Melanoma-immune cell co-culture three-dimensional model and methods of constructing and uses thereof

Abstract

The present application relates to a melanoma-immune cell co-culture three-dimensional model, a construction method thereof and use thereof. The melanoma-immune cell co-culture three-dimensional model provided by the present application can not only simulate a three-dimensional environment for growth of melanoma cells, but also simulate interaction of the melanoma cells with a tumor microenvironment (TME) (including an extracellular matrix thereof), and has a wide application prospect in the field of scientific research and drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a three-dimensional model of melanoma-immune cell co-culture, its construction method and application, and a culture medium. Background Technology

[0002] Melanoma (MM) is a highly malignant tumor originating from melanocytes. It commonly occurs in the skin, but can also occur in mucous membranes and internal organs, accounting for approximately 3% of all tumors. Cutaneous malignant melanoma ranks third among malignant skin tumors (approximately 6.8%–20%). It is most common in adults, with a higher incidence in fair-skinned Caucasians and a lower incidence in dark-skinned Asians and Africans. In recent years, the incidence and mortality rates of malignant melanoma have been increasing annually, and compared to other solid tumors, it has a lower age of onset.

[0003] Several treatments exist for melanoma, including checkpoint blockade immunotherapy, targeted therapy, and triglyceride endothelial cell (TIL) therapy, all of which have proven effective. However, most patients do not respond quickly and develop resistance, primarily due to intratumoral and intertumoral heterogeneity. Good preclinical tumor models are a crucial cornerstone of tumor research. The research, development, and validation of novel targets and therapies for various tumors, including mucosal melanoma, rely heavily on preclinical tumor models. In vivo and in vitro tumor models that preserve the patient's tumor genetic background and biological behavioral characteristics while mimicking the heterogeneity of clinical tumors are effective tools for tumor research. Currently, there are no particularly comprehensive models that fully represent the tumor immune microenvironment. Commonly used animal models have limitations in replicating the natural tumor immune microenvironment, the long tumor formation time, and the infiltration of host mouse immune cells into the tumor. Consequently, the success rate of translating cancer therapies from animal models into human patients is less than 10%. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a three-dimensional model of melanoma-immune cell co-culture. This method produces a novel three-dimensional (3D) model that bridges the gap between overly simplified 2D experiments and overly complex animal models. This 3D model not only simulates the three-dimensional environment of melanoma cell growth but also the interaction between melanoma cells and the tumor microenvironment (TME) (including its extracellular matrix), making it an ideal tool for drug screening research. In particular, this invention uses cells derived from primitive melanoma tumor tissue to construct the 3D model, preserving specific tumor characteristics and showing broad application prospects in scientific research and drug development.

[0005] Specifically, in a first aspect, the present invention provides a method for constructing a three-dimensional model of melanoma-immune cell co-culture, comprising: Provides containers for primary melanoma tissue and cell culture; The primary melanoma tissue was washed, broken, digested, centrifuged, and resuspended in melanoma co-culture medium to obtain a cell suspension containing melanoma cells and infiltrating immune cells in the tumor microenvironment of the melanoma cells. An anti-adhesion agent was added to the cell culture vessel; After adding IL-2 and extracellular matrix material to the cell suspension, it was seeded into the cell culture container and cultured to obtain a three-dimensional model of melanoma-immune cell co-culture. The melanoma co-culture medium is a mixture of Advanced DMEM / F12 medium and AIM V medium at a volume ratio of (1-3):(1-3), and contains the following components: GlutaMAX 1-10 mM, HEPES 10-25 mM, Wnt3a 50-500 ng / mL, RSPO1 50-500 ng / mL, A83-01 0.1-1 μM, B27 additive 1-5% (v / v), gastrin 5-20 nM, SB202190 5-20 μM, N-acetylcysteine ​​0.5-5 mM, nicotinamide 5-10 mM, epidermal growth factor 50-500 ng / mL, and CTS. TM Immune cell serum substitute SR 5%-20% of volume, penicillin-streptomycin bispecific antibody 1%-5% of volume.

[0006] In this invention, the functions achieved by adding specific amounts of components to the melanoma co-culture medium are described below:

[0007] In this invention, there are no limitations on the cell culture container, as long as it can perform the function of cell culture. For example, the cell culture container can be a cell culture plate or a cell culture chip. In some specific embodiments, the cell culture container is a 24-well cell culture plate. In other specific embodiments, the cell culture container is a 48-well cell culture plate. In other specific embodiments, the cell culture container is a 96-well cell culture plate. In still other specific embodiments, the cell culture container is one of those disclosed in application numbers CN2023105307566, CN2023211335836, or those disclosed in CN2023105307068, CN2023105302350, or CN2023302759312, and these applications are incorporated herein by reference in their entirety.

[0008] In this invention, the primary melanoma tissue is derived from melanoma tissue from a melanoma patient. The melanoma patient can be any mammal suffering from melanoma, such as humans, domestic animals, farm animals, and zoo, sports, or pet animals, including dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0009] In this invention, a cell suspension containing melanoma cells and infiltrating immune cells in the tumor microenvironment of the melanoma cells is obtained by washing, breaking, digesting, centrifuging, and resuspending the primary melanoma tissue in a melanoma co-culture medium.

[0010] In some embodiments, the washing includes washing primary melanoma tissue with a primary tissue washing solution to remove surface-attached mucosal and adipose tissue. The primary tissue washing solution is a basal buffer (e.g., PBS), preferably with an antibiotic added, the antibiotic being selected from one or more of penicillin-streptomycin solution and gentamicin.

[0011] In some embodiments, the fragmentation includes: fragmenting the cleaned primary melanoma tissue to a diameter of approximately 1-2 mm. 3 The tissue fragments were resuspended in primary tissue washing solution, centrifuged, and the supernatant was removed.

