Abstract P3-05-21: An integrative approach to the discovery of triple-negative breast cancer markers derived from extracellular vesicles
Authors: Igyártó B , Sharma R , Garcia K , David-Dirgo V , Pathak K , Kroll M , Terraf P , Van Keuren-Jensen K , Pirrotte P 
Published: February 2018

Abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype accounting for nearly 20% of all breast cancer (BC) cases with disproportionally poor prognosis. Treatment options are limited by the lack of expression of common BC receptors targeted by FDA-approved small molecule inhibitors, resulting in an unmet need for efficient and cost effective treatments. Immunotherapies are emerging as a powerful alternative to standard chemo- and radiation therapy. Our objectives are to generate vaccines targeting TNBC and to identify circulating biomarkers capable of monitoring their therapeutic efficacy in a human transgenic mouse model (C57BL/6) compatible with EO771.LMB, a murine cell line of TNBC with a reported basal-like phenotype.
Our current study characterized the cancer-related proteomic and metabolic signatures derived from extracellular vesicles isolated from EO771.LMB. It has been shown that extracellular vesicles (EV) shed by tumors carry complex cargos comprised of proteins, metabolites and nucleic acids and may exhibit cancer-specific signatures with a potential as prognostic markers or predictors of therapy response. EVs released by TNBC tumors into circulation might provide non-invasive and highly actionable insight into the tumor biology of this aggressive cancer.
To further our understanding of cancer-specific EV proteomes, we used mass-spectrometry based proteomics and identified a total of 2265 unique proteins from EVs isolated from conditioned media from EO771.LMB. Our survey across the EV proteome, the expression proteome (5096) and phosphoproteome (2728) identified 6461 unique proteins overall. Within EV sub-fractions, microvesicles and apoptotic bodies were characterized by proteomic cargos different from exosomes. Over 90% of proteins identified in EVs shed by cultured EO771.LMB mapped with proteins curated in ExoCarta. Of interest, exploration of the EV proteome identified 47 GPI-anchored surface proteins. These candidate membrane proteins included Glypican-1 and CD109, previously reported as highly specific to cancer, as well as potential TNBC-specific markers. Our multipronged strategy for deep proteomic profiling of EO771.LMB enhanced identification by ˜25% compared to global whole cell proteomics alone. We also identified ˜3150 metabolites corresponding to lysophosphatidyl choline (35%), fatty-acids, -esters, -amides, -alcohols (30%), glycerophospholipids and sphingolipids (16%), sugar (1%), amino acids and biogenic amines (4%), organic acids and derivatives, cyclic alcohols, aromatic compounds and steroids (13%). Of these, 1450 metabolites were shared between cell line and EVs.
A proteogenomic approach expanded identification to onco-proteoforms and tumor-specific peptides not represented in canonical databases. Custom, cell-specific protein databases derived from whole transcriptome sequencing of EO771.LMB elucidated novel protein variants and mutations. Pan-cancer markers vs. TNBC-specific markers will be verified in other murine cell lines, across multiple cancer types and fibroblast controls. Optimal membrane markers for downstream immune-affinity purification from plasma will be proposed, and their ability to diagnose disease or monitor tumor load during treatment will be discussed.
Citation Format: Igyártó B, Sharma R, Garcia K, David-Dirgo V, Pathak K, Kroll M, Terraf P, Van Keuren-Jensen K, Pirrotte P. An integrative approach to the discovery of triple-negative breast cancer markers derived from extracellular vesicles [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-21.