Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 14:14:155.
doi: 10.1186/s12943-015-0426-x.

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

Alice Conigliaro  1 , Viviana Costa  2 , Alessia Lo Dico  3 , Laura Saieva  3 , Simona Buccheri  3   4 , Francesco Dieli  3 , Mauro Manno  5 , Samuele Raccosta  5 , Carmine Mancone  6   7 , Marco Tripodi  7   8 , Giacomo De Leo  3 , Riccardo Alessandro  9   10
Affiliations

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

Alice Conigliaro et al. Mol Cancer. .

Abstract

Background: CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.

Methods: Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.

Results: Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.

Conclusions: Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
CD90+ population. a Huh7 and sorted CD90+ Huh7 were stained for hepatocytic (HNF4alpha), epithelial (E-Cadherin) and mesenchymal (Vimentin) markers, in blue the nuclear staining with DAPI. Characterization of isolated exosomes. b Dynamic light scattering of vesicles isolated from Huh7 (in black) and from CD90 + Huh7 cells (in red). c Western blot for endosomal markers Alix, Tsg101 and HSC70 in Huh7 and CD90+Huh7 population with their relative exosomes. d Confocal microscopy analysis on HUVECs treated for 1, 3, and 6 h with 5 μg/ml of exosomes from CD90+ or Huh7 cells. HUVECs were stained with phalloidin Alexa Fluor488 (green), nuclear counterstaining was done using DAPI (blue), exosomes were labelled with PKH26 (red)
Fig. 2
Fig. 2
HUVECs characterization after exosomes treatment: a RT-PCR analyses for VEGF, VEGF-R and ICAM1 were done on HUVECs 18 h after treatment with CD90+ or Huh7-derived exosomes (5 μg/ml). ΔΔct expressed as fold of induction (FOI) compared with control (untreated cells). ***p < 0.001; *p < 0.05. b Left panel: ELISA for VEGF released by HUVECs 18 h after treatment with CD90 + exo or Huh7exo. Untreated cells were used as control. *p < 0.05. Middle-right panels: Tubulogenesis analysis. Phase contrast micrographs (20×) and quantification of matrigel assay expressed as length of cable as arbitrary unit. c FACS analysis for ICAM-1 on HUVECs 18 h after treatment with Huh7exo or CD90 + exo, respectively. d Adhesion capacity. Left panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer pre-treated with Huh7exo or CD90 + exo. Right panel: Quantification of adhesion established by counting the number of adherent CD90 + cells (violet) per field; *p < 0.05
Fig. 3
Fig. 3
a. LncRNAs expressed in CD90+ cells and their exosomes (left and middle panel). Data are expressed as fold induction compared with Huh7 mix population. Of the 90 lncRNAs analyzed, only those over-expressed more than ten-fold in CD90+ cells were considered. Listed on the right the lncRNA up-regulated in HCC. Right panel: LncRNA Profile in exosomes released by CD90 + Huh7. Data are expressed as fold of induction compared with exosomes from Huh7 parental cells. b H19 analysis. Real-time PCR analysis for H19 expression in exosomes derived from Huh7 or CD90+ cells. Exosomes were treated with RNase and subsequently processed for RNA extraction and retrotrascription. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with Huh7-derived exosomes. ***p < 0.001. c Real-time PCR for H19 on HUVEC 18 h after treatment with CD90 + exo or Huh7exo. Data were normalized for β-actin and ΔΔct indicated as fold of induction compared with control (untreated cells). ***p < 0.001
Fig. 4
Fig. 4
H19 overexpression. a Left panel: Real-time PCR performed on HUVECs 18 h post-transfection. Data were normalized for β-actin and ΔΔct expressed as fold of induction pH19 vs. pEmpty **p < 0.01; *p < 0.05. Right panel: ELISA assay for VEGF level in supernatant from HUVECs 18 h after transfection. ***p < 0.001. b Left Panel: Phase contrast (20×) of tubulogenesis assay performed 18 h after transfection. Right panel: quantification of matrigel assay expressed as length of cable as arbitrary unit*p<0.05. c FACS analysis for ICAM expression in HUVEC transfected cells. d Left Panel: Phase contrast micrographs (20×) showing the adhesion of CD90 + cells on HUVEC monolayer transfected with pEmpty or pH19. Right Panel Quantification was calculated by counting the number of adherent CD90+ cells (violet) per field. **p < 0.01
Fig. 5
Fig. 5
a Real-time PCR for H19, VEGF, VEGFR1 and ICAM1 from HUVECs transfected with H19 siRNA or negative scramble and treated with CD90 + exo. Data were normalized for β-actin and ΔΔct expressed as fold of induction siRNA H19 versus negative control. **p < 0.01, ***p < 0.001 b ELISA assay for VEGF detection on the supernatant from HUVECs treated as indicated above. ***p < 0.001
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379(9822):1245–55. doi: 10.1016/S0140-6736(11)61347-0. - DOI - PubMed
    1. Uchino K, Tateishi R, Shiina S, Kanda M, Masuzaki R, Kondo Y, et al. Hepatocellular carcinoma with extrahepatic metastasis: clinical features and prognostic factors. Cancer. 2011;117(19):4475–83. doi: 10.1002/cncr.25960. - DOI - PubMed
    1. Yoo DJ, Kim KM, Jin YJ, Shim JH, Ko GY, Yoon HK, et al. Clinical outcome of 251 patients with extrahepatic metastasis at initial diagnosis of hepatocellular carcinoma: does transarterial chemoembolization improve survival in these patients? J Gastroenterol Hepatol. 2011;26(1):145–54. doi: 10.1111/j.1440-1746.2010.06341.x. - DOI - PubMed
    1. Roncalli M, Park YN, Di Tommaso L. Histopathological classification of hepatocellular carcinoma. Digestive and liver disease. Off J Italian Soc Gastroenterol Italian Assoc Study Liver. 2010;42(Suppl 3):S228–34. - PubMed
    1. Suzuki A, Zheng Y, Kondo R, Kusakabe M, Takada Y, Fukao K, et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology. 2000;32(6):1230–9. doi: 10.1053/jhep.2000.20349. - DOI - PubMed

Publication types