Characterization of CD45 /CD31 /CD105 Circulating Cells in The

Published OnlineFirst August 6, 2013; DOI: 10.1158/1078-0432.CCR-12-3685

Clinical Cancer Human Cancer Biology Research

Characterization of CD45/CD31þ/CD105þ Circulating Cells in the Peripheral Blood of Patients with Gynecologic Malignancies

Hyun-Kyung Yu1,2, Ho-Jeong Lee1,5, Ha-Na Choi1, Jin-Hyung Ahn1, Ji-Young Choi3, Haeng-Seok Song3, Ki-Heon Lee3, Yeup Yoon1, Lee S. H. Yi2, Jang-Seong Kim4, Sun Jin Kim5, and Tae Jin Kim3

Abstract Purpose: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells. þ Experimental Design: The frequency of representative CEC subsets (i.e., CD45 /CD31 , CD45 / þ þ þ þ CD31 /CD146 , CD45 /CD31 /CD105 ) was analyzed in the peripheral blood of patients with gyne- þ cologic cancer (n ¼ 56) and healthy volunteers (n ¼ 44). CD45 /CD31 cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry. þ þ Results: CD45 /CD31 /CD105 cells were significantly increased in the peripheral blood of patients þ þ with cancer, whereas evaluation of CD45 /CD31 /CD146 cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry þ þ analyses showed that these CD45 /CD31 /CD105 cells did not express vWF and CD146 but rather þ þ CD144. Furthermore, CD45 /CD31 /CD105 cells uniformly expressed the monocyte-specific markers þ þ CD14 and CD68. These results suggest that CD45 /CD31 /CD105 cells carry the characteristics of monocytes rather than endothelial cells. þ þ Conclusions: Our data indicate that CD45 /CD31 /CD105 circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis. Clin Cancer Res; 1–11. 2013 AACR.

Authors' Affiliations: 1Mogam Biotechnology Research Institute, Yongin; Introduction 2Department of Biological Science, Sungkyunkwan University, Suwon; 3Department of Obstetrics and Gynecology, Cheil General Hospital and Overcoming resistance to therapy is the ultimate goal of Women's Healthcare Center, Kwandong University College of Medicine, Seoul; 4Biomedical Translational Research Center, Korea Research Insti- the development of novel treatment modalities in cancer tute of Bioscience and Biotechnology, Daejeon, Republic of Korea; and (1). The biologic heterogeneity and genetic instability of 5Department of Cancer Biology, The University of Texas MD Anderson cancer cells are significant barriers for the design of effective Cancer Center, Houston, Texas therapies. Therefore, relatively more homogenous and Note: Supplementary data for this article are available at Clinical Cancer genetically stable host factors have been suggested as alter- Research Online (http://clincancerres.aacrjournals.org/). native targets (2). Angiogenesis, which is one of the com- Corresponding Authors: Jang-Seong Kim, Biomedical Translational mon and crucial steps in the development and progression Research Center, Korea Research Institute of Bioscience and Biotechnol- ogy, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea. of solid tumors, is a host-dependent process and, conse- Phone: 82-42-860-4270; Fax: 82-42-879-8498; E-mail: quently, has been introduced as an attractive target of cancer [email protected]; Sun Jin Kim, Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, treatment (3). A significant number of drugs designed to Houston, TX 77030. Phone: 1-713-563-4653; Fax: 1-713-563-5489; interrupt the establishment of tumor-associated vasculature E-mail: [email protected]; and Tae Jin Kim, Department of Obstet- by neutralizing vasculogenic factors is currently in clinical rics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University College of Medicine, 1-19 Mukjeong-dong, trials and some of them have been approved for clinical use Jung-gu, Seoul 100-380, Republic of Korea. Phone: 82-2-2000-7577; Fax: in patients with cancer (4, 5). However, understanding the 82-2-2000-7183; E-mail: [email protected] mechanisms of angiogenesis and establishing validated doi: 10.1158/1078-0432.CCR-12-3685 markers that accurately reflect the pharmacologic effects of 2013 American Association for Cancer Research. antiangiogenic therapeutics remain major challenges (6).

