Cancer Cells Under Oxidative Stress Secrete Extracellular Vesicles Containing Damaged Mitochondrial Components That Can Alter the Death Rate and Mitochondrial Function of Recipient Cells

Abstract

Mitophagy is a critical survival mechanism in which damaged mitochondria are separated into mitophagosomes for degradation via the lysosomes and proteasomes. Extracellular vesicles (EVs) are membrane-bound structures that are secreted into the extracellular space by cells and function as transporters of proteins and nucleic acids between cells. In cancer, EVs have been shown to carry bioactive molecules that impact the biology and function of cancer cells. Under stress, EVs are released by cells, which can bring the damaged mitochondria and signals to other cells. However, no research has been conducted about how EVs from oxidatively stressed cancer cells affect recipient cancer cells. This study addresses this gap in knowledge by examining how EVs from 4T1 breast cancer cells treated with CCCP impact mitochondrial health, ATP production, and caspase-3 activity. We hypothesized that the EVs could transfer damaged mitochondria from the stressed cells to the recipient cells. EVs were isolated from both the CCCP-treated and untreated cells by 2K and 2K+ spins and further purified with IZON columns. The EVs from both treated and untreated cells were used to treat the 4T1 cells, and the MitoTox and Caspase-3/7 assays were performed at the time points 1H, 3H, 6H, ON, and 48H. Compared to the control group, all EV-treated groups showed increased mitochondrial membrane depolarization overnight. ATP production decreased slightly across all EV-treated groups, which suggests less mitochondrial energy output. Caspase-3 activity dropped significantly following overnight treatment. These results support that EVs from oxidatively stressed cancer cells can encourage mitochondrial dysfunction in surrounding cells while also modulating cell death. Understanding the effect that these EVs have on cancer cells can offer insight into how cancer cells adapt under therapeutic pressure.