Abstract 958: Exosomes from EGFR-mutated adenocarcinoma induce a hybrid EMT and MMP9-dependant tumour invasion
Authors: Jouida Amina , McCarhty Cormac , Fabre Aurelie , Kelly Allan , Nadarajan Parthiban , Callaghan Marissa O , Keane Michael P. 
Published: June 2022

Abstract
Exosomes are a class of extra cellular vesicles (EVs), with a multi vesicular endosomal origin, that are released by all cell types, with sizes ranging from 30-150nm and a lipid bilayer membrane. Recent studies document that tumour cells shed exosomes at considerably higher rates, and these exosomes play critical roles in several early and late events associated with tumour development and metastasis. Thus, circulating exosomes are emerging as a new paradigm of ‘liquid biopsy’ for non-invasive cancer diagnosis. They have been reported to influence epithelial mesenchymal transition (EMT) in pathological states such as metastatic lung cancer. We successfully isolated exosomes from an Epidermal Growth Factor Receptor (EGFR) mutated cell line (HCC827) and from serum of different lung cancer patients: EGFR-mutated adenocarcinoma (N=5), wild-type adenocarcinoma (N=5), squamous (N=3) and serum of patients with no cancer (N=5) and analysed their effects on cells from an A549 cell line. By zymography, we observed that exosomes derived from the EGFR-mutated adenocarcinoma patients contains significantly more MMP-9 and pro-MMP-9 than those derived from other patients. When incubated with A549 cells, all exosomes, derived from the EGFR mutated cell line and from patients (regardless of patient origin), induced an increase in MMP2 activity. However, the increase of MMP9 activity was seen only in A549 cells treated with exosomes derived from EGFR-mutated adenocarcinoma patients and from the EGFR mutated cell line. MMPs are one of the major attributes that epithelial cells acquire after undergoing EMT. Respectively by a modified Boyden chamber assay and by wound healing assay, we showed, that EGFR-mutated exosomes treatment increases the invasive capacity (fold change of 1.7; p-value=0.0008) and the migratory capacity [CM1] of A549 and HBE4 E6/E7 cells in an MMP-9 dependant pathway. By western blot and qPCR, we observed a significant increase of vimentin expression, a mesenchymal marker while retaining the epithelial characteristics as evidenced by the unaltered levels of E-cadherin and EPCAM. EMT is not a binary process but instead, cells often exhibit a spectrum of epithelial/mesenchymal phenotype(s). Such hybrid cells can move collectively as clusters which is thought to enhance their invasive properties. By qPCR, we observed an increase of NRF2, P-cadherin and cytokeratin 14, markers of hybrid-EMT and collective migration. Our results suggest that exosomes derived from EGFR-mutated adenocarcinoma serum could be a potential mediator of hybrid-EMT and tumour invasion and may provide insights into the mechanisms of lung cancer progression and metastasis. Further studies are required to precisely delineate these mechanisms and explore novel therapeutic avenues.
Citation Format: Amina Jouida, Cormac McCarhty, Aurelie Fabre, Allan Kelly, Parthiban Nadarajan, Marissa O Callaghan, Michael P. Keane. Exosomes from EGFR-mutated adenocarcinoma induce a hybrid EMT and MMP9-dependant tumour invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 958.