Cholangiocarcinoma (CCA) constitutes a diverse group of malignancies emerging in the biliary tree. CCAs are divided into three subtypes depending on their anatomical site of origin: intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA) CCA 1,2 (Fig. 1). Of note, considered as an independent entity, mixed HCC-CCA tumours are a rare type of liver malignancy sharing features of both iCCA and HCC and presenting an aggressive disease course and poor prognosis 3,4. iCCAs arise above the second-order bile ducts, whereas the point of anatomical distinction between pCCA and dCCA is the insertion of the cystic duct. pCCA and dCCA can also be collectively referred to as 'extrahepatic' (eCCA) 5. In the USA, pCCA is the single largest group, accounting for approximately 50-60% of all CCAs, followed by dCCA (20-30%) and iCCA (10-20%) 1,6,7. CCA is the second most common primary hepatic malignancy after hepatocellular carcinoma (HCC), comprising approximately 15% of all primary liver tumours and 3% of gastrointestinal cancers 1,6,7. CCAs are usually asymptomatic in early stages and, therefore, often diagnosed when the disease is already in advanced stages, which highly compromises therapeutic options, resulting in a dismal prognosis 1,8. CCA is a rare cancer, but its incidence (0.3-6 per 100,000 inhabitants per year) 1 and mortality (1-6 per 100,000 inhabitants per year, globally 9 , not taking into account specific regions with incidence >6 per 100,000 habitants such as South Korea, China and Thailand) have been increasing in the past few decades worldwide, representing a global health problem. Despite advances in

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HGF/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3, 5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by PCR analysis of FACS- sorted EpCam-positive cells. Loss of c-Met receptor decreased sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration and decreased hepatocytic differentiation in vivo as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (HNF-4α) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche which was associated with greatly reduced MMP9 activity and decreased expression of SDF1. Using a combination of double immunofluorescence of cell type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response as compared to the selective inactivation in the epithelial cell lineages achieved in c-Metfl/fl; Alb-Cre+/- mice. However, in both models, genetic loss of c-met triggered a similar cascade of events leading to failure of HSCs mobilization and death of the mice. Conclusion: These results establish a direct contribution of c-Met in regulation of HSC response, and support a unique role for HGF/c-Met as an essential growth factor signaling pathway for regeneration of diseased liver.

Stratification of intrahepatic cholangiocarcinoma patients based on occurrence of mutations in three classifier genes (IDH, KRAS, TP53) revealed unique oncogenic programs (mutational, structural, epimutational) that influence pharmacologic response in drug repositioning protocols; this genome dissection approach highlights the potential of individual mutations to induce extensive molecular heterogeneity and could facilitate advancement of therapeutic response in this dismal disease. (Hepatology 2018).

BACKGROUND & AIMS Human primary liver cancer (PLC) is classified into biologically distinct subgroups, based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of PLC. METHODS We transduced mouse primary hepatic progenitor cells (HPC), lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and simian virus 40 large-T antigen. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells co-expressed hepatocyte (HNF4A), biliary progenitor cell (keratin 19, EpCAM, A6), and mesenchyme (vimentin) markers and showed disregulation of genes that control the epithelial–mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage-stage dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSC and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. CONCLUSIONS Any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC, by activating different cell type-specific pathways. Identification of common and cell-of-origin specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer.

Background & Aims A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. Methods CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. Results CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p = 0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n = 23) and correlated with a high level of serum cancer antigen 19.9 (n = 17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n = 12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n = 104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. Conclusion CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. Lay summary Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like ...

The idea that tumor initiation and progression are driven by a subset of cells endowed with stem-like properties was first described by Rudolf Virchow in 1855. 'Cancer stem cells', as they were termed more than a century later, represent a subset of tumor cells that are able to generate all tumorigenic and nontumorigenic cell types within the malignancy. Although their existence was hypothesized >150 years ago, it was only recently that stem-like cells started to be isolated from different neoplastic malignancies. Interestingly, Virchow, in suggesting a correlation between cancer and the inflammatory microenvironment, also paved the way for the 'Seed and Soil' theory proposed by Paget a few years later. Despite the time that has passed since these two important concepts were suggested, the relationships between Virchow's 'stem-like cells' and Paget's 'soil' are far from being fully understood. One emerging topic is the importance of a stem-like niche in modulating the biological properties of stem-like cancer cells and thus in affecting the response of the tumor to drugs. This review aims to summarize the recent molecular data concerning the multilayered relationship between cancer stem cells and tumor-associated macrophages that form a key component of the tumor microenvironment. We also discuss the therapeutic implications of targeting this synergistic interplay.

Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene silencing are characteristic features of human cancer. Here, we report a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in human liver cancer. Using transcriptomic and epigenomic profiling, we identified a zebularine signature that classified liver cancer cell lines into two major subtypes with different drug-responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines were associated with upregulation of oncogenic networks (e.g. E2F1, MYC, and TNF) driving liver cancer growth in vitro and in preclinical mouse models. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularineresistant liver cancer cells leading to increased survival and decreased pulmonary metastasis. Integration of zebularine gene expression and demethylation response signatures differentiated patients with HCC according to their survival and disease recurrence and identified a subclass of patients within the poor survivors likely to benefit from therapeutic agents that target the cancer epigenome.