Supplementary Material for: Two distinct characteristics of immune microenvironment in human hepatocellular carcinoma with Wnt/β-catenin mutations

Introduction Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. Methods This study includes 152 resected HCCs; mutations in the catenin beta-1, adenomatous polyposis coli, or AXIN1, or AXIN2 genes were defined as Wnt/β-catenin mutations. With hierarchical cluster analyses, TME were classified into inflamed or non-inflamed classes based on the gene expressions associated with T-cell activation. Expression profiles of molecules related to cell differentiation and biliary-stem cell markers were compared between the TME classes to investigate whether differences in tumor traits were associated with TME. Results Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of AXIN2 and GLUL, which are involved in the canonical Wnt/β-catenin signaling and hepatocyte differentiation, respectively. Non-inflamed tumors showed higher enhancement on the hepatobiliary-phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) compared to inflamed tumors. HCCs classified as inflamed class are revealed to have high numbers of CD3+, CD4+, and CD8+ tumor infiltrating lymphocytes on immunostaining. This class is associated with increased expression of anti-epithelial cell adhesion molecule (EpCAM) and FOXM1 accompanied by upregulation of genes related to interferon-gamma signaling, dendritic cell migration, regulatory T cells and MDSC activation and recognized as low enhancement nodule on Gd-EOB-DTPA-enhanced MRI. Conclusion Heterogeneity of tumor traits and TME was observed in HCC with Wnt/β-catenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the HNF4A but also by FOXM1, both of which are downstream transcription factor of the Wnt/β-catenin signaling pathway.

引言 免疫治疗对于不可切除肝细胞癌（hepatocellular carcinoma, HCC）正成为极具前景的治疗策略；抗肿瘤应答效果受肿瘤微环境（tumor microenvironment, TME）调控。尽管已有研究证实Wnt/β-连环蛋白（Wnt/β-catenin）突变可诱导肿瘤形成非炎症表型，但其对肿瘤微环境的具体作用仍存在争议。本研究旨在阐明携带Wnt/β-连环蛋白突变的肝细胞癌中免疫表型的异质性。
方法 本研究纳入152例手术切除的肝细胞癌标本；将β-连环蛋白1（catenin beta-1）、腺瘤性息肉病 coli（adenomatous polyposis coli）、AXIN1或AXIN2基因发生突变定义为Wnt/β-连环蛋白突变。基于与T细胞活化相关的基因表达谱，通过分层聚类分析将肿瘤微环境分为炎症型与非炎症型两类。对比两类肿瘤微环境中细胞分化相关分子及胆管干细胞标志物的表达差异，以探究肿瘤特征的异质性是否与肿瘤微环境相关。
结果 152例肝细胞癌标本中共有40例（26.3%）携带Wnt/β-连环蛋白突变。其中33例被归类为非炎症型（33/40，82.5%），7例为炎症型（7/40，17.5%）。非炎症型肿瘤的免疫组织化学染色显示CD3+、CD4+及CD8+阳性细胞数量较少，且AXIN2与GLUL的mRNA表达水平较高——前者参与经典Wnt/β-连环蛋白信号通路，后者与肝细胞分化过程密切相关。与炎症型肿瘤相比，非炎症型肿瘤在钆塞酸二钠（gadolinium-ethoxybenzyl-diethylenetriamine, Gd-EOB-DTPA）增强磁共振成像（magnetic resonance imaging, MRI）的肝胆期呈现更高的强化程度。免疫染色结果显示，炎症型肝细胞癌可见大量CD3+、CD4+及CD8+肿瘤浸润淋巴细胞。该类肿瘤的抗上皮细胞黏附分子（epithelial cell adhesion molecule, EpCAM）与FOXM1表达水平升高，同时伴随干扰素-γ信号通路、树突状细胞迁移、调节性T细胞及髓系来源抑制细胞（myeloid-derived suppressor cells, MDSC）活化相关基因的上调，且在Gd-EOB-DTPA增强MRI上表现为低强化结节。
结论 携带Wnt/β-连环蛋白突变的肝细胞癌中存在肿瘤特征与肿瘤微环境的异质性。本研究提示，肿瘤特征及肿瘤微环境的调控不仅依赖于肝细胞核因子4α（hepatocyte nuclear factor 4 alpha, HNF4A）的活化，还与FOXM1密切相关——二者均为Wnt/β-连环蛋白信号通路的下游转录因子。