Immune cell organoid co-cultures

Abstract

This invention provides co-cultures of organoids and immune cells, and methods for using these co-cultures to identify reagents for treating diseases.

Description

[0001] All references cited in this article are incorporated in their entirety through citation. Technical Field

[0002] This invention relates to organoid co-cultures and their use in disease research. Background Technology

[0003] While in vitro systems used for such research remain fundamental, clinical research on physiologically based diseases, such as cancer and immune diseases, remains the cornerstone of medical progress. Similarly, modern protocols for treating such diseases typically involve rigorous testing systems during development to ensure efficacy and safety. Although recent advances in these areas have improved the effectiveness of research and therapeutic testing systems, improvements are needed in terms of system efficiency, accuracy, and cost-effectiveness. An ideal testing system would precisely replicate the physiological condition of a patient or patient population at the biochemical, cellular, tissue, organ, and organismal levels without requiring direct testing on the patient and minimizing the use of patient samples. A wide variety of processing reagents and schedules must be accommodated within a single system.

[0004] Screening candidate compounds requires in vitro models to identify new regimens for researching and treating cancer and immune diseases at the population level. Furthermore, there is a growing interest in personalized medicine, where in vitro models can be used to test (sometimes pre-approved) regimens in patient subgroups with specific characteristics, or even samples from individual patients, to determine the optimal regimen for that particular subgroup or patient.

[0005] The field of organoid technology is revolutionizing our understanding of developmental biology. Organoids are cellular structures derived from the expansion of epithelial cells and are composed of tissue-specific cell types that are self-organized through lineage guarantees of cell sorting and spatial constraints (Clevers, Cell. 2016-06-16; 165(7):1586-1597). A limitation of existing organoid-based models is that they contain only epithelial cells and therefore cannot fully represent in vivo tissue systems containing multiple cell types. In particular, there is no description of 'co-cultures' of human cancer organoids (“tumor-like”) and immune cells, and certainly no description of cases where both cancer and immune cells are derived from the same patient. Immune cells improve the accuracy of organoids as test systems, replicate the patient's physiological state, and ensure that the immune system is represented in the test system.

[0006] Previous attempts have demonstrated the co-culture of murine intraepithelial lymphocytes (IELs) with murine intestinal epithelial organoids to understand the spatiotemporal behavior of IELs and intestinal epithelial cells—Nozaki et al. (J Gastroenterol. 2016 Mar; 51(3):206-13) and Rogoz et al. (J Immunol Methods. 2015 Jun; 421:89-95)—but there has been no report on the progress in developing human organoid co-cultures and their applications in cancer research and treatment. So-called 'tumor-like' cells have been prepared from samples derived from colorectal cancer patients (Drost et al., Nature. 2015 May 7; 521(7550):43-7; van de Wetering et al., Cell. 2015 May 7; 161(4):933-45), but have not yet been co-cultured with immune cells to investigate cancer treatment options.

[0007] There is a need for improved methods for preparing organoid and tumor-like cocultures and for using these cocultures in drug screening, particularly in systems where the interaction between disease cells and immune cells can be utilized to study an increased range of drugs with high throughput capabilities. Invention Overview

[0009] The inventors of this invention have developed organoid co-cultures that can be used for research related to diseases such as cancer and immune diseases, including the identification of suitable treatments for such diseases. In some embodiments, this involves preparing co-cultures of organoids and immune cells (particularly disease organoids such as tumor-like cells) and immune cells, which can be exposed to candidate reagents for treating the disease and any changes can be detected for the identification of suitable candidate reagents.

[0010] Therefore, the present invention provides a method for identifying reagents suitable for treating cancer, wherein the method comprises:

[0011] A tumor-like co-culture is contacted with one or more candidate reagents, wherein the tumor-like co-culture comprises immune cells and at least one tumor type.

[0012] Detecting the presence of one or more alterations in tumor-like cocultures that indicate a candidate reagent is suitable for treating the cancer, and

[0013] If the presence or absence of one or more of the aforementioned changes is detected in the tumor-like coculture, the candidate reagent is identified as suitable for treating the cancer.

[0014] In some embodiments, the method further includes comparing the presence or absence of said one or more variations in the tumor-like co-culture with a reference organoid or reference tumoroid, and wherein said method further includes:

[0015] Contact a reference organoid co-culture or a reference tumor-like co-culture with one or more candidate reagents, wherein the reference organoid co-culture or the reference tumor-like co-culture comprises immune cells and at least one organoid or tumor-like cell.

[0016] Detect the presence of one or more variations in the reference organoid co-culture or the reference tumor co-culture that indicate the suitability of candidate reagents for treating the cancer.

[0017] The present invention also provides a method for identifying reagents suitable for treating immune diseases, wherein the method comprises:

[0018] The organoid co-culture is contacted with one or more candidate reagents, wherein the organoid co-culture comprises diseased immune cells and at least one organoid.

[0019] Detecting the presence of one or more changes in the organoid co-culture indicating that candidate reagents are suitable for treating the immune disease, and

[0020] If the presence or absence of one or more of the aforementioned changes is detected in the organoid co-culture, the candidate reagent is identified as suitable for treating the immune disease.

[0021] In some embodiments, the method further includes comparing the presence or absence of one or more variations of the organoid co-culture with reference immune cells (e.g., from a control patient lacking the immune disease), and wherein the method further includes:

[0022] Contact a reference organoid co-culture with one or more candidate reagents, wherein the reference organoid co-culture comprises immune cells and at least one organoid.

[0023] Detect the presence of one or more variations in the reference organoid co-culture that indicate the suitability of candidate reagents for treating the immune disease.

[0024] Methods for testing the efficacy and / or safety of CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors in the treatment of epithelial cancer are also provided, the methods comprising:

[0025] Tumor epithelial cells, normal epithelial cells, and immune cells can be optionally provided from the same patient.

[0026] The tumor epithelial cells are expanded in a tumor-like culture medium to form a tumor-like organism, and the tumor-like organism and the immune cells are cultured in a tumor-like co-culture medium containing interleukin to form a tumor-like co-culture.

[0027] The normal epithelial cells were expanded in organoid culture medium to form organoids, and the organoids and the immune cells were cultured in organoid co-culture medium containing interleukin to form a reference organoid co-culture.

[0028] The tumor-like co-culture and the reference organoid co-culture are contacted with the CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors. The presence or absence of one or more changes in the tumor-like co-culture and the reference organoid co-culture is detected, wherein the presence or absence of such one or more changes indicates the efficacy and / or safety of the CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors.

[0029] Compare the tumor-like co-culture with the reference organoid co-culture.

[0030] Methods for testing the efficacy and / or safety of candidate compounds in the treatment of epithelial cancer are also provided, the methods comprising:

[0031] Tumor epithelial cells, normal epithelial cells, and immune cells may be provided from the same patient. The tumor epithelial cells are expanded in a tumor-like culture medium to form a tumor-like organism, and the tumor-like organism and the immune cells are cultured in a tumor-like co-culture medium containing interleukin to form a tumor-like co-culture.

[0032] The normal epithelial cells were expanded in organoid culture medium to form organoids, and the organoids and the immune cells were cultured in organoid co-culture medium containing interleukin to form a reference organoid co-culture.

[0033] The tumor-like co-culture and the reference organoid co-culture are then contacted with the candidate compound.

[0034] The presence or absence of one or more changes in the tumor-like co-culture and the reference organoid co-culture, wherein the presence or absence of said one or more changes indicates the efficacy and / or safety of the candidate compound, and

[0035] Compare the tumor-like co-culture with the reference organoid co-culture.

[0036] A method for preparing organoid immune cell co-cultures is also provided, wherein the method includes:

[0037] Optionally, epithelial cells in contact with the extracellular matrix are cultured in organoid culture medium to obtain organoids;

[0038] Remove the extracellular matrix and the organoid culture medium from the organoid;

[0039] The organoids were resuspended in a culture medium for immune cells supplemented with interleukin;

[0040] Prepare an immune cell suspension comprising immune cells, an immune cell culture medium supplemented with interleukin, and collagen at a concentration of at least 5-10% in the suspension; and

[0041] The immune cell suspension containing immune cells is mixed with the resuspended organoids.

[0042] A method for preparing a tumor immune cell co-culture is also provided, wherein the method includes:

[0043] Optionally, tumor epithelial cells in contact with the extracellular matrix are cultured in tumor-like culture medium to obtain organoids;

[0044] Remove the extracellular matrix and the tumor-like culture medium from the tumor-like tumor;

[0045] The tumor-like cells were resuspended in a culture medium supplemented with interleukin;

[0046] Prepare an immune cell suspension comprising immune cells, an immune cell culture medium supplemented with interleukin, and collagen at a concentration of at least 5-10% in the suspension; and

[0047] The immune cell suspension containing immune cells is mixed with a resuspended tumor-like substance.

[0048] A method for testing therapeutic agents is also provided, wherein the method includes:

[0049] The organoid co-culture is contacted with one or more candidate reagents, wherein the organoid co-culture comprises immune cells and at least one organoid.

[0050] Detecting the presence of one or more changes in the organoid co-culture that indicate therapeutic efficacy, and

[0051] If the presence or absence of one or more of the aforementioned changes is detected in the organoid co-culture, the candidate reagent is identified as a therapeutic agent.

[0052] Organoid co-cultures that can be obtained or acquired by the method of the present invention are also provided.

[0053] Tumor-like cocultures that can be obtained or acquired by the method of the present invention are also provided.

[0054] Organoids that can be obtained or acquired by the method of the present invention are also provided.

[0055] The invention also provides tumor-like groups that can be obtained or acquired through the method of the present invention.

[0056] An organoid co-culture medium suitable for use in the method of the present invention is also provided.

[0057] Tumor-like co-culture media and organoid co-culture media suitable for use in the methods of the present invention are also provided. Tumor-like or organoid cultures in a medium containing interleukin are also provided, optionally wherein the interleukin is selected from IL-2, IL-7, and IL-15.

[0058] The present invention also provides a kit comprising the tumor-like, organoid, tumor-like co-culture or organoid co-culture of the present invention. Brief description of the attached diagram

[0060] Figure 1 Organoids, tumor-like cells, and T cells are derived from the primary patient's tissue.

[0061] Figure 1 A. Schematic diagram of the procedure. Biopsies of normal colonic mucosa and tumor tissue were obtained from the resected colon and / or rectum of patients with colorectal cancer. Peripheral blood was also collected during the procedure. Normal colonic mucosa was treated with EDTA to release crypts for the derivation of normal colonic organoids and further digested to prepare a single-cell suspension containing intraepithelial lymphocytes (IELs) for T-cell culture. Tumor tissue was digested to prepare a single-cell suspension containing epithelial tumor cells for the derivation of tumoroids and tumor-infiltrating lymphocytes (TILs) for T-cell culture. Peripheral blood was treated to purify peripheral blood mononuclear cells rich in peripheral blood lymphocytes (PBLs) and T cells. Preliminary analysis was performed by T-cell receptor (TCR) sequencing and immunophenotyping of T cells, as well as single-cell messenger RNA (mRNA) sequencing of cells present in the single-cell suspensions of normal colonic epithelium and tumor epithelium. Organoid cultures were analyzed using whole-genome sequencing, mRNA sequencing, and peptidome mapping.

[0062] Figure 1B. Representative bright-field images of normal colonic organoids and tumor-like cells derived from patient samples. Colonic crypts were embedded in basement membrane extract (BME) and cultured in a medium containing: R-vertebral protein 1, cephalin, Wnt3A conditioned medium, vitamin A-free B27 supplement, nicotinamide, N-acetylcysteine, EGF, TGF-β inhibitor A-83-01, gastrin, p38MAPK inhibitor SB202190, and prostaglandin E2. Normal colonic organoids developed within 1 week and were then passaged weekly (above). Single-cell suspensions from colorectal cancer samples were embedded in basement membrane extract (BME) and cultured in a medium containing: R-vertebral protein 1, cephalin conditioned medium, vitamin A-free B27 supplement, nicotinamide, N-acetylcysteine, EGF, TGF-β inhibitor A-83-01, gastrin, p38MAPK inhibitor SB202190, and prostaglandin E2. The tumor-like structure forms within one week and is then passaged weekly (see figure below).

[0063] Figure 1 C. Representative bright-field images of clonal growth halos of intraepithelial lymphocytes (IEL) and tumor-infiltrating lymphocytes (TIL) derived from patient samples (left panel). Flow cytometry analysis showed robust expansion of CD4+ helper T (Th) cells and CD8+ cytotoxic T cells (CTLs). Single-cell suspensions from normal colonic mucosa or colorectal cancer tissue were maintained in T cell culture medium containing interleukin-2 (IL-2). Clonal growth halos of T cells were significant within 1–2 weeks (left panel).

[0064] Figure 1 As further described in Example 1.

[0065] Figure 2 Validation of the principle of co-culturing normal colon organoids and allogeneic CD3+ T cells in basement membrane extract (BME) droplets.

[0066] Figure 2A. Schematic diagram of the procedure. Normal colonic organoids were released from BME droplets using cell recovery solution and washed in fully advanced DMEM / F12. Expanded CD3+ T cells were harvested from the culture and labeled with green dye (Vybrant CFDA SE cell tracer). The colonic organoids and labeled T cells were mixed in human colonic organoid medium and embedded in BME droplets. The co-culture was maintained in human colonic organoid medium containing IL-2 for 60 hours. The co-culture was released from BME using cell recovery solution and fixed in 4% paraformaldehyde. The fixed whole-mounts were stained with phalloidin to label polymerized actin and the nuclei were labeled with DAPI. The whole-mounts were mounted onto a slide in ProLong Gold anti-quenching mounting medium and imaged on a Leica SP8X confocal microscope.

[0067] Figure 2 B. Maximum projection of z-stack image of colonic organoid coculture. F-actin in the organoids is labeled in dark gray, while T cells are labeled in light gray. The inset in the right figure shows T cells infiltrating the colonic epithelium.

[0068] Figure 2 C. Three-dimensional reconstruction of normal colonic organoids and T cells.

[0069] Figure 2 This is further described in Example 8.

[0070] Figure 3 In vivo imaging of tumor-like co-cultures was performed to assess optimal T cell motility.

[0071] Figure 3 A. Schematic diagram of the procedure. Tumor-like cells were released from BME droplets using a cell recovery solution and washed in fully advanced DMEM / F12. Allogeneic CD8+ T cells isolated from peripheral blood samples were labeled with a green dye (Vybrant CFDA SE cell tracer). The tumor-like cells and T cells were mixed with human colon organoid culture medium containing IL-2 and 10% BME or rat tail type I collagen and in vivo imaging was performed for 80 hours at 37°C and 5% CO2 using a Leica SP8X confocal microscope equipped with an in vivo imaging chamber.

[0072] Figure 3 B. Representative composite image of tumor-like co-cultures. Bright-field and green fluorescence channels were combined to produce composite images. T cell migration pathways were traced using Imaris software.

[0073] Figure 3C. Quantitative analysis of T cell trajectory length under both conditions showed that T cells co-cultured in 10% collagen had significantly longer trajectory paths compared to those in 10% BME.

[0074] Figure 3 This is explained in more detail in Example 10.

[0075] Figure 4 The generation of human leukocyte antigen (HLA) A2 positive and negative clonal tumors.

[0076] Figure 4 A. Schematic diagram of the procedure. Tumor-like cells are isolated into single cells using TrypLE enzyme digestion. Single cells are stained with anti-HLA-A2 antibody and purified based on anti-HLA-A2 immunoreactivity. HLA-A2... +ve and HLA-A2 -ve Tumor cells are embedded and maintained to produce tumor-like structures.

[0077] Figure 4 B. Flow cytometry analysis revealed the establishment of pure HLA-A2+ve or HLA-A2-ve tumor cell lines. Controls included the HLA-A2+ve JY cell line and a normal colonic organoid line derived from the same patient sample as the HLA-A2+ve or HLA-A2-ve tumor cell lines.

[0078] Figure 4 This is further explained in Example 11.

[0079] Figure 5 The cytotoxicity of T cells that have undergone antigen-mediated antitumor response was measured.

[0080] Figure 5 A. Schematic diagram of the procedure. HLA-A2+ve or HLA-A2-ve tumors were pulsed with HLA-A2-restricted Wilms tumor 1 (WT1) peptide for 2 hours. Then, TCR transgenic CD8+ T cells carrying WT1 peptide-specific TCRs were co-cultured with HLA-A2+ve or HLA-A2-ve tumors pulsed with WT1 peptide for 48 hours.

