EXOSOMAL ISOLATION AND CHARACTERIZATION TO IMPROVE TRIPLE NEGATIVE BREAST CANCER OUTCOMES.

Purpose: Triple negative breast cancer (TNBC) is an aggressive form of breast cancer associated with poor clinical outcomes and lack of targetable molecular signatures. It was previously reported that serum exosomal-annexin A2 (Exo-ANXA2) is higher in TNBC patients. Additionally, higher levels were reported in African-Americans compared to Caucasian-Americans. Therefore, we propose to examine novel exosome isolation and characterization techniques to measure exo-AnxA2 levels in serum samples of TNBC patients that would be easily adaptable in a clinical setting to improve treatment strategies in African-American TNBC patients. Methods: A novel magnet-activated cell sorting (MACS) technology which employs microbeads (diameter=50nm) to isolate exosomes will be used to determine the exo-AnxA2 levels in serum samples. Exosomes isolated will be subsequently characterized through bead-associated exosomal flow cytometry, exo-ELISA, western blot, RNA isolation, and proteomics to analyze exo-AnxA2 levels and other cargo proteins. Results: Protein estimation of MACS-isolated exosomes revealed an average of 1.75 µg/µl of intact exosomes from 1.0 ml serum sample. These results suggest that approximately 200µl of patient serum sample would yield approximately 2.0 µg of exosomes for exosomal flow cytometry. Technical challenges include creating a clump-free exosome isolate for cytometry analysis without and adapting a non-specialized flow cytometer for exosomal analysis. Conclusion: We expect the results to identify significant co-expressive molecules with exo-ANXA2 that could improve detection, prognosis, and treatment choice of aggressive forms of TNBC. Funding: Award Number R01CA220273 (JKV).