Exosomal Proteins as Potential Markers for Breast Cancer Disparity
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PURPOSE: Breast cancer is the most common non-cutaneous malignancy and the second most lethal form of cancer among women in the United States. However, the mortality differs among different races, with Black women having significantly higher mortality rates than White women, even when factors like socioeconomic status are controlled. One explanation for this is the fact that Black women are diagnosed at higher rates with a particularly aggressive subtype known as triple-negative breast cancer (TNBC), which lacks the receptors that chemotherapy drugs classically target. It is important to recognize the various biological factors that contribute towards this health disparity. The overarching goal of this research is to identify differentially expressed exosomal proteins as potential makers to understand this disparity. Proteins under investigation include anti-apoptotic proteins and transcription factors. The specific goal for this project is to optimize protocol for exosome isolation and characterization. METHODS: MDA-MB-231 and MDA-MB-468 metastatic TNBC cell lines were cultured in media supplemented with exosome-depleted serum. After 24h, the culture media was centrifuged to remove cell debris and processed for exosome isolation using ExoQuick-TC kit (System Biosciences). The protocol involves precipitation of exosomes using the ExoQuick reagent. The exosomal pellet was resuspended in PBS and used for Western blot to analyze proteins and Nanoparticle Tracking Analysis (NTA) for determining exosomes size and concentration. The NTA calculates the size of the particles based on their movement. For Western blotting the antibodies used were against standard exosomal markers. RESULTS: Western blot analysis for exosomes showed that all samples were positive for standard exosomal protein markers such as Flotillin, CD54 and EpCAM and negative for non-exosomal marker GM130. The average size of the exosomes isolated from the MDA-MB-231 and MDA-MB-468 were 147.8 nm, and 146.6 nm respectively as determined by NTA. The concentration of exosomes from the two cells lines was determined to be 3.01e+8 ± 7.96e+6 particles/mL for MDA231 and 1.61e+8 ± 8.35e+6 particles/mL for MDA468. CONCLUSIONS: This research project streamlined a method to isolate and characterize exosomes from TNBC cell lines using the ExoQuick kit. The next step would be to isolate exosomes from a panel of breast cancer cell-lines as well from serum or plasma derived from White and Black breast cancer patients. Exosomes from these racial groups will be probed to investigate the differential expression of any potential biomarker. Such markers can be developed into novel diagnostic tools or as potential therapeutic targets, which will help clarify and reduce the current mortality gap between the two populations.