[0081] Figure 5 B. Representative bright-field images of the co-cultures after 48 hours showed significant death of HLA-A2+ve tumors treated with WT1 peptide pulses alone. All other cases (i.e., HLA-A2+ve or HLA-A2-ve tumors without WT1 peptide pulses and HLA-A2-ve tumors treated with WT1 peptide pulses) showed normal growth.

[0082] Figure 5 This is explained in more detail in Example 12.

[0083] Figure 6 A cell viability assay for antitumor-like reactivity of antigen-experienced T cells with and without checkpoint inhibition.

[0084] Figure 6 A. A schematic diagram of the program. (e.g.) Figure 5 As described in section A, co-culture was performed, but only for 12 hours, with and without anti-PD1 checkpoint inhibitors. Cell viability was determined using the CellTiter Glo luminescent cell viability assay kit (Promega) according to the manufacturer's instructions.

[0085] Figure 6 B. Normalize the viability of tumor-like cells compared to the peptide-free control.

[0086] Figure 6 This is explained in more detail in Example 13.

[0087] Figure 7 Determining the differential effects on T cell activation through organoid / tumoroid co-culture.

[0088] Figure 7 A. A schematic diagram of the procedure. Using dispersing enzymes from... Tumor-like organisms were released from droplets and subsequently passed through 70 μm and 20 μm filters. Organoids were recovered from the 20 μm filter, counted, and plated. Tumor-like organisms and T cells were then mixed with RPMI, IL-2, and 5% [a specific drug / injection]. The human colon organoid culture medium was mixed and incubated at 37°C and 5% CO2. After 24 hours of incubation, the organoids were imaged using a bright-field inverted microscope.

[0089] Figure 7 B. Representative images of tumor-like co-cultures.

[0090] Figure 7 C. Representative images of organoid co-cultures.

[0091] Figure 7 D. Quantification of IFN-γ levels in co-cultures.

[0092] Figure 7 This is further explained in Example 14.

[0093] Figure 8 In vivo imaging of tumor-like co-cultures used to assess correlation and cell-killing capabilities.

[0094] Figure 8 A. A schematic diagram of the procedure. Using dispersing enzymes from... Tumor-like organisms were released from droplets and subsequently passed through 70 μm and 20 μm filters. Organoids were recovered from the 20 μm filter, counted, and plated. Cultured T cells were labeled with far-infrared stain (CellVue deep purple-red). Tumor-like organisms and T cells were then coated with RPMI, IL-2, and 5% [a specific drug / treatment]. The culture medium of human colon organoids was mixed and in vivo imaging was performed for 68 hours at 37°C and 5% CO2 on a Leica SP8X confocal microscope equipped with an in vivo imaging chamber.

[0095] Figure 8 B. Representative composite images of tumor-like and non-targeted T cell co-cultures. Bright-field and far-infrared fluorescence channels were combined to generate composite images.

[0096] Figure 8 C. Representative composite image of tumor-like and targeted T-cell co-cultures. Bright-field and far-infrared fluorescence channels were combined to generate the composite image.

[0097] Figure 8 This is further described in Example 15.

[0098] Figure 9 CRC organoids express immunomodulatory molecules. Normal colon and CRC organoids were generated in a patient-specific manner, and RNA was extracted and analyzed using an Affymetrix single-transcriptional microarray.

[0099] Figure 9 A. Mean gene expression of different immunomodulators in normal colon and CRC organoids; ns, not significant; *, p<0.05.

[0100] Figure 9 B. Displays hierarchical clusters of normal colon and CRC organoids from an individual in a 'live biobank' showing the expression of selected immunomodulator genes. The color gradient represents the z-value of each row (gene transcript).

[0101] Figure 9 C. Genetically engineered human colon organoids carrying one or more mutations found in CRC. CD274 (PD-L1) expression levels in the organoids (n=2) were assessed by quantitative PCR in steady state (control) and after stimulation with 20 ng / mL recombinant human IFN-γ. A, APC KO / KO ;ND, not detected;K,KRAS G12D / + :P,P53 KO / KO S,SMAD4 KO / KO WT, Wild type.

[0102] Figure 9D. Genetically engineered human colon organoids carrying one or more mutations found in CRC. CD274 (PD-L1) expression levels in the organoids (n=2) were assessed by flow cytometry in steady state (control) and after stimulation with 20 ng / mL recombinant human IFN-γ. A, APC KO / KO ;ND, not detected;K,KRAS G12D / + :P,P53 KO / KO S,SMAD4 KO / KO WT, Wild type.

[0103] Figure 10 HLA-A2 in clonal amplification of HLA-A2 + and HLA-A2 - Expression in CRC organoids. This figure shows representative curves from multiple repeated experiments. Normal controls (left) and HLA-A2 controls were stimulated with and without 20 ng / mL recombinant human IFN-γ. + CRC (Chinese image) and HLA-A2 - Flow cytometry analysis of HLA-A2 expression in the CRC (right figure) lineage.

[0104] Figure 11 CRC organoids as an assessment of CD8 + Tools for T cell antigen-specific killing

[0105] Figure 11 A. Experimental protocol.

[0106] Figure 11 B. Cloned HLA-A2 + and HLA-A2 - Flow cytometry analysis of HLA-A2 expression in the lineage.

[0107] Figure 11 C. Bright-field image of CRC organoids co-cultured with WT1 peptide-specific T cell receptor-specific transgenic T cells for 48 hours; scale bar: 1 mm.

[0108] Figure 11 D. HLA-A2 levels of peptide pulses at the beginning and end of co-culture with specified peptide-specific T cells. + Images of CRC organoids; scale bar: 70 μm.

[0109] Figure 11 E. For example, by using HLA-A2 with the indicated peptide pulse. + The ELISA measurement of the supernatant collected after 18 hours of co-culture of CRC organoids showed that IFN-γ was produced by T cells specific to WT1 (top) and EBV (bottom) peptides.

[0110] Figure 11 F. HLA-A2 using EBV peptide pulses + Still images of live cells from an 18-hour co-culture experiment of CRC organoids and EBV-specific T cell clones.

[0111] Figure 11 G. Quantitative analysis of the cytotoxic effect of specific T cells on CRC organoids. The figure represents multiple replicate experiments using co-cultures of EBV peptide and EBV T cells or co-cultures of WT1 peptide and WT1 T cells.

[0112] Figure 11 H. A representative projected image (blue) of T cells infiltrating peptide pulses in CRC organoids, as recorded during live-cell imaging experiments.

[0113] Figure 11 I. Quantitative analysis of the organotoxic effects of specific T cells on IFN-γ-treated CRC organoids in the presence or absence of a blocking antibody against PD-1. The figure represents multiple replicate experiments using EBV peptide and EBV T cell co-culture or WT1 peptide and WT1 T cell co-culture.

[0114] Figure 11 J. Quantitative pulsed or non-pulsed HLA-A2 peptides + Cell viability of organoids co-cultured with antigen-specific T cells for 18 hours. The figure represents the ratio between peptide-pulsed and non-peptide-pulsed conditions. Invention Details

[0116] definition

[0117] "Allogeneic" refers to entities (e.g., cells, tumor-like organisms, co-cultures) derived from different patients. In the case of cells, this can refer to cells derived from samples from different patients or healthy controls. Examples of suitable samples include, but are not limited to, peripheral blood or tissue biopsies.

[0118] As used herein, “about” or “approximately” is equivalent. Any number used herein, whether or not it has “about” or “approximately”, is intended to cover any normal fluctuations as understood by those skilled in the art. As used herein, when applied to one or more intended values, the term “about” or “approximately” refers to a value similar to the reference value. In some embodiments, unless otherwise stated or clearly apparent from the context (other than such a number would exceed 100% of the possible value), the term “about” or “approximately” refers to a value falling within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the reference value.

[0119] "Biologically active" refers to the characteristic of any agent that is active in a biological system, particularly an organism. For example, an agent that has a biological effect on an organism when applied to that organism is considered biologically active.

[0120] "Co-culture" refers to two or more cell types maintained under conditions suitable for their co-growth. In the context of this disclosure, "organoid co-culture" refers to epithelial organoids, as defined elsewhere, cultured together with non-epithelial cell types (particularly immune cell types). In some embodiments, the cell types in the co-culture exhibit structural, biochemical, and / or phenomenological relationships that they do not exhibit individually. In some embodiments, the cell types in the co-culture mimic the structural, biochemical, and / or phenomenological relationships between cell types observed in vivo.

[0121] "comprise / comprises / comprising" will be understood to mean including the said step or element, or a group of steps or elements, but does not exclude any other steps or elements, or a group of steps or elements.

[0122] "Dosage" refers to the specified amount of pharmaceutical reagent provided in a single administration. In some embodiments, the dosage may be administered in two or more doses, including pills, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dosage requires a volume that is not easily contained in a single injection. In such embodiments, the desired dosage may be achieved using two or more injections. In some embodiments, the dosage may be administered in two or more injections to minimize injection site response in an individual. In some embodiments, the dosage is administered via slow infusion.

[0123] "Immune disease" refers to any disorder of the immune system. Immune diseases often have a genetic component and include autoimmune diseases (in which the immune system incorrectly acts on its own components) and immune-mediated diseases (in which the immune system exhibits excessive function).

[0124] "Immunotherapy" refers to any medical intervention that induces, suppresses, or enhances a patient's immune system to treat a disease. In some implementations, immunotherapy activates a patient's innate and / or adaptive immune responses (such as T cells) to more effectively target and eliminate pathogens or cure diseases (such as cancer or immune disorders).

[0125] "Intestine" and "intestinal" refer to the gastrointestinal tract, which includes the mouth, oral cavity, esophagus, stomach, large intestine, small intestine, rectum, and anus.

[0126] "Organoids" refers to cellular structures obtained by expanding adult (post-embryonic) epithelial stem cells (preferably characterized by Lgr5 expression) and composed of tissue-specific cell types that are self-organized through lineage assurance via cell sorting and spatial confinement (as described in Clevers, Cell. 16 June 2016; 165(7):1586-1597, with particular reference to the section entitled "Organoids derived from adult stem cells" starting on page 1590). In this application, the term "organoid" may be used to refer to normal (e.g., non-tumor) organoids. When an organoid is described as a "disease" organoid, this means that the organoid has a disease phenotype, for example, usually because the organoid has been derived from one or more epithelial stem cells with a disease phenotype, or in some embodiments, because the organoid has been genetically modified to exhibit specific characteristics of a disease phenotype.

[0127] A “population” refers to a group of entities that share common characteristics. In some embodiments, a “population” refers to patients who share a set of relevant clinical characteristics. Preferably, a “population” may refer to a group of patients who share the same cancer and / or are being treated with the same reagent, and / or are susceptible to successful treatment with the same reagent. During treatment, a population may differ in one or more characteristics, including genotype and / or specific reagent response characteristics. A population may also refer to a group of cells, organs, and / or co-cultures that share one or more genotypes, phenotypes, or biochemical characteristics. A “subpopulation” refers to a group of entities that share a greater number of common characteristics or a smaller number of different characteristics compared to a larger population in which the entities of the subpopulation are also classified.

[0128] "Safety" refers to the treatment of a disease in which, according to standard clinical practice, the treatment has no side effects or only has side effects at a tolerable level.

[0129] "Side effects" or "adverse effects" refer to physiological responses to treatment that are not attributable to the intended effects.

[0130] The terms "object," "patient," or "individual" can refer to a human being or any non-human animal (such as any mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In a preferred embodiment, the patient is a mammal, more preferably a human being. "Human" can refer to prenatal and / or postnatal forms. An object can be a patient, which refers to a person presented to a healthcare provider for the diagnosis or treatment of a disease. The term "object" is used interchangeably with "individual" or "patient" herein. A patient may have a disease or condition or be susceptible to a disease or condition, but may or may not exhibit symptoms of the disease or condition.

[0131] "Having" refers to a patient who has been diagnosed with or is exhibiting one or more symptoms of a disease, condition, and / or illness.

[0132] "Susceptible to" refers to a patient who has not been diagnosed with a disease, condition, and / or illness. In some embodiments, a patient susceptible to a disease, condition, and / or illness may not exhibit symptoms of the disease, condition, and / or illness. In some embodiments, a patient susceptible to a disease, condition, illness, or event may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, condition, and / or illness; (2) a genetic polymorphism associated with the development of the disease, condition, and / or illness; (3) increased and / or decreased expression and / or activity of proteins associated with the disease, condition, and / or illness; (4) habits and / or lifestyles associated with the development of the disease, condition, and / or illness; and / or (5) having received, planning to receive, or needing a transplant. In some embodiments, a patient susceptible to a disease, condition, and / or illness will develop the disease, condition, and / or illness. In some embodiments, a patient susceptible to a disease, condition, and / or illness will not develop the disease, condition, and / or illness.

[0133] "Therapeutic effective amount" means an amount of therapeutic agent sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, condition, and / or illness when administered to a person suffering from or susceptible to such a disease, condition, and / or illness. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0134] "Treatment" refers to any method used to partially or completely relieve, improve, reduce, suppress, prevent, or delay the onset of one or more symptoms or features of a particular disease, condition, and / or symptom, or symptom, or to reduce the severity and / or incidence of one or more symptoms or features of a particular disease, condition, and / or symptom, or ...

[0135] "Tumor-like" refers to an organoid comprising cells exhibiting one or more genetic, phenotypic, or biochemical characteristics classified as cancerous. In this application, the term "tumor-like" includes "organoids" derived from cancerous tissue. The term "tumor-like" may also include tumor progression organoids (TPOs), which are engineered tumor organoid cultures in which normal organoids have been engineered (e.g., using Cas9 technology) to contain cancerous mutations.

[0136] Reagents for identifying suitable treatment

[0137] Summary. This invention relates to co-cultures of organoids and immune cells ('organoid co-cultures') and / or co-cultures of disease organoids (such as tumor-like organisms) and immune cells ('disease organoid co-cultures' or more specifically 'tumor-like organism co-cultures'), and their suitability as candidate agents for studying the physiology of diseases and / or treating diseases. Suitability for treating diseases may include efficacy and / or safety in treating diseases. Diseases of particular interest include cancer and immune diseases.

[0138] Therefore, the present invention provides a method for identifying reagents suitable for treating cancer, wherein the method comprises:

[0139] A tumor-like co-culture is contacted with one or more candidate reagents, wherein the tumor-like co-culture comprises immune cells and at least one tumor type.

[0140] Detecting the presence of one or more alterations in tumor-like cocultures that indicate a candidate reagent is suitable for treating the cancer, and

[0141] If the presence or absence of one or more of the aforementioned changes is detected in the tumor-like coculture, the candidate reagent is identified as suitable for treating the cancer.

[0142] A method for testing therapeutic agents is also provided, wherein the method includes:

[0143] The organoid co-culture is contacted with one or more candidate reagents, wherein the organoid co-culture comprises immune cells and at least one organoid.

[0144] Detecting the presence of one or more changes in the organoid co-culture that indicate therapeutic efficacy, and

[0145] If the presence or absence of one or more of the aforementioned changes is detected in the organoid co-culture, the candidate reagent is identified as a therapeutic agent.

[0146] In some embodiments, the organoids are disease organoids (e.g., organoids exhibiting an immune disease phenotype). Because of the presence of immune cells in the co-cultures of the present invention, these co-cultures are particularly suitable for studying the suitability of candidate immunotherapeutic agents.

[0147] The method of this invention has high-throughput (HTP) capability. In some embodiments, the method of this invention can be performed on a 96-well plate and / or a 384-well plate.

[0148] Contact steps. This may involve exposing organoid co-cultures to therapeutic levels of known or unknown therapeutic agents. Typically, the agent is dissolved in a solution to a (predicted) therapeutically effective concentration and administered to the co-cultures by injection (or other appropriate application) into the containers in which the co-cultures are maintained.

[0149] Detection steps. In some embodiments, the invention includes the step of detecting the presence of one or more variations in a tumor-like coculture indicating suitability for treatment with a candidate reagent.

[0150] In principle, any biochemical, genetic, phenotypic, or phenotypical changes in the co-culture can be detected. In some embodiments, the one or more changes may be changes in one or more disease biomarkers (such as cancer biomarkers). In some embodiments, the one or more changes may include decreased cell viability, reduced cell proliferation, increased cell death, changes in cell or organoid size, changes in cell motility, dissociation or disruption of the intact / dense epithelial cell layer (i.e., cell dissociation from the dense epithelial cell layer), changes in the production of cytokines and cytotoxic molecules by co-cultured immune cells, and changes in the expression of one or more genes.

