Developing liver spheroids for elucidating the role of colorectal cancer-derived smallextracellular vesicles in the pre-metastatic niche formation

Introduction: Colorectal cancer (CRC) is the third most common tumor in the world frequently associated with liver metastasiscausing unfavorable prognosis. A recent study performed in our laboratory has demonstrated that CRC small extracellular vesi-cles (SEVs) induce an epithelial to mesenchymal transition (EMT) of hepatocytes (heps) driving them to actively participate inthe pre-metastatic niche formation, probably contributing to form a liver fibrotic microenvironment. Since 2D cell cultures par-tially reflect the structural complexity of the in vivo microenvironment to give more power to our functional model, we switchedto use hepatocyte spheroids (HeSPHs), which can give us more proper information on the consequences of CRC_SEV-inducedhepatocytes EMT on cell-cell interactions and extracellular matrix remodelling which can drive and support the invasion oftumor cells.Methods: We isolated SEVs from SW480 CRC cells, through differential centrifugation followed by ultracentrifugation. HeSPHswere obtained by seeding normal human liver cells (THEL-2) in ultra-low attachment 96 well plates. After treating HeSPHs withCRC_SEVs, we analyzed the modulation of expression of structural and functional hepatocyte markers. By co-culturing HeSPHswith SW620-GFP cells, we evaluated the ability of CRC_SEV to increase the invasion potential of tumor cells.Results: Our data on HeSPHs confirmed the ability of CRC_SEVs to alter the expression of hepatocyte structural and functionalmarkers (ApoE, albumin and cytokeratins 8/18) observed in the 2D model. Moreover, we found that in CRC-SEVs-treated HeS-PHs the invasive capability of tumor cells increased, indicating that injured-SEV heps can have driving and supporting tumorliver colonization.Summary/Conclusion: Overall, the HeSPHs represent a promising model to study the role that tumor-derived SEVs can havein rendering heps able to actively drive the formation of an environment conducive to metastasis.