The Role Of Inflammatory Cancer-associated Fibroblasts And FN14 Indicated By Single-cell Transcriptomic Analysis In Papillary Thyroid Carcinoma

Background: Despite being a cancer with a good prognosis,a small proportion of papillary thyroid carcinomas(PTCs)still exhibit a strong invasive ability.It is important to cultivate a mechanism to identify these carcinomas in the early stage.In multiple cancers,a series of studies have suggested that cancer-associated fibroblasts,especially one of the subtypes called inflammatory cancer-associated fibroblasts(i CAFs),play an important role in worse prognosis.Method: Through single-cell sequencing,we investigated the cell types,identified DEGs in different cells,and identified interactions between them.TNFRSF12 A,a gene in cancer cells,was found to interact significantly with fibroblasts.The biological function and mechanism of FN14 in PTC were investigated by immunohistochemistry,CCK8 assay,Western bolt assay,cell scratch assay and Transwell migration assay.The interaction between fibroblasts and cancer cells was preliminarily investigated by coculture experiments and colony formation experiments.Results: We identified 8 cell populations,including epithelial cells,ECs,myeloid cells,and fibroblasts,and obtained their subtypes by further reclustering.For epithelial cells,we characterized them into cancer cells and follicular cells.Subsequently,we identified a series of DEGs between these two cell types,including FN1 and CST6.More importantly,we identified two subtypes of fibroblasts that were principally enriched in an invasive sample and found that they have a strong interaction between cancer cells,macrophages,and ECs.TNFRSF12A(FN14),plays an important role in the connection between cancer cells and fibroblasts.The results showed that 1.FN14 was highly expressed at both cellular and tissue levels.2.FN14 up-regulated the expression of P65.After overexpression of FN14,WB results showed that the expression of P65 protein was significantly increased.3.FN14 promotes the proliferation and migration of PTC cells.The efficiency of overexpression and silencing in TPC1 and BCPAP was demonstrated using Q-RT-PCR,and WB.CCK8 assay showed that overexpression of FN14 significantly enhanced the proliferation of PTC cell lines.The scratch assay and Transwell assay showed that the migration ability of the PTC1 cell line was significantly enhanced after overexpression of FN14.4,Co-culture results showed that PDGFRB expression of MRC-5 cells was significantly increased after co-culture with PTC cell lines.Cell colony assay showed that CAF promoted the proliferation of PTC cell line.Conclusion: In invasive PTC,more fibroblasts were infiltrated.These fibroblasts,especially i CAFs,have a significant interaction with macrophages,cancer cells,and ECs.i CAF can act on FN14,which is highly expressed in cancer tissues and cancer cells and promote the proliferation of PTC.CAF can provide a new direction for predicting the prognosis and treating PTC.