Exosomes and their role in tumorigenesis and a.pdf

Contents lists available at ScienceDirectDrug Resistance Updatesjournal homepage: and their role in tumorigenesis and anticancer drug resistanceNeta Milman,Lana Ginini,Ziv GilThe Laboratory for Applied Cancer Research,Department of Otolaryngology Head and Neck Surgery,The Clinical Research Institute at Rambam Healthcare Campus,Technion Integrated Cancer Center,Rappaport Institute of Medicine and Research,Technion,Israel Institute of Technology,Haifa,IsraelA R T I C L E I N F OKeywords:ExosomesDrug resistanceTumor microenvironmentDrug deliveryA B S T R A C TExosomes are a class of extracellular vesicles ranging in size from 40 to 100 nm,which are secreted by bothcancer cells and multiple stromal cells in the tumor microenvironment.Following their secretion,exosomespartake in endocrine,paracrine and autocrine signaling.Internalization of exosomes by tumor cells influencesseveral cellular pathways which alter cancer cell physiology.Tumor-derived exosomes secreted by cancer orstromal cells can also confer anticancer drug-resistant traits upon cancer cells.These exosomes promote che-moresistance by transferring their cargo which includes nucleic acids,proteins,and metabolites to cancer cells oract as a decoy for immunotherapeutic targets.Depletion of exosomes can reverse some of the detrimental effectson tumor metabolism and restore drug sensitivity to chemotherapeutic treatment.Herein we discuss variousapproaches that have been developed to deplete exosomes for therapeutic purposes.The natural composition,low immunogenicity and cytotoxicity of exosomes,along with their ability to specifically target tumor cells,render them an appealing platform for drug delivery.The ability of exosomes to mediate autocrine and paracrinesignaling in target cells,along with their natural structure and low immunogenicity render them an attractivevehicle for the delivery of anticancer drugs to tumors.1.IntroductionExosomes are a heterogeneous class of extracellular vesicles rangingin size from 40 to 100nm,which are essentially released from all celltypes.Exosomes are secreted to the extracellular environment whenlate endosomal or endosomal multivesicular body(MVB)membranesfuse with the plasma membrane.Involved in numerous biologicalprocesses and pathological conditions,exosomes serve as a form ofintercellular communication,by delivering their cargo of proteins,li-pids,nucleic acids and metabolites to cells in their immediate sur-roundings as well as at distant organs.All cells in the tumor micro-environment(TME)are known to secrete exosomes,including:cancercells,fibroblasts and immunocytes.The cargo of these exosomes pro-motes angiogenesis,modulates stromal reaction and regulates immuneresponse.In addition,exosomes modify signaling pathways of cancercells,thus promoting tumor progression and drug resistance.In thecurrent review,we describe the mechanisms by which exosomesmodulate tumor physiology and drug resistance and discuss strategiesto target their activity on tumors.2.Exosome biogenesisThe term exosome was first used to describe a group of membrane-bound,30100nm vesicles,secreted from reticulocytes during theirmaturation(Wolf,1967).Exosomes are one of two broad categories ofextracellular vesicles;the other category is microvesicles.Microvesicles(also referred to as ectosomes)are defined as extracellular vesiclesformed directly from the plasma membrane by outward budding(Steinand Luzio,1991).Exosomes are formed as intraluminal vesicles(ILV)inMVBs,by the inward invagination of the plasma membrane(Hardinget al.,1983;Johnstone et al.,1987).MVBs,which are components ofthe endosomal system,can either fuse with lysosomes,leading to thedegradation of their cargo,or alternatively,fuse with the plasmamembrane(Mobius et al.,2002;Buschow et al.,2009).Fusion with theplasma membrane leads to the release of ILV as exosomes to the ex-tracellular space(Kowal et al.,2014).The first step in exosome biogenesis is the recruitment of cargomolecules to microdomains that are comprised of specialized lipids andproteins in the MVB membrane(Berson et al.,2001).Cytosolic com-ponents are subsequently recruited to these microdomains(Geminardet al.,2004).The endosomal sorting complex required for transport(ESCRT)are protein complexes involved in the recruitment and loadinghttps:/doi.org/10.1016/j.drup.2019.07.003Received 16 May 2019;Received in revised form 19 July 2019;Accepted 21 July 2019Corresponding author at:Department of Head and Neck Surgery,Rambam Healthcare Campus,Haalia St No 8,Haifa,Israel.E-mail address:zivbaseofskull.org(Z.Gil).Drug Resistance Updates 45(2019)1121368-7646/2019 Elsevier Ltd.All rights reserved.Tof cargo proteins to exosomes and in the later steps of membranebudding.In the canonical pathway,ESCRT 0 and ESCRT I proteincomplexes participate in the cargo recruitment steps,by recognizingubiquitin on cargo proteins(Henne et al.,2011).ESCRT II and IIIprotein complexes participate in the later steps