A secretory form of Parkin-independent mitophagy contributes to the repertoire of extracellular vesicles released into the tumour interstitial fluid in vivo

Abstract

We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labelling confirmed the IF EV tumour 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 (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination state 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 mitochondrial function and cellular immunology.

Keywords: autophagosome; autophagy; breast cancer; extracellular vesicle; mitochondria; mitophagy.

FIGURE 1

Methodology of collecting and characterizing tumour interstitial fluid EVs. (a) Tumour cells shed EVs into the interstitial fluid (IF) of the tumour microenvironment. The IF becomes lymphatic drainage that carries the EVs to the sentinel lymph node (SLN). (b) IF resident EVs (green particles) are harvested from fresh, solid tumour tissue by low speed centrifugation. (c) Molecular characterization of EVs includes isolation and purification by differential ultracentrifugation, density gradient ultracentrifugation, and immunoprecipitation with downstream analysis by western blot, mass spectrometry, electron microscopy, and fluorescent imaging of cellular molecular tags that become EV cargo

FIGURE 2

All the major categories of IF and in vitro EVs contain the tumour cell specific eGFP marker, verifying EV tumour cell origin in vivo. (a) eGFP‐4T1 mammary carcinoma cells cultured to confluency release GFP‐expressing EVs into all the common EV subtypes (2K, 10K, and 100K). The GFP‐expressing EVs contain specific EV markers on their surface with the highest expression levels in the 100K fraction. (b) 10⁶ GFP‐4T1 cells/ml were injected into the mammary fat pad of the syngeneic BALB/c mouse model. After 2 weeks, tumours were excised and placed in an EconoSpin column lined with glass wool and gently centrifuged at 8160 × g for 10 min to isolate bulk EVs from the tumour IF. (c) Bulk EVs isolated from the tissue were fractionated by ultracentrifugation at 2000 × g (2K) for 45 min and 10,000 × g (10K) for 45 min. Smaller diameter EVs (100K*) were isolated via nanoparticle capture technology from the resulting 10K fraction supernatant. Expression of GFP and common EV markers were Western Blotted

FIGURE 3

High number and diversity of in situ IF EV cargo proteins compared to in vitro culture. (a) Culture‐derived EVs were analysed by subpopulation via mass spectrometry. The proteins returned were analysed via InteractiVenn software (Heberle et al., 2015). A majority of culture‐derived EVs’ proteins were shared among all subpopulations. (b) Analysis of IF proteins returned from the T1 tumour show that the 10K and 100K subpopulations share a greater number of proteins than between any other set of subpopulations. The 100K EVs contained the greatest proportion and number of unique proteins. (c) Comparison of culture EVs and T1 derived EVs indicate that tumour‐derived EVs of the 10K and 100K populations contain greatest number of distinct, different proteins by a factor of five. (d) Total number of in vitro versus in vivo EV proteins derived via MS demonstrate IF EVs rich repertoire of proteomic contents

FIGURE 4

Protein pathways analysis reveal glycolytic, neurodegenerative, and pro‐angiogenic pathways are the most similar comparing in vitro to in vivo. (a) Fewer proteins are shared between the culture (green) and tumour‐derived (orange) EVs for the 2K population; however, the shared pathways enriched are similar to the 10K and 100K populations. (b) 10K tumour‐derived EVs contain a majority of unique peptides compared to their culture counterparts. However, the 10K and 100K EVs are both enriched for peptides associated with T cell activation and Ras pathways. (c) Similar to the 10K comparison, the 100K EVs from the tumour have a majority of unique peptides. Nevertheless, all shared pathways enriched between the EVs are indicative of neurodegenerative processes, as seen by the Parkinson's and Huntington's disease pathways, and all subpopulations are enriched for pro‐angiogenic factors as seen by the fibroblast growth factor signalling pathway and angiotensin‐II stimulated signalling

FIGURE 5

IF EVs and in vitro EVs exhibit characteristics of autophagosomes and mitophagy by TEM and Western Blotting. (a) Western Blot analysis of culture‐derived EVs reveal secreted autophagy and mitophagy related structures within all EVs. The presence of LC3‐I/II and p62 indicate that these EVs are autophagosomes. Moreover, PINK‐1 is the central initiator of the mitophagy process additionally found in the EVs. (b) Tumour‐derived EVs confirm the results of the culture EVs and further indicate that the EVs released are involved in the secretory autophagy and mitophagy process. (c) Transmission Electron Microscopy of culture 4T1 EV subpopulations visualizes classical autophagosome characteristics such as double‐membraned vesicles with internal contents for each subpopulation. By population, the 2K EVs display a heterogenous population with internal vesicle structures, and the 100K EVs are homogenous in size and structure

