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: Kaplan–Meier survival analysis based on the PTK7 expression. Kaplan–Meier plots demonstrate the correlation between PTK7 IHC expression and patients in OSCC (n = 80). Redline, high expression (n = 45); black line, low expression (n = 35). *, a statistically significant difference. IHC, immunohistochemistry; OSCC, oral squamous cell carcinoma

Fig 4: POSTN–PTK7 regulated Wnt/ß-Catenin signaling in HNSCC.a After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 cells were treated with 100 ng/mL rhPOSTN for 0, 15, 30, 60, 120, 240 min. The lysates were prepared, and the expression of PTK7, p-Lrp6, Dvl2, Naked2, GSK3ß, p-GSK3ß, and ß-Catenin was analyzed by western blot. Tubulin was used as a loading control. b After being starved with serum-free DMEM overnight, SCC-25 shNC and shPTK7 cells were treated with 100 ng/mL rhPOSTN for 0, 15, 30, 60, 120, and 240 min. The lysates were prepared, and the expression of PTK7, p-Lrp6, Dvl2, Naked2, GSK3ß, p-GSK3ß, and ß-Catenin was analyzed by western blot. Tubulin was used as a loading control. c After being starved with serum-free DMEM overnight, CAL 27 Lv-NC and Lv-PTK7 cells were treated with 100 ng/mL rhPOSTN for 0, 15, 30, 60, 120, and 240 min. The lysates were prepared, and the expression of PTK7, p-Lrp6, Dvl2, Naked2, GSK3ß, p-GSK3ß, and ß-Catenin was analyzed by western blot. Tubulin was used as a loading control. d After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 cells were treated with control DMEM medium or 100 ng/mL rhPOSTN for 30 min; the location of ß-Catenin was detected by laser scanning microscopy. Scale bar: 10 µm

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