Fig. 5: Activated fibroblasts promote lung metastasis in ACC.

A Schematic diagram of exosome-modulated fibroblasts regulating tumor cells and generating a feedback loop for adjacent tumor cells. The fibroblasts were pretreated with Vector-exo or S100A9-exo for 48 h; these activated fibroblasts were subsequently co-cultured with SACC-83 cells for follow-up experiments. B CCK-8 assay of SACC-83 cells co-cultured with pretreated fibroblasts was performed every 24 h. C Migration and invasion of these co-cultured samples were assessed by the Transwell assay. Scale bar = 200 μm. D Representative images and quantification of colony number in colony formation assays after 10 days of co-culture. E Representative fluorescence images and quantification displaying EdU incorporation (green) in the co-cultured samples. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. F Western blot analysis of E-cadherin and N-cadherin expression in co-cultured SACC-83 cells, with GAPDH used as a loading control. G Formation of lung metastases in mice after injection of SACC-83 cells harvested from co-culture with exosomes/fibroblasts, as represented by bioluminescence signals (left) and normalized photon flux (right) (n = 7 per group). H Representative images (left) and quantification of metastatic tumor nodules (right) in H&E-stained lung sections from NOD/SCID mice (n = 7 per group). Scale bar = 100 μm. GO (I) and KEGG (J) enrichment analyses of differentially expressed genes in SACC-83 cells co-cultured with the indicated fibroblasts, determined by RNA sequencing. K ELISA quantification of IL-17 protein levels in conditioned medium (CM) from NFs treated with Vector-exo (Vector-exo-NF-CM) or S100A9-exo (S100A9-exo-NF-CM). Control IgG or an anti-IL-17 neutralizing antibody (IL-17 Ab) was added to S100A9-exo-NF-CM as indicated. L Western blot analysis of N-cadherin, E-cadherin, MMP9, and MMP2 expression in SACC-83 cells. The cells were co-cultured with S100A9-exo-activated fibroblasts, with either control IgG or an IL-17 neutralizing antibody added to the co-culture medium. M Western blot analysis of EMT markers in SACC-83 cells treated with increasing concentrations of rhIL-17A. Data are presented as the mean ± SD for (B, C right, D, E right, K) and as the mean ± SEM for (G right). p values were calculated using two-way ANOVA for (B), two-tailed Student’s t test for (C–E, G right, K), and Mann–Whitney U test for (H right). *p < 0.05, **p < 0.01, ***p < 0.001.