of exosome formation,budding and fusion.The proteins Alix Syntenin and Vsp-4 cooperatewith the ESCRT proteins in the process of exosome biogenesis.Exo-somes can also be formed in an ESCRT-independent manner,as de-monstrated by the formation and secretion of exosomes even after de-pletion of components of the ESCRT complex(Colombo et al.,2013).Inoligodendroglial cell lines,for instance,exosome biogenesis is depen-dent on ceramide formation by sphingomyelinase(Trajkovic et al.,2008).The ceramide-rich domains form spontaneous curvatures thatlead to invaginations.Tetraspanins were also implicated in the ESCRT-independent formation of exosomes.Tetraspanins form specializedstructural domains in membranes similar to caveolae,by homotypicand heterotypic protein-protein interactions and by associations withlipids including cholesterols and gangliosides(Andreu and Yanez-Mo,2014).Tetraspanins,specifically CD9,CD81 and CD82,are also in-volved in the sorting of cargo to exosomes.After interaction with acognate antigen-specific T cell,MHCII molecules in dendritic cells wereincorporated into CD9 containing ILVs that were secreted as exosomes.Unlike lysosomal targeting,this process was independent of MHC IIubiquitination(Buschow et al.,2009;Chairoungdua et al.,2010).After their secretion,exosomes can act on their cells of origin in anautocrine manner,on cells in their vicinity in a paracrine manner aswell as on cells in distant organs.To exert their effect,exosomes maybind to the surface of their target cells and signal through cell surfacereceptors.Alternatively,they may enter target cells via endocytosis orby direct fusion with the plasma membrane of the target cells,andthereby release their cargo.In the sections below,we discuss the effectsof exosomes secreted by cancer cells and stromal components,on tumorprogression and on the development of resistance to therapy.3.The role of exosomes in the tumor microenvironmentThe TME consists of tumor and stromal compartments.The stromais heterogeneous in nature,and comprises of the extracellular matrix(ECM)and various cell types.The stromal cellular compartment in-cludes infiltrated immune cells,fibroblasts and vascular cells,all con-tributing functional and structural support to the tumor(Schiavoniet al.,2013).Constant crosstalk occurs between stromal and tumorcells,and as demonstrated below,a large part of this communication ismediated by extracellular vesicles.3.1.Immunocyte-tumor communication mediated by exosomes3.1.1.The effect of tumor-derived exosomes on immunocytesImmune cells are a major component of the TME(Hanahan andWeinberg,2011).Tumor-associated macrophages(TAM)promote theprogression and metastasis of tumors and are associated with poorprognosis(Schiavoni et al.,2013).Following their recruitment from thecirculation,factors in the TME,including tumor-derived exosomes(TDX),drive the differentiation of the macrophages towards an anti-inflammatory M2 phenotype.Transfer of miR-940 by exosomes fromhypoxic human epithelial ovarian cancer cells to human macrophagesresulted in the polarization of the macrophages to an M2-like pheno-type(Chen et al.,2017).Likewise,triple-negative breast cancer-derivedexosomes promoted the M2 differentiation of RAW264.4 human mac-rophages(Piao et al.,2018).The protein cargo of TDX from metastaticmouse colon carcinoma cells polarized bone marrow-derived macro-phages towards the M2-like phenotype,by upregulation of MCP-1 andTNF(Chen et al.,2016).Interestingly,human gastric cancer TDX werereported to increase the transcript levels of pro-inflammatory factors(IL-6,TNF-and CCL2);however,these inflammatory macrophagesalso promoted tumor growth and dissemination(Piao et al.,2018).The presentation of neoplastic cell antigens by dendritic cells(DC)in the draining lymph nodes leads to T cell stimulation(Gardner andRuffell,2016).Tumor associated DC were shown to be defective in Tcell stimulation(Veglia and Gabrilovich,2017).The transfer of miR-203 by pancreatic cancer-derived exosomes to neighboring DC resultedin reduced expression of TNF-and IL-2,which is necessary for DCmaturation(Zhou et al.,2014).A considerable fraction of tumor CD8+cytotoxic T cells(CTL)becomes exhausted and unable to exert their cytolytic activity.Headand neck squamous cell carcinoma-derived exosomes mediated the lossof expression of the co-stimulatory molecule CD27/CD28 and atte-nuated antitumor IFN-cytokine production from responder T cells(Maybruck et al.,2017).In addition,TDX from breast and bladdercancer generated high levels of adenosine by ATP hydrolysis;this ne-gatively regulated high-affinity A2A positive T cell functions throughthe inhibition of responses to IL-2 and TCR ligation(Clayton et al.,2011).3.1.2.The effect of immune cell-derived exosomes on tumorsImmune cell-derived exosomes are also important contributors toTME immune regulation.By transmission of miR-21-5p and miR-155-5p,exosomes derived from CD163+(M2)macrophages,isolated fromhuman colon cancer specimens,promoted invasion