Construction method and application of gastric cancer organoid co-culture model

Abstract

The invention discloses a construction method and application of a gastric cancer organoid co-culture model, and the method comprises the following steps: S1, pretreating a gastric cancer tumor tissue, and then extracting TRM cells from the gastric cancer tumor tissue by adopting a cell screen scaffold climbing-out method; s2, acquiring epithelial cells from the gastric cancer tumor tissues, and constructing gastric cancer organs through three-dimensional culture; s3, mixing the TRM cells and the gastric cancer organs according to different proportions, co-culturing in a culture medium containing IL-15 and IL-2, and maintaining basic metabolism of the TRM cells to obtain the gastric cancer organ co-culture model. According to the invention, the immune microenvironment in TME can be better simulated, so that the TME can be better used for developing immunotherapy drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing a gastric cancer organoid co-culture model and application thereof. Background Art

[0002] Gastric cancer (GC) is a common malignant tumor. Although surgical resection combined with adjuvant chemotherapy is still the mainstream treatment, molecular classification (such as TCGA classification) reveals that there is a high degree of heterogeneity among gastric cancer patients, resulting in limited targeted treatment options (such as HER2 inhibitors) being effective only for specific populations. In recent years, although the combination of immune checkpoint inhibitors (ICIs) represented by PD-1 / PD-L1 target inhibitors has brought hope to some patients, the overall response rate is still low, suggesting that the overall suppression state of the tumor immune microenvironment (TME) is the core reason for the limited efficacy.

[0003] Previous studies have shown that the immune escape mechanism of GC involves multiple factors, such as insufficient immune cell infiltration. Some GC patients show a "cold tumor" phenotype, that is, the density of tumor-killing T cell infiltration is low, and the expression of PD-L1, a molecule related to tumor immune escape, is high. Inhibitory immune cells are also enriched in GC, such as tumor-associated macrophages (TAMs) and regulatory T cells (Tregs), which inhibit the function of tumor-killing T cells by secreting factors such as IL-10 and TGF-β. In addition, metabolic reprogramming often occurs in the TME of tumors. Tumor cells can interfere with the metabolism of cells in the microenvironment and reshape their epigenetic modifications by competitively consuming glucose, releasing lactate, and manipulating abnormal lipid localization, thereby inhibiting the activity of tumor-killing T cells. The above mechanisms together lead to the exhaustion of effector immune cell function in the TME, and existing preclinical models are difficult to simulate this complex interaction network, which severely limits the development of immunotherapy drugs. Summary of the invention

[0004] The present invention provides a method for constructing a gastric cancer organoid co-culture model and its application, so as to better simulate the immune microenvironment in TME, thereby better being used for the development of immunotherapy drugs.

[0005] One aspect of the present invention provides a method for constructing a gastric cancer organoid co-culture model, comprising: Step S1, pre-treating the gastric cancer tumor tissue, and then extracting TRM cells from the gastric cancer tumor tissue using a cell screen scaffold crawling method; Step S2, obtaining epithelial cells from gastric cancer tumor tissue and constructing gastric cancer organoids through three-dimensional culture; Step S3, mixing TRM cells and gastric cancer organoids in different proportions, co-culturing them in a culture medium containing IL-15 and IL-2, maintaining the basal metabolism of TRM cells, and obtaining a gastric cancer organoid co-culture model.

[0006] In the method for constructing the gastric cancer organoid co-culture model, in step S1, the gastric cancer tumor tissue is pretreated, specifically comprising: Gastric cancer tumor tissue was obtained, and the basal muscle layer, serosa, and adipose tissue were grasped with sterile forceps, and non-target components were trimmed and removed; The tissue was repeatedly rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal blood vessel residues. The antibiotics added included 1500 U / mL penicillin, 1500 μg / mL streptomycin, 500 μg / mL gentamicin, and 1 μg / mL amphotericin. The tissue was cut into small pieces for subsequent TRM cell isolation.

[0007] The method for constructing the gastric cancer organoid co-culture model, wherein in step S1, TRM cells are extracted from gastric cancer tumor tissue using a cell screen scaffold crawling method, specifically comprising: Place the tissue piece in a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, making sure the side in contact with the gastric contents faces upwards; Pre-fill 7 mL of cytokine-free culture medium in a culture dish. The components of the cytokine-free culture medium are: RPMI1640, 10% fetal bovine serum and Primocin. Place the cell screen-tissue piece complex in the dish and culture at 37°C for 36-48 hours. After removing the sieve, collect the cells that have fallen off the bottom of the sieve and are placed on the bottom of the culture dish, centrifuge and discard the supernatant, and freeze them using a freezing solution with a gradient cooling method.