[0012] In some embodiments, the digestion is performed using a primary melanoma tissue digestion solution, the base medium of which is Advanced DMEM / F12 medium supplemented with collagenase, and optionally at least one of hyaluronidase and Rho kinase inhibitor. Preferably, the collagenase is selected from at least one of collagenase I, collagenase II, and collagenase IV, wherein the concentration of collagenase I is 1-2 mg / mL, the concentration of collagenase II is 1-2 mg / mL, and the concentration of collagenase IV is 1-4 mg / mL; preferably, the concentration of hyaluronidase is 100-500 μg / mL; preferably, the Rho kinase inhibitor is Y27632; more preferably, the concentration of Y27632 is 5-15 μM.

[0013] In some embodiments, the digestion includes: adding the primary melanoma tissue digestion solution to the tissue fragments, mixing by pipetting, and then placing the mixture in a metal bath shaker or other equivalent equipment for digestion at 37°C for 40 minutes, until the tissue fragments are digested into single cells or small cell clusters. After digestion, a digestion termination solution is added to stop the digestion. Preferably, the digestion termination solution is Advanced DMEM / F12 medium containing 10-20% (v / v) fetal bovine serum and 1-5% (v / v) penicillin-streptomycin antibiotics.

[0014] In some embodiments, the digestion and centrifugation process includes a step of filtering the digested cell suspension. After filtration, the cells are resuspended in the digestion termination solution.

[0015] In some implementations, the centrifugation conditions are 2000 rpm, 4°C for 5 minutes.

[0016] Optionally, the centrifugation process may further include a red blood cell lysis step. For example, if a significant amount of red blood is visible in the centrifuged cell pellet, 1 mL of red blood cell lysis buffer is added and mixed thoroughly, and the mixture is allowed to stand at room temperature for 2 minutes. Subsequently, a digestion termination solution is added to terminate the lysis reaction, and the mixture is then centrifuged at 2000 rpm and 4°C for 5 minutes, and the supernatant is discarded.

[0017] In some embodiments, an anti-adhesion agent is added to the cell culture vessel for anti-adhesion treatment. Preferably, the anti-adhesion agent is Poly-HEMA or a commercially available anti-adhesion solution. In some specific embodiments, the anti-adhesion agent is added to the cell culture vessel, allowed to stand at room temperature, the anti-adhesion agent is discarded, and then washed with DPBS to completely remove residual agent.

[0018] In some implementations, the amount of IL-2 added is 50-500 IU / mL.

[0019] In some embodiments, the extracellular matrix material is selected from Matrigel and hydrogel materials (including natural biomaterials, synthetic biomaterials and their mixtures).

[0020] In some embodiments, the amount of the extracellular matrix material added is 5-30% by volume.

[0021] In some embodiments, the cell suspension is prepared at a concentration of 0.5 × 10⁻⁶. 5 -1×10 6 Seeding density of cells / well.

[0022] In some implementations, the culture time for inoculation into the cell culture container is 1-7 days.

[0023] In some specific implementations, the culture temperature is 37°C.

[0024] Secondly, the present invention provides a three-dimensional model of melanoma-immune cell co-culture, which is constructed by the construction method described in the present invention.

[0025] In some embodiments, the melanoma-immune cell co-culture three-dimensional model replicates the tumor microenvironment characteristics of natural human melanoma, expresses tumor-infiltrating immune cell markers, and has at least one of the following characteristics: (1) It has a broad spectrum of immune cell lineage markers, including CD45; (2) Adaptive lymphocyte lineage-specific markers, including: CD3, CD4, CD8, CD19; among which CD3, CD4, and CD8 represent T lymphocyte subsets, and CD19 represents B lymphocytes; (3) It contains markers of immune cell activation and markers of T cell function / exhaustion; among which, the activation markers include CD25 and CD69; the T cell exhaustion markers include PD-1; (4) Optionally, it may have one or more of the following cell markers: NK cell marker CD56 / CD16; tumor-associated neutrophil marker CD15; tumor-associated macrophage marker CD68 / CD163; regulatory T cell marker FoxP3 / CTLA-4; myeloid-derived suppressor cell (MDSC) marker CD11b / CD33; (5) It can respond to drug stimulation; (6) It can respond to radiotherapy, thermotherapy, oncolytic virus therapy and photodynamic therapy.

[0026] In some embodiments, the drug includes chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; preferably, the chemotherapy drugs include dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; preferably, the targeted drugs include BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; preferably, the immunotherapy drugs include PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; preferably, the tumor angiogenesis inhibitors include bevacizumab and axitinib.

[0027] Thirdly, the present invention provides a culture medium comprising a melanoma co-culture medium, wherein the melanoma co-culture medium is a mixture of Advanced DMEM / F12 medium and AIM V medium at a volume ratio of (1-3):(1-3), and is supplemented with the following components: GlutaMAX 1-10 mM, HEPES 10-25 mM, Wnt3a 50-500 ng / mL, RSPO1 50-500 ng / mL, A83-01 0.1-1 μM, B27 additive 1-5% by volume, gastrin 5-20 nM, SB202190 5-20 μM, N-acetylcysteine ​​0.5-5 mM, nicotinamide 5-10 mM, epidermal growth factor 50-500 ng / mL, CTSTM Immune cell serum substitute SR 5%-20% of volume, penicillin-streptomycin bispecific antibody 1%-5% of volume.

[0028] In some embodiments, the culture medium is used in the method for constructing the three-dimensional model of melanoma-immune cell co-culture of the present invention, or for culturing the three-dimensional model of melanoma-immune cell co-culture of the present invention.

[0029] Fourthly, the present invention provides the use of the method for constructing the three-dimensional model of melanoma-immune cell co-culture, or the three-dimensional model of melanoma-immune cell co-culture, in drug detection and / or effectiveness detection of treatment methods.

