Methods and compositions for measuring composition of tumor microenvironment

Abstract

To provide methods and compositions that can measure the composition of the TME of a solid tumor and can be used to determine what immunomodulators, either alone or in combination with other anticancer drugs, may be effective in treating a tumor in a given subject.SOLUTION: A method of measuring the composition of a solid tumor microenvironment comprises the steps of: (a) permitting agents to simultaneously bind to cancer cells, immune cells or both cancer cells and immune cells present in a single cell suspension to produce labeled cells, where cell surface markers comprise cell-type markers, IMRs and IMR-Ls; and (b) measuring the presence and / or amount of the labeled cells by cytometry thereby to (i) measure the presence and amount of cancer cells, immune cells or both cancer cells and immune cells present in the solid tumor microenvironment and (ii) measure whether the cancer cells, immune cells or both the cancer cells and immune cells express the IMRs and the IMR-Ls, thereby measuring the solid tumor microenvironment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 758,393, filed November 9, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to the field of cancer immunology. More particularly, the present disclosure relates to methods and compositions for isolating and analyzing tumor samples to measure the phenotypic composition of the tumor microenvironment, assess the functional status of infiltrating immune cells, quantify the expression of immunoregulatory receptors and ligands, and predict response to immunotherapy. [Background technology]

[0003] background The discovery of checkpoint inhibitors in cancer therapy has provided new therapies for the treatment of cancers that, until this discovery, had eluded successful treatment because cancers had evolved mechanisms to evade the subject's immune system. However, despite the significant progress achieved to date in treating various cancers with checkpoint inhibitors, currently available biomarkers and diagnostic methods are poorly predictive of response, and therefore a significant number of patients remain unresponsive to immunotherapy treatment. Furthermore, given the complexity of the tumor microenvironment (TME) of solid tumors, which contains epithelial cells, endothelial cells, mesenchymal cells, stromal cells, cancer cells, and immune cells, solid tumors can be more difficult to evaluate for potential treatment options. In certain solid tumors, cancer cells influence the activity or inactivity of immune cells within the TME. Thus, despite the progress made to date, there remains a need for methods and compositions that can measure the composition of the TME of solid tumors and thus determine which immunomodulatory agents, alone or in combination with other anti-cancer agents, may be effective in treating a given subject's tumor. Summary of the Invention

[0004] The present invention is based, in part, on the discovery that the present invention can measure the composition of the solid tumor microenvironment (TME) and perform single-cell phenotypic and functional analysis of immune cells and cancer cells located within the TME. More specifically, the methods and compositions described herein facilitate quantitative characterization of immunosuppressive phenotypes and cellular expression of targetable immunoregulatory (e.g., checkpoint) receptors and their ligands within the TME. The methods and compositions can be used to determine whether a subject with a solid tumor is likely to respond to a particular immunomodulatory drug. The methods and compositions also facilitate treatment of a given subject's solid tumor by aiding in the selection of an appropriate immunomodulatory drug for treating the subject's solid tumor. Additionally, the methods and compositions of the present invention provide drug discovery / evaluation tools for (a) early stage immunotherapy discovery research; (b) providing mechanism-based proof of principle in preclinical studies; (c) patient stratification in clinical trials; and (d) selecting immunomodulatory agents most likely to be effective in treating a subject's cancer. The methods described herein also facilitate phenotypic analysis of tumor-infiltrating lymphocytes (TILs), thereby providing accurate and highly reproducible data sets under validated conditions. Thus, the breadth and quality of data collected by the methods described herein offers advantages over traditional cellular evaluation techniques, such as traditional immunohistochemistry.

[0005] In one aspect, the present disclosure relates to a method for measuring the composition of a solid tumor microenvironment, comprising combining a single-cell suspension of cells derived from a solid tumor with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, where the cell surface markers include cell-type markers, immunomodulatory receptors (IMRs), and IMR-ligand markers (IMR-Ls), and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single-cell suspension to generate labeled cells. The method further comprises measuring the presence and / or quantity of the labeled cells by cytometry to (i) determine the presence and / or quantity of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor microenvironment, and (ii) determine whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L, thereby measuring the solid tumor microenvironment.

[0006] In another aspect, the disclosure relates to a method for determining whether a subject with a solid tumor is likely to respond to an immunomodulatory drug, comprising combining a single-cell suspension of cells derived from the solid tumor with a plurality of labeling agents capable of binding to a plurality of corresponding cell surface markers expressed on cancer cells and / or immune cells, where the cell surface markers include cell-type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls), and allowing the agents to bind to cancer cells, immune cells, or both cancer cells and immune cells simultaneously present in the single-cell suspension to generate labeled cells. The method further includes combining at least a portion of the single cell suspension with an immunomodulatory drug; and (i) measuring the presence and / or amount of labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer and immune cells present in the solid tumor, and determining whether the cancer cells, immune cells, or both cancer and immune cells express at least one IMRs and / or at least one IMR-Ls; and (ii) measuring the effect of the immunomodulatory drug on cellular markers on or in the cancer cells, immune cells, or both cancer and immune cells, thereby determining whether the subject is likely to respond to the immunomodulatory drug.

[0007] In another aspect, the present disclosure relates to a method for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunomodulatory agent, thereby treating the solid tumor. The immunomodulatory agent is selected by a method comprising combining a single-cell suspension of cells derived from the solid tumor with a plurality of labeling agents capable of binding to a plurality of corresponding cell surface markers expressed on cancer cells and / or immune cells, where the cell surface markers include cell-type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls), and allowing the agent to bind to cancer cells, immune cells, or both cancer cells and immune cells simultaneously present in the single-cell suspension to generate labeled cells. The method further includes combining at least a portion of the single cell suspension with an immunomodulatory drug; and (i) measuring the presence and / or amount of labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer and immune cells present in the solid tumor, and determining whether the cancer cells, immune cells, or both cancer and immune cells express at least one IMRs and / or at least one IMR-Ls; and (ii) measuring the effect of the immunomodulatory drug on cellular markers on or in the cancer cells, immune cells, or both cancer and immune cells, thereby determining whether the subject is likely to respond to the immunomodulatory drug.

[0008] In particular embodiments of the above aspects, the cytometry is flow cytometry, mass cytometry, image cytometry, and / or single cell technology (SCT). In particular embodiments of the above aspects, the labeling agent is selected from the group consisting of an oligonucleotide, a fluorophore, an infrared label, and a heavy metal label. In particular embodiments of the above aspects, the immune cells include lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or combinations thereof.

[0009] In certain embodiments, the cell surface markers further comprise a cell activation marker. In particular embodiments, cell type markers include markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells) and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells). In particular embodiments, cell type markers are cancer cell markers, such as, for example, CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin. In particular embodiments, cell activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197), and / or functional markers such as IFNγ, TNFα, and / or other cytokines and / or granzyme B. In particular embodiments, the IMR or IMR-L markers are PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), ID O, TIGIT, CD73, CD39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and / or CD115 (CSF-1R).

[0010] In certain embodiments, the method further comprises combining the cells with an immunomodulatory agent. In certain embodiments, the method comprises measuring the effect of the immunomodulatory agent on the expression of at least some of the cell markers on the cancer cells and / or immune cells. The immunomodulatory agent can be combined with the single cell suspension before, during, or after combining the single cell suspension of solid tumor-derived cells with a plurality of labeling agents capable of binding to a plurality of corresponding cell surface markers expressed on the cancer cells and / or immune cells. In particular embodiments, multiple different labeled cells are detected simultaneously during cytometry. In particular embodiments, multiple different cell surface markers are detected simultaneously during cytometry. In particular embodiments, at least 14 different cell surface markers are detected simultaneously. In certain embodiments, receptor-ligand interactions between labeled cells can be detected and, optionally, quantified. In certain embodiments, the receptor-ligand interaction includes the interaction between a checkpoint inhibitor and its cognate ligand. In certain embodiments, the receptor-ligand interaction can be selected from the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM-3 and Gal9, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, and / or CD-40L and CD40. In particular embodiments, the presence and / or amount of a cell activation marker, an IMR marker, an IMR-L marker, or a combination of an activation marker and an IMR and / or IMR-L marker expressed on cancer cells and / or immune cells is measured.

