Introduction: Heparin sulfate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signaling, modulated by Sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localisation of SULF2 was analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68 and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cells phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type-2-diabetes, SULF2 was more commonly expressed in cancer associated fibroblasts (CAFs)(52%) and independently associated with shorter survival (7.2 versus 29.2 months, p=0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in-vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In Human PBMCs, SULF2 preferentially induced migration of macrophage precursors (monocytes), inducing a phenotypic chance consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2 induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling, but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

The Author(s). Published by S. Karger AG, Basel