Methods and systems for evaluation of cell samples

Abstract

To provide a method of evaluating a candidate molecule.SOLUTION: There is provided a method of evaluating a candidate molecule comprising: (a) administering the candidate molecule to a cell sample of a tumor tissue derived from a subject; (b) culturing the cell sample under serum-free conditions, at a liquid-air interface, in the presence of the candidate molecule for at least 1 day; and (c) evaluating a response of the cell sample to the candidate molecule multiple times after administration of the candidate molecule.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 820,719, filed March 19, 2019, which is incorporated by reference in its entirety. Summary of the Invention

[0002] Disclosed herein in some embodiments is a method for evaluating a candidate molecule, comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample in the presence of the candidate molecule for at least 1 day; and (c) evaluating the response of the cell sample to the candidate molecule. In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 12 days. In some embodiments, in step (b), the cell sample is cultured for 16 days. In some embodiments, in step (b), the cell sample is cultured for 20 days. In some embodiments, the method further comprises the step of (d) administering an additional candidate molecule to the cell sample and culturing the cell sample for at least 24 hours. In some embodiments, in step (d), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (d), the cell sample is cultured for 4 days. In some embodiments, in step (d), the cell sample is cultured for 8 days. In some embodiments, in step (d), the cell sample is cultured for 12 days. In some embodiments, the method further comprises the step of (e) evaluating the response of the cell sample to the additional candidate molecule.In some embodiments, the method further comprises (d) administering a second dose of the additional candidate molecule to the cell sample and culturing the cell sample for at least 24 hours. In some embodiments, in step (d), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, the method further comprises (e) evaluating the response of the cell sample to the second dose of the candidate molecule. In some embodiments, in step (d), the cell sample is cultured for 4 days. In some embodiments, in step (d), the cell sample is cultured for 8 days. In some embodiments, in step (d), the cell sample is cultured for 12 days.

[0003] In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a cellular tissue. In some embodiments, the cell sample is a cancer cell. In some embodiments, the cell sample comprises a cell from the microenvironment of a tumor tissue. In some embodiments, the cell from the microenvironment of a tumor tissue is an immune cell, a fibroblast, an endothelial cell, a pericyte, an adipocyte, a mesenchymal stem cell, or a bone marrow-derived cell. In some embodiments, the immune cell is a lymphoid cell or a myeloid cell. In some embodiments, the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell. In some embodiments, the myeloid cell is a basophil, an eosinophil, a mast cell, a neutrophil, a monocyte, an erythrocyte, a macrophage (e.g., tissue-resident and peripherally derived macrophages), a myeloid suppressor cell, or a dendritic cell. In some embodiments, the cell sample is cultured in serum-free conditions. In some embodiments, the cell sample is cultured with cytokines or growth factors. In some embodiments, the cell sample is cultured at a liquid-air interface. In some embodiments, the cell sample is submerged in cell culture medium. In some embodiments, the response is a viability response, a gene expression response, a protein expression response, a protein modification response, a cell signaling response, a morphological response, an immunophenotypic response, or a metabolic response.

[0004] In some embodiments, the step of assessing the viability response comprises measuring a metabolite from the cell sample. In some embodiments, measuring a metabolite from the cell sample comprises isolating a metabolite from the cell culture medium and measuring the metabolite in the absence of the cell sample. In some embodiments, the metabolite from the cell sample is a product of a reduction reaction. In some embodiments, the metabolite from the cell sample is derived from resazurin. In some embodiments, the metabolite from the cell sample is resorufin. In some embodiments, the metabolite from the cell sample is derived from tetrazole. In some embodiments, the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof. In some embodiments, the metabolite from the cell sample is formazan. In some embodiments, the cell sample is from a xenograft derived from a patient. In some embodiments, the cell sample is a primary tumor sample. In some embodiments, the primary tumor sample is a surgical sample obtained from a patient. In some embodiments, the primary tumor sample is a biopsy sample obtained from a patient. In some embodiments, the biopsy sample is obtained by core needle biopsy.

[0005] In some embodiments, the method further comprises the step of recommending treatment of the subject with the candidate molecule. In some embodiments, the method further comprises the step of recommending not treating the subject with the candidate molecule. In some embodiments, the candidate molecule is selected from the group consisting of a biological molecule and a chemical molecule. In some embodiments, the biological molecule is selected from the group consisting of a recombinant protein, an enzyme, an antibody, a growth factor, a receptor, and any portion thereof.

[0006] In some embodiments, step (a) further comprises providing one or more additional candidate molecules, where step (c) comprises evaluating the response of the cell sample to the candidate molecules and the one or more additional candidate molecules. In some embodiments, the one or more additional candidate molecules are provided simultaneously with the candidate molecules. In some embodiments, the candidate molecules and the one or more additional candidate molecules are provided sequentially. In some embodiments, the method further comprises (d) administering the candidate molecules to an additional cell sample of tumor tissue from the subject; (e) culturing the additional cell sample in the presence of the candidate molecules for at least 5 days; (f) evaluating the response of the additional cell sample to the candidate molecules; and (g) comparing the response of the additional cell sample to the response of the cell sample. In some embodiments, the cell sample is from a first location of the tumor tissue and the additional cell sample is from a second location of the tumor tissue. In some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue at different times. In some embodiments, the different times are at least 1, 2, 3, 4, 5, 10, 15, or 20 days apart. In some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously. In some embodiments, performing a biological assay on the cell sample comprises permeabilizing the cell sample. In some embodiments, performing a biological assay on the cell sample comprises lysing the cell sample. In some embodiments, the biological assay is selected from the group consisting of flow cytometry, nucleic acid sequencing, polymerase chain reaction, histological analysis, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and any combination thereof.

[0007] Disclosed herein in some embodiments is a method for evaluating a candidate molecule, comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of tumor tissue for at least one day; (c) evaluating the response of the cell sample of tumor tissue to the candidate molecule; and (d) repeating steps (a)-(c). In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 4 days. In some embodiments, step (d) comprises repeating (a)-(d) at least once, at least twice, at least three times, at least four times, at least five times, or at least six times. In some embodiments, step (d) comprises repeating (a)-(d) three times. In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a cellular tissue. In some embodiments, the cell sample is a cancer cell. In some embodiments, the cell sample comprises a cell from the microenvironment of a tumor tissue. In some embodiments, the cell from the microenvironment of a tumor tissue is an immune cell, a fibroblast, an endothelial cell, a pericyte, an adipocyte, a mesenchymal stem cell, or a bone marrow-derived cell. In some embodiments, the immune cell is a lymphoid cell or a myeloid cell. In some embodiments, the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell. In some embodiments, the myeloid cells are basophils, eosinophils, mast cells, neutrophils, monocytes, macrophages (eg, tissue-resident and peripherally-derived macrophages), myeloid suppressor cells, erythrocytes, or dendritic cells.

[0008] In some embodiments, the cell sample is cultured in serum-free conditions. In some embodiments, the cell sample is cultured at a liquid-air interface. In some embodiments, the cell sample is submerged in cell culture medium. In some embodiments, the response is a viability response, a gene expression response, a protein expression response, a protein modification response, a cell signaling response, a morphological response, or a metabolic response. In some embodiments, evaluating the response comprises measuring a metabolite from the cell sample. In some embodiments, measuring a metabolite from the cell sample comprises isolating the metabolite from the cell culture medium and measuring the metabolite in the absence of the cell sample. In some embodiments, the metabolite from the cell sample is a product of a reduction reaction. In some embodiments, the metabolite from the cell sample is derived from resazurin. In some embodiments, the metabolite from the cell sample is resorufin. In some embodiments, the metabolite from the cell sample is derived from tetrazole. In some embodiments, the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof. In some embodiments, the metabolite from the cell sample is a formazan.

[0009] In some embodiments, the cell sample is from a xenograft derived from a patient. In some embodiments, the cell sample is a primary tumor sample. In some embodiments, the primary tumor sample is a surgical sample obtained from the patient. In some embodiments, the primary tumor sample is a biopsy sample obtained from the patient. In some embodiments, the biopsy sample is obtained by core needle biopsy. In some embodiments, the method further comprises the step of recommending treatment of the subject with the candidate molecule. In some embodiments, the method further comprises the step of recommending not treating the subject with the candidate molecule. In some embodiments, the candidate molecule is selected from the group consisting of a biological molecule and a chemical molecule. In some embodiments, step (a) further comprises providing one or more additional candidate molecules, wherein step (c) comprises evaluating the response of the cell sample to the candidate molecule and the one or more additional candidate molecules. In some embodiments, the one or more additional candidate molecules are provided simultaneously with the candidate molecule. In some embodiments, the candidate molecule and the one or more additional candidate molecules are provided sequentially. In some embodiments, the method further comprises (e) administering the candidate molecule to an additional cell sample of tumor tissue from the subject; (f) culturing the additional cell sample of tumor tissue for at least one day; (f) evaluating the response of the additional cell sample of tumor tissue to the candidate molecule; and (h) repeating steps (e)-(g); and (i) comparing the response of the additional cell sample to the response of the cell sample. In some embodiments, the cell sample is from a first location of the tumor tissue and the additional cell sample is from a second location of the tumor tissue. In some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue at different times. In some embodiments, the different times are at least 1, 2, 3, 4, or 5 days apart; in some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously. In some embodiments, the step of performing a biological assay on the cell sample comprises fixing the cell sample. In some embodiments, the step of performing a biological assay on the cell sample comprises permeabilizing the cell sample.In some embodiments, performing a biological assay on the cell sample comprises lysing the cell sample, hi some embodiments, the biological assay is selected from the group consisting of flow cytometry, nucleic acid sequencing, polymerase chain reaction, histological analysis, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and any combination thereof.

[0010] Disclosed herein, in some embodiments, is a method of assessing cell viability, the method comprising: (a) culturing a tumor cell sample in cell culture medium; (b) isolating metabolites from the cell culture medium; and (c) measuring the metabolites in the absence of the cell sample, thereby assessing cell viability of the cell sample. In some embodiments, the cell sample is from a cell line. In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a tumor sample. In some embodiments, the cell sample is a cancer cell. In some embodiments, the cell sample comprises a cell from the microenvironment of the tumor tissue. In some embodiments, the cell from the microenvironment of the tumor tissue is an immune cell, a fibroblast, an endothelial cell, a pericyte, an adipocyte, a mesenchymal stem cell, or a bone marrow-derived cell. In some embodiments, the immune cell is a lymphoid cell or a myeloid cell. In some embodiments, the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell. In some embodiments, the myeloid cells are basophils, eosinophils, mast cells, neutrophils, monocytes, erythrocytes, macrophages (eg, tissue-resident and peripherally-derived macrophages), myeloid suppressor cells, or dendritic cells.

[0011] In some embodiments, the cell sample is cultured in serum-free conditions. In some embodiments, the cell sample is cultured at a liquid-air interface. In some embodiments, the cell sample is submerged in cell culture medium. In some embodiments, the step of measuring the metabolic product comprises fluorescence measurement or chemiluminescence measurement. In some embodiments, the cell sample is cultured for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, the cell sample is cultured for 12 days. In some embodiments, the cell sample is cultured for 16 days. In some embodiments, the cell sample is cultured for 20 days. In some embodiments, the metabolic product from the cell sample is a product of a reduction reaction. In some embodiments, the metabolic product from the cell sample is derived from resazurin. In some embodiments, the metabolic product from the cell sample is resorufin. In some embodiments, the metabolite from the cell sample is derived from tetrazole. In some embodiments, the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof. In some embodiments, the metabolite from the cell sample is formazan. In some embodiments, the method further comprises administering a candidate molecule to the cell sample. In some embodiments, the cell sample is from a xenograft derived from a patient. In some embodiments, the cell sample is from a primary tumor sample.In some embodiments, the primary tumor sample is a surgical sample obtained from the patient. In some embodiments, the primary tumor sample is a biopsy sample obtained from the patient. In some embodiments, the biopsy sample is obtained by core needle biopsy. In some embodiments, the method further comprises recommending treatment of the subject with the candidate molecule. In some embodiments, the method further comprises recommending not treating the subject with the candidate molecule.