[0008] The method for constructing the above-mentioned gastric cancer organoid co-culture model, wherein the centrifugation conditions are 400g for 5 minutes, and the composition of the freezing solution is 10% DMSO and 90% FBS.

[0009] In the method for constructing the gastric cancer organoid co-culture model, step S2 specifically comprises: Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrix gel, and cultured in a human gastric cancer organoid culture medium for 7 days to obtain gastric cancer organoids. The components and contents of the human gastric cancer organoid culture medium were as follows: GlutaMAX, 1×; HEPES Buffer, 10mM; Primocin, 1×; N-acetylcysteine, 1mM; N2, 1×; B27, 1×; Wnt3a, 100-300ng / mL; R-spondin 1, 1μg / mL; bone morphogenetic protein antagonist Noggin, 100ng / mL; gastrin protein Gastrin, 10nM; epidermal growth factor EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5μM; nicotinamide, 10mM; ROCK pathway inhibitor Y-27632, 10μM.

[0010] In the method for constructing the gastric cancer organoid co-culture model, step S3 specifically comprises: The stable gastric cancer organoids were scraped from the wells, washed, digested into single cells using TrypLE and counted; TRM cells were revived and counted, and TRM cells were mixed with gastric cancer organoid epithelial cells digested into single cells at a ratio of 1:20 and resuspended in Matrigel; The basal activity of TRM cells was maintained using a mixed medium, which was a 1:1 volume mixture of RPMI 1640 and human gastric cancer organoid medium without Y-27632, supplemented with 5 IU / mL of IL-2 and 1 ng / mL of IL-15; Replace the mixed culture medium every 48-72 hours to maintain TRM activity and gastric cancer organoid growth; For co-culture organoids, the mixed medium was discarded, the co-cultures were gently washed with PBS and collected, and centrifuged at 400 g for 4 min to avoid loss of TRM cells.

[0011] Another aspect of the present invention provides the use of the gastric cancer organoid co-culture model constructed by the above construction method in immunotherapy drug screening.

[0012] Beneficial effects: (1) The gastric cancer organoid co-culture model constructed by the method of the present invention comprises tumor-derived epithelial cells and tissue-resident memory T cells (TRM) resident in the tumor tissue, thereby constructing an in vitro organoid model with immune-active components, which can better simulate the immune microenvironment of the tumor in situ and provide a microenvironmental basis for the efficacy of immune checkpoint inhibitors.

[0013] (2) Compared with PBMC cells, TRM cells derived from tumor tissues have the characteristics of specific recognition of tumor antigens, which can better restore the individual immune microenvironment. Compared with a larger number of TIL populations, they have fewer exhausted cells, better activity, and clear subpopulations. The characteristics of TRM itself as a T cell enable it to respond to immune checkpoint inhibitors, making the in vitro reaction more controllable.

[0014] (3) The present invention uses a cell mesh scaffold crawling method to obtain TRM cells, which does not require digestive enzymes, only mechanical operations, can retain TRM surface molecular markers to the greatest extent, can achieve sufficient purity without flow sorting, and can grow stably and long-term with organoids, which is conducive to in vitro observation of epithelial cell-immune cell interactions. Experiments show that the gastric cancer organoid co-culture model constructed in the present invention can maintain activity for up to 50 days and can be passaged, supporting long-term drug efficacy observation.

[0015] (4) The construction method of the present invention is easy to operate, has a short cycle, and has good repeatability. The gastric cancer organoid co-culture model responds to low-dose inflammatory factors and immune checkpoint inhibitors. It can be operated in large quantities in vitro to achieve high-throughput experiments and support high-sensitivity drug screening (such as immune checkpoint inhibitor detection). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of obtaining cells under an inverted microscope using the cell screen scaffold crawling method for different gastric cancer tissues; Figure 2 Schematic diagram of flow cytometric analysis of crawling out cells using TRM surface markers CD69 and CD103; Figure 3 This is a simplified diagram of the co-culture process of TRM cells and gastric cancer organoids; Figure 4 Panoramic immunofluorescence staining and monolayer magnification of TRM cells and gastric cancer organoid co-culture chimeric growth on days 7, 14, and 50; Figure 5 It is a statistical diagram of the proportion of TRM and epithelial cells; Figure 6 A comparison of bright field images of gastric cancer epithelial organoids cultured alone and gastric cancer organoid co-culture models during long-term culture; Figure 7 Bright field images of gastric cancer organoid co-culture model before and after cryopreservation-thawing and panoramic scanning immunofluorescence images of TRM surface markers after thawing; Figure 8 The results of apoptosis in gastric cancer organoids cultured alone and co-cultured after low-dose TNF-α induction; Fig. 9This is a graph showing the results of testing the apoptosis-inducing ability of different types of PD-1 / PD-L1 monoclonal antibodies in a gastric cancer organoid co-culture model at low doses. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0018] An embodiment of the present invention provides a method for constructing a gastric cancer organoid co-culture model, comprising steps S1 to S3: Step S1, pre-treating the gastric cancer tumor tissue, and then extracting TRM cells from the gastric cancer tumor tissue using a cell screen scaffold crawling method.

