Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects by Epidermal growth factor inhibitor treatments

Extracellular Vesicles

Abstract Precision cancer medicine have changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by tyrosine kinase inhibitors (TKIs) towards mutated Epidermal growth factor receptor (EGFR). Yet, responses to such TKIs e.g., erlotinib and osimertinib among patients are heterogenous and there is a need for non-invasive blood-based analytics to follow treatment response and reveal resistance to improve patient’s treatment outcome. Recently, extracellular vesicles (EVs) have been identified as an important source of tumor biomarkers promising to revolutionize liquid biopsy-based diagnosis of cancer. However, high heterogeneity has been a major bottleneck. The pathological signature is often hidden in the differential expression of membrane proteins in a subset of EVs which are difficult to identify with bulk techniques. Using a fluorescence-based approach, we for the first time demonstrate that the single-EV technique can be used to monitor the treatment response of targeted cancer therapies such as TKIs towards EGFR. To test the hypothesis, we analyzed the membrane proteins of native EVs extracted from EGFR-mutant NSCLC cell line, both prior and post treatment with EGFR-TKIs erlotinib or osimertinib. The selected cell line being refractory to erlotinib and responsive to osimertinib makes it a suitable model system. The expression level of five surface proteins; two common tetraspanins (CD9, CD81) and three markers of specific interest in lung cancer (EGFR, PD-L1, HER2) were studied. The data suggest that in contrast to erlotinib, the osimertinib treatment increases the population of PD-L1, EGFR and HER2 positive EVs while the expression level per EV decreases for all the three markers. The PD-L1 and HER2 expressing EV population seems to increase by several fold because of osimertinib treatment. The observations agree with the previous reports performed on cellular level indicating the biomarker potential of EVs for liquid-biopsy based monitoring of targeted cancer treatments. Highlights Membrane protein analyses of single EVs may reveal distinct differences when lung cancer cells are refractory vs responsive under different EGFR-TKI treatments. Comparison of 1 st generation erlotinib and 3 rd generation osimertinib shows clear signature on the expression of PD-L1, EGFR, HER2 on single EVs Colocalization showed a change in common marker combinations before after treatment. PD-L1 expression per vesicle decreases while the number of PD-L1 positive EVs increases as a result of osimertinib treatment, indicating that such signature may not be detectable under bulk analysis

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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