[0012] In some embodiments, the method further comprises (d) culturing the additional cell sample with the additional cell culture medium; (e) isolating metabolites from the additional cell culture medium; (f) measuring metabolites from the additional cell culture medium in the absence of the additional cell sample, thereby assessing cell viability of the additional cell sample; and (g) comparing the cell viability of the additional cell sample to the cell viability of the cell sample. In some embodiments, the cell sample is from a first location of the tumor tissue, and the additional cell sample is from a second location of the tumor tissue. In some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue at different times. In some embodiments, the different times are at least 1, 2, 3, 4, 5, 10, 15, or 20 days apart. In some embodiments, the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously. In some embodiments, the tumor tissue is a tissue sample from a subject.

[0013] Disclosed herein is a method of assessing radiation dose in some embodiments, comprising: (a) administering a radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample for at least 1 day; and (c) assessing the response of the cell sample to the radiation dose. In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, or at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 12 days. In some embodiments, in step (b), the cell sample is cultured for 16 days. In some embodiments, in step (b), the cell sample is cultured for 20 days.

[0014] Disclosed herein is a method for evaluating a tumor treatment field in some embodiments, comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample for at least 1 day; and (c) evaluating the response of the cell sample to the tumor treatment field. In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 12 days. In some embodiments, in step (b), the cell sample is cultured for 16 days. In some embodiments, in step (b), the cell sample is cultured for 20 days.

[0015] Disclosed herein in some embodiments is a method for assessing radiation dose, comprising: (a) administering a radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of tumor tissue for at least 1 day; (c) assessing the response of the cell sample of tumor tissue to the radiation dose; and (d) repeating steps (a)-(c). In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 4 days. In some embodiments, step (d) comprises repeating (a)-(d) at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In some embodiments, step (d) comprises repeating (a)-(c) three times.

[0016] Disclosed herein in some embodiments is a method for evaluating a tumor treatment field, comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample of tumor tissue for at least 1 day; (c) evaluating the response of the cell sample of tumor tissue to the tumor treatment field; and (d) repeating steps (a)-(c). In some embodiments, in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days. In some embodiments, in step (b), the cell sample is cultured for 4 days. In some embodiments, step (d) comprises repeating (a)-(d) at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In some embodiments, step (d) comprises repeating (a)-(c) three times.

[0017] Disclosed herein, in some embodiments, is a method for evaluating a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to the tumor treatment field; (b) culturing the cell sample of tumor tissue for at least one day; (c) evaluating the response of the cell sample of tumor tissue to the tumor treatment field; and (d) repeating steps (a)-(c).

[0018] Disclosed herein, in some embodiments, is a method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue derived from a subject; (b) culturing the cell sample in the presence of the candidate molecule for at least 12 days; and (c) evaluating the response of the cell sample to the candidate molecule.

[0019] Disclosed herein is a method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue derived from a subject; (b) culturing the cell sample of tumor tissue for at least 12 days; (c) evaluating the response of the cell sample of tumor tissue to the candidate molecule; and (d) repeating steps (a)-(c).

[0020] Disclosed herein, in some embodiments, is a method for assessing cell viability, the method comprising: (a) culturing a tumor cell sample in cell culture medium for at least 4 days; (b) isolating metabolic products from the cell culture medium; and (d) measuring the metabolic products in the absence of the cell sample, thereby assessing cell viability of the cell sample.

[0021] Disclosed herein, in some embodiments, is a method of evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of tumor tissue for at least four days; (c) evaluating the response of the cell sample of tumor tissue to the candidate molecule; and (d) repeating steps (a)-(c) at least three times.

[0022] Disclosed herein, in some embodiments, is a method of assessing a radiation dose, the method comprising: (a) administering a radiation dose to a cell sample of tumor tissue derived from a subject; (b) culturing the cell sample for at least 12 days; and (c) assessing the response of the cell sample to the radiation dose.

[0023] Disclosed herein, in some embodiments, is a method for evaluating a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue derived from a subject to the tumor treatment field; (b) culturing the cell sample for at least 12 days; and (c) evaluating the response of the cell sample to the tumor treatment field.

[0024] Disclosed herein, in some embodiments, is a method of assessing a radiation dose, the method comprising: (a) administering a radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of tumor tissue for at least 12 days; (c) assessing the response of the cell sample of tumor tissue to the radiation dose; and (d) repeating steps (a)-(c).

[0025] Disclosed herein, in some embodiments, is a method of evaluating a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to the tumor treatment field; (b) culturing the cell sample of tumor tissue for at least 12 days; (c) evaluating the response of the cell sample of tumor tissue to the tumor treatment field; and (d) repeating steps (a)-(c). [Brief description of the drawings]

[0026] The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized and the accompanying drawings. [Figure 1A] In vitro viability measurements at days 0, 4, 8 and 12 (div) are shown for breast (FIG. 1A), pancreatic (FIG. 1B), and glioblastoma (FIG. 1C) tumor samples. [Figure 1B] In vitro viability measurements at days 0, 4, 8 and 12 (div) are shown for breast (FIG. 1A), pancreatic (FIG. 1B), and glioblastoma (FIG. 1C) tumor samples. [Figure 1C]In vitro viability measurements at days 0, 4, 8 and 12 (div) are shown for breast (FIG. 1A), pancreatic (FIG. 1B), and glioblastoma (FIG. 1C) tumor samples. [Figure 1D] Shown is histological analysis (H&E staining) of PA0170 pancreatic patient-derived xenograft (PDX) tissue after 0, 1, 3, 5, and 13 days in vitro. [Figure 1E] H&E staining of human subject tumor tissues from breast (FIG. 1E), lung (FIG. 1F), and kidney (FIG. 1G) cancers is shown. [Figure 1F] H&E staining of human subject tumor tissues from breast (FIG. 1E), lung (FIG. 1F), and kidney (FIG. 1G) cancers is shown. [Figure 1G] H&E staining of human subject tumor tissues from breast (FIG. 1E), lung (FIG. 1F), and kidney (FIG. 1G) cancers is shown. [Figure 1H] Correlation between viability readings (FIG. 1H) and manually counted cell numbers (FIG. 1I) when measured in the same tumor slice is shown. [Figure 1I] Correlation between viability readings (FIG. 1H) and manually counted cell numbers (FIG. 1I) when measured in the same tumor slice is shown. [Figure 1J] Viability readings are shown from GBM tissues cultured submerged in medium or at the air-medium interface, as indicated. [Figure 2A] Shows a readout of BR0851 TNBC PDX tumor cell viability when treated with cisplatin or doxorubicin. [Figure 2B] Shows a readout of viability of BR1126 TNBC PDX tumor cells when treated with cisplatin or paclitaxel. [Figure 2C] Shows immunohistochemistry analysis of BR0851 TNBC PDX slices treated with cisplatin or alternatively doxocyclin for 6 days in vitro. [Figure 2D]Shown is a viability analysis of SN289 GBM PDX tissues treated with radiation alone, temozolomide (TMZ) plus radiation, or procarbazine, lomustine, and vincristine (PCV) plus radiation at days 0, 4, 8, and 12 of culture. [Figure 2E] Shown is a viability analysis of colon cancer PDX models treated with oxaliplatin, 5-fluorouracil (5-FU), or the combination (Ox+5FU) on days 0, 4, and 8 of culture. [Figure 3A] Viability analysis of CN0458 (FIG. 3A) and CNSTG colon PDX (FIG. 3B) tissues treated with vehicle, oxaliplatin (OXP), or 5-fluorouracil (5FU) at the indicated concentrations for the indicated times is shown. [Figure 3B] Viability analysis of CN0458 (FIG. 3A) and CNSTG colon PDX (FIG. 3B) tissues treated with vehicle, oxaliplatin (OXP), or 5-fluorouracil (5FU) at the indicated concentrations for the indicated times is shown. [Figure 3C] Shown is a viability analysis of BR11267 TNBC PDX (Figure 3A) and BR0851 TNBC PDX (Figure 3B) cells treated with vehicle or cisplatin (Cis) at the indicated concentrations for the indicated times. [Figure 3D] Shown is a viability analysis of BR11267 TNBC PDX (Figure 3A) and BR0851 TNBC PDX (Figure 3B) cells treated with vehicle or cisplatin (Cis) at the indicated concentrations for the indicated times. [Figure 4A-1] Shows survival measurements of CN0458 colon PDX tumors treated with one of 119 drugs as indicated. [Figure 4A-2] Shows survival measurements of CN0458 colon PDX tumors treated with one of 119 drugs as indicated. [Figure 4B] Viability measurements of CN0458 colon PDX tumors treated with 7 of the 119 drugs shown in Figure 4A are shown at higher magnification. [Figure 5A]Shown is the viability response of PDX tumor slices from four different patients (CN1571, CN1572, CN1473, and CN1574) to dabrafenib plus trametinib treatment. [Figure 5B] Correlation of treatment response to trametinib plus dabrafenib treatment in matched patients, PDX mouse models, and PDX tumor slices. [Figure 6] 1 shows an exemplary experimental paradigm for measuring viability from tumor tissue. [Figure 7] Exemplary growth curves and treatment responses using 18-gauge needle biopsy cores from MMTV-PyMT breast tumors and CT26 colon tumors are shown. MMTV-PyMT biopsy core slices were treated with vehicle control (DMSO), cyclophosphamide (CPP, 20 μM), doxorubicin (DOX, 100 nM), or combination (CPP (20 μM) + DOX (100 nM) + TAXOL (1 nM)). CT26 colon tumor biopsy core slices were treated with control (IgG + DMSO), IgG + 5-fluorouracil (5FU, 1 μM), anti-PD1 + DMSO, or anti-PD1 + 5FU + oxaliplatin (anti-PD1 + 5FU (1 μM) + OX (1 μM)). [Figure 8] Figure 1 shows the emergence of therapeutic resistance with long-term culture in some tumor tissues. Human mCRC biopsy cores treated with encorafenib (E, 100nM, BRAFV600E inhibitor), cetuximab (C, 10μg / ml, EGFR inhibitor), binimetinib (B, 100nM, MEK inhibitor) and the combination show the emergence of encorafenib resistance after 16 days of culture, which is suppressed by treatment with the MEK inhibitor binimetinib (compare E vs B and E+C vs E+C+B on days 16 and 20). [Figure 9] 1 shows the maintenance of immune cells in tumor slices on day 4 of culture. [Figure 10]We demonstrate the use of tumor slices to measure differential immunotherapy responses in human tumors. Needle biopsy cores from two different mCRC patients were treated with control (DMSO), encorafenib (100 nM) + cetuximab (E (100 nM) + C (10 μg / ml)), immune checkpoint inhibitor nivolumab (N, 10 μg / ml), or combination (E + C + N). See also Figure 7 for mouse mammary tumor tissue response to anti-PD1 treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Although many cytotoxic and targeted therapies show promising activity in cultured cells, most have not demonstrated comparable efficacy in patients, suggesting that the predictive power of existing cell culture models is suboptimal. Cell lines (e.g., cancer cell lines) cultured in isolation may not adequately recapitulate the complexity of human tissues (e.g., tumor tissues) at multiple levels. For example, cancer initiation and progression can be regulated by the tumor microenvironment, including immune cells (e.g., via inflammatory cytokines) and non-cancerous cells such as stromal cells. Complex model systems such as patient-derived xenografts (PDX) and other animal models are often expensive and time-consuming. Established cell lines, freshly dissociated primary cells, or organoids lack native cells, i.e., cell-cell communication and extracellular matrix, and also lack important stromal cells, including immune cells.

[0028] Disclosed herein, in some embodiments, are methods and systems for evaluating cell samples (e.g., intact cancer tissue) that recapitulate cellular heterogeneity, preserve cell:cell interactions, and are amenable to cost-effective and timely experimental manipulation. Disclosed herein, in certain embodiments, are methods and systems for evaluating cell samples as intact systems. Disclosed herein, in certain embodiments, are methods and systems for evaluating cell samples from a subject.