[0011] The above description sets forth several aspects and embodiments of the present invention. This patent application specifically contemplates all combinations and permutations of such aspects and embodiments. These and other aspects and features of the present invention are set forth in the following detailed description and claims. The foregoing and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings, in which like reference elements identify common features in the following corresponding drawings. [Brief explanation of the drawings]

[0012] [Figure 1] Gating trees for automated metric extraction from predefined gating routines for identification of Tregs in a Treg staining panel and expression of granzyme B or cytokines are shown. Flow cytometry data were analyzed using a combination of the Qognit software package Ryvett and the Verity Software House software package Winlist. The gating tree represents the Boolean logic used to identify cell populations, capture all cell populations, and further separate them into distinct populations as gating tree branches. [Figure 2] Gating trees for automated metric extraction from predefined gating routines for identification of T cells, B cells, NK cells, and monocyte / macrophage cells and expression of IMRs and IMR-Ls on identified cell subsets are shown. Flow cytometry data were analyzed using a combination of the Qognit software package Ryvett and the Verity Software House software package Winlist. The gating tree represents the Boolean logic used to identify cell populations, capture all cell populations, and further separate them into distinct populations as gating tree branches. [Figure 3] 1 shows immunomodulatory receptor (IMR) / immunomodulatory ligand (IMR-L) pairs detectable according to the methods of the present invention. [Figure 4A] FIG. 1 shows surface and intracellular phenotypic flow cytometry plots for dissociated tumor samples showing the presence of CD3+, CD4+, CD8+ and CD4+CD25hiFoxP3+ Treg cells. [Figure 4B]Associated numerical plots (expressed as percent positive) showing the prevalence of CD3+, CD4+, CD8+, and CD4+CD25hiFoxP3+ Treg cells are shown. Elevated levels of CD4+CD25hiFoxP3+ Tregs suggest (a) an immunosuppressive signature due to tumor dissemination and (b) poor prognosis and objective response. (RD-PBMC = PBMC reference donor was used throughout all assays.) [Figure 5] Flow cytometry histogram plots and associated numerical plots of gated histograms for one or more basal or induced intracellular detectable antigens (expressed as percent positive) are shown. Intracellular cytokine expression (IFNγ and TNFα) was measured in CD4+ and CD8+ T cells isolated from the tumor microenvironment of breast, lung, and kidney tumors in the presence or absence of Leukocyte Activation Cocktail with BD GolgiPlug™. Triangles represent breast tumors, squares represent lung tumors, diamonds represent kidney tumors, and circles represent data from the PBMC reference donor (RD-PBMC). [Figure 6] Flow cytometry histogram plots showing Granzyme B CD8+ cytotoxic T cell expression in breast, lung, and kidney tumors compared to control (FMO) and healthy donor PBMCs and associated numerical plots of gated histograms (expressed as percent positive). Triangles represent breast tumors, squares represent lung tumors, diamonds represent kidney tumors, and circles represent data from the PBMC reference donor. MFI = mean fluorescence intensity. [Figure 7] FIG. 1 shows a surface phenotype flow cytometry plot for a dissociated tumor sample showing the presence of CD45+ leukocytes, CD326+ epithelial tumor cells, CD14+ monocytes / macrophages, and CD4+ and CD8+ T cells. [Figure 8]Flow cytometry histogram plots showing expression of the immunosuppressive checkpoint TIGIT (T cell immunoreceptor with Ig and ITIM domains) expressed on CD4+, CD8+, and CD14+ cells, but not on CD326+ epithelial tumor cells. FMO = control. [Figure 9] Numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed in CD326+ epithelial tumor cells from a cohort of dissociated breast, lung, and kidney tumors are shown. Medium gray boxes represent expression levels in breast tumors, light gray boxes represent expression levels in lung tumors, and dark gray boxes represent expression levels in kidney tumors. [Figure 10] Numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed on CD14+ myeloid cells from cohorts of dissociated breast, lung, and kidney tumors are shown. Medium gray boxes represent expression levels in breast tumors, light gray boxes represent expression levels in lung tumors, and dark gray boxes represent expression levels in kidney tumors. [Figure 11] Numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed on CD8+ T cells obtained from cohorts of dissociated breast, lung, and kidney tumors are shown. Medium gray boxes represent expression levels in breast tumors, light gray boxes represent expression levels in lung tumors, and dark gray boxes represent expression levels in kidney tumors. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description The present invention is based, in part, on the discovery that the present invention can measure the composition of the solid tumor microenvironment and perform single-cell phenotypic and functional analysis of immune and cancer cells located within the solid tumor microenvironment (TME). More specifically, the methods and compositions described herein facilitate quantitative characterization of immunosuppressive phenotypes and cellular expression of targetable immunoregulatory (e.g., checkpoint) receptors and their ligands within the TME. The methods and compositions can be used to determine whether a given subject with a solid tumor is likely to respond to treatment with a particular immunomodulatory drug, such that the subject is treated with one of many drugs selected to produce a positive outcome in the subject, without exposure to drugs that are unlikely to produce a positive outcome. The methods and compositions described herein can be used to rapidly and quantitatively analyze the TME of solid tumors to determine the immune status within the TME and provide clinically relevant information based on phenotypic and functional analysis of tumor-infiltrating lymphocytes (TILs) within the TME. Characterization of TILs in the context of the TME can be important for targeted immunotherapy. For example, phenotypic subset analysis of TILs can correlate with clinical outcome and can be used to guide immunotherapy assays. For example, elevated CD8+ and CD56+ TIL densities predict a positive response to cancer immunotherapy.

[0014] Similarly, the methods and compositions described herein facilitate the assessment of tumor-infiltrating NK cells, T cells (Tregs, CD8+ cytotoxic T cells, exhausted T cells), myeloid-derived suppressor cells (MDSCs), and various subsets of dendritic cells (DCs) and macrophages (MFs) within the TME. These techniques further facilitate the assessment of the proportion of immunomodulatory receptor (IMR) / immunomodulatory receptor ligand (IMR-L)-positive immune cells and tumor cells. The methods also allow for the analysis of activation / exhaustion markers, such as cytokine and granzyme expression. Additionally, the methods and compositions described herein can be used to measure the functional status of TILs, which can aid in predicting the clinical response of a given tumor-bearing subject. For example, the techniques described herein can be used to determine, for a given solid tumor sample, whether immune cells are activated or exhausted, and whether they are suppressed by Tregs, M2 macrophages, and MDSCs within the TME. Furthermore, the methods and compositions also facilitate quantification of the (co)expression profiles of targetable IMRs and their cognate ligands in TILs and tumor cells, which provides healthcare providers with information about whether a particular immunomodulatory drug has a positive effect on the TME and can be used alone or in combination with another anti-cancer drug to promote a positive therapeutic outcome. For example, the selection of a particular immunomodulatory drug can reduce or eliminate resistance that certain cancer cells may have to treatment with a particular anti-cancer drug.

[0015] In general, the techniques described herein involve combining a single-cell suspension of solid tumor-derived cells with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer and immune cells present in the single-cell suspension. The methods involve measuring the presence and / or quantity of the labeled cells by cytometry, thereby determining not only the presence and / or quantity of cancer cells, immune cells, or both cancer and immune cells present within the solid tumor microenvironment, but also phenotypic information about whether the cells express specific phenotypic markers, such as IMRs, IMR-Ls, and cell activation markers. In certain embodiments, the method further comprises combining the cells with an immunomodulatory drug, alone or in combination with an anti-cancer drug, and then determining whether the immunomodulatory drug affects the expression of cellular markers on the cancer cells and / or immune cells.

[0016] The methods and compositions described herein can be used to determine whether a subject with a solid tumor is likely to respond to an immunomodulatory drug. For example, a single-cell suspension of cells derived from a solid tumor can be combined with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer and immune cells present in the single-cell suspension, generating labeled cells. Furthermore, at least a portion of the single-cell suspension of cells can be contacted with an immunomodulatory drug (alone or in combination with an anticancer drug), and the labeled cells can then be analyzed by cytometry to determine the presence and / or quantity of cancer cells, immune cells, or both cancer and immune cells present in the solid tumor. Furthermore, the effect of the immunomodulatory drug on cell markers in cancer cells, immune cells, or both cancer and immune cells can be measured. This information can be used to determine whether a subject is likely to respond positively to the immunomodulatory drug.

[0017] The methods and compositions described herein can be used to treat a solid tumor in a subject in need thereof by administering to the subject an effective amount of an immunomodulatory agent selected by the techniques described herein, thereby treating the solid tumor. The immunomodulatory agent is selected using a method comprising the steps of: (a) combining a single-cell suspension of cells derived from the solid tumor with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, allowing the agents to simultaneously bind to the cancer cells, immune cells, or both cancer and immune cells present in the single-cell suspension to produce labeled cells; (b) combining at least a portion of the single-cell suspension of cells with the immunomodulatory agent; and (c) (i) measuring the presence and / or quantity of labeled cells by cytometry, thereby determining the presence and quantity of cancer cells, immune cells, or both cancer and immune cells present in the solid tumor, and (ii) determining the effect of the immunomodulatory agent on cellular markers on or in the cancer cells, immune cells, or both cancer and immune cells, thereby determining whether the subject is likely to respond to the immunomodulatory agent. The sections below provide further details on how the methods and compositions described herein can be used to analyze the TME of solid tumors, and how the methods and compositions can determine whether a subject is likely to respond favorably to a given immunomodulatory agent.

[0018] I. Cell type It is recognized that the TME includes both tumor cells and immune cells as described herein. (a) Tumor cells The term "tumor cell" is used interchangeably herein with "cancer cell." Tumor cell types that can be examined using the methods and compositions described herein include epithelial-, endothelial-, and mesenchymal-derived cells. Tumors that can be investigated using the techniques described herein include, but are not limited to, anal cancer, bladder cancer, intestinal (large and small intestine) cancer, brain cancer, breast cancer, oral cancer, cervical cancer, esophageal cancer, fallopian tube cancer, head and neck cancer, colon cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, urinary tract cancer, uterine cancer, and vulvar cancer.

