microcancers ( Figures 31 A-31D), and that increased expression of genes related to PI3K signaling in NR PT431 microcancers can be targeted to increase efficacy of JQ1 treatment ( Figure 3 IE).
  • Figures 32A-32B show that JQ1 treatment resulted in alterations of pathway -based transcriptional subtypes in non-responder (NR) PT431 and responder (R) PT440 microcancers ( Figure 32A) and the protein interaction network enriched in responder (R) PT440 microcancers_( Figure 32B).
  • This document provides methods and materials for assessing cancer treatments and/or treating mammals (e.g., humans) having a brain cancer (e.g., a brain cancer such as glioma).
  • mammals e.g., humans
  • a brain cancer e.g., a brain cancer such as glioma
  • the methods and materials provided herein can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating a particular mammal's cancer (e.g., a particular human’s cancer) such as a brain cancer (e.g., a glioma).
  • one or more samples obtained from a mammal (e.g., a human) having a cancer (e g., a brain cancer such as glioma) can be used to generate an ex vivo 3D microcancer culture model (i.e., a microcancer model) that can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating that mammal’s cancer (e.g., that human’s cancer).
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • an ex vivo 3D microcancer culture model i.e., a microcancer model
  • a sample can be a biological sample.
  • a sample can contain one or more cancer cells (e.g.. one or more brain cancer cells such as glioma cells).
  • cancer cells e.g.. one or more brain cancer cells such as glioma cells.
  • samples that can be obtained and used to generate microcancer models as described herein include, without limitation, tissue samples and blood samples.
  • a sample that can be obtained and used to generate a microcancer model as described herein can be a cancer biopsy sample.
  • a sample can be a fresh sample.
  • a sample can be a frozen sample.
  • a sample e.g., a tissue sample obtained from a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) for use in generating a collection of microcancer models as described herein can be dissociated into a single cell suspension.
  • a sample e.g., a tissue sample obtained from a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) into a single cell suspension.
  • a sample can be dissociated into a single cell suspension by mechanical dissociation.
  • Examples of methods for mechanical dissociation of a sample (e.g., a tissue sample) that can be used as described herein include, without limitation, crushing, cutting, and scrapping of the sample.
  • a sample e.g., a tissue sample
  • An example of a method for enzymatic dissociation of a sample (e.g., a tissue sample) that can be used as described herein includes, without limitation, application of an enzy me (e.g., trypsin or collagenase) to the sample.
  • a sample e.g., a tissue sample
  • An example of a method for chemical dissociation of a tissue sample that can be used as described herein includes, without limitation, application of a chemical (e.g.. EGTA or egtazic acid) to the sample.
  • a sample e.g. a tissue sample
  • a sample can be dissociated into a single cell suspension by a combination of mechanical, enzymatic, and/or chemical dissociation.
  • a sample e.g., a tissue sample obtained from a mammal (e.g., a human) having a cancer (e g., a brain cancer such as glioma) that has been dissociated into a single cell suspension as described herein can be added to the locations (e.g.. wells) of any appropriate cell culture vessel (e.g., a microtiter plate having 96, 384, or 1536 wells) to generate microcancer models.
  • an appropriate cell culture vessel is a hanging drop plate (e.g., a hanging drop plate having 96, 384, or 1536 wells).
  • an appropriate cell culture vessel is an ultra-low attachment plate (e.g., an ultra-low attachment plate having 96, 384, or 1536 wells). Any appropriate number of cells can be added to a location (e.g., a well) of a cell culture vessel (e.g., a microtiter plate having 96, 384, or 1536 wells) to generate a microcancer model.
  • a cell culture vessel e.g., a microtiter plate having 96, 384, or 1536 wells
  • from about 100 cells to about 20000 cells can be added to a single location (e.g., a single well) of a cell culture vessel to generate a microcancer model.
  • 500 cells can be added to a single location (e.g., a single well) of a cell culture vessel to generate a microcancer model.
  • a microcancer model from a sample (e.g., a tissue sample) obtained from a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) that has been dissociated into a single cell suspension and added to a cell culture vessel as described herein, the cells can be incubated in an appropriate cell culture medium.
  • a sample e.g., a tissue sample
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • an appropriate cell culture medium that can be used to make a microcancer model described herein can include, without limitation, a medium such as DMEM/F-12 medium, Advanced DMEM/F12 medium, DMEM medium, or MEM medium supplemented with serum, one or more serum-free medias, insulin, one or more steroids, one or more growth factors, one or more growth regulators, one or more antibiotics, and one or more kinase inhibitors.
  • serum that can be included within a culture medium for a microcancer model described herein include, without limitation, heat inactivated horse serum, heat inactivated fetal bovine serum, fetal bovine serum, and heat inactivated adult human serum.
  • Examples of serum-free media that can be included within a culture medium for a microcancer model described herein include, without limitation, B-27 (with or without vitamin A), N-2, and Gem21.
  • Examples of insulin that can be included within a culture medium for a microcancer model described herein include, without limitation, human insulin and bovine insulin.
  • Examples of steroids that can be included wi thin a culture medium for a microcancer model described herein include, without limitation, hydrocortisone and dexamethasone.
  • grow th factors examples include, without limitation, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) , neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and hormones including, without limitation, 3,3',5'-Triiodo-L- thyronine (T3) and its precursor thyroxine (T4), P-estradiol, progesterone, gastrin I, prostaglandin El, and prostaglandin E2.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • BDNF brain-derived neurotrophic factor
  • HGF hepatocyte growth factor
  • T3 3,3',5'-Triiodo-L- thyronine
  • T4 precursor thyroxine
  • P-estradiol progesterone
  • growth regulators examples include, without limitation, N-Acetyl-L-cysteine, nicotinamide, and glutamine supplements (e.g., Glutamax).
  • examples of antibiotics that can be included within a culture medium for a microcancer model described herein include, without limitation, penicillin, streptomycin, amphotericin B. tetracycline, primocin. gentamicin, kanamycin, neomycin, and ciprofloxacin.
  • kinase inhibitors examples include, without limitation, a rho kinase (ROCK) inhibitor (e.g., Y27632 and CAS 871543-07-6), a GSK-3a/p inhibitor (e.g. CHIR-99021), a myosin II ATPase inhibitor (e.g. blebbistatin), an ALK4/5/7 inhibitor (e.g. A83-01), and a Rael Inhibitor (e.g. CAS 1177865-17-6).
  • a rho kinase (ROCK) inhibitor e.g., Y27632 and CAS 871543-07-6
  • GSK-3a/p inhibitor e.g. CHIR-99021
  • myosin II ATPase inhibitor e.g. blebbistatin
  • an ALK4/5/7 inhibitor e.g. A83-01
  • Rael Inhibitor e.g. CAS 1177865-17
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing heat inactivated horse serum, insulin, hydrocortisone, EGF, penicillin, and streptomycin.
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing heat inactivated horse serum, insulin, EGF, penicillin, streptomycin, and ROCK inhibitor, without any hydrocortisone or without any steroid.
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing heat inactivated horse serum, insulin. EGF.
  • the serum can be included in the range of 5 percent to 15 percent (e.g., from 8 percent to 12 percent). For example, when including serum, the serum can be included at a level of 10 percent.
  • the insulin can be included in the range of 5 ng/mL to 5000 ng/mL (e.g., from 50 ng/mL to 1000 ng/mL). For example, when including insulin, 5 pg/ml of insulin can be included.
  • the steroid when including a steroid such as hydrocortisone, can be included in the range of 5 ng/mL to 5000 ng/mL (e.g., from 50 ng/mL to 1000 ng/mL). For example, when including hydrocortisone, 5 pg/ml of hydrocortisone can be included.
  • a growth factor such as EGF. the growth factor can be included in the range of 1 ng/mL to 100 ng/mL (e g., from 1 ng/mL to 20 ng/mL). For example, when including EGF, 10 ng/ml of EGF can be included.
