Different subpopulations of endothelial cells present in the circulation are candidate biological surrogate markers for the effects of anti-angiogenic cancer therapeutics on the tumor vasculature. In general two types of circulating endothelial cells (CECs) are distinguished, mature CECs and endothelial progenitors (CEPs), which based on animal models studies would increase or decrease during anti-angiogenic therapy. In addition, a colony assay after 5 days of culturing blood mononuclear cells in endothelial medium (CFU-EC, Endocult) has been suggested to reflect endothelial precursor function in vivo. We measured simultaneously CECs and CEPs by multiparameter flow cytometry, as well as CFU-EC in volunteers and cancer patients. A reproducibility study in 10 untreated cancer patients to determine the frequencies and stability of the numbers of circulating cells with endothelial marker profiles, consistent with CECs, including CD45low to exclude hematopoetic cells, CD31bright, CD146 (P1H12)+ and VEGFR2 (KDR)+ using a newly available directly labelled antibody. In addition, CEPs were defined as VEGFR2+ positive hematopoetic stem cells (HSCs). From each patient two blood samples were obtained on two days of the same week and the stability of the frequencies was determined. The number (cells /mL blood) of CD45low /CD31bright cells (CECs) was 8890 (990-25250) of which 1320 (160-2880) were VEGFR2+ compared to IgG controls. A similar number of CD45low / P1H12+ / VEGFR2+ cells were present: 1180 (80-3610) suggesting at least partial overlap between the VEGFR2+ populations. CD45dim / CD34bright (HSCs) were 2397 (161-7726) and 20 (0-119) per mL were VEGFR2+ (CEPs). Almost 100% of all the gated cells were viable as determined by staining with the viable marker 7-AAD. Preliminary data indicate a higher number of CEPs in cancer patients compared to volunteers. The mean number of CFU-EC in 10 cancer patients was 28.7 ± 40.1, compared to 18.91 ± 28.3 in 10 volunteers with no apparent correlation to CEPs. In conclusion, by using 4-color flow cytometry, CD45low /CD31bright cells with a low expression of the endothelial markers, CD146 and VEGFR2 can be measured reproducibly in the blood from cancer patients. CEPs can be measured reliably in human blood using this approach, but because of their low numbers a sufficient number of cells (from at least one ml of blood) has to be measured. This methodology is now ready to be used for monitoring the effects of anti-angiogenic therapy in cancer patients but it can also readily be extended to include other markers by 6-color flow cytometry. Further studies are essential to reveal functional properties of the gated as well as the cultured cell populations.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]