[0019] Typical tumors include, for example, ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma), ovarian granulosa cell tumor, fallopian tube adenocarcinoma, peritoneal carcinoma, uterine (endometrial) adenocarcinoma, sarcomatoid carcinoma, cervical cell carcinoma, endocervical adenocarcinoma, vulvar cancer, breast cancer, primary and metastatic (ductal carcinoma, mucinous carcinoma, lobular carcinoma, malignant phyllodes tumor), head and neck cancer, oral cancer including tongue cancer, primary and metastatic, esophageal cancer, adenocarcinoma, gastric adenocarcinoma, primary small intestine cancer, colon adenocarcinoma, primary and metastatic (adenocarcinoma, mucinous carcinoma, large cell neuroendocrine carcinoma, mucinous carcinoma), appendix adenocarcinoma, colorectal cancer, rectal cancer, anal cancer (squamous, basaloid), carcinoid tumor, primary and metastatic (appendix, small intestine, colon), pancreatic cancer, liver cancer (hepatocellular carcinoma, cholangiocarcinoma), liver metastatic cancer to the liver, lung cancer, primary and metastatic (squamous cell, adenocarcinoma, adenosquamous carcinoma, giant cell carcinoma, non-small cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, neuroendocrine carcinoma, large cell carcinoma, bronchopulmonary carcinoma), renal cell (kidney) carcinoma, primary and metastatic bladder cancer, primary and metastatic, prostate adenocarcinoma, primary and metastatic, brain tumors, primary and metastatic (glioblastoma, pleomorphic neuroectodermal tumor, neuroectodermal tumor, oligodendroglioma, malignant astrocytoma), skin tumors (malignant melanoma, sebaceous cell carcinoma), thyroid cancer (papillary and follicular), thymic carcinoma, shenoid carcinoma, carcinoma of unknown primary origin, neuroendocrine carcinoma, testicular malignant tumors (seminoma, embryonal carcinoma, malignant mixed tumor), etc.

[0020] (b) Immune cells Immune cells that can be detected and analyzed according to the present invention include lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells (granulocyte- and monocyte-derived)).

[0021] II. Sample Processing and Cell Counting A solid tumor sample can be obtained from a subject and processed by any means known in the art to form a single cell suspension of cells. As used herein, a "single cell suspension" is a suspension of one or more cells in a liquid sample, wherein the cells are primarily in the form of single cells rather than clusters or aggregates of cells. In certain embodiments, single cells represent 60%, 70%, 80%, 90%, or 95% of the cells in the cell suspension. In certain embodiments, solid tumors are digested manually and / or enzymatically. For example, solid tumor samples can be cut into smaller fragments and subjected to enzymatic digestion using trypsin, collagenase, deoxyribonuclease (DNAse), dispase, and / or hyaluronidase. In certain embodiments, the digested tissue is filtered to remove larger, undigested fragments. Additionally or alternatively, tissue dissociation can be performed using an automated tissue homogenizer, such as the gentleMACS™ Octo Dissociator (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). For example, cells can be filtered through a 70 μM filter to remove undigested tissue.

[0022] The resulting single cell suspension may contain all of the cell types present in the tumor microenvironment, including epithelial cells, endothelial cells, mesenchymal cells, stromal cells, tumor / cancer cells, and immune cells. After dissociation, the cells can be washed, pelleted, resuspended, and / or counted. Cells can be counted using any means known in the art, for example, using a manual cell counter or an automated cell counter. For example, for solid tumors, nucleated cell counts can be obtained using an automated cell counter that utilizes bright-field imaging and / or fluorescent imaging (e.g., dual fluorescent imaging). Exemplary automated cell counters include the Nexcelom Cellometer 2000 (Nexcelom, Lawrence, MA), the Countess II FL Automated Cell Counter (ThermoFisher, Waltham, MA), and the TC20™ Automated Cell Counter (BioRad, Hercules, CA). For whole blood, bone marrow, PBMCs, or BMMCs, an automated cell counter such as a Coulter counter, for example, the Beckman Coulter Act2 Diff (Beckman Coulter, Inc., Brea, CA), can be used.

[0023] After determining the approximate number of cells in the sample, the cells can be pelleted and resuspended in a buffer at a desired concentration based on the cell count. Suitable buffers include RPMI 1640 + 10% FBS + 1% penicillin-streptomycin and / or RPMI 1640 + 10% FBS or 1x PBS + 0.5% BSA. Approximately 0.5 to 5x10 cells can be resuspended. 6 cells / mL, e.g., about 0.5 to about 1 x 10 6 cells / mL, approximately 0.5 to approximately 2×10 6 cells / mL, approximately 0.5 to approximately 3×10 6 cells / mL, approximately 0.5 to approximately 4×10 6 cells / mL, about 1 to about 2×10 6 cells / mL, about 1 to about 3×10 6 cells / mL, about 1 to about 4×10 6 cells / mL, about 1 to about 5×10 6 cells / mL, about 2 to about 3 x 10 6 cells / mL, about 2 to about 4×10 6 cells / mL, about 2 to about 5×106 cells / mL, about 3 to about 4 x 10 6 cells / mL, about 3 to about 5×10 6 cells / mL, about 4 to about 5 × 10 6 In particular embodiments, the cells can be resuspended at a concentration of about 1.2 to about 2.4 x 10 cells / mL. 6 cells / mL, approximately 1.2 to approximately 2×10 6 cells / mL, approximately 1.2 to approximately 1.5×10 6 cells / mL, approximately 1.5 to approximately 2.4×10 6 cells / mL, approximately 1.5 to approximately 2×10 6 Resuspend at a concentration of 100 cells / mL.

[0024] III. Cell analysis To analyze cells initially disposed with the TME, the cells, once converted into a single cell suspension, are combined with multiple (e.g., 5, 6, 7, 8, 9, 10, 11, 20, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more) labeling agents that bind to select cell surface markers to determine whether the cells are cancer cells or immune cells or subtypes and / or whether the cells or subtypes express, for example, IMRs, IMR-Ls, or cell activation markers. Specifically, cells can be plated and stained with a vital dye (e.g., the amine-reactive dye Alexa750) to distinguish between live and dead cells. The cells can be washed with a staining buffer (e.g., 1x PBS + 0.5% BSA). For example, the washing process can be automated using a Biotek ELx450 deep-well plate washer. The cells can be fixed and permeabilized using standard methods in the art, for example, using the FOXP3 / Transcription Staining Buffer Kit from eBioscience (ThermoFisher, Waltham, MA). The cells can then be washed with a staining buffer (e.g., 1x PBS + 0.5% BSA) and stained with one or more labeling agents to detect one or more cell markers. The method for staining the cells will depend on the labeling agent used.

[0025] (a) Cell surface markers The methods disclosed herein can be used to detect any cell surface marker, such as a cancer cell marker, an activation marker, or an IMR or IMR-L marker. Cell type markers are proteins or other molecules present in or on specific cell types, e.g., cancer cells or immune cells. In certain embodiments, the presence of specific cancer cell markers can indicate the type of cancer. In certain embodiments, the presence of specific cancer cell markers provides a target for cancer treatment. Exemplary cancer cell markers include CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin. In certain embodiments, the presence of specific immune cell markers identifies immune cells as a particular immune cell type or subtype. Exemplary immune cell markers are provided in Table 1 below.

[0026] [Table 1]

[0027] Activation markers can be proteins or other molecules. The presence of an activation marker in or on a cancer cell or immune cell indicates that a particular pathway (e.g., an immune pathway) is active. Exemplary activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197), and / or functional markers (e.g., IFNγ, TNFα, and / or other cytokines and / or granzyme B). Exemplary IMR or IMR-L markers include PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, and T These include IGIT, CD73, CD39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and / or CD115 (CSF-1R).

[0028] In certain embodiments, other markers of signal transduction can be detected according to the methods described herein. In certain embodiments, the methods include detecting receptors or transporters (e.g., CD3, CD4, CD5, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD20, CD25, CD27, CD28, CD31, CD34, CD38, CD45, CD45RA, CD45RO, CD56, CD69, CD71, CD80, CD86, CD90, CD117, CD123, CD133, CD135, CD235, cytokines, cytokines, cytokine receptors, cytokine signaling, cytokine ... Cytokaritin, EPCAM, FOXP3, HLA-DR, IgD, IgG, IgM, MDR1, ABCG2), DNA damage or apoptosis signaling molecules (e.g., Bcl-2, Bcl-xL, cytochrome C, caspase 3 or 8, cPARP, annexin V, DNMT1, 3a, 3b, p-H2AX, p-53BP1, p-ATM, p-DNA-PKcs, p-p53, p53, p21, p-Chk2 , p-RPA2, p-BRCA1), immune signaling molecules (e.g., p-Akt, p-Blnk, p-Erk, p-Gsk3b, p-Lyn, p-NFkB, p-Plcg2, p-S6, p-Stat5, p-Syk, p-SLP-76, p-ZAP-70, p-Lck, p-CD3z, p-Vav, p-Lat, p-Pyk2), differentiation, maturation and / or cytokine / chemokine response signaling molecules (e.g., p-Sta t1, p-Stat3, p-Stat4, p-Stat5, p-Stat6, p-Erk, p-p38, p-NFkB, IkB, pRelB), intracellular cytokines (e.g., IL-2, IL-4, IL-6, IL-8, IL-10, IL17A, IFNa, IFNg, TNFa), measures of cytotoxic effector function (e.g., CD107a, granzymes, perforin, annexin V), PKC, CA ++These include detecting signaling molecules (e.g., p-Akt, p-Erk, p-PLCg2, p-PKCa, p-S6, p-p38), survival, proliferation, cell cycle, and pattern recognition receptor signaling molecules (e.g., p-Akt, p-NFkB, p-S6, IkB, p-Erk, p-p38, cyclin A2, cyclin B1, p-CDK1, p-HH3, p-MK2, p21, p-CREB, pc-JUN). In particular embodiments, a panel of cellular markers is measured to identify cell type, IMR and / or IMR-L, and / or exhaustion markers. An exemplary panel of cellular markers is shown in Table 2 below.

