Multiomic analysis uncovers a continuous spectrum of differentiation and Wnt-MDK-driven immune evasion in hepatoblastoma [RNA-seq]. Multiomic analysis uncovers a continuous spectrum of differentiation and Wnt-MDK-driven immune evasion in hepatoblastoma [RNA-seq]

Background & Aims: Hepatoblastoma is the most common pediatric cancer of the liver and the majority of cases display activating mutations in the Wnt/β-catenin pathway. Understanding the complex milieu of the tumor microenvironment has resulted in promising new therapies for adult cancers, but similar approaches in pediatric cancers are still lacking. We aimed to provide a comprehensive analysis of the tumor microenvironment of hepatoblastoma unveiling its spatial architecture and key signaling mechanisms. Methods: Single-cell/-nucleus RNA-seq (n=15), spatial transcriptomics (n=22), and multiplex immunofluorescence stainings (n=7) of treated, untreated, and metastasized pediatric hepatoblastomas were performed. An RNA-seq validation cohort (n=110) including hepatoblastoma, non-tumor and fetal liver samples and single-cell RNA-seq data of healthy immune cells were used for further analysis. Western blotting and RNA-seq of hepatoblastoma and macrophage cell lines were conducted for experimental validation. Results: Of four identified transcriptional tumor programs, “Developmental” and “Metabolic” reflected different hepatic differentiation stages, while “Cycling” was enriched in undifferentiated cells and relapsed samples, and “Intermediate” displayed high activity in samples from patients with poor outcomes. We discovered an increased ratio of anti- to pro-inflammatory immune cells and evidence of immune exclusion from tumor areas. Wnt-responsive upregulation of the immunomodulator midkine in hepatoblastoma cells was associated with a change in macrophage phenotype, which could be partially reversed through midkine inhibition. Conclusions: Hepatoblastoma cells exist along a continuous spectrum of hepatic differentiation and inhabit an altered immune environment. Wnt signaling augments midkine expression, which appears to be involved in shaping the immune environment by modifying macrophages to enable immune evasion, thereby providing a potential therapeutic target. Overall design: To investigate the importance of Wnt signaling and MDK signaling in shaping the immune environment of hepatoblastoma, we performed RNA-seq of HepG2 and Huh6 hepatoblastoma cell lines treated with different concentrations of a Wnt inhibitor (PKF118-310) and an MDK inhibitor (iMDK) and of THP-1-derived macrophages co-cultured with HepG2 and Huh6 cells that had or had not been treated with iMDK. Per cell line, there is a control condition treated with the empty vehicle. PKF118-310 was used at concentrations of 0.2 µM, 0.3 µM and 0.4 µM (0.4 µM only for Huh6 cells). iMDK was used at concentrations of 5 µM and 10 µM. Each condition was tested in triplicates (except for HepG2 PKF118-310 0.3 µM due to low RNA concentrations in two out of the three samples).

背景与目的：肝母细胞瘤（hepatoblastoma）是儿童最常见的肝脏恶性肿瘤，多数病例存在Wnt/β-连环蛋白通路（Wnt/β-catenin pathway）的激活突变。学界对肿瘤微环境复杂微环境的解析已为成人恶性肿瘤带来了颇具前景的新型治疗方案，但儿童恶性肿瘤领域仍缺乏类似的研究策略。本研究旨在对肝母细胞瘤的肿瘤微环境开展全面分析，以揭示其空间结构与关键信号调控机制。
方法：本研究对接受过治疗、未接受治疗及发生转移的儿童肝母细胞瘤样本开展了单细胞/单细胞核RNA测序（single-cell/-nucleus RNA-seq，n=15）、空间转录组测序（spatial transcriptomics，n=22）及多重免疫荧光染色（multiplex immunofluorescence stainings，n=7）。此外，本研究纳入了包含肝母细胞瘤、非肿瘤组织及胎儿肝脏样本的RNA-seq验证队列（n=110），并结合健康免疫细胞的单细胞RNA-seq数据开展后续分析。同时，通过对肝母细胞瘤细胞系及巨噬细胞系进行蛋白质印迹实验与RNA测序，完成了实验验证。
结果：本研究共鉴定出四种肿瘤转录程序：其中“发育型”与“代谢型”分别对应不同的肝脏分化阶段；“增殖型”在未分化细胞及复发样本中富集；“中间型”则在预后不良患者的样本中呈现高活性。我们发现，肿瘤区域内抗炎免疫细胞与促炎免疫细胞的比值升高，且存在肿瘤区域免疫排斥的现象。肝母细胞瘤细胞中受Wnt信号调控的免疫调节因子中期因子（midkine, MDK）表达上调，该现象与巨噬细胞表型改变相关，而通过抑制中期因子可部分逆转这一表型变化。
结论：肝母细胞瘤细胞的肝脏分化状态呈连续谱系分布，且肿瘤微环境存在免疫状态异常。Wnt信号可上调中期因子的表达，后者通过调控巨噬细胞表型以塑造免疫微环境、促进肿瘤免疫逃逸，因此可作为潜在的治疗靶点。
实验整体设计：为探究Wnt信号通路与中期因子（MDK）信号通路在塑造肝母细胞瘤免疫微环境中的作用，本研究对经不同浓度Wnt抑制剂（PKF118-310）与中期因子抑制剂（iMDK）处理的HepG2、Huh6肝母细胞瘤细胞系，以及与经iMDK处理或未处理的HepG2、Huh6细胞共培养的THP-1源性巨噬细胞开展了RNA测序。每个细胞系均设置空载体处理的对照条件。PKF118-310的使用浓度为0.2 μM、0.3 μM及0.4 μM（仅Huh6细胞使用0.4 μM浓度）；iMDK的使用浓度为5 μM与10 μM。除HepG2细胞经0.3 μM PKF118-310处理的组因3份样本中2份RNA浓度过低被排除外，其余各组均设置3次生物学重复。