[0151] In principle, any suitable laboratory method known to those skilled in the art can be used for detection. In some embodiments, detection of one or more changes may include cell proliferation assays, viability assays, flow cytometry analysis, ELISA of IFN-γ (interferon-γ), etc. Figure 8 (D) gene expression analysis and / or cell imaging performed in D.

[0152] Decreased cell viability can be detected by the CellTiter Glo luminescent cell viability assay kit (Promega), intracellular flow cytometry staining (BD) of active caspase 3, or positive staining of dead cells. Positive strains of dead cells include non-cell membrane-permeable DNA staining (e.g., NucRed Dead 647 ReadyProb).

[0153] Increased cell death can be detected using bright-field imaging.

[0154] Qualification steps. Qualification may include identifying changes of a specific magnitude and may be an automated and / or high-throughput process.

[0155] Comparison Steps. In some embodiments, the invention may include a step of comparing the organoid co-culture or tumoroid co-culture with a control, which may or may not be related to the identification step. This may involve comparing the presence or absence, or magnitude, of one or more changes in the tumoroid co-culture with a reference organoid or reference tumoroid, and may further include:

[0156] Contact a reference organoid co-culture or a reference tumor-like co-culture with one or more candidate reagents, wherein the reference organoid co-culture or the reference tumor-like co-culture comprises immune cells and at least one organoid.

[0157] Detect the presence of one or more variations in the reference organoid coculture or the reference tumor coculture that indicate the suitability of candidate reagents for cancer treatment.

[0158] In some implementations, a candidate reagent is identified as a suitable reagent if the presence or absence of a change is detected in a tumor-like coculture but not in a reference coculture.

[0159] In some implementations, reference organoid cocultures or reference tumor-like cocultures are used as controls (such as negative or positive controls).

[0160] Selection Step. In some embodiments, the method of the present invention includes the step of selecting candidate reagents suitable for treating cancer. Selection differs from identification because selection can involve considerations of the presence or absence, or magnitude, of one or more variations of the provided method. For example, selection may include additional considerations (such as reagent bioavailability, suitability for patient subgroups, or reagent delivery mechanism) that may or may not be tested in the method.

[0161] In some embodiments, this step may be the final step of the method of the present invention. In other embodiments, further steps are contemplated. For example, the method of the present invention may also include the step of using a selected candidate reagent during treatment.

[0162] Reagents. Any reagent can be tested according to the method of the present invention. This includes any biological, chemical, physical, or other reagents, or multiple reagents applied simultaneously or sequentially.

[0163] The suitability of reagents (or 'candidate reagents') for testing the treatment of cancer can be selected from one or more of the following therapeutic agent categories: immunotherapeutic agents, tumor-specific peptides, checkpoint inhibitors, alkylating agents, antimetabolites, metabolic agonists, metabolic antagonists, plant alkaloids, mitotic inhibitors, antitumor antibiotics, topoisomerase inhibitors, radiotherapy agents, chemotherapy agents, antibodies, photosensitizers, stem cell grafts, vaccines, cytotoxic agents, cell inhibitors, tyrosine kinase inhibitors, proteasome inhibitors, cytokines, interferons, interleukins, intercalating agents, targeted therapeutic agents, small molecule drugs, hormones, steroids, cell therapy agents, viral vectors, and nucleic acid therapy agents.

[0164] Preferably, the reagent is a tumor-specific peptide, a checkpoint inhibitor, or an immunotherapeutic agent.

[0165] More preferably, the reagents are immunotherapeutic agents, such as chimeric antigen receptor (CAR)-T cell therapeutic agents, therapeutic TCR transgenic T cells, or neoantigens. Other reagents include those associated with antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP).

[0166] Context. The claimed method of the invention can be performed in vivo, in vitro, in situ, out-of-situ, or any combination thereof. Preferably, the method is performed in vitro.

[0167] Personalized medicine

[0168] Summary. One approach to testing different treatment options can be described as a 'personalized medicine' approach to testing. Personalized medicine approaches may include testing one or more candidate agents known to be suitable for treatment, and / or identifying one or more candidate agents as suitable agents for a particular patient.

[0169] Personalized medical applications of this invention may require that both the tumor-like coculture and the reference organoid coculture or the reference tumor-like coculture are derived from specific patients for whom the suitability of the candidate reagent for treating cancer is being identified.

[0170] The inventors of this invention have demonstrated that immune cells, normal (e.g., non-tumor) epithelial cells, and tumor epithelial cells can be obtained from a single tissue from a single patient, and that organoid immune cell co-cultures and tumor immune cell co-cultures can be obtained from these cells. These co-cultures provide particularly useful models for testing individual patient responses to candidate reagents.

[0171] The candidate reagents thus identified can then be used to treat patients for whom the candidate reagents have been identified as suitable for treating cancer.

[0172] filter

[0173] Summary. Another approach to testing different treatment options can be described as a 'screening' method of testing. Screening methods may involve testing one or more candidate agents with unknown suitability for treatment, and / or identifying a subset of one or more candidate agents as suitable agents for treatment.

[0174] The screening application of the present invention may require one or more candidate reagents to have known suitability for treating a first type of cancer and unknown suitability for treating a second type of cancer, wherein the screening includes identifying a subset of the one or more candidate reagents as suitable reagents for treating the second type of cancer.

[0175] In some implementations, screening methods identify agents suitable for treating cancer at the 'population' level rather than the subpopulation level. In other implementations, screening methods identify agents suitable for treating cancer at the subpopulation level. In some implementations, screening methods are not used to identify agents suitable for treating cancer at the individual patient level (which is typically included in personalized medicine approaches).

[0176] Cell types and diseases

[0177] Species. The cells, cancer cells, organoids, and / or co-cultures of the present invention, or cells, cancer cells, organoids, and / or co-cultures suitable for use with the methods of the present invention, can be primarily any multicellular organism, preferably a cancer-susceptible multicellular organism. In some embodiments, the cells, cancer cells, organoids, and / or co-cultures of the present invention are mammalian (meaning derived from mammals), such as murine cells, primate cells, or human cells, cancer cells, organoids, and / or co-cultures. In a preferred embodiment, the cells, cancer cells, organoids, and / or co-cultures of the present invention are human (meaning derived from humans).

[0178] Epithelial cells. The organoids and / or organoid co-cultures of the present invention are derived from epithelial cells. Organoids and / or organoid co-cultures may be derived from normal (i.e., non-disease) epithelial cells or from diseased epithelial cells (sometimes specifically referred to as 'disease organoids' or 'disease co-cultures'). The tumor-like and / or tumor-like co-cultures of the present invention are derived from tumor epithelial cells. Any epithelial cells from which organoids or tumor-like structures can be generated are suitable for the present invention. Preferred tumor epithelial cells and / or normal epithelial cells include lung cells, hepatocytes, breast cells, skin cells, intestinal cells, crypt cells, rectal cells, pancreatic cells, endocrine cells, exocrine cells, ductal cells, kidney cells, adrenal cells, thyroid cells, pituitary cells, parathyroid cells, prostate cells, gastric cells, esophageal cells, ovarian cells, fallopian tube cells, or vaginal cells. Particularly preferred epithelial cells are intestinal cells (e.g., colorectal cells). Epithelial cells may be epithelial stem cells, preferably epithelial stem cells characterized by Lgr5 expression.

[0179] In some embodiments, tumor epithelial cells and / or normal epithelial cells are obtained from samples from cancer patients. In specific embodiments, tumor epithelial cells and normal epithelial cells are obtained from samples from the same cancer patient, optionally from the same sample. Suitable samples for obtaining epithelial cells include tissue biopsies, such as ascites from patients with colorectal or ovarian cancer; urine from patients with kidney cancer; or tissue biopsies from resected colon and / or rectum from patients with colorectal cancer.

[0180] Immune cells. Any immune cells that can be incorporated into the co-culture are suitable for use with the method of the present invention. Preferred immune cells include one or more cell types selected from: intraepithelial lymphocytes (IEL), tumor-infiltrating lymphocytes (TIL), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), T cells, cytotoxic T lymphocytes (CTL), B cells, NK cells, mononuclear phagocytes, α / β receptor T cells, and γ / δ receptor T cells. Preferred immune cells also include bone marrow-derived suppressor cells.

[0181] Immune cells can be obtained from established cell lines available in the art (e.g., from ATCC or similar cell line libraries). Alternatively, immune cells can be purified from impure samples from the subject. There is an advantage to obtaining tumor-like cells in co-cultures from the same patient as the tumor epithelial cells, as the resulting co-cultures are the most representative (and therefore the most reliable) of the patient from whom the cells originated. This is particularly useful in the context of personalized medicine.

[0182] Impure immune samples from which immune cells can be obtained may include tumor-like samples, normal (non-tumor) colon tissue, and / or peripheral blood. In some embodiments, immune cells are obtained from samples from cancer patients. In some embodiments, immune cells are obtained from peripheral blood samples and / or tissue biopsies. For example, peripheral blood lymphocytes (PBLs) and / or T cells may be obtained from peripheral blood samples, respectively; or tumor-infiltrating lymphocytes (TILs) and / or intraepithelial lymphocytes (IELs) may be obtained from tumor or healthy tissue biopsies, respectively.

[0183] The immune cells suitable for use in the methods of the present invention may be allogeneic to tumor-like and / or organoids. In some embodiments, the immune cells are HLA matched to the tumor-like and / or organoids, i.e., the immune cells may be compatible with the patient antigens from which the tumor-like and / or organoids are derived (Shiina et al. (2016), MHC Genotyping in Human and Nonhuman Species by PCR-based Next-Generation Sequencing, Next-Generation Sequencing-Advances, Applications and Challenges, Dr. Jerzy Kulski (ed.), InTech, DOI:10.5772 / 61842)(Choo, Yonsei Med J. Feb. 28, 2007; 48(1):11-23).

[0184] T-cell engineering. An important aspect of this invention is the use of engineered T cells (such as chimeric antigen receptor (CAR)-T cells) (Sadelain et al., Nature. May 24, 2017; 545(7655):423-431). This invention provides methods and co-cultures that can be used to assess the suitability of different CAR-T cell types for different tumor phenotypes and tumor microenvironments. Compared to existing methods, this invention offers a simplified approach to CAR-T cell selection and performance enhancement, with improved scalability and reduced cost. In particular, this invention is well-suited for use with γδT cells (unconventional T cells with strong antitumor responses to a broad spectrum of tumors of different tissue origins) (Sebestyen et al., CellRep. May 31, 2016; 15(9):1973-85). Thus, in some embodiments, the immune cells in the co-culture are engineered T cells (such as CAR-T cells).

[0185] Organoids and tumor-like cells. Organoids can be prepared by culturing normal epithelial cells in an organoid culture medium. Tumor-like cells can be prepared by culturing tumor epithelial cells in a tumor-like culture medium. Normal epithelial cells can be autologous to tumor epithelial cells (i.e., derived from the same patient). The organoids / tumor-like cells of the present invention can be characterized by Lgr5 expression. In some embodiments, the organoids / tumor-like cells are three-dimensional cellular structures. In some embodiments, the organoids / tumor-like cells include a lumen surrounded by epithelial cells. In some embodiments, the epithelial cells surrounding the lumen are polarized. Polarization can be disrupted in the tumor-like cells. The epithelial cells from which the organoids / tumor-like cells are obtained are preferably primary epithelial cells.

[0186] Cancer Types. The method of this invention can be applied to any cancer. In some embodiments, the cancer can be one or more of the following: adenoma, adenomatous polyp, renal cancer, adrenal adenoma, thyroid adenoma, pituitary adenoma, parathyroid adenoma, hepatocellular adenoma, fibroadenoma, cystic adenoma, bronchial adenoma, sebaceous adenoma, prostate adenoma, adenocarcinoma, bile duct carcinoma, squamous cell carcinoma, ductal carcinoma, lobular carcinoma, carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, spindle cell carcinoma, sarcomatoid carcinoma, pleomorphic carcinoma, carcinosarcoma, basal cell carcinoma, vasoactive intestinal peptide tumor, linitis plastic, adenoid cystic carcinoma, renal cell carcinoma, mucoepidermoid carcinoma, intestinal cancer, small bowel cancer, colon cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, rectal cancer, vaginal cancer, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, and melanoma.

[0187] The method of the present invention is particularly applicable to cancers including epithelial cancers, such as gastrointestinal or colorectal cancer, pancreatic cancer, and breast cancer.

[0188] Cancer Stages. This invention is applicable to any stage of cancer progression. Cancer progression can be characterized in several systems. The TNM (tumor, nodule, metastasis) system comprises three categories, each assigned a numerical degree. T refers to the size of the cancer and the extent to which it has spread to nearby tissues—it can be 1, 2, 3, or 4, where 1 is small and 4 is large. N refers to whether the cancer has spread to lymph nodes—it can be between 0 (no lymph nodes containing cancer cells) and 3 (numerous lymph nodes containing cancer cells). M refers to whether the cancer has spread to another part of the body—it can be 0 (cancer has not spread) or 1 (cancer has spread). A second system is a numerical staging system comprising four stages. Stage 1 typically means the cancer is relatively small and contained within the organ from which it originated. Stage 2 typically means the cancer has not yet begun to spread to surrounding tissues, but the tumor is larger than a stage 1 tumor. Sometimes stage 2 means the cancer cells have spread to lymph nodes near the tumor. This depends on the specific type of cancer. Stage 3 typically means the cancer is larger. It may have begun to spread to surrounding tissues, and cancer cells are present in the lymph nodes in that area. Stage 4 means that the cancer has spread from its origin to another organ in the body. This is also known as secondary or metastatic cancer. The grading system is the third system characterizing the degree of cancer progression. In Stage I, cancer cells resemble normal cells and do not grow rapidly. In Stage II, cancer cells do not look like normal cells and grow faster than normal cells. In Stage III, cancer cells look abnormal and may grow or spread more aggressively.

[0189] Some of the agents tested in the methods of this invention (such as immunotherapy) are more relevant in later stages (metastatic) of cancer (such as colorectal cancer) because surgical resection is often sufficient when metastasis is absent. Therefore, this invention can be applied to cancers at or below stage III, grade III, or T2 N1 M1.

[0190] Immunotherapy can also be relevant in the early stages of other cancers that are difficult to surgically remove. Furthermore, the use of this invention in tumor progression organoids (TPOs) enables the study of treatments for cancers in more readily resectable stages. Therefore, this invention can be applied to cancers at or below stage II, grade II, or T2N1 M0.

[0191] Immune diseases. Besides cancer, the methods of this invention can also be used to study diseases affecting immune cells. In principle, any condition of the immune system that affects immune cells can be studied in co-culture. Preferred immune diseases include immune diseases of the digestive and respiratory systems, particularly the intestines and lungs. Exemplary immune diseases include irritable bowel disease (IBD), ulcerative colitis (UC), chronic obstructive pulmonary disease (COPD), and asthma.

[0192] When testing for immune disorders using the method of the present invention, organoids can be cultured separately from diseased immune cells and immune cells from healthy control patients.

[0193] Biopsy and sample sourcing. Organoid and / or tumor-like samples can be obtained during surgery from normal mucosa and tumor tissue, such as from resected colon, rectum, small intestine, and / or ileum from colorectal cancer patients and / or healthy controls. Immune cells can be derived from peripheral blood collected during surgery.

[0194] Organoids, tumor-like organisms and co-cultures

[0195] Tumor-like co-culture formulations. In one aspect, the present invention provides a method for preparing a tumor-like immune cell co-culture. The method includes the step of mixing tumor-like cells with immune cells as described herein in vitro. Mixing may include sequentially layering T cells and organoids into the same wells in a multi-well plate, or may include sequentially pipetting T cells and organoids into a gel. In a preferred embodiment, the tumor-like co-culture is maintained in a co-culture medium as described herein.

[0196] In some embodiments, the method for preparing tumor-like immune cell co-cultures further includes one or more of the following preparation steps:

[0197] The at least one tumor-like cell is prepared by culturing tumor epithelial cells in a tumor-like culture medium; and / or

[0198] Immune cells are prepared by culturing them in an immune cell expansion medium.