[0029] [Table 2]

[0030] For example, in certain embodiments, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more different cell surface markers are simultaneously detected, or in certain embodiments, 5 to 19, 6 to 18, 7 to 17, 8 to 16, 9 to 15, 10 to 14, 10 to 13, 11 to 12, 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 12 to 14, 13 to 14, 5 to 16, 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16, or 15 to 16 different cell surface markers are simultaneously detected. In particular embodiments of the methods described herein, at least 14 different cell surface markers are detected simultaneously. In particular embodiments, receptor-ligand interactions between labeled cells can be detected, such as the interaction between a checkpoint inhibitor and its cognate ligand. Exemplary receptor-ligand pairs are shown in Figure 3. For example, the receptor-ligand interaction can be selected from the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM-3 and Gal9, TIGIT / CD112-CD155, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, LAG3 / MHC, KIR-MHC, and / or CD-40L and CD40.

[0031] (b) Labeling agent mixed with single cell suspension Once prepared, the single cell suspension could be distributed into multiple receiving vessels, eg, the wells of a multi-well plate, eg, a 96 or 364 well plate, and prepared for cytometric analysis. Cell plating and subsequent handling steps can be automated, for example, using Hamilton robotics liquid handlers (Hamilton Company, Reno, NV). Cells can be fixed and permeabilized using any method known in the art prior to exposure to the labeling agent. In certain embodiments, if the labeling agent is sensitive to fixation, the cells are exposed to the labeling agent without fixation and permeabilization or before fixation and permeabilization. In certain embodiments, the labeling agent comprises a binding agent (e.g., a member of a binding complex, e.g., an antibody, protein, aptamer, avimer, Adnectin and Affibody® ligand, member of a ligand-receptor pair, small molecule inhibitor) that is conjugated, e.g., covalently attached, to the label, which binds to a cell surface marker (e.g., a cancer cell marker, an activation marker, or an immunomodulatory receptor (IMR) or IMR-ligand (IMR-L) marker). In certain embodiments, cells are exposed to a binder (e.g., an antibody) that binds to the cell surface marker to form a cell surface marker / binder complex, and the cell surface marker / binder complex is then exposed to a labeling agent that binds to the cell surface marker / binder complex. Certain embodiments use oligonucleotide conjugates (i.e., oligonucleotide labels) in which a binder (e.g., an antibody) is bound to a first oligonucleotide and a second oligonucleotide that is complementary to (i.e., capable of binding to (hybridizing with)) the first oligonucleotide is directly or indirectly bound to one or more labels (e.g., one or more fluorophores). Annealing of the first and second oligonucleotides links the binder to the one or more labels. The annealed oligonucleotide conjugate constitutes a binder-label conjugate, which can then be used in cytometry applications (e.g., flow cytometry).

[0032] (c) Binder Binding agents suitable for use in accordance with the methods herein include any substance capable of preferentially binding to a cell surface marker described herein (e.g., a cancer cell marker, an activation marker, or an immunomodulatory receptor (IMR) or IMR-ligand (IMR-L) marker). For example, binding agents can include antibody (e.g., monoclonal antibody) proteins, peptide aptamers, avimers, Adnectin and Affibody® ligands, members of ligand-receptor pairs, and small molecule inhibitors. Exemplary binders include CD44 binders, CD47 binders, CD49f binders, CD271 binders, CD326 binders, cytokeratin binders, E-cadherin binders, vimentin binders, CD25 binders, CD26 binders, CD27 binders, CD28 binders, CD38 binders, CD40 binders, CD44 binders, CD62L binders, CD69 binders, CD80 binders, CD86 binders, CD95 binders, CD95L binders, CD127 binders, CCR7 (CD197) binders, IFNγ binders, TNFα binders, granzyme B binders, PD-1 (CD279) binders, PD-L1 (CD274) binders, CTLA-4 (CD152) binders, LAG3 (CD223) binders, OX40 (CD1 34) binders, TIM3 (CD366) binders, GITR (CD357) binders, 4-1BB (CD137) binders, KIR (CD158B) binders, 2B4 (CD244) binders, ICOS (CD278) binders, IDO binders, TIGIT binders, CD73 binders, CD39 binders, CD172a (SIRPa) binders, B7H4 (B7S1) binders, VISTA (B7-H5) binders, CD355 (CRTAM) binders, KLRG1 binders, CD160 (BY55, NK1, NK28) binders, CD30 (TNFRSF8) binders, CD224 (GGT1) binders, CD226 binders, CD272 (BTLA) binders, and / or CD115 (CSF-1R) binders.

[0033] Antibodies suitable for use in accordance with the methods herein include anti-CD44 antibodies, anti-CD47 antibodies, anti-CD49f antibodies, anti-CD271 antibodies, anti-CD326 antibodies, anti-cytokeratin antibodies, anti-E-cadherin antibodies, anti-vimentin antibodies, anti-CD25 antibodies, anti-CD26 antibodies, anti-CD27 antibodies, anti-CD28 antibodies, anti-CD38 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD62L antibodies, anti-CD69 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-CD95 antibodies, anti-CD95L antibodies, anti-CD127 antibodies, anti-CCR7 (CD197) antibodies, anti-IFNγ antibodies, anti-TNFα antibodies, Anti-granzyme B antibody, anti-PD-1 (CD279) antibody, anti-PD-L1 (CD274) antibody, anti-CTLA-4 (CD152) antibody, anti-LAG3 (CD223) antibody, anti-OX40 (CD134) antibody, anti-TIM3 (CD) 366) Antibodies, anti-GITR (CD357) antibodies, anti-4-1BB (CD137) antibodies, anti-KIR (CD158B) antibodies, anti-2B4 (CD244) antibodies, anti-ICOS (CD278) antibodies, anti-IDO antibodies, anti-TIGIT antibodies, Anti-CD73 antibody, anti-CD39 antibody, anti-CD172a (SIRPa) antibody, anti-B7H4 (B7S1) antibody, anti-VISTA (B7-H5) antibody, anti-CD355 (CRTAM) antibody, anti-KLRG1 antibody, anti-CD160 (BY5) 5, NK1, NK28) antibody, anti-CD30 (TNFRSF8) antibody, anti-CD224 (GGT1) antibody, anti-CD226 antibody, anti-CD272 (BTLA) antibody, and / or anti-CD115 (CSF-1R) antibody.

[0034] (d) Labeling agent (i) Fluorophores (including visible and infrared fluorophore labels) Fluorophores suitable for use in accordance with the methods herein include, but are not limited to, Cy5.5, Cy5, and Cy7 (GE Healthcare); AlexaFluor488, AlexaFluor594, AlexaFluor647, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677, Dy682, Dy752, and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health), PE, PE-Cy7, PerCP, PerCP-Cy5.5, FITC, BV421, BV510, BV605.

[0035] (ii) Heavy metal labeling Heavy metal labels, such as lanthanides, can be used in certain embodiments of the methods described herein, such as mass cytometry. Lanthanides include, but are not limited to, lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). In certain embodiments, for example, 139 La, 141 Pr, 142 Nd, 144 Nd, 145 Nd,146 Nd, 147 Nd, 147 Sm, 152 Sm, 151 EU, 153 EU, 156 Gd, 159 Tb, 164 Dy, 165 Ho, 166 Er, 169 Tm, 171 Yb, 174 Yb, and 176 Lanthanide isotopes, including Yb, are used.

[0036] (e) Cytometry (i) Flow cytometry In certain embodiments, labeled cells are analyzed using flow cytometry. For example, once labeled cells are obtained using the staining procedures described herein, the labeled cells are analyzed by flow cytometry. Exemplary flow cytometers useful for the methods described herein include, for example, Attune NxT flow cytometers (ThermoFisher, Waltham, MA), CytoFLEX flow cytometers (Beckman Coulter, Indianapolis, IN), FACSVerse or FACSCanto II flow cytometers (Becton Dickinson, San Jose, CA), or Aurora flow cytometers (Cytek, Oakland, CA), which can simultaneously measure multiple, e.g., 16, fluorescent dyes. Data obtained from the flow cytometer can be analyzed, for example, using standard settings on the flow cytometer.

