Abstract 4433: P-gp-overexpressing MDR cells shed larger extracellular vesicles than their drug-sensitive counterparts

Abstract

Abstract P-glycoprotein (P-gp) overexpression is a major cause of multidrug resistance (MDR) and presents a serious challenge to the efficient treatment of cancer1. In spite of being a major clinical challenge, to date there are no non-invasive means to diagnose the MDR phenotype, using non-tumour biological samples. Recently, the non-genetic acquisition of functional P-gp has been described as being possibly mediated by extracellular vesicles (EVs). Indeed, P-gp may be transferred by EVs from MDR cells to drug-sensitive (DS) recipient cells3. Collectively, EVs may refer to exosomes (with endosomal origin and sizes ranging from 30-100nm), microvesicles (with plasma membrane origin and sizes ranging from 50-2000nm) or even apoptotic bodies (ranging from 50-5000nm)4. This study aimed at characterizing and comparing EVs from P-gp-overexpressing MDR cancer cells and their respective DS counterparts, as an attempt to identify MDR biomarkers. Two pairs of MDR cancer cell lines and their DS counterparts were used: a chronic myeloid leukaemia pair (K562Dox, a kind gift from Dr. Jean-Pierre Marie and K562, from EACC) and a non-small cell lung cancer pair (RH460 and NCI-H460, a kind gift from Dr. Milica Pesic). EVs shed by these cells were characterized in terms of size and molecular markers. In addition, their protein content was investigated by proteomic analysis and by Western blot. This study describes, for the first time, that P-gp overexpressing MDR cells produce larger EVs than their DS counterparts. In addition, a distinct protein signature was found between the EVs shed by DS cells and their MDR counterparts, consisting mainly in differences in the relative abundances of particular proteins. As predicted, P-gp was found present only in the EVs shed by MDR cells. Interestingly, proteins involved in the biogenesis of exosomes, such as Syntenin-1, TSG-101, CD63, Clathrin LCB and CHMP4, were found decreased in EVs shed by MDR cells. These results were in agreement with the identified size differences between the EVs from MDR and DS cells mentioned above, which may suggest that MDR cells produce more microvesicles and that DS cells produce more exosomes. In conclusion, it is proposed that EVs shed by the P-gp overexpressing MDR cells may have a specific size and protein signature, consisting in P-gp presence and a lower abundance of proteins involved in the biogenesis of exosomes. This may allow the development of a non-invasive way of detecting and predicting MDR, by analysing EVs shed into the circulation of cancer patients. 1. Lopes-Rodrigues, V. et al. The network of P-glycoprotein and microRNAs interactions. Int J Cancer 135, 253-263, (2014). 2. Bebawy, M. et al. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells. Leukemia 23, 1643-1649, (2009). 3. Akers, J. C. et al. Biogenesis of extracellular vesicles (EV). J Neurooncol 113, 1-11, (2013). Citation Format: Vanessa Lopes-Rodrigues, Alessio Di Luca, Diana Sousa, Hugo Seca, Paula Meleady, Michael Henry, Raquel T. Lima, Robert O'Connor, M. Helena Vasconcelos. P-gp-overexpressing MDR cells shed larger extracellular vesicles than their drug-sensitive counterparts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2015-4433