[0199] In a preferred embodiment, the tumor-like culture medium (optionally including any extracellular matrix) is removed from the at least one tumor-like tumor before mixing it with immune cells. The extracellular matrix can be obtained using a commercially available kit (such as a cell recovery solution). TM (Cell Recovery Solution TM Corning) is used to break down the matrix. Alternative matrix materials (such as collagen) can be used to replace the removed matrix.

[0200] In some embodiments, the method further includes the step of obtaining immune cells from impure immune samples. Methods for isolating immune cells from impure immune samples are known in the art. An exemplary method for isolating lymphocytes from single-cell suspensions and T-cell expansion cultures is described in Example 5.

[0201] This invention provides a tumor immune cell co-culture obtained by the above method. This invention also provides the use of the said tumor-like immune cell co-culture in drug screening, toxicology screening, research, and reagent development.

[0202] Tumor-like cocultures can be ectopic, in vitro, and / or in vitro. In vitro is preferred.

[0203] Organoid co-culture formulations. In one aspect, the present invention provides a method for preparing organoid-immune cell co-cultures. The method includes the step of mixing organoids as described herein with immune cells in an in vitro culture. In a preferred embodiment, the organoid co-culture is maintained in a co-culture medium as described herein.

[0204] In some embodiments, the method for preparing the organoid immune cell co-culture includes one or more of the following steps:

[0205] The at least one organoid is prepared by culturing normal epithelial cells in an organoid culture medium; and / or

[0206] Immune cells are cultured in an immune cell expansion medium.

[0207] In a preferred embodiment, the organoid culture medium (optionally including any extracellular matrix, such as basement membrane matrix 'BME' or Matrigel) is removed from at least one organoid before mixing it with immune cells. The extracellular matrix can be obtained using a commercially available kit (such as a cell recovery solution). TM (Corning) is used to break down the matrix. Alternative matrix materials (such as collagen) can be used to replace the removed matrix.

[0208] In some embodiments, the method further includes the step of obtaining immune cells from impure immune samples. Methods for isolating immune cells from impure immune samples are known in the art. An exemplary method for isolating lymphocytes from single-cell suspensions and T-cell expansion cultures is described in Example 5.

[0209] This invention also provides organoid immune cell co-cultures obtained by the above method. Furthermore, this invention provides applications of the said organoid immune cell co-cultures in drug screening, toxicology screening, research, and drug development.

[0210] Organoid co-cultures can be ectopic, in vitro, and / or in vitro. In vitro is preferred.

[0211] Preliminary analysis. In some embodiments, the method of the present invention further includes one or more preliminary analysis steps. Preliminary analysis of tumor-like and / or organoids may include whole-genome sequencing, mRNA sequencing, peptidomimetics, and / or microscopy. Preliminary analysis can be used to ensure, in the form of information discovery and / or information verification, that the tumor-like and / or organoids are homogeneous and / or meet expectations. For example, preliminary analysis can be used to determine differences in mRNA transcription between organoids and tumor-like organisms, and whether these differences in mRNA transcription are reflected in differences in protein expression. The presence of specific antigens on organoids / tumor-like organisms can also be confirmed, and whether any new antigens are generated only on tumor-like organisms. The upregulation of immunosuppressive factors in tumor cells within the tumor microenvironment can also be investigated.

[0212] Immune cells can undergo one or more preliminary analytical steps. For example, preliminary analysis of immune cells may include immunophenotyping and / or T-cell receptor sequencing. Preliminary analysis can be used to examine CAR-T cells for expressing receptors necessary for recognizing tumor cells. It can also investigate the upregulation of tumor-specific receptors.

[0213] In a specific implementation, the method of the present invention includes the step of determining the HLA type of a cell, organoid, or tumoroid.

[0214] Preliminary analyses, either one or more, can be performed on co-cultures. Preliminary analyses of tumor-like co-cultures and / or organoid co-cultures may include imaging analysis, flow cytometry analysis, and / or cytokine secretion analysis. These preliminary analyses can be used to ensure that the co-cultures are homogeneous and / or meet expectations.

[0215] Sources of tumor-like cells and organoids. The tumor-like cells and / or organoids of the present invention may comprise or consist of autologous cells (i.e., cells obtained from the same patient). For example, tumor-like cells can be obtained by culturing tumor cells (e.g., colorectal cancer cells), while organoids can be obtained by culturing normal (non-tumor) cells (e.g., normal colon cells) from the same tissue of the same patient. This is particularly useful in the case of reference organoids.

[0216] The present invention also provides tumor-like and / or organoids in a culture medium containing interleukins (such as IL-2, IL-7, or IL-15). In some embodiments, the at least one tumor-like or at least one organoid comprises or is composed of mammalian cells (preferably human cells).

[0217] Separation of tumor-like and organoids. In some embodiments, tumor-like and / or organoids are separated into populations sharing one or more genotypic, phenotypic, and / or epigenetic markers before being mixed with immune cells. Preferably, the genotypic, phenotypic, and / or epigenetic markers facilitate (i) the interaction between tumor-like and / or organoids and (ii) immune cells.

[0218] Populations separated from tumor-like or organoid groups can share the presence or absence of HLA haplotypes, such as HLA-A2.

[0219] This separation step allows for the identification of relevant patient groups and subgroups.

[0220] culture medium

[0221] Immune cell culture medium. Immune cell culture medium can be used to prepare immune cells for co-culture, for example, by promoting the growth and division (expansion) and / or differentiation of immune cells to produce a population suitable for co-culture.

[0222] In a preferred embodiment, the immune cell culture medium contains interleukin. In some embodiments, the interleukin is selected from IL-2, IL-7, and IL-15. In a preferred embodiment, the interleukin in the immune cell culture medium is IL-2.

[0223] In some embodiments, the concentration of interleukin is 2000-6000 IU / mL. The preferred concentration of IL-2 in the immune cell culture medium is 50 μM.

[0224] The immune cell culture medium may also contain RPMI medium (e.g., RPMI 1640, Gibco), optionally supplemented with penicillin / streptomycin and / or hepes and / or glutamate. TM And / or sodium pyruvate and / or serum (e.g., 5% human AB serum, Sigma-Aldrich). In principle, any mammalian basal cell culture medium can be used instead of RPMI medium, such as DMEM / 12.

[0225] Organoid and tumor-like culture media. Tumor-like and organoid culture media can be used to prepare organoids and tumor-like organisms for co-culture, for example, by promoting growth, division (expansion), structural tissue or other development to produce tumor-like organisms and / or organoids suitable for co-culture.

[0226] Suitable tumor-like and organoid cultures for different tissues are known in the art (e.g., Clevers, Cell. 16 June 2016; 165(7):1586-1597). Preferred organoid / tumor-like cultures contain a Wnt agonist (e.g., any one of R-vertebral proteins 1-4), mitotic growth factors (e.g., selected from EGF, FGF, HGF, and BDNF), and a BMP inhibitor (e.g., head proteins) (e.g., as described in WO2010 / 090513). In some embodiments, the organoid / tumor-like culture medium also contains a TGF-β inhibitor (e.g., A83-01, Tocris) (e.g., as described in WO2012 / 168930). The addition of a TGF-β inhibitor is particularly suitable for the culture of human cells. The TGF-β inhibitor preferably inhibits the Alk4 / 5 / 7 signaling pathway.

[0227] In some embodiments, certain culture medium components are optional for tumor-like media because some tumor cells contain mutations in constitutively activated or inactivated pathways (such as the Wnt pathway), thus eliminating the need for exogenous factors designed to regulate those pathways. Therefore, for example, in some embodiments, the tumor-like medium does not contain a Wnt agonist.

[0228] Preferred organoid culture media, particularly suitable for culturing colonic organoids, comprise one or more (or preferably all) basal media (such as advanced DMEM / F12 medium, Gibco), Wnt ligands (such as Wnt-3a), Wnt agonists (such as any one of R-vertebral proteins 1-4), BMP inhibitors (such as cephalin), EGF and TGF-β inhibitors (such as A83-01, Tocris), and optionally also comprise one or more (or all) of the following: p38 MAPK inhibitors, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or antimicrobial agents (such as primary cell antibiotics (primocin)).

[0229] Preferred tumor-like culture media, particularly suitable for culturing colorectal cancer tumor-like tumors, contain one or more (or preferably all) of the following: a basal medium (such as advanced DMEM / F12 medium, Gibco), a Wnt agonist (such as any one of R-vertebral proteins 1-4), a BMP inhibitor (such as cephalin), EGF and TGF-β inhibitors (such as A83-01, Tocris), and optionally also contain one or more (or all) of the following: p38MAPK inhibitors, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27, and / or antimicrobial agents (such as primary cell antibiotics). The tumor-like culture medium may optionally contain a Wnt ligand (such as Wnt-3a), which is particularly useful for colorectal tumors most sensitive to immunotherapy (e.g., MSI tumors that typically lack Wnt pathway mutations).

[0230] In some embodiments, tumor or organoids are cultured in an immune cell expansion medium or a mixture of an immune cell expansion medium and a preferred tumor or organoid culture medium.

[0231] Those skilled in the art know of culture media that are specific to other types of organoids and tumoroids, and the present invention can be applied accordingly to other organoids and tumoroids.

[0232] Co-culture media. This invention provides culture media for co-culturing tumor-like cells and immune cells (e.g., as described in the examples). This invention also provides culture media for co-culturing organoids and immune cells (e.g., as described in the examples). Any of the above-described immune cell culture media or tumor-like / organoid culture media can be used as a co-culture medium to culture immune cell-organoid / tumor-like co-cultures.

[0233] The co-culture medium of the present invention advantageously allows for the co-culture of immune cells and organoids / tumoroids. In the case of tumoroids, such co-culture is difficult or even impossible without using the medium adaptation method employed in the co-culture medium of the present invention. The inventors of the present invention have observed for the first time that the medium used for co-culturing between tumoroids and immune cells benefits from a reduced Wnt component (relative to organoid medium) to maintain immune cell function. This can be achieved by co-culturing in 100% immune cell medium or in a mixture of immune cell medium and organoid / tumoroid medium. The same medium can be used for the co-culture of organoids and immune cells, although the reduced Wnt component is not as beneficial for organoid co-culture.

[0234] Therefore, in some embodiments, the co-culture medium comprises a portion of immune cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) and a portion of organoid / tumor-like cell culture medium (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). For example, in a preferred embodiment, the co-culture medium comprises about 50% immune cell culture medium and about 50% tumor / organoid culture medium. In some embodiments, the Wnt component in the tumor-like culture medium is depleted before being used in the mixture between the immune cell culture medium and the organoid / tumor-like culture medium.

[0235] In some embodiments, immune cell culture media (such as T cell culture media, for example RPMI 1640 (Gibco)) are used as co-culture media. This medium is particularly useful for supporting the maintenance of immune cells (especially human immune cells) in co-culture. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the co-culture medium consists of immune cell culture media.

[0236] Extracellular matrix. Cells are preferably cultured in a microenvironment that at least partially mimics the cellular niche in which the cells naturally reside. The cellular niche is determined in part by the cells and the extracellular matrix (ECM) secreted by the cells in the niche. A cellular niche can be mimicked by culturing the cells in the presence of biological or synthetic materials that interact with cell membrane proteins such as integrins. Therefore, the extracellular matrix as described herein is, for example, any biological or synthetic material, or a combination thereof, that mimics an in vivo cellular niche by interacting with cell membrane proteins such as integrins. Any suitable extracellular matrix can be used.

[0237] In a preferred method of the invention, cells are cultured in contact with the extracellular matrix (ECM). "Contact" refers to physical, mechanical, or chemical contact, meaning that force is required to separate the resulting organoid or epithelial cell population from the extracellular matrix. In some embodiments, the ECM is a three-dimensional matrix. In some embodiments, cells are embedded in the ECM. In some embodiments, cells are attached to the ECM. The culture medium of the invention can diffuse into a three-dimensional ECM.

[0238] In another embodiment, the ECM is in suspension, meaning the cells are in contact with the ECM in a suspension system. In some embodiments, the concentration of ECM in the suspension is at least 1%, at least 2%, or at least 3%. In some embodiments, the concentration of ECM in the suspension is from 1% to about 10% or from 1% to about 5%. The suspension method can have the advantages of the upscale method.

[0239] One type of ECM is secreted by epithelial cells, endothelial cells, luminal wall endodermal-like cells (e.g., Englebreth Holm Swarm luminal wall endodermal-like cells described in Hayashi et al. (2004) Matrix Biology 23:4762), and connective tissue cells. This ECM comprises various polysaccharides, water, elastin, and glycoproteins, wherein the glycoproteins include collagen, entactin / nidogen, fibronectin, and laminin. Therefore, in some embodiments, the ECM used in the methods of the present invention comprises one or more components selected from the following list: polysaccharides, elastin, and glycoproteins, such as glycoproteins including collagen, entactin / nidogen, fibronectin, and / or laminin. For example, in some embodiments, collagen is used as the ECM. Different types of ECM are known, including different compositions containing different types of glycoproteins and / or different combinations of glycoproteins.

[0240] Examples of commercially available extracellular matrix include: extracellular matrix proteins (Invitrogen) and basement membrane formulations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g. Basement membrane extract (Trevigen) or Matrigel™ (BD Biosciences).

[0241] In some embodiments, the ECM is an ECM containing laminin (such as Matrigel™ (BD Biosciences)). In some embodiments, the ECM is Matrigel™ (BD Biosciences) which contains laminin, nestin, and type IV collagen. In some embodiments, the ECM contains laminin, nestin, type IV collagen, and heparan sulfate proteoglycan (e.g. Type 2 basement membrane extract (Trevigen). In some embodiments, the ECM contains at least one glycoprotein (such as collagen and / or laminin). A mixture of naturally occurring or synthetic ECM materials may be used if desired. In some embodiments, the ECM is BME ('basement membrane extract'), which is a soluble form of the basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumors (e.g., BME).

[0242] In another embodiment, the ECM can be a synthetic ECM. For example, a synthetic ECM (such as ProNectin (Sigma Z378666)) can be used. In another instance, the ECM can be a plastic (such as polyester) or a hydrogel. In some embodiments, the synthetic matrix can be coated with a biological material (such as one or more glycoproteins, such as collagen or laminin).

[0243] Three-dimensional extracellular matrix (ECM) supports the culture of three-dimensional epithelial organoids. The extracellular matrix material is typically a droplet of cells suspended at the bottom of a culture dish. Generally, culture medium is added and diffused into the ECM once the matrix has solidified at 37°C. Cells in the culture medium adhere to the ECM through interactions with their surface structures, such as integrins.

[0244] Culture medium and / or cells can be placed on ECM, embedded in ECM, or mixed with ECM.

[0245] Preferred ECMs for culturing tumor-like / organoids include BME and Matrigel.

[0246] The preferred ECM for culturing cocultures is collagen (such as rat tail type I collagen). Rat tail type I collagen has been shown to improve immune cell motility during coculture – see Example 11. Collagen may constitute at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% (v / v) of the coculture.

[0247] Interleukin. The co-culture medium may contain interleukin (IL), optionally including one or more of interleukin or IL-2 (concentration of 100-200 IU / mL), IL-7 (concentration of 10-100 ng / mL), and IL-15 (concentration of 10-100 ng / mL). The preferred interleukin concentration used in the co-culture medium is 25 μM. These concentrations used for co-culture are the opposite of the IL concentrations used for amplification, which are higher (e.g., IL-2 at a concentration of 2000-6000 IU / mL for immune cell amplification).

[0248] IL-2 is the preferred interleukin for tumor-associated immune cells. For other immune cells or diseases such as irritable bowel syndrome (IBD) or ulcerative colitis (UC), IL-7 and / or IL-15 are preferred (Rabinowitz et al., Gastroenterology, March 2013; 144(3):601-612.e1).

[0249] In some embodiments, the tumor-like co-culture medium and / or organoid co-culture medium comprises a mixture of (a) an immune cell expansion medium and (b) a tumor-like medium or organoid medium, optionally wherein the medium is present in a 50:50 (v / v) ratio.

[0250] Motility and protein concentration. In some embodiments, co-culture and / or co-culture media advantageously confer improved motility on immune cells. As described above, such co-cultures and / or co-culture media may contain extracellular matrix (ECM). The extracellular matrix may be matrix gel or BME. In a preferred embodiment, the extracellular matrix is ​​collagen or rat tail type I collagen.