[0037] In certain embodiments, prior to acquisition of flow cytometry data, information such as sample ID, labeling agent, and flow cytometer instrument settings and acquisition parameters is assembled into a plate layout file using Ryvett software from Qognit, Inc. (Qognit, San Carlos, CA). This plate layout file can be imported into the flow cytometry software prior to acquisition. After generation of flow cytometry data, a flow cytometry standard (FCS) file can be analyzed and gated in the Ryvett software using a desired gating routine. After manual review of the automated gating, the raw data and metric calculations can be exported to a CSV file using predetermined criteria and data extraction routines. In specific embodiments, flow cytometry data can be analyzed using gating from WinList (Software House, Topsham, ME), Ryvett (Qognit, Redwood City, CA), FlowJo (FloJo LLC, Ashland, OR), or Kaluza (Beckman Coulter, Indianapolis, IN). This software can be used to examine cell populations within the TME to determine what cancer and immune cells are present in the TME, their state (e.g., inactive, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls.

[0038] As is understood in the art, gating trees can be used to analyze cell populations from flow cytometry experiments and further separate them into distinct populations as gating tree branches. Figures 1 and 2 show gating trees for automated metric extraction from predefined gating routines for identifying Tregs in a staining panel and for granzyme B or cytokine expression. Each gate (defined as G4, G9, G10, G17, etc. in Figures 1 and 2) consists of multiple regions that are combined using Boolean logic (e.g., "and," "or," and "not" statements) to create a gating scheme that includes, excludes, or combines the cell populations represented by the gates. In addition to measuring the presence of a given cell surface marker (e.g., a cell-type marker, immunomodulatory receptors (IMRs), or IMR ligands (IMR-L)) on a cell, the methods described herein can include quantitatively measuring the amount of the marker. For example, the amount of a marker can be measured directly as the number of equivalents of reference fluorophores (ERFs). See, e.g., Gaigalas et al. (2016) Journal of Research of the National Institute of Standards and Technology 121:264-281. In certain embodiments, if the amount of the measured marker (e.g., IMR or IMR-L, activation and / or exhaustion marker) exceeds a certain threshold, the subject will be determined to be likely to respond to an immunomodulatory agent.

[0039] (ii) Mass cytometry In certain embodiments, the labeled cells are analyzed using mass cytometry. Mass cytometry combines flow cytometry with mass cytometry. In contrast to flow cytometry, which distinguishes signals by measuring the fluorescence spectra of various reporters, mass cytometry utilizes probes (e.g., antibodies) conjugated to stable heavy metal isotopes using a mass cytometer such as the Time-Of-Flight (CyTOF®) system (Fluidigm, San Francisco, CA) (Bandura et al. (2009) Anal Chem. 81:6813-6822; Bjornson et al. (2013) Current Opinion in Immunology 25:484-494). For example, once labeled cells are obtained using methods known in the art, the labeled cells are analyzed by mass cytometry. Exemplary mass cytometers useful in the methods herein include, for example, the Helios® (Fluidigm, San Francisco, CA). Data acquired from the mass cytometer can be analyzed using, for example, mass cytometer software, such as CyTOF® software version 7.0 (Fluidigm, San Francisco, Calif.).

[0040] In certain embodiments, mass cytometry data can be analyzed using Conditional-Density Resampled Estimate of Mutual Information (DREMI), which can optionally be coupled with Conditional-Density Rescaled Visualization (DREVI) (Krishnaswamy et al. (2014) Science 346(6213): 1250689). Thus, similar to flow cytometry, mass cytometry can be used to interrogate cell populations within the TME to determine what cancer and immune cells are present in the TME, their state (e.g., inactive, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls. Additionally, mass cytometry can be used to perform single-cell genome sequencing to reveal, for example, mutations (e.g., somatic mutations) in single cells (e.g., immune or cancer cells).

[0041] (iii) Image cytometry In certain embodiments, the labeled cells are analyzed using image cytometry. Image cytometry can be used to measure many of the same parameters as flow cytometry, but in addition, image cytometry involves three-dimensional imaging using automated microscopy and computerized image processing and analysis, allowing the acquisition and identification of tens of thousands of cellular events based on fluorescence and / or morphological parameters. Thus, in contrast to flow cytometry, image cytometry can also evaluate cellular events by their actual images. An overview of imaging cytometry is provided by Barteneva et al. (2012) J Histochem Cytochem 60(10): 723-733. Exemplary imaging cytometers include the FlowSight® Imaging Flow Cytometer and the ImageStream® X Mk II Imaging Flow Cytometer (Luminex Corp., Austin, TX).

[0042] In certain embodiments, image cytometry can be used to examine cell populations within the TME to determine which cancer and immune cells are present in the TME, their state (e.g., inactive, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls. Furthermore, in certain embodiments, image cytometry can be used to assess morphological characteristics of cells (e.g., cancer cells and / or immune cells), colocalization of two proteins, binding of two cells (e.g., cancer cells and / or immune cells), visualization of immune synapse formation, and nuclear translocation. Furthermore, image cytometry can be used to perform single-cell genomic sequencing, e.g., to reveal mutations (e.g., somatic mutations) in single cells (e.g., immune cells or cancer cells). Image cytometry can also be combined with laser capture microdissection (LCM) for laser ablation mass cytometry and for proteomic and genomic or mRNA analysis.

[0043] (iv) Single Cell Technology (SCT) Single cell technology (SCT) can also be used according to the methods disclosed herein under various conditions (e.g., in the presence of immunomodulatory and / or anti-cancer drugs) to assess individual cells within the TME and their interactions with other cells. Single cells can be isolated and manipulated from the TME using several different methods, including fluidics-based, physics-based, electric-field-driven (e.g., dielectrophoresis (DEP)), optoelectronic tweezers (OET), and optical techniques, such as optical tweezers (see, e.g., Skelley et al. (2009) Nat. Methods 6:147-152; Thieleche et al. (1999) IEEE Eng. Med. Biol. Mag. 18:48-52; Taff et al. (2005) Anal Chem. 77:7976-7983; Juan et al. (2011) Nat. Photonics 5:349-356; and Mirsaidov et al. (2008) Lab Chip 8:2174-2181).

[0044] In certain embodiments, SCT is performed using a microfluidic chip. For example, SCT can be performed using a hydrodynamic-based microfluidic chip. In other embodiments, dielectrophoretic digital sorting captures single cells in dielectrophoretic (DEP) cages using a semiconductor-controlled array of electrodes on a microfluidic chip. In certain embodiments, SCT is performed using microfluidic slides (e.g., nCounter® by NanoString Technologies, Inc., Seattle, WA; Genesis System by Celsee®, Ann Arbor, MI). Single cells can be analyzed using several techniques, such as assessing growth rate (Cermak et al. (2016) Nat. Biotech 34:1052-1059), measuring cell membrane potential (Liu et al. (2017) Nano Lett. 17:2757-2764), assessing the cell's genome and / or transcriptome (Horgan (2011) Obstet. Gynaecol. 13:189-195), proteomics (Horgan, (2011), supra), and mass cytometry (Li et al. (2000) Trends Biotechnol. 18:151-160). In certain embodiments, cells are assessed using one or more of the aforementioned SCT techniques to determine their cell type, their state (e.g., inactive, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls.

[0045] V. Immunomodulatory Agents The system can also be used to determine whether cells isolated from the TME respond to the addition of immunomodulatory drugs alone or in combination with other anti-cancer drugs. Immunomodulatory agents suitable for use herein include any agent that can modulate immune cells, for example, by activating the immune system to kill tumor cells or by removing immune cell inhibitory signals from tumor cells. In certain embodiments, the cells are exposed to the immunomodulatory agent before being combined with the labeling agent. In certain embodiments, the immunomodulatory agent is a checkpoint inhibitor, which can be chosen from, for example, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0046] In certain embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that acts as an immune system checkpoint, suppressing or otherwise regulating T cell activity in a timely manner to prevent an overactive immune response. However, cancer cells can exploit this checkpoint by expressing a ligand, e.g., PD-L1, which interacts with PD-1 on the surface of T cells to shut down or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutics. Exemplary therapeutic methods utilizing PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994 and EP Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0047] In particular embodiments, the immunomodulatory agent is a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (e.g., CD80, also known as B7-1 and CD86) on the surface of antigen-presenting cells (but not cancer cells) leads to T cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Typical anti-CTLA-4 antibodies include U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; No. 109,003, No. 7,132,281, No. 6,207,156, No. 7,807,797, No. 7,824,679, No. 8,143,379, No. and 8,883,984, International (PCT) Publication Nos. WO 98 / 42752, WO 00 / 37504, and WO 01 / 14424, and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab. In certain embodiments, an immunomodulatory agent is administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (also known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205. Other immunomodulatory agents include, for example, anti-CD20 antibodies, such as Arzerra® (ofatumumab, GlaxoSmithKine), Rituxan® (rituximab, Genentech, Biogen), and Mabthera® (rituximab, Roche); and anti-CD52 antibodies, such as Campath® (alemtuzumab, Genzyme). Additional antibody-based immunomodulatory agents include those listed below in Table 3. For each specific antibody in Table 3, the type of cancer that the antibody or antibody-drug conjugate targets is also indicated.

