Abstract 5548: Targeted nanoparticle drug delivery to radiation inducible GRP78 in tumor vasculature
Authors: Ralph J. Passarella, Daniel Spratt, Alice Van Der Ende, John G. Phillips, Hongmei Wu, Dennis E. Hallahan, Eva Harth, Roberto Diaz
Journal: Cancer Research (2010)

Abstract Purpose/Objectives: GRP78 is a major endoplasmic reticulum chaperone that suppresses stress-induced apoptosis. High GRP78 levels have been shown to correlate with a worse pathologic grade and poor patient survival in various cancer types. GRP78 is also known to confer chemoresistance to tumors and tumor-associated endothelial cells. The goal of this study is to elucidate the mechanism responsible for radiation-mediated neoantigen expression in tumor microvasculature. GIRLRG, a peptide already shown to bind specifically to radiation (XRT) treated, responding tumors, was used in tumor protein extraction to identify its possible cellular targets. GRP78 was identified from the protein extraction as a possible receptor for GIRLRG. We tested the hypothesis that GRP78 is upregulated in response to radiation and can be used as a radiation-inducible drug delivery target. Methods: Proteins from untreated and XRT treated GL261 gliomas were extracted and incubated with agarose beads coated with GIRLRG and then analyzed by gel electrophoresis and mass spectrometry. A fluorescently labeled antibody to GRP78 was used to assess location of GRP78 expression post-XRT in vitro. WB analysis, immunohistochemistry (IHC), and in vivo imaging of GRP78 expression levels was performed in XRT treated and untreated GL261 and MDA-MB-231 breast tumors 48 hours post-XRT. In vivo blocking experiments were performed to assess GRP78 as the radiation inducible receptor for the GIRLRG peptide. Finally, GIRLRG was conjugated to a slow release nanoparticle drug delivery system encapsulating paclitaxel to target GRP78 in vivo. Results: Mass spectrometry analysis revealed that GIRLRG specifically extracted a 78k-dalton protein, GRP78 from XRT treated tumor samples. Similarly, WB, IHC, and in vivo imaging analysis showed an upregulation of GRP78 expression in XRT treated GL261 and MDA-MB-231 samples. In vitro imaging showed GRP78 to be upregulated at the cell surface in response to XRT. A blocking antibody to GRP78 was able to significantly abrogate GIRLRG binding post-XRT (p &amp;lt; 0.05). The GIRLRG-nanoparticle drug delivery system produced a significant tumor growth delay relative to all control groups (p &amp;lt; 0.05). Conclusions: GRP78 has been identified as GIRLRG's binding partner. We found that GRP78 is induced by XRT. This finding correlates with GRP78's known stress induced anti-apoptotic function, as XRT is a known cellular stressor. Furthermore, we utilized upregulation of GRP78 post-XRT by combining chemotherapeutic agents to a peptide ligand, GIRLRG, that recognizes the XRT-induced receptor, providing a targeting system that increases chemotherapeutic efficacy for irradiated tumors in vivo. In the future we plan to investigate if blocking GRP78 could potentially radiosensitize tumors by promoting an apoptotic state. Overall the regulation of GRP78 has the potential to provide exciting new targets for anti-cancer strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5548.