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Validation of a standardized method for enumerating circulating endothelial cells and progenitors: flow cytometry and
Patrizia Mancuso1, Pierluigi Antoniotti, Jessica Quarna
1Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology, Milan, Italy.
Insights
A new flow cytometry method accurately quantifies circulating endothelial cells (CEC) and progenitors (CEP) in blood. This reliable technique allows for sample freezing, crucial for large-scale clinical trials and antiangiogenic therapy research.
Area of Science:
- Cell Biology
- Hematology
- Oncology
Background:
- Circulating endothelial cells (CEC) and progenitors (CEP) are vital biomarkers.
- Antigenic overlap with other blood cells complicates accurate quantification.
- CEC/CEP are crucial for assessing antiangiogenic therapies and patient stratification.
Purpose of the Study:
- To develop and validate a standardized flow cytometry method for precise CEC and CEP quantification.
- To differentiate CEC/CEP from platelets and other blood cell populations.
- To assess CEC/CEP viability and enable sample cryopreservation for clinical trials.
Main Methods:
- Experimental validation of a novel flow cytometry technique.
- Precise isolation of CEC/CEP using specific cell markers (DNA/Syto16(+)CD45(-)CD31(+)CD146(+)).
- Electron microscopy and VE-cadherin mRNA analysis confirmed cell identity.
Main Results:
- The method reliably distinguishes CEC/CEP from other blood components.
- CEC counts were significantly higher in cancer patients (951/mL) than healthy subjects (140/mL).
- Apoptotic CECs were lower in cancer patients (43%) compared to healthy subjects (77%).
- Low intrareader and interreader variability demonstrated method standardization.
- Standardized freezing protocol showed acceptable variability for sample storage.
Conclusions:
- The validated method accurately enumerates a pure endothelial cell population.
- The procedure exhibits limited variability, making it suitable for clinical applications.
- Sample cryopreservation is feasible, supporting large-scale CEC enumeration in clinical trials.
- This approach can be extended to study other angiogenic cell populations.
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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