Circulating endothelial cells in patients with colorectal cancer: flow cytometry analysis of cell marker CD146-PE and CD45-FITC.

Introduction: Colorectal cancer (CRC) remains the third most common cancer both in men and women worldwide, and its incidence rates continue to increase in the USA and Western Europe. Angiogenesis is crucial for growth of all tumors, and mature and immature endothelial cells, such as circulating endothelial cells (CECs) and endothelial progenitor cells, are present in the human blood. CECs are extremely rare in normal peripheral blood, but it has been shown that their number is significantly increased in many pathological conditions, including cancer. Some Among CECs display characteristics of terminally differentiated and mature cells, while others express specific surface antigens, suggesting a progenitor-like or stem-like phenotype. Thus, vasculogenesis is not restricted to embryonic development, and there are putative circulating endothelial progenitors which can participate in the generation of new vessels. CECs measurement is a promising biomarker to assess the efficacy of antiangiogenic therapies, and their increase may suggest the possible presence of metastatic disease, including in patients with CRC. Different cell surface markers have been tested to detect CECs, and flow cytometry represents a well suited method for their detection and quantitation. The aim of this study was to evaluate the levels of the microenvironmental cell marker CD146 +CD45- phenotype, which is linked to angiogenesis, vessel damage, and disease progression, in patients with pT0-1 CRC. Methods: Blood samples from 41 patients (26 men, 15 women, median age 64 years, range 39-74 years) with confirmed colorectal adenocarcinoma undergoing curative surgery were collected for analysis of CECs. Patients with distant metastases (negative F-18 FDG PET/CT) have been excluded, as well as those who had undergone adjuvant chemotherapy. Written informed consent was obtained from all the participants. All samples were stained with antihuman CD146 phycoerythrin (CD146-PE) and CD45 fluorescein isothiocyanate (CD45-FITC). Immunophenotyping was obtained using a Beckman Coulter XL-MCL flow cytometer to assess levels of CD146+CD45- CECs, using a 600 s acquisition time for each sample. According to final pathology, two groups of patients were obtained: Group A (28 patients, 68.3%) with negative lymph nodes (pN0), and Group B (13 patients, 31.7%) with regional lymph node metastasis (pN1). Results: N1 patients (Group B) had a level of CD146+/CD45- cells significantly increased (19.2±9.1 vs. 12.4±8.6 CECs, 95% CI 0.72-12.88, p=0.029) in respect to N0 patients (Group B). No correlation (R=0.10, p=0.22) was found between number of the N1 and level of putative CECs. There was no difference (p=NS) between men and women. Conclusion: Although CECs are a rare cell population (less than one in 1000 circulating blood cells) and their not yet completely established phenotype represent a technical challenge, our preliminary data suggest a possible increased angiogenic activity in patients with N1 CRC, which can be demonstrated using flow cytometry analysis of CECs. Further endothelial cell markers, alone or in combination, should be studied to confirm this hypothesis.