  • the antibiotic when including an antibiotic such as penicillin and/or streptomycin, the antibiotic can be included in the range of 5000 units/mL to 20000 units/mL (e.g., from 10000 units/mL to 15000 units/ml) or 5 mg/ml to 20 mg/ml. (e.g., from lOmg/mL to 10 mg/mL).
  • penicillin 10,000 units/ml of penicillin can be included
  • streptomycin 10 mg/ml of streptomycin can be included.
  • a kinase inhibitor such as ROCK inhibitor
  • the kinase inhibitor can be included in the range of 2pM to 20pM (e.g., from 5 pM to 15 pM).
  • ROCK inhibitor when including ROCK inhibitor, lOpM of ROCK inhibitor can be included.
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing 10 percent heat inactivated horse serum, 5 pg/mL insulin, 5 pg/mL hydrocortisone, 10 ng/mL EGF, 10,000 units/ml penicillin, and 10 mg/mL streptomycin.
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing 10 percent heat inactivated horse serum, 5 pg/mL insulin, 10 ng/mL EGF, 10,000 units/ml penicillin, 10 mg/mL streptomycin, and 10 pM ROCK inhibitor, with zero hydrocortisone and with zero steroids.
  • an appropriate cell culture medium that can be used to make and maintain a microcancer model described herein can be DMEM/F-12 medium containing 10 percent heat inactivated horse serum, 5 pg/mL insulin, 10 ng/mL EGF, 10,000 units/ml penicillin, and 10 mg/mL streptomycin, with zero hydrocortisone and with zero steroids, and with zero ROCK inhibitor and with zero kinase inhibitors.
  • a collection of microcancer models can be generated as described elsewhere (see. e.g., U.S. Patent No. 11,845,084).
  • a sample e.g., a tissue sample
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • one or more of those microcancer models and/or a portion of the originally obtained sample e.g., a portion of a cancer biopsy
  • a microcancer model described herein can be assessed by live imaging.
  • a microcancer model described herein can be assessed by live imaging to monitor cell death (e.g.. apoptosis, autophagic cell death, and necrosis) in the microcancer model.
  • using live imaging to monitor cell death (e.g., apoptosis, autophagic cell death, and necrosis) in a microcancer model described herein can include incubating the microcancer model with one or more dyes specific for different types of cell death (e.g., apoptosis, autophagic cell death, and necrosis).
  • using live imaging to monitor cell death (e.g., apoptosis, autophagic cell death, and necrosis) in a microcancer model described herein can include incubating the microcancer model with one or more dyes specific for mitochondria membrane potential and/or mitochondrial superoxide production to assess apoptosis.
  • a microcancer model described herein can be incubated with annexin V fluorescent conjugates and/or caspase 3/7 specific fluorescent dyes to assess apoptosis.
  • a microcancer model described herein can be incubated with an RFP-GFP-LC3B tandem sensor such as a Fluorescent PremoTM Autophagy' Tandem Sensor RFP-GFP-LC3B or a luminescent LC3 reporter system such as an Autophagy LC3 HiBiT Reporter Assay System to visualize pH changes and quantitatively measure autophagic flux to assess autophagic cell death.
  • an RFP-GFP-LC3B tandem sensor such as a Fluorescent PremoTM Autophagy' Tandem Sensor RFP-GFP-LC3B or a luminescent LC3 reporter system such as an Autophagy LC3 HiBiT Reporter Assay System to visualize pH changes and quantitatively measure autophagic flux to assess autophagic cell death.
  • a microcancer model described herein can be incubated with propidium iodide, a green fluorescent nuclear and chromosome counterstain that is impermeant to live cells such as SYTOXTM green fluorescent dye, and/
  • an assay that assesses annexin V binding and DNA release such as RealTime-GloTM Annexin V Apoptosis and Necrosis Assay 7 can be used to assess necrosis in a microcancer model described herein.
  • using live imaging to monitor cell death in a microcancer model described herein can include incubating the microcancer model with one or more dyes that are agnostic to the mechanism of cell death.
  • a microcancer model described herein can be incubated with CellToxTM, PKH26, Cytotox, and/or C.Live Tox to monitor cell death agnostic to the mechanism of cell death.
  • a microcancer model described herein can be assessed by live imaging to monitor cellular senescence in the microcancer model.
  • a microcancer model described herein can be incubated with Beta-Glow to monitor cellular senescence.
  • multiple different dyes can be incubated with the same microcancer model.
  • a dye for assessing apoptosis, a dye for assessing autophagic cell death, and a dye for assessing necrosis can be used together with one microcancer model.
  • a single dye can be incubated with a microcancer model to assess multiple characteristics.
  • a microcancer model described herein can be incubated with ApoTox-GloTM to assess cell viability, cytotoxicity, and apoptosis.
  • one or more of those microcancer models and/or a portion of the originally obtained sample can be assessed by multi-omic analysis.
  • a microcancer model described herein and/or a portion of the originally obtained sample can be assessed by any appropriate multi-omic analysis technique.
  • Examples of multi-omic analyses that can be performed to assess a microcancer model and/or a portion of the originally obtained sample include, without limitation, next generation sequencing (NGS), mate-pair whole-genome DNA sequencing (MPseq), RNA sequencing (RNAseq), whole exome sequencing (WES), whole genome sequencing (WGS), single cell or single nucleus RNA sequencing, epigenomic sequencing, spatial profiling, proteomics, and metabolomics.
  • NGS next generation sequencing
  • MPseq mate-pair whole-genome DNA sequencing
  • RNAseq RNA sequencing
  • WES whole exome sequencing
  • WGS whole genome sequencing
  • single cell or single nucleus RNA sequencing epigenomic sequencing
  • epigenomic sequencing epigenomic sequencing
  • spatial profiling proteomics
  • metabolomics and metabolomics.
  • a microcancer model described herein can be assessed by whole genome sequencing.
  • a portion of the originally obtained sample e.g., a portion of a cancer biopsy
  • a portion of the originally obtained sample e.g., a portion of a cancer biopsy
  • both a microcancer model described herein and a portion of the originally obtained sample e.g.. a portion of a cancer biopsy
  • that microcancer model can be assessed by whole genome sequencing.
  • a microcancer model or a collection of microcancer models described herein can be used to identify one or more candidate anti-cancer treatments that are likely to be effective in treating a particular mammal’s cancer (e.g.. a particular human’s brain cancer such as a glioma) based, at least in part, on the ability to promote reduced cell viability in one or more of that mammal’s microcancer models (as compared to the cell viability in one or more of the mammal’s control microcancer models not contacted with any potential anti-cancer treatments).
  • a particular mammal’s cancer e.g. a particular human’s brain cancer such as a glioma
  • a mammal’s microcancer models e.g., one or more of a human’s microcancer models
  • a candidate anti-cancer treatment can be classified as being likely to be effective in treating that mammal’s cancer.
  • the cell viability in one or more of a mammal’s microcancer models contacted with a potential anti-cancer treatment is not reduced (e.g., as compared to the cell viabi li ty in one or more of that mammal’s control microcancer models not contacted with any potential anticancer treatments), then that potential anti-cancer treatment can be classified as being unlikely to be effective in treating that mammal’s cancer.
