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

Abstract

Background: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown.

Methods: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment.

Results: Our study shows for the first time that TGFβ1-carrying CRC_sEVs impair the morphological and functional properties of healthy human 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-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Keywords: Colorectal cancer; Hepatocytes; Liver metastasis; Small extracellular vesicles; Transforming growth factor‑β1 (TGFβ1).

Fig. 1

Characterization of sEVs isolated from CRC cell-conditioned media by ultracentrifugation. a NTA of the sizes of SW480-derived sEVs (SW480_sEVs) and SW620-derived sEVs (SW620_sEVs). b Representative TEM micrographs of sEVs isolated from CRC cell culture medium in which clusters (upper panel) and single vesicles (lower panel) are shown. c sEVs were compared to whole cell lysates by Western blot for the presence of EV markers

Fig. 2

CRC_sEVs alter the functional properties and morphology of hepatocytes. a CellTox assay showed that treatment for 24 and 48 h with CRC_sEVs did not alter the viability of hepatocytes (RFU: relative fluorescence units). b Gene expression levels of ALBU, CYP3A4, and APOE were measured in THLE-2 cells treated with CRC_sEVs. c ELISA of ALBU released in the conditioned medium of hepatocytes treated with CRC_sEVs for 24 h. In all reported graphs, the asterisks indicate significant differences vs untreated control cells (Ctrl) (*p < 0.05; **p < 0.01; ***p < 0.01). d Fluorescent confocal microscope images showing HNF4 expression and localization in hepatocytes treated for 24 h with CRC_sEVs; e Fluorescent confocal microscope images showing the morphological changes induced by treatment with CRC_sEVs for 24 h; Actin Green (green) was used to stain actin fibers; Hoechst (blue) was used to stain the nuclei; the yellow arrows indicate the CRC_SEV-induced holes in the hepatocyte monolayer. Ctrl untreated control cells

Fig. 3

Confocal microscopy analysis of Vimentin (red) and CK8/18 (green) in hepatocytes treated with CRC_sEVs for 24 h; nuclei are in blue. Untreated cells are indicated as a control (Ctrl)

Fig. 4

CRC_sEVs carry TGFβ1 and induce TGFβ1 signaling pathways in hepatocytes. a Western blot analysis of TGFβ1 in SW480 and SW620 cells and derived sEVs. b dSTORM imaging of SW480_ and SW620_sEVs. The graphs on the right report the ratios of the number of each sEV group to the total number of counted sEVs expressed as a percentage. c ELISA showing the effect of trypsin treatment on the presence of TGFβ1 in CRC_sEVs

Fig. 5

Characterization of sEVs isolated from the plasma of CRC patients (CRC_P/sEVs) and healthy subjects (HS/sEVs). Representative TEM micrograph (a) and NTA (b) of sEVs isolated from a CRC patient plasma. c Western blot analysis of EV markers (CD81 and Alix) and of proteins expected to be underrepresented in EVs (Calnexin and Cytochrome C). d Western blot analysis of TGFβ1 in CRC_P/sEVs and HS/sEVs

Fig. 6

CRC_sEVs induce the modulation of TGFβ/SMAD targets associated with EMT in hepatocytes. Western blot assays were performed to verify the ability of SW480_ and SW620_sEVs to modulate SMAD 2/3 phosphorylation in hepatocytes (a), the expression of SNAIL and SLUG (b), and mesenchymal (c) and epithelial markers (d). Each Western blot is associated with the corresponding densitometric analysis where the reported values are the mean of at least 2 independent experiments (± SD) of the protein normalized vs loading control (Tubulin or GAPDH). e Western blot analyses of SNAIL, SLUG, and Vimentin in hepatocytes treated with CRC_P/sEVs and HS/sEVs; Ctrl: untreated control cells. *p ≤ 0.05; **p ≤ 0.01

Fig. 7

sEVs isolated from CRC biopsies modulate the expression of mesenchymal and epithelial markers in hepatocytes. a Representative TEM micrograph of sEVs isolated from CRC biopsy (CRC/B1 in Table 2). Scale bars: 1 µm and 100 nm. b Confocal microscopy analysis of Vimentin (red) and CK8/18 (green) in hepatocytes treated with sEVs isolated from a CRC biopsy (CRC/B2 in Table 2; CRC/B2_sEVs) and from the corresponding non-CRC mucosa (NCRC/B2 in Table 2; NCRC/B2_sEVs) for 24 h; nuclei are in blue. Untreated cells are indicated as control (Ctrl)