Validation of a Standardized Method for Enumerating Circulating Endothelial Cells and Progenitors: Flow Cytometry and Molecular and Ultrastructural Analyses

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.+ 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 F 171/mL in healthy subjects (n = 37) and 951 F1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 F 14% in healthy subjects and 43 F 23% in cancer patients (P < 0.0001). CEPs were 181 F 167/mL in healthy donors and 429 F 507/mL in patients (P = 0.00019). Coefficients of variation were 4 F 4% (intrareader), 17 F 4% (interreader), and 17 F 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 F 8% (intrareader), 16 F 10% (interreader), and 26 F 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.

Circulating perivascular progenitors: A target of PDGFR inhibition

“…The combination of Syto16 and 7AAD was used to gain insight into PPC viability according to van der Pol et al 13,14 Necrotic cells were identified as Syto16low/7AADþ, apoptotic cells as Syto16low/7AADÀ and viable cells as Syto16bright/7AADÀ.…”

“…Sections were analyzed with a Philips CM-10 or a FEI Tecnai 12G2 electron microscopes (Einhdoven, the Netherlands). 13,15 Molecular characterization of sorted PPCs…”

Role of endothelial progenitor cells in breast cancer angiogenesis: from fundamental research to clinical ramifications

“…These conflicting results may be explained by the very limited number of patients analyzed, as well as different markers, used for enumerating EPC (Table 3). Accordingly, a recent report by Mancuso et al [54], who used a more standardized method, with limited intrareader and interreader variability, showed more EPC in metastatic breast cancers than locally advanced ones. Interestingly, in patients with pediatric solid malignancies, including medulloblastoma, neuroblastoma, sarcoma, and lymphomas [59], the levels of circulating bone marrow-EPC were found to be significantly higher, compared to no metastatic diseases.…”

“…In light of the aforementioned results, showing the role of EPC in mammary tumor angiogenesis in animal models, [51,52] and in breast cancer patients [53,54]. As shown in Table 3, 3 of 6 studies failed to show significant difference in EPC among varying stages of breast cancer [53,55,56].…”

Simultaneous characterization of progenitor cell compartments in adult human liver

“…As a result, FCM has become the gold standard to characterize clinical grade cell preparations in several fields of regenerative medicine. In our own laboratories, it has allowed the identification of rare stem cells and progenitors in peripheral blood and parenchymal tissues (12)(13)(14), thus prompting us to refine FCM protocols for liver stem cell analysis with a view to future clinical applications.…”