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
免疫治疗正逐渐成为不可切除性肝细胞癌（HCC）的潜在治疗手段；肿瘤微环境（TME）对抗肿瘤反应的影响显著。尽管报道指出Wnt/β-catenin突变可导致非炎症表型，但其对TME的作用尚存争议。本研究旨在阐明携带Wnt/β-catenin突变的HCC中免疫表型的异质性。

方法
本研究纳入了152例切除的HCC病例；猫眼小带β-1、腺瘤性息肉病结肠癌基因或AXIN1、AXIN2基因的突变被定义为Wnt/β-catenin突变。通过层次聚类分析，根据与T细胞激活相关的基因表达，将TME分为炎症和非炎症两大类。比较TME类别中与细胞分化和胆管干细胞标志物相关的分子表达谱，以探究肿瘤性状的差异是否与TME相关。

结果
在152例HCC中，有40例（26.3%）携带Wnt/β-catenin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%）。非炎症型以免疫染色中CD3+、CD4+和CD8+细胞数量少、AXIN2和GLUL mRNA表达高为特征，AXIN2和GLUL分别参与经典的Wnt/β-catenin信号传导和肝细胞分化。与炎症型肿瘤相比，非炎症型肿瘤在钆-乙氧基苯甲基二乙三胺（Gd-EOB-DTPA）增强磁共振成像（MRI）的肝胆期中显示出更高的增强。归类为炎症型的HCC在免疫染色中显示出高数量的CD3+、CD4+和CD8+肿瘤浸润性淋巴细胞。此类肿瘤与抗上皮细胞粘附分子（EpCAM）和FOXM1表达的升高相关，伴随干扰素-γ信号传导、树突状细胞迁移、调节性T细胞和MDSC激活相关基因的上调，并在Gd-EOB-DTPA增强MRI上表现为低增强结节。

结论
在携带Wnt/β-catenin突变的HCC中观察到肿瘤性状和TME的异质性。研究表明，肿瘤性状和TME不仅由HNF4A的激活决定，还由Wnt/β-catenin信号传导途径下游转录因子FOXM1决定。