Abstract 2208: Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer

Abstract

Abstract Objective: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Method: A recently described isolation method, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). The authors showed comparable results for their method (abbreviated HSP-EV here) and UC-EV, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium. Subsequently we applied this technique on secretomes of fresh human colorectal cancer (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue. Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs. Conclusions: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting. Citation Format: Meike De Wit, Jaco Knol, Inge de Reus, Tim Tim Schelfhorst, Logan Bishop-Currey, Nicole van Grieken, Sander Piersma, Thang V. Pham, Remond J. Fijneman, Gerrit A. Meijer, Henk Verheul, Connie R. Jimenez. Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2017-2208