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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

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)
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Fig1: 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)

Mentions: 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 40th 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).Fig. 1


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)

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4536801&req=5

Fig1: 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)
Mentions: 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 40th 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).Fig. 1

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