CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA

As reported by Yang and colleagues [11], highly positive CSC-like CD90+ cells were isolated by cell sorting, starting from Huh7 cell line presenting a mean of 4 % CD90+ cells and 2 % CD90 high-expressing cells. After sorting, the purity of the selected CD90+ population was monitored during cell passages by FACS analysis, and the cells were kept in culture until they maintained a positivity for CD90 of over 90 % (at approximately the 40^th passage). Isolated CD90+ cells, in contrast to the parental Huh7 and as already described by others [12], showed a mesenchymal phenotype, revealing a delocalized E-Cadherin and a lack of expression of HNF4α, a master regulator of hepatocytic differentiation (Fig. 1a). On the contrary, most of the cells were positive for vimentin, a component of intermediate filaments in mesenchymal cells (Fig. 1a). In order to evaluate the ability of Huh7 and its CD90+ subpopulation to release nanovesicles, the conditioned medium was collected, and the vesicles isolated as described by members of our group [26, 27]. Measures obtained by DLS revealed, in the ultracentrifuged cell culture medium, vesicles with an average size in diameter of 50 nm and 100 nm from CD90+ or Huh7 cell medium, respectively (Fig. 1b). This in line with the exosomes dimensions between 30 and 150 nm [28]. Moreover, Western blot analyses showed that Alix and Tsg101 markers are expressed but not enriched in exosomes released by CD90 + Huh7 (Fig. 1c).

CD90 + CSCs have been associated with metastasis and early recurrence in HCC [12, 20]. In order to evaluate whether the CD90+Huh7  cells were able to influence the tumor microenvironment, we treated HUVECs with exosomes released by CD90+ Huh7 cells or Huh7 parental cells (CD90 + exo and Huh7exo). Endothelial cells rapidly internalized exosomes from both cell types; uptake was evident after one-hour of incubation at 37 °C, and increased over the course of six hours (Fig. 1d). Eighteen hours after exosome treatment, real-time PCR analysis revealed that the addition of CD90 + exo, but not of Huh7exo, highly increased the mRNA levels of the pro-angiogenic factor VEGF and its receptor VEGF-R1 in endothelial cells (Fig. 2a). ELISA assay showed that HUVECs treated with CD90 + exo released three-fold more VEGF (Fig. 2b, left panel). Moreover, a significant increase in the number and the length of tubular-like structures was observed when HUVECs were treated with CD90 + exo compared with Huh7exo (Fig. 2b, middle and right panels).

Liver CD90+ CSCs were found circulating in HCC patients and in metastatic colonies [19]. For this reason, we tested the ability of exosomes released by the hepatoma cell line or by sorted CD90+ cells to modulate the adhesion to an endothelial cell monolayer, a crucial event for intra- or extra-vasation. As revealed by real-time PCR (Fig. 2a), and confirmed by FACS analysis (Fig. 2c), treatment of HUVECs with CD90 + -derived exosomes modulated intercellular adhesion molecules, inducing an increase in the expression of ICAM-1. No significant differences were found in VCAM and VE-Cadherin gene expression (data not shown). To validate our data, we did an adhesion assay, pre-treating endothelial cells with exosomes. As shown in Fig. 2d, CD90 + exo caused a two-fold increase in adhering cells compared with pre-treatment Huh7 exo.

To further confirm our observation, the same experiments were performed with SkHep, a hepatoma cell line already characterized as 100 % CD90+, and displaying mesenchymal stem cell characteristics [29]. Additional file 1 illustrates the characterization of exosomes released by SkHep and their uptake by HUVECs, that present different features compared to CD90+ Huh7 derived exosomes. In addition, measures obtained by DLS revealed, in the ultracentrifuged SKHep culture medium, exosomes with an average size in diameter of 70 nm expressing high level of the exosomal markers TSG101 and HSC70. As observed for CD90 + exo, the SkHep-derived exosomes induced a pro-angiogenic stimulus in endothelial cells, modifying their transcriptional profile and enhancing tube formation in matrigel, as well as increasing the adhesive properties of HUVECs.