[0029] In some embodiments, evaluating the cell sample includes evaluating a candidate molecule (e.g., a chemical molecule, a biological molecule, etc.) provided to the cell sample for a given duration, thereby evaluating the response of the sample to the candidate molecule. In some embodiments, a candidate molecule is administered to the cell sample (e.g., tumor tissue), the cell sample is cultured in the presence of the candidate molecule for at least five days, and the response to the candidate molecule (e.g., cell viability) is evaluated. In some embodiments, the disclosed methods of evaluating the response of a cell sample to a candidate molecule are useful, for example, to ascertain the long-term effectiveness of one or more compounds in treating tumor cells from a subject. In some embodiments, the disclosed methods are useful to compare the long-term effectiveness of multiple compounds in treating cells from the same tumor sample. In some embodiments, the disclosed methods are useful to determine changes in cellular states (e.g., cancer stem cell maturation, immune cell repolarization, etc.) of a cell sample from a tumor sample. In some embodiments, the disclosed methods are useful to detect patient-specific responses to a candidate molecule or combination of candidate molecules.

[0030] In some embodiments, evaluating the cell sample includes evaluating the candidate molecule in a longitudinal manner. In some embodiments, the candidate molecule is administered to a cell sample (e.g., tumor tissue), the cell sample is cultured in the presence of the candidate molecule for at least one day, the response to the candidate molecule (e.g., cell viability) is evaluated, and the process is repeated at least once. In some embodiments, the response of the cell sample to the candidate molecule is accomplished without destroying the cell sample, thereby allowing for continuous culturing and evaluation of the cell sample. In some embodiments, the disclosed methods of evaluating the response of a cell sample to a candidate molecule are useful, for example, to determine the effectiveness of one or more compounds in treating tumor cells from a subject over time. For example, in some embodiments, the disclosed methods are useful for identifying a compound that has minimal efficacy early in treatment of a tumor sample and high efficacy later in treatment. In some embodiments, the disclosed methods are useful for comparing the efficacy of multiple compounds in treating cell samples from the same tumor sample over time.

[0031] In some embodiments, assessing the cell sample includes assessing cell viability. In some embodiments, disclosed herein is a method of assessing cell viability. In some embodiments, assessing cell viability includes culturing the cell sample in cell culture medium, isolating metabolites from the cell culture medium, and measuring the metabolites in the absence of the cell sample. In some embodiments, the disclosed methods for assessing cell viability are useful, for example, for evaluating the response of a cell sample (e.g., a tumor cell) to a candidate compound. In some embodiments, measuring metabolites from a cell sample in the absence of a cell sample is useful for obtaining measurements with less variability compared to measuring metabolites in a cell sample.

[0032] definition As used herein, the term "subject" is used to mean an animal, preferably a mammal, including a human or non-human. The terms patient, subject, and individual are used interchangeably. None of these terms are intended to be construed as requiring the supervision of a medical professional (e.g., a doctor, a nurse, a physician's assistant, a nursing assistant, a hospice worker).

[0033] The term "about," when referring to a measurable value, such as an amount, a time duration, and the like, is intended to encompass a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods. Moreover, "about" can mean plus or minus less than 1 percent, or plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 percent, or more than 30 percent, which may vary depending on the circumstances and are known or knowable to those of skill in the art. "About" also includes the exact amount. Thus, "about 10 kDa" means "about 10 kDa" and also "10 kDa."

[0034] Long-term evaluation of candidate molecules Disclosed herein, in certain embodiments, are methods for long-term evaluation of one or more candidate molecules on a cell sample. In some embodiments, the disclosed methods include administering one or more candidate compounds to a cell sample, culturing the cell sample in the presence of one or more candidate molecules for at least 5 days, and evaluating the response of the cell sample to one of the plurality of candidate molecules. In some embodiments, the cell sample comprises cells derived from a tumor tissue. In some embodiments, the cell sample is cultured for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more, before evaluating the response of the cell sample to the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 5 days before evaluating the response. In some embodiments, the cell sample is cultured for at least 6 days before evaluating the response. In some embodiments, the cell sample is cultured for at least 7 days before assessing the response. In some embodiments, the cell sample is cultured for at least 8 days before assessing the response. In some embodiments, the cell sample is cultured for at least 10 days before assessing the response. In some embodiments, the cell sample is cultured for at least 12 days before assessing the response. In some embodiments, the cell sample is cultured for at least 15 days before assessing the response. In some embodiments, the cell sample is cultured for at least 16 days before assessing the response. In some embodiments, the cell sample is cultured for at least 20 days before assessing the response.

[0035] In some embodiments, the cell sample comprises a cell. In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a tumor tissue. In some embodiments, the cell sample comprises cells from the microenvironment of the tumor tissue. Examples of cells from the microenvironment of the tumor tissue include, but are not limited to, immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, and bone marrow-derived cells. In some embodiments, the immune cells are lymphoid or myeloid cells. Examples of lymphoid cells include T cells, B cells, natural killer cells, and dendritic cells. Examples of myeloid cells include macrophages, tissue-resident monocytes (e.g., microglial cells), monocyte-derived suppressor cells, basophils, eosinophils, mast cells, neutrophils, monocytes, erythrocytes, macrophages (e.g., tissue-resident macrophages and peripherally derived macrophages), myeloid suppressor cells, and dendritic cells. In some embodiments, the cell sample is from a xenograft sample from a patient. In some embodiments, the cell sample is from a primary tissue sample (e.g., a primary tumor sample). In some embodiments, the cell sample comprises cancer cells. In some embodiments, the cell sample comprises glioblastoma cells. In some embodiments, the cell sample comprises colon cancer cells. In some embodiments, the cell sample comprises pancreatic cancer cells. In some embodiments, the cell sample comprises breast cancer cells. In some embodiments, the cell sample comprises prostate cancer cells. In some embodiments, the cell sample comprises kidney cancer cells. In some embodiments, the cell sample comprises lung cancer cells. In some embodiments, the cell sample comprises head and neck cancer cells. In some embodiments, the cell sample comprises skin cancer cells. In some embodiments, the cell sample comprises ovarian cancer cells. In some embodiments, the cell sample comprises uterine cancer cells. In some embodiments, the cell sample comprises sarcoma cells. In some embodiments, the cell sample comprises liver cancer cells. In some embodiments, the cell sample comprises gastric cancer cells.In some embodiments, the cell sample includes cancer cells that have metastasized to different organ sites (eg, breast cancer cells that have metastasized to the lungs).

[0036] In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 5 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 6 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 8 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 10 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 12 days. In some embodiments, the cell sample is cultured for at least 15 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cells are cultured in serum-free medium. In some embodiments, the cells are cultured at a liquid-air interface. In some embodiments, the cells are cultured submerged in cell culture medium. In some embodiments, the cells are cultured in a low oxygen environment (e.g., in a hypoxic chamber). In some embodiments, the cells are cultured at a low pH (e.g., acidic conditions) relative to standard culture conditions. In some embodiments, the cells are cultured at a high pH (e.g., alkaline conditions) relative to standard culture conditions. In some embodiments, the cells are cultured in conditions with high concentrations of amino acids, metabolites, and / or carbon sources relative to standard culture conditions.In some embodiments, the cells are cultured at reduced concentrations of amino acids, metabolites, and / or carbon sources relative to standard culture conditions. In some embodiments, the disclosed methods include administering a second dose of one or more candidate molecules to the cell sample. In some embodiments, the cell sample is cultured for at least 24 hours after administering the second dose of one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more after administering the second dose of one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 1 day after administering the second dose of one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 3 days after administering the second dose of the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 5 days after administering the second dose of the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 8 days after administering the second dose of the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 10 days after administering the second dose of the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 12 days after administering the second dose of the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 15 days after administering the second dose of the one or more candidate molecules. In some embodiments, the response of the cell sample to the second dose of the one or more candidate molecules is evaluated.

[0037] In some embodiments, the disclosed methods include administering one or more additional candidate molecules to the cell sample. In some embodiments, the cell sample is cultured for at least 24 hours after administering the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more after administering the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 1 day after administering the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 3 days after administering the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 5 days after administering the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 8 days after administration of the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 10 days after administration of the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 12 days after administration of the one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 15 days after administration of the one or more additional candidate molecules. In some embodiments, the response of the cell sample to the one or more additional candidate molecules is evaluated.

[0038] In some embodiments, the response of the cell sample to one or more candidate molecules is assessed (e.g., via viability analysis) and then a biological assay (e.g., nucleic acid sequencing, histological analysis, etc.) is performed on the cell sample. In some embodiments, assessing the response of the cell sample to one or more candidate molecules does not include destroying (e.g., lysing, permeabilizing, fixing, etc.) the cell sample. In some embodiments, performing a biological assay on the cell sample includes destroying (e.g., lysing, permeabilizing, fixing, etc.) the cell sample.

[0039] In some embodiments, the response of the cell sample to one or more candidate molecules is compared to the response of an additional cell sample to one or more candidate molecules. For example, in some embodiments, a candidate molecule is provided to each of two cell samples, each cell sample is cultured for at least five days, the response of each cell sample to the candidate molecule is evaluated, and the responses of each cell sample are compared. In some embodiments, the cell sample and the additional cell sample are obtained from different tumor tissues. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue at different times. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue at substantially the same time. In some embodiments, evaluating the response of the candidate molecule to two cell samples obtained from the same tumor tissue is useful in assessing heterogeneity within the tumor tissue. For example, the cell sample and the additional cell sample may be obtained from different locations of the same tumor tissue at substantially the same time, and the response from each cell sample to the candidate molecule may be compared, thereby assessing specific heterogeneity in the response to the candidate molecule within the tumor tissue. In another example, a cell sample and an additional cell sample may be obtained from the same tumor tissue at different times and the response from each cell sample to the candidate molecule may be compared, thereby evaluating specific heterogeneity in the response to the candidate molecule within the tumor tissue over time. In another example, a cell sample from tumor tissue and a cell sample from healthy tissue may be obtained from the same subject and the response from each cell sample to the candidate molecule may be evaluated.

[0040] In some embodiments, the disclosed methods include administering one or more candidate molecules to a cell sample in combination with one of a plurality of radiation doses. In some embodiments, the disclosed methods include administering one or more candidate molecules to a cell sample in combination with exposure to a tumor treatment radiation field. In some embodiments, the disclosed methods include administering one or more candidate molecules to a cell sample in combination with immunotherapy.

[0041] In some embodiments, the disclosed methods include administering one or more radiation doses to a cell sample, culturing the cell sample for at least five days, and evaluating the response of the cell sample to the one or more radiation doses. In some embodiments, the cell sample is exposed to one or more radiation doses at least once, at least twice, at least three times, at least four times, at least five times, or more, before evaluating the response. In some embodiments, the disclosed methods include exposing the cell sample to a tumor treatment field, culturing the cell sample for at least five days, and evaluating the response of the cell sample to the tumor treatment field.

[0042] Longitudinal evaluation of candidate molecules Disclosed herein, in some embodiments, is a method for long-term evaluation of one or more candidate molecules on a cell sample. In some embodiments, the disclosed method includes administering one or more candidate molecules to the cell sample, culturing the cell sample, evaluating the response of the cell sample to the one or more candidate molecules, and repeating the administering, culturing, and evaluating steps with a second dose of the same one or more candidate molecules or one or more additional candidate molecules. In some embodiments, the cell sample is cultured for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 24 hours, or more before evaluating the response of the cell sample to the one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 12 hours before evaluating the response. In some embodiments, the cell sample is cultured for at least 24 hours before evaluating the response.

[0043] In some embodiments, the cell sample is cultured for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more, before assessing the response of the cell sample to one or more candidate molecules. In some embodiments, the cell sample is cultured for at least 1 day before assessing the response. In some embodiments, the cell sample is cultured for at least 2 days before assessing the response. In some embodiments, the cell sample is cultured for at least 3 days before assessing the response. In some embodiments, the cell sample is cultured for at least 4 days before assessing the response. In some embodiments, the cell sample is cultured for at least 5 days before assessing the response. In some embodiments, the cell sample is cultured for at least 10 days before assessing the response. In some embodiments, the cell sample is cultured for at least 12 days before assessing the response. In some embodiments, the cell sample is cultured for at least 16 days before assessing the response. In some embodiments, the cell sample is cultured for at least 20 days before assessing the response.

