Cultivation, Authentication, And Epithelial-mesenchymal Transition Related Origin Analysis Of Cancer-associated Fibroblasts In A Human Laryngeal Xenografted Tumor Model

BackgroundThe progression, metastasis, and even initiation of cancer are no longer recognized as independent events that are solely caused by genetic mutations and the uncontrollable growth of malignant cancer cells. The microenvironment of the local host tissue, which contains various types of stromal cells, has been recognized as an essential participant. As the most abundant cell type in the tumor stroma, cancer-associated fibroblasts (CAFs) are recognized as playing a crucial role in cancer development by various mechanisms. Recently, CAFs-targeted therapy has emerged as a new hotspot in anti-cancer research. However, the origin of CAFs remains obscure. Epithelial-mesenchymal transition (EMT) from cancer cells is considered an important origin of CAFs, and whether the laryngeal cancer cells can generate their own CAFs via EMT remains unknown.ObjectiveTo explore whether the laryngeal cancer cells in a laryngeal xenografted tumor model can generate their own CAFs via EMT.MethodsThe laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in BALB/c nude mice. The CAFs from the tumor nodules, which were pathologically examined, were primary cultured, purified and morphologically observed by phase-contrast microscope. The identity of CAFs was further confirmed by in vitro immunocytochemical staining, proliferation, migration, invasion assay, and in vivo cancer-promoting assay. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. The matched normal fibroblasts (NFs) from the adjacent connective tissues served as a control consistently.ResultsA pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. The CAFs were successfully primary cultured and purified in vitro. The CAFs and NFs showed negative staining of pan-CK and positive staining of vimentin, indicating their fibroblast identities. Compared with the NFs, the CAFs showed positive staining of a-SMA and Fap, two markers of activated fibroblasts. Although the CAFs manifested higher proliferation, migration, invasion, and cancer-promoting capacities compared with the NFs (P<0.05), an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes, which were distinct from the karyotype of HEp-2 cells.ConclusionThe CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model. Further exploration is needed to identify the exact origin of CAFs.