  • determining if a potential anti-cancer treatment is likely to be effective in treating a mammal (e.g., a human) having a cancer can include comparing the cell viability in one or more of that mammal’s microcancer models contacted with the potential anti-cancer treatment to the cell viability in one or more of that mammal’s microcancer models not contacted with that potential anti-cancer treatment; and classifying the potential anti-cancer treatment as being likely to be effective in treating that mammal’s cancer if the cell viability in one or more of that mammal’s microcancer models contacted with the potential anti -cancer treatment is reduced compared to the cell viability in one or more of that mammal’s microcancer models not contacted with that potential anticancer treatment; or classifying the potential anti-cancer treatment as being unlikely to be effective in treating that mammal’s cancer if the cell viability in one or more of that mammal
  • determining if a potential anti-cancer treatment is likely to be effective in treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) can include (a) contacting one or more of that mammal’s microcancer models with the potential anti-cancer treatment; (b) comparing the cell viability in one or more of that mammal’s microcancer models contacted with the potential anti-cancer treatment following the contacting step (a) to the cell viability in one or more of that mammal’s microcancer models not contacted with that potential anti-cancer treatment; and (c) classifying the potential anti-cancer treatment as being likely to be effective in treating that mammal’s cancer if the cell viability in one or more that that mammal’s microcancer models contacted with the potential anti-cancer treatment is reduced compared to the cell viability in one or more of that mammal's microcancer models not contacted with that potential anti-
  • any appropriate method can be used to contact a microcancer model described herein with a potential anti-cancer treatment.
  • a potential anti-cancer treatment includes administration of one or more anti-cancer compounds
  • contacting one or more microcancer models described herein can include culturing the one or more microcancer models in a solution (e.g., a culture medium) containing each of the one or more anti-cancer compounds.
  • a microcancer model described herein can be contacted with a potential anticancer treatment for any appropriate amount of time.
  • a microcancer model described herein can be contacted with a potential anti-cancer treatment for the amount of time necessary 7 to determine whether the cell viability in the microcancer model has been reduced.
  • contacting a microcancer model described herein with a potential anticancer treatment can occur over several hours, several days, or several weeks.
  • contacting a microcancer model described herein with a potential anti-cancer treatment can occur from about 12 hours to about 10 weeks (e.g., from about 1 day to about 8 weeks, from about 3 days to about 8 weeks, from about 4 days to about 8 weeks, from about 5 days to about 8 weeks, from about 7 days to about 8 weeks, from about 8 days to about 8 weeks, from about 9 days to about 8 weeks, from about 10 days to about 8 weeks, from about 11 days to about 8 weeks, from about 12 days to about 8 weeks, from about 13 days to about 8 weeks, from about 14 days to about 8 weeks, or from about 3 weeks to about 8 weeks).
  • contacting a microcancer model described herein with a potential anti-cancer treatment can occur over 12 hours. In some cases, contacting a microcancer model described herein with a potential anti-cancer treatment can occur over 3 days. In some cases, contacting a microcancer model described herein with a potential anti-cancer treatment can occur over 6 days. In some cases, contacting a microcancer model described herein with a potential anticancer treatment can occur over 3 weeks.
  • Any appropriate mammal having a cancer can be assessed and/or treated as described herein.
  • mammals that can have cancer e.g., a glioma
  • can be assessed and/or treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats.
  • a human having a cancer e.g., a brain cancer such as glioma
  • a human having a cancer can be assessed and/or treated as described herein.
  • the cancer can be any type of cancer.
  • the cancer can include one or more solid tumors.
  • the cancer can be a blood cancer.
  • the cancer can be a primary' cancer.
  • the cancer can be a metastatic cancer.
  • the cancer can be a refractory cancer.
  • the cancer can be a relapsed cancer.
  • cancers that can be assessed and/or treated as described herein include, without limitation, brain cancers, breast cancers, lung cancers, liver cancers, skin cancers, colon cancers, renal cancers, head and neck cancers, hepatocellular carcinomas, endometrial cancers, cholangiocarcinomas, rhabdomyosarcomas, gallbladder cancers, ovarian cancers, uterine cancers, mesotheliomas, renal cancers, sarcomas, pancreas cancers, bladder cancers, anus cancers, bone marrow cancers, peritoneum cancers, prostate cancers, testis cancers, thyroid cancers, ureter cancers, and vaginal cancers.
  • Examples of brain cancers that can assessed and/or treated as described herein include, without limitation, gliomas (e.g., GBMs), acoustic neuromas (schwannomas), pituitary adenomas, medulloblastomas, meningiomas, astrocytomas, oligodendrogliomas, and pediatric gliomas.
  • gliomas e.g., GBMs
  • acoustic neuromas e.g., acoustic neuromas
  • the glioma can be any grade glioma (e.g., grade 1 glioma, grade 2 glioma, grade 3 glioma, or grade 4 glioma (GBM)).
  • the methods described herein can include identifying a mammal (e.g., a human) as having a cancer (e.g., a brain cancer such as glioma). Any appropriate method can be used to identify a mammal as having a cancer (e.g., a brain cancer such as glioma).
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • Any appropriate method can be used to identify a mammal as having a cancer (e.g., a brain cancer such as glioma).
  • Examples of techniques that can be used to confirm the presence of cancer include, without limitation, neurological examinations (e.g., checking vision, hearing, balance, coordination, strength, and/or reflexes), imaging tests (e.g., MRI, computerized tomography (CT), and/or positron emission tomography (PET)), and laboratory tests based on blood or tissue biopsy samples.
  • neurological examinations e.g., checking vision, hearing, balance, coordination, strength, and/or reflexes
  • imaging tests e.g., MRI, computerized tomography (CT), and/or positron emission tomography (PET)
  • PET positron emission tomography
  • a potential anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective in treating a particular mammal (e.g., a particular human) having a cancer (e.g., a brain cancer such as glioma) by comparing the cell viability in one or more of the mammal’s microcancer models generated as described herein and contacted with the potential anti-cancer treatment to the cell viability in one or more of that mammal’s microcancer models not contacted with that potential anti-cancer treatment. Any appropriate method can be used to assess cell viability . For example, cell viability can be assessed by examining live versus dead cells in a microcancer model.
  • cell viability' can be assessed by examining cellular mechanisms such as cell proliferation, membrane integrity, mitochondrial membrane integrity, enzy me activity, and/or metabolic activity'.
  • nucleic acid binding dyes, amine-reactive viability dyes, enzyme activity assays, Ki67 staining, and/or metabolic activity assays such as measurement of ATP concentration can be used to assess cell viability in a microcancer model.
  • Such assays can be performed on any appropriate detection platform including, without limitation, light microscopy, fluorescence microscopy, immunohistochemistry’, flow cytometry, and microplate reader.
  • cell viability can be assessed by quantifying cell viability'.
  • a luminescent assay can be used to quantify cell viability in a microcancer model.
  • any appropnate level of reduced cell viability can be detected and used to identify candidate anti-cancer treatments for that particular mammal (e.g., that particular human).
  • the cell viability' in a particular mammal’s microcancer model described herein that was contacted with a potential anti-cancer treatment can be reduced by at least 5% (e.g., about 5%, about 10%, about 20%, about 30%. about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more such as 100%) as compared to the cell viability in a control microcancer model for that mammal not contacted with the potential anti-cancer treatment.
  • the cell viability in a particular mammal’s microcancer model described herein that was contacted with a potential candidate anti-cancer treatment can be reduced by at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold as compared the cell viability in a control microcancer model for that mammal not contacted with the potential anti -cancer treatment.
  • the effectiveness of a potential anti-cancer treatment can be represented as a percentage of reduced cell viability in a particular mammal’s collection of microcancer models as compared to the cell viability in one or more control microcancer models for that mammal not contacted with the potential anti-cancer treatment.
  • the effectiveness of a potential anti -cancer treatment e g., represented as a percentage of reduced cell viability in a mammal’s collection of microcancer models as compared the cell viability in one or more control microcancer models for that mammal not contacted with the potential anti-cancer treatment
  • the effectiveness of a potential anti-cancer treatment can be assessed at the anticipated Cmax of a drug of a potential anti-cancer treatment.