[0044] In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response of the cell sample to the one or more candidate molecules are repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more. In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response are repeated at least once. In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response are repeated at least twice. In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response are repeated at least three times. In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response are repeated at least four times. In some embodiments, the steps of administering one or more candidate molecules to the cell sample, culturing the cell sample, and evaluating the response are repeated at least five times. In some embodiments, the administering, culturing, and evaluating steps are repeated with additional doses of the same one or more candidate molecules, e.g., to evaluate the long-term effect of the one or more candidate molecules on the cell sample (e.g., a tumor tissue sample). In some embodiments, the administering, culturing, and evaluating steps are repeated with one or more different candidate molecules, e.g., to evaluate the effect of multiple different candidate molecules.

[0045] In some embodiments, the cell sample comprises a cell. In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a tumor tissue. In some embodiments, the cell sample comprises cells from the microenvironment of the tumor tissue. Examples of cells from the microenvironment of the tumor tissue include, but are not limited to, immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, and bone marrow-derived cells. In some embodiments, the immune cells are lymphoid or myeloid cells. Examples of lymphoid cells include T cells, B cells, natural killer cells, and dendritic cells. Examples of myeloid cells include macrophages, tissue-resident monocytes (e.g., microglial cells), monocyte-derived suppressor cells, basophils, eosinophils, mast cells, neutrophils, monocytes, erythrocytes, and dendritic cells. In some embodiments, the cell sample is derived from a xenograft sample from a patient. In some embodiments, the cell sample is derived from a primary tissue sample (e.g., a primary tumor sample). In some embodiments, the cell sample comprises cancer cells. In some embodiments, the cell sample comprises glioblastoma cells. In some embodiments, the cell sample comprises colon cancer cells. In some embodiments, the cell sample comprises pancreatic cancer cells. In some embodiments, the cell sample comprises breast cancer cells. In some embodiments, the cell sample comprises prostate cancer cells. In some embodiments, the cell sample comprises kidney cancer cells. In some embodiments, the cell sample comprises lung cancer cells. In some embodiments, the cell sample comprises head and neck cancer cells. In some embodiments, the cell sample comprises skin cancer cells. In some embodiments, the cell sample comprises ovarian cancer cells. In some embodiments, the cell sample comprises uterine cancer cells. In some embodiments, the cell sample comprises sarcoma cells. In some embodiments, the cell sample comprises liver cancer cells. In some embodiments, the cell sample comprises gastric cancer cells.In some embodiments, the cell sample includes cancer cells that have metastasized to different organ sites (eg, breast cancer cells that have metastasized to the lungs).

[0046] In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 5 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 6 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 8 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 10 days. In some embodiments, the cell sample is cultured under conditions sufficient to allow for continued viability of the cell sample for at least 12 days. In some embodiments, the cell sample is cultured for at least 20 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured in serum-free medium. In some embodiments, the cell sample is cultured in the presence of cytokines or growth factors. In some embodiments, the cell sample is cultured at a liquid-air interface. In some embodiments, the cell sample is cultured submerged in cell sample culture medium. In some embodiments, the cell sample is cultured in a low oxygen environment (e.g., in a hypoxic chamber). In some embodiments, the cell sample is cultured at a low pH (e.g., acidic conditions) relative to standard culture conditions. In some embodiments, the cell sample is cultured at a high pH (e.g., alkaline conditions) relative to standard culture conditions. In some embodiments, the cell sample is cultured in conditions with high concentrations of amino acids, metabolites, and / or carbon sources relative to standard culture conditions.In some embodiments, the cell sample is cultured in conditions that provide reduced concentrations of amino acids, metabolites, and / or carbon sources relative to standard culture conditions.

[0047] In some embodiments, the response of a cell sample to one or more candidate molecules is evaluated (via viability analysis), and then a biological assay (e.g., nucleic acid sequencing, histological analysis, etc.) is performed on the cell sample. In some embodiments, a biological assay is performed to evaluate one or more cell characteristics, such as cell morphology, genetic abnormalities, gene expression, protein expression, epigenetic characteristics, protein modifications, immunophenotype, metabolite expression, and / or histological characteristics. In some embodiments, the biological assay is an assay for evaluating cell morphology (e.g., light microscopy, electron microscopy, flow cytometry, etc.). In some embodiments, the biological assay is an assay for evaluating genetic abnormalities (e.g., karyotyping, DNA sequencing, fluorescent in situ hybridization (FISH), etc.). In some embodiments, the biological assay is an assay for evaluating gene expression (e.g., RNA-sequencing, microarray analysis, Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), etc.). In some embodiments, the biological assay is an assay for assessing protein expression (e.g., immunoblotting, mass spectrometry, flow cytometry, high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunofluorescence, immunohistochemistry, etc.). In some embodiments, the biological assay is an assay for assessing post-translational modifications of proteins (e.g., immunoblotting, mass spectrometry, ELISA, immunofluorescence, immunohistochemistry, etc.). In some embodiments, the biological assay is an assay for assessing epigenetic signatures (e.g., ATAC-sequencing, DNase-sequencing, MNase-sequencing, bisulfite sequencing, chromatin immunoprecipitation (ChIP), etc.). In some embodiments, the biological assay is an assay for assessing protein modifications (e.g., immunoblotting, mass spectrometry, etc.). In some embodiments, the biological assay is an assay for assessing metabolite expression (e.g., mass spectrometry, HPLC, NMR, etc.).In some embodiments, the biological assay is an assay for evaluating secreted proteins (e.g., mass spectrometry, cytokine arrays, growth factor arrays, ELISA, bead-based assays, etc.). In some embodiments, the biological assay is an assay for evaluating histological characteristics. In some embodiments, evaluating the response of a cell sample to one or more candidate molecules does not include disrupting (e.g., lysing, permeabilizing, fixing, etc.) the cell sample. In some embodiments, the biological assay is an assay for evaluating changes in immune cell composition, activity state, or function. In some embodiments, performing a biological assay on a cell sample includes disrupting (e.g., lysing, permeabilizing, fixing, etc.) the cell sample.

[0048] In some embodiments, the response of the cell sample to one or more candidate molecules is compared to the response of an additional cell sample to one or more candidate molecules. For example, in some embodiments, a candidate molecule is provided to each of two cell samples, each cell sample is cultured in the presence of the candidate molecule, the response of each cell sample to the candidate molecule is evaluated, the process is repeated at least once, and the longitudinal response of each cell sample to the candidate sample is compared. In some embodiments, the cell sample and the additional cell sample are obtained from different tumor tissues. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue at different times. In some embodiments, the cell sample and the additional cell sample are obtained from the same tumor tissue at substantially the same time. In some embodiments, evaluating the response of a candidate molecule to two cell samples obtained from the same tumor tissue is useful in assessing heterogeneity within the tumor tissue. For example, the cell sample and the additional cell sample may be obtained from different locations of the same tumor tissue at substantially the same time, and the response to the candidate molecule may be compared for each cell sample, thereby evaluating the specific heterogeneity in the response to the candidate molecule within the tumor tissue. In another example, the cell sample and the additional cell sample may be obtained from the same tumor tissue at different times, and the response to the candidate molecule may be compared for each cell sample, thereby evaluating the specific heterogeneity in the response to the candidate molecule within the tumor tissue over time. In another example, a cell sample from tumor tissue and a cell sample from healthy tissue may be obtained from the same subject, and the response from each cell sample to the candidate molecule may be evaluated.

[0049] In some embodiments, the disclosed methods include administering one or more candidate molecules to a cell sample in combination with one of a plurality of radiation doses, hi some embodiments, the disclosed methods include administering one or more candidate molecules to a cell sample in combination with exposure to a tumor treatment radiation field.

[0050] In some embodiments, the disclosed methods include administering a radiation dose to a cell sample, culturing the cell sample, evaluating the response of the cell sample to one of a plurality of radiation doses, and repeating the administering, culturing, and evaluating steps, for example, with a second radiation dose. In some embodiments, the cell sample is exposed to one or more radiation doses at least once, at least twice, at least three times, at least four times, at least five times, or more, before evaluating the response. In some embodiments, the disclosed methods include exposing the cell sample to a tumor treatment field, culturing the cell sample, evaluating the response of the cell sample to the tumor treatment field, and repeating the administering, culturing, and evaluating steps, for example, with the same tumor treatment field or a different tumor treatment field.

[0051] Evaluation of responses In some embodiments, the disclosed methods include evaluating the response of a cell sample (e.g., cells) to one or more candidate molecules. In some embodiments, evaluating the response of a cell sample to one or more candidate molecules does not include disrupting the cell sample (e.g., lysing, permeabilizing, fixing, etc.). In some embodiments, the response is a change in one or more cellular conditions in response to exposure to one or more candidate molecules. In some embodiments, the response is, for example, a viability response, a gene expression response, a protein expression response, a protein modification response, a cell signaling response, a morphological response, or a metabolic response. In some embodiments, the response is a viability response. In some embodiments, the response is a change in cell viability in response to exposure to one or more candidate molecules.

[0052] In some embodiments, evaluating the response of the cell sample to the one or more candidate molecules comprises measuring gene expression of the cell sample exposed to the one or more candidate molecules. Methods for measuring gene expression include, but are not limited to, nucleic acid sequencing and microarray analysis. In some embodiments, evaluating the response of the cell sample to the one or more candidate molecules comprises measuring protein expression of the cell sample exposed to the one or more candidate molecules. Methods for measuring protein expression include, but are not limited to, mass spectrometry, Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, mass cytometry, and enzyme-linked immunosorbent assay (ELISA). In some embodiments, evaluating the response of the cell sample to the one or more candidate molecules comprises measuring protein modification of the cell sample exposed to the one or more candidate molecules. Methods for measuring protein modification include, but are not limited to, mass spectrometry, Western blotting, and ELISA. In some embodiments, evaluating the response of the cell sample to the one or more candidate molecules comprises measuring cellular signaling of the cell sample exposed to the one or more candidate molecules. Methods for measuring cell signaling include, but are not limited to, mass spectrometry, Western blotting, nucleic acid sequencing, and microarray analysis. In some embodiments, evaluating the response of a cell sample to one or more candidate molecules includes measuring morphological changes in a cell sample exposed to one or more candidate molecules. Methods for measuring morphological changes include, but are not limited to, optical microscopy and electron microscopy. In some embodiments, evaluating the response of a cell sample to one or more candidate molecules includes measuring metabolite expression in a cell sample exposed to one or more candidate molecules. Methods for measuring protein modifications include, but are not limited to, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR).

[0053] In some embodiments, evaluating the response of the cell sample to the one or more candidate molecules includes measuring the viability of cells of the cell sample exposed to the one or more candidate molecules. For example, evaluating the response of the cell sample to the one or more candidate molecules may include measuring the viability of cells of the cell sample after culturing the cell sample for a period of time in the presence of the one or more candidate molecules. In some embodiments, measuring the viability of cells of the cell sample includes measuring a metabolite from the cell sample. In some embodiments, measuring the metabolite includes exciting the metabolite and measuring the resulting fluorescence (e.g., measuring the fluorescence intensity and wavelength). In some embodiments, the metabolite is measured while inside the cell sample. In some embodiments, the metabolite from the cell sample is isolated from the cell culture medium and measured in the absence of the cell sample. In some embodiments, the metabolite from the cell sample is the result of a reduction reaction. In some embodiments, the metabolite from the cell sample is derived from a molecule provided to the cell sample. In some embodiments, the metabolite from the cell sample is derived from resazurin. In some embodiments, the metabolite is resorufin. In some embodiments, the metabolite from the cell sample is derived from tetrazole. Examples of tetrazole include 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or its salt, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or its salt, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or its salt. In some embodiments, the metabolite from the cell sample is formazan.

[0054] Measurement of cell viability Disclosed herein, in some embodiments, are methods of measuring cell viability. In some embodiments, the disclosed methods include measuring cell viability in the absence of a cell sample. In some embodiments, the disclosed methods include culturing a cell sample (e.g., cells) in a cell culture medium, isolating a metabolite from the cell culture medium, and measuring the metabolite in the absence of a cell sample. In some embodiments, measuring the metabolite includes exciting the metabolite and measuring the resulting fluorescence (e.g., measuring fluorescence intensity and wavelength).

