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/ß-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).