[0030] In some embodiments, the drug detection includes drug sensitivity testing; preferably, the drugs in the drug detection include chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; more preferably, the chemotherapy drugs include dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; more preferably, the targeted drugs include BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; more preferably, the immunotherapy drugs include PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; more preferably, the tumor angiogenesis inhibitors include bevacizumab and axitinib; preferably, the treatment methods include radiotherapy, thermotherapy, oncolytic viruses, and photodynamic therapy.

[0031] In some embodiments, the drug detection method includes flow cytometry endpoint detection. In some specific embodiments, after treating the melanoma-immune cell co-culture three-dimensional model with the drug, all cells in the cell culture container are collected for flow cytometry detection to assess the effect of the drug on the immune cell population and tumor cell apoptosis. Recommended detection indicators include: Changes in the number and subsets of immune cells: CD45 + (White blood cells), CD3 + (T cells), CD4 + (Helper T cells), CD8 + (Cytotoxic T cells), CD19 + (B cells), CD25 + (Activated T cells), CD69 + (Early activation of T cells), PD-1 + (Programmed death receptor 1 positive cells), etc.; Apoptosis / death: Annexin V (early apoptosis marker), Caspase-3 (cysteine ​​aspartate protease 3, activated apoptosis-executing enzyme), PI (propidium iodide) or 7-AAD (7-aminoactinomycin D, late apoptosis / necrosis marker). Optional extended markers: Cell proliferation: Ki-67 (proliferation-associated nuclear antigen); Immune activation / inhibition balance: Granzyme B, IFN-γ, TNF-α, IL-2 (interleukin-2, intracellular staining); Exhaustion markers: Tim-3 (T cell immunoglobulin mucin 3), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immunoglobulin and ITIM domain protein); Tumor cell markers: SOX10 (sex-determining region Y-box protein 10), Melan-A / MART-1 (melanoma antigen A / melanoma antigen recognized by T cells-1), S100 (S100 protein, distinguishing tumor cells from immune cells).

[0032] In some embodiments, the drug detection method includes cell viability assay. In some specific embodiments, the overall cell metabolic activity is detected using methods such as the MTT assay (tetramethylazazole salt colorimetric method), the CCK-8 assay (cell counting kit method 8), or the CellTiter-Glo (CTG, cell viability luminescence assay), and the half-maximal inhibitory concentration (IC50) of the drug is calculated. 50 (half-inhibitory concentration) and relative survival rate.

[0033] In some embodiments, the drug detection method includes immunofluorescence / confocal imaging. In some specific embodiments, the three-dimensional model is fixed and stained, and immunofluorescence staining is used to observe: expression of apoptotic proteins: Annexin V, Cleaved Caspase-3; immune cell infiltration and spatial distribution: co-localization of CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), etc., with tumor markers; tumor microenvironment remodeling: ECM (extracellular matrix) components (collagen I, fibronectin), vascular mimicry (CD31, platelet-endothelial cell adhesion molecule), etc.

[0034] In some embodiments, the drug detection method includes cytokine secretion detection. In some specific embodiments, cytokine secretion detection includes methods such as CBA (cell counting microsphere array), Luminex (liquid phase chip), or ELISA (enzyme-linked immunosorbent assay) to detect the secretion levels of cytokines, such as IL-2 (interleukin-2), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), perforin, and granzyme B.

[0035] Fifthly, the present invention provides a drug detection method specifically designed for detecting drugs using the melanoma-immune cell co-culture three-dimensional model provided by the present invention. In some embodiments, the drug detection method includes at least one or more of the following: drug sensitivity testing, flow cytometry endpoint detection, cell viability testing, immunofluorescence / confocal imaging, and cytokine secretion detection.

[0036] In some embodiments, the drug in the drug detection includes at least one or more of chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; preferably, the chemotherapy drugs include at least one or more of dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; more preferably, the targeted drugs include at least one or more of BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; more preferably, the immunotherapy drugs include at least one or more of PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; further preferably, the tumor angiogenesis inhibitors include bevacizumab and / or axitinib; preferably, the treatment methods include at least one or more of radiotherapy, thermotherapy, oncolytic viruses, and photodynamic therapy.

[0037] In some embodiments, the flow cytometry endpoint detection includes: after treating the melanoma-immune cell co-culture three-dimensional model with drugs, collecting all cells in the cell culture container for flow cytometry detection to assess the effect of the drugs on the immune cell population and tumor cell apoptosis; more preferably, the detection indicators include at least one or more of the following: (1) changes in the number and subsets of immune cells: CD45 + (White blood cells), CD3 + (T cells), CD4 + (Helper T cells), CD8 + (Cytotoxic T cells), CD19 + (B cells), CD25 + (Activated T cells), CD69 + (Early activation of T cells), PD-1 +(Programmed death receptor 1 positive cells) etc.; (2) Apoptosis / death: Annexin V (early apoptosis marker), Caspase-3 (cysteine ​​aspartate protease 3, activated apoptosis execution enzyme), PI (propidium iodide) or 7-AAD (7-aminoactinomycin D, late apoptosis / necrosis marker); (3) Optional extended indicators: cell proliferation: Ki-67 (proliferation-associated nuclear antigen); immune activation / inhibition balance: Granzyme B (granzyme B), IFN-γ (interferon γ), TNF-α (tumor necrosis factor α), IL-2 (interleukin 2, intracellular staining); exhaustion markers: Tim-3 (T cell immunoglobulin mucin molecule 3), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immunoglobulin and ITIM domain protein); tumor cell markers: SOX10 (sex-determining region Y-box protein 10), Melan-A / MART-1 (melanoma antigen A / melanoma antigen recognized by T cells-1), S100 (S100 protein, distinguishing tumor cells from immune cells).