[0251] The inventors of this invention demonstrated that the greatest improvement in motility was observed using collagen (particularly rat tail type I collagen). Specifically, immune cells (e.g., T cells) in a BME-based culture medium showed an average trajectory length of 43.635 μm, while immune cells (e.g., T cells) in a rat tail type I collagen-based culture medium showed an average trajectory length of 135.08 μm. This is a three-fold increase in motility. The co-culture medium may contain at least 0.15 mg / (ml) ) to 0.95 mg / (ml) Protein concentrations, used for containing 2% to 10% Culture medium.

[0252] In some implementation schemes, such as using Figure 3 As determined by the assay in Example 10, at least 20%, at least 30%, at least 40%, or at least 50% of the immune cells in the co-culture were able to move a distance of at least 200 μM, at least 250 μM, at least 300 μM, at least 350 μM, or at least 400 μM within 80 hours.

[0253] Durability and duration of activity. In some embodiments, the culture medium of the present invention allows immune cells to remain in the immune cell expansion medium for at least 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, or 240 hours.

[0254] In some embodiments, the culture medium of the present invention allows immune cells to remain active for at least 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after co-culture formation (i.e., after the point at which immune cells are mixed with organoids / tumoroids).

[0255] In some embodiments, the culture medium of the present invention allows tumor-like cocultures to remain in tumor-like coculture medium, or reference organoid cocultures or reference tumor-like cocultures to remain in organoid coculture medium for at least 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, or 240 hours. In some embodiments, the coculture can be for 10 days or longer, or for the same number of days as the coculture can remain in the culture without passage.

[0256] The activity of immune cells can be detected based on cell morphology (e.g., the absence of rounded cells and the presence of cell protrusions indicate that the cells are still active).

[0257] Disclaimer. In some embodiments, IL-2 has not been used in any of the culture media claimed in this invention.

[0258] Other methods and products of the present invention

[0259] Kits. This invention provides kits comprising any organoid, tumoroid, or co-culture of this invention.

[0260] In some embodiments, the kit comprises one or more of the following: a syringe, an alcohol swab, a cotton ball, a gauze pad, and an instruction manual for carrying out the method of the present invention. Example

[0261] Other features, objects, and advantages of the invention will become apparent from the following embodiments. However, it should be understood that these embodiments are given by way of illustration only and not by way of limitation, while indicating embodiments of the invention. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the embodiments. The invention is illustrated using tumor-like bodies as disease organoids, but it is contemplated that other disease organoids, particularly those related to immune diseases, can be used in the same manner. Therefore, where this disclosure refers to "tumor-like bodies," it is intended to be replaced by "disease organoids" (such as "immune disease organoids").

[0262] The following culture media were used in the examples:

[0263] Human colon organoid culture medium.

[0264] The medium is supplemented with 50% WNT3A conditioned medium (internal), 20% R-vertebral protein 1 conditioned medium (internal), 10% head protein conditioned medium (internal), and 1× B27 supplement (Gibco). TM Complete advanced DMEM / F12 medium containing 1.25 mM N-acetylcysteine ​​(Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 50 ng / mL human epidermal growth factor (EGF; Peprotech), 10 nM gastrin (Sigma-Aldrich), 500 nM TGF-β inhibitor A-83-01 (Tocris), 3 μM p38MAPK inhibitor SB202190 (Sigma-Aldrich), 10 nM prostaglandin E2 (Tocris), and 100 mg / mL primary cell antibiotic (Invivogen).

[0265] Culture medium for human colorectal cancer tumors.

[0266] Supplemented with 20% R-vertebral protein 1 conditioned medium, 10% head protein conditioned medium, and 1× B27 supplement (without vitamin A) (Gibco) TM Complete advanced DMEM / F12 medium containing 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 50 ng / mL human EGF, 10 nM gastrin, 500 nM TGF-β inhibitor A-83-01, 3 μM p38MAPK inhibitor SB202190, 10 nM prostaglandin E2 and 100 mg / mL primary cell antibiotics.

[0267] Human T cell culture medium.

[0268] RPMI 1640 (Gibco) supplemented with penicillin / streptomycin and 5% AB serum (Sigma-Aldrich) TM ).

[0269] Ijssel medium.

[0270] The supplement contains penicillin / streptomycin, 1% human AB serum (Sigma-Aldrich), bovine serum albumin, insulin, oleic acid, linoleic acid, transferrin, and ethanolamine (all Sigma-Aldrich).

[0271] In the following examples, the generation and characterization of organoid co-cultures and tumor-like co-cultures are described in Examples 1-9. Examples 10-15 illustrate the application of these methods and the co-cultures themselves.

[0272] Example 1. Collection of normal colon and colorectal cancer biopsies from a hospital.

[0273] This example demonstrates the isolation of a cell sample, which was used in subsequent examples to prepare organoid, tumor-like, and immune cell samples.

[0274] Biopsies of normal colonic mucosa and tumor tissue were taken from the resected colon and / or rectum of patients with colorectal cancer. Peripheral blood was also taken during the operation.

[0275] Specifically, biopsies from human colorectal cancer tissue and normal (adult) human colonic mucosal epithelium were collected in 50 mL standard tubes containing 10–15 mL of ice-cold advanced DMEM / F12 medium. The medium was fully prepared with penicillin / streptomycin (from 10,000 U / mL penicillin and 10 KμM / mL streptomycin stock solution at 100×), HEPES (from 1M 100× stock solution), and GlutaMAX (from 100× stock solution; all from Gibco). TM ) and the Rho kinase inhibitor Y-27632 (Sigma-Aldrich). The biopsy specimens should be stored on ice and processed immediately, or stored at 4°C for up to 24 hours until separation begins.

[0276] exist Figure 1 The process is illustrated schematically in A.

[0277] Example 2. Crypts and derived normal colon organoids were isolated from normal colon tissue; intraepithelial T cells were isolated from normal colon tissue for T cell culture.

[0278] This embodiment illustrates the processing of normal colon samples for the development of organoid cultures and for the isolation of immune cells from normal colon samples.

[0279] Normal colonic mucosa was treated with EDTA to release crypts for the derivation of normal colonic organoids, and then further digested to prepare a single-cell suspension containing intraepithelial lymphocytes (IELs) for T-cell culture.

[0280] Recesses were isolated from normal colon tissue, and normal colon organoids were derived from them.

[0281] Muscle and fat layers were removed under a dissecting microscope using surgical scissors and forceps. The cleaned tissue was cut into strips approximately 1–2 mm in diameter. One strip was fixed in 4% formaldehyde (Sigma-Aldrich) for histological analysis, and another strip was rapidly frozen (in dry ice or liquid nitrogen) and stored at -80°C for gene and / or protein analysis. The remaining strips were washed three times with fresh chelation solution (5.6 mM Na₂HPO₄, 8.0 mM KH₂PO₄, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose, and 54.9 mM D-sorbitol dissolved in sterile water; all from Sigma-Aldrich). The washed strips were incubated for 30 minutes at 4°C in a rotating wheel (cold chamber) in a chelation solution containing 2 mM EDTA (internal) and 0.5 mM DL-dithiothreitol (DTT; Sigma-Aldrich). Shake the tube vigorously to release the colonic crypts from the stroma. If no crypts are seen, repeat the incubation with fresh, fully chelated solution. Allow tissue fragments to settle for 1–2 minutes and transfer the supernatant containing the crypts to a new tube. Add 5–10 mL of fetal bovine serum (FCS; Sigma-Aldrich) and centrifuge the crypts at 300 × g for 5 minutes at 4°C. Keep the remaining tissue fragments on ice for dissociation of intraepithelial T cells. Wash the crypts three times in fully purified high-grade DMEM / F12. Resuspend the crypts in basement membrane extract (BME; The cells were plated at different densities and placed in a humidified incubator at 37°C and 5% CO2 for 30 minutes. After BME solidification, human colon organoid culture medium supplemented with the Rho kinase inhibitor Y-27632 was added and replaced every 3-4 days. Organoids formed from crypts were passaged every 7-10 days.

[0282] Subsequently, the organoid cultures were preliminarily analyzed using whole-genome sequencing, mRNA sequencing, and peptidomimetics.

[0283] Intraepithelial T cells were isolated from normal colon tissue for T cell culture.

[0284] Tissue fragments isolated and preserved from the colonic crypts were placed in a Petit's dish and cut into very fine slices (<1 mm) using forceps, scissors, and a scalpel. The tissue fragments were then transferred to 50 mL standard tubes and incubated in 20 mL of RPMI 1640 medium (Gibco). TMWash three times in a medium completely containing 10% FCS and penicillin / streptomycin to remove any residual EDTA and inhibitors. After the tissue slides settle to the bottom of the beaker, remove the medium with a pipette. Then, incubate the tissue slides in 10 mL RPMI 1640 medium containing 1 mg / mL collagenase 1A, 10 U / mL deoxyribonuclease I (both Sigma-Aldrich), and the Rho kinase inhibitor Y-27632 at 37°C for 1 hour with shaking. Add 2 mL of FCS to the cell suspension and filter the entire suspension through a 100 μm cell filter. Centrifuge the single-cell suspension at 300 × g for 5 minutes at 4°C. Remove the supernatant and wash the cell pellet twice in complete RPMI 1640 medium. Freeze the single-cell suspension in Recovery medium. TM Cell culture cryopreservation medium or a 1:1 mixture of FCS and 10% DMSO in advanced DMEM / F12, both from Gibco. TM It is cryopreserved in liquid nitrogen or further processed for T cell culture.

[0285] Example 3. Digestion of colorectal cancer tissue for tumor organoid cell and T cell culture; deriving colorectal cancer tumor-like cells.

[0286] This embodiment illustrates the processing of cancerous colon samples for the development of tumor-like cultures and the isolation of immune cells from cancerous colon samples.

[0287] Tumor tissue is digested to prepare single-cell suspensions containing epithelial tumor cells for use in the development of tumor-derived tumors and for the culture of tumor-infiltrating lymphocytes (TILs) for T-cell culture.

[0288] Digestion of colorectal cancer tissue for tumor and T cell culture.

[0289] Tumor biopsies were cut into strips approximately 1–2 mm in size. One strip was fixed in 4% formaldehyde for histological analysis, and each strip was rapidly frozen (in dry ice or liquid nitrogen) and stored at -80°C for gene and / or protein analysis. The remaining strips were further cut using forceps until the tumor mass appeared viscous. The tumor mass was incubated for 1 hour at 37°C with shaking in 10 mL of complete advanced DMEM / F12 medium containing 1 mg / mL type II collagenase, 10 μg / mL hyaluronidase, and the Rho kinase inhibitor Y-27632. After incubation, 2 mL of FCS was added to the serous tumor mass, and the cell suspension was filtered through a 100 μm cell filter and centrifuged at 300 × g for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was washed twice in complete advanced DMEM / F12 medium. The single-cell suspension was then incubated in Gibco medium.TM Recovery TM Cell culture cryopreservation in liquid nitrogen in a 1:1 mixture of FCS and 10% DMSO of advanced DMEM / F12, or further processing to derive colorectal cancer tumors and T cell cultures.

[0290] Derivative colorectal cancer-like tumors.

[0291] A portion of the tumor single-cell suspension was resuspended in BME and plated at different dilutions. The BME was then immobilized in a humidified incubator at 37°C and 5% CO2 for 30 minutes. Cells embedded in the BME were cultured in human colorectal cancer tumor-like culture medium supplemented with the Rho kinase inhibitor Y-27632. The culture medium was changed every 3–4 days. Organoids formed from single tumor cells were passaged every 7–10 days.

[0292] Example 4. Analysis of organoids and tumoroids.

[0293] Bright-field optical microscopy was used for analysis, and single-cell suspensions of successful organoid and tumor-like samples were confirmed. Figure 1 Image B shows representative bright-field images of normal colonic organoids and tumor-like structures derived from patient samples.

[0294] As described above, colonic crypts were embedded in normal colonic organoid culture medium (basement membrane extract (BME)) and cultured in a medium containing R-vertebral protein 1, cephalin, Wnt3A conditioned medium, vitamin A-free vitamin B27 supplement, nicotinamide, N-acetylcysteine, EGF, TGF-β inhibitor A-83-01, gastrin, p38MAPK inhibitor SB202190, and prostaglandin E2. Normal colonic organoids developed within one week and were then passaged weekly (see above figure).

[0295] Single-cell suspensions from colorectal cancer samples were embedded in basement membrane extract (BME) and cultured in a medium containing tumor-like culture medium (R-vertebral protein 1, head protein conditioned medium, vitamin A-free B27 supplement, nicotinamide, N-acetylcysteine, EGF, TGF-β inhibitor A-83-01, gastrin, p38MAPK inhibitor SB202190, and prostaglandin E2). Tumor-like cells formed within one week and were then passaged weekly (see figure below).

[0296] As in Figure 1 As seen in each figure of B, there are single-cell suspensions of organoids, tumoroids, and immune cells that have achieved good decomposition.

[0297] Example 5. Isolation of lymphocytes from single-cell suspensions and T-cell expansion cultures.

[0298] This example demonstrates further processing of the immune cells, which subsequently produces an expanded culture of the immune cells.

[0299] Add 5 mL of pure Ficoll-Paque PLUS (GE Healthcare) to a 15 mL standard tube. Resuspend the single-cell suspension obtained from digestion of normal colon or colorectal cancer tissue in 5 mL of complete RPMI 1640 medium and carefully place it on top of a clear Ficoll-Paque PLUS layer. Centrifuge the sample at 800 × g for 20 min at room temperature. Collect cells from the layer above the clear Ficoll-Paque PLUS layer containing T cells, resuspend in 10 mL of complete RPMI 1640 medium, and centrifuge at 300 × g for 5 min. Resuspend the cell pellet in complete RPMI 1640 medium and count the cells. Refrigerate the single-cell suspension in Gibco medium. TM Recovery TM Cell culture cryopreservation medium (either a 1:1 mixture of FCS and 10% DMSO of advanced DMEM / F12) in liquid nitrogen can be used immediately for expansion culture. For T cell expansion cultures, in a humidified incubator at 37°C and 5% CO2, in 1 mL of RPMI 1640 medium, at a concentration of 1×10⁻⁶. 6 Lymphocytes were cultured at a total viable cell concentration on anti-CD28 (Miltenyi) coated cell culture plastic in RPMI 1640 medium completely containing penicillin / streptomycin, 5% human AB serum, and 6000 IU recombinant human IL-2 (Miltenyi). The medium was replaced after 1 week.

[0300] Alternatively, peripheral blood may be processed to purify peripheral blood mononuclear cells rich in peripheral blood lymphocytes (PBLs) and T cells.

[0301] Primary analysis was performed using T-cell receptor (TCR) sequencing and T-cell immunophenotyping (comparative) Figure 1 C and Example 6 below).

[0302] Example 6. Analysis of isolated immune cells.

[0303] Figure 1 C shows a representative bright-field image of clonal growth of intraepithelial lymphocytes (IEL) and tumor-infiltrating lymphocytes (TIL) derived from a patient sample (left image).

[0304] Flow cytometry analysis showed robust expansion of CD4+ helper T cells (Th) and CD8+ cytotoxic T cells (CTLs). Single-cell suspensions from normal colonic mucosa or colorectal cancer tissue were maintained in T cell culture medium containing interleukin-2 (IL-2). Clonal growth of T cells was significant within 1–2 weeks (left panel).

[0305] Therefore, analysis of isolated immune cells reveals that they maintain their function and biological representativeness.

[0306] Example 7. Passage of epithelial organs and tumor-like structures.

[0307] This example demonstrates the maintenance of organoid and tumor-like cultures.

[0308] Organoid cultures were disrupted ('isolated') by pipetting drops of BME onto the growth medium using a 1 mL micropipette (P1000 Gilson). The disrupted organoids were centrifuged at 500 × g for 5 minutes. The precipitated organoids were resuspended in TrypLE (Gibco). TM Incubate the organoids in a water bath at 37°C for 5–15 minutes. Isolate the organoids into single cells using pre-wetted, flame-polished glass Pasteur pipettes. Aspirate the isolated organoids into excess, complete, high-quality DMEM / F12 and centrifuge at 500 × g for 5 minutes. Re-plate the epithelial single cells into BME at the desired density and incubate in a humidified incubator at 37°C and 5% CO2. After BME solidification, add the appropriate medium (human colon organoid medium or human colorectal cancer tumor-like medium) supplemented with the Rho kinase inhibitor Y-27632. Replace the medium every 3–4 days. Passage the organoids formed from single tumor cells every 7–10 days.