[0055] In some embodiments, the cell sample comprises a cell. In some embodiments, the cell sample comprises a plurality of cells. In some embodiments, the cell sample is from a cell line. In some embodiments, the cell sample is an intact tissue. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is isolated from a tumor tissue. In some embodiments, the cell sample comprises cells from the microenvironment of the tumor tissue. Examples of cells from the microenvironment of the tumor tissue include, but are not limited to, immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, and bone marrow-derived cells. In some embodiments, the immune cells are lymphoid or myeloid cells. Examples of lymphoid cells include T cells, B cells, natural killer cells, and dendritic cells. Examples of myeloid cells include macrophages, tissue-resident monocytes (e.g., microglial cells), monocyte-derived suppressor cells, basophils, eosinophils, mast cells, neutrophils, monocytes, erythrocytes, and dendritic cells. In some embodiments, the cell sample is from a xenograft sample from a patient. In some embodiments, the cell sample is from a primary tissue sample (e.g., a primary tumor sample). In some embodiments, the cell sample comprises cancer cells. In some embodiments, the cell sample comprises glioblastoma cells. In some embodiments, the cell sample comprises colon cancer cells. In some embodiments, the cell sample comprises pancreatic cancer cells. In some embodiments, the cell sample comprises breast cancer cells. In some embodiments, the cell sample comprises prostate cancer cells. In some embodiments, the cell sample comprises kidney cancer cells. In some embodiments, the cell sample comprises lung cancer cells. In some embodiments, the cell sample comprises head and neck cancer cells. In some embodiments, the cell sample comprises skin cancer cells. In some embodiments, the cell sample comprises ovarian cancer cells. In some embodiments, the cell sample comprises uterine cancer cells. In some embodiments, the cell sample comprises sarcoma cells. In some embodiments, the cell sample comprises liver cancer cells. In some embodiments, the cell sample comprises gastric cancer cells.In some embodiments, the cell sample comprises cancer cells that have metastasized to a different organ site (e.g., breast cancer cells that have metastasized to the lung). In some embodiments, the cell sample is cultured for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 25 days, at least 30 days, or more, under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 5 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 6 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 8 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 10 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 12 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured for at least 15 days under conditions sufficient to allow continued viability of the cell sample. In some embodiments, the cell sample is cultured in serum-free medium. In some embodiments, the cell sample is cultured at a liquid-air interface. In some embodiments, the cell sample is cultured submerged in cell culture medium.

[0056] In some embodiments, the metabolites from the cell sample are derived from molecules provided to the cell sample. In some embodiments, the molecules are provided to the cell sample for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 24 hours, or more, before measuring the metabolites from the cell sample. In some embodiments, the molecules are provided to the cell sample for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, about 16 hours, about 24 hours, before measuring the metabolites from the cell sample. In some embodiments, the molecules are provided to the cell sample for about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, before measuring the metabolites from the cell sample. In some embodiments, the molecules are provided to the cell sample for about 12 hours, before measuring the metabolites from the cell sample. In some embodiments, the molecule is provided to the cell sample for about 13 hours prior to measuring a metabolic product from the cell sample. In some embodiments, the molecule is provided to the cell sample for about 14 hours prior to measuring a metabolic product from the cell sample. In some embodiments, the molecule is provided to the cell sample for about 15 hours prior to measuring a metabolic product from the cell sample. In some embodiments, the molecule is provided to the cell sample for about 16 hours prior to measuring a metabolic product from the cell sample.

[0057] In some embodiments, the metabolite from the cell sample is the result of a reduction reaction. In some embodiments, the metabolite from the cell sample is derived from resazurin. In some embodiments, the metabolite is resorufin. In some embodiments, the metabolite from the cell sample is derived from tetrazole. Examples of tetrazoles include 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof. In some embodiments, the metabolite from the cell sample is formazan.

[0058] In some embodiments, measuring metabolites in the absence of a cell sample results in reduced measurement variability compared to measuring metabolites in the presence of a cell sample (while inside the cell sample). For example, in some embodiments, measurements of multiple metabolites from multiple cells from the same cell sample have less variability when measured in the absence of a cell sample (e.g., isolated from cell culture medium) than when measured in the presence of a cell sample (intracellular).

[0059] Cell samples In some embodiments, the disclosed methods include the use of one or more cell samples. In some embodiments, the cell sample includes one or more cells. In some embodiments, the cell sample is a cell. In some embodiments, the cell includes a cancer cell. In some embodiments, the cell sample includes a cell from a tumor microenvironment. Examples of cells from the tumor tissue microenvironment include, but are not limited to, immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, and bone marrow-derived cells. In some embodiments, the immune cells are lymphoid or myeloid cells. Examples of lymphoid cells include T cells, B cells, natural killer cells, and dendritic cells. Examples of myeloid cells include macrophages, tissue-resident monocytes (e.g., microglial cells), monocyte-derived suppressor cells, basophils, eosinophils, mast cells, neutrophils, monocytes, erythrocytes, and dendritic cells. In some embodiments, the cell sample is a tissue sample. In some embodiments, the cell sample is derived from tumor tissue. In some embodiments, the cell sample is derived from a subject (e.g., tissue from the subject). In some embodiments, the cell sample is derived from a cell line. In some embodiments, the cell sample is an organoid. In some embodiments, the cell sample is derived from an organoid. In some embodiments, the cell sample is derived from a primary tissue (e.g., primary tumor tissue). In some embodiments, the cell sample is derived from a patient-derived xenograft (PDX). In some embodiments, the use of a cell sample derived from a patient (e.g., from a primary tumor tissue or a patient-derived xenograft) allows for detection of a patient-specific response to a candidate molecule. In some embodiments, the cell sample comprises a single type of cell (e.g., cancer cells). In some embodiments, the cell sample comprises multiple types of cells (e.g., cancer cells and cells from the tumor microenvironment). In some embodiments, the cell sample is a primary tumor sample. In some embodiments, the cell sample is a method according to claim 1, wherein the cell sample is from a metastatic tumor sample.

[0060] In some embodiments, the primary tumor sample is a surgical sample from a subject. In some embodiments, the primary tumor sample is derived from a patient biopsy (e.g., removal of tissue from any part of a patient's body, such as a core biopsy, an excision biopsy, an incision biopsy, or a punch biopsy). In some embodiments, the biopsy is a core needle biopsy (e.g., a biopsy in which a hollow needle is inserted into a tumor to remove a tissue sample from the tumor). In some embodiments, the needle for obtaining the core biopsy is a 14-gauge, 15-gauge, 16-gauge, 17-gauge, 18-gauge, 19-gauge, 20-gauge, 21-gauge, or 22-gauge needle. In some embodiments, the needle is an 18-gauge needle.

[0061] In some embodiments, multiple cell samples are obtained via core needle biopsy from different anatomical locations on the same tumor. In some embodiments, multiple samples are obtained at the same time point. In some embodiments, the samples are obtained at different time points. For example, in some embodiments, the disclosed methods are useful for identifying tumor samples that have minimal efficacy early in treatment and high efficacy later in treatment.

[0062] Candidate molecules In some embodiments, the disclosed methods include evaluating one or more candidate molecules. In some embodiments, the candidate molecule is a therapeutic agent. In some embodiments, the therapeutic agent is a cancer therapeutic agent. In some embodiments, the candidate molecule is a compound that is suspected to be a therapeutic agent. In some embodiments, the candidate molecule is not a therapeutic agent. In some embodiments, the candidate molecule is a molecule that is suspected to be capable of eliciting a response from a cell sample.

[0063] In some embodiments, the candidate molecule is a chemical molecule. In some embodiments, the candidate molecule is a small molecule. In some embodiments, the small molecule is at most 900 daltons, at most 800 daltons, at most 700 daltons, at most 600 daltons, at most 500 daltons, at most 400 daltons, at most 300 daltons, at most 200 daltons, at most 100 daltons, or smaller in size. In some embodiments, the candidate molecule is a biological molecule. Examples of biological molecules include, but are not limited to, recombinant proteins, enzymes, antibodies, engineered antibodies, engineered antibody-drug chimeras, growth factors, cytokines, chemokines, receptors, and any portion thereof.

[0064] Biological assays In some embodiments, the disclosed methods include performing one or more biological assays on the cell sample. In some embodiments, the biological assays are performed on the cell sample after evaluating the response of the cell sample to one or more candidate molecules. For example, the candidate molecules are provided to the cell sample, the cell sample is cultured for at least five days in the presence of the candidate molecules, the viability response of the cell sample is evaluated, and the biological assay is subsequently performed. In some embodiments, performing the biological assay on the cell sample includes disrupting (e.g., lysing, permeabilizing, fixing, etc.) the cells of the cell sample. In some embodiments, the biological assays are performed on the cell sample after the long-term evaluation of the candidate molecules. In some embodiments, the biological assays are performed on the cell sample after the long-term evaluation of the candidate molecules.

[0065] In some embodiments, the biological assay comprises fixing the cells. In some embodiments, the biological assay comprises permeabilizing the cells. In some embodiments, the biological assay comprises lysing the cells. In some embodiments, the biological assay is performed to assess one or more cellular characteristics, for example, cell morphology, genetic abnormalities, gene expression, protein expression, epigenetic characteristics, protein modifications, metabolite expression, and / or histological characteristics. In some embodiments, the biological assay is an assay for assessing cell morphology (e.g., light microscopy, electron microscopy, flow cytometry, etc.). In some embodiments, the biological assay is an assay for assessing genetic abnormalities (e.g., karyotyping, DNA sequencing, fluorescent in situ hybridization (FISH), etc.). In some embodiments, the biological assay is an assay for assessing gene expression (e.g., RNA-sequencing, microarray analysis, Northern blotting, reverse transcription polymerase chain reaction (RT-PCR), etc.). In some embodiments, the biological assay is an assay for assessing protein expression (e.g., immunoblotting, mass spectrometry, flow cytometry, high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunofluorescence, immunohistochemistry, etc.). In some embodiments, the biological assay is an assay for assessing epigenetic characteristics (e.g., ATAC-sequencing, DNase-sequencing, MNase-sequencing, bisulfite sequencing, chromatin immunoprecipitation (ChIP), etc.). In some embodiments, the biological assay is an assay for assessing protein modifications (e.g., immunoblotting, mass spectrometry, etc.). In some embodiments, the biological assay is an assay for assessing metabolite expression (e.g., mass spectrometry, HPLC, etc.). In some embodiments, the biological assay is an assay for assessing histological features. In some embodiments, the biological assay is an assay for assessing secreted molecules. EXAMPLES

[0066] Example 1: Long-term culture and longitudinal monitoring of PDX tumor slices We developed a slice culture system that allows for the continued growth of intact tumor tissue ex vivo for more than 20 days. Four parameters were tested in the development of optimal culture conditions, including five culture media conditions, four different viability assays, three different culture conditions, and five different tumor sizes. These parameters and final culture conditions are shown in Table 1.

[0067] [Table 1]

[0068] Culture conditions consisted of generating tumor slices of defined size (3 mm diameter, 250–350 μM thickness) cultured at the liquid-air interface in serum-free culture medium.

[0069] Under these conditions, tumor tissue originating from multiple organ sites (eg, brain, breast, colon, pancreas) was maintained with continuous growth ex vivo for up to 16 days.

[0070] The growth of each sample was measured longitudinally using a fluorescent metabolic viability assay, CellTiter-Blue (CTB), allowing multiple real-time measurements from the same tissue before and after different perturbations. Figures 1A-1C show viability measurements at 0, 4, 8, and 12 days (div) in vitro for breast (Figure 1A), pancreatic (Figure 1B), and glioblastoma (Figure 1C) tumor samples. These results demonstrate the viability and continued growth of cultured tissues under these conditions. Figure 1D shows histological analysis (H&E staining) of PA0170 pancreatic cells after 0, 1, 3, 5, and 13 days in vitro. Figures 1E-1G show H&E staining of human patient tumors from breast (Figure 1E), lung (Figure 1F), and kidney (Figure 1G) after the indicated days in vitro. These results show that tumor tissues from different organ sites maintain their original histological characteristics even after 13 days of culture.