[0038] In some embodiments, the cell viability assay includes: detecting overall cell metabolic activity using methods such as the MTT assay (tetramethylazazole salt colorimetric method), the CCK-8 assay (cell counting kit method 8), or the CellTiter-Glo (CTG, cell viability luminescence assay), and calculating the half-maximal inhibitory concentration (IC50) of the drug. 50 (half-inhibitory concentration) and relative survival rate.

[0039] In some embodiments, the immunofluorescence / confocal imaging includes: fixing and staining a three-dimensional model, and observing at least one or more of the following by immunofluorescence staining: expression of apoptotic proteins: Annexin V, Cleaved Caspase-3; immune cell infiltration and spatial distribution: co-localization of CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), etc., with tumor markers; tumor microenvironment remodeling: ECM (extracellular matrix) components (collagen I, fibronectin), vascular mimicry (CD31, platelet-endothelial cell adhesion molecule), etc.

[0040] In some embodiments, the cytokine secretion detection includes: detecting the secretion level of cytokines using the CBA method (cell counting microsphere array), Luminex (liquid phase chip), or ELISA (enzyme-linked immunosorbent assay); more preferably, the cytokines include at least one or more of IL-2 (interleukin-2), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), perforin, and Granzyme B.

[0041] The three-dimensional model for co-culture of melanoma and immune cells provided by this invention has the following beneficial effects: (1) The three-dimensional model of melanoma-immune cell co-culture provided by the present invention can reproduce the dynamic environment of cancer cell growth, including the cell-cell and cell-extracellular matrix (ECM) interactions that affect cancer progression and drug resistance.

[0042] (2) The three-dimensional model of melanoma-immune cell co-culture provided by the present invention enables melanoma cells and immune cells to coexist stably and be spatially ordered in a three-dimensional scaffold (extracellular matrix material), and the immune cells have the ability to infiltrate the tumor area in a directional manner, thereby truly restoring the cell composition and interaction relationship of the tumor immune microenvironment.

[0043] (3) The specific melanoma co-culture medium provided by this invention is designed specifically for the three-dimensional model of this application. It differs from traditional organoid culture media, and can simultaneously support melanoma cell proliferation and maintain immune cell activity, avoiding the problem of immune cell inability to proliferate in traditional culture media and methods. Furthermore, this invention adds IL-2 and CTS to the melanoma co-culture medium. TM The immune cell serum substitute SR works synergistically to provide essential activation signals and nutritional support for immune cells in the co-culture system, solving the technical problem of rapid death and functional loss of immune cells during in vitro culture in existing technologies.

[0044] (4) The method for constructing the three-dimensional model of melanoma-immune cell co-culture provided by the present invention adopts the suspension culture method with anti-adhesion treatment, combined with low concentration of extracellular matrix material, to avoid the physical barrier of traditional high concentration extracellular matrix material embedding to immune cell infiltration, and promote the spatial interaction between immune cells and tumor cells.

[0045] (5) The three-dimensional model of melanoma-immune cell co-culture provided by the present invention can provide a good platform for drug detection, screening and development, treatment method and effect evaluation, mechanism research, etc. Attached Figure Description

[0046] Figure 1 Bright-field micrographs of three-dimensional models prepared from melanoma tissues (tissue 1 and tissue 2) are shown.

[0047] Figure 2A and Figure 2B The changes in immune cell phenotypes in the three-dimensional melanoma-immune cell co-culture model (tissue 1) obtained in Example 1 after 5 days of culture were shown in the control group and the 100 μg / mL pembrolizumab treatment group.

[0048] Figure 3 This study demonstrates the apoptotic status of melanoma organoids after 5 days of culture in the control group and the 100 μg / mL pembrolizumab treatment group.

[0049] Figure 4 CD45 was compared between the "original tissue", the "culture system of this invention", and the "traditional culture system". + White blood cells, CD3 + Distribution of T cells and CD4 / CD8 T cell subsets. Detailed Implementation

[0050] The materials, methods, and embodiments described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification, the drawings, and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] The culture media used in the following examples are as described above.

[0052] Example 1 In this embodiment, primary cells were extracted from surgically removed tumor tissue of melanoma patients and co-cultured in three dimensions, and a three-dimensional model of melanoma-immune cell co-culture was constructed.

[0053] (1) Reagent preparation ① Remove the melanoma co-culture medium from the 4°C freezer and allow it to return to room temperature for later use; ② Preparation of primary tissue washing solution: Add 4% penicillin-streptomycin and 200 μg / mL gentamicin to phosphate-buffered saline (PBS), mix well and set aside. ③ Remove the primary tissue digestion solution and digestion termination solution from the 4°C refrigerator and allow them to return to room temperature for later use.

[0054] (2) Tissue cleaning Take three sterile 60 mm culture dishes and add 6 mL of primary tissue washing solution to each dish. Wash the fresh melanoma tissue removed surgically three times in each of the three culture dishes, gently turning the tissue with sterile forceps each time to remove the attached mucosa, fat and necrotic tissue.

[0055] (3) Shred the tissue Using a sterile scalpel handle, scalpel blade, and disposable forceps, the cleaned tissue was repeatedly minced in a clean 60 mm petri dish until it resembled a paste (tissue blocks approximately 1-2 mm in diameter). 3 Transfer the shredded tissue to a 15 mL centrifuge tube, add 10 mL of primary tissue washing buffer to resuspend, centrifuge at 2000 rpm and 4℃ for 5 minutes, and discard the supernatant.

[0056] (4) Tissue digestion Add 10 mL of melanoma primary tissue digestion solution preheated to 37°C to a centrifuge tube. The solution consists of 1 mg / mL collagenase I, 1 mg / mL collagenase II, and 10 μM Y27632 (Rho kinase inhibitor).

