Molecular mechanisms underlying the ability of colorectal cancer derived small extracellular vesicles to induce epithelial to mesenchymal transition of normal hepatocytes: new insight on liver metastases formation

Colorectal cancer (CRC) is one of the most common tumor diseases, accounting for two million new cases every year [1]. Epidemiological data show that almost 50% of the CRC patients will develop liver metastases, which are still the leading cause of CRC related deaths [2]. Many literature data have shown the role of Tumor derived small extracellular vesicles (TD_sEVs) in the definition of the pre-metastatic niche (PMN) in the liver, promoting morpho-functional alterations which pave the way for the establishment of a favorable microenvironment for future organ colonization by metastatic cells [3]. Although the studies have highlighted the effect of TD_sEVs on the PMN formation in the liver, most of them focused on the intervention of the non-parenchymal component of the liver (Hepatic Stellate Cells and Kupffer cells), while the active role of the parenchymal cells, the hepatocytes (which account for almost 80% among the liver cell population), has been mostly neglected [4]. Our research group already demonstrated the effects of CRCderived small extracellular vesicles (CRC_sEVs) in the promotion of the epithelial-to mesenchymal transition (EMT) of human healthy hepatocytes, particularly focusing on TGF-β1 signaling activation, but the inhibition of this pathway only partially blocked the EMT program on the hepatocytes [5]. Non-coding RNA cargo in the EVs is gaining particular interest, due to their activity in gene expression regulation [6]. I focused my attention on the role of lncH19 carried by CRC_sEVs and its involvement in biological processes associated with the establishment of a favorable microenvironment for metastatic cell organ colonization. Moreover, in the context of EV research, the current methodology deployed in the field can rarely reproduce the chronic effects of the sEVs on recipient cells. In fact, in most studies focusing on the role of TD_sEVs in PMN formation, the effect of the vesicles on recipient cells is evaluated through immediate and acute treatment, which due to high lipid dose delivery can trigger unspecific responses in target cells, altering the downstream results [7]. Recently, new microfluidic devices are gaining the attention of EV-researchers, partly because of the possibility of interconnecting the different organ cytotypes through different chambers and channels in these innovative culture systems. These Organ-on-a-Chip platforms (OoCs) also offer the opportunity to perform chronic delivery of the EVs to the recipient cells, using syringe or peristaltic pumps, mimicking a condition more similar to the in vivo condition, contextually reducing the number of animal models deployed for biomedical research, in accordance with the 3R ethical guidelines [8]. The data reported in this thesis show, for the first time, the role of Colorectal cancer derived sEVs (CRC_sEVs) in promoting alternative splicing (AS) mechanisms of mRNAs related to the Epithelial-ToMesenchymal transition (EMT), a biological process required for the establishment of organ fibrosis, with a particular focus on EV-associated lncH19, which role in the PMN definition has not been deeply investigated [9, 10]. Moreover, I developed an advanced OoC culture system which offers the possibility to increase hepatocyte differentiation, prolonging their maintenance in vitro, and performing a chronic delivery of the CRC_sEVs, which overcomes the limitations of the acute treatment normally performed for the majority of EV studies.