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Therefore, establishing a method to validate the identity Translational Relevance of CECs detected by flow cytometry and to assess their Circulating endothelial cells (CEC) are recognized as a biologic significance is critical before expanding their marker of tumor angiogenesis and a predictor of prog- clinical use. nosis, as well as a target for therapy. However, their Considering the angiogenic role of monocytes and the presence in the peripheral blood of patients with cancer technical hurdles of flow cytometry, we hypothesized that and role in angiogenesis remain controversial. Our þ þ a subset of circulating cells detected by flow cytometry results showed that CD45 /CD31 /CD105 cells were þ using conventional CEC markers might be monocytes rath- significantly increased, whereas CD45 /CD31 / þ er than endothelial cells. To show this, we first evaluated CD146 cells were not detected in the peripheral blood the flow-cytometric techniques and markers currently in of patients with gynecologic cancer. In addition, the use and analyzed the frequency of representative CEC þ þ þ expression of monocyte-specific markers such as CD14 subsets (i.e., CD45 /CD31 , CD45 /CD31 /CD146 , þ þ þ þ and CD68 in CD45 /CD31 /CD105 cells suggested CD45 /CD31 /CD105 ; refs. 25–28) in the peripheral that they are monocytes rather than endothelial cells. blood of patients with gynecologic cancer and healthy Collectively, our data suggest that the accuracy of con- volunteers. To identify the genuine lineage of those cells, þ ventional flow-cytometric analyses for identifying CECs we isolated CD45 /CD31 cells (a common denominator should be meticulously reevaluated in its technical and of CECs) and assessed the expression of various markers for biologic aspects. Moreover, further investigation is nec- endothelial cells or monocytes by immunocytochemistry. essary to establish the biologic significance of the pres- þ þ ence of CD45 /CD31 /CD105 monocytes and their Materials and Methods function in relation to angiogenesis. Subjects Peripheral blood samples (1–2 mL) were collected from 44 healthy donors (12 men and 32 women; age, 28–54 years) and 56 patients with gynecologic cancer including 8 Because circulating endothelial cells (CEC) are likely to patients with endometrial cancer (age, 39–59 years), 24 contribute to new vessel formation (7) and their levels in the with cervical cancer (age, 30–71 years), and 24 with ovarian blood change in response to pro- or antiangiogenic drugs cancer (age, 23–67 years; Supplementary Table S1). All (8–10), the measurement of CECs (total CECs including healthy volunteers were free of any medications and had progenitor cells) has been regarded as a promising strat- no cardiovascular disease. The Institutional Review Board at egy for monitoring tumor angiogenesis. Several studies Kwandong University College of Medicine (Seoul, Republic reported a significant increase in the number of CECs in of Korea) approved all protocols, and informed consent was patients with cancer with progressive disease (i.e., lym- obtained from all subjects. phoma, breast cancer, renal cancer, etc.; refs. 11–13). In addition, studies have shown that CEC kinetics and Antibodies for flow cytometry viability correlate well with clinical outcomes of patients The following monoclonal antibodies directly conjugat- with cancer undergoing antiangiogenic treatment (14, ed with fluorescein isothiocyanate (FITC), phycoerythrin 15). (PE), peridinin chlorophyll A protein (PerCP), or allophy- However, the presence and role of CECs have been cocyanin (APC) were used for flow-cytometric analysis: controversial for decades because trials using CECs as a anti-CD31 FITC (WM-59 clone), anti-CD61 FITC (VI-PL2 diagnostic parameter or a therapeutic target did not clone), anti-CD3 PE (SK7 clone), anti-CD19 PE (HIB19 produce consistent results. Moreover, markers that accu- clone), anti-CD31 PE (WM-59 clone), anti-CD41a PE rately identify CECs have not been established because (HIP8 clone), anti-CD56 PE (MY31 clone), anti-CD146 PE many circulating cells such as monocytes express many of (P1H12 clone), anti-CD45 PerCP (2D1 clone), and anti- the same markers as endothelial cells (16). Consequently, CD14 APC (M5E2 clone). Isotype-matched FITC-, PE-, the detection and estimation of CECs have remained PerCP-, and APC-conjugated control antibodies were pur- challenging as they comprise a small proportion of chased from BD Biosciences. Anti-CD105 PE (SN6 clone) peripheral blood cells, and there is no consensus in the and isotype-matched PE-conjugated control antibodies immunophenotype of CEC (17). The application of var- were purchased from Serotec. Anti-CD31 APC (WM-59 ious combinations of markers or different enumeration clone) and isotype-matched APC-conjugated control anti- techniques to the detection of CECs has produced dis- bodies were purchased from eBioscience. crepant results in the amount and immunophenotype of CECs (18–20). Furthermore, certain authors have ques- Preparation of peripheral blood mononuclear cells tioned whether CECs detected by flow cytometry are Peripheral blood was collected from healthy volunteers authentic endothelial cells and their function in angio- and patients with cancer using EDTA as an anticoagulant genesis, if any (17, 21). Recent studies have suggested and processed within several hours after collection as fol- that the actual angiogenic cell types incorporated into lows: whole blood was diluted 1:1 (vol/vol) with PBS newly formed vessels are myeloid cells such as monocytes containing 0.5% bovine serum albumin (BSA) and 2 (22–24) rather than CECs and/or their progenitor cells. mmol/L EDTA and overlaid onto an equal volume of Ficoll