[0019] Among them, the pretreatment of gastric cancer tumor tissue specifically includes: Gastric cancer tumor tissue was obtained, and the basal muscle layer, serosa, and adipose tissue were grasped with sterile forceps, and non-target components were trimmed and removed; The tissue was repeatedly rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal blood vessel residues. The antibiotics added included 1500 U / mL penicillin, 1500 μg / mL streptomycin, 500 μg / mL gentamicin, and 1 μg / mL amphotericin. The tissue was cut into small pieces for subsequent TRM cell isolation.

[0020] Among them, the cell screen scaffold crawling method was used to extract TRM cells from gastric cancer tumor tissue, specifically including: Place the tissue piece in a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, making sure the side in contact with the gastric contents faces upwards; Pre-fill 7 mL of cytokine-free culture medium in a 6 cm culture dish. The composition of the cytokine-free culture medium is: RPMI 1640, 10% fetal bovine serum and Primocin, place the cell screen-tissue small piece complex, and culture at 37°C for 36-48 hours; After removing the sieve, collect the cells that fall off the bottom of the sieve and on the bottom of the culture dish, centrifuge at 400g for 5 minutes, then discard the supernatant and freeze in a freezing solution with a gradient cooling. The freezing solution consists of 10% DMSO and 90% FBS. At the same time, take a portion and identify the purity and activity of TRM cells by flow cytometry.

[0021] The process of TRM cell purity and activity testing is as follows: The obtained TRM cells were resuspended with an appropriate amount of flow cytometry blocking solution (PBS containing 0.5% BSA), passed through a 40μm sieve to remove mucus and cell clumps, and CD69, CD103 flow cytometry antibodies and DAPI were added at a dilution ratio of 1:50. The cells were incubated on ice in the dark for 1 hour for staining, and the TRM cell proportion and overall viability were identified by flow cytometry analysis after washing with flow cytometry buffer.

[0022] The cell screen support crawling method can be used to easily and efficiently extract TRM cells resident in gastric cancer tissue. Figure 1 , Figure 1 In the table, 06, 14, 18, and 20 represent four different human tissue samples, N represents normal tissue, and T represents tumor tissue. Figure 1 It can be seen that the crawling cells are about 20μm in diameter under an inverted microscope and have tentacles, which is consistent with the description of T cell morphology. Figure 2 Flow cytometry analysis showed that CD69 + / CD103 + The purity of TRM cells can reach 80%.

[0023] Step S2, obtaining epithelial cells from gastric cancer tumor tissue and constructing gastric cancer organoids through three-dimensional culture.

[0024] Wherein, step S2 specifically includes: Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrix gel, and cultured in a human gastric cancer organoid culture medium for 7 days to obtain gastric cancer organoids. The components and contents of the human gastric cancer organoid culture medium were as follows: GlutaMAX, 1×; HEPES Buffer, 10mM; Primocin, 1×; N-acetylcysteine, 1mM; N2, 1×; B27, 1×; Wnt3a, 100-300ng / mL; R-spondin 1, 1μg / mL; bone morphogenetic protein antagonist Noggin, 100ng / mL; gastrin protein Gastrin, 10nM; epidermal growth factor EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5μM; nicotinamide, 10mM; ROCK pathway inhibitor Y-27632, 10μM.

[0025] Step S3, mixing TRM cells and gastric cancer organoids in different proportions, co-culturing them in a culture medium containing IL-15 and IL-2, maintaining the basal metabolism of TRM cells, and obtaining a gastric cancer organoid co-culture model.

