Abstract 3335: Identification of candidate proteins for the targeted imaging and therapy of glioma stem cells: Effect of antibody-conjugated magnetic nanoparticles and proteasomal inhibitor bortezomib on GBM-derived neurospheres and glioma stem cell marker CD133

Abstract

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common and lethal malignant primary brain tumor in adults. Glioma stem cells (GSCs) have properties of self renewal, pluripotency and high tumorigenicity. Magnetic iron oxide nanoparticles (IONPs) have emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as therapy by cancer cell targeted delivery of agents. In this study, we have studied differential activation of signaling pathways in purified CD133-positive GSCs in an effort to identify candidate proteins for the targeted imaging and therapy of GSCs by magnetic nanoparticles. We have also studied the therapeutic effect of IONPs conjugated with an EGFRvIII antibody against GSCs and the regulation of the GSC marker, CD133, by the proteasomal inhibitor bortezomib. Methods: Glioblastoma tumor specimens were used to isolate neurospheres. Neurospheres of an EGFRvIII-negative and EGFRvIII-positive GBM tumors were further enriched for CD133-positive cells using Indirect CD133 MicroBeads kit or FACS analysis to ∼90% purity. Magnetic nanoparticles were conjugated with the EGFRvIII antibody using Ocean NanoTech kit. Neurospheres were treated with 200 nM bortezomib. Results: Using Western blot analysis of CD133-positive GSCs from different patients, we identified nestin, wt EGFR, EGFRvIII, PDGFRß, carbonic anhydrase IX (CAIX) podoplanin as well as CD133 marker as potential candidates for the targeted imaging and therapy of GSCs. Hypoxia (1% O2) greatly enhanced expression of CD133 antigen in EGFRvIII positive neurospheres in a HIF-2α-independent manner. Treatment with proteasomal inhibitor bortezomib decreased expression of CD133. EGFRvIII antibody conjugated to IONPs (EGFRvIIIAb-IONPs) promoted apoptosis of not only EGFRvIII-positive stem cells, but also CD133-negative cells. Conclusion: Candidate proteins found in CD133-positive GSCs include nestin, wt EGFR, EGFRvIII, PDGFRβ, CAIX, and podoplanin. Hypoxia activated expression of CD133 and CAIX, a well-established hypoxia marker. The proteasomal inhibitor bortezomib downregulated hypoxia-induced expression of CD133 and CAIX. Antibodies against the candidate proteins, conjugated to magnetic nanoparticles, may form the basis of targeted imaging and therapy of GSCs. IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant can promote apoptosis in neurospheres, CD133-positive GSCs, and CD133-negative neurospheres. In conclusion, a combination therapy in which GSCs are targeted a) with antibody-conjugated IONPs b) with proteasomal inhibitors to downregulate GSCs markers may have a synergistic effect against GSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3335.