Supplementary Material for: Sulfatase-2 from cancer associated fibroblasts – an environmental target for hepatocellular carcinoma?

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.

引言：肝肿瘤微环境（Tumor Microenvironment, TME）中的硫酸乙酰肝素蛋白聚糖（Heparin Sulfate Proteoglycans）是细胞信号转导的关键调控因子，其活性可被硫酸酯酶2（Sulfatase-2, SULF2）调控。肝细胞癌（Hepatocellular Carcinoma, HCC）中存在SULF2过表达现象。本研究旨在明确SULF2在肝细胞癌肿瘤微环境中的作用本质与生物学影响。
方法：对60例肝细胞癌患者的肝脏活检组织进行分析，检测SULF2的表达与定位情况，并结合临床参数与预后进行关联分析。通过免疫组化（Immunohistochemistry, IHC）、基于癌症基因组图谱（The Cancer Genome Atlas, TGCA）的生物信息学分析、原代分离的癌症活化成纤维细胞单培养、三维球体培养以及20例接受索拉非尼治疗的独立患者队列，对其功能与机制进行评估。免疫组化检测靶点包括αSMA、磷脂酰肌醇蛋白聚糖-3（Glypican-3）、β-连环蛋白（β-catenin）、磷酸化Ser536位点的RelA、CD4、CD8、CD66b、CD45、CD68以及CD163。通过迁移实验评估SULF2对外周血单个核细胞的影响，并采用荧光激活细胞分选术对免疫细胞表型进行鉴定。
结果：本研究发现，尽管15%（9/60）的病例中SULF2表达于肿瘤细胞，且与肿瘤晚期分期及2型糖尿病相关，但SULF2更常见于癌症相关成纤维细胞（Cancer Associated Fibroblasts, CAFs）中（占比52%），且其独立与更短的总生存期相关（中位生存期7.2个月 vs 29.2个月，p=0.003）。基质SULF2可在体外调控磷脂酰肌醇蛋白聚糖-3/β-连环蛋白信号通路，尽管体内关联分析提示，癌症相关成纤维细胞来源的SULF2对预后的影响还存在其他潜在机制。从人肝细胞癌中分离的癌症相关成纤维细胞可释放SULF2。该蛋白可被转化生长因子β1（Transforming Growth Factor β1, TGFβ1）诱导，促进肝细胞癌增殖与索拉非尼耐药，且在癌症基因组图谱队列中，癌症相关成纤维细胞来源的SULF2与TGFβ1及免疫耗竭状态相关。基质细胞中存在PDGFRβ/STAT3信号通路的自激活现象，且体外实验可释放强效单核细胞/巨噬细胞趋化因子CCL2。在人外周血单个核细胞中，SULF2可优先诱导巨噬细胞前体（单核细胞）的迁移，诱导表型改变，符合免疫耗竭特征。在人肝细胞癌组织中，癌症相关成纤维细胞来源的SULF2与巨噬细胞招募增加相关，肿瘤类器官研究显示基质来源的SULF2可通过旁分泌激活IKKβ/NF-κB通路，促进肿瘤细胞增殖、侵袭及索拉非尼耐药。
结论：癌症相关成纤维细胞来源的SULF2不仅可调控磷脂酰肌醇蛋白聚糖-3/β-连环蛋白信号通路，还可通过激活TAK1/IKKβ/NF-κB通路影响肝细胞癌免疫微环境，与肿瘤进展及治疗耐药相关。其有望成为肝细胞癌患者联合治疗的潜在靶点。