The role of CAF in chemotherapy resistance of gastric cancer: organoid models study
10.3760/cma.j.cn115355-20250208-00065
- VernacularTitle:CAF在胃癌化疗耐药中的作用：类器官模型研究
- Author:
Ruoxin LI
1
;
Linteng ZHANG
1
;
Lingyu ZHANG
1
Author Information
1. 福建医科大学肿瘤临床医学院 福建省肿瘤医院 福建省肿瘤转化医学重点实验室，福州 350014
- Publication Type:Journal Article
- Keywords:
Stomach neoplasms;
Organoids;
Cancer-associated fibroblasts;
Drug sensitivity;
Tumor microenvironment
- From:
Cancer Research and Clinic
2025;37(8):597-605
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects of carcinoma-associated fibroblasts (CAF) in gastric cancer microenvironment on the proliferation of gastric cancer organoids and the sensitivity to chemotherapeutic drugs, as well as the underlying mechanisms.Methods:Tissue specimens of 6 patients with gastric cancer who underwent surgical resection in Fujian Cancer Hospital from June 2022 to January 2024 were collected from the biobank. Organoids derived from gastric cancer and isolated tumor tissue CAF and paracancerous tissue normal fibroblasts (NF) were constructed by the primary tissue culture method. The morphology of organoids and fibroblasts was observed under microscope. Immunohistochemistry (IHC) method was used to detect the expressions of gastric cancer markers in organoids. The expression levels of fibroblast related markers were detected by using immunofluorescence, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting methods. A co-culture model of gastric cancer organoids and fibroblasts was constructed by using the Transwell chamber and then was divided into 3 groups: NF and gastric cancer organoids co-culture group (NF co-culture group), CAF and gastric cancer organoids co-culture group (CAF co-culture group), and gastric cancer organoid separate culture group (separate culture group). Different concentrations of oxaliplatin, paclitaxel, and 5-fluorouracil (5-FU) were applied to the co-culture system of fibroblasts and gastric cancer organoids. The morphology of gastric cancer organoids in each group was observed under microscope, and the relative viability of the organoids was detected by using the CellTiter-Glo 3D luminescence method. CAF and NF of 3 patients who successfully constructed organoids were collected for transcriptome sequencing, and the differentially expressed genes (with |log 2 fold change| > 0 and P < 0.05) between CAF and NF were analyzed. Pathway enrichment analysis of the upregulated differentially expressed genes in CAF was performed by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results:The organoids of 4 patients were successfully constructed, among which 3 organoids with relatively consistent results and covering the heterogeneity of the disease were performed for further analysis. After HE staining, microscopic observation showed that the morphological characteristics of the organoids were homogeneous with those of gastric cancer tissues. IHC method detection showed that both organoids and primary tumor tissues both expressed gastric cancer markers CDX2, Villin, and CK20. Inverted microscopic observation revealed that NF was morphologically similar to CAF, but NF had fewer cytoplasmic protrusions. RT-qPCR method, immunofluorescence and Western blotting detections showed that the mRNA and protein expression levels of α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP) and Vimentin in CAF were all higher than those in NF, and the differences were statistically significant (all P < 0.05). Inverted microscope observation showed that after co-cultured for 96 h, the size of organoids in CAF co-culture group [(308±61) μm] was larger than that in NF co-culture group [(155±33) μm] and separate culture group [(91±17) μm], and the differences were statistically significant (all P < 0.05). CellTiter-Glo 3D luminescence assay showed that the relative viability of organoids in the CAF co-culture group was enhanced after 96 h of co-culture compared with NF co-culture group and separate culture group, and the differences were statistically significant (all P < 0.05); there was no statistically significant difference in the relative viability between NF co-culture group and separate culture group ( P > 0.05). With the increase of the concentrations of oxaliplatin, 5-FU and paclitaxel, the relative activity of organoids after drug effect for 96 h in the 3 groups gradually decreased. The relative viability of the organoids in CAF co-culture groups treated with 6.25, 12.5, 25, 50, and 100 μmol/L oxaliplatin, 12.5, 25, 50, and 100 μmol/L 5-FU, and 0.625, 1.25, 2.5, and 5 μmol/L paclitaxel was higher than that of the other 2 groups, and the differences were statistically significant (all P < 0.05). Transcriptome sequencing analysis revealed that compared with NF, there were 893 differentially expressed genes with upregulated mRNA expression and 424 differentially expressed genes with downregulated mRNA expression in CAF. KEGG pathway enrichment analysis showed that the upregulated differentially expressed genes were mainly involved in the calcium signaling pathway. Conclusions:CAF can promote the proliferation ability of gastric cancer organoids and decrease the sensitivity to chemotherapy drugs, which may be related to the calcium signaling pathway.