[0309] Preliminary analysis was performed by sequencing single-cell messenger RNA (mRNA) from single-cell suspensions of cells present in normal colonic epithelium and tumor epithelium.

[0310] Example 8. Generation of organoid co-cultures and tumor-like co-cultures.

[0311] This example demonstrates the co-culture of organoids and tumoroids from Example 5 with immune cell cultures from Example 4.

[0312] After isolation (as in Example 7 above), 5000 cells were plated in BME and cultured for 3-4 days in human colonic culture medium or human colorectal cancer tumor-like culture medium. After culture, the culture medium was removed, and the cells were incubated on ice for 25 minutes before being recovered using the cell recovery solution. TM (Cell Recovery Solution TMCorning) Broken BME / Droplets. Subsequently, before mixing with T cells, the cells were centrifuged (500×g for 5 minutes) and resuspended in T cell culture medium supplemented with 100 IU / mL recombinant human IL-2.

[0313] T cells were counted and a concentration of 100,000 cells / mL was achieved in complete T cell culture medium supplemented with 100 IU / mL recombinant human IL-2. 100 μL of epithelial carcinoma-like tumor suspension was mixed with 100 μL of T cell suspension in 96-well plates. 22 μL of rat tail collagen (Gibco) was added. TM The cells were dissolved in the mixture to achieve a collagen concentration of 10% in the suspension. The cells were then allowed to stand at 37°C and 5% CO2 for 30 minutes to allow the cells and collagen to settle before analysis.

[0314] exist Figure 2 The study demonstrates the principle-validating co-culture of normal colonic organoids and allogeneic CD3+ T cells in basement membrane extract (BME) droplets.

[0315] Figure 2 A schematic diagram of the procedure is shown. As described above, normal colonic organoids were released from BME droplets using cell recovery solution and washed in fully advanced DMEM / F12. Expanded CD3+ T cells were harvested from the culture and labeled with green dye (Vybrant CFDA SE cell tracer). The colonic organoids and labeled T cells were mixed in human colonic organoid culture medium and embedded in BME droplets. The co-culture was maintained in human colonic organoid culture medium containing IL-2 for 60 h. The co-culture was released from BME using cell recovery solution and fixed in 4% paraformaldehyde. The fixed encapsulated whole was stained with phalloidin to label polymerized actin and the cell nuclei were labeled with DAPI. The whole encapsulated whole was mounted onto a glass slide in ProLong Gold anti-quenching mounting medium and imaged on a Leica SP8X confocal microscope.

[0316] exist Figure 2 Figure B shows the maximum projection of the z-stack image of the colon organoid co-culture. F-actin in the organoids is labeled in dark gray, while T cells are labeled in light gray. The inset in the right figure shows T cells infiltrating the colonic epithelium.

[0317] exist Figure 2 C shows a three-dimensional reconstruction of normal colonic organoid cells and T cells.

[0318] As shown in the figure, the organoids exhibit the expected level of structural organization and interact with immune cells, showing significant similarity to in vivo systems.

[0319] Example 9. Analysis of co-cultures by imaging, flow cytometry and cytokine secretion.

[0320] This embodiment analyzes the organoid co-cultures and tumor-like co-cultures generated in Example 6 to study the mechanism of interaction between co-culture components.

[0321] Imaging analysis.

[0322] Imaging analysis was used to determine the percentage of dead cells in co-cultures.

[0323] Before culturing, use cell tracer dyes (e.g., CFSE, molecular probes) TM (Molecular Probes TM T cells were labeled. Organoids were labeled with a directly conjugated mouse anti-human EPCAM (BD Bioscience) antibody or a cell-tracing dye (different from the dye used for T cell labeling). The dye used to label apoptotic cells (NucRed Dead) was applied using a confocal laser scanning microscope (e.g., Leica SP8X; or any type of live-cell imaging time-lapse fluorescence microscope). TM Cells were imaged overnight (12–18 hours) at 37°C and 5% CO2 in the presence of Molecular Probes. Subsequently, time-lapse images were analyzed using Imaris software (Bitplane), and the percentage of dead organoids was calculated by assessing the percentage of colocalizing voxels of EPCAM, and markers of dead cells could be visualized.

[0324] Flow cytometry analysis

[0325] Flow cytometry analysis was used to assess surface markers present on immune cells in co-cultures.

[0326] After isolation (as in Example 7 above), 5000 cells were plated in BME and cultured for 3-4 days in human colonic culture medium or human colorectal cancer tumor-like culture medium. After culture, the culture medium was removed, and the cells were incubated on ice for 25 minutes before being recovered using the cell recovery solution. TM (Corning) Broken BME / Droplets. Subsequently, before mixing with T cells, the cells were centrifuged (500×g for 5 minutes) and resuspended in T cell culture medium supplemented with 100 IU / mL recombinant human IL-2.

[0327] T cells were counted and their concentration was increased to 500,000 / mL in complete T cell culture medium supplemented with 100 IU / mL recombinant human IL-2. 100 μL of epithelial carcinoma-like tumor suspension was mixed with 100 μL of T cell suspension in 96-well plates. Cells were co-cultured overnight, harvested, and processed using TripLE (Gibco)... TM Prepare single-cell suspensions. Fix the single-cell suspensions with 4% paraformaldehyde (Sigma-Aldrich) and permeabilize with a buffer containing 0.5% saponin (BD Bioscience). Alternatively, use a commercially available kit (e.g., the BD Cytofix / Cytoperm Plus Fixation / Permeabilization Kit, BD Bioscience). Cells are then incubated with flow cytometry antibodies against CD3, EPCAM, interferon (IFN)γ, and / or tumor necrosis factor (TNF)α, as well as an antibody recognizing active caspase-3 (all from BD Bioscience), before flow cytometry analysis.

[0328] Cytokine secretion analysis.

[0329] Organoids for co-culture were isolated, plated, cultured, and prepared as described above. T cells were counted and their concentration was increased to 500,000 / mL in complete T cell culture medium supplemented with 100 IU / mL recombinant human IL-2. 100 μL of epithelial carcinoma tumoroid suspension was mixed with 100 μL of T cell suspension in 96-well plates. After 72 h of culture, the supernatant was harvested for ELISA to assess T cell cytokine production (e.g., IFNγ, TNFα). The culture supernatant was stored at -20°C until analysis.

[0330] Example 10. In vivo imaging of tumor-like cocultures co-cultured with rat tail type I collagen showed increased motility of T cells.

[0331] This example tested the effects of different structural components used in the developmental coculture on the motility of the resulting immune cells.

[0332] Figure 3 A schematic diagram of the procedure is shown in Figure A. As described above, tumor-like cells are released from BME droplets using a cell recovery solution and washed in fully advanced DMEM / F12. Allogeneic CD8+ T cells isolated from the peripheral blood sample are labeled with a green dye (Vybrant CFDA SE cell tracer).

[0333] Tumor-like cells and T cells were mixed with human colon organoid culture medium containing IL-2 and 10% BME or rat tail type I collagen, and in vivo imaging was performed for 80 h at 37°C and 5% CO2 using a Leica SP8X confocal microscope equipped with an in vivo imaging chamber.

[0334] Figure 3 B shows a representative composite image of the tumor-like co-culture. Bright-field and green fluorescence channels were combined to produce the composite image. T cell migration pathways were traced using Imaris software.

[0335] like Figure 3 As shown in Figure C, quantitative analysis of T cell trajectory length under both conditions reveals significantly longer trajectory paths for T cells co-cultured in 10% collagen compared to those in 10% BME. These results indicate that the use of rat tail type I collagen in co-culture can develop more in vivo-like systems, which produce longer trajectories and thus maintain the motility of immune cells.

[0336] Example 11: Generation of clonal tumor-like co-cultures.

[0337] This example illustrates clonal tumors that produce human leukocyte antigen (HLA) A2 type positive and negative results.

[0338] Figure 4 Figure A shows a schematic diagram of the procedure. Tumor-like cells are isolated into single cells using TrypLE enzyme digestion. Single cells are stained with anti-HLA-A2 antibody and purified based on anti-HLA-A2 immunoreactivity. HLA-A2... +ve It encapsulates and maintains HLA-A2-VE tumor cells to produce tumor-like structures.

[0339] Figure 4 Flow cytometry analysis in cell B showed that pure HLA-A2+ve or HLA-A2-ve tumor cell lines were established. Controls were the HLA-A2+ve JY cell line and a normal colonic organoid line derived from the same patient sample as the HLA-A2+ve or HLA-A2-ve tumor cell lines.

[0340] Example 12. An experiment on antigen-specific killing of epithelial carcinoma-like tumors mediated by cytotoxic T cells.

[0341] This embodiment relates to a 'cell killing assay' performed on a tumor-like co-culture. This is an example of applying the method of the present invention to αβT cells that have undergone neoantigen treatment for cancer therapy.

[0342] As described above, colorectal cancer tumor-like cells or normal tissue organoids were isolated and maintained as single cells. In the presence of αCD28 stimulating antibody, 10,000–50,000 T cells (TIL or PBMC-derived) were co-cultured with 50,000 tumor-like / organoid-derived single cells for 2 weeks in human T cell culture medium and 200 IU / mL recombinant human IL-2. The culture medium was changed every 2–3 days. Subsequently, in complete Ijssel medium supplemented with 200 IU / mL recombinant human IL-2, feeder cells (1 × 10⁻⁶) were introduced. 6 Cells / mL, from PBMCs of 3 different donors and 1×10 5 Cells were clonally expanded in the presence of a mixture of JY and / or LAZ509 cells (1 × 10⁶ cells / mL). Alternatively, T cells were directly FACS-sorted from TIL or IEL single-cell preparations into plates containing complete Ijssel medium (1 × 10⁶ cells / mL). 6 Cells / mL, from PBMCs of 3 different donors and 1×10 5 (a mixture of JY and / or LAZ509 cells / mL). The expanded clones were then co-cultured with neoantigen pulsed tumor organoids as described above.

[0343] The identified putative tumor neoantigen was then loaded onto epithelial carcinoma organoids. The BME / BME cultured in the plate was disrupted by resuspending the culture medium in the plate. Droplets. Relevant peptides were added to organoids, which were then cultured at 37°C and 5% CO2 for 2 hours. Clonal expansion of T cells was then co-cultured with the autologous organoids for imaging, flow cytometry analysis, and / or cytokine secretion analysis as described above.

[0344] exist Figure 5 The study demonstrated the killing activity of antitumor T cells that had undergone antigen exposure, and in Figure 5 A schematic diagram of the procedure is shown in Figure A. HLA-A2+ve or HLA-A2-ve tumors were pulsed with HLA-A2-restricted Wilms tumor (WT)1 peptide for 2 hours. Then, TCR transgenic CD8+ T cells carrying WT1 peptide-specific TCRs were co-cultured with HLA-A2+ve or HLA-A2-ve tumors pulsed with WT1 peptide for 48 hours.

[0345] Figure 5 Figure B shows representative bright-field images of the co-cultures after 48 hours. HLA-A2+ve tumors treated with WT1 peptide pulses alone showed significant death. All other cases (i.e., HLA-A2+ve or HLA-A2-ve tumors without WT1 peptide pulses and HLA-A2-ve tumors treated with WT1 peptide pulses) showed normal growth.

[0346] The results showed that the neoantigen WT1 peptide was effective in killing tumor-like organisms (and potentially in cancer treatment) with the HLA-A2+ve phenotype, but not with other phenotypes.

[0347] Example 13. Cell viability assay for antitumor activity of antigen-exposed T cells with and without checkpoint inhibition.

[0348] Figure 6 Cell viability assays demonstrating antitumor activity of antigen-exposed αβT cells with and without checkpoint inhibition are shown. This is an example of the application of the method of the present invention to chemical reagents for cancer treatment.

[0349] Figure 6 A diagram of the program is shown in section A. (For example...) Figure 5 Co-culturing was performed as described in section A, but only for 12 hours, with and without anti-PD1 checkpoint inhibitors. Cell viability was determined using the CellTiter Glo luminescent cell viability assay kit (Promega) according to the manufacturer's instructions.

[0350] Figure 6 B shows the normalization of tumor-like cell viability relative to the peptide-free control. Therefore, co-cultures were successfully used to show the lowest tumor-like cell viability when the combination of HLA-A2, IL-2, and anti-PD1 checkpoint inhibitors was present, suggesting that anti-PD1 checkpoint inhibitor therapy may be most effective when applied to patient subgroups showing IL-2 and HLA-A2 cancer types.

[0351] Example 14. Determination of differential effects on T cell activation using organoid / tumoroid co-cultures.

[0352] This example illustrates how the presence of γδT cells activates tumor-like organisms in a co-culture in an antigen-nonspecific manner, where they do not activate organoids above a T-cell-free baseline during co-culture. IFN-γ was used to determine activation.

[0353] exist Figure 7 A diagram illustrates the procedure. Dispersing enzymes are used from... Tumor-like organisms were released from droplets and subsequently passed through 70 μm and 20 μm filters. Organoids were recovered from the 20 μm filter, counted, and plated. Tumor-like organisms and T cells were then mixed with RPMI, IL-2, and 5% [a specific drug / injection]. The human colon organoid culture medium was mixed and incubated at 37°C and 5% CO2. After 24 hours of incubation, the organoids were imaged using a bright-field inverted microscope.

[0354] Representative bright-field images (respectively) of tumor-like co-cultures and organoid co-cultures are shown in Figure 7 B and Figure 7 C.

[0355] exist Figure 7 D shows the quantification of IFN-γ levels in the co-culture.

[0356] Example 15. In vivo imaging of tumor-like co-cultures for assessing correlation and cell-killing ability.

[0357] This study investigates the cytotoxic ability of T cells and how it varies with different T cell subtypes and different T cell / tumor antigen combinations.

[0358] exist Figure 8 A diagram illustrates the procedure. Using dispersing enzymes from... Tumor-like organisms were released from droplets and subsequently passed through 70 μm and 20 μm filters. Organoids were recovered from the 20 μm filter, counted, and plated. Cultured T cells were labeled with far-infrared stain (CellVue deep purple-red). Tumor-like organisms and T cells were then coated with RPMI, IL-2, and 5% [a specific drug / treatment]. The culture medium of human colon organoids was mixed and in vivo imaging was performed for 68 h at 37°C and 5% CO2 on a Leica SP8X confocal microscope equipped with an in vivo imaging chamber.

[0359] exist Figure 8 Image B shows a representative composite image of a tumor-like co-culture containing non-targeted T cells. The bright-field and far-infrared fluorescence channels were combined to generate the composite image.

[0360] exist Figure 8 Figure C shows a representative composite image of a tumor-like co-culture containing targeted T cells. The bright field channel and far-infrared fluorescence channel were combined to generate the composite image.

[0361] Example 16. Using epithelial organoid cultures to mimic cancer immune regulation.

[0362] Here, we utilize organoid technology to investigate immune-cancer interactions and assess immunomodulation in colorectal cancer (CRC). Transcriptional profiling and flow cytometry revealed that organoids maintain differential expression of immunomodulatory molecules present in the primary tumor. Finally, we established a method for in vitro mimicking antigen-specific epithelial cell killing and cancer immunomodulation using CRC organoids co-cultured with cytotoxic T cells (CTLs).

[0363] CRC is one of the most common cancers worldwide. While early-stage CRC is highly treatable with surgical removal, later stages are often incurable. CRC arises from a multi-step process originating from small lesions in the colonic epithelium. These lesions grow into adenomas with low-grade dysplasia, which develop into high-grade dysplasia, eventually leading to invasive carcinoma. Genetic mutations in signaling pathways, such as the classic Wnt signaling pathway, are the molecular basis of CRC4. However, the interaction between the tumor and its microenvironment is another key hallmark. Cancer cells remodel their microenvironment (e.g., fibroblasts, vascular system, and immune cells) to support tumor growth. Infiltrating immune cells (ICs) (such as CTLs or macrophages) play a crucial role by generating different immune responses (e.g., anti-tumor cytotoxicity (the former) or promoting chronic inflammation of the tumor (the latter)). Therefore, escape from the surveillance immune system has been considered one of the hallmarks of cancer. Cancer cells undergo a process known as immune editing and silencing, for example, by preventing activation of T cells stimulated by inhibitory cell surface receptors such as CTL-associated antigen (CTLA)-4 or programmed cell death (PD)1. Overcoming this active immune regulation by tumor cells has become a major therapeutic target. However, tumor heterogeneity (such as different CTL infiltration or varying expression of immunosuppressive factors) can affect the efficacy of antitumor drugs by mediating drug resistance. Therefore, developing in vitro model systems that characterize the communication between tumors and their environment is crucial. Organoid cultures grown from different epithelial tissues serve as excellent tools for studying tissue homeostasis and disease in vivo. Furthermore, organoid biobanks of various epithelial organoid systems have been established, and tumor-derived organoids have been successfully used as a platform for screening different drugs to predict patient responses. Here we describe the establishment of a method for mimicking antigen-specific epithelial cell killing and cancer immune regulation in vitro using tumor-like organisms co-cultured with immune cells (specifically, CRC organoids co-cultured with CTLs).

