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.
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 cancerpatients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 +/- 14% in healthy subjects and 43 +/- 23% in cancerpatients (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.
Authors: Sarika Jain; Maureen M Ward; Jennifer O'Loughlin; Marissa Boeck; Naomi Wiener; Ellen Chuang; Tessa Cigler; Anne Moore; Diana Donovan; Christina Lam; Marta V Cobham; Sarah Schneider; Paul Christos; Rebecca N Baergen; Alexander Swistel; Maureen E Lane; Vivek Mittal; Shahin Rafii; Linda T Vahdat Journal: Breast Cancer Res Treat Date: 2011-12-09 Impact factor: 4.872
Authors: John F de Groot; Yuji Piao; Hai Tran; Mark Gilbert; Hua-Kang Wu; Jun Liu; B Nebiyou Bekele; Tim Cloughesy; Minesh Mehta; H Ian Robins; Andrew Lassman; Lisa DeAngelis; Kevin Camphausen; Alice Chen; W K A Yung; Michael Prados; Patrick Y Wen; John V Heymach Journal: Clin Cancer Res Date: 2011-06-01 Impact factor: 12.531
Authors: Charles F Bellows; Yan Zhang; Jinyun Chen; Marsha L Frazier; Mikhail G Kolonin Journal: Cancer Epidemiol Biomarkers Prev Date: 2011-09-19 Impact factor: 4.254
Authors: Claudine Isaacs; Pia Herbolsheimer; Minetta C Liu; Mary Wilkinson; Yvonne Ottaviano; Gina G Chung; Robert Warren; Jennifer Eng-Wong; Philip Cohen; Karen L Smith; Karen Creswell; Antonella Novielli; Rebecca Slack Journal: Breast Cancer Res Treat Date: 2010-10-26 Impact factor: 4.872
Authors: Margaret M Tropea; Bonnie J A Harper; Grace M Graninger; Terry M Phillips; Gabriela Ferreyra; Howard S Mostowski; Robert L Danner; Anthony F Suffredini; Michael A Solomon Journal: Thromb Haemost Date: 2014-07-24 Impact factor: 5.249