[0026] Wherein, step S3 specifically includes: The stable gastric cancer organoids were scraped from the wells, washed, digested into single cells using TrypLE and counted; TRM cells were revived and counted, and TRM cells were mixed with gastric cancer organoid epithelial cells digested into single cells at a ratio of 1:20 and resuspended in Matrigel; The basal activity of TRM cells was maintained using a mixed medium, which was a 1:1 volume mixture of RPMI 1640 and human gastric cancer organoid medium without Y-27632, supplemented with 5 IU / mL of IL-2 and 1 ng / mL of IL-15; Replace the mixed culture medium every 48-72 hours to maintain TRM activity and gastric cancer organoid growth; For co-culture organoids, the mixed medium was discarded, the co-cultures were gently washed with PBS and collected, and centrifuged at 400 g for 4 min to avoid loss of TRM cells.

[0027] After obtaining the gastric cancer organoid co-culture model, co-culture collection and downstream analysis can be performed as follows: (1) Sample collection: Gently collect the co-cultured organoids and surrounding residual cells, centrifuge at 50g for 1 minute, remove the supernatant, and fix with 4% paraformaldehyde for subsequent immunofluorescence staining.

[0028] (2) Short-term stimulation: Add low-dose inflammatory factors or PD-1 / PD-L1 monoclonal antibodies (1 μM) and observe the changes in organoid apoptosis rate within 24 hours using the GreenNu live cell Caspase-3 activity detection kit.

[0029] (3) Long-term observation: The killing effect of TRM on organoids can be continuously monitored by regular bright field image capture.

[0030] The following is an observation of the construction results and stability of the gastric cancer organoid co-culture model. Figure 3 , gastric cancer organoid co-culture model can be stimulated by factors or drugs within a certain number of days to achieve the effect of in vitro simulation. Figure 4 , Figure 5 and Figure 6 In the gastric cancer organoid co-culture model, the organoids and TRM cells grow in a chimeric manner, and as the number of days increases, the number of TRM cells in a single tumor organoid continues to increase. The gastric cancer organoid co-culture model can maintain activity for up to 50 days, and the tumor organoids are gradually killed under the action of low-dose inflammatory factors (IL-2 and IL-15) in the culture medium. Figure 7 The gastric cancer organoid co-culture model is still usable after freezing and thawing, and can continue to maintain the activity of TRM mosaic growth, which suggests that the constructed gastric cancer organoid co-culture model can provide an in vitro model for long-term observation of epithelial-immune cell interactions.

[0031] Next, we tested the response of the gastric cancer organoid co-culture model to low-dose inflammatory factors and immune checkpoint inhibitors.

[0032] In the gastric cancer organoid co-culture model, TNF-α, an inflammatory factor that can activate T cells, was added, and the apoptosis level of the organoids was displayed using a green fluorescent probe of the apoptosis marker active Caspase-3. Figure 8 , Figure 8 The green in the middle is the fluorescent probe of active Caspase-3, a marker of apoptosis. The results showed that at low doses of TNF-α, the gastric cancer organoid co-culture model can produce a dose-dependent apoptotic response to it, while the single organoid under the same culture conditions will not. For further information, please refer to Fig. 9 When a low dose (1 μM) of immune checkpoint inhibitor PD-1 / PD-L1 monoclonal antibody was added to the culture system, the apoptosis rate of the organoids increased significantly compared with the PBS control group. In existing studies, the immune checkpoint inhibitors used in in vitro experiments are often more than ten times this dose, which suggests that the gastric cancer organoid co-culture model can respond sensitively to immune checkpoint inhibitors in vitro. The above results show that the gastric cancer organoid co-culture model can be used for immunotherapy drug screening.

[0033] In summary, the method for constructing and applying the gastric cancer organoid co-culture model provided by the present invention has the following beneficial effects: (1) The gastric cancer organoid co-culture model constructed by the method of the present invention comprises tumor-derived epithelial cells and memory T cells (TRM) resident in the tumor tissue, thereby constructing an in vitro organoid model with immune-active components, which can better simulate the immune microenvironment of the tumor in situ and provide a microenvironmental basis for the efficacy of immune checkpoint inhibitors.

[0034] (2) Compared with PBMC cells, TRM cells derived from tumor tissues have the characteristics of specific recognition of tumor antigens, which can better restore the individual immune microenvironment. Compared with a larger number of TIL populations, they have fewer exhausted cells, better activity, and clear subpopulations. The characteristics of TRM itself as a T cell enable it to respond to immune checkpoint inhibitors, making the in vitro reaction more controllable.

[0035] (3) The present invention uses a cell mesh scaffold crawling method to obtain TRM cells, which does not require digestive enzymes, only mechanical operations, can retain TRM surface molecular markers to the greatest extent, can achieve sufficient purity without flow sorting, and can grow stably and long-term with organoids, which is conducive to in vitro observation of epithelial cell-immune cell interactions. Experiments show that the gastric cancer organoid co-culture model constructed in the present invention can maintain activity for up to 50 days and can be passaged, supporting long-term drug efficacy observation.