[0057] After gently mixing with a pipette, place the centrifuge tube in a metal bath shaker and digest at 37°C for 40 minutes. During digestion, remove the centrifuge tube every 5 minutes and invert it twice to ensure thorough dispersion of the tissue.

[0058] (5) Digestion monitoring and termination Twenty minutes after digestion began, a small amount of digestion solution was taken every 5 minutes and observed under an inverted microscope. When a large number of cell clusters were observed in the field of view, three volumes of pre-cooled digestion termination solution (Advanced DMEM / F12 + 10% fetal bovine serum) were immediately added to the centrifuge tube and gently mixed to terminate digestion.

[0059] (6) Filtration and initial centrifugation The cell suspension after digestion termination was filtered through a 100 μm cell sieve into a new sterile centrifuge tube. The original centrifuge tube and sieve were rinsed with 3 mL of basal buffer, and the remaining cells were collected. Three volumes of digestion termination solution were added to the collected cell suspension, and the mixture was centrifuged at 2000 rpm and 4°C for 5 minutes. The supernatant was discarded.

[0060] (7) Red blood cell lysis (depending on the situation) If a significant amount of red blood is visible to the naked eye in the resulting cell pellet, add 1 mL of erythrocyte lysis buffer, gently mix with a pipette, and allow to stand at room temperature for 2 minutes. Then, add 5 times the volume of digestion termination solution to terminate the lysis reaction, centrifuge at 2000 rpm and 4°C for 5 minutes, and discard the supernatant.

[0061] (8) Cell resuspension and counting Resuspend the cell pellet in melanoma co-culture medium. Mix 10 μL of the cell suspension with an equal volume of trypan blue, observe the cell state under a microscope, and count the cells. Record the total number of viable cells and the viability (the viability should be ≥60% to continue the experiment).

[0062] (9) Anti-adhesion treatment Take a 48-well plate and add 100 μL of anti-adhesion reagent (such as Poly-HEMA or a commercial anti-adhesion solution) to each well. Gently shake the plate to evenly cover the bottom of the plate with the reagent. After standing at room temperature for 20 minutes, discard the anti-adhesion reagent. Add 250 μL of LPBS to each well and wash for 5 minutes. Repeat the washing twice to completely remove residual reagent.

[0063] (10) Cell plating and three-dimensional co-culture The medium consisted of 250 μL of melanoma co-culture medium per well, 200 IU / mL IL2, and 10% Matrigel gel, with a concentration of 0.5 × 10⁻⁶ ppm. 6 Cells were seeded at a density of 100 cells per well and cultured in a cell culture incubator at 37°C with 5% CO2 to obtain a three-dimensional model of melanoma-immune cell co-culture.

[0064] The melanoma co-culture medium was a 1:1 volume mixture of Advanced DMEM / F12 and AIM V media, supplemented with the following components: GlutaMAX 2 mM, HEPES 10 mM, Wnt3a 300 ng / mL, RSPO1 300 ng / mL, A83-01 0.2 μM, B27 additive 2 volumes, gastrin 10 nM, SB202190 5 μM, N-acetylcysteine ​​1 mM, nicotinamide 10 mM, epidermal growth factor 50 ng / mL, and CTS. TM Immune cell serum substitute SR 10% (volume) %, penicillin-streptomycin bispecific antibody 1% (volume).

[0065] Another melanoma patient underwent surgical removal of tumor tissue, and primary cells were extracted and three-dimensionally co-cultured according to steps (1)-(10) of this embodiment to obtain another three-dimensional model of melanoma-immune cell co-culture.

[0066] Figure 1 These are bright-field micrographs of three-dimensional models prepared from melanoma tissues (tissue 1 and tissue 2) in this embodiment. Figure 1The left image corresponds to tissue 1, showing numerous densely distributed, dark brown or brownish-yellow granular structures of varying sizes. The dark areas represent organoids formed by the aggregation of melanoma cells, while smaller, lighter-colored, round structures are scattered throughout, representing co-cultured immune cells. Overall, the tissue exhibits high cell density and multicentric aggregation. Figure 1 The right image corresponds to tissue 2, where the cell structure is relatively sparse and scattered. Several large, well-defined round or oval structures are visible, which are melanoma organoids. Some immune cells are also distributed there.

[0067] Example 2 This embodiment uses primary cells derived from tumor tissue surgically removed from a melanoma patient as an example to illustrate the application of this model in drug sensitivity testing.

[0068] (1) Cell preparation and culture The single-cell suspension, whose immune cell content in step (8) of Example 1 was confirmed by primary flow cytometry, was then subjected to a 1×10⁻⁶ ppm concentration. 5 Cells were seeded at a density of 100 cells / well in 48-well plates and cultured in suspension using the melanoma co-culture medium described in Example 1, following the method described in Example 1. After 48 hours of culture, once the cells had fully adapted to the three-dimensional microenvironment, drug administration was performed.

[0069] (2) Drug preparation The groups were divided into a control group and a pembrolizumab group (100 μg / mL, final concentration). The concentration of the stock solution for the current batch of pembrolizumab was determined according to the drug instructions. The drug was diluted to twice the target concentration (i.e., 2× concentration) using complete culture medium to ensure that the final concentration in the wells after drug addition was the set value.

[0070] (3) Dosing procedure Set the following groups in the 48-well plate: Control group: group without medication; Drug administration group: 100 μg / mL (final concentration) pembrolizumab group.

[0071] Each group was set up with 3 replicates. 250 μL of the corresponding concentration of 2× drug working solution (or control culture medium) was added to each well, and the cells were gently mixed. The cells were then placed in a 37°C, 5% CO2 saturated humidity incubator for further culture.

[0072] (4) Morphological photographic record On the day of drug administration (Day 0) and on the 1st (Day 1), 3rd (Day 3), and 5th (Day 5) after drug administration, bright-field photography was performed on each well using an inverted microscope to record changes in cell morphology and organoid growth.

