Macrophages Promote Glioma Stem Cells To Induce Angiogenesis

Malignant glioma is one of the most common primary tumors in the centralnervous system (CNS), and is characterized by highly vascularization. Angiogenesisis crucial for glioma growth and invasion, which may lead to its resistance toradio-/chemo-therapy and high mortality rate.Cancer stem cells (CSCs), because of their unique biological characteristics andimportant role in tumors, have increasingly been attracting more attention. Previously,Prof. Bian and his colleagues in our department performed a systemic study on gliomastem cells (GSCs) and found that CD133could be a unique biomarker for GSCs.Furthermore, they demonstrated that CD133+GSCs possessed strong capabilities ofself-renewal and differentiation as well as invasion.It has been reported that tumor microenvironment can strongly influence theinitiation and progression of tumor. For example, a huge amount of infiltratedinflammatory cells are found within tumor tissue during the early stage of tumordevelopment. Among them, macrophages are the dominant one and participate inmany aspects of tumor progression. However, how macrophages cast their impacts onthe GSCs-promoted angiogenesis to be further explored. In vitro culture system hasbeen widely applied by many researchers to study glioma angiogenesis, which is,however, unable for us to directly observe how macrophages affect GSCs-promotedangiogenesis in vivo.Zebrafish, as an ideal model organism, has been widely applied for tumorresearch. It has been known that macrophages/monocytes are the earliest blood cellsthat appear in the zebrafish embryos. Besides, the establishment of fli: greenfluorescent protein (GFP) transgenic zebrafish that stably expresses GFP in all bloodvessels enables us to observe angiogenesis in the live zebrafish embryos. Therefore, taking advantage of fli:GFP transgenic zebrafish embryos as a glioma model is usefulfor our understanding of the effect of macrophages on the angiogenesis in the earlydevelopment of tumors.We first established a stable red fluorescent protein (RFP) expressing humanprimary malignant glioma cells091214, and isolated CD133+GSCs from them. Then,both CD133+and CD133-glioma cells expressing RFP were successfully transplantedinto the yolk sac region of fli:GFP transgenic zebrafish embryos. I found that theangiogenesis could be significantly induced by GSCs, and applied neutral red stainingto directly visualize macrophages in the live zebrafish embryos. I observed that moremacrophages were accumulated in the region of injection of GSCs than the region ofinjection of CD133-glioma cells. Finally, whole mount in situ hybridization (WISH)of zebrafish embryos was employed to detect the expression of L-plastin, which hasbeen characterized as a molecular marker for macrophages in zebrafish. Using WISH,we could locate the macrophages in the zebrafish at the mRNA level, and accuratelystudy the impacts of macrophages on the angiogenesis promoted by GSCs.In summary, we established an in vivo zebrafish model for GSCs-inducedangiogenesis in zebrafish and found that GSCs-induced angiogenesis had a correlationwith the distribution of macrophages, which might facilitate the angiogenesis inducedby GSCs. Our current study will provide experimental evidence for design a noveltherapy to target macrophages for interrupting GSCs-induced angiogenesis to treatglioma.