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Paque (GE healthcare). Samples were centrifuged at 1,800 FcR blocking reagent and stained with fluorescence-labeled rpm for 25 minutes at room temperature with no brake. The monoclonal antibodies against CD45 and CD31. The cells mononuclear cell layer was carefully collected and washed were fixed with 4% PFA and then sorted with a FACS Aria twice with cold PBS containing 0.5% BSA and 2 mmol/L flow cytometer. A 70-mm nozzle (BD Biosciences), a sheath EDTA at 4C. Red blood cells were lysed with 0.38% pressure of 20 to 25 pounds per square inch, and an ammonium chloride solution. The final mononuclear cell acquisition rate of 2,000 to 3,000 events per second were preparation was resuspended with PBS containing 0.5% used according to the guidelines for FACS Aria users (BD BSA and 2 mmol/L EDTA and then subjected to flow- Biosciences). cytometric analysis. The viability of the mononuclear cells used for the analyses was determined by the dye exclusion Immunofluorescence staining test, and cells with a viability of 99% or more were used for Immunocytochemical fluorescence labeling of cells was further experiments. conducted as previously described (24). Briefly, CD45 / þ CD31 cells isolated by flow cytometry were cytospun onto Flow cytometry and cell sorting glass slides and washed 3 times with PBS for 3 minutes. To Isolated peripheral blood mononuclear cells (PBMC; 107 stain intracellular antigens, cells were permeabilized with cells per mL of blood) were pretreated with FcR blocking 0.5% Triton X-100 (Sigma Chemical Co.). To prevent cross- reagent (Miltenyi Biotec) to block nonspecific antibody reaction with antibodies used to stain cells in the flow- binding and incubated on ice for 25 minutes with a panel cytometric analysis, Fab-fragment blocking was conducted of monoclonal antibodies (summarized in Table 1). Cells overnight at 4C with an antibody from the same host were washed with PBS containing 0.5% BSA and 2 mmol/L species of antibody, which was a F(ab0)2 fragment from EDTA and fixed with 4% paraformaldehyde (PFA; Electron goat anti-mouse immunoglobulin G (IgG) (Jackson Immu- Microscopy Sciences). The antibody-labeled cells were ana- noResearch Laboratories) in this study. After the blocking lyzed using a FACS Aria flow cytometer (BD Biosciences) step, cells were stained with the following antibodies: equipped with 2 lasers (488 nm and 633 nm). Data were mouse anti-human CD105 monoclonal antibody (mAb; analyzed with FlowJo software (Tree Star, Inc.,) or FACS 1:100, Serotec), mouse anti-human CD146 mAb (1:100 Diva (BD Biosciences). For the analysis of CEC candidates, dilution, Chemicon), mouse anti-human CD144 mAb at least 100,000 singlet lymphocytes were isolated and (1:100, Reliatech GmbH), mouse anti-human CD68 mAb þ þ the frequencies of CD45 /CD31 , CD45 /CD31 / (1:100 dilution, DAKO), polyclonal rabbit anti-human þ þ þ CD146 , or CD45 /CD31 /CD105 cells were analyzed vWF Ab (1:100 dilution, DAKO), or mouse anti-human and expressed as a percentage of the singlet lymphocyte CD14 mAb (1:20 dilution, DAKO), followed by labeling þ population. For sorting of CD45 /CD31 cells, PBMCs with the corresponding secondary antibodies conjugated prepared from patients with cancer were pretreated with with FITC or Texas Red. For double-staining experiments,

Table 1. Antibody panels used for ﬂow-cytometric analysis and isolation of CD45/CD31þ cells

FITC PE PerCP APC For immunotyping Controls for compensation IgG1 IgG1 IgG1 IgG2a CD31 IgG1 IgG1 IgG2a IgG1 CD31 IgG1 IgG2a IgG1 IgG1 CD45 IgG2a IgG1 IgG1 IgG1 CD14 Samples CD31 CD3 CD45 CD14 CD31 CD19 CD45 CD14 CD31 CD56 CD45 CD14 CD31 CD146 CD45 CD14 For CEC detection Controls for compensation IgG1 IgG1 IgG1 - CD31 IgG1 IgG1 - IgG1 CD31 IgG1 - IgG1 IgG1 CD45 - FMO control CD31 IgG1 CD45 - Samples CD31 CD146 CD45 - CD31 CD105 CD45 - For isolation of CD45/CD31þ cells Controls for compensation - - IgG1 IgG1 - - IgG1 CD31 - - CD45 IgG1 Sample - - CD45 CD31

