Abstract 322: Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer
Authors: Sohal Ikjot Singh , Shaw Sydney N. , Kasinski Andrea L. 
Published: April 2023

Abstract
Extracellular vesicles (EVs) act as key mediators of cell-to-cell communication in several pathophysiological processes including cancer. EVs inherit a cocktail of molecular cues (mRNAs, miRNAs, ncRNAs, proteins, metabolites, etc.) from cancer cells that mediate tumor microenvironment changes to facilitate cancer progression and metastasis. The heterogenous EV populations and their content dynamically change during cancer, which is why, there has been great progress in utilizing them as prognostic and diagnostic biomarkers. However, harnessing EVs for therapeutic applications requires greater understanding of the fundamental EV biology such as their origin, intracellular trafficking, cargo loading, and export mechanisms. Here, we utilized a genetically-encoded electron dense tag, APEX2 (an engineered peroxidase derived from soybean ascorbate peroxidase) and electron microscopy to elucidate that EVs, in the form of multivesicular bodies, can also originate at the trans-Golgi interface. We also identified a novel subpopulation of EVs, which expressed a classic trans-Golgi network protein (referred as TGNP) on their surface. While EVs of late endosomal origin are well known, and there are reports of mitochondrial origin as well, a trans-Golgi EV biogenesis pathway has not been reported before. Intriguingly, we determined that certain cells hijack the trans-Golgi pathway for EV biogenesis, which we hypothesize contributes to differential cancer cell EV cargo and phenotypic consequences. Initially, using confocal microscopy in non-small cell lung cancer (NSCLC) cells, we identified that some of the tetraspanins historically thought to be involved in late endosomal trafficking were instead enriched in the trans-Golgi compartments. We found that this colocalization pattern was conserved in several other NSCLC cell lines. To further elucidate this phenomenon at the trans-Golgi interface, we tagged TGNP to APEX2. After verifying the expression and localization of TGNP-APEX2 by immunostaining, we performed electron microscopy. TGNP-APEX2 was found to be present on the membrane of intraluminal vesicles inside multivesicular bodies. Interestingly, the multivesicular bodies containing TGNP-APEX2 vesicles were not of late endosome origin. Furthermore, TGNP was also found to be present in EVs of all NSCLC cell lines tested. Inhibiting Golgi trafficking using small molecule inhibitors (Brefeldin A) significantly reduced EV secretion and levels of trans-Golgi-enriched tetraspanins in the EV lysates. The data elucidates that EVs can also originate from trans-Golgi interface and identified novel subpopulation that expresses TGNP. The broader questions regarding the functional relevance of trans-Golgi-mediated EV biogenesis, the type of cargo exported, and mediators of loading and export in the context of cancer are currently under investigation.
Citation Format: Ikjot Singh Sohal, Sydney N. Shaw, Andrea L. Kasinski. Electron microscopy using electron dense genetic tag elucidates novel extracellular vesicle biogenesis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 322.