A Secretory Form of Parkin-Independent Mitophagy Contributes to the Repertoire of Extracellular Vesicles Released Into the Tumor Interstitial Fluid in Vivo

Abstract

Extracellular vesicles (EVs) released from healthy and diseased tissue interstitial fluid (IF) into the lymphatic drainage are poorly understood aspects of EV biology. EVs circulating in the lymph constitute an information highway between tissues where the lymph is a portal of entry for tissue EVs to reach the blood. We characterized the interstitial fluid (IF) content of EVs using a GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labeling confirmed the IF EV tumor cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (Parkin-independent mitophagy required), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination triggers mitophagy away from apoptosis),VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for assessing mitochondrial function in health and disease, for cancer immunotherapy, and cellular immunology.