About CD45−/CD31+/CD105+ Circulating Cells in Patients with Gynecologic Malignancies—Response
Authors: Hyun-Kyung Yu, Sun Jin Kim, Jang-Seong Kim
Journal: Clinical Cancer Research (2014)

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).The main purpose of our study was 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, Patrizia Mancuso, 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 per mL 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−/CD31+/CD146+ cells; this number is equivalent to more than 500,000 PBMCs (Fig. 1A in ref. 1). Based on the gated singlet lymphocytes, the median frequency of CD45−/CD31+/CD146+ cells in patients with gynecologic malignancies was determined to be 0.001%, with a range of 0% to 0.016%, 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−/CD31+/CD146+ cells and FMO control group increased simultaneously. Therefore, our data indicate that an evaluation of the frequency of CD45−/CD31+/CD146+ 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 use DNA 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−/CD31+/CD146+ or CD45−/CD31+/CD105+ 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−/CD31+/CD105+ 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−/CD146+ CECs (4) and that Weibel–Palade bodies and high levels of CD144 gene expression were found in CD45−/CD146+ 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−/CD31+/CD105+ 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 CD14+ monocytes (10). Therefore, the expression of VEGFR2 and CD144 and the presence of Weibel–Palade bodies seem to not fully confirm that these cells are endothelial cells (11). In addition, we found that CD45−/CD31+/CD105+ cells did not express vWF. We attempted to determine the cellular structure of CD45−/CD31+/CD105+ cells by electron microscopy, but we were unable to perform the electron microscopy experiments. This failure resulted from technical difficulties in the detection of rare cells with Weibel–Palade bodies because an immense volume of whole blood was needed to prepare the cells for electron microscopic examination, as had been previously speculated (12).CD45 has long been regarded as a hallmark of leukocytes, including monocytes. However, this does not mean that all monocytes express CD45 antigens. Catellier and colleagues reported that CD45 antigens were detected in only 94.4% of monocytes (13), indicating the possibility of the presence of CD45− monocytes.In conclusion, we consider this debate to reflect current scientific and technical issues, particularly the lack of a consensus about the detection and enumeration of CECs. Especially in the quest for clinically applicable and practical methodologies for analyzing CECs to establish significant, interpretable, and predictive parameters, rather than laboratory-based complicated and restricted procedures, there remains a need for significant improvements in sample processing methods, the selection of surface markers, and analysis tools. Although further investigations of the biologic and clinical significance of our findings are critical, we suggest that the state-of-the-art CEC detection technology should be reevaluated to identify the exact nature of the cells in question.See the original Letter to the Editor, p. 1393No potential conflicts of interest were disclosed.This study was financially supported by a grant from the Korea Health 21 R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A050905), a grant from the KRIBB Research Initiative Program and by Basic Science Research Programs through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0073284 and 2012R1A1A2007994).