Several studies have shown that bone marrow-derived endothelial cells (EC) may contribute to tumor angiogenesis and that in the peripheral blood of cancer patients there is an increased amount of circulating ECs (CECs) that may participate to new vessel formation. Recent data also showed that in B-cell neoplasms ECs are in part tumor-related reflecting a novel aspect of tumor angiogenesis. All together these observations suggest that tumors can elicit the sprouting of new vessels from existing capillaries through the secretion of angiogenic factors and that, in some cases, cancer cells can also mimic the activities of ECs by participating in the formation of vascular-like networks. In this study we aim to characterize the clinical and biological role of CECs in a series of 85 chronic lymphocytic leukemia (CLL) patients. CEC levels were evaluated by multiparameter flow cytometry and correlated with known clinical and biological parameters. For biological studies, CECs were first isolated by immunomagnetic sorting and then characterized by phenotypic studies with antibodies recognizing endothelial and CLL antigens, by FISH analyses with specific probes and by gene expression profiling comparing CLL CECs with CECs from normal subjects, and with monocytes and lymphocytes from the same CLL patient. We found that CEC levels were significantly higher in CLL patients in comparison to normal healthy subjects (p=0.037). Higher CEC levels were associated with advanced disease stage (p=0.012) and with lack of response to treatment or progressive disease (p=0.005). No association was demonstrated with CD38/ZAP70 expression and FISH/cytogenetic abnormalities. In all experiments more than 95% of immunomagnetically sorted cells were of EC origin as demonstrated by phenotypic analyses (VEGFR2+, vWF+, CD144+, UEA1 lectin+, CD45–, CD14–, CD5–, CD19–). FISH analysis showed that a significant proportion of sorted CECs was tumor-derived because they harbored the same genetic lesion as observed in neoplastic CLL cells. The fraction of CECs showing cytogenetic aberrations averaged 40.7% (range, 20-78%). More then 85% of CECs presented features of EPCs because they expressed CD133, a marker gradually lost during EC differentiation and absent in mature ECs. CLL CEC had a similar gene expression pattern for several genes characterizing CEC function such as CD144, CD34, CD133, CD146, CD31, VEGFR2, VEGFR3, VWF, and TIE2. Moreover, CLLCEC showed a strongly different gene expression pattern compared to normal CEC characterised by increased cell survival and proliferation including activation of Wnt and inhibition of Notch signalling pathways, reduction of cell adhesion to extracellular matrix and enhanced pro-angiogenic function. Gene expression profiling analysis also suggested that similarities exist with clonal CLL lymphocytes. In conclusion these findings suggest that in CLL CECs are in part tumor related and with a gene expression profile that may indicate their contribution to tumor neovasculogenesis and possibly to the spreading and progression of the disease.

CLINICAL AND BIOLOGICAL CHARACTERIZATION OF CIRCULATING ENDOTHELIAL CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA

RIGOLIN, Gian Matteo;RIZZOTTO, Lara;CICCONE, Maria;SOFRITTI, Olga;DAGHIA, Giulia;CAVAZZINI, Francesco;CASTOLDI, Gianluigi;CUNEO, Antonio
2008

Abstract

Several studies have shown that bone marrow-derived endothelial cells (EC) may contribute to tumor angiogenesis and that in the peripheral blood of cancer patients there is an increased amount of circulating ECs (CECs) that may participate to new vessel formation. Recent data also showed that in B-cell neoplasms ECs are in part tumor-related reflecting a novel aspect of tumor angiogenesis. All together these observations suggest that tumors can elicit the sprouting of new vessels from existing capillaries through the secretion of angiogenic factors and that, in some cases, cancer cells can also mimic the activities of ECs by participating in the formation of vascular-like networks. In this study we aim to characterize the clinical and biological role of CECs in a series of 85 chronic lymphocytic leukemia (CLL) patients. CEC levels were evaluated by multiparameter flow cytometry and correlated with known clinical and biological parameters. For biological studies, CECs were first isolated by immunomagnetic sorting and then characterized by phenotypic studies with antibodies recognizing endothelial and CLL antigens, by FISH analyses with specific probes and by gene expression profiling comparing CLL CECs with CECs from normal subjects, and with monocytes and lymphocytes from the same CLL patient. We found that CEC levels were significantly higher in CLL patients in comparison to normal healthy subjects (p=0.037). Higher CEC levels were associated with advanced disease stage (p=0.012) and with lack of response to treatment or progressive disease (p=0.005). No association was demonstrated with CD38/ZAP70 expression and FISH/cytogenetic abnormalities. In all experiments more than 95% of immunomagnetically sorted cells were of EC origin as demonstrated by phenotypic analyses (VEGFR2+, vWF+, CD144+, UEA1 lectin+, CD45–, CD14–, CD5–, CD19–). FISH analysis showed that a significant proportion of sorted CECs was tumor-derived because they harbored the same genetic lesion as observed in neoplastic CLL cells. The fraction of CECs showing cytogenetic aberrations averaged 40.7% (range, 20-78%). More then 85% of CECs presented features of EPCs because they expressed CD133, a marker gradually lost during EC differentiation and absent in mature ECs. CLL CEC had a similar gene expression pattern for several genes characterizing CEC function such as CD144, CD34, CD133, CD146, CD31, VEGFR2, VEGFR3, VWF, and TIE2. Moreover, CLLCEC showed a strongly different gene expression pattern compared to normal CEC characterised by increased cell survival and proliferation including activation of Wnt and inhibition of Notch signalling pathways, reduction of cell adhesion to extracellular matrix and enhanced pro-angiogenic function. Gene expression profiling analysis also suggested that similarities exist with clonal CLL lymphocytes. In conclusion these findings suggest that in CLL CECs are in part tumor related and with a gene expression profile that may indicate their contribution to tumor neovasculogenesis and possibly to the spreading and progression of the disease.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/532855
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