  • each drug of a potential anti-cancer treatment will be administered to a mammal (e.g., a human) at an acceptable dose per administration. In some cases, that dose is set by a regulatory agency such as the U.S. Federal Drug Administration or determined by a practicing clinician. After administering a drug at a particular dose to a mammal, that amount of administered drug will achieve a maximum concentration within the mammal’s blood, plasma, cerebral fluid, and/or tissues. That maximum concentration is referred to the C max for that dose of that drug. In general, Cmax values are know n or can be measured for the typical drug dosages used to treat mammals (e.g., humans).
  • the anticipated Cmax values of drugs can be used to identify candidate anti-cancer treatments that are likely to be effective against a particular mammal’s cancer.
  • a first potential anti-cancer treatment can be a treatment that involves administering 100 mg of drug 1 to a human having brain cancer with that dose achieving a plasma Cmax of 10 ng/mL.
  • the mammal’s microcancer models can be exposed to that Cmax value and cell viability assessed and compared to one or more of that mammal’s control microcancer models.
  • that first potential anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective against that particular mammal’s cancer. If the Cmax value of drug 1 within brain tissue is known, then the mammal’s microcancer models can be exposed to that Cmax value and cell viability assessed and compared to one or more of that mammal’s control microcancer models.
  • a second potential anti-cancer treatment can be a treatment that involves administering 200 mg of drug 1 to a human having brain cancer with that dose achieving a plasma Cmax of 15 ng/mL.
  • the mammal’s microcancer models can be exposed to that C max value and cell viability assessed and compared to one or more of that mammal’s control microcancer models. If cell viability' assessed at that C max value results in promoting a reduction in cell viability, then that second potential anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective against that particular mammal’s cancer.
  • a third potential anti-cancer treatment can be a treatment that involves administering 100 mg of drug 2 to a human having brain cancer with that dose achieving a C max within the brain of 5 ng/mm 3 .
  • the mammal’s microcancer models can be exposed to that C max value and cell viability assessed and compared to one or more of that mammal's control microcancer models. If cell viability assessed at that C ma x value results in promoting a reduction in cell viability, then that third potential anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective against that particular mammal’s cancer.
  • a fourth potential anti-cancer treatment can be a treatment that involves administering a combination of 100 mg of drug 1 and 100 mg of drug 2 to a human having brain cancer with that dose of drug 1 achieving a plasma C max of 10 ng/mL and with that dose of drug 2 achieving a C max within the brain of 5 ng/mm 3 .
  • the mammal’s microcancer models can be exposed to that Cmax value for drug 1 and that C max value for drug 2 and cell viability assessed and compared to one or more of that mammal's control microcancer models. If cell viability assessed at those C max values results in promoting a reduction in cell viability, then that fourth potential anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective against that particular mammal’s cancer.
  • potential anti-cancer treatments that include two different drugs can be identified as being a candidate anti-cancer treatment that is likely to be effective against a particular mammal’s cancer by assessing each of the two drugs individually and each together and then comparing effectiveness in reducing cell viability of each drug individually to the effectiveness when used in combination.
  • a potential anti-cancer treatment includes a combination of two different drugs (e.g., drug 1 plus drug 2)
  • one or more of a mammal’s microcancer models can be exposed the first drug (e.g., drug 1)
  • one or more of that mammal’s microcancer models can be exposed the second drug (e.g., drug 2).
  • the first drug and the second drug e.g., drug 1 and drug 2
  • cell viability can be assessed. If the combination outperforms both drugs when used individually, then that particular anti-cancer treatment can be identified as being a candidate anti-cancer treatment that is likely to be effective against that particular mammal’s cancer.
  • the anticipated C max values of each drug used in a potential drug combination treatment can be used to identify candidate anti-cancer treatments involving the use of two or more drugs that are likely to be profoundly synergistically effective against a particular mammal’s cancer.
  • a potential anti-cancer treatment involves administering a combination of 100 mg of drug 1 and 100 mg of drug 2 to a human having brain cancer with that dose of drug 1 achieving a plasma C max of 10 ng/mL and with that dose of drug 2 achieving a C max within the brain of 5 ng/mm’
  • one or more of that human’s microcancer models can be exposed to that C max value for drug 1.
  • one or more of that human’s microcancer models can be exposed to that C max value for drug 2, and one or more of that human’s microcancer models can be exposed to both that Cmax value for drug 1 and that Cmax value for drug 2.
  • the reduction in cell viabilities resulting from exposure to the Cmax value of drug 1 alone, the reduction in cell viabilities resulting from exposure to the Cm x value of drug 2 alone, and the reduction in cell viabilities resulting from exposure to both the Cmax value of drug 1 and the Cmax value of drug 2 can be compared to determine if the combination resulted in a meaningful additional benefit against that particular human’s cancer.
  • combination treatment efficacy is depicted using an xy plot, where the x axis is the percent inhibition of cell viability of drug 1 at drug l’s Cmax and the y axis is the percent inhibition of cell viability of drug 2 at drug 2’s Cmax (see, e.g., Figure 28).
  • the first (x,y) data point can be plotted based on the percent inhibition of cell viability of drug 1 alone at its Cmax (x) and the percent inhibition of cell viability of drug 2 tested alone at its Cmax (y), while the second (X,Y) data point can be plotted based on the percent inhibition of cell viability' for the combined exposition to both drug 1 and drug 2 at the Cmax of drug 1 (X) and the Cmax of drug 2 (Y).
  • An example of such plotting is shown in Figure 28.
  • any position from after 12:00 to before 1 :00 or from after 2:00 to before 3:00 can be used to identify that potential anticancer treatment as being a candidate anti-cancer treatment involving a combination of two drugs likely to have an added level of effectiveness against that particular human’s cancer.
  • any position from 1 :00 to 2:00 can be used to identify that potential anti-cancer treatment as being a candidate anti-cancer treatment involving a combination of two drugs likely to be profoundly synergistically effective against that particular human’s cancer.
  • Any other direction of the line from the first data point to the second data point can indicate that the combined use of the two drugs provides little, if any, added benefit for that particular mammal having cancer.
  • any position from after 3:00 to 12:00 can be used to identity' that potential anti-cancer treatment as involving a combination of two drugs unlikely to have an added level of effectiveness against that particular mammal’s cancer.
  • Generating this type of data for each drug individually and in combination and plotting that data in this manner can allow clinicians to quickly identify candidate anti-cancer treatments, from among tens, hundreds, and even thousands of potential anti-cancer treatments, that are meaningful candidates for the particular mammal (e.g., human) being assessed and treated.
  • This approach also allows clinicians to avoid administering a potential anti-cancer treatment that may be very' effective in other mammals (e.g., other humans) to a particular mammal (e.g., a particular human) that is unlikely to experience a beneficial outcome with that otherwise effective treatment.
  • This type of individually tailored approach can help prolong cancer survival for patients.
  • a candidate anti-cancer treatment identified as being likely to be effective in treating a particular mammal e.g., a particular human
  • a cancer e.g., a brain cancer such as glioma
  • a cancer e.g., a brain cancer such as glioma
  • a candidate anti-cancer treatment identified as being likely to be effective in treating a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • a reduction of cell viability in one or more of that mammal’s microcancer models contacted with that candidate anti-cancer treatment as compared to cell viability’ in one or more control microcancer models for that mammal that were not contacted with the candidate anti-cancer treatment can be selected to be administered to that mammal to treat that mammal’s cancer.
  • any appropriate anti-cancer treatment or suspected anti-cancer treatment can be assessed as described herein to determine if it is likely to be effective against a particular mammal’s cancer.
  • individual chemotherapeutic agents, individual targeted cancer drugs, individual immunotherapy drugs, radiation therapy, antibodies, antibody-drug conjugates, bispecific antibodies, oncolytic viruses, cellular therapies, PROTACs, two or more chemotherapeutic agents, two or more targeted cancer drugs, two or more immunotherapy drugs, and any combination chemotherapeutic agents, targeted cancer drugs, and/or immunotherapy drugs can be assessed as described herein to identify anti-cancer treatments 1 i kely to be effective against a particular mammal’s cancer.