FIGURE 6

EV subpopulation localization of key autophagy, mitophagy, and checkpoint markers. (a) Autophagosome specific p62, LC3‐I/II, and checkpoint inhibitor PD‐L1 are localized to the 100K CD81‐enriched EVs. (b) Density gradient fractions reveal co‐localization of PINK1 and CD81. (c) Immunoprecipitation of CD81+ EVs after density gradient ultracentrifugation were probed for Western Blot. (d) Anti‐CD81 immunoprecipitated EVs were enriched in PINK1. (e) Five‐day‐old eGFP cultures were collected and differentially centrifugated to remove the 2K and 10K populations. The resulting supernatant was mixed with ExoMax solution to enrich for EVs and exclude free protein from the sample. Furthermore, to isolate the separate EV populations within the ExoMax EV concentrate, the sample was placed onto an IZON 35 nm size exclusion column. The resulting fractions were pooled into sets of 5, Nanotrapped, and analysed by Western blot. (f) The proteomic content of each set of pooled fractions found two distinct groups of EV populations (fractions 6–10 and fractions 16–20). Western blot for PINK1 revealed that this marker was preferentially within the 16–20 fraction group enriched in 35–50 nm EVs and tracked with CD9 and eGFP. (g) TEM images of eGFP‐4T1 100K EVs contained two distinct populations of larger EVs (> 100 nm, red) and smaller EVs (< 100 nm, blue) by TEM

FIGURE 7

Mitophagy inducement of eGFP 4T1 EVs by CCCP correlates with PINK1 expression. (a) GFP‐4T1 cells treated with increasing levels of CCCP lead to a release of increased Parkin‐independent mitophagy‐containing 100K EVs (ARIH1+/PINK1+/CD81+). (b) Inducing mitophagy (10 nM CCCP) within sub confluent eGFP‐4T1 cells that are labelled with the mitochondrial membrane fluorescent tag MitoTracker Deep Red (MTDR) lead to an increased release of 100K EVs containing active mitochondrial molecules. MTDR fluorescence intensity was normalized to GFP fluorescence intensity by EV subpopulation

FIGURE 8

Functional Role of EV associated Parkin‐independent mitophagy‐related proteins. (a) Hypothetical depiction of autophagy and mitophagy processes within the cell and their potential relation to interstitial EVs based on the results of this study. Inside the cell, unconventional protein secretion is taking place, such as secretory autophagy and mitophagy, where organelles associated with each of these types of unconventional protein secretion can be participants in the repertoire of EVs that are passively or actively secreted into the tumour or in vivo interstitium. The legend in the upper center shows the sequence‐specific proteins found in high abundance in the tumour‐derived 100K EVs that are related to the Parkin‐independent process of mitophagy. (b) Known functional location of this complex of proteins that play in the mediation of mitophagy. EV, extracellular vesicle; AP, autophagosome; MVB, multivesicular body; ILV, intraluminal vesicle; PINK1, PTEN‐induced kinase 1; DRP1, dynamin‐related protein 1; FIS1, mitochondrial fission 1 protein; ARIH1, Ariadne RBR E3 Ubiquitin Protein Ligase 1; HUWE1, HECT, UBA And WWE Domain Containing E3 Ubiquitin Protein Ligase 1; SMURF1, Smad ubiquitin regulatory factor 1;, Ub, ubiquitin; p62, sequestome 1; LC3, Microtubule‐associated protein 1A/1B‐light chain 3; PE, phosphatidylethanolamine; Sec22B, SEC22 Homolog B, Vesicle Trafficking Protein; Rab33B, Ras‐Related Protein Rab‐33B; HSP90/CDC37, 90 kDa heat shock protein and Cell Division Cycle 37; VDAC, Voltage dependent anion‐selective channel 1 (Camara et al., 2017 ); TOM34, Translocase Of Outer Mitochondrial Membrane 34 (Faou and Hoogenraad, 2012 ); TOM20, Translocase Of Outer Mitochondrial Membrane 20 VPS‐35,Vacuolar protein sorting ortholog 35