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Outline
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™ 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™ 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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