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the protein and fragment blocking steps were repeated event frequencies. In line with the study by Cui and collea- before treating with the second primary antibody to gues (31), gating with FMO controls was shown to be a prevent cross-reaction. Cell nuclei were counterstained more efficient method to increase the accuracy and speci- with Hoechst 33342 dye (Invitrogen). The slides were ficity of the positive signals in polychromatic flow-cyto- then washed 3 times with PBS for 3 minutes each and metric detection of rare events, such as CECs, than the use of mounted in Vecta-shield (Vector Laboratories). Images isotype controls. were acquired with a LSM510 Meta DuoScan confocal On the basis of these results, we established a gating system (Zeiss). strategy to determine the frequencies of CECs in PBMCs, as described in Fig. 2B. In brief, cells were stained with Statistical analysis a panel of antibodies in parallel with FMO controls. The The Mann–Whitney U test was used to determine the singlet lymphocyte population was identified on a FSC/ statistical significance of differences in the frequencies of SSC plot and subgated onto a bivariant antigen plot to þ CEC candidates between the peripheral blood collected identify CD45 /CD31 cells. These cells were further sub- þ þ from patients with cancer and healthy volunteers. All sta- gated to identify the corresponding CD146 or CD105 tistical tests were two-sided. P values less than 0.05 were subpopulation. considered significant. Flow-cytometric analysis of CECs in PBMCs Results A number of protein markers including CD31, CD34, Establishment of flow cytometry gating strategies for CD105, CD146, CD144, and VEGF receptor-2 have been the measurement of circulating endothelial cells used to define CECs. However, there is no truly specific First, we found that monocytes showed higher levels of marker to identify CECs because those markers are also autofluorescence than lymphocytes (data not shown), indi- expressed in other type of cells (32). A generally accepted þ cating that they should be analyzed separately for fluores- definition of CECs is CD45 /CD31 cells expressing cence compensation. In flow-cytometric analysis, the detec- CD146 or CD105 (11), but this definition needs to be tion of equal levels of autofluorescence in positive and modified. According to the gating strategy described above, þ negative populations for each single stain indicates that the we examined the frequencies of CD45 /CD31 , CD45 / þ þ þ þ fluorescent compensation is appropriate (29). As an initial CD31 /CD146 , and CD45 /CD31 /CD105 cells in the step to establish the efficient gating strategies for the detec- PBMCs of patients with cancer (n ¼ 56) and healthy tion of CECs by flow cytometry, we attempted to determine volunteers (n ¼ 44) by flow cytometry. The frequency of þ þ the subset(s) of PBMCs expressing the CD45 /CD31 CD45 /CD31 cells was significantly higher in the singlet phenotype. PBMCs were plotted according to the forward FSClow/SSClow population of patients with cancer (median, scatter (FSC) versus side scatter (SSC) profiles and FSClow 1.365%; range, 0.110–26.85%) than in that of healthy /SSClow (Fig. 1A, left), FSChigh/SSCmid (Fig. 1B, left), and volunteers (median, 0.183%; range, 0.027–3.980%; P < FSCmid/SSChigh (data not shown) fractions were gated as 0.0001) as shown in Fig. 3A. In contrast with a previous þ þ lymphocytes, monocytes, or granulocyte subpopulations, report (11), the frequency of CD45 /CD31 /CD146 cells respectively. Subpopulations of cells with different CD45 in healthy volunteers (median, 0%; range, 0–0.003%) and and CD31 expression patterns were further analyzed for the patients with cancer (median, 0.001%; range, 0–0.016%) expression of CD146 or lineage-specific markers including was lower than the cutoff values of the FMO control group CD3 (T lymphocyte), CD14 (monocyte), CD19 (B lym- (median, 0.007%; range, 0–0.021%), indicating that esti- phocyte), and CD56 (NK cells). The FSClow/SSClow subset mation of the frequency of those cells is not possible both in þ was mostly composed of CD45 /CD31 cells expressing cancer patients and healthy controls due to the limited CD3, CD19, or CD56 antigens (Fig. 1A), whereas the resolution of the flow cytometry. Actually, when the isotype þ þ þ FSChigh/SSCmid subset mainly included CD45 /CD31 control was used, the frequencies of CD45 /CD31 / þ þ cells expressing the CD14 antigen (Fig. 1B). CD45 /CD31 CD146 cells were significantly higher (median, 0.041%; cells were detected only in the FSClow/SSClow fraction (Fig. range, 0.007–0.132%; Fig. 2C), underscoring the impor- 1A) but neither in the FSChigh/SSCmid (Fig. 1B) nor FSCmid/ tance of using FMO controls. SSChigh fractions (data not shown). Meanwhile, CD146 expression was detected only in the þ In the polychromatic flow-cytometric analysis used in the CD45 /CD31 subpopulation of the FSClow/SSClow popu- present study, adequate threshold was assessed by fluores- lations (Fig. 1A). These CD146-positive cells also expressed cence-minus-one (FMO) gating, which consists of analyz- CD3 (data not shown). These results, together with those of ing cells stained with all antibodies except the one being previous studies showing that CD146 is present in a subset þ tested (30). To investigate the effects of the gating controls of activated T lymphocytes (21, 33), indicate that CD146 þ on the actual event frequencies, the flow-cytometric analysis cells in the CD45 /CD31 lymphocyte subpopulation are T of the singlet lymphocyte fraction was conducted using lymphocytes. þ þ either the isotype control or the FMO control (Fig. 2A). The frequency of CD45 /CD31 /CD105 cells in Because the negative threshold of the FMO control (Fig. 2A, healthy volunteers (median, 0.003%; range, 0–0.027%) b) was higher than that of the isotype control (Fig. 2A, a), showed no significant differences statistically when com- gating with FMO controls could decrease the false-positive pared with FMO control (P > 0.05). On the other hand, the

