CEC a heterogenous and rare cell type of the peripheral blood
CEC have been shown to contribute significantly to angiogenesis in ischemia, inflammation, wound healing and tumor progression [1]. In cancer patients CEC measurement in peripheral blood has been proposed a non-invasive tool to assess tumor angiogenesis [2] and monitor antiangiogenic therapies [3] CEC comprise a heterogenous cell population consisting of endothelial cells shed from the vessel wall [4], bone marrow-derived CEP [5] and endothelial precursors originating from monocytic cells [6] (figure 1). CEC are only a tiny subset of the mononuclear cell fraction of peripheral blood rendering their quantification a challenging task. Endothelial colony formation assays are labor-intensive, time-consuming, poorly standardised and may give rise preferentially to monocytic cells [7]. Currently, the most common technique applied for CEC quantification is multicolor flow cytometry. Based on this method absolute numbers of CEC reported in the literature vary greatly ranging from 0 to 7.900 CEC/mL in peripheral blood of healthy controls and from 5 to 39.000 CEC/mL under pathological conditions [2, 8]. Thus, there is an urgent need for more reliable and standardized methods for CEC quantification.
In this study both methods, flow cytometry and qPCR, were evaluated to compare the ability to detect mature peripheral blood-derived endothelial colony forming cells (ECFC; CEC phenotype) and cord blood-derived progenitors (CEP phenotype) spiked into PBMNC of healthy volunteers.
Phenotype analysis of cord blood progenitor cells and ECFC by flow cytometry
At first, the phenotypes of cord blood derived progenitor cells and ECFC was determined by flow cytometry (figure 2). As expected, ECFC were CD45- CD31+ (figure 2A). Further subtyping revealed that cells were CD34-KDR+. Moreover, cells were CD133- and CD144+ (data not shown). Due to the two-step isolation procedure cord blood progenitor cells displayed a CD45-/low CD34+ phenotype (figure 2B). The majority of CD34+ cells displayed a CD31+ CD133- phenotype corresponding to hematopoietic precursor cells. In accordance with previous reports only a small subset of cells displayed a CD31+ CD133low phenotype (5–10% of the CD34+ cells)[9]. Therefore, only few cord blood-derived progenitor cells were detectable in the CEP window during the subsequent spiking experiments. Moreover, progenitor cells were KDR- and CD144- (data not shown).
Detection of progenitors spiked into peripheral blood samples
In five independent experiments cord blood derived progenitors from different donors were spiked in triplicates into PBMNC from healthy volunteers at concentrations ranging from 0.001 to 1%. As depicted in figure 3A, flow cytometry allowed detection of progenitors with a CEP phenotype at concentrations ≥ 0.01% (mean: 4 cells/106 PBMNC vs 1 cell in unspiked controls, p = 0.017).
Quantification of both CD34 and CD133 gene transcripts proved to be a reliable approach for detecting spiked progenitor cells in PBMNC samples with 10-fold greater sensitivity than flow cytometry. At a frequency of only 0.001% progenitor cells were detectable by a 3.9-fold increased gene expression for CD133 (SD 2.2) and a 3.4-fold increase for CD34 (SD 2.3) in comparison to unspiked controls (p = 0.03; figure 3B). Although linear, the increase of CD34 and CD133 gene transcripts was not proportional to the number of cells spiked, possibly due to technical reasons (e.g., cell clumping) or cell death as progenitor cells are more fragile compared to mature cells. Due to their low expression on progenitors as described above, CD144 and KDR gene expression was not analyzed.
Detection of ECFC spiked into peripheral blood samples
In five independent experiments freshly detached autologous ECFC were spiked into PBMNC samples of the respective donor at concentrations ranging from 0.001 to 1%. Using flow cytometry ECFC spiked into PBMNC samples were detectable at frequencies ≥ 0.01% (mean: 68 cells/106 PBMNC vs 20 cells in unspiked controls, p = 0.004; figure 4A).
QPCR was at least 10-fold more sensitive than flow cytometry for ECFC detection. Indeed, at a frequency of 0.001% relative gene expression of KDR was increased 48.3-fold (SD 52.1; p = 0.027) and that of CD144 40.8-fold (SD 58.5; p = 0.04) compared to unspiked controls (figure 4B). Due to their low expression level on ECFC as described above, CD34 and CD133 gene transcripts were not determined in this setting.
Detection of CEC/CEP in peripheral blood of cancer patients and healthy volunteers
Applying 4-color flow cytometry no significant differences concerning CEC/CEP numbers were found between cancer patients and age-matched healthy controls (figure 5A). We then applied qPCR for quantifying gene transcripts of endothelial progenitor cell markers CD34, CD133 and endothelial cell markers KDR and CD144. Overall, gene transcripts for CD34 and CD144 were found abundantly expressed (i.e., >100 copies/μg RNA) in the total study cohort whereas only very few KDR or CD133 gene transcripts were detectable (figure 5B). Subgroup analysis showed no influence of gender, age or tumor stage on any of the markers studied. No significant elevation of any of the endothelial marker gene transcripts determined was found in locally advanced rectal cancer patients. In contrast, in the cohort of patients with metastatic NSCLC a significantly increased CD34 and KDR gene expression was found (p = 0.028 and p = 0.002, respectively).