Abstract 2653: Usefulness of plasma exosomes to characterize hypoxic phenotype in primary prostate tumors
Authors: Panigrahi Gatikrushna , Su Yixin , Rios-Colon Leslimar , Peak Taylor , Sai Kiran , Praharaj Prakash , Lee Jingyun , Furdui Cristina , Hemal Ashok , Kumar Deepak , Deep Gagan 
Published: August 2019

Abstract
Hypoxia in primary prostate cancer (PCa) selects aggressive clones and causes treatment failure. However, there are several clinical challenges in measuring hypoxia in PCa. For example, MRI needs sophisticated instruments and highly trained personnel; needle electrodes could cause bleeding; and pimonidazole and immuno-histochemistry (IHC) for hypoxia biomarkers require surgical removal of tumor. Hence, novel non-invasive measures are needed to assess hypoxia. Exosomes are nano-vesicles (40-150 nm in size) secreted by cells for intercellular communication and are present in all biofluids loaded with unique cargo that could predict the cell of their origin and cellular physiologic state. Our primary goal here was to use exosomes to non-invasively assess hypoxia in PCa. We employed ultracentrifugation, mass spectrometry (MS), immunogold labeling, electron microscopy, PET, IHC, xenograft and immune-pull down methods. First, we isolated exosomes from the conditioned media of African American PCa MDA PCa 2b and E006AA-hT cells cultured under normoxia (21% O2) and hypoxia (1% O2) by ultracentrifugation. Next, through a novel method involving exosomal surface shaving and LC-MS/MS, we identified 13 unique proteins present only on the surface of exosomes secreted by PCa cells under hypoxia. Among the 13 unique proteins, Annexin A6 expression was highly correlated with 44 hypoxia signature genes in TCGA-PRAD. Next, Annexin A6 expression was confirmed on the surface of exosomes isolated from the blood of nude mice implanted with human PCa 22Rv1 xenografts. Hypoxia in xenografts was confirmed by F18MISO-PET imaging, and pimonidazole staining in xenograft tissues. Next, we pull-down Annexin A6+ exosomes using biotin-tagged human Annexin A6 antibody and streptavidin tagged agarose resin, and analyzed proteins loading by MS. Interestingly, Annexin A6+ exosomes showed a hypoxia signature as 35/172 loaded proteins were hypoxia and HIF1α regulated. We next isolated Annexin A6+ exosomes from the blood of African American and Caucasian PCa patients (n=5 each) with high grade disease (Gleason score &gt;7) and confirmed Annexin A6 expression on their surface. Importantly, for similar Gleason score disease, Annexin A6+ exosomes concentration was 2.6 times higher in African American patients, and could be associated with disease aggressiveness. Together, these results suggest that exosomes could be useful to non-invasively assess hypoxia in PCa.
Citation Format: Gatikrushna Panigrahi, Yixin Su, Leslimar Rios-Colon, Taylor Peak, Kiran Sai, Prakash Praharaj, Jingyun Lee, Cristina Furdui, Ashok Hemal, Deepak Kumar, Gagan Deep. Usefulness of plasma exosomes to characterize hypoxic phenotype in primary prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2653.