[0036] (4) The construction method of the present invention is easy to operate, has a short cycle, and has good repeatability. The gastric cancer organoid co-culture model responds to low-dose inflammatory factors and immune checkpoint inhibitors. It can be operated in large quantities in vitro to achieve high-throughput experiments and support high-sensitivity drug screening (such as immune checkpoint inhibitor detection).

[0037] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a gastric cancer organoid co-culture model, characterized in that: include: Step S1, pre-treating the gastric cancer tumor tissue, and then extracting TRM cells from the gastric cancer tumor tissue using a cell screen scaffold crawling method; Step S2, obtaining epithelial cells from gastric cancer tumor tissue and constructing gastric cancer organoids through three-dimensional culture; Step S3, mixing TRM cells and gastric cancer organoids in different proportions, co-culturing them in a culture medium containing IL-15 and IL-2, maintaining the basal metabolism of TRM cells, and obtaining a gastric cancer organoid co-culture model.

2. The method for constructing a gastric cancer organoid co-culture model according to claim 1, characterized in that: In step S1, the gastric cancer tumor tissue is pretreated, specifically including: Gastric cancer tumor tissue was obtained, and the basal muscle layer, serosa, and adipose tissue were grasped with sterile forceps, and non-target components were trimmed and removed; The tissue was repeatedly rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal blood vessel residues. The antibiotics added included 1500 U / mL penicillin, 1500 μg / mL streptomycin, 500 μg / mL gentamicin, and 1 μg / mL amphotericin. The tissue was cut into small pieces for subsequent TRM cell isolation.

3. The method for constructing a gastric cancer organoid co-culture model according to claim 2, characterized in that: In step S1, TRM cells are extracted from gastric cancer tumor tissue using a cell screen scaffold crawling method, which specifically includes: Place the tissue piece in a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, making sure the side in contact with the gastric contents faces upwards; Pre-fill 7 mL of cytokine-free culture medium in a culture dish. The components of the cytokine-free culture medium are: RPMI1640, 10% fetal bovine serum and Primocin. Place the cell screen-tissue piece complex in the dish and culture at 37°C for 36-48 hours. After removing the sieve, collect the cells that have fallen off the bottom of the sieve and are placed on the bottom of the culture dish, centrifuge and discard the supernatant, and freeze them using a freezing solution with a gradient cooling method.

4. The method for constructing a gastric cancer organoid co-culture model according to claim 3, characterized in that: The centrifugation condition was 400 g for 5 minutes, and the composition of the freezing solution was 10% DMSO and 90% FBS.

5. The method for constructing a gastric cancer organoid co-culture model according to claim 1, characterized in that: Step S2 specifically includes: Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrix gel, and cultured in a human gastric cancer organoid culture medium for 7 days to obtain gastric cancer organoids. The components and contents of the human gastric cancer organoid culture medium were as follows: GlutaMAX, 1×; HEPES Buffer, 10 mM; Primocin, 1×; N-acetylcysteine, 1 mM; N2, 1×; B27, 1×; Wnt3a, 100-300 ng / mL; R-spondin 1, 1 μg / mL; bone morphogenetic protein antagonist Noggin, 100 ng / mL; gastrin protein Gastrin, 10 nM; epidermal growth factor EGF, 50 ng / mL; fibroblast growth factor FGF-10, 100 ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5 μM; nicotinamide, 10 mM; ROCK pathway inhibitor Y-27632, 10 μM.

6. The method for constructing a gastric cancer organoid co-culture model according to claim 1, characterized in that: Step S3 specifically includes: The stable gastric cancer organoids were scraped from the wells, washed, digested into single cells using TrypLE and counted; TRM cells were revived and counted, and TRM cells were mixed with gastric cancer organoid epithelial cells digested into single cells at a ratio of 1:20 and resuspended in Matrigel; The basal activity of TRM cells was maintained using a mixed medium, which was a 1:1 volume mixture of RPMI 1640 and human gastric cancer organoid medium without Y-27632, supplemented with 5 IU / mL of IL-2 and 1 ng / mL of IL-15; Replace the mixed culture medium every 48-72 hours to maintain TRM activity and gastric cancer organoid growth; For co-culture organoids, the mixed medium was discarded, the co-cultures were gently washed with PBS and collected, and centrifuged at 400 g for 4 min to avoid loss of TRM cells.

7. Application of the gastric cancer organoid co-culture model constructed by the construction method according to any one of claims 1 to 6 in immunotherapy drug screening.

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