Construction and application of gastric cancer organoid co-culture model

Abstract

The present invention discloses a method for constructing a gastric cancer organoid co-culture model and its application, the method comprising: step S1, pre-treating gastric cancer tumor tissue, and then extracting TRM cells from the gastric cancer tumor tissue using a cell sieve scaffold crawling method; step S2, obtaining epithelial cells from the gastric cancer tumor tissue, and constructing gastric cancer organoids by three-dimensional culture; step S3, mixing TRM cells with gastric cancer organoids in different proportions, co-culturing 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. The present invention can better simulate the immune microenvironment in TME, thereby being better used in the development of 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 its application. Background Art

[0002] Gastric cancer (GC) is a common malignancy. While surgical resection combined with adjuvant chemotherapy remains the mainstream treatment, molecular classifications (such as the TCGA classification) reveal a high degree of heterogeneity among GC patients, resulting in limited targeted therapies (such as HER2 inhibitors) that are effective only in specific populations. In recent years, while combination therapies with immune checkpoint inhibitors (ICIs), particularly those targeting PD-1 / PD-L1, have offered hope for some patients, overall response rates remain low, suggesting that the overall suppressive state of the tumor immune microenvironment (TME) is a key factor limiting efficacy.

[0003] Previous studies have shown that the immune escape mechanisms of GC involve multiple factors, such as insufficient immune cell infiltration. Some GC patients exhibit a "cold tumor" phenotype, characterized by low infiltration density of tumor-killing T cells and high expression of the tumor immune escape molecule PD-L1. GC is also enriched with suppressive immune cells, such as tumor-associated macrophages (TAMs) and regulatory T cells (Tregs), which suppress the function of tumor-killing T cells by secreting factors such as IL-10 and TGF-β. Furthermore, metabolic reprogramming often occurs within the tumor's TME. Tumor cells can interfere with the metabolism of cells in the microenvironment and reshape their epigenetic modifications through competitive glucose consumption, lactate release, and manipulation of lipid localization abnormalities, thereby inhibiting the activity of tumor-killing T cells. These mechanisms collectively lead to the exhaustion of effector immune cell function in the TME. Existing preclinical models struggle to simulate this complex network of interactions, severely limiting 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 the 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:

[0006] 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;

[0007] Step S2, obtaining epithelial cells from gastric cancer tumor tissue and constructing gastric cancer organoids through three-dimensional culture;

[0008] Step S3: TRM cells and gastric cancer organoids are mixed in different proportions and co-cultured in a culture medium containing IL-15 and IL-2 to maintain the basal metabolism of TRM cells and obtain a gastric cancer organoid co-culture model.

[0009] In the method for constructing the gastric cancer organoid co-culture model, in step S1, the gastric cancer tumor tissue is pretreated, specifically comprising:

[0010] 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.

[0011] The tissue was rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal vascular residues. The antibiotics added included 1500 U / mL penicillin, 1500 μg / mL streptomycin, 500 μg / mL gentamicin, and 1 μg / mL amphotericin.

[0012] The tissue was cut into small pieces for subsequent TRM cell isolation.

[0013] 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 sieve scaffold crawling method, specifically comprising:

[0014] Place the tissue piece on a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, ensuring that the side in contact with gastric contents faces upwards.

[0015] 7 mL of cytokine-free culture medium was pre-filled in a culture dish. The composition of the cytokine-free culture medium is: RPMI1640, 10% fetal bovine serum and Primocin. The cell strainer-tissue piece complex was placed in the dish and cultured at 37°C for 36–48 hours.

[0016] 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. Use freezing solution to cool the cells in a gradient manner.

[0017] The above-mentioned method for constructing a gastric cancer organoid co-culture model comprises the following steps: centrifugation at 400 g for 5 minutes, and the freezing solution comprises 10% DMSO and 90% FBS.

[0018] In the method for constructing the gastric cancer organoid co-culture model, step S2 specifically comprises:

[0019] Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrigel, 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, 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; and ROCK pathway inhibitor Y-27632, 10 μM.

[0020] In the method for constructing the gastric cancer organoid co-culture model, step S3 specifically comprises:

[0021] Stable gastric cancer organoids were scraped from the well plate, washed, digested into single cells using TrypLE, and counted;

[0022] TRM cells were revived and counted, and then mixed with gastric cancer organoid epithelial cells digested into single cells at a ratio of 1:20 and resuspended in Matrigel.

[0023] The basal activity of TRM cells was maintained using a mixed medium consisting of RPMI 1640 mixed with human gastric cancer organoid culture medium without Y-27632 at a volume ratio of 1:1 and supplemented with 5 IU / mL of IL-2 and 1 ng / mL of IL-15.

[0024] Replace the mixed culture medium every 48-72 hours to maintain TRM activity and gastric cancer organoid growth;

[0025] For co-cultured organoids, the mixed medium was discarded, and the co-culture was gently washed with PBS and collected, and centrifuged at 400 g for 4 minutes to avoid loss of TRM cells.

[0026] Another aspect of the present invention provides the use of the gastric cancer organoid co-culture model constructed by the above-mentioned construction method in the screening of immunotherapy drugs.