[0048] [Table 3] JPEG2025172732000004.jpg248162

[0049] Once one or more immunomodulatory agents, alone or in combination with an anti-cancer drug (e.g., a compound described below), have been identified as conferring a positive outcome on cancer cells and / or immune cells within the TME, the immunomodulatory agent(s) can be administered to a subject, alone or in combination with the anti-cancer drug.

[0050] IX. Pharmaceutical Compositions and Administration of Immunomodulatory Agents For therapeutic uses, the immunomodulatory agent is preferably combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to such compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. As used herein, the term "pharmaceutically acceptable carrier" refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media or agents for pharmaceutical active substances is well known in the art.

[0051] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., sorbent ... agents) (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavors, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine glucan ... sorbic acid or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (e.g., sucrose or sorbitol); osmolality enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0052] In certain embodiments, the pharmaceutical composition may contain nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) Bioeng. Transl. Med. 1: 10-29). In certain embodiments, the pharmaceutical composition may comprise a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery vehicles, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Sustained-release formulations may comprise, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also comprise liposomes, which can be prepared by any of several methods known in the art.

[0053] Pharmaceutical compositions containing the immunomodulatory agents disclosed herein can be provided in unit dosage form and prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. A preferred route of administration is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as EDTA; a buffer, such as acetate, citrate, or phosphate; and an agent for adjusting osmolality, such as sodium chloride or glucose.

[0054] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Pharmaceutical preparations are preferably sterile. Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization by filtration can be performed before or after lyophilization and reconstitution. The compositions described herein may be administered locally or systemically. Administration will generally be parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, it is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0055] Generally, a therapeutically effective amount of an active ingredient, e.g., an immunomodulator, is in the range of 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 10 mg / kg. The dosage will depend on variables such as the type and severity of the disease or symptom being treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper limit to rapidly achieve the desired blood or tissue levels. Alternatively, the initial dosage may be suboptimal, with the daily dosage gradually increased over the course of treatment. Human dosages can be optimized in conventional Phase I dose-escalation studies designed to range, for example, from 0.5 mg / kg to 20 mg / kg. Dosage frequency can vary depending on factors such as the route of administration, dosage, serum half-life of the immunomodulator, and the disease being treated. Typical dosing frequencies are once daily, once weekly, and once every two weeks. A preferred route of administration is parenteral, eg, intravenous infusion. In certain embodiments, the immunomodulatory agent is lyophilized and then reconstituted with buffered saline at the time of administration.

[0056] VI. Therapy Once an appropriate immunomodulatory agent is selected for a given subject, alone or in combination with an anti-cancer drug, the subject can be treated according to normal healthcare practices. In particular, an effective amount of an immunomodulatory agent can be administered to a subject, alone or in combination with an effective amount of another anti-cancer drug. As used herein, the term "effective amount" refers to the amount of an active agent (e.g., an immunomodulatory agent) sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., halting its progression; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism to be treated with the methods and compositions described herein. The organism preferably includes, but is not limited to, a mammal (e.g., mouse, monkey, horse, cow, pig, dog, cat, etc.), and more preferably includes a human.

[0057] Examples of cancers that can be treated using the techniques described herein are provided in Section I. In particular embodiments, the cancer is a metastatic cancer. In particular embodiments, the cancer is a refractory cancer. As noted above, it is contemplated that an immunomodulatory agent can be administered alone or in combination with another anti-cancer or therapeutic agent. The term "administered in combination" herein is understood to mean delivering two (or more) different therapies to a subject so that the effects of the therapies overlap at some point during the course of the subject's suffering from a disorder. In certain embodiments, delivery of one treatment is still occurring when delivery of a second treatment begins, resulting in an overlap in administration. This is sometimes referred to herein as "co-delivery." In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In certain embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or than would be seen in a similar situation with the first treatment. In certain embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed with one treatment delivered in the absence of the other treatment. The effects of the two treatments can be partially additive, or wholly additive, or greater than additive. Delivery can be such that the effect of the first treatment is still detectable when the second treatment is delivered.

[0058] In certain embodiments, the immunomodulatory agent is administered in combination with one or more additional therapies, such as surgery, radiation therapy, or administration of another therapeutic agent. In certain embodiments, the additional therapy includes chemotherapy, such as a cytotoxic agent. In certain embodiments, the additional therapy includes a targeted therapy, such as a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy includes an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, such as a steroid, a biological immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In certain embodiments, the additional therapy includes a combination of therapeutic agents from different classes.

[0059] Exemplary anti-cancer drugs that can be administered in combination with the methods or compositions described herein include, for example, microtubule inhibitors, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA), These include: tamoxifen (MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating drugs, drugs capable of interfering with signal transduction pathways, drugs that promote apoptosis and radiation, or antibody molecule conjugates that bind to surface proteins and deliver toxins. In one embodiment, the cytotoxic agent that can be administered in the methods or compositions described herein is a platinum agent (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, a taxane (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomatin, thiazol-3, thiazol-4, thiazol-5, thiazol-6, thiazol-7, thiazol-8, thiazol-9, thiazol-10, thiazol-11, thiazol-12, thiazol-13, thiazol-14, thiazol-15, thiazol-16, thiazol-17, thiazol-18, thiazol-19, thiazol-20, thiazol-19 ... isin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.

[0060] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism to be treated with the methods and compositions of the invention, preferably a mammal (e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, non-human primate such as monkey, chimpanzee, baboon, and rhesus monkey), more preferably a human. As used herein, the term "treating" includes any effect that results in the improvement of a condition, disease, disorder, etc., for example, palliating, alleviating, regulating, arresting, slowing the progression of, ameliorating, or eliminating the condition, disease, disorder, etc., or ameliorating the symptoms thereof. For example, treating a cancer or tumor means reducing the growth of the cancer or tumor, modulating the cancer or tumor, stopping the growth of the cancer or tumor, slowing the progression of the cancer or tumor growth, or ameliorating or eliminating the growth of the cancer or tumor. Treating can be curing, ameliorating, or at least partially ameliorating the disorder, e.g., cancer. In particular embodiments, treating is curing a disease, e.g., cancer. The term "disorder" refers to, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0061] In applications where an element or component is included in and / or selected from a recited list of elements or components, it should be understood that it can be any one of the recited elements or components, or that the element or component can be selected from a group consisting of two or more of the recited elements or components. Throughout the description where compositions are described as having, including, or comprising specified ingredients, or processes and methods are described as having, including, or comprising specified steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the listed ingredients, and processes and methods of the invention that consist essentially of or consist of the listed processing steps. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Throughout the description where compositions and kits are described as having, including, or comprising specific ingredients, or processes and methods are described as having, including, or comprising specific steps, it is further contemplated that there are compositions and kits of the invention that consist essentially of or consist of the listed ingredients, and processes and methods of the invention that consist essentially of or consist of the listed processing steps. Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether or not expressly stated herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular compound is referenced, that compound can be used in various embodiments of the compositions of the invention and / or methods of the invention, unless the context clearly dictates otherwise. In other words, within the scope of this application, while embodiments have been described and depicted in a manner that allows a clear and concise application to be written or drawn, it is intended and will be recognized that the embodiments may be combined or separated in various ways without departing from the present teachings and invention. For example, it will be recognized that all features described and depicted herein are applicable to all aspects of the invention as described and depicted herein.

[0062] In this disclosure, unless the context is inappropriate, the parts of speech "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of that part of speech. By way of example, "an element" means one element or more than one element. In this disclosure, unless otherwise indicated, the term "and / or" is used to mean either "and" or "or." The phrase "at least one of" should be understood to include each of the listed objects following this phrase individually and various combinations of two or more of the listed objects, unless the context and usage require otherwise. The phrase "and / or" in connection with more than two listed objects should be understood to have the same meaning, unless the context requires otherwise. The terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should be understood as generally open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless specifically stated otherwise or the context requires otherwise. When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0063] For example, when the molecular weight of a polymer is provided and no absolute value is provided, the molecular weight should be understood to be an average molecular weight unless otherwise indicated or the context requires otherwise. Generally, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of that variable controls. It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously. For example, the use of any and all examples or exemplary language in this specification, such as "such as" or "including," is intended merely to better describe the invention and does not impose limitations on the scope of the invention, except as claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0064] The following examples further illustrate the present disclosure and should not be construed as limiting the disclosure to either the scope or spirit of the specific procedures described therein. It should be understood that the examples are provided to illustrate particular embodiments and are not intended to limit the scope of the disclosure thereby.