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A – /CD31 + CD45 CD56 CD45 CD45 CD45 CD3 CD14 CD19 CD146 + /CD31 + CD45 CD56 CD45 CD45

+ –

CD45 /CD31 CD45 CD3 CD14 CD19 CD146 + CD45+/CD31+ /CD31 – CD45 CD56 CD45 CD45 CD45–/CD31+ CD45

CD3 CD14 CD19 CD146

B – /CD31 + CD45 CD56 CD45 CD45 CD45 CD3 CD14 CD19 CD146 + /CD31 + CD45 CD56 CD45 CD45 CD45+/CD31–

CD45 CD3 CD14 CD19 CD146

CD45+/CD31+ + /CD31 – CD45–/CD31+ CD45 CD56 CD45 CD45 CD45

CD3 CD14 CD19 CD146

Figure 1. CD45 and CD31 expression in PBMCs obtained from patients with cancer. PBMCs were initially gated into (A) FSClow/SSClow and (B) FSCmid/SSCmid fractions to include mainly lymphocytes and monocytes, respectively, and singlet cells were selected on the basis of FSC-Height versus FSC-Area plots. The cells were then subdivided into CD45þ/CD31, CD45þ/CD31þ, and CD45/CD31þ cells. The cells were further analyzed for the expression of lineage-speciﬁc markers (CD3, CD14, CD19, and CD56) and CD146. Lymphocytes gated on FSC/SSC plots were mostly CD45þ/CD31 (A, left), whereas monocytes were mostly CD45þ/CD31þ (B, left). In addition, CD45þ/CD31 cells were composed of CD3þ, CD19þ, CD56þ, and CD146þ cells (A and B, top right), whereas CD45þ/CD31þ cells were mostly CD14þ (A and B, middle right), regardless of lymphocyte and monocyte gating based on the FSC/SSC plot. In particular, FSClow/SSClow gating showed CD45/CD31þ cells (A and B, left) that did not express lineage-speciﬁc markers or CD146 (A and B, bottom right).

þ þ frequency of CD45 /CD31 /CD105 cells in patients There was no signiﬁcant statistical difference in the fre- þ þ þ with cancer (median, 0.012%; range, 0–0.461%) was signi- quency of CD45 /CD31 , CD45/CD31 /CD146 , and þ þ ﬁcantly higher than in healthy volunteers (P < 0.0001) CD45/CD31 /CD105 cells between healthy male and as shown in Fig. 3B. female volunteers.

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A Isotype control FMO Control IgG1-PE IgG1-PE

Figure 2. Gating strategy for the IgG1-FITC CD31-FITC detection of CEC candidates. A, B quantile contour plots showed an increased ﬂuorescence background in the FMO control (right) compared with the isotype control (left). B, gating strategy for CD45–/CD31+ cells þ FSC-H CD45-PerCP SSC-A the detection of CD45 /CD31 , CD45/CD31þ/CD146þ,and CD45/CD31þ/CD105þ cells by FSC-A FSC-A CD31-FITC low ﬂow cytometry. First, FSC /SSClow fractions were gated on the basis of FSC-A/SSC-A plots, and singlet cells were selected on the basis of FSC-H/FSC-A plots. Next, CD45/CD31þ cells were þ

CD146-PE initially gated. CD45 /CD31 / CD105-PE CD146þ or CD45/CD31þ/ CD105þ cells were further CD31-FITC CD31-FITC analyzed on the basis of FMO control gating. C, graphs showed that the percentage of false- C 0.16 positive cells was signiﬁcantly 0.14 increased in using isotype control 0.12 as compared with FMO control. 0.10 Median value was indicated as a horizontal bar. 0.08 0.06 % of cells 0.04 0.02 0.00 Using Using isotype controlFMO control

þ Characterization of anucleated CD45 /CD31 cells microscopy, anucleated cells (2–6 mm in diameter), which To date, several methods have been used to quantify were smaller than lymphocytes, and nucleated cells (8–10 CECs. However, there is currently no consensus on the mm in diameter; data not shown). Strijbos and colleagues þ most accurate markers for their identification. Moreover, reported that the vast majority of CD45 /CD31 cells are, CEC quantification methods have not been adequately in fact, large platelets rather than endothelial cells (17). To validated or standardized. For these reasons, there has been investigate the possibility of false-positive results of the a significant variation in the CEC numbers reported, includ- flow-cytometric quantification of CECs, we assessed þ ing those of the present study. To determine whether the CD45 /CD31 cells for the expression of platelet markers CEC candidate cells identified by flow cytometry have true such as CD41 and CD61 by flow cytometry and found that þ þ endothelial phenotypes, we isolated CD45 /CD31 cells most CD45 /CD31 cells (more than 98%) stained posi- from the PBMCs of patients with cancer by FACSAria sorter tive for CD41 and CD61 (Fig. 4A and B, respectively). þ and examined their morphologic and immunologic char- Furthermore, anucleated CD45 /CD31 cells stained neg- þ acteristics. CD45 /CD31 cells (mostly lymphocytes) and ative for anti-CD146, anti-CD105, and anti-CD144 anti- þ þ CD45 /CD31 cells (mostly monocytes) were also isolated bodies but positive for vWF, a common marker of endo- and used as controls. thelial cells and platelets (Fig. 5B, top) as assessed by þ Two different populations of CD45 /CD31 cells were immunocytochemical staining and confocal microscopy. observed by confocal microscopy and scanning electron These results together with the morphologic phenotypes of

