Fig 2: Effects of anlotinib on CAFs and Cervical Cancer Cells. (A) Anlotinib concentration-dependently inhibited the proliferation of CAFs. High concentrations of anlotinib inhibit the proliferation of NFs (n = 5). (B) Anlotinib inhibited proliferation of Hela and Siha only at high concentrations (n = 5). (C) The protein expression of α-SMA was evaluated by western blotting in anlotinib-treated CAFs(n=3). (D) The protein expression of FAP was evaluated by western blotting in anlotinib-treated CAFs (n=3). *P<0.05, **P<0.01, ***P<0.001. Compared with the 0uM group.

Fig 3: Atg7 deletion converts fibroblasts into activated fibroblasts exhibiting features of CAFs.A The growth numbers of WT MEFs and Atg7−/− MEFs were recorded at 1 day, 2 days and 3 days. B Scratch assays of WT MEFs and Atg7−/− MEFs. C Western blot analysis of ATG7, ACTA2, Vimentin, PDGFR-α, FAP protein levels in WT MEFs and Atg7−/− MEFs. D Immunofluorescence staining of Vimentin and ACTA2 in WT MEFs and Atg7−/− MEFs. DAPI-stained nuclei are blue. Scale bar = 15 μm. E Collagen contraction assay of WT MEFs and Atg7−/− MEFs. Representative images of three replicates of each group at 3 days are shown. F The morphology and outline of WT MEFs and Atg7−/− MEFs in the collagen lattice are described by 2D and 3D culture. G. Flow cytometry analysis of CD44 expression in WT MEFs and Atg7−/− MEFs. H. Quantitative PCR analysis of TGF-β1, MMP2, SDF-1 and IL-6 expression in WT MEFs and Atg7−/− MEFs. Data are presented as mean ± s.d. of three independent experiments and analyzed by Student’s t test. NS not significant, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Effects of fibroblasts derived from breast cancer patients on cancer cells.A Western blot analysis of ATG7, ACTA2, FAP, and PDGFR protein levels in fibroblasts from breast cancer patient samples. Hyperplasia indicates patients with hyperplasia of the breast, I indicates patients with TNM stage I breast cancer, II indicates patients with TNM stage II breast cancer. B Collagen contraction assay of fibroblasts from patient samples. C Flow cytometry analysis of CD44 expression in patient fibroblasts. D Growth numbers of 4T1 cells were recorded after treatment with the indicated exosomes. E Transwell assays of 4T1 cells treated with the indicated exosomes. Representative images are shown, and the migrated cells were counted. F Western blot analysis of E-cadherin, Vimentin, Occludin, and Snail protein levels in the indicated exosomes treated with 4T1 cells. G Western blot analysis of ATG7, ACTA2, Vimentin, PDGFR-α and FAP protein levels in immortalized patient fibroblasts with ATG7 overexpression (2#) or knockdown (4#). H QPCR analysis of miR-6803b in exosome from immortalized patient fibroblasts with ATG7 overexpression (2#) or knockdown (4#). I Growth numbers of MDA-MB-231 cells (with Scarb1 overexpression or knockdown) were recorded after treatment with the indicated exosomes (immortalized patient fibroblasts 2# and 4#), miR-6803b mimic or miR-6803b inhibitor. J Western blot analysis of E-cadherin and Vimentin protein levels in MDA-MB-231 cells (with Scarb1 overexpression or knockdown) that treated with the indicated exosomes (immortalized patient fibroblasts 2# and 4#), miR-6803b mimic or miR-6803b inhibitor. K Transwell assays of MDA-MB-231 cells (with Scarb1 overexpression or knockdown) with the indicated exosomes (immortalized patient fibroblasts 2# and 4#), miR-6803b mimic or miR-6803b inhibitor. L A proposed working model. Data are presented as mean ± s.d. of three independent experiments and analyzed by Student’s t test. NS not significant, *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: CAF induces osimertinib resistance of NSCLC. (A) Western blotting showed the level of α-SMA, Vimentin and FAP in patient-derived tumor with stroma tissue and tumor tissue. (B) CCK-8 assay showed the effect of CAF on the cell proliferation of tumor following the treatment of osimertinib. (C) Colony formation assay showed the effect of CAF on the single cell proliferation potential of tumor cells following the treatment of osimertinib. (D) Transwell assay showed the effect of CAF on the invasive ability of tumor cells. Data are presented as mean ± standard deviation. P values were calculated using unpaired two-tailed Student’s t-test. **P < 0.01.