and migration inSW48 colorectal cancer cells in vitro.Moreover,the pre-treatment ofSW48 cells with CD163+macrophages in an experimental metastasisassay using immunodeficient mice,resulted in increased cell invasionand migration(Lan et al.,2018).Similarly,exosomes secreted frommurine M2 polarized bone marrow-derived macrophages(BMDM)promoted the migration of mouse gastric carcinoma MFC cells.Me-chanistically,BMDM-derived exosomes transmitted apolipoprotein E,which activated the PI3K-Akt signaling pathway in recipient gastriccancer cells(Zheng et al.,2018).In contrast to TAM,DC-derived exo-somes(DEX)were shown to promote anti-tumorigenic processes.Murine DEX expressed high levels of transmembrane TNF and oftransmembrane and soluble Fas ligand(FasL),which mediated thekilling of melanoma,colon adenocarcinoma and squamous cell carci-noma cells(Munich et al.,2012).Natural killer(NK)cells appear to kill cancer cells also via secretedexosomes.The addition of NK-exosomes,enriched with FasL and per-forin,to human melanoma cells,had a lethal effect,mediated by FasL.Interestingly,these exosomes had no effect when added to healthykidney cells(Zhu et al.,2017).The role of mast cells in the TME is still not clear(Aponte-Lopezet al.,2018).However,internalization of mast cell-derived exosomes bylung cancer cells led to enhanced migration and a high proliferationrate.This effect was mediated by delivery of the mast/stem cell growthfactor receptor kit,ultimately leading to increased cyclin D1 expression(Xiao et al.,2014)(Figs.1 and 2).3.2.Cancer-associated fibroblast-tumor communication mediated byexosomes3.2.1.Tumor-derivedexosomesaffectcancer-associatedfibroblastdifferentiationAs major producers of extracellular matrix,fibroblasts are involvedin regulation of tissue remodeling and repair.Cancer-associated fibro-blasts(CAF)were differentiated from tissue resident fibroblasts andmesenchymal stem cells,and shown to promote tumor growth(Ohlundet al.,2014).By transmission of telomerase reverse transcriptase(hTERT)mRNA,TDX from T cell leukemia,breast cancer cells,coloncarcinoma and chronic myeloid leukemia induced the transformation offibroblasts to CAF and the formation of tumor-supporting niches(Gutkin et al.,2016).Proteomics profiling of exosomes derived from malignant me-sothelioma(MM)revealed that their cargo is enriched with proteinsthat function in immune response,angiogenesis and cell transformationN.Milman,et al.Drug Resistance Updates 45(2019)1122Fig.1.Exosomes participate in the reciprocal crosstalk between tumor and stromal cells in the tumor microenvironment.At the tumor microenvironment,cancer and stromal cells secrete exosomes that affect cellular processes which promote tumor progression.The figure shows the type of cells and the effect on cancer:TAM Tumor Associated Macrophages,CAF Cancer Associated Fibroblasts,DC-Dendritic Cells,TC-T cells,NK Natural Killer Cells,ER-Endoplasmic Reticulum,MVB Multivesicular Body,FasL-Fas ligand,EMT-Epithelial to mesenchymal transformation,M2-Macrophages type 2.Fig.2.Mechanisms of promotion of anticancer drug resistance by exosomal cargo.A.Transmission of nucleic acid cargo that elicits gene regulation in therecipient cell.B.Transmission of protein cargo by exosomes induces signaling events inducing overexpression of multidrug efflux transporters in the recipient cell.C.Expression of immune therapy targets by exosomes,directly competing with cancer cells.ROS reactive oxygen species,RTK-receptor tyrosine kinase.N.Milman,et al.Drug Resistance Updates 45(2019)1123during cancer progression.MM-derived exosomes were reported to in-duce the migration of CAF of human and mouse origin by transportingtheir cargo(Greening et al.,2016).Moreover,constitutively active TGF-cargo from prostate cancer exosomes and mesothelioma cell-derivedexosomes triggered the differentiation of fibroblasts to myofibroblasts(Webber et al.,2010).3.2.2.The effects of CAF-derived exosomes on tumorsThe transfer of miRs-21,-378e and-143 by CAF-derived exosomesto the human breast cancer cell lines BT549,MDA-MB-231 and T47Dpromoted stemness of the cells,epithelial-mesenchymal transition andanchorage-independent growth(Donnarumma et al.,2017).In prostatecancer,exosomes were shown to regulate metabolism.Specifically,theyenhanced proliferation,increased glucose uptake and lactate secretion,and reduced the oxygen consumption rate in prostate cancer cells.Thecargo of CAF-derived exosomes contained“offthe shelf“metabolites,including lactate,acetate,amino acids,tricarboxylic acid cycle inter-mediates and lipids that were utilized by the cancer cells(Zhao et al.,2016).4.The role of exosomes in the emergence of resistance to anti-tumor therapyA major obstacle in fighting cancer continues to be chemoresistance.Resistance can be inherent to the tumor cells,or emerge after an initialresponse of the tumor to the treatment(acquired resistance)(Gonenand Assaraf,2012;Niewerth et al.,2015;Taylor et al.,2015;Zhitomirsky and Assaraf,2016;Li et al.,2016a;Ra