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vWF (36), and CD144 (37). Consistent with the flow- þ A P < 0.0001 B P < 0.0001 cytometric results, most nucleated CD45 /CD31 cells expressed CD105 but not CD146 (Fig. 5A, top). These þ þ 30 0.5 CD45 /CD31 /CD105 cells also expressed CD144 (Fig. 0.4 5A, bottom). 20 0.3 0.2 P < 0.0001 An increasing body of evidence indicates that monocytes 0.1 10 share several functional and immunophenotypic character- 0.050 istics with endothelial cells (38, 39). Moreover, endothelial 5.5 % of cells % of cells progenitor cells or circulating angiogenic cells derived from 0.025 3.0 monocyte/macrophage lineage were reported to promote angiogenesis by secreting angiogenic growth factors (40). 0.5 0.000 þ þ Healthy Cancer FMO Healthy Cancer To determine whether CD45 /CD31 /CD105 cells pos- subjects patients Control subjects patients sess monocyte/macrophage characteristics in addition to those of endothelial cells, cells were costained with an anti- CD68 antibody (macrophage marker) and the anti-vWF Figure 3. Differential levels of CEC candidates in patients with þ þ gynecologic cancer (n ¼ 56) and healthy volunteers (n ¼ 44). PBMCs were antibody. Interestingly, CD45 /CD31 /CD105 cells analyzed by three-color flow cytometry followed by the gating strategy stained positive for CD68 (Fig. 5B, bottom) but negative þ described in Fig. 2B. The frequencies of (A) CD45 /CD31 and (B) CD45 for vWF (Fig. 5B, top). Furthermore, these cells expressed þ þ low low þ /CD31 /CD105 cells in singlet FSC /SSC populations from the monocyte-specific antigen CD14 (Fig. 5C). CD45 / patients with cancer and healthy volunteers are depicted as a scatter plot. þ þ þ CD31 cells (mostly lymphocytes) were used as a negative The numbers of CD45 /CD31 and CD45/CD31 /CD105 cells were fi control for CD68, vWF, and CD14 expression, whereas signi cantly higher in patients with cancer than in healthy volunteers þ þ (A and B, respectively). Differences in CEC candidate levels between CD45 /CD31 cells (mostly monocytes) were used as a patients with cancer and healthy volunteers were assessed by the Mann– positive control for CD68 and CD14 expression (Fig. 5C for Whitney U test. Median value was indicated as a horizontal bar. CD14 expression). Human umbilical vein endothelial cells

þ and human dermal microvascular endothelial cells were these cells suggest that anucleated CD45 /CD31 cells are used as positive controls for vWF, CD146, CD105, and mainly platelets. CD144 (data not shown). þ þ The speciﬁcity of sorted CD45 /CD31 cell population Characterization of nucleated CD45 /CD31 cells could be conﬁrmed and the possibility of contamination of þ þ To determine whether endothelial cells are included sorted cell population with CD45 /CD31 cells could be þ þ þ in the nucleated CD45 /CD31 cell population, CD45 / excluded because CD45 /CD31 cells were not detected þ þ CD31 cells were isolated as described previously and when the sorted cells by CD45 /CD31 gate were analyzed stained with antibodies against CD146 (34), CD105 (35), by the CD45/CD31 expression (Supplementary Fig. S1). Collectively, these data indicate that anucleated CD45 / þ þ CD31 cells were mainly platelets, and CD45 /CD31 / þ A CD105 cells were derived from monocytes/macrophages rather than endothelial cells. Discussion CECs in the peripheral blood have been widely recog- CD45-PerCP

CD41-PE nized as a marker of angiogenesis. Most studies have relied on multiparametric flow cytometry to identify CD31-FITC CD31-FITC endothelial cells because of the limited specificity of the markers used for CEC detection, which can also be B expressed by other hematopoietic cells (16, 39). CECs are currently defined as cells that express CD31 and other markers such as CD146, CD34, or CD105, but not CD45, a pan-leukocyte marker (7, 11). However, their rareness and phenotypic overlap with other hematopoietic cells CD45-PerCP CD61-FITC have led to controversies about the identification and determination of CECs in peripheral blood. Therefore, CD31-PE CD61-PE the present study focused on assessing the accuracy of current flow-cytometric techniques for the identification

þ of CECs and the immunofluorescence phenotyping of Figure 4. CD41 and CD61 expression in CD45 /CD31 cells. The CECs using different markers. Our results suggest that expression of the platelet markers CD41 and CD61 in CD45/CD31þ cells was analyzed by three-color flow cytometry, followed by the gating those cells that meet the conventional criteria to be strategy shown in Fig. 2. Quantile contour plots indicated that most defined as CECs are derived from a monocyte lineage CD45/CD31þ cells expressed CD41 and CD61. rather than having an endothelial origin.

