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Clonal evolution of a tumor ecosystem depends on different selection pressures that are principally immune and treatment mediated. We integrate RNA-seq, DNA sequencing, TCRseq and SNP array data across multiple regions of liver cancer specimens to map spatio-temporal interactions between cancer and immune cells. We investigate how these interactions reflect intra-tumor heterogeneity (ITH) by correlating regional neo-epitope and viral antigen burden with the regional adaptive immune response. Regional expression of passenger mutations dominantly recruits adaptive responses as opposed to hepatitis B virus and cancer-testis antigens. We detect different clonal expansion of the adaptive immune system in distant regions of the same tumor. An ITH-based gene signature improves singlebiopsy patient survival predictions and an expression survey of 38,553 single cells across 7 regions of 2 patients further reveals heterogeneity in liver cancer. These data quantify transcriptomic ITH and how the different components of the HCC ecosystem interact during cancer evolution.

Purpose Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt-signaling. Underlying biological mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer. Experimental Design The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs) and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt-signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model. Results Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1-class [138 cases (21.5%)] or Wnt-TGFβ-class [177 cases (27.6%)]. CTNNB1-class was characterized by up-regulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, while Wnt-TGFβ-class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt-signaling and β-catenin protein in HepG2 (CTNNB1-class), SNU387 (Wnt-TGFβ-class), SNU398 (CTNNB1-mutation) and Huh7 (Lithium-chloride-pathway activation) cell lines. Additionally, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1-class-specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals. Conclusions Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt-signaling in experimental models that harbor the CTNNB1-class-signature.