[0065] Example 1 - Determining the composition of the tumor microenvironment This example demonstrates the ability of the claimed method to measure the composition of the tumor microenvironment from solid tumors of the lung, breast, and kidney using both surface and intracellular markers. hi Tumors showing elevated levels of FoxP3+ Tregs suggest a tumor-mediated immunosuppressive signature and therefore a poor prognosis and objective response, which may aid in the development of patient treatment strategies. In this example, lung, breast, and kidney solid tumor samples were obtained and transported to Pierian Biosciences in Miltenyi Tissue Transport Buffer using a Therapak controlled-rate carrier maintained at 2-8°C. Upon receipt, tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI 1640 plus pen / strep). Tumor fragments were then mechanically dissociated into a single-cell suspension using enzymes from the Miltenyi Human Tumor Dissociation Kit and a Miltenyi gentleMACS™ Octo Dissociator and heater. The single-cell suspension was subsequently filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended at the desired concentration in RPMI 1640 + 10% FBS or 1x PBS + 0.5% BSA.

[0066] After resuspending the cells in the appropriate buffer, 80 μL was plated into designated wells of a deep-well 96-well plate (2 mL volume). Cells were then first stained with the amine-reactive dye Alexa750 for 15 minutes to distinguish between live and dead cells. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Subsequently, cells were fixed and permeabilized using the FOXP3 / Transcription Staining Buffer Kit from eBioscience, following the manufacturer's recommendations for processing and staining cells in deep-well plates. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Cells were then stained with a fluorescently labeled antibody cocktail to identify CD3+, CD4+, CD8+, and Tregs (CD4+CD25). hi FoxP3+). Stained cells were incubated for 1 hour at ambient temperature in the dark. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer and then fixed with 1% final concentration of PFA.

[0067] Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information, such as sample ID, antibody staining panel, and predefined routine flow cytometer instrument settings and acquisition parameters, was incorporated into a 96-well plate layout using Ryvett software (Qognit, Inc.). This plate layout file was imported into the Attune NxT software prior to acquisition. After acquisition, FCS files were automatically analyzed and gated using predefined gating routines in the Ryvett software. After manual review of the automated gating, raw data and metric calculations were exported to a CSV file using predefined criteria and data extraction routines. The results are shown in Figures 4A-4B. Figure 4A shows the distribution of CD3+, CD4+, CD8+, and Tregs (CD4+CD25+). hi Figure 4B shows a representative flow cytometry plot showing the detection of CD3+ T cells (expressed as a percentage of CD45+ leukocytes), CD4+ and CD8+ T cells (expressed as a percentage of CD3+ T cells), and Tregs (expressed as a percentage of CD4+ T cells). These results demonstrate that the methods described herein can be used to detect the presence and quantity of specific types of immune cells within the tumor microenvironment. Understanding the composition of immune cells within the tumor microenvironment, for example, the ratio of CD4+ / CD8+ cells and CD8+ / Tregs, is important for determining the likelihood of a patient responding to a given immunotherapy.

[0068] Example 2 - Functional capacity of cells from dissociated tumors is assessed by surface and intracellular staining of Targets in basal and induced states This example demonstrates the ability of the claimed method to measure functional capacity by measuring both basal and induced levels of functional readouts in immune cells identified from solid tumors of the breast, lung, and kidney, using both surface and intracellular markers. In this example, lung, breast, and kidney solid tumor samples were obtained and transported to Pierian Biosciences in Miltenyi Tissue Transport Buffer using a Therapak controlled-rate carrier maintained at 2-8°C. Upon receipt, tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI 1640 plus pen / strep). Tumor fragments were then mechanically dissociated into a single-cell suspension using enzymes from the Miltenyi Human Tumor Dissociation Kit and a Miltenyi gentleMACS™ Octo Dissociator and heater. The single-cell suspension was subsequently filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended at the desired concentration in RPMI 1640 + 10% FBS or 1x PBS + 0.5% BSA.

[0069] After resuspending the cells in the appropriate buffer, 80 μL was plated into designated wells of a deep-well 96-well plate (2 mL volume). Cells were then first stained with the amine-reactive dye Alexa750 for 15 minutes to distinguish live from dead cells. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Cells were then conditioned for 3 hours with Leukocyte Activation Cocktail with BD GolgiPlug™ from Becton Dickinson. Leukocyte Activation Cocktail with BD GolgiPlug™ is a ready-to-use polyclonal cell activation mixture containing phorbol ester, PMA (phorbol 12-myristate 13-acetate), calcium ionophore (ionomycin), and the protein transport inhibitor BD GolgiPlug™ (brefeldin A). After 3 hours, cells were subsequently fixed and permeabilized using the FOXP3 / Transcription Staining Buffer Kit from eBioscience according to the manufacturer's recommendations for processing and staining cells in deep-well plates. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Cells were then stained with a fluorescently labeled antibody cocktail to identify CD3+, CD4+, CD8+, and Tregs (CD4+CD25). hi FoxP3+). Stained cells were incubated for 1 hour at ambient temperature in the dark. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer and then fixed with 1% final concentration of PFA.

[0070] Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information, such as sample ID, antibody staining panel, and predefined routine flow cytometer instrument settings and acquisition parameters, was incorporated into the 96-well plate layout using Ryvett software (Qognit, Inc.). After acquisition, FCS files were automatically analyzed and gated using predefined gating routines in the Ryvett software. After manual review of the automated gating, raw data and metric calculations were exported to a CSV file using predefined criteria and data extraction routines. The results are shown in Figures 5-6. Figure 5 shows representative flow cytometry plots demonstrating the detection of basal and induced IFNγ and TNFα in CD4+ and CD8+ T cells, as well as associated dot plots showing the expression of IFNγ and TNFα (expressed as percent positive). FIG. 6 shows representative flow cytometry plots demonstrating the detection of granzyme B expression in CD4+ and CD8+ T cells from breast, lung, and kidney tumors.

[0071] IFNγ and TNFα are cytokines that suggest an "inflammatory" tumor with the potential for a greater immune response, with elevated levels of IFNγ and TNFα during initial immunotherapy and subsequent follow-up associated with primary and secondary responses, respectively. Granzyme B is an effector molecule found on CD8+ cells and is part of the granzyme B-perforin complex used by CD8+ cells to kill target tumor cells. Decreased levels of granzyme B are associated with reduced CD8+ cytotoxic potential, suggesting T cell exhaustion. Measuring the percentage of CD8+ cells expressing granzyme B in tumors and quantitative levels of granzyme B expression are useful in predicting response to immunotherapy and developing appropriate treatment guidelines for patients.

[0072] Example 3 - Evaluation of immune checkpoint and exhaustion markers and their cognate ligands from solid tumors In this example, we measured the expression levels of immune checkpoint / exhaustion markers and their cognate ligands on TILs and tumor epithelial cells. Immunotherapy treatment currently relies on a single IHC measurement of the IMR ligand PD-L1 expression on tumor cells as a treatment guide, but this does not fully identify IMR and IMR-L expression on any other cellular components within the tumor microenvironment. The holistic approach described herein can improve patient stratification and treatment guidance. In this example, lung, breast, and kidney solid tumor samples were obtained and transported to Pierian Biosciences in Miltenyi Tissue Transport Buffer using a Therapak controlled-rate carrier maintained at 2-8°C. Upon receipt, tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI 1640 plus pen / strep). Tumor fragments were then mechanically dissociated into a single-cell suspension using enzymes from the Miltenyi Human Tumor Dissociation Kit and a Miltenyi gentleMACS™ Octo Dissociator and heater. The single-cell suspension was subsequently filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended at the desired concentration in RPMI 1640 + 10% FBS or 1x PBS + 0.5% BSA.

[0073] After resuspending the cells in the appropriate buffer, 80 μL was plated into designated wells of a deep-well 96-well plate (2 mL volume). Cells were then first stained with the amine-reactive dye Alexa750 for 15 minutes to distinguish live from dead cells. Cells were then washed twice with staining buffer (1x PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Cells were then stained with a fluorescently labeled antibody cocktail to detect CD326+, CD45+, CD3+, CD4+, CD8+, CD19+, CD56+, and CD14+ cells. Additional labeled antibodies were used to identify the following IMRs or IMR-Ls: CD73, CD112, CD155, CD172ab, CD274, CD279, CD366, TIGIT, and TIM-3. The stained cells were incubated in the dark at ambient temperature for 20 minutes. Cells were then washed twice with staining buffer (1× PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer before being fixed in PFA at a final concentration of 1%. Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information, such as sample ID, antibody staining panel, and predefined routine flow cytometer instrument settings and acquisition parameters, was incorporated into the 96-well plate layout using Ryvett software (Qognit, Inc.). After acquisition, FCS files were automatically analyzed and gated using predefined gating routines in the Ryvett software. After manual review of the automated gating, raw data and metric calculations were exported to a CSV file using predefined criteria and data extraction routines.