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Figure 5. Immunocytochemical þ staining of CD45 /CD31 cells. CD45/CD31þ cells sorted by flow cytometry were cytospun on glass slides, fixed with 4% PFA, permeabilized with 0.1% Triton X-100 for CD68 and vWF staining, and then labeled with antibodies against the endothelial cell markers CD105, CD146, CD144, and vWF or the monocyte markers CD68 and CD14. A, double staining of CD45/CD31þ cells with anti- CD146 or CD144 and anti-CD105 Abs. CD45/CD31þ cells expressed CD105 and CD144 (bottom) but not CD146 (top). Nuclei were counterstained with Hoechst 33342 dye (original magnification, 630). B, double staining of CD45/CD31þ cells with anti-vWF or CD68 and anti- CD105 Abs. Nucleated CD45/ CD31þ cells expressed CD68 (bottom) but not vWF (top), whereas anucleated CD45/ CD31þ cells expressed only vWF. Nuclei were counterstained with Hoechst 33342 dye (original magnification, 630). C, single staining of CD45/CD31þ cells with anti-CD14 Ab. Nucleated CD45/CD31þ cells expressed CD14. CD45þ/CD31 and CD45þ /CD31þ cells were used as negative and positive controls for the detection of CD14 expression, respectively. The control slide was stained with only fluorochrome- conjugated secondary Ab. Nuclei were counterstained with Hoechst 33342 dye (original magnification, 630).

Autofluorescence is an important consideration when Several studies have shown evidences that CECs, defined þ þ þ þ conducting polychromatic flow-cytometric analyses. as DNA cells with CD34 /CD45 /CD146 or CD31 / þ Because monocytes have higher levels of autofluorescence CD45 /CD146 immunophenotypes, are significantly than lymphocytes, these 2 types of cells should be analyzed elevated in the peripheral blood of patients with cancer separately (29). The frequency of CECs has mostly been than healthy subjects (41, 42). The origin of the cells was analyzed by gating the lymphocyte and monocyte popula- confirmed as endothelial cells by morphology, immuno- tions as a whole without separation, which can lead to false- histochemistry, gene expression, and the presence of Wei- negative or false-positive results. To overcome this potential bel–Palade bodies. However, there have been scientific defect, we first gated FSClow/SSClow fraction (mostly lym- issues to be improved that the effects of various fluorescent þ phocytes) and then subgated CD45 /CD31 cells for a compensation methods on the detection of CECs were not more detailed and accurate characterization. In addition, considered. Moreover, expression of several lineage markers we also showed that FMO is a more accurate control to set other than endothelial cell origin should be fully investi- the boundaries for the analysis of rare cells such as CECs as gated to identify the genuine origin of those cells, because described previously (31). the immunophenotypes can be overlapped among cells

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Characterization of Circulating Endothelial Cells

