Fig 2: CAF-secreted CTHRC1 mediates the migration, invasiveness and EMT of BC cells via activation of the Wnt/ß-catenin signaling pathway. (A) Protein expression of ß-catenin, cyclin D1 and c-Myc in MDA-MB-468 BC cells induced by CAF-CM in the presence of CTHRC1 neutralizing or IgG isotype control antibody was analyzed by western blotting. (B) Quantitative analysis of ß-catenin, cyclin D1 and c-Myc expression level. **P<0.01 vs. the control group; ##P<0.01 vs. the CAF-CM+lgG group. (C) proliferation and (D) ß-catenin expression was concentration-dependent. *P<0.05 and **P<0.01 vs. the control group (0 ng/ml DKK1). Effect of CAF-CM induction on MDA-MB-468 cell (E and F) migration and (G and H) invasiveness was evaluated 24 h after Wnt/ß-catenin pathway inhibition via neutralizing antibody or DKK1 administration. **P<0.01 vs. the CAF-CM+Ab-CTHRC1 group; ##P<0.01 vs. the CAF-CM+DKK1 group. (I and J) Protein expression of E-cadherin, N-cadherin, vimentin, w-catenin, cyclin D1 and c-Myc in MDA-MB-468 BC cells induced by CAF-CM in the presence of CTHRC1 neutralizing antibody or DKK1. **P<0.01 vs. the CAF-CM+Ab-CTHRC1 group; ##P<0.01 vs. the CAF-CM+DKK1 group. CTHRC1, collagen triple helix repeat containing-1; CAF, cancer-associated fibroblast; CM, conditioned media; DKK1, Dickkopf-1.

Fig 3: CTHRC1 is highly expressed in CAFs in the BC tumor microenvironment. Expression of CTHRC1 in (A) serum and (B) cancer tissues of patients with BC was detected by ELISA. **P<0.01. (C) Expression of CTHRC1 in adjacent and BC tissues was detected by immunohistochemical staining; arrows indicate CTHRC1-positive cells; magnification, ×200. (D) CTHRC1 protein expression level in MCF10A normal human mammary epithelial cells, BC cell lines (HCC1937, Hs578T, MCF7 and MDA-MB-468), CAFs and NFs was determined by ELISA. **P<0.01 vs. MCF10A cells; ##P<0.01 vs. the NFs. (E) CTHRC1 protein expression level in CAF-CM and NF-CM was determined by western blotting. **P<0.01. CTHRC1, collagen triple helix repeat containing-1; CAF, cancer-associated fibroblast; NF, normal fibroblast; BC, breast cancer; CM, conditioned media.

Fig 4: CAFs enhance the migration and invasion abilities of breast cancer cells via the secretion of CTHRC1. Effect of CAF-CM induction on MDA-MB-468 cells (A and B) migration and (C and D) invasiveness was determined following addition of a CTHRC1 neutralizing antibody or IgG isotype control antibody for 24 h. **P<0.01 vs. the control group; ##P<0.01 vs. the CAF-CM+lgG group. CTHRC1, collagen triple helix repeat containing-1; CAF, cancer-associated fibroblast; CM, conditioned media.