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Detection of Circulating Endothelial Cells: CD146-Based Magnetic Separation Enrichment or Flow Cytometric Assay?

Laboratoire d'Hématologie et d'Immunologie, INSERM UMR-S 608, UFR de Pharmacie, Marseille, France

Andrew Blann

Haemostasis Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom

Alexander Woywodt

Division of Nephrology, Department of Medicine, Hannover Medical School, Hannover, Germany

To the Editor:

In their recent article entitled, "Differential CD146 Expression on Circulating Versus Tissue Endothelial Cells in Rectal Cancer Patients: Implications for Circulating Endothelial and Progenitor Cells As Biomarkers for Antiangiogenic Therapy," Duda et al¹ evaluated the potential interest of these emerging biomarkers to monitor antiangiogenic therapies. They concluded that CD146 was expressed on vascular endothelium and on a subset of T lymphocytes but not on circulating endothelial cells or progenitor cells.

CD146 has never been considered as a specific marker of vascular endothelium. CD146, also known as S-Endo-1, Mel-CAM, MUC18, was first described as a marker of melanoma cells.² It was also detected on other cell types, such as pericytes, bone marrow fibroblasts, nerve fibers, and T-lymphocyte subsets. Therefore, any approach using CD146 to discriminate endothelial cells from other blood cells has to deal with the low expression of this marker on a subset of activated T lymphocytes as described by Pickl et al³ and more recently by Elshal et al.⁴ This low expression should not be considered as a technical limitation for the detection of circulating endothelial cells (CECs), since the methodology used to detect CECs, CD146-based immunomagnetic separation (IMS), has been adapted to cope with it. In our early flow cytometric-based trials of CEC detection,⁵ we first described the prototypic CD146 Mab S-Endo 1 as being detectable on endothelial cells with an expression level 1000-fold higher than the mean intensity observed on leukocytes. This is illustrated in Figure 1 of this article dated 1991 showing that its mean expression level on the whole lymphocyte population was far below the mean level measured on human umbilical vein endothelial cells used as a model of endothelial cells. Although CD146 is indeed detectable on a subset of T lymphocytes 1, 3, 4 the expression level on the few positive T lymphocytes is still very low (< 5,000 CD146 molecules per positive cell, unpublished data, P. Poncelet, Biocytex, Marseille, France) as compared with endothelial cells. Such a striking difference in antigen density between CD146+ lymphocytes and EC (between 100,000 and 1,000,000 CD146 molecules per positive cell, according to the type of EC⁶) explain the typical images obtained when EC are sorted using CD146-driven IMS. The IMS-enriched cell suspension is made of large endothelial cells surrounded with numerous beads (> five beads per CEC and size > 20 µm in consensus protocol, see Woywodt et al⁷) and small size lymphocytes bearing very few beads (< 5 beads and size < 10 µm) that have been captured in variable amounts.

It is undisputed that CD146-based magnetic separation rarely provides an absolutely pure fraction of endothelial cells and that the procedure includes a visual recognition step made under the microscope. Since this step relies on the skills and experience of the operator, this is best accompanied by an additional specific characterization step, such as UEA-1 staining^7,8 to confirm that all separated cells are of endothelial origin and provide a more precise quantitation. Specifically, a positive stain for Europaeus lectin-1 (UEA-1) excludes lymphocytes. This methodology has been used successfully in a large number of studies showing increased levels of CEC in various pathological situations, including ischemic, inflammatory, immune, and infectious disease.^9-11 These studies have validated the endothelial origin of CD146-positive cells in peripheral blood by staining with endothelial markers, such as CD 31, vWF and UEA-1, and by demonstration of Weibel-Palade bodies. In our opinion, there is no doubt that CD146-driven IMS yields endothelial cells from peripheral blood. Although enumeration of CEC is not a standardized procedure, there is a good degree of agreement between laboratories using the CD146-driven IMS with values in the order of less than 10 cells/mL in most normal individuals. This finding gives us further confidence in this technique.¹⁰

The lack of a single marker for differentiated endothelial cells necessitates that a combination of different criteria are used to define CEC. In a recent collaborative multicentric study based on the experience of several groups,⁸ the following definition of CEC was proposed: a CEC is a cell that exceeds 10 µm in size and has more than five immunomagnetic beads attached. The rosetted cell stains positive with at least two endothelial markers (for example, CD146 and UEA-1) and is negative for leukocyte markers (for example, CD14 and CD45). The lack of consensus for the definition of CECs and EPCs is also reflected in the article by Duda and colleagues. The term viable CECs used in their study must surely indicate a progenitor phenotype. In contrast, CECs are sloughed off the vessel wall, negative for CD133, and cannot give rise to colonies with a high proliferative potential.