[0073] (5) Flow cytometry detection 5.1 Cell Collection After drug treatment, collect cells from each group. Prepare the corresponding number of flow cytometry tubes, place them on an ice box, and label the cell information.

[0074] 5.2 Multicolor Staining The required antibody volume and FACS buffer (phosphate buffer + 2% fetal bovine serum) volume were calculated based on a 100 μL staining system per sample. The antibody combination used in this assay included CD45, CD3, CD4, CD8, and PD-1. Additionally, Annexin V and PI antibody staining were performed to detect cell apoptosis.

[0075] Centrifuge the collected cells at 2000 rpm and 4°C for 5 minutes, and discard the supernatant. Add 100 μL of the pre-prepared multicolor antibody mixture (a combination of CD45, CD3, CD4, CD8, and PD-1), gently pipette to mix, and then transfer the cell suspension to labeled flow cytometry tubes. Wrap the tubes in aluminum foil to protect them from light and incubate at 4°C for 30 minutes for staining.

[0076] 5.3 Preparation of Single-Dye Compensation Tubes Vortex UltraComp eBeads Plus compensating microspheres (BioLegend, #302606) for 1 minute. Prepare the corresponding number of flow cytometry tubes for each antibody required for staining (CD45, CD3, CD4, CD8, PD-1), place them on an ice box, and label them with antibody information. Add 1 drop of microsphere suspension to each tube, followed by 1 test antibody suspension. Wrap with aluminum foil to protect from light and incubate at 4°C for 30 minutes for staining.

[0077] 5.4 Cleaning After staining, add 1 mL of FACS buffer to each tube (including cell tubes and single-stain compensation tubes), centrifuge at 600g, 4℃ for 5 minutes, and discard the supernatant. Repeat the washing once. After discarding the supernatant, resuspend each tube in 300 μL of FACS buffer, ready for instrumental analysis.

[0078] 5.5 On-machine testing After the flow cytometer completed self-cleaning and quality control (QC) testing, the corresponding fluorescence channels and gating strategies for each antibody were set. First, the voltage and compensation were adjusted using a single-stain compensation tube, and then the cell sample tubes and blank control tubes were loaded sequentially. The changes in the proportions of different immune cell subsets under different drug concentrations were recorded and analyzed.

[0079] Figure 2A and Figure 2BThe changes in immune cell phenotypes after 5 days of culture in the three-dimensional melanoma-immune cell co-culture model (tissue 1) obtained in Example 1 in the control group and the 100 μg / mL pembrolizumab treatment group are shown. Figure 2A In the middle, from left to right, the following are detected: CD45 + White blood cells, single-cell screening, lymphocyte expression; Figure 2B In the middle, from right to left, the following CD3 values ​​are tested: + T cells, CD4 + / CD8 + T cell subsets and PD-1 expression were also observed. Results showed that pembrolizumab treatment reduced CD8 expression. + The decrease in PD-1 expression in T cells indicates that the drug effectively inhibits PD-1 expression in T cells.

[0080] Figure 3 This is a flow cytometry two-parameter scatter plot showing the apoptosis status of melanoma organoids after 5 days of culture in the control group and the 100 μg / mL pembrolizumab treatment group. The X-axis represents Annexin V (early apoptosis marker), the Y-axis represents PI (late apoptosis / necrosis marker), and the four quadrants represent different apoptosis stages. Results showed that the total apoptosis rate was increased in the pembrolizumab group (63.2% vs. 58.0%), mainly driven by a significant increase in late apoptosis (Q2), while early apoptosis (Q3) decreased, suggesting that the drug may accelerate tumor cells into an irreversible death stage. The proportion of viable cells decreased (41.4% vs. 34.9%), indicating a direct killing effect of the drug. These results support the anti-tumor effect of pembrolizumab in co-culture models by inducing apoptosis.

[0081] Example 3 This embodiment compares the maintenance of immune cell components in the culture system and construction method of the present invention with that in the traditional melanoma organoid culture system, and uses the immune cell components in the original tumor tissue as a reference standard to verify the superiority of the culture system and construction method of the present invention in preserving the authenticity of the tumor immune microenvironment.

[0082] 1. Cell source and experimental grouping Tumor tissue was surgically removed from the same melanoma patient as Tissue 1, and tissue digestion and single-cell preparation were performed according to the method described in Example 1. The resulting single-cell suspension was divided into the following three groups for detection and culture: Original tumor group: Uncultured original tumor tissue was digested to obtain single-cell suspensions, which were directly analyzed by flow cytometry and used as a reference.

[0083] Experimental group: The melanoma-immune cell co-culture three-dimensional model was constructed and cultured using the method and culture system described in Example 1, and the melanoma co-culture medium described in Example 1 was used.

[0084] Control group: Organoids were cultured using traditional methods and systems, with the following culture media: 40% ADMEM / F12 basal medium, 50% Wnt3a / RSPO1 / Noggin conditioned medium (L-WRN, ATCC), and 10% inactivated fetal bovine serum, supplemented with the following components: 1 mM HEPES, 1× GlutaMax, 10 mM nicotinamide, 1 mM N-acetylcysteine, 1× vitamin A-free B27 supplement, 0.5 μM A83-01, 1× penicillin-streptomycin-glutamine, 10 nM gastrin, 10 μM SB-202190, 50 ng / mL epidermal growth factor (EGF), and 50 ng / mL fibroblast growth factor (FGF-1).

[0085] 2. Cultivation conditions The experimental group followed the formula 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / well in anti-adhesion treated 48-well plates, and cultured in suspension at 37°C in a 5% CO2 incubator. The culture period was 5 days.

