IF EVs: a new molecular measurement of mitochondrial health

Abstract

Tumor interstitial fluid (IF) is the wet matrix bathing all tumor cells. Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space. EVs released from healthy and diseased tissue IF into the lymphatic drainage are poorly understood. PINK1 is a mitochondrial membrane sensor to initiate mitophagy, the necessary process of removing damaged mitochondria. Healthy mitochondria break down PINK1 into fragments by protease cleavage of the full length (FL) PINK1. In contrast, stressed/damaged mitochondria fail to cleave PINK1, thereby leaving FL-PINK1 protein to trigger mitophagy. We hypothesize PINK1 cleavage status is a novel measure of a cell’s mitochondrial health. We discovered that PINK1, during high mitophagy demand, exports damaged mitochondrial pieces within EVs. We found that tumor IF EVs contain different ratios of FL-PINK1 to c-PINK1. We compared ratios of the PINK1 levels in tumor IF vs normal IF and found non-tumor IF contained only c-PINK1 whereas tumor IF contained a greater ratio of FL-PINK1 indicative of oxidative stress within the tumor tissue. During in vitro assays using mitophagy-inducing drug CCCP on 4T1 breast cancer cells we found greater levels of FL-PINK1 EV export. Thus IF EV PINK1 fragmentation ratio is a novel sensitive indicator of cellular mitochondrial health