[0071] To verify that the viability measurements accurately reflected changes in tumor cell numbers, the viability readings were compared to actual cell counts from the same tissue. Viability was measured using multiple different PDX models. Viable cells were manually counted on the same tissue immediately after taking the viability reading. The tissue was dissociated and viable cells were counted from each well using a hemocytometer by Trypan blue exclusion. Figure 1H and Figure 1I show the correlation between cell counts and viability readings in SN211 GBM PDX tissues. Figure 1J shows viability measurements from GBM tissues cultured submerged (left) or at the air-medium interface (right). Measurements were taken on days 1, 7, or 14, as indicated. These data show a significant improvement in viability and proliferation for tissues cultured at the air-medium interface compared to tissues cultured submerged.

[0072] Materials and Methods Patient-derived xenografts Early passage (P0-P1) patient-derived xenografts (PDXs) were obtained from Jax-West and expanded in 2-3 month old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ; strain: 005557), NSG-Hprt mice (NOD.Cg-Prkdcscid Hprtem1Mvw Il2rgtm1Wjl / MvwJ; strain: 026222), or NGS-EGFP mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CAG-EGFP)1Osb / SzJ; strain: 021937). They tested two triple-negative breast cancer (TNBC) samples, five glioblastoma multiforme (GBM) samples, three pancreatic cancer samples, and three colon cancer samples. GBM and TNBC models were grown orthotopically by injection into the brain or fourth breast fat pad, respectively. Pancreatic and colon tumor models were grown by subcutaneous injection.

[0073] Tumor tissue harvesting and organotypic slice culture Tumor 500-1000mm 3 Tumor tissues were harvested when the tumor tissue reached a median diameter of 1 mm. Samples were embedded in 4% low melting point agarose (Bio-Rad®) in KREB buffer or phosphate-buffered saline (PBS) and precisely cut into slices 250-350 μM thick using a Vibratome VT1200S (Leica Microsystems). Slicing speed and size were adjusted according to the density and integrity of the tumor tissue. Tissues with diameters of 1 mm, 2 mm, or 3 mm were generated from tumor-containing areas using a biopsy needle, avoiding necrotic regions. Each slice punch was cultured on a thin porous membrane (Nucleopore™ Track-Etched-Whatman®) in 12- or 48-well culture dishes using 1 ml or 0.2 ml of serum-free slice culture medium (Slice Medium: DMEM / F-12 (Hyclone®), B27 supplement (Gibco®), 100 U / mL penicillin (Gibco®), and 100 μg / mL streptomycin (Gibco®).

[0074] Viability assays and histological analysis 5 μl of Cell-Titer Blue (CTB) (Promega®) substrate was added to 1 ml of culture medium and incubated at 37°C for 15 or 24 hours. 100 μl of medium was removed from each sample culture and fluorescence was measured (Ex560 / Em590) using an Enspire® multimode plate reader (Perkin Elmer®). Background fluorescence of the culture medium was subtracted from each sample reading every day. Medium was changed immediately after CTB reading and drugs were added to the new medium to continue the culture. At desired time points, additional viability measurements were performed to collect longitudinal data and conditioned medium was collected and analyzed. After the final time point, tissues were fixed in formalin and embedded in paraffin or OCT for histological analysis or dissolved in RIPA buffer or Trizol and conditioned medium was frozen. To account for variability in the tissue composition of each slice punch, viability readings for each sample were normalized to the CTB reading immediately after slicing (day 0 ex vivo: 1 dex CTB reading), and the relative fold change in CTB measurements at each time point was analyzed.

[0075] Example 2: Using long-term cultures of PDX slices to test drug responses Thirteen drugs were tested alone or in combination with radiation in 12 different PDX models from brain, breast, colon, and pancreas (Table 2). Combinations tested included procarbazine, lomustine, and vincristine ("PCV") with 5FU, leucovorin, irinotecan, and OXP ("FLCO").

[0076] [Table 2]

[0077] Figures 2A and 2B show examples from two triple-negative breast cancer (TNBC) PDXs depicting observed responses. Two separate TNBC PDX slices were treated with standard care chemotherapy, cisplatin (Cis), doxorubicin (Dox), or paclitaxel (Taxol). Results were observed via viability measurements as described in Example 1. Viability assay readouts indicate that BR0851 TNBC PDX tumors are insensitive to cisplatin treatment but sensitive to Dox treatment, as demonstrated by elevated viability readouts in vehicle and cisplatin-treated slices and decreased viability readouts in Dox-treated slices at 12 days ex vivo ("div") compared to 0 div (Figure 2A). On the other hand, BR1126 TNBC PDX tumors are sensitive to both Cis and Taxol treatment (Figure 2B).

[0078] Immunohistochemistry of BR0851 TNBC PDX slices at 13 div showed that total tissue counts were similar in vehicle and Cis-treated tissues, but that Dox-treated tissues had an overall decrease in cell counts, increased nuclear pyknosis and abnormalities, and increased areas of necrosis compared to vehicle and cisplatin-treated tissues (Figure 2C). Thus, the immunochemistry analysis was consistent with the CTB readout, i.e., Dox treatment resulted in complete remission by 13 div. In contrast, cisplatin-treated tissues continued to grow and exhibit a progressive disease profile, even after prolonged exposure to the drug.

[0079] Using a PDX GBM model (SN289), the two treatment regimens were compared side-by-side on the same tumor. Multiple slice punches were generated from a single PDX tumor and exposed to the different treatments in multiple replicates. Radiation therapy (IR) by itself inhibited growth initially but not in long-term culture, while combining TMZ with radiation resulted in a decline in viability from 4 to 13 div (Figure 2D). PCV also showed a similar response when combined with radiation, resulting in a stable disease profile up to 13 div (Figure 2D).

[0080] The current standard of care for metastatic colorectal cancer (mCRC) includes oxaliplatin and 5-fluorouracil (5-FU) treatment. Figure 2E shows viability measurements observed from cells treated with oxaliplatin (0.1 μM, 1 μM, and 10 μM, as shown from left to right), 5-FU (1 μM, 10 μM, and 100 μM, as shown from left to right), or in combination for the indicated days in vitro (div), as described in Example 1. The data demonstrate that the response of mCRC to oxaliplatin is dose and time dependent. In contrast, 5-FU treatment shows no decrease in cell viability, even at the highest dose. These results indicate that such tissue culture and response measurements can be used to eliminate ineffective chemotherapies from combination treatments in individual patients.

[0081] Materials and Methods Histological analysis and immunohistochemistry Formalin-fixed, paraffin- or OCT-embedded tissue sections were stained with hematoxylin and eosin (H&E) for histological evaluation. Specific markers of proliferation (KI67: Abcam®, ab15580, 1:300) and apoptosis (cleaved caspase 3: Cell Signaling®, #9661, 1:300) were analyzed using standard immunohistochemistry analysis protocols. In addition, the human origin of PDX tumors was confirmed by using a human-specific antibody (HuNu: EMD Millipore®, MAB1281, (clone 235-1), 1:300).

[0082] Example 3: Ex vivo slice cultures to predict inter-model and inter-patient differences in drug sensitivity Inter- and intratumor heterogeneity plays a crucial role in drug response and the emergence of drug resistance. Just as different patients respond differently to the same drug, different PDX models are expected to show differential sensitivity to the same drug. To test whether the slice culture system can report differential drug sensitivity of different PDX models, we compared the treatment response of separate tumors of the same clinical type. For example, two different colon PDX tumors were treated with standard of care agents oxaliplatin (OXP) and 5-fluorouracil (5FU), and both colon tumor models showed different responses to OXP and 5FU (Figure 3A and Figure 3B). Based on the viability readout, 100uM of OXP was more effective in suppressing the viability of CNSTG colon tumor slices compared to CN0458 colon tumor slices, while 100uM of 5FU was more effective in suppressing the viability of CN0458 tumor slices compared to CNSTG colon tumor slices (Figure 3A and Figure 3B).

[0083] To determine whether differences in ex vivo drug response are consistent with in vivo drug sensitivity, we compared the response of two different TNBC PDX models for which in vivo drug response data exist (BR1126 and BR0851, JAX PDX Resource). Ex vivo cisplatin treatment resulted in a partial response in BR1126 TNBC PDX slices but not in BR0851 TNBC PDX slices (Figures 3C and 3D). This pattern was similar for in vivo treatment, with BR1126 showing a partial response while BR0851 showed no response (Figures 3A and 3B). This correlation was evident, even though treatment doses and scheduling differed significantly between the in vivo and ex vivo studies. These results suggest that the ex vivo slice system can be used to predict relative in vivo sensitivity in a cost- and time-efficient manner.

[0084] Example 4: Genomics-informed, unbiased pharmacological screening using tumor slices Although there are over 100 FDA approved tumor drugs, the number of drugs used as standard of care for the treatment of a particular tumor type in a clinical setting is only a small fraction of all available drugs. To perform an unbiased drug efficacy screen with limited amounts of tumor tissue from individual patients, a high content drug screen was performed with a modification of the ex vivo slice culture system described herein. Approved tumor drug set VI, which contains 119 FDA approved tumor drugs, was obtained from the National Cancer Institute (NCI). PDX tumor slices were obtained as described in Example 1, and tumor slice punches with a diameter of 2 mm were seeded in 96 well plates and submerged in 200 ul of TSC medium. On days 1 and 3, viability was measured using a long-term Cell-Titer Blue (CTB) viability assay as described in Example 1, and on day 1, the medium was replaced after CTB measurement. Drugs were added at a final concentration of 100 μM on day 1. Viability readings for samples on day 3 (i.e., 2 days of drug exposure) were normalized to the day 1 viability readings for each well and expressed as the fold change on day 3 compared to the day 1 readings.

[0085] 2 mm punches were generated from two CN0458 colon PDX tumors so that each of the 119 FDA-approved drugs from the NCI drug panel could be tested in triplicate. Cells were cultured for 3 days. Baseline viability of each slice was assessed by performing a CTB reading immediately after setting up the cultures (0 div). Each of the 119 drugs was added to replicate wells after the baseline CTB reading. CTB readings were performed after day 1 of drug treatment (1 div) and again after day 3 of drug treatment (3 div) to determine early response. The same experiment was performed twice to test the reproducibility of the procedure and highly consistent results were observed. Figure 4A shows the average (n=6 total) of CTB measurements from these two experiments for each drug at 3 div, normalized to the baseline reading. Seven drugs reproducibly suppressed viability of >50% of the starting tissue. Two of these seven drugs have inhibitory activity against the same protein, ALK. None of the seven drugs are currently standard of care for mCRC patients, suggesting that this method may be used to repurpose FDA-approved oncology drugs. Figure 4B shows the mean CTB measurements for these seven drugs at higher magnification, normalized to baseline readings.

[0086] Example 5: Tumor slices accurately predict patient response To validate the clinical utility of the tumor slice system, tissue samples from mCRC PDX models generated from four patients (CN1571, CN1572, CN1573, and CN1574) enrolled in the clinical trial were obtained, cultured, and evaluated using the methods described in Example 1. Figure 5A shows the viability analysis of tissue samples in response to the combination treatment of dabrafenib (Dab) plus trametinib (Tra) or DMSO control at the indicated days ex vivo (dev). Tumor slice responses were compared to previously published data on patient response and to the response of in vivo PDX models from the same four patients using RECIST criteria with modifications outlined in Table 3, showing the correlation of tumor slice responses. The results of this comparison are shown in Figure 5B. Tumor slice responses were consistent with the corresponding PDX and patient responses.

[0087] [Table 3]

[0088] Example 6: Variability in response in cells from different locations of tumor samples Tumor tissue samples from two locations of the tumor of the same patient were obtained and cultured as described in Example 1. Each sample was treated with cisplatin (Cis), doxorubicin (Dox), or paclitaxel (Taxol). Viability measurements were obtained on days 0, 3, 5, 9, and 12 in vitro. Significant differences between the viability measurements of each of the two samples were observed, demonstrating heterogeneity in drug response within the tumor tissue.