  • anticancer treatments that can be assessed as described herein include, without limitation, temozolomide, carboplatin, cisplatin, doxorubicin, gemcitabine, paclitaxel, abemaciclib, vorinostat, neratinib. osimertinib, sunitinib, pimasertib, paxalisib. capivasertiv, idasanutlin.
  • ulixertinib foretinib, navitoclax, niraparib, JQ1, trotabresib, CC-115, vorasidenib, vistusertib, afatinib, alisertib, alpelisib, pembrolizumab, durvalumab, nivolumab, atezolizumab, sacituzumab govitecan, enfortumab vedotin.
  • tisotumab vedorin T-DM1, T-DXd, amivantamab-vmjw, teclistamab-cqyv.
  • blinatumomab VSV, Morreton virus, VMG, CAR T- cells), MZ-1, and MET-PROTAC.
  • a candidate anti-cancer treatment e.g., a particular human having a cancer (e.g.. a brain cancer such as glioma) as described herein and administering that identified candidate anti-cancer treatment to that particular mammal, the effectiveness of that administered candidate anti-cancer treatment can be monitored over time using one or more of that mammal's microcancer models.
  • one or more of a mammal's microcancer models can be exposed to the candidate anti-cancer treatment administered to the mammal in a similar manner (e.g., at the same timing and at the predicted or measured C ma x for the cancer target) and those microcancer models can be monitored over time for the emergence of drug resistance or drug escape within the microcancer models.
  • a large collection of a mammal's (e.g., a human’s) microcancer models can be exposed to the candidate anti -cancer treatment being administered to that mammal in a similar manner such that that the large collection of microcancer models potentially mimics the evolution of the actual cancer cells and/or cancer tissue within the treated mammal.
  • Such microcancer models can be referred to herein as treatment-exposed microcancer models. If resistant cancer cells are observed as remaining and/or emerging across some, most, or all the treatment-exposed microcancer models, then the clinician can stop or taper off the administration of that candidate anti-cancer treatment for that mammal and a similar stopping or tapering can be carried out for the collection of treatment-exposed microcancer models.
  • potential anticancer treatments can be assessed using one or more of the treatment-exposed microcancer models for that mammal to identify one or more candidate anti-cancer treatments likely to be effective against that particular mammal’s potentially resistant cancer so that the clinician can select an effective second anti-cancer treatment for that particular mammal. Additional rounds of this ex vivo mimicking of in vivo cancer treatments can be carried out as well.
  • This document also provides methods for treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma).
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • microcancer models for that mammal as described herein (e.g.. to identify one or more candidate anti-cancer treatment likely to be effective in treating that mammal ’s cancer) can be administered or instructed to self-administer a candidate anti-cancer treatment identified as being likely to be effective in treating that mammal.
  • a mammal e.g., a human having a cancer (e.g., a brain cancer such as glioma) can be administered or instructed to self-administer an anti-cancer treatment that is selected based, at least in part, on its identification as being a candidate anti-cancer treatment likely to be effective in treating that mammal’s (e.g., that human’s) cancer (e.g., brain cancer such as glioma) using that mammal’s microcancer models as described herein.
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • an anti-cancer treatment that is selected based, at least in part, on its identification as being a candidate anti-cancer treatment likely to be effective in treating that mammal’s (e.g., that human’s) cancer (e.g., brain cancer such as glioma) using that mammal’s microcancer models
  • a candidate anti-cancer treatment when identified as being likely to be effective in treating a mammal (e g., a human) having a cancer (e.g., a brain cancer such as glioma) based, at least in part, on the reduction of cell viability in one or more of that mammal’s microcancer models as described herein, the mammal can be administered or can be instructed to self-administer that candidate anti-cancer treatment.
  • a mammal e g., a human
  • a cancer e.g., a brain cancer such as glioma
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • that anti-cancer treatment can be the sole anti-cancer treatment used to treat that mammal’s cancer.
  • an anti-cancer treatment when an anti-cancer treatment is identified as being unlikely to be effective in treating a mammal’s cancer (e g., a human’s cancer) based, at least in part, on an absence of a reduction of cell viability in one or more of that mammal’s microcancer models contacted with that potential anti-cancer treatment, the mammal can be administered or instructed to self-administer an alternative anti-cancer treatment.
  • a mammal e a human’s cancer
  • This document also provides methods for treating a mammal (e.g., a human) having a brain cancer (e g., a glioma).
  • a mammal e.g., a human having a brain cancer (e g., a glioma).
  • any one or more of the compounds set forth in Table 1 can be administered to a mammal (e.g., a human) having a brain cancer (e.g., a glioma) to treat that brain cancer.
  • a mammal (e.g., a human) having a brain cancer (e.g., a glioma) can be treated with any combination of compounds set forth in Table 2.
  • one of the compounds set forth in Table 1 can be administered to a mammal (e.g., a human) having a brain cancer (e.g., a glioma) as the sole active ingredient to treat the cancer.
  • a mammal e.g., a human
  • a brain cancer e.g., a glioma
  • one of the combinations of compounds set forth in Table 2 can be administered to a mammal (e.g., a human) having a brain cancer (e.g., a glioma) as the sole active ingredients to treat the cancer.
  • a mammal e.g., a human
  • a brain cancer e.g.. a glioma
  • the one or more compounds can be administered to the mammal having cancer in any appropriate amount (e.g., any appropriate dose).
  • an effective dose of one or more compounds can be a flat dose.
  • effective dose of one or more compounds can be based on the body of a mammal (e.g., a human) to be treated as described herein.
  • an effective amount of one or more compounds can be from about 0.001 mg of compound per kg body weight of a mammal (mg/kg) to about 100 mg/kg (e.g., from about 0.005 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.5 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 100 mg/kg, from about 1.5 mg/kg to about 100 mg/kg, from about 5 mg/kg to about 100 mg/kg, from about 0.005 mg/kg to about 75 mg/kg, from about 0.005 mg/kg to about 50 mg/kg, from about 0.005 mg/kg to about 25 mg/kg, from about 0.005 mg/kg to about 10 mg/kg.
  • the effective amount of one or more compounds can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.
  • the one or more compounds when treating a mammal (e.g., a human) having a brain cancer (e.g., a glioma) as described herein (e.g., by administering (a) any one or more of the compounds set forth in Table 1 or (b) any combination of compounds set forth in Table 2), the one or more compounds can be administered to a mammal having brain cancer at any appropriate frequency.
  • the frequency of administration can be any frequency that can treat a mammal having cancer without producing significant toxicity to the mammal.
  • the frequency of administration can be from about twice a day to about once every 7 other day, from about once a day to about once a week, from about once a day to about once a month, from about once a week to about once a month, or from about twice a month to about once a month.
  • the frequency of administration can remain constant or can be variable during the duration of treatment.
  • various factors can influence the actual frequency of administration used for a particular application.
  • the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
  • an effective duration can be any duration that can treat a mammal having cancer without producing significant toxicity 7 to the mammal.
  • the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment.
  • an effective duration can vary with the frequency ⁇ of administration, effective amount, use of multiple treatment agents, and/or route of administration.
  • the methods and materials provided herein can include monitoring the mammal (e.g., the human) being treated as described herein (e.g.. by administering (a) any one or more of the compounds set forth in Table 1 or (b) any combination of compounds set forth in Table 2).
  • the size of the cancer e.g., the number of cancer cells and/or the volume of one or more tumors
  • Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced.
  • imaging techniques can be used to assess the size of the cancer present within a mammal (e.g., a human).
  • the treatment when treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) as described herein, the treatment can be effective to treat the cancer.
  • the number of cancer cells present within a mammal can be reduced using the methods and materials described herein.