It should be noted that earlier flow cytometric measurement (FCM) studies, which spiked blood with human umbilical vein endothelial cells as model cells for CECs, showed a clear detection limit of about 0.18 CEC/µL blood (ie, 180 CEC/mL blood).⁵ As stated in the discussion section of that article, the level of sensitivity of FCM may not suffice to precisely determine the level of endothelial cells potentially present in normal peripheral blood.⁵ Among the different laboratories using the CD146 driven IMS, there is a good degree of agreement with values in the order of less than 10 cells/mL in most normal individuals. Such a range (ie, 0 to 10 cells/mL in blood samples) is a real challenge for any technique, including FCM despite its increasing potential for multicolor detection.

Therefore, we would like to suggest to Duda and colleagues to investigate whether the sensitivity limit of the flow cytometry method they used is low enough to determine the level of CECs in peripheral blood. Moreover, we would like to take issue with their use of CD31 as an endothelial marker since like CD146, CD31 is not specific for the endothelium and can be detected on leukocytes subsets or platelet/leukocytes aggregates. Indeed, on the basis of CD31-bright/CD45-negative labeling as a combination to identify CEC, the amount of cells recorded per milliliter of blood is about 1,000- to 100,000-fold higher than the level of CEC consensually reported in normal and pathological settings using CD146 based immunomagnetic separation. This huge discrepancy rises the question of the nature of this CD31-bright/CD45-negative population. Accordingly, we also suggest to the authors to perform cell sorting of this population, in order to confirm its endothelial origin on the basis of well-established criteria such as size, UEA-1 Lectin labeling or intracellular detection of von Willebrand factor.

Regarding the conclusion of Duda et al¹ that CECs need to be integrated into larger phase II-III clinical trials, we support the authors' statement that "Improving the analytic methodology will be critical for the clinical development and optimal use of this emergent biomarker. Flow cytometric enumeration of CECs is far from being a standardized procedure and many efforts are still needed to get easy-to-use appropriate assays." Whether current multiparameter flow cytometry methodologies address the desired level of sensitivity and reproducibility remains controversial. The most important question to address is whether or not the very low frequency of CEC necessitates enrichment of these cells (or depletion of unwanted cells) to be in the range of the current sensitivity limit of FCM assays.

CECs are now considered a good marker of endothelial injury. There is a growing belief that CECs may evolve into a surrogate biomarker for detecting and monitoring vascular disease. In this regard, consensus on the appropriate technique(s) will be a key issue before CECs can be evaluated in large cohorts of patients.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The authors indicated no potential conflicts of interest.

REFERENCES

1. Duda DG, Cohen KS, di Tomaso E, et al: Differential CD146 expression on circulating versus tissue endothelial cells in rectal cancer patients: Implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. J Clin Oncol 24:1449-1453, 2006[Abstract/Free Full Text]

2. Dignat-George F: CD146 (S-Endo/Muc18) workshop panel report, in Oxford University Press (eds): Leucocyte Typing VI. New York, Garland Publishers, 1997, pp 757-759

3. Pickl WF, Majdic O, Fisher GF, et al: MUC18/MCAM (CD146) an activation antigen of human T lymphocytes. J Immunol 158:2107-2115, 1997[Abstract]

4. Elshal MF, Khan SS, Takahashi Y, et al: CD146 (Mel-CAM), an adhesion marker of endothelial cells, is a novel marker of lymphocyte subset activation in normal peripheral blood. Blood 106:2923-2924, 2005[Free Full Text]

5. George F, Poncelet P, Laurent JC, et al: Cytofluorometric detection of human endothelial cells in whole blood using S-Endo 1 monoclonal antibody. J Immunol Methods 139:65-75, 1991[CrossRef][Medline]

6. George F, Mutin M, Poncelet P, et al: Antigenic densities defined by workshop endothelial section mAbs, in Schlossman SF, Boumsell L, Gilles W (eds), Leucocyte Typing V. Oxford University Press, 1995, pp 1798-1801

7. Woywodt A, Blann A, Kirsch T, et al: Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: Proposal of a definition and a consensus protocol. J Thromb Haemost 4:671-677, 2006[CrossRef][Medline]

8. Woywodt A, Goldberg C, Scheer J, et al: An improved assay for enumeration of circulating endothelial cells. Ann Hematol 83:491-494, 2004[Medline]

9. Dignat-George F, Sampol J: Circulating endothelial cells in vascular disorders: New insights into an old concept. Eur J Haematol 65:215-220, 2000[CrossRef][Medline]

10. Blann AD, Woywodt A, Bertolini F, et al: Circulating endothelial cells: Biomarker of vascular disease. Thromb Haemost 93:228-235, 2005[Medline]

11. Woywodt A, Streiber F, de Groot K, et al: Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis. Lancet 361:206-210, 2003[CrossRef][Medline]

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This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dignat-George, F. Articles by Woywodt, A. Search for Related Content PubMed PubMed Citation Articles by Dignat-George, F. Articles by Woywodt, A. Social Bookmarking                            What's this?