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About CD45−/CD31+/CD105+ Circulating Cells in Patients with Gynecologic Malignancies—Response

We appreciate the opportunity to reply to the comments by Drs. Francesco Bertolini, Patrizia Mancuso, and John V. Heymach about the technical and biologic details of our article (1). Themain purpose of our studywas to validate a variety of clinically applicable and feasible methods for the detection and enumeration of circulating endothelial cells (CEC) by analyzing the phenotypes of cells identified using current parameters for CECs. Accordingly, we performed analyses using widely accepted standards for the detection and enumeration of CECs, such as Ficoll gradient separation, minimum number of cells sorted (100,000 cells per sample), and a panel of antibodies (i.e., CD45, CD31, CD146, or CD105; ref. 2). Our key aim was to determine whether these methods are feasible for clinical applications. First, they raised concerns about technical details, including the Ficoll procedure, whether an adequate number of cells were analyzed to detect CECs, and the need for DNA staining procedures. Ficoll density gradient separation is a standard procedure for peripheral blood mononuclear cell (PBMC) enrichment (3). Moreover, many groups, including those of Drs. Francesco Bertolini, PatriziaMancuso, and John V. Heymach (4, 5), have shown that PBMCs isolated using the Ficoll procedure can be successfully used to identify CECs. Studies using the Ficoll procedure have reported the detection of a wide range of numbers of CECs in healthy subjects and patients with cancer, depending on the type of cancer and whether apoptotic cells were present. Next, they suggested that an analysis of 100,000 cells was not adequate for detecting and enumerating rare events, such as the presence of CECs (usually <30 permL of blood). In a flow cytometric evaluation of rare events, the frequency of positive cells can be considered informative when it is significantly higher than that of a background control. In our study, we analyzed at least 100,000 singlet lymphocytes for the enumeration of CD45 /CD31þ/CD146þ cells; this number is equivalent tomore than 500,000 PBMCs (Fig. 1A in ref. 1). Based on the gated singlet lymphocytes, the median frequency of CD45 /CD31þ/CD146þ cells in patients with gynecologic malignancies was determined to be 0.001%,with a range of 0% to0.016%,or 0 to 16 cells per 500,000 PBMCs. These values were significantly lower than the cut-off values of the fluorescence minus one (FMO) control group (median, 0.007%; range, 0%–0.021%). The enumeration of those cells would be unreliable even if we were to analyze more cells because the frequency of both CD45 /CD31þ/CD146þ cells and FMO control group increased simultaneously. Therefore, our data indicate that an evaluation of the frequency of CD45 /CD31þ/CD146þ cells in the experimental setting of our study is not feasible due to the limited resolution of flow cytometry. They also suggested that we should useDNA staining or a viability dye to improve the quality of the CEC enumeration by flow cytometry (5, 6). According to our preliminary data, in which the analyses were optimized, the use of 7-aminoactinomycin D (7-AAD) to screen for dead cells produced no difference in the numbers of CD45 /CD31þ/CD146þ or CD45 /CD31þ/CD105þ cells measured in fresh blood. Moreover, we used fresh blood samples, and analyses were performed only on samples with >99% viable cells to exclude the dead cell population. For these reasons, we omitted the DNA and viability staining steps to reduce risks such as the loss of cells due to the additional experimental steps. Second, they did not agree with the statement that CD45 /CD31þ/CD105þ circulating cells are monocytes rather than endothelial cells because these cells express monocyte markers such as CD14 and CD68 but lack the pan-hematopoietic marker CD45 and express the endothelial-restricted antigen CD144. It was previously reported that VEGFR2 was expressed on 50% of CD45 / CD146þ CECs (4) and that Weibel–Palade bodies and high levels of CD144 gene expression were found in CD45 /CD146þ CECs (5), supporting the endothelial origin of these cells. Accordingly, they suggested that we should add electron microscopy and RNA transcriptome data to our phenotypic investigation data to more accurately evaluate the origin of CD45 /CD31þ/CD105þ cells. Monocytes have been reported to constitutively express CD31 (7) and other markers, including CD144, VEGFR2 kinase insert domain receptor (KDR), Tie-2, and CD105 (8, 9). Therefore, the presence of Weibel–Palade bodies, which are endothelial cell storage granules that primarily contain von Willebrand factor (vWF), can be regarded as a unique feature of endothelial cells. However, structures resembling Weibel–Palade bodies have also been observed in endothelial-like cells derived from human CD14þ monocytes (10). Therefore, the expression of VEGFR2 and CD144 and the presence ofWeibel–Palade bodies seem to not fully confirm that these cells are endothelial cells (11). In addition, we found that CD45 /CD31þ/CD105þ cells did not express Authors' Affiliations: Cancer Biology Team, Mogam Biotechnology Research Institute, Yongin; Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; and Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas