Characterization of Large EVs From Breast Cancer Cells

Abstract

Cancer is one of the leading causes of death worldwide. Breast cancer alone kills over 43,000 women each year2. While methods of diagnostics and identification of this disease have improved greatly, the realm of treatment leaves much to be desired. Extracellular vesicles (EVs) are small membrane-bound particles secreted from all eukaryotic cells3. They have recently come to focus as a new means of cancer diagnosis and treatment as they are heavily involved in intercellular communication4. It is our hypothesis that larger extracellular vesicles contain genetic material, such as DNA and proteins, that can act as biomarkers to diagnose and determine means of breast cancer treatment as well as act as a means of therapy themselves.
In this experiment, we took EVs from breast cancer cells spun in the centrifuge at 2,000xg (2K), 10,000xg (10K), and 100,000xg (100K). We then analyzed their contents through western blotting. Additionally, a qPCR was performed to observe the presence of GAPDH DNA. Mass spectrometry was also used to further break down and identify the proteins as well as cross-reference them with other EVs. The results of the qPCR showed that DNA was consistently present within the 2K EVs. Our western blot confirmed the presence of CDK6, AKT, and Actin. This information opens up the possibility of diagnosing breast cancer based on an analysis of the composition of EVs as they mirror the composition of their mother cells. Additionally, the presence of certain proteins and kinases suggests that EVs play an important role in influencing recipient cells and creating a pro-tumor environment. Our cross-referenced mass spectrometry showed 5 new clusters of unique proteins that are all involved in the transcription and translation of the EVs’ DNA and RNA. Future methods of treatment can tailor therapies to patients based on the composition of the EVs from their particular cancer, leading to personalized medicine.