Validation of a standardized method for enumerating circulating endothelial cells and progenitors: Flow cytometry and molecular and ultrastructural analyses

Patrizia Mancuso, Pierluigi Antoniotti, Jessica Quarna, Angelica Calleri, Cristina Rabascio, Carlo Tacchetti, Paola Braidotti, Hua Kang Wu, Amado J. Zurita, Luca Saronni, John B. Cheng, David R. Shalinsky, John V. Heymach, Francesco Bertolini

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16 +CD45 -CD31 +CD146 + CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n - 78; P « 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P « 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

Original languageEnglish
Pages (from-to)267-273
Number of pages7
JournalClinical Cancer Research
Volume15
Issue number1
DOIs
Publication statusPublished - Jan 1 2009

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Flow Cytometry
Endothelial Cells
Population
Healthy Volunteers
Blood Platelets
Weibel-Palade Bodies
Clinical Trials
Blood Cells
Neoplasms
Electron Microscopy
Research Design
Tissue Donors
Messenger RNA
Endothelial Progenitor Cells
DNA
Genes
Therapeutics

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Validation of a standardized method for enumerating circulating endothelial cells and progenitors : Flow cytometry and molecular and ultrastructural analyses. / Mancuso, Patrizia; Antoniotti, Pierluigi; Quarna, Jessica; Calleri, Angelica; Rabascio, Cristina; Tacchetti, Carlo; Braidotti, Paola; Wu, Hua Kang; Zurita, Amado J.; Saronni, Luca; Cheng, John B.; Shalinsky, David R.; Heymach, John V.; Bertolini, Francesco.

In: Clinical Cancer Research, Vol. 15, No. 1, 01.01.2009, p. 267-273.

Research output: Contribution to journalArticle

Mancuso, Patrizia ; Antoniotti, Pierluigi ; Quarna, Jessica ; Calleri, Angelica ; Rabascio, Cristina ; Tacchetti, Carlo ; Braidotti, Paola ; Wu, Hua Kang ; Zurita, Amado J. ; Saronni, Luca ; Cheng, John B. ; Shalinsky, David R. ; Heymach, John V. ; Bertolini, Francesco. / Validation of a standardized method for enumerating circulating endothelial cells and progenitors : Flow cytometry and molecular and ultrastructural analyses. In: Clinical Cancer Research. 2009 ; Vol. 15, No. 1. pp. 267-273.
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AU - Antoniotti, Pierluigi

AU - Quarna, Jessica

AU - Calleri, Angelica

AU - Rabascio, Cristina

AU - Tacchetti, Carlo

AU - Braidotti, Paola

AU - Wu, Hua Kang

AU - Zurita, Amado J.

AU - Saronni, Luca

AU - Cheng, John B.

AU - Shalinsky, David R.

AU - Heymach, John V.

AU - Bertolini, Francesco

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N2 - Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16 +CD45 -CD31 +CD146 + CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n - 78; P « 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P « 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

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