Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic representation of prostate non-tumor and tumor endothelial cell isolation and enrichmentClinically examined and representative samples were mounted in OCT. Prostate tissues obtained from robotic radical prostectomy specimens from each patient were macrodissected for prostatic adenocarcinoma (tumor) and matched histologically benign regions. Portion of freshly macrodissected prostate tissues (tumor and non-tumor) were immediately digested and cultured in endothelial cell selection medium. Given that enough tissues were obtained, a portion of the tissues used for ECs isolation were embedded in OCT for frozen sections. ECs were isolated by CD31 magnetic Dynabead® and further enriched by CD31 fluorescent activated cell sorting (FACS).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3824530&req=5

Figure 1: Schematic representation of prostate non-tumor and tumor endothelial cell isolation and enrichmentClinically examined and representative samples were mounted in OCT. Prostate tissues obtained from robotic radical prostectomy specimens from each patient were macrodissected for prostatic adenocarcinoma (tumor) and matched histologically benign regions. Portion of freshly macrodissected prostate tissues (tumor and non-tumor) were immediately digested and cultured in endothelial cell selection medium. Given that enough tissues were obtained, a portion of the tissues used for ECs isolation were embedded in OCT for frozen sections. ECs were isolated by CD31 magnetic Dynabead® and further enriched by CD31 fluorescent activated cell sorting (FACS).

Mentions: As shown in Figure 1, prostate NdECs and TdECs were isolated using both Dynabead-based and fluorescent activated cell sorting methodologies. CD31 expression was the primary endothelial cell marker used for purification and enrichment of primary cultures of prostate NdEC and TdECs. By using the two-step Dynabead-based and FACS purification approaches, TdECs and NdECs showed >90% enrichment in primary culture.


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)

Schematic representation of prostate non-tumor and tumor endothelial cell isolation and enrichmentClinically examined and representative samples were mounted in OCT. Prostate tissues obtained from robotic radical prostectomy specimens from each patient were macrodissected for prostatic adenocarcinoma (tumor) and matched histologically benign regions. Portion of freshly macrodissected prostate tissues (tumor and non-tumor) were immediately digested and cultured in endothelial cell selection medium. Given that enough tissues were obtained, a portion of the tissues used for ECs isolation were embedded in OCT for frozen sections. ECs were isolated by CD31 magnetic Dynabead® and further enriched by CD31 fluorescent activated cell sorting (FACS).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of prostate non-tumor and tumor endothelial cell isolation and enrichmentClinically examined and representative samples were mounted in OCT. Prostate tissues obtained from robotic radical prostectomy specimens from each patient were macrodissected for prostatic adenocarcinoma (tumor) and matched histologically benign regions. Portion of freshly macrodissected prostate tissues (tumor and non-tumor) were immediately digested and cultured in endothelial cell selection medium. Given that enough tissues were obtained, a portion of the tissues used for ECs isolation were embedded in OCT for frozen sections. ECs were isolated by CD31 magnetic Dynabead® and further enriched by CD31 fluorescent activated cell sorting (FACS).
Mentions: As shown in Figure 1, prostate NdECs and TdECs were isolated using both Dynabead-based and fluorescent activated cell sorting methodologies. CD31 expression was the primary endothelial cell marker used for purification and enrichment of primary cultures of prostate NdEC and TdECs. By using the two-step Dynabead-based and FACS purification approaches, TdECs and NdECs showed >90% enrichment in primary culture.

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