Ovarian Cancer Stem-like Cells Differentiate Into Endothelial Cells And Participate In Tumor Angiogenesis Through Autocrine CCL5 Signaling

BackgroundCancer stem cells(CSCs)are well known for their self-regeneration and tumorigenesis potential.In addition,the multi-differentiation potential of CSCs has become a popular issue and continues to attract increased research attention.Recent studies demonstrated that,in glioblastoma and breast cancer,CSCs are able to differentiate into functional endothelial cells(ECs)and participate in tumor angiogenesis.Many studies have reported that angiogenesis is considered a hallmark of cancer because it is indispensable to fueling tumor and leads to the malignancy development,invasion,and metastasis.Therefore,exploring the underlying mechanism of CSCs-derived angiogenesis would benefit to the improvement of tumor occurrence and development theory.Our previous studies demonstrated that ovarian cancer stem like cells(CSLCs)abundantly self-produce the chemokine CCL5 and its receptors.It has been reported that CCL5 is a pro-malignant factor relevant to tumor angiogenesis,and play a critical role in tumor growth,metastasis,and progression.ObjectiveAngiogenesis play a critical role in development,invasion,and metastasis of ovarian cancer.However,traditional anti-angiogenesis has a limited efficacy,as well has many side effects.In this study,we found that ovarian CSLCs differentiate into ECs both morphologically and functionally.Furthermore,we studied function and mechanism of CCL5 in the differentiation of CSLCs into ECs.We expected that our data can help improve the tumor angiogenesis theory and provide a novel anti-angiogenesis strategy for clinical ovarian cancer therapy.Methods1.On the basis of previous studies on ovarian cancer CSLCs,the vitro tube formation experiment and immunological techniques were used to study whether the ovarian CSLCs and non-CSLCs(NCSLCs)possess the function of endothelial differentiation and angiogenesis.In addition,immunofluorescence analysis performed to detect source of vascular ECs of nude mice xenografts tissue and prove ovarian CSLCs are capable of ECs differentiation and participate in tumor angiogenesis.2.Several methods,such as anti-CCL5 nuetralizing antibodies,CCL5-shRNA,recombinant human-CCL5 and blocking of CCL5 specific receptors,performed to study whether chemokine CCL5 mediate ovarian CSLCs differentiate into ECs.Nude mice xenograft experiment and immunohistochemistry technique are used to prove whether ECs differentiation of CSLCs depend on CCL5 signal.3.Accordance with results of Western blot and immunofluorescence analysis to find downstream pathways of CCL5 signal,and prove whether CCL5 through NF-?B?STAT3 pathways to mediate ovarian CSLCs differentiate into ECs.Tube formation experiment and immunological techniques were performed to demonstrate mechanism of CCL5 modulate ECs differentiation of ovarian CSLCs.Results1.Matrigel angiogenesis model showed that the ovarian cancer CSLCs can form the microtubule network,which was similar to microvascular network formed by human umbilical vein endothelial cells(HUVECs),but NCSLCs did not form the microtubule network under the same conditions.Immunofluorescence analyses the expression of vascular endothelial cell specific markers of cells derived from tube formation assay and nude mice xenograft tissue,showed that ovarian CSLCs can differentiate into ECs and participate in tumor angiogenesis,while NCSLCs do not have this capacity.2.The results of antibody block CCL5 function show that anti-CCL5 antibody treatment could partially inhibit microtube formation in a dose-dependent manner.In addition,the result of CCL5-silenced CSLCs is similar to the results obtained with anti-CCL5 antibody treatment.Moreover,recombinant human CCL5 significantly promoted the differentiation of CSLCs into ECs and tube formation.The blocking of CCR1,CCR3 or CCR5 partially decreased the tube network formed from CSLCs compared with the results obtained with the isotype antibody control.Immunohistochemistry staining performed to detect CD31 expression of nude mice xenograft,the results showed that CCL5 silencing in CSLCs lead to decreasing of human derived CD31,and have no influence on mouse derived CD31.3.Western blots and immunofluorescence analysis showed that CCL5 has capacity of activating the NF-?B and STAT3 signaling pathways in ovarian CSLCs.Furthermore,blocking of the NF-?B or STAT3 signaling pathways can inhibit ECs differentiation and tube formation of ovarian CSLCs.Conclusion1.Ovarian cancer stem-like cells differentiate into endothelial cells with endothelial morphology and function.2.Ovarian cancer stem-like cells induced differentiation into endothelial cells through self-production of CCL5.3.CCL5 activates the NF-?B or STAT3 pathways to control endothelial differentiation of ovarian cancer stem-like cells.