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
免疫治疗已成为不可切除肝细胞癌（hepatocellular carcinoma, HCC）颇具前景的治疗手段，而抗肿瘤应答受肿瘤微环境（tumor microenvironment, TME）影响。尽管已有研究报道Wnt/β-连环蛋白（Wnt/β-catenin）突变可诱导非炎症表型，但其对肿瘤微环境的作用仍存在争议。本研究旨在阐明携带Wnt/β-连环蛋白突变的肝细胞癌患者免疫表型的异质性。

方法
本研究纳入152例经手术切除的肝细胞癌标本；将β-连环蛋白1（catenin beta-1）、结肠腺瘤性息肉病（adenomatous polyposis coli, APC）、AXIN1或AXIN2基因发生突变定义为Wnt/β-连环蛋白突变。通过分层聚类分析，基于与T细胞活化相关的基因表达谱，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谱，以探究肿瘤表型差异是否与肿瘤微环境相关。

结果
152例肝细胞癌标本中，40例（26.3%）携带Wnt/β-连环蛋白突变。其中33例（82.5%，33/40）被归为非炎症型，7例（17.5%，7/40）归为炎症型。非炎症型肿瘤的免疫组化特征为CD3+、CD4+及CD8+细胞数量较少，且AXIN2与谷氨酰胺合成酶（GLUL）的mRNA表达水平较高，二者分别参与经典Wnt/β-连环蛋白信号通路及肝细胞分化过程。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（MRI）的肝胆期呈更高强化。免疫组化结果显示，归为炎症型的肝细胞癌标本中CD3+、CD4+及CD8+肿瘤浸润淋巴细胞数量较多。该类肿瘤的特征为上皮细胞黏附分子（epithelial cell adhesion molecule, EpCAM）与FOXM1表达上调，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）活化相关基因的表达上调，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，存在肿瘤表型与肿瘤微环境的异质性。研究提示，肿瘤表型与肿瘤微环境不仅受肝细胞核因子4α（hepatocyte nuclear factor 4 alpha, HNF4A）活化的调控，同时也受FOXM1的调控，二者均为Wnt/β-连环蛋白信号通路的下游转录因子。