Fig. 4: The level of TGFα secreted by rCAFs is elevated and correlates with chemoresistance in HNSCC patients.

a Cytokine XL array analysis of sCAFs and rCAFs. Boxes indicate the selected cytokines in rCAFs. b, c RT-PCR and western blot analysis of the indicated cytokines in rCAFs relative to sCAFs (n = 3 chemo-sensitive or chemo-resistant patients). d Co-immunofluorescent staining of TGFα and α-SMA expression in tumor sections from chemo-sensitive and -resistant patients respectively. Arrow indicate TGFα and α-SMA double positive cells. e Violin plot showing the mean staining intensity of CAF-TGFα in each group (n = 20 patients, our cohort). A.U. stands for arbitrary unit. f Kaplan–Meier survival study of the HNSCC patients with high or low CAF-TGFα expression (n = 91 patients, our cohort). g The relationship between TGFα expression and overall survival in HNSCC patients (n = 499 patients, KM plotter database). h The corelation between CAF-TGFα expression and cancer progression in HNSCC patients (n = 91 patients, our cohort). i Schematics diagram represents the blood sampling strategy of HNSCC patients who received NACT treatment. j Violin plots show the level of serum TGF in entire cohort, chemosensitive or chemoresistant patients before, during and after receiving NACT treatment. k Table shows the putative p65 binding sites on human TGFα promoter. l Western blot analysis of the expression of p-p65 and total p65 in sCAFs and rCAFs. β-actin was used as a loading control. m Western blot and RT-PCR analysis of rCAFs after treated with or without 20 μM Maslinic acid as indicated. n Western blot and RT-PCR analysis of sCAFs transfected with p65 overexpression vector or empty vector (ctrl). o ChIP assays. Bar charts show the relative enrichment of p65 binding on TGFα promoter in rCAFs as compared to sCAFs (n = 3 independent experiments). p Luciferase assay of sCAFs after co-transfected with a luciferase reporter vector containing TGFα promoter and an escalated amount of p65 overexpression plasmid (n = 3 independent experiments). q Co-immunofluorescent staining of p-p65 and α-SMA in tumor sections from each group (n = 20 patients, our cohort). Arrow indicates p-p65 and α-SMA double positive cells. Data are shown as means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. non-significant. b, e, h, q Student’s t test. f, g Log-rank (Mantel-Cox) test. j, o, p One-way ANOVA. Scale bars in (d, q) represent 100 μm.