To construct an in vitro co-culture model of gastric cancer organoids and cancer-associated fibroblasts (CAFs), and investigate the role of cancer-associated fibroblasts in the proliferation and chemotherapy resistance of gastric cancer organoids.

Tumor tissues from 12 gastric cancer patients undergoing surgical treatment in Department of General Surgery of Second Affiliated Hospital of Army Medical University from February 2023 to March 2024 were collected to construct gastric cancer organoids using 3D culture. HE staining was used to observe the morphology, and immunohistochemical assay was employed to determine the expression of cytokeratin CK7, carcinoembryonic antigen (CEA), and proliferation marker Ki-67. After CAFs derived from the same patient were cultured, observed for their morphology under a light microscope, and detected for the phenotype by flow cytometry, the cells were co-cultured with gastric cancer organoids in a 1:1 ratio. Phase-contrast microscopy was applied to observe the growth of the organoids and analyze the number, average diameter, and total area. Then, organoids cultured alone served as the control group. After the control and co-culture groups were treated with chemotherapy drugs, 5-fluorouracil and oxaliplatin, for 48 h, the viability and apoptosis of organoids were assessed with CellTiter-Glo^® 3D assay and CellEvent^TM Caspase 3/7 activity, respectively.

Gastric cancer organoids and CAFs were successfully established from 10 gastric cancer patient-derived samples. The gastric cancer organoids exhibited morphological characteristics consistent with the corresponding primary tumors, and showed positive expression of CK7, CEA, and Ki-67. CAFs displayed typical spindle-shaped morphology and exhibited the phenotypic markers CD326^-, CD45^-, CD31^-, α-SMA⁺, CD73⁺, CD90⁺, and CD105⁺. Compared to the organoids cultured alone, the organoids co-cultured with CAFs showed more formation of organoids, in larger average diameter, and taking larger total area (P<0.05). After the treatment of 5-fluorouracil and oxaliplatin, the half-maximal inhibitory concentration (IC₅₀) was 10.66 and 3.26 μmol/L, respectively in the control group, while was 46.23 and 91.11 μmol/L in the co-culture group.Additionally, the number of CellEvent^TM Caspase 3/7 positive apoptotic cells was significantly less in the co-culture group than the control group.

Compared with individually cultured gastric cancer organoids, the co-culture model of gastric cancer organoids and CAFs better simulates the pro-tumor proliferation and drug resistance effects of in vivo tumor microenvironment.

To detect the infiltration level of CD103⁺CD39⁺CD8⁺ T cell subsets in gastric cancer tissues and analyze their response characteristics, clarifying the clinical relevance of this subset.

Surgical specimens (tumor tissues, adjacent tissues, and peripheral blood) were collected from gastric cancer patients at the Department of General Surgery, Second Affiliated Hospital of Army Medical University between January 2023 and May 2024. Flow cytometry and immunofluorescence staining quantified the proportion and number of CD103⁺CD39⁺CD8⁺ T cells in tumor versus adjacent tissues. Flow cytometry further analyzed CD69, PD-1, and IFN-γ expression in this subset. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured in vitro to assess TGF-β1-induce-induced differentiation. Correlation with disease stage was analyzed by detecting the infrltration level of CD103⁺CD39⁺CD8⁺ T Cell subsets via flow cytometry.

Compared with adjacent tissues, gastric cancer tissues showed significantly increased proportions of CD103⁺CD39⁺CD8⁺ T cells (P<0.05); immunofluorescence confirmed higher numbers in tumor tissues. Relative to CD103⁺CD39^- and CD103^-CD39^-CD8⁺ T subsets, CD103⁺ CD39⁺CD8⁺ T cells exhibited elevated CD69 and PD-1 expression (P<0.05) but reduced IFN-γ (P<0.05). In vitro TGF-β1 stimulation upregulated co -expression of CD103 and CD39 in CD8⁺ T cells (P<0.05). Moreover, advanced gastric cancer patients had higher tumor infiltration of this subset than early-stage patients (P<0.05).

CD103⁺CD39⁺CD8⁺ T cell subsets display increased infiltration and functionally impaired responses in gastric cancer, facilitating tumor immune escape.