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
免疫治疗已成为治疗不可切除性肝细胞癌（HCC）的一种有前景的方法；肿瘤微环境（TME）对抗肿瘤反应有显著影响。尽管Wnt/β-catenin突变被报道导致非炎症表型，但其在TME中的作用仍存在争议。本研究旨在阐明具有Wnt/β-catenin突变的HCC免疫表型的异质性。

方法
本研究纳入了152例切除的HCC病例；猫烯蛋白β-1、腺瘤性息肉病基因APC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分为炎症和非炎症类别。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，以研究肿瘤特性差异是否与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例被归类为非炎症性（33/40，82.5%），7例为炎症性（7/40，17.5%）。非炎症性类别在免疫染色中表现出CD3+、CD4+和CD8+细胞数量较少，AXIN2和GLUL（分别参与经典Wnt/β-catenin信号传导和肝细胞分化）的mRNA表达较高。与炎症性肿瘤相比，非炎症性肿瘤在钆-乙氧基苯甲基二乙三胺（Gd-EOB-DTPA）增强磁共振成像（MRI）的肝胆相上显示更高的增强。归类为炎症性的HCC在免疫染色中表现出CD3+、CD4+和CD8+肿瘤浸润淋巴细胞数量较高。此类与抗上皮细胞粘附分子（EpCAM）和FOXM1的表达增加相关，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
在具有Wnt/β-catenin突变的HCC中观察到肿瘤特性和TME的异质性。表明肿瘤特性和TME不仅由HNF4A的激活决定，还由下游转录因子FOXM1决定，后者也是Wnt/β-catenin信号通路的一部分。