[0027] Beneficial effects:

[0028] (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 onset of immune checkpoint inhibitors.

[0029] (2) Compared with PBMC cells, TRM cells derived from tumor tissue have the characteristics of specific recognition of tumor antigens and can better restore the individual immune microenvironment. Compared with the 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 response more controllable.

[0030] (3) The present invention uses a cell mesh scaffold crawling method to obtain TRM cells. This method does not require digestive enzymes, only mechanical manipulation, and can retain TRM surface molecular markers to the greatest extent possible. It can achieve sufficient purity without flow cytometry, and can grow stably and long-term in a chimeric manner with organoids, facilitating in vitro observation of epithelial cell-immune cell interactions. Experiments have shown 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.

[0031] (4) The construction method of the present invention is easy to operate, has a short cycle, and good reproducibility. The gastric cancer organoid co-culture model responds to low-dose inflammatory factors and immune checkpoint inhibitors. It can be operated on a large scale in vitro, achieving high-throughput experiments and supporting high-sensitivity drug screening (such as immune checkpoint inhibitor detection). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of obtaining cells from different gastric cancer tissues using the cell mesh scaffold crawl-out method under an inverted microscope;

[0033] Figure 2 Schematic diagram of flow cytometric analysis of crawling-out cells using TRM surface markers CD69 and CD103;

[0034] Figure 3 A simplified diagram of the co-culture process of TRM cells and gastric cancer organoids;

[0035] Figure 4 The figure shows panoramic immunofluorescence staining and monolayer magnification of TRM cells and gastric cancer organoid co-culture chimeric growth on days 7, 14, and 50.

[0036] Figure 5 Statistical graph of the ratio of TRM and epithelial cells;

[0037] Figure 6 A comparison of bright field images of individually cultured gastric cancer epithelial organoids and a gastric cancer organoid co-culture model during long-term culture.

[0038] Figure 7 Bright field images of the gastric cancer organoid co-culture model before and after cryopreservation and thawing, and panoramic scanning immunofluorescence images of TRM surface markers after thawing;

[0039] Figure 8 The results of apoptosis in gastric cancer organoids cultured alone and co-cultured after low-dose TNF-α induction;

[0040] Figure 9 The figure shows the test results of the apoptosis-inducing ability of different types of PD-1 / PD-L1 monoclonal antibodies in the gastric cancer organoid co-culture model at low doses. DETAILED DESCRIPTION

[0041] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The drawings illustrate several embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0042] An embodiment of the present invention provides a method for constructing a gastric cancer organoid co-culture model, comprising steps S1 to S3:

[0043] 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.

[0044] Among them, pretreatment of gastric cancer tumor tissue specifically includes:

[0045] 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.

[0046] The tissue was rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal vascular residues. The antibiotics added included 1500 U / mL penicillin, 1500 μg / mL streptomycin, 500 μg / mL gentamicin, and 1 μg / mL amphotericin.

[0047] The tissue was cut into small pieces for subsequent TRM cell isolation.

[0048] Among them, TRM cells were extracted from gastric cancer tumor tissue using a cell screen scaffold crawling method, specifically including:

[0049] Place the tissue piece on a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, ensuring that the side in contact with gastric contents faces upwards.

[0050] Pre-fill 7 mL of cytokine-free culture medium (RPMI 1640, 10% fetal bovine serum, and Primocin) in a 6 cm dish, place the cell strainer-tissue fragment complex, and culture at 37°C for 36–48 hours.

[0051] After removing the sieve, collect the cells that have fallen onto the bottom of the culture dish and centrifuge at 400g for 5 minutes. Discard the supernatant and cryopreserve in a gradient of 10% DMSO and 90% FBS. Simultaneously, remove a portion of the cells and verify TRM cell purity and viability by flow cytometry.

[0052] The process of TRM cell purity and activity testing is as follows:

[0053] The obtained TRM cells were resuspended in an appropriate amount of flow cytometry blocking buffer (PBS containing 0.5% BSA), passed through a 40μm sieve to remove mucus and cell clumps, and CD69 and 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. After washing with flow cytometry buffer, the TRM cell proportion and overall viability were analyzed by flow cytometry.

[0054] The cell mesh 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 antennae, 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%.

[0055] Step S2: Obtain epithelial cells from gastric cancer tumor tissue and construct gastric cancer organoids through three-dimensional culture.

[0056] Wherein, step S2 specifically includes:

[0057] Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrigel, 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, 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; and ROCK pathway inhibitor Y-27632, 10 μM.

[0058] Step S3: TRM cells and gastric cancer organoids are mixed in different proportions and co-cultured in a culture medium containing IL-15 and IL-2 to maintain the basal metabolism of TRM cells and obtain a gastric cancer organoid co-culture model.

[0059] Wherein, step S3 specifically includes:

[0060] Stable gastric cancer organoids were scraped from the well plate, washed, digested into single cells using TrypLE, and counted;

[0061] TRM cells were revived and counted, and then mixed with gastric cancer organoid epithelial cells digested into single cells at a ratio of 1:20 and resuspended in Matrigel.

