ITGA2 as a potential nanotherapeutic target for glioblastoma

Abstract

High grade gliomas, including glioblastoma (GBM), are the most common and deadly brain cancers in adults. Here, we performed a quantitative and unbiased screening of 70 cancer-related antigens using comparative flow cytometry and, for the first time, identified integrin alpha-2 (ITGA2) as a novel molecular target for GBM. In comparison to epidermal growth factor receptor (EGFR), a well-established GBM target, ITGA2 is significantly more expressed on human GBM cells and significantly less expressed on normal human glial cells. We also found that ITGA2 antibody blockade significantly impedes GBM cell migration but not GBM cell proliferation. To investigate the utility of ITGA2 as a therapeutic target in GBM, we designed and engineered an ITGA2 antibody-directed liposome that can selectively deliver doxorubicin, a standard-of-care chemotherapeutic agent, to GBM cells. This novel approach significantly improved antitumor efficacy. We also demonstrated that these ITGA2 antibody-directed liposomes can effectively breach the blood-brain tumor barrier (BBTB) in vitro via GBM-induced angiogenesis effects. These findings support further research into the use of ITGA2 as a novel nanotherapeutic target for GBM.

Figure 1

Identification of ITGA2 as a molecular target for GBM. (a) Surface protein expression profile of 70 cancer-related antigens in three human GBM cell lines and normal SVG-P12 cells. (b) Venn diagram indicating ITGA2 co-overexpressed by three GBM cell lines in reference to SVG-P12 cells. (c) Cell membrane expression levels of ITGA2 and EGFR in GBM and SVG-P12 cells using flow cytometry analysis, showing increased tumor-specificity of ITGA2 expression on GBM cells as compared to both control cells and also to the current GBM marker, EGFR. (d) Representative microscopic images of immunofluorescent staining of ITGA2 in GBM and SVG-P12 cells. Scale bars represent 50 µm. (e) ITGA2 mRNA expression levels in human GBM tumor tissues and normal brain tissues. **P < 0.001. (f) Correlation between overall survival and ITGA2 mRNA expression levels in GBM patients as determined via Kaplan-Meier analysis (Log-rank test). Data of (e) and (f) were obtained from the R2: Genomics Analysis and Visualization Platform database.

Figure 2

Therapeutic functions of ITGA2 blockade. (a) The effect of ITGA2 antibody on GBM cell proliferation. Representative microscope images (b) and quantitative analyses (c) of migrated GBM cells under ITGA2 antibody blockade treatment in a transwell migration assay. Significant reductions in GBM migration were observed with ITGA2 blockade. IgG was used as a control. ***P < 0.001, NS not significant.

Figure 3

Development of an ITGA2 antibody-directed nanomedicine. (a) Schematic illustration of ITGA2 antibody-conjugated, doxorubicin-encapsulating liposomes (ITGA2-Dox-LP). (b) The surface density of ITGA2 antibody or IgG-conjugated on ITGA2-Dox-LP or IgG-Dox-LP quantified using a microbead assay. Hydrodynamic diameter (c) and zeta-potential (d) of ITGA2-Dox-LP and IgG-Dox-LP quantified using dynamic light scattering measurement. (e) The encapsulating efficiency of doxorubicin in ITGA2-Dox-LP and IgG-Dox-LP. (f) Representative fluorescent images of GBM and SVG-P12 cells uptaking fluorescent-labeled ITGA2-FITC-LP and IgG-FITC-LP. The scale bars represent 50 µm. (g) Quantitative GBM cell uptake of ITGA2-FITC-LP in comparison to non-specific IgG-FITC-LP. NS, not significant; **P < 0.01; ***P < 0.001.

Figure 4

GBM-targeted therapy of ITGA2-Dox-LP. (a) Cytotoxicity of ITGA2-LP (vehicle), IgG-Dox-LP, and ITGA2-Dox-LP in A172, U87, and SVG-P12 (normal) cells. (b) Cell migration trajectories of A172 (upper panel) and U87 (lower panel) cells under the treatment of ITGA2-LP blockade. Significant reductions in cell migration were observed following ITGA2 blockade. IgG-LP was used as a control. (c) Quantified cell migration speed of A172 and U87 cells with or without ITGA2-LP blockade. (d) ITGA2-FITC-LPs transmitted across an in vitro BBTB (A172 and U87 cells) were quantified using flow cytometry analysis. Normal SVG-P12 cells were used as a control.