  • the methods and materials described herein can be used to reduce the number of cancer cells present within a mammal (e.g.. a human) having a cancer (e g., a brain cancer such as glioma) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the treatment when treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) as described herein, the treatment can be elfective to prevent an increase in the number of cancer cells present within the mammal receiving the treatment.
  • the number of cancer cells present within a mammal (e.g., a human) having a cancer (e.g., glioma) and being treated as described herein can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a mammal is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a mammal.
  • the treatment when treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) as described herein, the treatment can be effective to reduce the size of a tumor present in the mammal receiving the treatment.
  • the methods and materials described herein can be used to reduce the size of one or more tumors present within a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the size (e.g., volume) of one or more tumors present within a mammal does not increase. Any appropriate method can be used to determine whether or not the size of the tumor present within a mammal is reduced. For example, imaging techniques can be used to assess the size of the tumor present within a mammal.
  • the treatment when treating a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) as described herein, the treatment can be effective to improve survival of the mammal.
  • the methods and materials described herein can be used to improve disease-free survival (e.g., relapse-free survival).
  • the methods and materials described herein can be used to improve overall survival.
  • the methods and materials described herein can be used to improve the survival of a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) by, for example, 10. 20. 30. 40.
  • the methods and materials described herein can be used to improve the survival of a mammal (e.g., a human) having a cancer (e.g., a brain cancer such as glioma) by, for example, at least 4 months (e.g., about 4 months, about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years).
  • a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • at least 4 months e.g., about 4 months, about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years.
  • the treatment when treating a mammal (e.g., a human) having a cancer (e.g.. a brain cancer such as glioma) as described herein, the treatment can be effective to reduce or eliminate one or more symptoms of the cancer (e.g.. the ghoma).
  • symptoms of a cancer e.g., a brain cancer such as glioma
  • Examples of symptoms of a cancer (e g., a brain cancer such as glioma) that can be reduced or eliminated using the methods and materials described herein can include, without limitation, headache, nausea, vomiting, confusion, a decline in brain function, problems with thinking and understanding information, memory loss, personality changes, irritability, vision problems, blurred vision, double vision, loss of peripheral vision, speech difficulties, and seizures.
  • the methods and materials described herein can be used to reduce one or more symptoms of a cancer (e g., a brain cancer such as ghoma) within a mammal (e.g., a human) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • a cancer e.g., a brain cancer such as ghoma
  • a mammal e.g., a human
  • the number of cancer cells present within a mammal e.g., a human
  • a cancer e.g., a brain cancer such as glioma
  • imaging techniques can be used to assess the number of cancer cells present within a mammal.
  • Example 1 Generation and characterization of patient-derived microcancer models
  • This example describes the generation and characterization of microcancer models derived from glioma patients for use in functional drug screening and for the rapid interrogation of potential vulnerabilities to targeted treatments.
  • MPseq, WES, and RNAseq were performed after isolation of DNA and RNA from flash-frozen tumor tissue and analyzed (Vasmatzis et al. , Mayo Clin Proc, 95: 306 (2020)). Tumor was identified by gross and frozen section microscopic examination in the Mayo Clinic Frozen Section Laboratory. Fresh tumor in excess of diagnostic clinical material was placed in a plastic cassette and snap frozen in isopentane. A secondary review by an experienced surgical pathologist was performed using a toluidine blue-stained frozen section. A tumor cellularity of >40% in the sample was obtained that included the use of macrodissection or laser capture microdissection as needed (Murphy et al., DNA Res, 19: 395 (2012)).
  • DNA was extracted with the Qiagen AllPrep DNA/RNA mini kit (80204, Qiagen) or Qiagen DNeasy Blood and Tissue Kit (69504. Qiagen) following the manufacturer’s protocol. In cases where laser capture microdissection was done or initial DNA yield was too low' for sequencing (e g., PT427), whole genome amplification was performed using the Repli-G mini kit (150023, Qiagen) (Murphy et al., DNA Res, 19: 395 (2012)).
  • CNAs were determined by the comparison with the genome ploidy for each case.
  • Shallow deletion events included clonal hemizygous deletion and various degrees of subclonal hemizy gous or homozy gous deletion. Deep deletion indicated clonal homozy gous deletion.
  • Amplification was called when a significant copy number gain was observed (e.g., >7n for a diploid genome).
  • Molecular alterations were deemed pathogenic when TSGs exhibited LoF mutation(s) or double-hit, or when oncogenes exhibited GoF mutation(s), copy number amplification, and/or oncogenic fusion transcripts (N. Cancer Genome Atlas Research, Nature, 455: 1061 (2008)).
  • the TERT promoter sequence at the two known pathogenic sites C228 and C250 was amplified with M13-TERT genotype primers using Q5® high-fidelity DNA polymerase (M0492S, NEB) (Bell et al. . Science, 348: 459 (2016)). Amplified PCR segments were then gel purified for Sanger sequencing using standard Ml 3 sequencing forward and reverse primers (Genewiz). The PCR primers and their sequences are shown in Table 3.
  • Tissue processing was performed using Human Tumor Dissociation Kit (130-095- 929, Miltenyi Biotec) following the manufacturer’s instructions.
  • Dissociated cells were resuspended in microcancer medium, which contains DMEM/F12 media 1143 (SH30023.01, HyClone) supplemented with 10% heat-inactivated horse serum (16050-122. Gibco), 1% penicillin-streptomycin (MT30002CI, Coming), Y-27632 (10 pM; ALX-270-333, Enzo Life Sciences or S1049. Selleckchem), human EGF (10 ng/ml; AF-100-15, PeproTech) and insulin (5 pg/ml; 11882, Sigma).
  • Glioblastoma organoid (GBO) medium contained 50% advanced-DMEM:F12 (12634-010, ThermoFisher), 50% Neurobasal (10888022, ThermoFisher), 1X N-2 supplement (17502048, ThermoFisher), 1X B-27 supplement minus vitamin A (12587010, ThermoFisher).
  • IX GlutaMax 35050-06, ThermoFisher
  • 1% penicillin-streptomycin MT30002CI, Coming
  • 50 pM 2- mercaptoethanol 31350010, ThermoFisher
  • human insulin 2.5 pg/mL; 11882, Sigma
  • Y-27632 10 pM; ALX-270-333, Enzo Life Sciences.
  • Neurosphere medium (5) contained Neurobasal (10888022, ThermoFisher) supplemented with 1X N-2 supplement (17502048, ThermoFisher), IX B-27 supplement minus vitamin A (12587010, ThermoFisher), IX GlutaMax (35050-06, ThermoFisher), 1% penicillin-streptomycin (MT30002CI, Coming), Y-27632 (10 pM; ALX-270-333, Enzo Life Sciences), EGF (25 ng/mL; AF-100-15, PeproTech), and bFGF (25 ng/mL; AF-100-18B, Peprotech) (Jacob et al., Cell. 180: 188 (2020)).
  • MGMT promoter methylation was present in 9 glioblastoma cases, absent in 9, and not tested or indeterminate in 2 cases (Table 4).
  • CDKN2A and CDKN2B Biallelic inactivation of CDKN2A and CDKN2B were the most frequent alterations related to cell cycle signaling, present in 70% of glioblastomas and 30% of IDH-mut gliomas.
  • Low level copy number gain of CDK6 was present in most glioblastomas (80%), largely due to its location to the frequently gained chromosome 7.
  • Amplification of CCND1 was observed in one case, while RBI double-hit was found in three cases. Combined, deregulation of cell cycle signaling was observed in 73% of cases.