[0089] Example 7: Tumor slices can be established and examined from biopsy needle cores. Tumor tissue specimens from spontaneously developing mammary tumors of MMTV-PyMT mice and colon tumors of CT26 mice were extracted using an 18-gauge core biopsy needle. Samples were precision cut into slices 250-350 μM thick using a Vibratome VT1200S (Leica Microsystems). Each slice was cultured on a thin porous membrane (Nucleopore™ Track-Etched-Whatman®) in a 48-well culture dish using 200 μl of serum-free tumorsphere culture medium (TSC medium: DMEM / F-12 (Hyclone®), B27 supplement (Gibco®), 100 U / mL penicillin (Gibco®), and 100 μg / mL streptomycin (Gibco®).

[0090] MMTV-PyMT slices were treated with control (DMSO) and chemotherapy: cyclophosphamide (CPP), doxorubicin (DOX), and a combination of cyclophosphamide, doxorubicin, and paclitaxel (CPP+DOX+TAXOL). Drugs were added to MMTV-PyMT core slice cultures on day 1 at the following final concentrations: 20 μM CPP, 100 nM DOX, and 1 nM TAXOL, and medium changes were performed on days -1, 4, and 8.

[0091] CT26 core slices were then treated with control, immunoglobulin G (IgG) and DMSO (IgG+DMSO), and combinations of chemotherapy and immunotherapy, including IgG, fluorouracil (5FU), and oxaliplatin (Ox) (IgG+Ox+5FU), anti-PD1 and DMSO (anti-PD1+DMSO), and anti-PD1, 5FU, and oxaliplatin (anti-PD1+5FU+Ox). Drugs were added to CT26 core slice cultures on day 1 at the following final concentrations: 1 μM 5FU, 1 μM OX, and 10 μg / ml anti-PD1. Results were observed via viability measurements as described in Example 1. Representative results are shown in FIG. 7. Viability assay readouts indicate that MMTV-PyMT tumors are sensitive to Dox and CCP+Dox+Taxol treatments and insensitive to CPP treatment, as demonstrated by increased viability readouts in CCP-treated slices and decreased viability readouts in Dox and CCP+Dox+Taxol-treated slices at day 12 compared to day 0. Additionally, viability assay readouts indicate that CT26 tumors are more sensitive to IgG+OX+5FU and anti-PD1+OX+5FU treatments and less sensitive to anti-PD1+DMSO treatments.

[0092] Example 8: Detecting the emergence of therapeutic resistance using long-term tumor slice cultures Human metastatic colorectal cancer (mCRC) core needle biopsy samples were obtained from patients undergoing clinical trials. Samples were precision cut and cultured on slices as described in Example 7. Samples were treated with targeted therapy-encorafenib (enco), a BRAFV600E inhibitor; cetuximab (cetux) (EGFR inhibitor); binimetinib (bini) (MEK inhibitor); and combinations thereof, including combinations of encorafenib and cetuximab (enco+cetux), and combinations of encorafenib, cetuximab, and binimetinib (enco+cetux+bini). Drugs were added to tumor slice cultures on day 1 at the following final concentrations, with medium changes occurring on days -4, 8, 12, and 16: 100 nM encorafenib, 10 μg / mL cetuximab, and 100 nM binitetinib.

[0093] Results were monitored via viability counts as described in Example 1. Representative results are shown in Figure 8, which shows the emergence of encorafenib resistance at day 16 of culture, which is suppressed by treatment with the downstream MEK inhibitor binimetinib (compare enco vs bini, and enco_cetux vs enco+cetux+bini at days 16 and 20).

[0094] Example 9: Long-term maintenance of resident immune cells Flow cytometric analysis of spontaneous MMTV-PyMT mouse mammary tumor slice cultures analyzed with anti-CD45, CD3, CD8, and CD4 antibodies was performed to evaluate the long-term maintenance of resident immune cells in tumor slices exposed to the culture conditions described herein. Tissue specimens from mammary tumors of MMTV-PyMT mice were cultured ex vivo for 4 days with or without IL2. On the 4th day of ex vivo, MMTV-PyMT tumor slices were dissociated using a cocktail containing a 9:1 ratio of Accutase:Collagenase+Hyaluronidase. Dissociated single cells were washed, resuspended in blocking antibody (anti-CD16 / 32), and diluted in 2% BSA / PBS for 30 min on ice. Cells were then washed and resuspended in 45 μl of an antibody cocktail containing anti-CD45, CD3, CD8, and CD4 antibodies (PE-CY7, PE, APC, FITC, BV650, and BV711) in Brilliant Violet staining buffer and incubated on ice for 30 minutes. After incubation, cells were washed and stained with fixable viability dye for 30 minutes at room temperature. After staining, cells were washed and fixed with ICfix solution and then analyzed on an LSRII flow cytometer.

[0095] The results are shown in Figure 9A. CD3+, CD4+, and CD8+ cells in slice cultures were maintained at frequencies close to those of the parental tumor (without the addition of IL2), confirming that tumor slices represent the primary tumor tissue relative to the immune cell microenvironment. The addition of IL2 increased immune cell frequencies relative to those of the parental tumor.

[0096] Example 10: Measuring patient-specific responses to immunotherapy Core needle biopsy was used to obtain human metastatic colorectal cancer (mCRC) biopsy core samples from two patients in a clinical trial. Samples were precision cut onto slices and cultured as described in Example 7. Samples from each patient were then treated with control (DMSO), encorafenib and cetuximab (E+C), immune checkpoint inhibitor nivolumab (N), and a combination of encorafenib, cetuximab, and nivolumab (E+C+N). Drugs were added to the tumor slice cultures on days 1, 4, 8, 12, and 16 at the following final concentrations: 100 nM encorafenib, 10 μg / mL cetuximab, 100 nM binimetinib, and 10 μg / mL nivolumab. Samples were cultured for 14 days.

[0097] Results were monitored via viability measurements as described in Example 1. Results are shown in Figure 10. Two mCRC samples showed differential sensitivity to the checkpoint inhibitors nivolumab alone and the combination of encorafenib and cetuximab (E+C+N) at early and late time points.

[0098] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be utilized in implementing the present disclosure. The following claims are intended to define the scope of the disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby.

Claims

1. 1. A method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample in the presence of the candidate molecule for at least one day; (c) evaluating the response of the cell sample to the candidate molecules; A method comprising:

2. 2. The method of claim 1, wherein in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

3. The method of claim 2, wherein in step (b), the cell sample is cultured for 12 days.

4. 10. The method of claim 1, further comprising the step of: (d) administering an additional candidate molecule to the cell sample and culturing the cell sample for at least 24 hours.

5. 5. The method of claim 4, wherein in step (d), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

6. The method of claim 5, wherein in step (d), the cell sample is cultured for 12 days.

7. The method of claim 4, further comprising: (e) evaluating the response of the cell sample to the additional candidate molecules.

8. 10. The method of claim 1, further comprising the step of: (d) administering a second dose of said additional candidate molecule to said cell sample and culturing said cell sample for at least 24 hours.

9. 9. The method of claim 8, wherein in step (d), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

10. The method of claim 9, wherein in step (d), the cell sample is cultured for 12 days.

11. 9. The method of claim 8, further comprising: (e) evaluating the response of the cell sample to a second dose of the additional candidate molecule.

12. The method of claim 1 , wherein the cell sample comprises a plurality of cells.

13. The method of claim 1 , wherein the cell sample is an intact tissue.

14. 2. The method of claim 1, wherein the cell sample is an organoid.

15. The method of claim 1 , wherein the cell sample is isolated from the tumor tissue.

16. The method of claim 1 , wherein the cell sample comprises cancer cells.

17. The method of claim 1 , wherein the cell sample comprises cells from the microenvironment of the tumor tissue.

18. 18. The method of claim 17, wherein the cells from the microenvironment of the tumor tissue are immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, or bone marrow-derived cells.

19. 20. The method of claim 18, wherein the immune cell is a lymphoid or myeloid cell.

20. 20. The method of claim 19, wherein the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell.

21. 20. The method of claim 19, wherein the myeloid cell is a basophil, eosinophil, mast cell, neutrophil, monocyte, erythrocyte, macrophage, myeloid suppressor cell, or dendritic cell.

22. The method of claim 1 , wherein the cell sample is cultured in serum-free conditions.

23. The method of claim 1, wherein the cell sample is cultured at a liquid-air interface.

24. The method of claim 1 , wherein the cell sample is submerged in cell culture medium.

25. 2. The method of claim 1, wherein the response is a viability response, a gene expression response, a protein expression response, a protein modification response, a cell signaling response, a morphological response, or a metabolic response.

26. The method of claim 1 , wherein assessing a viability response comprises measuring a metabolic product from the cell sample.

27. 27. The method of claim 26, wherein measuring the metabolite from the cell sample comprises isolating the metabolite from a cell culture medium and measuring the metabolite in the absence of the cell sample.

28. 27. The method of claim 26, wherein the metabolic product from the cell sample is a product of a reduction reaction.

29. 27. The method of claim 26, wherein the metabolic product from the cell sample is derived from resazurin.

30. 30. The method of claim 29, wherein the metabolic product from the cell sample is resorufin.

31. 27. The method of claim 26, wherein the metabolic product from the cell sample is derived from tetrazole.

32. 32. The method of claim 31, wherein the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof.

33. 32. The method of claim 31 , wherein the metabolic product from the cell sample is formazan.

34. The method of claim 1 , wherein the cell sample is from a xenograft derived from a patient.

35. The method of claim 1 , wherein the cell sample is from a primary tumor sample.

36. 36. The method of claim 35, wherein the primary tumor sample is a surgical sample from the subject.

37. 36. The method of claim 35, wherein the primary tumor sample is a biopsy sample obtained from a patient.

38. 38. The method of claim 37, wherein the biopsy sample is obtained by core needle biopsy.

39. The method of claim 1, further comprising the step of recommending treatment of the subject with the candidate molecule.

40. The method of claim 1, further comprising the step of recommending not treating the subject with the candidate molecule.

41. The method of claim 1 , wherein the candidate molecule is selected from the group consisting of a biological molecule and a chemical molecule.

42. 42. The method of claim 41, wherein the biological molecule is selected from the group consisting of a recombinant protein, an enzyme, an antibody, a growth factor, a receptor, and any portion thereof.

43. The method of claim 1, wherein step (a) further comprises providing one or more additional candidate molecules, and step (c) comprises evaluating the response of the cell sample to the candidate molecules and the one or more additional candidate molecules.

44. 44. The method of claim 43, wherein the one or more additional candidate molecules are provided simultaneously with the candidate molecule.

45. 44. The method of claim 43, wherein the candidate molecule and the one or more additional candidate molecules are provided sequentially.

46. (d) administering the candidate molecule to an additional cell sample of the tumor tissue from the subject; (e) culturing the additional cell sample in the presence of the candidate molecule for at least 5 days; (f) evaluating the response of the additional cell sample to the candidate molecule; (g) comparing the response of the additional cell sample to the response of the cell sample; The method of claim 1 further comprising:

47. 47. The method of claim 46, wherein the cell sample is from a first location of the tumor tissue and the additional cell sample is from a second location of the tumor tissue.

48. 47. The method of claim 46, wherein the cell sample and the additional cell sample are obtained from the tumor tissue at different times.

49. 49. The method of claim 48, wherein the different times are at least 1, 2, 3, 4, 5, 10, 15, or 20 days apart.

50. 47. The method of claim 46, wherein the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously.

51. The method of claim 1 , further comprising the step of performing a biological assay on the cell sample.

52. 52. The method of claim 51, wherein performing the biological assay on the cell sample comprises fixing the cell sample.

53. 52. The method of claim 51, wherein performing the biological assay on the cell sample comprises permeabilizing the cell sample.

54. 52. The method of claim 51, wherein performing the biological assay on the cell sample comprises lysing the cell sample.

55. 52. The method of claim 51 , wherein the biological assay is selected from the group consisting of flow cytometry, nucleic acid sequencing, polymerase chain reaction, histological analysis, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and any combination thereof.