[0364] We first assessed whether CRC organoids expressed immunomodulatory molecules in established long-term expanded cultures. To this end, we used transcriptomic datasets generated from our 'living organoid biobank' of CRC patients to compare gene expression of T cell-specific immunomodulators in CRC organoids with expression levels found in normal colonic organoids (van de Wetering, M. et al., Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi:10.1016 / j.cell.2015.03.053(2015)). On average, transcription of T cell-stimulation-related genes (such as TNFSF4 or TNFSF9) was not altered in CRC organoids compared to normal colonic organs. Figure 9 A). However, the expression of human leukocyte antigen (HLA) genes HLA-A and HLA-C (which encode major histocompatibility complex (MHC) class I molecules that present antigens to T cells) was significantly downregulated in CRC organoid cells. Figure 9 A) A well-described phenomenon found in cancer. The expression of genes associated with the suppression of T cell function is significantly upregulated (e.g., BTLA), significantly downregulated (e.g., CD80, CD86, or LGALS9), or not altered at all (e.g., CD274 (encoding PD-L1), PDCD1LG2 (encoding PD-L2)). Figure 9 A). When assessing the expression levels of immune regulatory molecules across various organs, CRC organoids clustered predominantly, indicating heterogeneous downregulation of HLA-A, HLA-C, and LGALS9 compared to healthy colon organoids. Figure 9 B). However, the expression of the immunosuppressive genes CD274 and PDCD1LG2, for example, was highly upregulated in some CRC organoids compared with matched normal colon organoid cultures, reflecting the previously reported preservation of tumor heterogeneity in organoids. Figure 9 B). These molecular features provide a foundation for further research on tumor immunogenicity and its association with other tumor characteristics.

[0365] Four of the most commonly mutated genes in CRC are APC, P53, KRAS, and SMAD4, reflecting the progressive progression from normal intestinal epithelium to metastatic cancer. Introducing these cancer mutations into human intestinal organoid cultures using clustered, regularly spaced short palindromic repeats (CRISPR / Cas9) demonstrated that this process could be mimicked in vitro and after xenografting into mice. Using colon organoids carrying one or more of these cancer mutations, we investigated whether the upregulation of PD-L1 was associated with a specific mutational state. Furthermore, we exposed the mutated organoids and their wild-type control organoid lines to interferon (IFN)-γ, which is secreted by T cells and can trigger increased expression of immunomodulatory molecules such as PD-L1. We then assessed PD-L1 expression by quantitative polymerase chain reaction (qPCR) and flow cytometry. Figure 9 CD). In the absence of IFN-γ, compared with the control wild-type organoids, it carries a triple mutation (APC). KO / KO P53 KO / KO KRAS G12D / + ) and quadruple mutation (APC) KO / KO P53 KO / KO KRAS G12D / + and SMAD4 KO / KO Organoids showed lower CD274 gene expression. Figure 9 C). In general, PD-L1 expression was low in untreated organoid cell lines ( Figure 9 CD). However, PD-L1 expression was significantly upregulated at both the transcript and protein levels in IFN-γ-treated organoids. Figure 9 These data demonstrate that CRC organoid cells express immunomodulators, and that this expression is regulated in a similar manner to previously shown for in vivo tissues.

[0366] Our next goal is to establish a co-culture system of CRC organoids and CTLs to mimic antigen-specific killing of tumor cells in vitro. For this purpose, we used αβT cells carrying a transgenic T cell receptor (TCR) that recognizes an HLA-A2-restricted Wilms tumor (WT)1-derived peptide. We first used flow cytometry to screen CRC organoids from a 'live biobank' and newly generated CRC organoids for HLA-A2 expression. We identified three CRC organoid lines exhibiting partial downregulation of HLA-A2 (…). Figure 4 B). We were able to purify HLA-A2. + and HLA-A2 - CRC organoids and successfully established cultures from two populations. Figure 11 A). We confirmed HLA-A2- Stable downregulation of MHC-I in CRC organoids because IFN-γ stimulation does not trigger HLA-A2 reexpression. Figure 10 , Figure 11 B). Next, we pulsed these CRC organoid lines with the WT1 peptide, followed by co-culturing them with peptide-specific T cells for 48 hours. After co-culture, we found that regardless of whether peptide pulses were used, HLA-A2... - CRC organoids can survive. Figure 11 C). However, only HLA-A2 + CRC organoids survived in co-culture without prior peptide incubation. Figure 11 C). HLA-A2 of peptide pulses + CRC organoids were effectively killed by peptide-specific T cells, providing a validation of the principle that organoids can be used in vitro to study the antitumor response of cytotoxic T cells. To further confirm the antigen specificity of our 'killing' assay system, we improved our co-culture method by transfecting HLA-A2 with a construct expressing mNeonGreen-labeled histone H2B. + CRC organoids were generated and T cells were stained with CellTracker violet to allow for long-term tracking of both cell types (methods, below). We then pulsed HLA-A2 with either WT1 peptide or EBV-derived peptide. + CRC organoids (methods) were co-cultured with T cells carrying WT1-specific TCRs or EBV-specific TCRs. Here, only organoids pulsed with homologous peptides were effectively killed by T cells. Figure 11 D). The use of enzyme-linked immunosorbent assay (ELISA) to test IFN-γ production in T cells during co-culture confirmed the killing effect of T cells on antigen-specific organoids. Figure 11 E). To better follow the kinetics of organoid killing, we applied a fluorescent dye (NucRed Dead 647; method) that specifically stained apoptotic cells and performed in vivo confocal imaging on co-cultures ( Figure 11 F). Then, we quantified organoid killing by evaluating the co-localization of NucRed Dead dye with H2B-mNeonGreen (methods). Only when the peptide pulsed HLA-A2... + Significant co-localization of the two markers was observed only when CRC organoids were co-cultured with corresponding peptide-specific T cells, leading to the observation of organoid killing (…). Figure 11 G). Furthermore, under these co-culture conditions, T cells infiltrating the organoid epithelium can be readily detected. Figure 11H). Finally, we investigated whether this co-culture system could mimic the regulation of the immune response to immunosuppressive tumors. Indeed, the addition of a blocking antibody against PD-1 (αPD-1) enhanced tumor killing and IFN-γ production in IFN-γ-stimulated organoids expressing PD-L1. Figure 11 This was not observed when the organoids were not stimulated by IFN-γ and therefore did not express PD-1. In summary, T cells effectively killed co-cultured CRC organoids in an antigen-specific manner. Furthermore, the remission of this suppression by T cell inhibition followed by the use of αPD-1 can be mimicked. Here, we have demonstrated that epithelial organoids can be used to faithfully summarize the interactions between tumor tissue and the immune system. In addition, using our co-culture assay, we set the first step in reconstructing the tumor microenvironment in vitro. Further addition of other components of this microenvironment (such as fibroblasts, natural killer cells, myeloid-derived suppressor cells, and B cells) can elucidate the complex interactions between different cell types leading to tumor immune escape. Finally, this co-culture system can be used as a tool for drug screening that tests the suitability of certain immunotherapies for different tumors and different patients, such as those induced by chimeric antigen receptor (CAR) or TCR transgenic T cells, antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent phagocytosis (ADCP) against tumors.

[0367] method

[0368] Human materials and informed consent

[0369] Colon tissue (normal colon and tumor tissue) was obtained from the Department of Surgery and Pathology of the Diakonessenhuis Hospital in Utrecht, Netherlands. All patients included in this study were diagnosed with CRC. Informed consent was obtained from all enrolled patients. The collection of tissues was approved by the Medical Ethics Committee (METC) of the Diakonessenhuis Hospital, in accordance with the Helsinki Declaration, and in accordance with Dutch and EU legislation.

[0370] Organoid generation and culture

[0371] Epithelial organoids are derived from healthy colon or tumor tissue (van de Wetering, M. et al., Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi:10.1016 / j.cell.2015.03.053(2015)). In short, healthy colonic crypts were isolated by digesting colonic mucosa in a chelating solution supplemented with dithiothreitol (0.5 mM, Sigma) and EDTA (2 mM, internal) (5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose, and 54.9 mM D-sorbitol, Sigma) at 4°C for 30 minutes. Subsequently, colonic crypts were plated on basement membrane extract (BME; type 2 Cultrex PC BME RGF, Amsbio) and the organoids were grown in human intestinal stem cell culture medium (HISC) composed of advanced Durbeco-modified Eagle medium / F12 supplemented with penicillin / streptomycin, 10 mM HEPES and Glutamax (both Gibco, Thermo Fisher Scientific), 50% Wnt3a conditioned medium (internal), 20% R-vertebral protein 1 conditioned medium (internal), 10% cephalin conditioned medium (internal), 1×B27, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 50 ng / mL human EGF, 10 nM gastrin, 500 nM A83-01, 3 μM SB202190, 10 nM prostaglandin E2 and 100 μg / mL primary cell antibiotic (Invivogen). Tumor samples were digested into single cells for 30 minutes at 37°C with shaking in a mixture of type II collagenase (1 mg / mL, Gibco, Thermo Scientific) supplemented with hyaluronidase (10 μg / mL) and LY27632 (10 μM). The single tumor cells were plated in BME and cultured as organoids at 37°C in HICS-free Wnt conditioned medium supplemented with 10 μM LY27632. When referring to the “internal” components of the culture medium, commercially available alternatives are readily available to those skilled in the art (e.g., Wnt agonist (ATCC CRL 2647)). TMR-vertebral protein (R&D, #3500-RS / CF), head protein (Peprotech, #120-10C), EDTA (Thermo Fisher, #AM9260G)) and the technicians will understand that these will achieve the same or equivalent effects.

[0372] Tumor-like transfection

[0373] The tumor-like structures (specifically, CRC organoids) were isolated into smaller pieces using TrypLE and then transduced with H2B-mNeonGreen (pLV-H2B-mNeonGreen-ires-Puro).

[0374] T cells

[0375] αβ T cells producing transgenic TCRs carrying a peptide that recognizes an HLA-A2-restricted WT1-derived peptide are described in Kuball, J. et al., Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood 109, 2331-2338, doi:10.1182 / blood-2006-05-023069 (2007). In short, TCR α and β chains were cloned from elevated tetramer-positive T cell clones. Subsequently, CD8+ was transduced using retroviral supernatant from Phoenix-Ampho packaging cells. + αβTCR T cells, the Phoenix-Ampho packaging cells, were transfected with gag-pol, env, and pBullet retroviral constructs containing clonal TCR genes.

[0376] Tumor-like T cell co-culture and live-cell imaging

[0377] Five to seven days prior to co-culture, tumor-like cells stably transfected with H2B-mNeonGreen were isolated and digested, and seeded at a density of 5000 cells per 10 μLBME (25,000 cells per well in a 12-well cell culture plate). Two days prior to co-culture, IL-2 was deprived of T cells. One day prior to co-culture, the tumor-like cells were stimulated with a specified concentration of IFN-γ.

[0378] Prior to co-culture, T cells were stained with the cell proliferation dye eFluor 450 (CellProliferation Dye eFluor 450, eBioscience) according to the manufacturer's instructions. Before co-culture, tumor cells were pulsed with a TCR-specific peptide (ProImmune) at 37°C for 2 hours. Tumor cells and T cells were harvested and placed in T cell culture medium supplemented with 10% BME, 100 IU / mL IL-2, and NucRed Dead 647 (Thermo Fischer). Anti-PD1 blocking antibody (2 μg / mL) was added to the co-culture as instructed. Cells were seeded in 96-well glass-bottomed plates, and the co-culture was imaged using an SP8X confocal microscope (Leica).

[0379] Flow cytometry

[0380] APCR-tagged pentamers of the EBV-derived HLA-2:02 restriction peptide FLYALALLL (ProImmune) were used to sort pentamers from PBMCs isolated from the yellow layer of blood clots from healthy individuals. + CD8 + CD3 + T cells. Cells were sorted as single cells into 96-well plates using the BD FACSAria (BD Biosciences) cytometer. For flow cytometry, the following antibodies (all anti-human) were used: CD8-PE (clone RPA-T8), CD45-PerCP-Cy5.5 (2D1), CD274 (PD-L1)-APC (MIH1) (all BD Biosciences), CD279 (PD-1)-PE (EH12.2H7, Biolegend), and HLA-A2-PE (BB7.2, Santa Cruz).

[0381] Quantitative polymerase chain reaction (qPCR)

[0382] For qPCR analysis, RNA was isolated from organoids / tumoroids using the RNAeasy kit (QIAGEN) according to the manufacturer's protocol. PCR analysis was performed using SYBR Green reagent (Biorad). PCR reactions were performed in duplicate using the standard curve for each primer. Primers were designed using the NCBI primer design tool. Primers used in this study were: GAPDH forward (GTC GGAGTC AAC GGA TT), GAPDH reverse (AAG CTT CCC GTT CTC AG), HPRT forward (GGC GTC GTG ATTAGT GAT), HPRT reverse (AGG GCT ACA ATG TGA TGG), CD274 forward (TGC AGG GCA TTC CAG AAAGAT), and CD274 reverse (CCG TGA CAG TAA ATG CGT TCAG).

[0383] Transcription mapping analysis

[0384] Microarray analysis of organoids in a biobank is described in van de Wetering, M. et al., Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi:10.1016 / j.cell.2015.03.053 (2015).

[0385] Enzyme-linked immunosorbent assay (ELISA)

[0386] Keep the culture supernatant at -20°C and perform ELISA on the indicated cytokines using the ELISA MA standard (Biolegend) according to the manufacturer's protocol.

[0387] Cell viability assay

[0388] According to the manufacturer's protocol, the viability of co-cultured cells was assessed using the CellTiter-Glo luminescent cell viability assay (Promega).

[0389] Image analysis

[0390] Image analysis was performed using the Imaris software package (Bitplane). In short, a threshold for positive staining was set on the negative control. Co-localization channels for H2B-neon and NucRed Dead 647 signals were established. Cell death was quantified as the percentage of H2B-mNeonGreen+ voxel co-localized with the NucRed Dead signal.

[0391] Bioinformatics Analysis

[0392] Bioinformatics analysis of normalized gene expression data from microarray experiments was performed using standard packages (i.e., gplots) in R version 3.4.0 (RFoundation, https: / / www.r-project.org) and RStudio version 1.0.143 (https: / / www.rstudio.com). (van de Wetering, M. et al., Prospective derivation of aliving organoid biobank of colorectal cancer patients. Cell 161, 933-945, doi:10.1016 / j.cell.2015.03.053 (2015).)

[0393] Statistical analysis

[0394] Unless otherwise stated, all experiments were repeated at least three times. All data are presented as mean ± SEM. Statistical significance was analyzed using Graphpad Prism 6 or Microsoft Excel 2010 via ANOVA or two-tailed Student's t-test.

Claims

1. A method for identifying reagents suitable for treating cancer, wherein the method comprises: A tumor-like co-culture is contacted with one or more candidate reagents, wherein the tumor-like co-culture comprises immune cells and at least one tumor type. Detecting the presence of one or more alterations in tumor-like cocultures that indicate a candidate reagent is suitable for treating the cancer, and If the presence or absence of one or more of the aforementioned changes is detected in the tumor-like co-culture, the candidate reagent is identified as suitable for treating the cancer. The following steps are performed before the method described above: The at least one tumor-like cell was prepared by culturing tumor epithelial cells in a tumor-like culture medium. Immune cells are prepared by obtaining immune cells from impure immune samples and culturing them in an immune cell expansion medium; and A tumor-like coculture is prepared by mixing the at least one tumor-like cell with the immune cells in a tumor-like coculture medium. The tumor-like co-culture medium mentioned above includes IL-2.

2. The method of claim 1, wherein the immune cells are incorporated into the co-culture, and wherein the immune cells are compatible with patient antigens derived from the at least one type of tumor.