[0086] The control group was cultured using the traditional organoid droplet method. Cells were mixed with 100% Matrigel and seeded into the center of a 24-well plate in 30 μL droplets. The plate was placed in a 37°C incubator for 30 min. After the droplets solidified, the control group culture medium was added, and the plate was cultured in a 37°C, 5% CO2 incubator for 5 days.

[0087] 3. Detection time point The original tumor group was tested immediately after digestion. Samples from the experimental and control groups were taken for testing on day 5 of culture.

[0088] 4. Detection Indicators Flow cytometry analysis of total immune cell content: CD45 + The proportion of cells positive for common leukocyte antigen (CLA) among total viable cells; detection of T cell subsets: CD3. + (T cell receptor complex positive), CD4 + (Positive helper T cell markers), CD8 + The percentage of cells positive for cytotoxic T cell markers among CD45+ (leukocyte common antigen positive) cells. The maintenance effect of the culture system of this invention on immune cell components was compared with that of the traditional culture system, using the immune cell components in the original tumor tissue as a standard.

[0089] Figure 4 These are three sets of parallel flow cytometry images, showing CD45 levels in "original tissue," "culture system of this invention," and "traditional culture system." + White blood cells, CD3 + The distribution of T cells and CD4 / CD8 T cell subsets visually demonstrates the significant advantages of the culture system of this invention in maintaining the immune cell composition in a melanoma-immune cell co-culture model. The results show that the system of this invention can highly replicate the immune cell composition of the original tumor tissue, while the traditional system leads to severe loss and imbalance of immune cells (especially T cell subsets).

Claims

1. A method for constructing a three-dimensional model of melanoma-immune cell co-culture, comprising: Provides containers for primary melanoma tissue and cell culture; The primary melanoma tissue was washed, broken, digested, centrifuged, and resuspended in melanoma co-culture medium to obtain a cell suspension containing melanoma cells and infiltrating immune cells in the tumor microenvironment of the melanoma cells. An anti-adhesion agent was added to the cell culture vessel; After adding IL-2 and extracellular matrix material to the cell suspension, it was seeded into the cell culture container and cultured in suspension to obtain a three-dimensional model of melanoma-immune cell co-culture. The melanoma co-culture medium is a mixture of Advanced DMEM / F12 medium and AIM V medium at a volume ratio of (1-3):(1-3), and contains the following components: GlutaMAX 1-10 mM, HEPES 10-25 mM, Wnt3a 50-500 ng / mL, RSPO1 50-500 ng / mL, A83-01 0.1-1 μM, B27 additive 1-5% (v / v), gastrin 5-20 nM, SB202190 5-20 μM, N-acetylcysteine ​​0.5-5 mM, nicotinamide 5-10 mM, epidermal growth factor 50-500 ng / mL, and CTS. TM Immune cell serum substitute SR 5%-20% of volume, penicillin-streptomycin bispecific antibody 1%-5% of volume.

2. The construction method according to claim 1, wherein, The amount of IL-2 added is 50-500 IU / mL; and / or The extracellular matrix material is selected from matrix gels, hydrogels, and / or other materials. The amount of extracellular matrix material added is 5-30% by volume.

3. The construction method according to claim 1 or 2, wherein, The cell suspension was prepared at a concentration of 0.5 × 10⁻⁶. 5 -1×10 6 Density seeding of cells / well; and / or The culture time for inoculating the cells into the cell culture container is 1-7 days.

4. The construction method according to any one of claims 1-3, wherein, The digestion was performed using a primary melanoma tissue digestion solution, the base medium of which was Advanced DMEM / F12 medium, with the addition of collagenase, and optionally at least one of hyaluronidase and Rho kinase inhibitor. Preferably, the collagenase is selected from at least one of collagenase I, collagenase II, and collagenase IV, wherein the concentration of collagenase I is 1-2 mg / mL, the concentration of collagenase II is 1-2 mg / mL, and the concentration of collagenase IV is 1-4 mg / mL. Preferably, the concentration of the hyaluronidase is 100-500 μg / mL; Preferably, the Rho kinase inhibitor is Y27632; more preferably, the concentration of Y27632 is 5-15 μM.

5. The construction method according to any one of claims 1-4, wherein, The cell culture container is a cell culture plate or a cell culture chip.

6. A three-dimensional model for co-culturing melanoma and immune cells, which is constructed by the construction method according to any one of claims 1-5.

7. The three-dimensional model for melanoma-immune cell co-culture according to claim 6, wherein, The melanoma-immune cell co-culture three-dimensional model replicates the tumor microenvironment characteristics of natural human melanoma, expresses tumor-infiltrating immune cell markers, and possesses at least one of the following characteristics: (1) It has a broad spectrum of immune cell lineage markers, including CD45; (2) Adaptive lymphocyte lineage-specific markers, including: CD3, CD4, CD8, CD19; among which CD3, CD4, and CD8 represent T lymphocyte subsets, and CD19 represents B lymphocytes; (3) It contains markers of immune cell activation and markers of T cell function / exhaustion; among which, the activation markers include CD25 and CD69; the T cell exhaustion markers include PD-1; (4) Optionally, it may have one or more of the following cell markers: NK cell marker CD56 / CD16; tumor-associated neutrophil marker CD15; tumor-associated macrophage marker CD68 / CD163; regulatory T cell marker FoxP3 / CTLA-4; myeloid-derived suppressor cell (MDSC) marker CD11b / CD33; (5) It can respond to drug stimulation; (6) It can respond to radiotherapy, thermotherapy, oncolytic virus therapy and photodynamic therapy.

8. The three-dimensional model for melanoma-immune cell co-culture according to claim 7, wherein, The drugs include chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; Preferably, the chemotherapy drugs include dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; Preferably, the targeted drug includes BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; Preferably, the immunotherapy drug includes PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; Preferably, the tumor angiogenesis inhibitor includes bevacizumab and axitinib.

