Fig 2: SULF2 secreted by HCC cells induced the transformation from HSCs to CAFs. (A) Both RT-PCR and western immunoblotting assays showed that there was more expression of a-SMA, FAP, and POSTN in LX2 cells co-cultured with Hep3B SULF2 cells (LX2 SULF2 cells) than those co-cultured with Hep3B Vector cells (the protein was extracted from total cells). (B) LX2 cells co-cultured with Huh7 Scr shRNA cells expressed more a-SMA, FAP, and POSTN than those with Huh7 SULF2 shRNA cells. Cell fractions: LX2 cells (the protein was extracted from total cells). SULF2, sulfatase 2; HCC, hepatocellular carcinoma; CAF, carcinoma-associated fibroblast.

Fig 3: CAFs driven by SULF2 induced HCC EMT phenotype via upregulating SNAI1 by activating SDF-1/CXCR4 signaling. (A) By western immunoblotting assay, it was found that Hep3B LX2 SULF2 cells had significantly more expression of SNAI1, N-cadherin, and Vimentin and less E-cadherin expression than Hep3B LX2 Vector cells, while co-culture with LX2 SULF2 cells did not alter the expression of E-cadherin, SNAI1, N-cadherin, and Vimentin after treatment of AMD3100. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (B) As assessed by wound healing assay, the migration ability of Hep3B cells was found to be accelerated by co-culture with LX2 SULF2 cells, which was weakened by treatment of AMD3100. (C) Transwell invasion assay showed that co-culture with LX2 SULF2 cells consolidated the invasion capacity of Hep3B cells and AMD3100 treatment attenuated the regulatory effect of co-culture with LX2 SULF2 cells on the Hep3B cell invasion ability. CAF, carcinoma-associated fibroblast; SULF2, sulfatase 2; HCC, hepatocellular carcinoma; EMT, epithelial-to-mesenchymal transition.

Fig 4: Working model of the mechanism by which CAFs activated by SULF2 in HCC microenvironment promoted tumor growth. CAF, carcinoma-associated fibroblast; SULF2, sulfatase 2; HCC, hepatocellular carcinoma.

Fig 5: CAFs activated SDF-1/CXCR4/PI3K/AKT pathway and consequently inhibited HCC cell apoptosis. (A) Both RT-PCR and western immunoblotting assays showed that LX2 SULF2 cells expressed significantly more SDF-1 than LX2 Vector cells. Cell fractions: LX2 cells. (B) It was found by ELISA assay that there was more SDF-1 protein in conditioned medium from LX2 SULF2 cells than that from LX2 Vector cells (the protein was extracted from total cells). (C) Microarray profiling of mRNA expression showed that most downstream genes of SDF-1/CXCR4 signaling were remarkably upregulated in Hep3B CAFs cells in contrast to Hep3B Vector cells. (D) Co-culture with CAFs was found by western immunoblotting to increase expression of SDF-1, CXCR4, p-PI3K, and p-AKT in Hep3B cells, while treatment of CXCR4 inhibitor (AMD3100) did not alter the expression of phosphorylation of PI3K, AKT, BAD, caspase 9, and FKHRL 1 in Hep3B cells. Cell fractions: Hep 3B cells (the protein was extracted from total cells). (E) By both DAPI staining and caspase 3/7 activity assay, it was found that co-culture with CAFs suppressed cell apoptosis, whereas AMD3100 treatment did not influence Hep3B cell apoptosis apparently. CAF, carcinoma-associated fibroblast; HCC, hepatocellular carcinoma; SULF2, sulfatase 2.