Colorectal cancer-derived small extracellular vesicles induce TGFβ1-mediated epithelial to mesenchymal transition of hepatocytes

Abstract Background: The liver is the main metastatic site for patients with colorectal cancer (CRC) and represents the most frequent cause of death for patients affected by this cancer. In recent years, a number of studies have highlighted the crucial role played by small extracellular vesicles (sEVs) released by cancer cells in initiating pre-metastatic niche formation in the liver, specifically affecting the activities of non-parenchymal cells as Kupffer cells and hepatic stellate cells, while the role of the hepatocytes still remains unknown. Even if this cell component is the most conspicuous in the liver and it is responsible for several physiological activities of this complex organ, its role in liver metastasis is only partly described and related to tumour cell colonization, while no data is available about its enrolment during the pre-metastatic niche formation. Methods: sEVs were isolated from SW480 and SW620 CRC cells through differential centrifugation, quantified using the Nanosight technology and characterized by transmission electron microscope and western blot. The presence of TGFβ1 on the surface of CRC_sEVs was assessed by western blot, super-resolution fluorescence microscope, and ELISA. Functional and epithelial to mesenchymal transition (EMT) markers were analyzed through RT PCR, ELISA assay, western blot, and confocal analysis in human healthy hepatocytes (THLE-2 cells) treated with sEVs derived from CRC cells (CRC_sEVs) at different time points. Results: Our study shows for the first time that CRC_sEVs carrying the transforming growth factor‑β1 (TGFβ1) impair the morphological and functional properties of human healthy hepatocytes by triggering their TGFβ1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. Conclusions: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV conditioned hepatocytes could have an active and until now neglected role during the liver metastasis formation.