Abstract 2015: Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration
Paul MK., Dutta S., Bisht B., Ramin S-R., Pagano P., Bitan G., Minna JD., Dubinett SM.
Abstract Lung cancer is a highly metastatic disease. Contrary to the common belief that metastasis is a late event, recent findings have suggested that micrometastasis may also occur as an early phenomenon. An increasing body of evidence supports a role for epithelial-mesenchymal transition (EMT) in inducing field cancerization. Reports suggest that cancer-derived exosomes can act via intra-cellular communication to induce EMT and migration. However, the role of premalignant cell-derived exosomes in EMT induction, cellular migration and establishment of field cancerization in unknown. Herein, we describe the role of premalignant cell-derived exosomes on early metastatic behavior. Our laboratory has recently discovered a unique highly motile (HM) subpopulation of human bronchial epithelial cells (HBECs), using a novel “constricted migration” selection strategy, with enhanced metastatic potential in vivo. The HBECs used for selection were modified with changes reciprocating premalignancy (p53null, activated Kras-G12D). Comparative RNA-seq datasets illustrate increased expression of key EMT genes in HM-HBECs. This unique subpopulation of HM-HBECs offer a unique model to investigate premalignant cell migration. We used exosomes derived from HM, SM (slow motile) and unselected (UN) HBECs to evaluate EMT induction, migration, and invasion in vitro and in vivo. Exosomes were isolated by ultracentrifugation and characterized for size distribution using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy, and characterized by western blotting with antibodies specific for Alix, Flotillin, CD9 and CD63. Exosome labeling for cell uptake studies was performed using Dil dye entrapment and by transient transfection with CD63-GFP, releasing GFP+ exosomes. 6X108 HBEC cells produced approximately 6 x 108 exosomes with size ranging from 50 to 130 nm. The exosome signature of HM-, SM-, and UN-HBECs were characterized by mass spectroscopy and micro RNA sequencing. Exosomes from HM-HBECs induce UN-HBECs to obtain a HM phenotype with enhanced EMT, faster migration and increased invasive capacity. Western blot and qPCR data suggest a time-dependent activation of an EMT signature. Further in vivo experiments are in progress to evaluate the role of HM-HBEC derived exosomes on metastatic homing, tumor formation and spread of premalignant HBEC cells and tumor cells. Our data suggest that premalignant cell-derived exosomes possess distinct molecular signatures and could potentially mediate early migration, resulting in the creation of a premalignant field. As such, exosomes afford novel targets in premalignancy for early intervention and cancer interception. Citation Format: Manash K. Paul, Suman Dutta, Bharti Bisht, Salehi-Rad Ramin, Paul Pagano, Gal Bitan, John D. Minna, Steven M. Dubinett. Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2015.