Measurement of circulating endothelial cells as a biomarker for antiangiogenic therapy: Surface marker specificity, cell viability, and origin of circulating endothelial cells

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Evaluation of circulating endothelial cells (CECs) is a potential biomarker of efficacy of antiangiogenic agents currently in clinical trials. CECs are primarily identified using the pan-endothelial marker CD146, often used as a single marker for analyses of CECs. We investigated the phenotype of CD146+ mononuclear blood cells and evaluated their kinetics during vascular endothelial growth factor (VEGF) blockade by bevacizumab in cancer patients. We analyzed the expression of CD146 on mononuclear blood cells, primary tissue endothelial cells, and malignant and normal tissues by flow cytometric analyses and immunohistochemistry. Furthermore, we measured the circulation kinetics of CD146+ cells following a single injection of bevacizumab prior to chemo-radiotherapy in rectal cancer patients enrolled in a Phase I trial. Similar phenotypic and quantitative studies were performed in mouse tumor models during VEGF blockade. In peripheral blood of cancer patients, CD146 marked primarily a subset of hematopoietic cells with lymphoid markers (CD3 and CD4). CD146 was not detectable on CD31brightCD45— endothelial cells or CD34+CD133+ progenitor cells. In contradistinction, CD146 was detectable in tissues on both cellular components of the vessel wall: CD31brightCD45— endothelial cells and alpha-smooth muscle actin+ pericytes. Unlike CD31brightCD45— cells (identified as viable CECs), CD146+ cell concentration in the peripheral blood of cancer patients did not decrease during VEGF blockade. In mice, we found that CD31brightCD45— cells originated from the bone marrow, while the CD31dimCD45— did not. The latter cells are most likely to be endothelial cells shed by the tumor during VEGF blockade. Thus, they should be explored as an independent biomarker of antiangiogenic therapy. In the peripheral blood of the cancer patients analyzed, CD146 primarily marked lymphocytes. The kinetics of CD146+ cells during VEGF blockade were different from those of CD31brightCD45— cells, previously identified as viable CECs. Finally, the CD31dimCD45— CECs may be an independent biomarker. Although the clinical study was not powered to detect statistically significant correlations, our results directly impact the development of CECs as biomarker.