  • EGFR amplification was found in 5 cases with one expressing wild-type EGFR, one expressing an ectodomain p. A289D mutation, and the remaining expressing EGFRvIII variant. Two EGFRvIII+ cases concurrently exhibited ectodomain mutations. Further, the cohort included 2 amplified PTPRZ1-MET fusion glioblastomas and 2 F’DGFT -positive astrocytomas, one amplified and one expressing a /.AA/-PDGF A fusion transcript. Downstream of receptor tyrosine kinases (RTKs), 3 cases exhibited NF1 double hits, 1 expressed an oncogenic KRAS mutation, and several exhibited gains in BRAF, which resides in chromosome 7.
  • RTKs receptor tyrosine kinases
  • genomic alterations related to PI3K signaling occurred more frequently (47%) than MEK pathway alterations (20%). with double hits of PTEN being the most common alterations (9 out of 30 cases). Overall, genomic alterations in RTK/MEK/PI3K signaling were observed in 73% of cases.
  • microcancers derived from GBM8 PDX tumors maintain the cellular diversity of the original tumor including the presence of glioma stem cells, differentiated glioma cells, and murine stroma (Ganguli et al., Sci Adv, 7 (2021)).
  • GBM8 microcancers also maintained a similar Ki-67 proliferation index to parental orthotopic tumors (Figure 5A).
  • microcancers derived from human glioblastomas maintained proliferative capacity, exhibiting a cell doubling time of ⁇ 7 days ( Figure 5B). This rate of growth suggests that both cytotoxic and cytostatic effects can be interrogated in microcancers over a 6-day drug treatment, and that short-term culture would minimize selective pressure, thus maintaining the genomic profile of the original tumor.
  • microcancers exhibited similar heterozygosity and the same single base substitution signature SBS1 (Figure 6), as well as key oncogenic events, including homozygous 9p21 deletions involving CDKN2A and CDKN2B, and PTEN double hit, with similar allele frequencies (Figure 7A).
  • Microcancers also preserved all 28 mutations identified in the original tumor with one subclonal de novo variant of unknown significance (VUS) found ( Figures 7B-7C) (Table 5). Whole genome copy number alterations and DNA rearrangements (95 of 98 events; 96.9%) were also preserved (Figure 8A).
  • microcancer TERT promoter mutation status was identical to that of the parental tumor (Figure 8B). Overall, several lines of evidence indicate that microcancers recapitulate the genomic landscape of parental tumors with high fidelity. Table 5. Full gene list identified by WES for PT311
  • Example 2 Functional drug screen in patient-derived pCancers
  • This example describes functional drug screening conducted in patient-derived microcancer models (also referred to as pCancers) as a method for interrogating potential vulnerabilities of cancers to targeted treatments and for identifying new 7 treatment strategies.
  • Apportioned fresh tissue specimen was cryopreserved in CryoStor® CS10 cell freezing medium (C2874, Sigma). Thawed tissues were subjected to mechanical and enzymatic dissociation and dissociated cells (5x10 3 per well) were cultured as hanging drops for 6 days to allow microcancer generation (Vasmatzis el al., Mayo Clin Proc, 95: 306 (2020)). Established microcancers were transferred to the corresponding well of 96-well clear round bottom ultra-low attachment microplates (7007, Coming) and subjected to microscopic evaluation to ensure size similarity across wells, before drug testing at eight serial dilutions with corresponding drug solvents (DMSO or media) as vehicle controls.
  • DMSO or media drug solvents
  • RNA-seq data were processed through the MAP-Rseq pipeline (Kalari et al. , BMC Bioinformatics, 15:224 (2014)) to generate “.count’' files which w ere then processed by the edgeR package in R to generate log 2 normalized gene expression values.
  • Normalized enrichment score (NES) for each sample was obtained by the “gsva” function (in GSVA R package) using single sample GSEA (ssgsea) method.
  • GSEA ssgsea
  • Drug data processing and statistical analysis were performed using the GraphPad Prism software. Unpaired t-test was performed when assessing the impact of pTERT status on microcancer response to JQ1. Correlation analysis was performed using the Spearman and/or Pearson methods as indicated in figure legends. Astrocytomas and glioblastomas with >50% of tumor purity were included for statistical correlation analyses. Tumor purity was inferred based on DNA content (mutation allele fraction and ploidy) and/or RNA expression data (ESTIMATE algorithm, (Y oshihara et al., Nat Commun 4: 2612 (2013)). Correlation analysis was performed using the “contest” function 595 in R software package with Pearson’s correlation coefficient outputs. Statistical significance was assessed at p-value ⁇ 0.05.
  • drugs in each class were selected based on their ability to penetrate the central nervous system (CNS) (Table 6). Alternatively, compounds targeting key pathways and/or approved for use in other clinical settings were chosen.
  • response profiles were segregated into three categories: ⁇ 35%, 35-70%, and > 70% of inhibition at Cmax that were defined as minimal response, partial response, and strong response, respectively (Figure 14). Samples with sufficient tissue were subjected to testing with all the drugs in the panel.
  • Glioma microcancers showed in general minimal response to RTK targeted agents, including the EGFR inhibitor osimertinib, pan-ERBB inhibitor neratinib, and multi-RTK inhibitors foretinib and sunitinib ( Figures 13A-13B, Figure 14, and Figure 15A-15C). Similar results were also obtained by testing select cases with additional EGFR-related inhibitors, alone or in combination, except for PT431, an EGFRvIII amplified tumor that exhibited a partial response to erlotinib and a strong response to the combination of erlotinib with the dual PI3K/mT0R inhibitor paxalisib.
  • the MEK inhibitor pimasertib, MAPK/ERK inhibitor ulixertinib, PI3K7mTOR dual inhibitor paxalisib, pan-AKT inhibitor capivasertib, and mTOR/DNA-PK dual inhibitor CC-115 were utilized. In general, this strategy was more effective than targeting upstream RTKs. Pimasertib achieved 1 strong and 5 partial responses, while ulixertinib achieved 4 partial responses ( Figure 14). Inhibitors targeting the PI3K pathway were overall more effective, with paxalisib achieving 8 partial responses, capivasertib 1 strong and 19 partial responses, and CC-115 2 strong and 11 partial responses. The extent to which the DNA-PK (DDR kinase) inhibitor activity of CC-115 contributes significantly to these responses is currently unclear, although another DDR- targeting agent, the PARP inhibitor niraparib exhibited no effect in the microcancer cohort.
  • DDR kinase DNA-PK
  • the epigenome is largely altered during gliomagenesis and disease progression (Stepniak et al., Nat Commun, 12: 3621 (2021); Mack et al., J Ex Med, 216: 1071 (2019)).
  • the epigenome was targeted by testing the activity of histone deacetylase (HD AC) and bromodomain and extraterminal domain (BET) inhibitors, both shown to modulate chromatin structure organization and to globally affect gene expression (Manzotti et al.. Cancers (Basel) 11 (2019)).
  • HD AC inhibition by vorinostat resulted in moderate activity’ with 10 partial responses ( Figure 14).
  • JQ1 a preclinical BET inhibitor
  • birabresib a preclinical BET inhibitor
  • mivebresib. a preclinical BET inhibitor
  • trotabresib the latter been actively explored in gliomas
  • These inhibitors are particularly active against the BET protein BRD4 and can displace it from chromatin leading to anti-tumor activity via gene expression modulation (Manzotti el al., Cancers (Basel) 11 (2019)).
  • the cohort included 15 proneural (PN) cases, 11 classical (CL) cases, and 4 mesenchymal (MES) cases (Figure 19A). Further, to quantify transcriptional heterogeneity, which could affect response to treatment, a well-established ‘simplicity scoring’ (Wang et al. , Cancer Cell, 32: 42 e46 (2017)) w'as applied onto the classification. This scoring system ranges from 0 to 1 with 1 showing the simplest tumor that contains solely one subtype and 0 presenting multiple subtypes being present in a single tumor.
