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Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue.

Luo W, Hu Q, Wang D, Deeb KK, Ma Y, Morrison CD, Liu S, Johnson CS, Trump DL - Oncotarget (2013)

Bottom Line: The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs.Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer.Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.

ABSTRACT
Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissue obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

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Characterization of primary cultures of endothelial cells isolated from NdECs and TdECs prostate tissue(A) Representative photomicrographs of hematoxylin and eosin stained frozen sections of benign (left) and malignant (right) prostatic tissues of macrodissected tissues used for ECs isolation. ECs are highlighted in the frozen sections by CD31 immunostaining (Original magnification, x200). (B) Representative immunofluorescent photomicrographs of CD31 (green) and vWF (red) expression and uptake of DiI-Ac-LDL (red) (original magnification, ×200) in CD31+ prostate TdECs and NdECs. Absence of pan-cytokeratin expression by immunofluorescence was observed in CD31+ prostate TdECs and NdECs. LNCaP was used as positive control for pan-cytokeratin immunofluorescence. Nuclei are stained with DAPI (blue). Endothelial tube network was formed by primary cultures of prostate TdECs and NdECs (original magnification, x100). (C) Representative reverse transcription-PCR analysis of RNA from primary cultures of TdECs and NdECs, HUVECs, or LNCaP cells using human-specific primers for human CD31, CD34, ICAM-1, VCAM-1, VEGFR1, VEGFR2, AR and PSA. GAPDH was used as a loading control.
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Figure 2: Characterization of primary cultures of endothelial cells isolated from NdECs and TdECs prostate tissue(A) Representative photomicrographs of hematoxylin and eosin stained frozen sections of benign (left) and malignant (right) prostatic tissues of macrodissected tissues used for ECs isolation. ECs are highlighted in the frozen sections by CD31 immunostaining (Original magnification, x200). (B) Representative immunofluorescent photomicrographs of CD31 (green) and vWF (red) expression and uptake of DiI-Ac-LDL (red) (original magnification, ×200) in CD31+ prostate TdECs and NdECs. Absence of pan-cytokeratin expression by immunofluorescence was observed in CD31+ prostate TdECs and NdECs. LNCaP was used as positive control for pan-cytokeratin immunofluorescence. Nuclei are stained with DAPI (blue). Endothelial tube network was formed by primary cultures of prostate TdECs and NdECs (original magnification, x100). (C) Representative reverse transcription-PCR analysis of RNA from primary cultures of TdECs and NdECs, HUVECs, or LNCaP cells using human-specific primers for human CD31, CD34, ICAM-1, VCAM-1, VEGFR1, VEGFR2, AR and PSA. GAPDH was used as a loading control.

Mentions: Frozen prostate specimens obtained from robotic radical prostatectomy were evaluated by hematoxylin and eosin to ascertain regions of benign, normal- appearing prostate and regions of prostate adenocarcinoma and examined for CD31 expression (Figure 2A). Both NdECs and TdECs in primary culture demonstrated endothelial cell morphology, functionality, and marker expression profiles comparable to human umbilical vein endothelial cells (HUVECs). The cells grew in monolayers with a cobblestone morphology that was tightly associated and demonstrated clear contact inhibition.


Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue.

Luo W, Hu Q, Wang D, Deeb KK, Ma Y, Morrison CD, Liu S, Johnson CS, Trump DL - Oncotarget (2013)

Characterization of primary cultures of endothelial cells isolated from NdECs and TdECs prostate tissue(A) Representative photomicrographs of hematoxylin and eosin stained frozen sections of benign (left) and malignant (right) prostatic tissues of macrodissected tissues used for ECs isolation. ECs are highlighted in the frozen sections by CD31 immunostaining (Original magnification, x200). (B) Representative immunofluorescent photomicrographs of CD31 (green) and vWF (red) expression and uptake of DiI-Ac-LDL (red) (original magnification, ×200) in CD31+ prostate TdECs and NdECs. Absence of pan-cytokeratin expression by immunofluorescence was observed in CD31+ prostate TdECs and NdECs. LNCaP was used as positive control for pan-cytokeratin immunofluorescence. Nuclei are stained with DAPI (blue). Endothelial tube network was formed by primary cultures of prostate TdECs and NdECs (original magnification, x100). (C) Representative reverse transcription-PCR analysis of RNA from primary cultures of TdECs and NdECs, HUVECs, or LNCaP cells using human-specific primers for human CD31, CD34, ICAM-1, VCAM-1, VEGFR1, VEGFR2, AR and PSA. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824530&req=5

Figure 2: Characterization of primary cultures of endothelial cells isolated from NdECs and TdECs prostate tissue(A) Representative photomicrographs of hematoxylin and eosin stained frozen sections of benign (left) and malignant (right) prostatic tissues of macrodissected tissues used for ECs isolation. ECs are highlighted in the frozen sections by CD31 immunostaining (Original magnification, x200). (B) Representative immunofluorescent photomicrographs of CD31 (green) and vWF (red) expression and uptake of DiI-Ac-LDL (red) (original magnification, ×200) in CD31+ prostate TdECs and NdECs. Absence of pan-cytokeratin expression by immunofluorescence was observed in CD31+ prostate TdECs and NdECs. LNCaP was used as positive control for pan-cytokeratin immunofluorescence. Nuclei are stained with DAPI (blue). Endothelial tube network was formed by primary cultures of prostate TdECs and NdECs (original magnification, x100). (C) Representative reverse transcription-PCR analysis of RNA from primary cultures of TdECs and NdECs, HUVECs, or LNCaP cells using human-specific primers for human CD31, CD34, ICAM-1, VCAM-1, VEGFR1, VEGFR2, AR and PSA. GAPDH was used as a loading control.
Mentions: Frozen prostate specimens obtained from robotic radical prostatectomy were evaluated by hematoxylin and eosin to ascertain regions of benign, normal- appearing prostate and regions of prostate adenocarcinoma and examined for CD31 expression (Figure 2A). Both NdECs and TdECs in primary culture demonstrated endothelial cell morphology, functionality, and marker expression profiles comparable to human umbilical vein endothelial cells (HUVECs). The cells grew in monolayers with a cobblestone morphology that was tightly associated and demonstrated clear contact inhibition.

Bottom Line: The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs.Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer.Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.

ABSTRACT
Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissue obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

Show MeSH
Related in: MedlinePlus