Role Of EPCs In Tumor Microvascular Architecture Phenotype Heterogeneity Within Glioma Xenografted Mice

Neovascularization is crucial for the growth and metastasis of malignant solid tumors. Tumor neovascularization is now believed to occur via at least two possible mechanisms: the sprouting of pre-existing resident endothelial cells, i.e., angiogenesis, and the recruitment of bone marrow–derived EPCs, known as vasculogenesis. Although the mechanism of neovascularization has been one of the frontiers in cancer research, therapeutic efficacy of antiangiogenesis appears to be quiet. The possible reason is heterogeneity of the newly-formed microvascular network by incorporation of EPCs in the pre-existing endothlieum of tumor vessels. Tumor microvascular architecture phenotype heterogeneity, we nominated it as T-MAPH, has been considered to have a key negative effect on therapeutic efficacy of antiangiogenesis. Thus, it is of great importance and necessary to explore wether and how to EPCs contribute to T-MAPH. Malignant gliomas are highly vascularized tumors and the microvessel densities within the tumors are related to behaviors such as invasiveness and recurrence of gliomas, therefore, malignant gliomas have been considered as useful models for studies of tumor neovacularization and therapeutics.EPCs derived from either bone marrow, peripheral blood or umbilical cord blood are CD34, VEGFR2, or CD133 antigen-positive cells, which may home to the site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis. Little is known about how to EPCs contribute to T-MAPH.Chemokine receptor CXCR4 is involved in the regulation of tumor vascularization via EPCs. It has been reported that CXCR4 contribute to tumor vascularization by mobilizing, recruiting, inducing differentiation and tubular formation of EPCS. We found that CXCR4 promote T-MAPH by inducing the expression of VEGF and IL-8. However, exact role and detailed mechanism of CXCR4 in EPCs contributing to T-MAPH remain unclear.For a better understanding of the possible role and mechanism of CXCR4 in the genesis of malignant glioma microvascular architecture phenotype via EPCs, in this study, we investigated contribution of EPCs to T-MAPH. Firstly, we isolated and identified EPCs from human umbilical cord blood. Then, we explored EPCs contribution to T-MAPH. Finally, we studied the functional expression of CXCR4 by EPCs promoting T-MAPH and AMD3100 abolished the effects induced by activation of CXCR4. The main results and conclusions are as follows:1. Two types of EPCs isolated and identified from human umbilical cord blood were found to have potentials of renewal, differentiation and tubulogenesis. (1) By flow cytometry assay, the percentage of CD34+/CD133+ in mononuclear cells isolated from human umbilical cord blood decreased from1.06% to 0.26%. (2) By immunostaning assay, we identified that two types of EPCs, referred to as early EPCs and late outgrow EPCs. The early EPCs showed to be spindle-shaped with a tendency to form colonies which weakly potential, and were positive for CD14, CD34 and KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.The late outgrowth EPCs demonstrated colony forming cells with typical cobblestone morphology, showed robust proliferative potential and gave rise to secondary colonies. During the 8 and 15 days, cells continued to be positively expressed CD34/CD133 and CD34/KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.Those cells became positive for vWF and CD31 after 40 days of culture. (3)By three-diamensional in vitro Matrigel assays, we found that two types EPCs are capable of in vitro tubulogenesis.2. By transplantation of EPCs into the impaired bone-marrow glioma model we evaluated incorporation of EPCs into the vascular architecture of xenograft and role of EPCs in T-MAPH. Our data suggested that: (1) by immunostaning assay, we found that CFSE+EPCs expressed human CD31 preferentially home to tumor angiogenic site, not to liver and spleen organs, p<0.01.(2) by immunostaning assay, we found that EPCs contributed to tumor vascularization via two possible pathways, including the incorporation into the vascular architecture of U87 cell xenograft and chimeric vascular network formation or formation of functional blood vessels by singe cell and multicellular clusters localized in the vicinity of mouse-derived endothelia. With quantitative immunofluorescence and flow cytometry analysis, the percentage of EPCs-derived the endothelial population ranged from 11.3% to 18.6%. (3) By immunostaning labeled three-dimensional reconstruction, we found that chimeric vascular network was chaos with different structures, such as a sinusoid structure or a branching pattern. EPCs linked mouse ECs in branches to form thickened vessels of multicellular clusters. (4) EPCs enhanced MVD of glioma in impaired bone-marrow mouse and progressed tumor growth.3. We examined the effect of functional CXCR4 expressed by EPCs on T-MAPH. (1) Immunostaning assay was used to evaluate expression of CXCR4 and SDF-1 in EPCs. MTT, chemotaxis and three-diamensional in vitro Matrigel assays were used to explore proliferation, migration and in vitro tubulogenesis of EPCs, respectively. When activation or inactivation of SDF-1/CXCR4 axis with CXCR4 ligand SDF-1 or CXCR4 inhibitor AMD3100,expressed high levels of CXCR4 on EPCs were found to induce proliferation, migration and tubulogenesis of EPCs in response to the CXCR4 ligand SDF-1. However, AMD3100 abolished the effects induced by SDF-1. (2) In xenografts with EPCs, xenografts expressed higher levels of SDF-1, VEGF localized in or near the newly-forming microvessel and higher microvessel densities labeled by CD31.In summary, the present results suggest that (1) EPCs isolated and identified from human umbilical cord blood have characteristics of renewal, differentiation and tubulogenesis. Implantation of EPCs formed chimeric vascular network incorporated of EPCs into the vascular architecture and enhanced MVD of U87 cell xenograft of impaired bone-marrow mouse. (2) CXCR4 of EPCs induced proliferation, migration and tubulogenesis of EPCs and promoted T-MAPH via EPCs, which might be of significant importance in antiangiogenic therapy for cancer.