  • the average simplicity score (SPL) of the cohort was 0.50 ⁇ 0. 18 (mean ⁇ SD) with the lowest and highest scores being 0.18 and 0.86, respectively. SPL of less than 0.5 were observed in 14 out of 30 (46.7%) cases, indicating that multiple subtypes are present in most cases in the cohort.
  • a pathway-based transcriptomic classifier was recently developed for glioblastoma subgrouping, proposed to predict biological activities underlining drug responses (Garofano et al., Nat Cancer, 2: 141 (2021)).
  • the cohort consisted of 10 glycolytic/plurimetabolic (GPM) cases, 4 mitochondrial (MTC) cases, 9 neuronal (NEU) cases, and 7 proliferative/progenitor (PPR) cases (Figure 19B), with MTC and GPM representing the best and worst prognostic subtypes, respectively (Garofano et al, Nat Cancer, 2: 141 (2021)).
  • the most significantly differentially expressed genes in the MES signature were matrix metalloproteinase MMP7 and glioma pathogenesis-related protein 1 (GLIPR1) (Rosenzweig et al., Cancer Res, 66: 4139 (2006)) ( Figures 23A-23D), while progranulin (GRN) and putative GTPase activator protein (TBC1D22A) were most highly differentially expressed in the GPM signature ( Figures 24A-24D).
  • GPN progranulin
  • THC1D22A putative GTPase activator protein
  • Microcancers derived from PN glioma with high expression of ERBB3 appeared to be less 365 responsive to vorinostat ( Figures 23A-23D).
  • Response to CC-115 correlated with higher expression of MES and GPM associated genes, with extracellular matrix remodelers (COL15A1, LUM, COL1A1) topping the list ( Figures 23A-23D, Figures 24A-24D).
  • Response to the pan-AKT inhibitor capivasertib was negatively correlated to the expression of CL associated genes ( Figures 23A-23D).
  • the data suggest that CL-enriched tumors, which encompass most of the EGFR altered gliomas (Wang et al., Cancer Cell. 32: 42 e46 (2017)), are less likely to respond to capivasertib.
  • YAP/TAZ is a master regulator driving MES characteristics in glioma and a key regulator of glioblastoma sternness and plasticity (Castellan et al. , Nat Med, 24: 1599 (2016)).
  • the targets of these three drugs are linked to Hippo-YAP/TAZ signaling.
  • the effect of JQ1 and other BET inhibitors on YAP/TAZ signaling may be direct, as their main target BRD4 is recruited by YAP/TAZ to chromatin to enhance gene expression and drive tumorigenesis (Zanconato et al., Nat Med, 24: 1599 (2016)).
  • vorinostat’s target HDACs can facilitate transcriptional repression of tumor suppressor genes by YAP/TAZ (Kim et al. , Cell Rep, 11 : 270).
  • CC-115 may affect YAP/TAZ signaling by inhibiting DNA-PK, which reportedly forms a functional complex with YAP/TAZ (Bhat et al.. Neuro Oncol, 24: vii91 (2022)). or by suppressing mTOR activity, which is upregulated by YAP signaling (Tumaneng etal., Nat Cell Biol, 14: 1322 (2012)).
  • GSCs glioma stem cells
  • Gliomas are molecularly complex and highly heterogeneous tumors, arguing that single agent treatment strategies will rapidly be overcome by resistance mechanisms. Despite potential for increased toxicity 7 , combination treatments are more likely to overcome resistance and increase efficacy (Settleman et al., Cancer Discov, 11 : 1016 (2021); Plana et al. , Cancer Discov, 12: 606 (2022)). Therefore, rational drug combination strategies that are more effective than single-agent treatment were sought.
  • apoptosis-inducing agents i.e., navitoclax and idasanutlin
  • cytotoxicity' in cancers Cameiro et al., Nat Rev Clin Oncol, 17; 395 (2020)
  • Navitoclax with idasanutlin were thus combined to promote apoptosis by distinct mechanisms, and vorinostat with navitoclax to simultaneously target gene expression and eliminate senescent cells.
  • Drug combinations can significantly increase therapeutic benefit without necessarily exhibiting pharmacologically additive or synergistic effects (Settleman et al. , Cancer Discov, 11: 1016 (2021 ); Plana et al. , Cancer Discov, 12: 606 (2022)).
  • % inhibition at each drug’s Cmax was used to compare the effect of single agent treatment versus that of drug combination.
  • single agent A exhibited 20% inhibition at its Cmax value
  • agent B exhibited 62% inhibition at its Cmax. Combination of the two resulted in 77% and 83% inhibition at Cmax for drug A and drug B, respectively.
  • a tissue sample containing cancer cells is obtained from a human having brain cancer (e.g., a brain cancer such as glioma).
  • the tissue sample is used to generate a collection of microcancer models.
  • One or more of the human’s microcancer models is contacted with a potential anti-cancer treatment.
  • the cell viability in one or more of the human’s microcancer models contacted with the anti-cancer treatment is assessed.
  • the anti-cancer treatment is administered to the human.
  • the treatment can delay relapse, reduce the number of cancerous cells in the human having the brain cancer, reduce the size of the tumor, or reduce the rate of increase in the size of the tumor.
  • Example 4 Treating glioma follow ing development of anti-cancer treatment resistance
  • a tissue sample containing cancer cells is obtained from a human having brain cancer (e.g., a brain cancer such as glioma).
  • the tissue sample is used to generate a collection of microcancer models.
  • One or more of the human’s microcancer models is contacted with a potential anti-cancer treatment.
  • the cell viability' in one or more of the human’s microcancer models contacted with the potential anti-cancer treatment is assessed.
  • the anti-cancer treatment is administered to the human.
  • the treatment can delay relapse, reduce the number of cancerous cells in the human having the brain cancer, reduce the size of the tumor, or reduce the rate of increase in the size of the tumor.
  • the clinician stops or tappers off the administration of that anti-cancer treatment in the human.
  • one or more of the human's microcancer models is contacted with an alternative potential anti-cancer treatment.
  • the cell viability in one or more of the human’s microcancer models contacted with the alternative potential anti-cancer treatment is assessed.
  • the alternative potential anti-cancer treatment is administered to the human.
  • the alternative treatment can delay relapse, reduce the number of cancerous cells in the human having the brain cancer, reduce the size of the tumor, or reduce the rate of increase in the size of the tumor.
  • This example describes the further characterization of microcancer models and the elucidation of underlying drug response mechanisms in these models.
  • microcancer models were explored, focusing on JQ1, a BET inhibitor, as it was the most effective single agent in previous studies (see, Example 1) and is particularly effective in the most aggressive mesenchymal (MES) enriched high-grade glioma (HGG) cases.
  • the analysis utilized microcancer models that previously exhibited a response to JQ1 treatment (R; PT440) or no response to JQ1 treatment (NR; PT431).
  • RNA-seq analysis was performed before (time 0), during (Day 2). and after (Day 4) treatment with JQ1.
  • Temporal changes in gene expression and pathway enrichment were subsequently assessed to elucidate the underlying molecular programs.

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Abstract

La présente invention concerne des méthodes et matériaux d'évaluation de traitements du cancer et/ou de traitement de mammifères (par exemple, des êtres humains) atteints d'un cancer (par exemple, un cancer du cerveau tel qu'un gliome). Par exemple, des méthodes et matériaux selon l'invention peuvent être utilisés pour identifier des traitements anticancéreux candidats comme étant susceptibles d'être efficaces dans le traitement d'un mammifère (par exemple, un être humain) atteint d'un cancer (par exemple, un cancer du cerveau tel qu'un gliome). Ce document concerne également des méthodes et matériaux de traitement destinés au traitement d'un mammifère (par exemple, un être humain) atteint d'un cancer (par exemple, un cancer du cerveau tel qu'un gliome).
PCT/US2025/030210 2024-05-21 2025-05-20 Méthodes et matériaux d'évaluation de traitements du cancer Pending WO2025245123A1 (fr)

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