In summary, our results showed that exosomes released by CSC-like CD90+ liver cells, but not from hepatoma cells, induce pro-angiogenic stimuli in HUVECs, and influence the adhesion between CD90+ cells and endothelial cells.

It has been confirmed that dysregulation of lncRNAs is associated with several human tumors and, recently, a contribution of lncRNAs to hepatocarcinogenesis was found [30‐32]. In order to clarify the molecular mechanism driving the modifications induced in HUVECs by CD90 + -derived exosomes we did an lncRNA profile study in CD90+ cells and parental Huh7 by analyzing the expression of 90 different lncRNAs. In Fig. 3a (left and middle panel), the RNAs over-expressed in CD90+Huh7 cells compared with Huh7 parental cells with at least a ten-fold increase are listed. Among these, Air, Hotair, LincRNA-ROR, Hulc, and H19 have already been identified as positively correlated with hepatocellular carcinoma [31, 33, 34]. We focused our interest on H19, expression of which has been previously associated with metastasis [35, 36]. In line with recent articles, which have demonstrated that hepatocellular carcinoma cells release exosomes containing lncRNA [25, 37], we investigated the expression in exosomes, of those LncRNAs that we found overexpressed in cells. As shown in Fig. 3a right panel the LncProfiler performed on CD90+ Huh7 and Huh7-derived exosomes evidences that H19 was 10-fold up-regulated in exosomes derived from CD90+ Huh7, compared to parental cell line. The Real-time PCR confirmed that vesicles released by CD90 + cells (both sorted or SkHep cells) are highly enriched in H19 transcript compared with vesicles from Huh7 parental cells (Fig. 3b, S1e). Moreover, treatment with CD90 + -derived exosomes induced in HUVECs an increase in H19 transcript (Fig. 3c, S1f). These data suggest a transport of H19 lncRNA from CD90+ cells to HUVECs, even if we cannot exclude a stimulation of endogenous lncRNA.

To investigate a possible role of H19 as mediator of pro-angiogenic and adhesive stimuli in HUVECs, we transfected endothelial cells with the entire sequence of the lncRNA H19 (pH19). As shown in Fig. 4a, H19 overexpression in HUVECs induced a transcriptional modulation similar to that obtained after CD90 + exo treatment. Real-time PCR indicated that the over expression of H19 induced a significant increase in the VEGF and ICAM1 transcripts, while, no modulation compared to controls was observed for the transcription of VEGF-R1, VCAM and VE-cadherin (Fig. 4a right panel). The ELISA assay (Fig. 4a left panel) found, for the first time to our knowledge, a substantial increase in VEGF release induced by lncH19. Moreover, a rise in the number and length of tubes was found in HUVECs transfected with pH19 (Fig. 4b), while FACS analysis (Fig. 4c) indicated an increase in the number of ICAM-1-expressing cells induced by H19 overexpression, thus explaining the more adhesive phenotype of HUVECs. The adhesion assay, in fact, revealed a two-fold increase in adhering CD90+ cells when HUVECs were transfected with pH19 (Fig. 4d).

Overall, these data demonstrate, for the first time to our knowledge, the ability of the lncRNA H19 to stimulate angiogenesis, and to favor cell-cell interaction, allowing us to postulate H19 as a possible mediator of pro-metastatic properties of exosomes released by CD90+ cells. To confirm our hypothesis, lncH19 was silenced in HUVECs concomitantly with CD90 + exo treatment. As shown in Fig. 5a, the silencing of H19 abrogated the exosome-mediated induction of VEGFR1, while no modulation was revealed in the expression of ICAM1. Concerning the VEGF, even if the reduction of transcript did not appear significant (5a), the release of VEGF protein induced by exosome treatment was totally inhibited by H19 silencing (5b).