Unravelling stemness dynamics in intrahepatic cholangiocarcinoma: insights into primary and metastatic stem-like cell evolution

Background: The aggressiveness of intrahepatic cholangiocarcinoma (iCCA) relies on key tumor features such as cellular plasticity, self-renewal capacity, and metastatic potential, collectively described as tumor stemness. Molecular mechanisms driving the maintenance and evolution of stemness features during iCCA progression remain poorly understood. This study aims to characterize the dynamics of stemness-related traits in primary and metastatic iCCA, identify key molecular pathways that sustain tumor cell plasticity, and explore their role in disease dissemination. By elucidating these features in vivo, we seek to uncover novel targets to disrupt tumor stemness and hinder disease progression. Methods: Taking advantage of a 3D sphere culture system (SPH) as a model for cancer stem cells, an orthotopic mouse model was developed through intrahepatic injection of Luciferase-GFP-transfected SPH cells obtained by both established and primary iCCA cells. Disseminated GFP+ tumor cells in target organs (lungs, spleen, peritoneum, mesenteric lymph nodes) and blood were analyzed via FACS. GFP+ cells from primary and metastatic lung tumors were isolated by FACS for molecular profiling using pathway-focused qRT-PCR arrays. These findings were validated in human metastatic and non-metastatic iCCA samples through immunohistochemistry and correlated with clinical and pathological data. Results: SPH cells were responsible for a great tumor spread in all secondary organs analyzed, especially those derived from MTCHC-01 primary cells. The absence of CD45-/GFP+ tumor cells in the blood of animals suggested that lymphatic circle is the preferential via to iCCA dissemination. Notably, lung SPH-disseminated cells showed up-regulation of many tested stem-like, epithelial-to-mesenchymal transition, angiogenesis and drug resist ance related genes compared to SPH-primary tumor cells. A panel of 34 genes commonly up-regulated was found in all SPH-disseminated cells. High expression of some of these genes were correlated with a significantly lower 5-years and disease-free survival of 289 iCCA patients. Among these, TSPAN13 and WNT5A are of particular importance. Expression levels of these 2 genes-derived proteins were significantly up-regulated metastatic CCA specimens compared to non-metastatic ones. Conclusion: Our data indicate that a core stemness program is maintained during iCCA progression, with TSPAN13 and WNT5A emerging as key markers of stemness and metastasis.