3. The method of claim 1 or 2, wherein suitability for treating the cancer includes efficacy in treating the cancer and / or safety in treating the cancer.

4. The method of claim 1 or 2, wherein the one or more changes are changes in one or more cancer biomarkers.

5. The method of claim 1 or 2, wherein the one or more changes are selected from: decreased cell viability, decreased cell proliferation, increased cell death, changes in cell or organoid size, changes in cell motility, changes in the production of cytokines and cytotoxic molecules by co-cultured immune cells, dissociation or destruction of intact / dense epithelial cell layers, and changes in the expression of one or more genes.

6. The method of claim 1 or 2, wherein the detection includes cell proliferation assay, viability assay, flow cytometry analysis, ELISA of IFN-γ, gene expression analysis and / or cell imaging.

7. The method of claim 1 or 2, wherein one or more of the changes are a reduction in cell viability.

8. The method of claim 7, wherein the reduction in cell viability is detected by CellTiter Glo luminescent cell viability assay kit (Promega), intracellular flow cytometry staining (BD) of active caspase 3, or positive staining of dead cells.

9. The method of claim 1 or 2, wherein one or more of the changes is an increase in cell death.

10. The method of claim 9, wherein the increase in cell death is detected by bright-field imaging.

11. The method of claim 1 or 2, wherein prior to the method... The tumor-like co-culture is prepared by removing the tumor-like culture medium from the at least one tumor-like organism.

12. The method of claim 1 or 2, wherein the method comprises comparing the presence or absence of the one or more variations of the tumor-like co-culture with a reference organoid or a reference tumor-like organism, and wherein the method further comprises: Contact a reference organoid co-culture or a reference tumor-like co-culture with one or more candidate reagents, wherein the reference organoid co-culture or the reference tumor-like co-culture comprises immune cells and at least one organoid or tumor-like cell. Detect the presence of one or more variations in the reference organoid co-culture or the reference tumor co-culture that indicate the suitability of candidate reagents for treating the cancer.

13. The method of claim 12, wherein if the presence or absence of a change is detected in the tumor-like co-culture, but the presence or absence of a change is not detected in the reference organoid co-culture or the reference tumor-like co-culture, the candidate reagent is identified as a suitable reagent.

14. The method of claim 12, wherein the following steps are performed prior to the method: The at least one organoid was prepared by culturing normal epithelial cells in an organoid culture medium; The immune cells are prepared by isolating immune cells from an impure immune sample and culturing the immune cells in an immune cell expansion medium. and Prepare the reference organoid co-culture or the reference tumor co-culture.

15. The method of claim 14, wherein the impure immune sample is a tumor sample, normal colon tissue, and / or peripheral blood.

16. The method of claim 14, wherein the reference organoid coculture or the reference tumoroid coculture is prepared by removing the tumoroid culture medium or organoid culture medium from the at least one tumoroid or at least one organoid, and then mixing the at least one reference organoid or at least one reference tumoroid with the immune cells in an organoid coculture medium or a tumoroid coculture medium.

17. The method of any one of claims 14-16, wherein the normal epithelial cells and the tumor epithelial cells are autologous.

18. The method of claim 12, wherein the reference organoid co-culture or the reference tumor-like co-culture is used as a control.

19. The method of claim 18, wherein the reference organoid co-culture or the reference tumor-like co-culture is used as a negative control.

20. The method of claim 12, wherein the tumor-like co-culture medium comprises an extracellular matrix.

21. The method of claim 20, wherein the extracellular matrix is ​​selected from collagen or any animal-derived or synthetic basement membrane matrix.

22. The method of claim 21, wherein the collagen is rat tail type I collagen.

23. The method of claim 1 or 2, wherein the co-culture comprises at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% collagen.

24. The method of claim 23, wherein the co-culture comprises 10% (v / v) collagen.

25. The method of claim 20, wherein the extracellular matrix is ​​Matrigel®.

26. The method of claim 1 or 2, wherein the immune cells of the tumor-like co-culture have motility of at least 40 μm / day, 60 μm / day, 80 μm / day, 100 μm / day, 120 μm / day, or 140 μm / day.

27. The method of claim 1 or 2, wherein at least 20%, at least 30%, at least 40%, or at least 50% of the immune cells in the tumor-like co-culture are capable of moving a distance of at least 200 μm, at least 250 μm, at least 300 μm, at least 350 μm, or at least 400 μm within 80 hours.

28. The method of claim 1 or 2, wherein the immune cells remain active for at least 4 h, 8 h, 12 h, 24 h, 48 h, or 72 h.

29. The method of claim 1 or 2, wherein the one or more candidate reagents have known suitability for treating the cancer, and the method further comprises identifying the one or more candidate reagents as suitable reagents for treating the cancer of a particular patient.

30. The method of claim 12, wherein the tumor-like co-culture and the reference organoid co-culture or the reference tumor-like co-culture are both derived from a specific patient.

31. The method of claim 29, wherein the method further comprises treating the patient with the candidate reagent identified as suitable for treating the cancer of the particular patient.

32. The method of claim 1 or 2, wherein the one or more candidate reagents are selected from one or more of the following therapeutic agent classes: immunotherapeutic agents, alkylating agents, antimetabolites, metabolic agonists, metabolic antagonists, antitumor antibiotics, radiotherapy agents, chemotherapeutic agents, photosensitizers, stem cell grafts, tyrosine kinase inhibitors, proteasome inhibitors, cytokines, intercalating agents, small molecule drugs, hormones, steroids, viral vectors, and nucleic acid therapeutic agents.

33. The method of claim 32, wherein the one or more candidate reagents are selected from one or more of the following: tumor-specific peptides, checkpoint inhibitors, plant alkaloids, mitosis inhibitors, topoisomerase inhibitors, antibodies, vaccines, cytotoxic agents, cell inhibitors, interferons, interleukins, targeted therapeutic agents, and cell therapeutic agents.

34. The method of claim 1 or 2, wherein the one or more candidate reagents are selected from one or more of the following therapeutic agent classes: tumor-specific peptides, checkpoint inhibitors, CAR-T cell therapeutic agents, therapeutic TCR transgenic T cells, and neoantigens.

35. The method of claim 1 or 2, wherein one or more candidate reagents are immunotherapeutic agents.

36. The method of claim 35, wherein the immunotherapeutic agent is a CAR-T cell therapy agent, a therapeutic TCR transgenic T cell, or a neoantigen.

37. The method of claim 1 or 2, wherein the one or more candidate reagents have unknown suitability for treating cancer, and the method further comprises identifying a subset of the one or more candidate reagents as suitable reagents for treating cancer.

38. The method of claim 1 or 2, wherein the one or more candidate reagents have known suitability for treating a first cancer and unknown suitability for treating a second cancer, and the method further comprises identifying a subset of the one or more candidate reagents as suitable reagents for treating the second cancer.

39. The method of claim 1 or 2, wherein the cancer is epithelial carcinoma.

40. The method of claim 1 or 2, wherein the cancer is a gastrointestinal cancer.

41. The method of claim 1 or 2, wherein the cancer is colorectal cancer.

42. The method of claim 1 or 2, wherein the cancer includes cancer that is at or below stage II, grade II or T2 N1 M0.

43. The method of claim 1 or 2, wherein the tumor epithelial cells are obtained from a sample from a cancer patient.

44. The method of any one of claims 14-16, wherein the tumor epithelial cells and the normal epithelial cells are obtained from the same cancer patient.

45. The method of claim 44, wherein the tumor epithelial cells and the normal epithelial cells are obtained from the same sample.

46. ​​The method of claim 1 or 2, wherein the sample is a tissue biopsy.

47. The method of claim 46, wherein the tissue biopsy is obtained from the resected colon and / or rectum of a patient with colorectal cancer, ascites of a patient with colorectal cancer, or urine of a patient with ovarian cancer.

48. The method of claim 1 or 2, wherein the tumor epithelial cells are selected from: lung cells, hepatocytes, breast cells, skin cells, intestinal cells, crypt cells, rectal cells, pancreatic cells, endocrine cells, exocrine cells, ductal cells, kidney cells, adrenal cells, thyroid cells, pituitary cells, parathyroid cells, prostate cells, gastric cells, esophageal cells, ovarian cells, fallopian tube cells, or vaginal cells.

49. The method of claim 1 or 2, wherein the tumor epithelial cells are intestinal cells.

50. The method of claim 49, wherein the tumor epithelial cells are colorectal cells.

51. The method of claim 1 or 2, wherein the tumor epithelial cells are epithelial stem cells.

52. The method of claim 51, wherein the tumor epithelial cells are epithelial stem cells characterized by Lgr5 expression.

53. The method of claim 1 or 2, wherein the immune cells comprise one or more cell types selected from the following: intraepithelial lymphocytes (IEL), tumor-infiltrating lymphocytes (TIL), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), T cells and cytotoxic T lymphocytes (CTL), αβ T cells, γδ T cells, B cells, NK cells and mononuclear phagocytes.

54. The method of claim 1 or 2, wherein the immune cells are obtained from a sample from a cancer patient.

55. The method of claim 1 or 2, wherein the immune cells are obtained from peripheral blood samples and / or tissue biopsies.

56. The method of claim 53, wherein the peripheral blood lymphocytes (PBLs) and / or T cells are obtained from a peripheral blood sample.

57. The method of claim 53, wherein the tumor-infiltrating lymphocytes (TILs) and / or intraepithelial lymphocytes (IELs) are obtained from tissue biopsies.

58. The method of claim 1 or 2, wherein the immune cells and the tumor epithelial cells are obtained from the same patient.

59. The method of claim 1 or 2, wherein the immune cells and the tumor-like organism are allogeneic.

60. The method of claim 59, wherein the immune cells and the tumor-like structure are derived from peripheral blood or tissue biopsies of different patients or healthy controls.

61. The method of claim 1 or 2, wherein the immune cells are HLA-matched to the tumor-like structure.

62. The method of claim 1 or 2, wherein the immune cells are retained in the immune cell amplification culture medium for at least 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h.

63. The method of claim 1 or 2, wherein the at least one tumor type and / or the at least one organoid comprises or is composed of autologous cells.

64. The method of claim 1 or 2, wherein the at least one tumor type and / or the at least one organoid are separated into populations sharing one or more genotypes, phenotypes, and / or epigenetic markers before being mixed with the immune cells.

65. The method of claim 64, wherein the genotype, phenotype and / or epigenetic markers contribute to the interaction between (i) the at least one tumor type and (ii) the immune cells.

66. The method of claim 64, wherein the population-shared HLA haplotype is present or absent.

67. The method of claim 66, wherein the HLA haplotype is HLA-A2.

68. The method of claim 1 or 2, wherein the at least one type of tumor comprises or is composed of mammalian cells.

69. The method of claim 68, wherein the at least one type of tumor comprises or is composed of human cells.

70. The method of claim 1 or 2, wherein the at least one tumor-like co-culture is cultured in an immune cell expansion medium or in a 50:50 (v / v) mixture of immune cell expansion medium and tumor-like medium.

71. The method of claim 1 or 2, wherein the tumor-like coculture is retained in the tumor-like coculture medium for at least 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h.

72. The method of any one of claims 14-16, wherein the organoid culture medium comprises one or more of the following: basal culture medium, Wnt ligand, Wnt agonist, BMP inhibitor, EGF and TGF-β inhibitor.

73. The method of claim 72, wherein the organoid culture medium further comprises one or more of the following: p38 MAPK inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial agent.

74. The method of any one of claims 14-16, wherein the organoid culture medium comprises one or more of the following: advanced DMEM / F12 medium, Wnt-3a, any one of R-vertebral proteins 1-4, head protein, EGF, and A83-01.

75. The method of claim 74, wherein the organoid culture medium further comprises one or more of the following: p38 MAPK inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or primary cell antibiotics.

76. The method of claim 1 or 2, wherein the tumor-like culture medium comprises one or more of the following: basal culture medium, Wnt agonist, BMP inhibitor, EGF and TGF-β inhibitor.

77. The method of claim 76, wherein the tumor-like culture medium further comprises one or more of the following: p38MAPK inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or an antimicrobial agent.

78. The method of claim 77, wherein the tumor-like culture medium further comprises Wnt ligand.

79. The method of claim 1 or 2, wherein the tumor-like culture medium comprises one or more of the following: advanced DMEM / F12 medium, any one of R-vertebral proteins 1-4, head protein, EGF, and A83-01.

80. The method of claim 79, wherein the tumor-like culture medium further comprises one or more of the following: p38MAPK inhibitor, gastrin, nicotinamide, prostaglandin E, N-acetylcysteine, B27 and / or primary cell antibiotics.

81. The method of claim 80, wherein the tumor-like culture medium further comprises Wnt-3a.

82. The method of claim 1 or 2, wherein the immune cell expansion culture medium comprises IL-2.

83. The method of claim 82, wherein the immune cell amplification culture medium contains IL-2 at a concentration of 2000-6000 IU / mL.

84. The method of claim 82, wherein the immune cell expansion culture medium further comprises IL-7 and / or IL-15.

85. The method of claim 1 or 2, wherein the immune cell expansion medium further comprises RPMI medium.

86. The method of claim 85, wherein the RPMI medium is supplemented with penicillin / streptomycin and / or serum.

87. The method of claim 1 or 2, wherein the immune cell expansion culture medium further comprises RPMI 1640.

88. The method of claim 87, wherein the RPMI 1640 is supplemented with penicillin / streptomycin and / or 5% human AB serum.

89. The method of claim 1 or 2, wherein the tumor-like co-culture medium contains IL-2 at a concentration of 100-200 IU / mL.

90. A method for testing the efficacy and / or safety of CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors in the treatment of epithelial cancer, said method comprising: Tumor epithelial cells are expanded in a tumor-like culture medium to form tumor-like structures, and the tumor-like structures are cultured with immune cells in a tumor-like co-culture medium to form a tumor-like co-culture. Normal epithelial cells were expanded in organoid culture medium to form organoids, and the organoids were cultured with immune cells in organoid co-culture medium containing interleukin to form a reference organoid co-culture. The tumor-like co-culture and the reference organoid co-culture are then brought into contact with the CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors. The presence or absence of one or more changes in the tumor-like co-culture and the reference organoid co-culture indicates the efficacy and / or safety of the CAR-T immunotherapy, TCR transgenic T cells, neoantigens, or checkpoint inhibitors. Compare the tumor-like co-culture with the reference organoid co-culture. Prior to the method described above: Immune cells are prepared by obtaining immune cells from impure immune samples and culturing them in an immune cell expansion medium. The tumor-like co-culture medium mentioned above includes IL-2.

91. The method of claim 90, further comprising providing tumor epithelial cells, normal epithelial cells, and immune cells from the same patient.

92. The method of claim 90 or 91, wherein the immune cells are incorporated into the co-culture, and wherein the immune cells are compatible with the patient antigens from which the tumor-like organism and the organoid are derived.

93. A method for testing the efficacy and / or safety of a candidate compound for the treatment of epithelial cancer, the method comprising: Tumor epithelial cells are expanded in a tumor-like culture medium to form tumor-like structures, and the tumor-like structures are mixed with immune cells in a tumor-like co-culture medium to form a tumor-like co-culture. Normal epithelial cells were expanded in organoid culture medium to form organoids, and the organoids were mixed with immune cells in organoid co-culture medium containing interleukin to form a reference organoid co-culture. The tumor-like co-culture and the reference organoid co-culture are then contacted with the candidate compound. The presence or absence of one or more changes in the tumor-like co-culture and the reference organoid co-culture, wherein the presence or absence of said one or more changes indicates the efficacy and / or safety of the candidate compound, and Compare the tumor-like co-culture with the reference organoid co-culture. Prior to the method described above: Immune cells are prepared by obtaining immune cells from impure immune samples and culturing them in an immune cell expansion medium. The tumor-like co-culture medium mentioned above includes IL-2.

94. The method of claim 93, further comprising providing tumor epithelial cells, normal epithelial cells, and immune cells from the same patient.

95. The method of claim 93 or 94, wherein the immune cells are incorporated into the co-culture, and wherein the immune cells are compatible with the patient antigens derived from the tumor-like organism and the organoid.

96. The method of any one of claims 1, 90, and 93, wherein: (a) The immune cell expansion medium includes IL-2; (b) The tumor-like medium includes IL-2; or (c) The tumor-like medium includes IL-2 and the immune cell expansion medium includes IL-2.