2022, Vol. 26 ›› Issue (24): 3808-3813

Establishment and identification of an immortalized oral cancer-related fibroblast line

Chen Sheng1, Yang Yan1, Ding Liang2, Ye Chuanjin1, Zhang Lei1, Xia Shu1, Li Huiling1, Huang Xiaofeng1   

Abstract: BACKGROUND: Cancer-associated fibroblasts are important cell components that constitute the tumor microenvironment, but they are prone to aging and phenotype loss during the primary culture, which greatly reduces the experimental reproducibility of cancer-associated fibroblasts function-related research.  
OBJECTIVE: To establish an immortalized oral cancer-related cancer-associated fibroblast line and provide stable experimental cells for exploring the function of cancer-associated fibroblasts in oral cancer.
METHODS:   Enzymatic digestion method was used to isolate and purify cancer-associated fibroblasts from human oral cancer tissues, and the lentivirus packaged with human telomerase reverse transcriptase (hTERT) (pBABE-hygro-hTERT) was used to infect primary cancer-associated fibroblasts; and their morphology and markers were identified; cell proliferation and aging functions were tested. Furthermore, chromosome karyotype analysis and short tandem repeat analysis were performed on primary cancer-associated fibroblasts and immortalized cancer-associated fibroblasts for cell line identification.  
RESULTS AND CONCLUSION: (1) After fibroblasts were immortalized, the expression levels of their markers α-SMA, PDGFRβ and FSP-1 were not significantly changed. (2) Even after 15 passages, their proliferation rate was significantly better than that of non-immortalized cells. (3) The results of β-galactosidase staining showed that with the increase of cell passage, compared with non-immortalized fibroblasts, immortalized fibroblasts did not present obvious senescence. (4) The karyotype of immortalized fibroblasts still retained the basic characteristics of normal cells without malignant transformation. (5) Short tandem repeat identification results demonstrated that immortalized fibroblasts were primary cells, and there was no overlap with the information in the known cell database. (6) These findings indicate that an immortalized fibroblast line derived from oral cancer has been successfully established, which provides a further experimental basis for studying the tumor microenvironment of oral cancer.
Key words: tumor-associated fibroblasts, oral squamous cell carcinoma, primary cells, immortalized cell lines, cellular senescence, karyotype identification