Downregulation Of CXCR4 Expression Of GSCs Inhibits Invasion And Angiogenic Potential Of Glioma

With the rapid progress in the research field of stem cells and the development in related methodology, cancer stem cells (CSCs) hypothesis has been confirmed by the isolation of CSCs from acute myeloid leukemia (AML) and various malignancies of the breast, brain, colon, liver and prostate. Now this conception has been widely accepted by many researchers. Recently, a small population of stem cell-like precursors or glioma stem cells (GSCs) had been successfully isolated from glioma cells. These cancer stem cells could form tumorspheres, possess the capacity of self-renewal, generate daughter cells of different phenotypes from one mother cell, and differentiate into the phenotypically diverse populations of cells similar to those present in the initial GBM. This suggests that glioma cells also contain CSCs. Furthermore, these cells display strong capability on cancer resistance to chemo- and/or radio-therapy. It has been recently suggested that resistance to therapies might not be a feature of all the cells of glioma but rather of GSCs. These cells might be the root of tumor recurrence. In order to eliminate these cells, the drugs targeting CSCs have been under investigation.CXCR4 has been known to have broad activities in different kinds of tumors. It could be activated by SDF-1. Extensive studies on CXCR4 indicate that CXCR4 is a prognostic marker in various types of cancer, such as acute myelogenous leukemia, breast carcinoma, prostate cancer, glioma and colon cancer. Anti-CXCR4 may have anti-tumor activity such as inhibition of migration, invasion, and angiogenic potential in patients with various malignancies and also in some tumors it could inhibit proliferation and resistance to apoptosis. In glioma, CXCR4 is an important molecule mainly involved in invasion, angiogenic potential. It is considered as an effective target for the treatment of malignant gliomas. Although CXCR4 is an important molecule in glioma, the detailed pathophysiologic roles of CXCR4 in GSCs have not been elucidated. But this has a great relationship with the treatment effectiveness to glioma by targerting CXCR4. In this article we investigated whether downregulation of CXCR4 expression could affect GSCs proliferation.In this study, we detected expression of CXCR4 by CD133+ cells in U87 cell line and their xenografts grown in the brain of nude mice by indirect immunofluorescence under laser scanning microscopy. Tumorspheres were isolated from control U87 cells and CXCR4-siRNA U87 cells. We then investigated the expression of CD133, nestin and CXCR4 in tumorspheres by laser scanning microscopy. Effects of CXCR4 downregulation on invasion and VEGF production of GSCs were detected by transwell assay and ELISA respectively. Besides, we studied the density of micro-vessels and production of VEGF in the xenografts grown in the brain of nude mice formed by GSCs. The main results and conclusions are as follows:1. CXCR4 was expressed by glioma stem cellsOur results demonstrate that some tumor cells were stained positive for CD133, a cell surface marker of GSCs ,and CXCR4.2. GSCs were separately obtained from CXCR4-siRNA U87 cells and control U87 cells(a) We down-regulated U87 cells expression of CXCR4 by RNA interference, and generated stable transformed tumor cells. Three siRNAs were developed specific for the CXCR4 mRNA. We detected the effectiveness of interference by western blot. The siRNA-1 was more efficient in reduction of CXCR4 expression than others. CXCR4 expression in siRNA1 relative to the control is 36%.(b) Tumorspheres were isolated from control U87 cells and CXCR4-siRNA U87 cells. We found that both of them could express CD133 and nestin. tumorspheres which were obtained from CXCR4-siRNA U87 cells have lower expression of CXCR4 than tumorspheres cultured from control U87 cells.3. Suppression of CXCR4 expression inhibited invasion of GSCsKnocking down CXCR4 expression in GSCs drastically inhibited cell migration in response to CXCL12(siRNA:60±7.3 /HPF, control:166±11.2 / HPF, P<0.05). We further ran an invasion assay by coating the transwell with matrigel. We found that the number of cell migrated through the membranes is lower in CXCR4-siRNA U87 cells(siRNA: 17±5.8 /HPF, control: 71±8.7 /HPF, P<0.05). 4. Downregulation of CXCR4 expression inhibited angiogenic potential of GSCsWe found that prevented GSCs expression of CXCR4 by RNA interference can inhibits secretion of VEGF in GSCs(siRNA: 24 h 690±24 pg/ml, 48 h 1504±53 pg/ml; control: 24 h 850±25 pg/ml, 48 h 2001±150 pg/ml; P<0.05). We futher convinced it in the in vivo test. We found that xenografts formed by CXCR4-siRNA U87 cells have lower density of microvascular and secretion of VEGF.In conclusion, GSCs could express CXCR4 and downregulation of CXCR4 expression attenuates migration, invasion and pro-angiogenic capacity of GSCs, which suggests that targeting CXCR4 expressed on GSCs may be of benefit in glioma patients.