[0062] The basal activity of TRM cells was maintained using a mixed medium consisting of RPMI 1640 mixed with human gastric cancer organoid culture medium without Y-27632 at a volume ratio of 1:1 and supplemented with 5 IU / mL of IL-2 and 1 ng / mL of IL-15.

[0063] Replace the mixed culture medium every 48-72 hours to maintain TRM activity and gastric cancer organoid growth;

[0064] For co-cultured organoids, the mixed medium was discarded, and the co-culture was gently washed with PBS and collected, and centrifuged at 400 g for 4 minutes to avoid loss of TRM cells.

[0065] After obtaining the gastric cancer organoid co-culture model, the co-culture can be collected and downstream analyzed as follows:

[0066] (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.

[0067] (2) Short-term stimulation: Add low-dose inflammatory factors or PD-1 / PD-L1 monoclonal antibody (1 μM) and observe the changes in organoid apoptosis rate within 24 hours using the GreenNu live cell Caspase-3 activity detection kit.

[0068] (3) Long-term observation: The killing effect of TRM on organoids can be continuously monitored by regular bright field image capture.

[0069] 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.

[0070] Next, we tested the response of the gastric cancer organoid co-culture model to low-dose inflammatory factors and immune checkpoint inhibitors.

[0071] In a gastric cancer organoid co-culture model, TNF-α, an inflammatory factor that activates T cells, was added, and a green fluorescent probe for active Caspase-3, a marker of apoptosis, was used to visualize the level of organoid apoptosis. Figure 8 , Figure 8 The green in the middle is a fluorescent probe for 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, while the single organoid under the same culture conditions will not. For further information, please refer to Figure 9When a low dose (1 μM) of the immune checkpoint inhibitor PD-1 / PD-L1 monoclonal antibody was added to the culture system, the apoptosis rate of the organoids was significantly increased compared to the PBS control group. Existing studies have often used immune checkpoint inhibitors at doses more than ten times this level in vitro. This suggests that the gastric cancer organoid co-culture model can be sensitive to immune checkpoint inhibitors in vitro. These results demonstrate that the gastric cancer organoid co-culture model can be applied to immunotherapy drug screening.

[0072] 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:

[0073] (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 onset of immune checkpoint inhibitors.

[0074] (2) Compared with PBMC cells, TRM cells derived from tumor tissue have the characteristics of specific recognition of tumor antigens and can better restore the individual immune microenvironment. Compared with the 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 response more controllable.

[0075] (3) The present invention uses a cell mesh scaffold crawling method to obtain TRM cells. This method does not require digestive enzymes, only mechanical manipulation, and can retain TRM surface molecular markers to the greatest extent possible. It can achieve sufficient purity without flow cytometry, and can grow stably and long-term in a chimeric manner with organoids, facilitating in vitro observation of epithelial cell-immune cell interactions. Experiments have shown 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.

[0076] (4) The construction method of the present invention is easy to operate, has a short cycle, and good reproducibility. The gastric cancer organoid co-culture model responds to low-dose inflammatory factors and immune checkpoint inhibitors. It can be operated on a large scale in vitro, achieving high-throughput experiments and supporting high-sensitivity drug screening (such as immune checkpoint inhibitor detection).

[0077] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended 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, and co-culturing them in a culture medium containing IL-15 and IL-2 to maintain the basal metabolism of TRM cells, thereby obtaining a gastric cancer organoid co-culture model; 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 on a 100 μm sterile cell sieve and use ophthalmic forceps to position it correctly, ensuring that the side in contact with gastric contents faces upwards. 7 mL of cytokine-free culture medium was pre-filled in a culture dish. The composition of the cytokine-free culture medium is: RPMI1640, 10% fetal bovine serum and Primocin. The cell strainer-tissue piece complex was placed in the dish and cultured at 37°C for 36–48 hours. After removing the sieve, collect the cells that fall off the bottom of the sieve and are placed on the bottom of the culture dish. Centrifuge and discard the supernatant. Use a freezing solution with a gradient cooling method to freeze the cells. Step S2 specifically includes: Epithelial cells were extracted from gastric cancer tumor tissue, mixed with matrigel, and cultured in a human gastric cancer organoid culture medium for 7 days to obtain gastric cancer organoids. The composition and content of the human gastric cancer organoid culture medium are 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, 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; Step S3 specifically includes: Stable gastric cancer organoids were scraped from the well plate, washed, digested into single cells using TrypLE, and counted; TRM cells were revived and counted, and then 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 consisting of RPMI 1640 mixed with human gastric cancer organoid culture medium without Y-27632 at a volume ratio of 1:1 and 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-cultured organoids, the mixed medium was discarded, and the co-culture was gently washed with PBS and collected, and centrifuged at 400 g for 4 minutes to avoid loss of TRM cells.

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 rinsed five times with HBSS buffer supplemented with antibiotics to remove mucosal mucus and basal vascular 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 1, characterized in that: The centrifugation condition was 400 g for 5 minutes, and the freezing solution consisted of 10% DMSO and 90% FBS.

4. Application of the gastric cancer organoid co-culture model constructed by the construction method according to any one of claims 1 to 3 in immunotherapy drug screening.

Images