derived from endothelial cells, hematopoietic progenitors, cytes express not only CD31 constitutively (48) but also or monocytes, etc. other markers including CD144, KDR, Tie-2, and CD105 On the basis of the cell sorting conditions used, our when they are activated (49). Therefore, we further analyzed þ þ results did not agree with previous studies (11) in which few CD45 /CD31 /CD105 cells with antibodies against þ þ CD45 /CD31 /CD146 cells, which are known to define monocyte-specific markers such as CD14 and CD68, and þ þ CECs, were present in the peripheral blood, even in that of showed that CD45 /CD31 /CD105 cells uniformly patients with cancer. Although the frequency of CD45 / expressed both CD14 and CD68. This result indicates that þ þ þ þ CD31 /CD146 cells seemed to be significant if isotype CD45 /CD31 /CD105 cells have the characteristics of controls were used, they remained at background levels monocytes rather than those of endothelial cells (Supple- in the presence of FMO controls. In addition, our immu- mentary Fig. S2). Prokopi and colleagues showed that the nocytochemical data showed that none of the isolated endothelial phenotype may arise in mononuclear cells þ CD45 /CD31 cells expressed CD146. CD146 expression through the uptake of platelet microparticles (derived from has been reported on activated T-cell subsets in healthy the disintegration of platelets during mononuclear cell individuals (33). Previously, Duda and colleagues reported preparation) that abundantly contain marker proteins such þ that CD146 marks endothelial cells in normal and neo- as CD31 and vWF (50). More importantly, monocytes in the plastic tissues, as well as a subset of T cells (21). These peripheral blood are known to exhibit versatile and flexible authors reported that CD146 expression was frequently differentiation potentials and functions, as they can differ- þ þ detected on pericytes, and CD45 /CD146 cells were occa- entiate into macrophages, dendritic cells, osteoclasts, sionally contained in the massive hematopoietic cell infil- microglia, or even endothelial-like cells (51). Myeloid line- tration in tumor tissues. Our results were in agreement with age cells including monocytes were reported to express þ those of Duda and colleagues in that CD146 cells were markers that were expressed in both endothelial cells and þ detected exclusively in the CD3 cell population. monocytes (22, 49, 52–54). Furthermore, circulating þ þ þ We could not detect the CD45 /CD31 /CD146 cells in CD31 cells, which can contribute to the development of the peripheral blood of patients with gynecologic cancer; new blood vessels, were shown to be monocytes in an however, it remains to be further tested and compared elaborate animal model (24). þ among patients with different types or sites of malignancies. In summary, our results showed that CD45 /CD31 / þ For instance, endothelial cells in hemangioma tissues sho- CD105 circulating cells detected by flow cytometry under wed negative immunoreactivity for CD146 (43), whereas gating conditions we established, which were significantly the expression of CD146 was highly increased in the blood increased in the peripheral blood of patients with gyneco- vessels of breast carcinoma (44). logic cancer, were not CECs but rather monocytes, suggest- Because flow cytometry is not sensitive enough to obtain ing that the conventional flow-cytometric techniques used reproducible results when analyzing rare cells such as CECs, for the identification of cell subpopulations could be the results of these analyses should be interpreted with improved by adjusting gating conditions. Further study is caution (45). The frequency of CECs in the peripheral blood needed to identify the biologic significance of these cells and of the healthy population is between 1 10 7 and 1 10 5 their function in relation to angiogenesis. per leukocyte (0–20 cells/mL of venous blood; refs. 20, 46). This level is below the detection threshold for conventional Disclosure of Potential Conflicts of Interest flow cytometry, which is approximately 1 10 4 (45). We No potential conflicts of interest were disclosed. therefore assumed that the frequency of CECs may be too low for detection by flow cytometry, despite their presence Authors' Contributions among PBMCs. Conception and design: H.-K. Yu, J.-S. Kim, S.J. Kim, T.J. Kim þ Development of methodology: H.-K. Yu, J.-H. Ahn, L.S.H. Yi, J.-S. Kim On the other hand, the frequency of CD45 /CD31 / Acquisition of data (provided animals, acquired and managed patients, þ CD105 cells (another potential CEC candidate) was sig- provided facilities, etc.): H.-K. Yu, H.-J. Lee, H.-N. Choi, J.-Y. Choi, H.-S. nificantly higher in patients with cancer than in the healthy Song, K.-H. Lee, S.J. Kim, T.J. Kim þ Analysis and interpretation of data (e.g., statistical analysis, biosta- population. Furthermore, the frequency of CD45 /CD31 / tistics, computational analysis): H.-K. Yu, H.-N. Choi, J.-Y. Choi, H.-S. þ CD105 cells was significantly increased by up to approx- Song, J.-S. Kim, S.J. Kim, T.J. Kim Writing, review, and/or revision of the manuscript: H.-K. Yu, L.S.H. Yi, imately 8-fold in patients with gynecologic cancer com- J.-S. Kim, S.J. Kim, T.J. Kim pared with healthy volunteers. CD105 (endoglin), a 180 Administrative, technical, or material support (i.e., reporting or orga- kDa homodimeric integral membrane glycoprotein and a nizing data, constructing databases): H.-N. Choi, H.-S. Song, J.-S. Kim, S.J. Kim commonly used marker for the detection of CECs next to Study supervision: Y. Yoon, L.S.H. Yi, J.-S. Kim, S.J. Kim, T.J. Kim CD146, was mainly expressed on endothelial cells of capillaries, veins, and arteries (35) but was also detectable Grant Support on activated monocytes, macrophages, erythroid precur- This work was financially supported by a grant from the Korea Health 21 sors, fibroblasts, mesangial cells, follicular dendritic cells, R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A050905), by a grant from the KRIBB Research Initiative Program, and syncytiotrophoblasts (47). Interestingly, our immuno- and by Basic Science Research Programs through the National Research þ cytochemistry data showed that these CD45 /CD31 / Foundation of Korea funded by the Ministry of Education, Science and þ Technology (2009-0073284 and 2012R1A1A2007994). CD105 cells did not express vWF (36), whereas they were The costs of publication of this article were defrayed in part by the positive for CD144 (VE-cadherin; ref. 37). Actually, mono- payment of page charges. This article must therefore be hereby marked

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Yu et al.

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Received November 30, 2012; revised June 27, 2013; accepted July 15, this fact. 2013; published OnlineFirst August 6, 2013.

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www.aacrjournals.org Clin Cancer Res; 2013 OF11

Characterization of CD45−/CD31+/CD105+ Circulating Cells in the Peripheral Blood of Patients with Gynecologic Malignancies

Hyun-Kyung Yu, Ho-Jeong Lee, Ha-Na Choi, et al.

Clin Cancer Res Published OnlineFirst August 6, 2013.

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