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

Conigliaro A, Costa V, Lo Dico A, Saieva L, Buccheri S, Dieli F, Manno M, Raccosta S, Mancone C, Tripodi M, De Leo G, Alessandro R - Mol. Cancer (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie Cellulari ed Ematologia, Sapienza University of Rome, c/o Policlinico Umberto I, V Clinica Medica Viale Regina Elena, Rome, 324-00161, Italy. alice.conigliaro@uniroma1.it.

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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig2: 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

Mentions: 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).Fig. 2


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

Conigliaro A, Costa V, Lo Dico A, Saieva L, Buccheri S, Dieli F, Manno M, Raccosta S, Mancone C, Tripodi M, De Leo G, Alessandro R - Mol. Cancer (2015)

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
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536801&req=5

Fig2: 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
Mentions: 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).Fig. 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biotecnologie Cellulari ed Ematologia, Sapienza University of Rome, c/o Policlinico Umberto I, V Clinica Medica Viale Regina Elena, Rome, 324-00161, Italy. alice.conigliaro@uniroma1.it.

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.

No MeSH data available.


Related in: MedlinePlus