Isolation and identification of cancer-associated fibroblasts from human lung adenocarcinoma

Abstract:

Objective：To isolate and identify cancer-associated fibroblasts（CAFs） from human lung adenocarcinoma tissue，to investi－gate the effect of CAFs on the proliferation and migration of lung cancer cells，and to lay a foundation for further research on the role and mechanism of CAFs in the development and progression of lung cancer. Methods：Surgical specimens were obtained from patients with lung adenocarcinoma，and fibroblasts were isolated and cultured. A phase-contrast optical microscope was used to observe cell morphology，and indirect immunofluorescence assay was used to measure the expression of key molecular markers. CCK-8 assay was used to evaluate cell proliferation，flow cytometry was used to analyze cell cycle，and Transwell assay was used to assess cell migration ability. The co-culture method and a conditioned medium were used to analyze the effect of CAFs on the proliferation and migration of lung adenocarcinoma A549 cells，and qRT-PCR was used to measure the change in the expression of key genes. Results：CAFs were isolated from fresh human lung adenocarcinoma tissue，with a spindle-like shape. Immunofluorescence assay showed the expression of the fibroblast markers fibronectin and Vimintin in CAFs，while epithelial keratin 8/18 was not found in CAFs. The CAFs isolated from lung adenocarcinoma tissue showed active proliferation and significantly promoted the proliferation and migration of A549 cells and remarkably upregulated the mRNA expression of c-Myc，PIM1，SOCS3，and CCND1 in A549 cells. Conclusion：CAFs are successfully isolated from human lung adenocarcinoma tissue and such CAFs can significantly promote the proliferation and migration of lung cancer cells.