Circulating endothelial cells as biomarkers in clinical oncology

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Abstract

Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) play a different role in cancer development, acting as possible markers of vascular turnover/damage (CECs) and vasculogenesis (CEPs). Preclinical and clinical data suggest that CEC enumeration might be useful to define the best treatment option for patients who are candidate to anti-angiogenic therapy, while CEPs seem to have a “catalytic” role in different steps of cancer progression and recurrence after therapy. The definition of CEC and CEP phenotype and the standardization of CEC and CEP enumeration procedures are highly warranted to use these cells as biomarkers in clinical trials in oncology, and to compare results from different studies.

Introduction

The endothelial cell turnover has always been thought to be very slow compared with other tissues. This notwithstanding, cells with endothelial morphology were found to circulate in the blood more than 35 years ago (Hladovec and Rossamn, 1973). In the following years, the endothelial nature of these cells was confirmed by immunohistochemistry (IHC) studies, and their enumeration by means of positive enrichment, IHC or flow cytometry (FC) indicated that circulating endothelial cells (CECs) are increased in a very wide spectrum of disorders encompassing vascular, autoimmune, infectious and ischemic diseases (Moldovan et al., 1994, Bertolini et al., 2006). Over the past 10 years, increased CEC counts were observed in some cancer patients (Mancuso et al., 2001, Farace et al., 2007), and these cells were studied as surrogate biomarkers of angiogenesis and anti-angiogenic drug activity in preclinical models and medical oncology (Monestiroli et al., 2001, Shaked et al., 2005a, Shaked et al., 2005b, Calleri et al., 2009). These studies also indicated that the endothelial phenotype was expressed by cells displaying a wide variety of different features (Blann et al., 2005, Bertolini et al., 2006). Some CECs had a phenotype compatible with terminally differentiated endothelial cells (EC), in some cases being apoptotic or necrotic and thus most likely derived from the turnover of vessel walls. Some other cells expressed progenitor-associated antigens in addition to endothelial antigens, and were considered as circulating endothelial progenitor (CEP) candidates.

Section snippets

CEP and CEC phenotype

There is a lack of consensus regarding the surface markers that identify CEPs and CECs, due to the lack of a marker that can unambiguously identify these cells.

By means of flow cytometry, the most widely used antigens to identify CEPs are CD34, VEGFR2 and CD133. Many investigators identify and enumerate CEPs by means of the co-expression of CD34 and VEGFR2 (Rosenzweig, 2005, Bertolini et al., 2006, Asahara et al., 1997, Shi et al., 1998), but these antigens are expressed also by mature CECs.

CEC number and viability in cancer and treatment

Endothelial cell enumeration (both CECs and CEPs) has led to the observation that these cells are increased and are more viable in some types of cancer patients compared to healthy controls (Mancuso et al., 2001, Farace et al., 2007) (Fig. 2). In breast cancer, CECs and CEPs demonstrated a strong relationship with the Nottingham prognostic index (NPI), but only CECs positively predicted higher NPI scores and correlated with tumor invasiveness and size, possibly reflecting total tumor vascular

CEPs in tumor grow and metastases

There are different lines of research studying the role of CEP in early and late stage of cancer development and metastasis. In the late '90s, Asahara et al. (1997) and Shi et al. (1998) demonstrated that human peripheral blood contains cells that are able to differentiate into endothelial cells. However, the first proof of the contribution of CEPs in tumor development was reported by Lyden et al. (2001) in Id1 deficient mice. These mice have a severe CEP defect and show impaired angiogenesis,

CEC and CEP as tools to monitor minimal residual disease

A number of investigators have suggested that vessel-lining ECs in tumors might express a specific antigenic profile. The St. Croix lab compared gene expression patterns of ECs obtained from normal resting tissues, tumors, and regenerating liver (Seaman et al., 2007). They identified 25 transcripts overexpressed in tumor versus normal ECs, including 13 that were not found in the angiogenic endothelium of regenerating liver. Those EC tumor-specific antigens were primarily cell surface molecules

CEC/CEP in the clinic

It seems now possible to anticipate two separate fields of clinical investigation for CEPs and CECs. Some CEPs (along with other pro-angiogenic cells of hematopoietic and/or mesenchymal origin) seem to have a “catalytic” role (Seande et al., 2008) in promoting angiogenesis during tumor growth, in stimulating growth of micro- and macrometastases and in rebound revascularization after certain therapies (Rafii et al., 2002, Nolan et al., 2007, Gao et al., 2008, Lyden et al., 2001, Ciarrocchi et

Conclusions

In the future, a great effort should be devoted to reach a consensus in the identification and enumeration of CECs and CEPs. Multi parametric FC seems to be a good tool to reach this goal, in spite of the lack of a single specific antigen/marker able to identify these cells and of the rarity of them.

Clinical data suggest that the identification and enumeration of CECs and CEPs might be useful to select the most appropriate therapy for patients who are candidates to anti-angiogenic treatments,

Acknowledgments

We apologize to the numerous investigators whose papers could not be cited because of space limitations. This work was supported in part by AIRC (Associazione Italiana per la Ricerca sul Cancro), ISS (Istituto Superiore di Sanità), and Ministero della Salute grant RF-IMI-2006-411189.

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