Fig 2: POSTN secreted by CAFs promotes the CSC-like phenotype and tumor initiation in HNSCC by interacting with PTK7. a After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and the expression of the CSC markers CD166, SALL4, CD271, CD90, OCT-4, ALDH1, SOX2, and NANOG was analyzed by RT-PCR. **p < 0.01, ***p < 0.001, ****p < 0.0001, based on Student’s t-test. b After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and then their sphere-forming capacity was assessed. The results are shown as the mean ± SD. **p < 0.01, ***p < 0.001, based on Student’s t-test. Scale bar: 20 µm. c After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and then colony formation assay was performed. The results are shown as the mean ± SD. **p < 0.01, ***p < 0.001, based on Student’s t-test. d After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to erlotinib at different concentrations for 72 h. The IC50 of erlotinib was evaluated on the basis of cell viability using CCK8 assay. e After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and then proliferation assay was performed. The results are shown as the mean ± SD. *p < 0.05, **p < 0.01, based on Student’s t-test. f After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and then scratch-wound-healing assay was performed. The results are shown as the mean ± SD. **p < 0.01, ***p < 0.001, based on Student’s t-test. Scale bar: 40 µm. g, h After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 were exposed to rhPOSTN for 0 or 72 h, and then migration and invasion assay was performed. The results are shown as the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001, based on Student’s t-test. Scale bar: 30 µm

Fig 3: Immunohistochemical staining of lung adenocarcinoma (LUAD) specimens. (A) Immunostaining images of POSTN in the adjacent normal tissues. Scale bar = 100 µm. (B) The distribution of POSTN in the LUAD tumor tissues in interstitial cells was denser than that in cancer cells. Scale bar = 100 µm. (C) The expression of POSTN in tumor tissues was higher than that in the adjacent normal tissues (p < 0.0001). (D) Immunohistochemical staining of CD34, which was used to mark endothelial cells and to count microvessel density (MVD) in the adjacent normal tissues. Scale bar = 100 µm. (E) Distribution of CD34 in the LUAD tumor tissues. Scale bar = 100 µm. (F) The expression of MVD in the LUAD tissues was higher than that in the adjacent normal tissues (p < 0.0001). (G) The correlation analyses revealed that the POSTN expression was positively correlated with MVD in lung adenocarcinoma.

Fig 4: Weighted gene correlation network analysis. (A) Soft threshold screening. The horizontal axis of the graph represents the weight parameter power value, and the left vertical axis represents the scale-free fit index, namely, signed R2 , the higher the square of the correlation coefficient, which shows that the network was closer to the scale-free distribution. The vertical axis of the right graph represents the mean of all gene adjacency functions in the corresponding gene module. (B) Module merging. Genes were divided into modules through hierarchical clustering, and different colors represent different modules. A total of 11 co-expressed modules were acquired using the dynamic cut-tree algorithm. (C) Heat map of the relationship between the module and trait. The black module displayed the highest correlation with POSTN. (D) Correlation analysis of the gene with module and POSTN in microenvironment black module. (E) Screening of POSTN-related differentially expressed genes (DEGs), which yielded a total of 112 genes that were recognized as POSTN-related-hub DEGs.

Fig 5: POSTN secreted by CAFs is a potential ligand of PTK7. a The eluates of the CAFs were subjected to co-IP with the PTK7 antibody, and the silver stain photo of control (IgG) and co-IP (anti-PTK7) group is shown. b The MS/MS spectrum of POSTN is shown. c Cell lysates from HN6 + CAFs and SCC-25 + CAFs were prepared and immunoprecipitated with IgG and anti-POSTN antibodies. The cell lysates and immunoprecipitates were analyzed by western blot with anti-PTK7 and anti-POSTN antibodies. d Cell lysates from HN6 + CAFs and SCC-25 + CAFs were prepared and immunoprecipitated with IgG and anti-PTK7 antibodies. The cell lysates and immunoprecipitates were analyzed by western blot with anti-PTK7 and anti-POSTN antibodies. e The localization of POSTN and PTK7 in the HNSCC tissues was detected by confocal laser scanning microscopy, as indicated. Scale bar: 50 µm. f The localization of POSTN and PTK7 in the co-cultured HN6 and CAFs cells was detected by confocal laser scanning microscopy, as indicated. Scale bar: 10 µm