[0074] As shown in Figure 7, CD45+ leukocytes, CD326+ epithelial tumor cells, CD14+ monocytes / macrophages, and CD4+ and CD8+ T cells were detected within the tumor microenvironment. An example of inhibitory checkpoint receptor expression is shown in Figure 8. In the upper figure, inhibitory IMR TIGIT was detected on a subset of cells within the tumor microenvironment. Specifically, TIGIT was detected on CD4+, CD8+, and CD14+ leukocytes, but not on CD326+ tumor cells. The functional status of TILs was characterized by measuring the levels of IMR / exhaustion markers and their ligands (CD279 / CD274 [PD1 / PD-L1], TIGIT / CD112-CD155, CD366 [TIM-3] / Galectin-9, CD172a [SIRPa] / CD47, and CD73). As shown in Figures 9–11, significant heterogeneity across cell types and tumor types (breast, lung, and kidney) was observed, with separation of IMR / IMR-L-positive TILs from negative TILs and separation of IMR / IMR-L-positive tumor cells from negative tumor cells. This included upregulation of several IMR-Ls on TILs and IMRs on endothelial and stromal cells, suggesting tumor- and TIL-intrinsic mechanisms regulating checkpoint interactions. These results demonstrate that the methods described herein can be used to identify IMR / exhaustion markers and their ligands on specific cell types within the tumor microenvironment. Understanding the composition of IMR / exhaustion markers and their ligands on specific cell types (e.g., immune cells) within the tumor microenvironment is important for determining a patient's likelihood of responding to prescribed immunotherapy.

[0075] Example 4 - Treatment of patients with immunomodulatory drugs Tumor samples are received from patients and evaluated according to the methods described in Examples 1-3. A panel of IMR / exhaustion markers and their ligands (CD279 / CD274 [PD1 / PD-L1], TIGIT / CD112-CD155, CD366 [TIM-3] / Galectin-9, CD172a [SIRPa] / CD47, CD73) is measured to characterize the functional status of TILs. Results indicate the presence of PD-1 (CD279) on T cells and PD-L1 (CD274) on one or more of dendritic cells, macrophages, and tumor cells. Treatment of subjects with anti-PD-L1 or anti-PD-1 antibodies is believed to result in tumor regression.

[0076] Example 5 - Treatment of patients with a combination of immunomodulatory drugs Tumor samples are received from patients and evaluated according to the methods described in Examples 1-3. IMR / exhaustion markers and their ligands (CD279 / CD274 [PD1 / PD-L1], TIGIT / CD112-CD155, CD366 [TIM-3] / Galectin-9, CD172a [SIRPa] / CD47, CD73) are measured to determine the functional status of TILs. Results indicate the presence of PD-1 (CD279) on T cells and PD-L1 (CD274) on one or more of dendritic cells, macrophages, and tumor cells. Results further indicate the presence of the immune checkpoint protein TIGIT on T cells and NK cells and the corresponding ligands CD112 and CD155 on one or more of dendritic cells, macrophages, and tumor cells. It is believed that tumor regression may occur if subjects are treated with anti-PD-L1 or both an anti-PD-1 antibody and an anti-TIGIT antibody.

[0077] Incorporation by Reference The entire disclosure of each of the patent and scientific documents referenced herein is incorporated by reference for all purposes.

[0078] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments, therefore, are to be considered in all respects as illustrative and not limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

Claims

1. 1. A method for measuring the composition of a solid tumor microenvironment, comprising the steps of: (a) combining a single cell suspension of cells derived from a solid tumor with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, allowing the agents to bind to cancer cells, immune cells, or both cancer cells and immune cells simultaneously present in the single cell suspension to generate labeled cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls); and (b) measuring the presence and / or amount of the labeled cells by cytometry, thereby (i) determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the microenvironment of the solid tumor, and (ii) determining whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one of the IMRs and / or at least one of the IMR-Ls, thereby determining the solid tumor microenvironment. The method comprising:

2. 2. The method of claim 1, wherein the cytometry is selected from the group consisting of flow cytometry, mass cytometry, image cytometry, and single cell technology (SCT).

3. 3. The method of claim 1, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

4. 4. The method of any one of claims 1 to 3, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

5. The method of any one of claims 1 to 4, wherein the cell surface markers further comprise a cell activation marker.

6. 6. The method of claim 5, wherein the cell type markers include markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

7. The method according to any one of claims 1 to 6, wherein the cell type marker is a cancer cell marker.

8. 8. The method of claim 7, wherein the cancer cell markers include CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

9. 6. The method of claim 5, wherein the cell activation markers comprise CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197), and / or functional markers such as IFNγ, TNFα, and / or other cytokines, and / or granzyme B.

10. The IMR or IMR-L marker is PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, 10. The method of any one of claims 1 to 9, comprising CD39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and / or CD115 (CSF-IR).

11. 11. The method of any one of claims 1 to 10, further comprising combining the cells with an immunomodulatory agent.

12. 12. The method of claim 11, further comprising measuring the effect of the immunomodulatory drug on the expression of at least some of the cell markers on the cancer cells and / or immune cells.

13. 1. A method for determining whether a subject having a solid tumor is likely to respond to an immunomodulatory drug, comprising the steps of: (a) combining a single cell suspension of cells derived from a solid tumor with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, allowing the agents to bind to cancer cells, immune cells, or both cancer cells and immune cells simultaneously present in the single cell suspension to generate labeled cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls); (b) combining at least a portion of said single cell suspension of cells with an immunomodulatory agent; and (c) (i) determining the presence and / or amount of the labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and determining whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one of the IMRs and / or at least one of the IMR-Ls; and (ii) determining the effect of the immunomodulatory agent on the cell markers on or in the cancer cells, immune cells, or both the cancer cells and immune cells, thereby determining whether the subject is likely to respond to the immunomodulatory agent. The method comprising:

14. 14. The method of claim 13, wherein the immunomodulatory agent is combined with the single cell suspension before, during, or after step (a).

15. 15. The method of claim 13 or claim 14, wherein the cytometry is selected from the group consisting of flow cytometry, mass cytometry, image cytometry, and single cell technology (SCT).

16. 16. The method of any one of claims 13 to 15, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

17. 17. The method of any one of claims 13 to 16, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

18. The method of any one of claims 13 to 17, wherein the cell surface markers further comprise a cell activation marker.

19. 19. The method of any one of claims 13 to 18, wherein the cell type markers comprise markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

20. The method of any one of claims 13 to 19, wherein the cell type marker is a cancer cell marker.

21. 21. The method of claim 20, wherein the cancer cell markers include CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

22. 19. The method of claim 18, wherein the cell activation markers comprise CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197), and / or functional markers such as IFNγ, TNFα, or other cytokines, and / or granzyme B.

23. The IMR or IMR-L marker is PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, 23. The method of any one of claims 13 to 22, comprising CD39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and CD115 (CSF-1R).

24. 1. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject an effective amount of an immunomodulatory agent, thereby treating the solid tumor, wherein the immunomodulatory agent comprises the steps of: (a) combining a single cell suspension of cells derived from a solid tumor with multiple labeling agents capable of binding to multiple corresponding cell surface markers expressed on cancer cells and / or immune cells, allowing the agents to bind to cancer cells, immune cells, or both cancer cells and immune cells simultaneously present in the single cell suspension to generate labeled cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls); (b) combining at least a portion of said single cell suspension of cells with an immunomodulatory agent; and (c) (i) determining the presence and / or amount of the labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and determining whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one of the IMRs and / or at least one of the IMR-Ls; and (ii) determining the effect of the immunomodulatory agent on the cell markers on or in the cancer cells, immune cells, or both cancer cells and immune cells, thereby determining whether the subject is likely to respond to the immunomodulatory agent. The method is selected by utilizing a method comprising:

25. 25. The method of claim 24, wherein the immunomodulatory agent is combined with the single cell suspension before, during, or after step (a).

26. 26. The method of claim 24 or claim 25, wherein the cytometry is selected from the group consisting of flow cytometry, mass cytometry, image cytometry, and single cell technology (SCT).

27. 27. The method of any one of claims 24 to 26, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

28. 28. The method of any one of claims 24 to 27, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

29. The method of any one of claims 24 to 27, wherein the cell surface markers further comprise a cell activation marker.

30. 30. The method of any one of claims 24 to 29, wherein the cell type markers comprise markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

31. The method of any one of claims 24 to 30, wherein the cell type marker is a cancer cell marker.

32. 32. The method of claim 31 , wherein the cancer cell markers include CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

33. 30. The method of claim 29, wherein the cell activation markers comprise CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197), and / or functional markers such as IFNγ, TNFα, and / or other cytokines, and / or granzyme B.

34. The IMR or IMR-L marker is PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, C 34. The method of any one of claims 24 to 33, comprising D39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and / or CD115 (CSF-IR).

35. The method of any one of claims 1 to 34, wherein a plurality of different labeled cells are detected simultaneously during cytometry.

36. The method of any one of claims 1 to 35, wherein a plurality of different cell surface markers are detected simultaneously during cytometry.

37. 37. The method of claim 36, wherein at least 14 different cell surface markers are detected simultaneously.

38. 38. The method of any one of claims 1 to 37, wherein receptor-ligand interactions between said labeled cells can be detected and optionally quantified.

39. 39. The method of claim 38, wherein the receptor-ligand interaction comprises the interaction between a checkpoint inhibitor and its cognate ligand.

40. 40. The method of claim 39, wherein the receptor-ligand interaction can be selected from the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM-3 and Gal9, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, and / or CD-40L and CD40.

41. 41. The method of any one of claims 1 to 40, wherein the presence and / or amount of a cell activation marker, an IMR marker, an IMR-L marker, or a combination of an activation marker and an IMR and / or an IMR-L marker expressed on the cancer cells and / or immune cells is measured.

Images