56. 1. A method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of the tumor tissue for at least one day; (c) evaluating the response of the cell sample of the tumor tissue to the candidate molecule; (d) repeating steps (a)-(c); A method comprising:

57. 57. The method of claim 56, wherein in step (b), the cell sample is cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

58. 58. The method of claim 57, wherein in step (b), the cell sample is cultured for 4 days.

59. 57. The method of claim 56, wherein step (d) comprises repeating (a)-(c) at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 6 times.

60. 60. The method of claim 59, wherein step (d) comprises repeating (a)-(d) three times.

61. 60. The method of claim 59, wherein the cell sample comprises a plurality of cells.

62. 60. The method of claim 59, wherein the cell sample is an intact tissue.

63. 60. The method of claim 59, wherein the cell sample is an organoid.

64. 60. The method of claim 59, wherein the cell sample is isolated from the tumor tissue.

65. 60. The method of claim 59, wherein the cell sample comprises cancer cells.

66. 60. The method of claim 59, wherein the cell sample comprises cells from the microenvironment of the tumor tissue.

67. 67. The method of claim 66, wherein the cells from the microenvironment of the tumor tissue are immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, or bone marrow-derived cells.

68. 68. The method of claim 67, wherein the immune cell is a lymphoid or myeloid cell.

69. 69. The method of claim 68, wherein the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell.

70. 69. The method of claim 68, wherein the myeloid cell is a basophil, eosinophil, mast cell, neutrophil, monocyte, erythrocyte, macrophage, myeloid suppressor cell, or dendritic cell.

71. 57. The method of claim 56, wherein the cell sample is cultured in serum-free conditions.

72. 57. The method of claim 56, wherein the cell sample is cultured at a liquid-air interface.

73. 57. The method of claim 56, wherein the cell sample is submerged in cell culture medium.

74. 57. The method of claim 56, wherein the response is a viability response, a gene expression response, a protein expression response, a protein modification response, a cell signaling response, a morphological response, or a metabolic response.

75. 57. The method of claim 56, wherein the step of evaluating the response comprises measuring a metabolic product from the cell sample.

76. 76. The method of claim 75, wherein measuring the metabolite from the cell sample comprises isolating the metabolite from a cell culture medium and measuring the metabolite in the absence of the cell sample.

77. 76. The method of claim 75, wherein the metabolic product from the cell sample is a product of a reduction reaction.

78. 76. The method of claim 75, wherein the metabolic product from the cell sample is derived from resazurin.

79. 79. The method of claim 78, wherein the metabolic product from the cell sample is resorufin.

80. 78. The method of claim 77, wherein the metabolic product from the cell sample is derived from tetrazole.

81. 81. The method of claim 80, wherein the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof.

82. 81. The method of claim 80, wherein the metabolic product from the cell sample is formazan.

83. 57. The method of claim 56, wherein the cell sample is from a xenograft derived from a patient.

84. 57. The method of claim 56, wherein the cell sample is from a primary tumor sample.

85. 85. The method of claim 84, wherein the primary tumor sample is a surgical sample obtained from the patient.

86. 85. The method of claim 84, wherein the primary tumor sample is a biopsy sample obtained from a patient.

87. 87. The method of claim 86, wherein the biopsy sample is obtained by core needle biopsy.

88. 57. The method of claim 56, further comprising the step of recommending treatment of the subject with the candidate molecule.

89. 57. The method of claim 56, further comprising the step of recommending not treating the subject with the candidate molecule.

90. 57. The method of claim 56, wherein the candidate molecule is selected from the group consisting of a biological molecule and a chemical molecule.

91. 57. The method of claim 56, wherein step (a) further comprises providing one or more additional candidate molecules, and step (c) comprises evaluating the response of the cell sample to the candidate molecules and the one or more additional candidate molecules.

92. 92. The method of claim 91, wherein said one or more additional candidate molecules are provided simultaneously with said candidate molecule.

93. 92. The method of claim 91, wherein the candidate molecule and the one or more additional candidate molecules are provided sequentially.

94. (e) administering the candidate molecule to an additional cell sample of the tumor tissue from the subject; (f) culturing the additional cell sample of the tumor tissue for at least one day; (g) evaluating the response of the additional cell sample of the tumor tissue to the candidate molecule; (h) repeating steps (e)-(g); (i) comparing the response of the additional cell sample to the response of the cell sample; 57. The method of claim 56, further comprising:

95. 95. The method of claim 94, wherein the cell sample is from a first location of the tumor tissue and the additional cell sample is from a second location of the tumor tissue.

96. 95. The method of claim 94, wherein the cell sample and the additional cell sample are obtained from the tumor tissue at different times.

97. 97. The method of claim 96, wherein the different times are at least 1, 2, 3, 4, or 5 days apart.

98. 96. The method of claim 95, wherein the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously.

99. 57. The method of claim 56, further comprising performing a biological assay on the cell sample.

100. 100. The method of claim 99, wherein performing the biological assay on the cell sample comprises fixing the cell sample.

101. 100. The method of claim 99, wherein performing the biological assay on the cell sample comprises permeabilizing the cell sample.

102. 100. The method of claim 99, wherein performing the biological assay on the cell sample comprises lysing the cell sample.

103. 100. The method of claim 99, wherein the biological assay is selected from the group consisting of flow cytometry, nucleic acid sequencing, polymerase chain reaction, histological analysis, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and any combination thereof.

104. 1. A method for assessing cell viability, the method comprising: (a) culturing a tumor cell sample in a cell culture medium; (b) isolating metabolites from the cell culture medium; and (c) measuring the metabolites in the absence of the cell sample, thereby assessing the cell viability of the cell sample; and A method comprising:

105. 105. The method of claim 104, wherein the cell sample is from a cell line.

106. 105. The method of claim 104, wherein the cell sample comprises a plurality of cells.

107. 105. The method of claim 104, wherein the cell sample is an intact tissue.

108. The method of claim 104, wherein the cell sample is an organoid.

109. The method of claim 104, wherein the cell sample is isolated from a tumor.

110. 105. The method of claim 104, wherein the cell sample comprises cancer cells.

111. The method of claim 104, wherein the cell sample comprises cells from the microenvironment of a tumor tissue.

112. The method of claim 111, wherein the cells from the microenvironment of the tumor tissue are immune cells, fibroblasts, endothelial cells, pericytes, adipocytes, mesenchymal stem cells, or bone marrow-derived cells.

113. 113. The method of claim 112, wherein the immune cell is a lymphoid or myeloid cell.

114. The method of claim 113, wherein the lymphoid cell is a T cell, a B cell, a natural killer cell, or a dendritic cell.

115. 114. The method of claim 113, wherein the myeloid cell is a basophil, eosinophil, mast cell, neutrophil, monocyte, erythrocyte, macrophage, myeloid suppressor cell, or dendritic cell.

116. The method of claim 104, wherein the cell sample is cultured in serum-free conditions.

117. The method of claim 104, wherein the cell sample is cultured at a liquid-air interface.

118. 105. The method of claim 104, wherein the cell sample is submerged in cell culture medium.

119. 105. The method of claim 104, wherein the step of measuring the metabolite comprises a fluorescent or chemiluminescent measurement.

120. 105. The method of claim 104, wherein the cell sample is cultured for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

121. 121. The method of claim 120, wherein the cell sample is cultured for 12 days.

122. 105. The method of claim 104, wherein the metabolic product from the cell sample is a product of a reduction reaction.

123. The method of claim 104, wherein the metabolic product from the cell sample is derived from resazurin.

124. The method of claim 123, wherein the metabolic product from the cell sample is resorufin.

125. The method of claim 104, wherein the metabolic product from the cell sample is derived from tetrazole.

126. 126. The method of claim 125, wherein the tetrazole is selected from the group consisting of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) or a salt thereof, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) or a salt thereof, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) or a salt thereof.

127. 126. The method of claim 125, wherein the metabolic product from the cell sample is formazan.

128. 105. The method of claim 104, further comprising the step of administering a candidate molecule to the cell sample.

129. 105. The method of claim 104, wherein the cell sample is from a xenograft derived from a patient.

130. 105. The method of claim 104, wherein the cell sample is from a primary tumor sample.

131. 131. The method of claim 130, wherein the primary tumor sample is a surgical sample.

132. 131. The method of claim 130, wherein the primary tumor sample is a biopsy sample obtained from a patient.

133. 133. The method of claim 132, wherein the biopsy sample is obtained by core needle biopsy.

134. 105. The method of claim 104, further comprising the step of recommending treatment of the subject with the candidate molecule.

135. 105. The method of claim 104, further comprising the step of recommending not treating the subject with the candidate molecule.

136. (d) culturing the additional cell sample with additional cell culture medium; (e) isolating metabolic products from the additional cell culture medium; and (f) measuring the metabolic products from the additional cell culture medium in the absence of the additional cell sample, thereby assessing cell viability of the additional cell sample; (g) comparing the cell viability of the additional cell sample to the cell viability of the cell sample; 105. The method of claim 104, further comprising:

137. 137. The method of claim 136, wherein the cell sample is from a first location of tumor tissue and the additional cell sample is from a second location of the tumor tissue.

138. 137. The method of claim 136, wherein the cell sample and the additional cell sample are obtained from the tumor tissue at different times.

139. 139. The method of claim 138, wherein the different times are at least 1, 2, 3, 4, 5, 10, 15, or 20 days apart.

140. 137. The method of claim 136, wherein the cell sample and the additional cell sample are obtained from the tumor tissue substantially simultaneously.

141. The method of claim 104, wherein the tumor tissue is a tissue sample derived from a subject.

142. 1. A method for assessing radiation dosage, the method comprising: (a) administering said radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample for at least one day; (c) evaluating the response of the cell sample to the radiation dose; A method comprising:

143. 1. A method for assessing a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample for at least one day; (c) evaluating the response of the cell sample to the tumor treatment field; A method comprising:

144. 1. A method for assessing radiation dosage, the method comprising: (a) administering said radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of the tumor tissue for at least one day; (c) evaluating the response of the cell sample of the tumor tissue to the radiation dose; (d) repeating steps (a)-(c); A method comprising:

145. 1. A method for assessing a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample of the tumor tissue for at least one day; (c) evaluating the response of the cell sample of the tumor tissue to the tumor treatment field; (d) repeating steps (a)-(c); A method comprising:

146. 1. A method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample in the presence of the candidate molecule for at least 12 days; (c) evaluating the response of the cell sample to the candidate molecules; A method comprising:

147. 1. A method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of the tumor tissue for at least 12 days; (c) evaluating the response of the cell sample of the tumor tissue to the candidate molecule; (d) repeating steps (a)-(c); A method comprising:

148. 1. A method for assessing cell viability, the method comprising: (a) culturing a tumor cell sample in cell culture medium for at least 4 days; (b) isolating metabolites from the cell culture medium; and (c) measuring the metabolites in the absence of the cell sample, thereby assessing the cell viability of the cell sample; and A method comprising:

149. 1. A method for evaluating a candidate molecule, the method comprising: (a) administering the candidate molecule to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of the tumor tissue for at least 4 days; (c) evaluating the response of the cell sample of the tumor tissue to the candidate molecule; (d) repeating steps (a)-(c) at least three times; A method comprising:

150. 1. A method for assessing radiation dosage, the method comprising: (a) administering said radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample for at least 12 days; (c) evaluating the response of the cell sample to the radiation dose; A method comprising:

151. 1. A method for assessing a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample for at least 12 days; (c) evaluating the response of the cell sample to the tumor treatment field; A method comprising:

152. 1. A method for assessing radiation dosage, the method comprising: (a) administering said radiation dose to a cell sample of tumor tissue from a subject; (b) culturing the cell sample of the tumor tissue for at least 12 days; (c) evaluating the response of the cell sample of the tumor tissue to the radiation dose; (d) repeating steps (a)-(c); A method comprising:

153. 1. A method for assessing a tumor treatment field, the method comprising: (a) exposing a cell sample of tumor tissue from a subject to a tumor treatment field; (b) culturing the cell sample of the tumor tissue for at least 12 days; (c) evaluating the response of the cell sample of the tumor tissue to the tumor treatment field; (d) repeating steps (a)-(c); A method comprising:

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