9. A culture medium comprising a melanoma co-culture medium, said melanoma co-culture medium being a mixture of Advanced DMEM / F12 medium and AIM V medium at a volume ratio of (1-3):(1-3), and supplemented with the following components: GlutaMAX 1-10 mM, HEPES 10-25 mM, Wnt3a 50-500 ng / mL, RSPO1 50-500 ng / mL, A83-01 0.1-1 μM, B27 additive 1-5% by volume, gastrin 5-20 nM, SB202190 5-20 μM, N-acetylcysteine ​​0.5-5 mM, nicotinamide 5-10 mM, epidermal growth factor 50-500 ng / mL, CTS TM Immune cell serum substitute SR 5%-20%; penicillin-streptomycin bispecific antibody 1%-5%; Preferably, the culture medium is used in the method for constructing the three-dimensional model of melanoma-immune cell co-culture according to any one of claims 1-5, or for culturing the three-dimensional model of melanoma-immune cell co-culture according to any one of claims 6-8.

10. The use of the three-dimensional model of melanoma-immune cell co-culture constructed by the method of any one of claims 1-5, or the three-dimensional model of melanoma-immune cell co-culture as described in any one of claims 6-8, in drug detection and / or efficacy detection of treatment methods; Preferably, the drug detection includes drug sensitivity detection and drug efficacy detection; more preferably, the drug detection method includes at least one of flow cytometry endpoint detection, cell viability detection, immunofluorescence / confocal imaging, and cytokine secretion detection. Preferably, the drugs in the drug detection include chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; more preferably, the chemotherapy drugs include dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; more preferably, the targeted drugs include BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; more preferably, the immunotherapy drugs include PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; more preferably, the tumor angiogenesis inhibitors include bevacizumab and axitinib. Preferably, the treatment methods include radiotherapy, thermotherapy, oncolytic virus therapy, and photodynamic therapy.

11. A drug detection method, comprising: The three-dimensional model of melanoma-immune cell co-culture, constructed using the method of any one of claims 1-5 or the three-dimensional model of melanoma-immune cell co-culture as described in any one of claims 6-8, is used to detect the drug. Preferably, the drug detection method includes at least one or more of the following: drug sensitivity detection, flow cytometry endpoint detection, cell viability detection, immunofluorescence / confocal imaging, and cytokine secretion detection. More preferably, the drugs in the drug detection include at least one or more of chemotherapy drugs, targeted drugs, immunotherapy drugs, and tumor angiogenesis inhibitors; even more preferably, the chemotherapy drugs include at least one or more of dacarbazine, temozolomide, paclitaxel, and platinum-based drugs; more preferably, the targeted drugs include at least one or more of BRAF inhibitors, MEK inhibitors, and c-Kit inhibitors; more preferably, the immunotherapy drugs include at least one or more of PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, CAR-T cells, and bispecific antibodies; even more preferably, the tumor angiogenesis inhibitors include bevacizumab and / or axitinib; preferably, the treatment methods include at least one or more of radiotherapy, hyperthermia, oncolytic viruses, and photodynamic therapy. More preferably, the flow cytometry endpoint detection includes: after treating the melanoma-immune cell co-culture three-dimensional model with drugs, collecting all cells in the cell culture container for flow cytometry detection to evaluate the effect of the drugs on the immune cell population and tumor cell apoptosis; further preferably, the detection indicators include at least one or more of the following: (1) changes in the number and subsets of immune cells: CD45 + (White blood cells), CD3 + (T cells), CD4 + (Helper T cells), CD8 + (Cytotoxic T cells), CD19 + (B cells), CD25 + (Activated T cells), CD69 + (Early activation of T cells), PD-1 + (Programmed death receptor 1 positive cells) etc.; (2) Apoptosis / death: Annexin V (early apoptosis marker), Caspase-3 (cysteine ​​aspartate protease 3, activated apoptosis execution enzyme), PI (propidium iodide) or 7-AAD (7-aminoactinomycin D, late apoptosis / necrosis marker); (3) Optional extended indicators: cell proliferation: Ki-67 (proliferation-associated nuclear antigen); immune activation / inhibition balance: Granzyme B (granzyme B), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), IL-2 (interleukin-2, intracellular staining); exhaustion markers: Tim-3 (T cell immunoglobulin mucin molecule 3), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immunoglobulin and ITIM domain protein); tumor cell markers: SOX10 (sex-determining region Y-box protein 10), Melan-A / MART-1 (melanoma antigen A / melanoma antigen recognized by T cells-1), S100 (S100 protein, distinguishing tumor cells from immune cells); More preferably, the cell viability assay includes: detecting overall cell metabolic activity using methods such as the MTT assay (tetramethylazazole salt colorimetric method), the CCK-8 assay (cell counting kit method 8), or the CellTiter-Glo (CTG, cell viability luminescence assay), and calculating the half-maximal inhibitory concentration (IC50) of the drug. 50 (half-inhibitory concentration) and relative survival rate; More preferably, the immunofluorescence / confocal imaging includes: fixing and staining the three-dimensional model, and observing at least one or more of the following through immunofluorescence staining: expression of apoptotic proteins: Annexin V, Cleaved Caspase-3; immune cell infiltration and spatial distribution: co-localization of CD3 (T cells), CD8 (cytotoxic T cells), CD68 (macrophages), etc., with tumor markers; tumor microenvironment remodeling: ECM (extracellular matrix) components (collagen I, fibronectin), vascular mimicry (CD31, platelet-endothelial cell adhesion molecule), etc. More preferably, the cytokine secretion detection includes: CBA (cell counting microsphere array), Luminex (liquid phase chip) or ELISA (enzyme-linked immunosorbent assay) method to detect the secretion level of cytokines; more preferably, the cytokines include at least one or more of IL-2 (interleukin-2), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), perforin, and Granzyme B.

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