UNRAVELLING THE ROLE OF COLORECTAL CANCER DERIVED EXTRACELLULAR VESICLES IN DRIVING HEPATOCYTES TO ACTIVELY SHAPE HEPATIC PRE-METASTATIC NICHE: FROM IN VITRO INSIGHTS TO IN VIVO EVALUATION.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide, with liver metastases representing the major cause of mortality. A crucial step preceding metastasis formation is the establishment of the hepatic pre-metastatic niche (h-PMN), a supportive microenvironment actively shaped by soluble factors released by the primary tumor, including CRC-derived small extracellular vesicles (CRC-sEVs). While much of the research to date has focused on the impact of CRC-sEVs on non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, endothelial sinusoidal cells, and infiltrating macrophages, the contribution of hepatocytes (Heps), the liver's main parenchymal and functional cell population, remains underexplored in this context.This project aims to unravel the multifaceted role of Heps educated by CRC-sEVs in the early stages of liver metastasis formation. First, we investigated the immunomodulatory effects of CRC-sEVs on THLE-2. Our results revealed that the CRC-sEVs’ treatment induced in Heps a rapid increase of the nuclear form of the checkpoint inhibitor protein PD-L1, which, consistent with literature data, was associated with the upregulation of the alternative immunecheckpoint proteins VISTA and PD-L2. The presence of PD-L1 protein within CRC-sEVs and the absence of its regulation at mRNA level in the target cells, suggested that the observed effects were due to a sEV-mediated mechanism of nuclear delivery. This hypothesis was confirmed by exposing Heps to CRC-sEVs carrying a tagged PD-L1, which was specifically detected in the treated Heps’ nuclei. According to this observation, the inhibition of nuclear import pathways of the sEV cargo abolished in the Heps the effects elicited by the CRC-sEVs treatment. Subsequently, we addressed the pro-fibrotic potential of CRC-sEVs-educated Heps, given the crucial role of fibrosis within h-PMN establishment, by using an innovative three-dimensional (3D) Heps spheroid model. Unlike traditional 2D cultures, which require collagen or fibronectin coatings that may bias fibrosis studies, the 3D model provides a physiologically relevant system that better preserves Heps morphology and intercellular interactions. This allowed us to more accurately assess CRC-sEVs-induced fibrotic transformations, including epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling. Finally, to validate the Heps contribution to h-PMN formation in vivo, we developed a zebrafish CRC xenograft model, offering a transparent, cost-effective, and ethically advantageous platform in accordance with the 3Rs (Reduction, Refinement, Replacement) and enabling real-time imaging of CRC-sEVs interactions and metastatic niche development within the liver.Overall, this project provides new evidence for a previously underestimated role of Heps in PMN formation. By dissecting both the immunomodulatory and profibrotic roles of CRC- sEVs-educated Heps, and developing alternative models, namely spheroids and zebrafish larval xenografts to validate these findings, this work opens new avenues for early intervention and therapeutic targeting of liver metastasis in CRC.