Communication by Extracellular Vesicles Where We Are and Where We Need to Go
Leading Edge
Review
Communication by Extracellular Vesicles:
Where We Are and Where We Need to Go
Mercedes Tkach1and Clotilde The′ry1,*
1Institut Curie,PSL Research University,INSERM U932,75005Paris,France
*Correspondence:clotilde.thery@curie.fr
https://www.wendangku.net/doc/ff9873430.html,/10.1016/j.cell.2016.01.043
In multicellular organisms,distant cells can exchange information by sending out signals com-posed of single molecules or,as increasingly exempli?ed in the literature,via complex packets stuf-fed with a selection of proteins,lipids,and nucleic acids,called extracellular vesicles(EVs;also known as exosomes and microvesicles,among other names).This Review covers some of the most striking functions described for EV secretion but also presents the limitations on our knowl-edge of their physiological roles.While there are initial indications that EV-mediated pathways op-erate in vivo,the actual nature of the EVs involved in these effects still needs to be clari?ed.Here,we focus on the context of tumor cells and their microenvironment,but similar results and challenges apply to all patho/physiological systems in which EV-mediated communication is proposed to take place.
Introduction
Cells can communicate with neighboring cells or with distant cells through the secretion of extracellular vesicles(EVs).EVs are composed of a lipid bilayer containing transmembrane pro-teins and enclosing cytosolic proteins and RNA.Cells can secrete different types of EVs that have been classi?ed accord-ing to their sub-cellular origin(Colombo et al.,2014).On one hand,EVs can be formed and released by budding from the cells’plasma membrane.These EVs display a diverse range of sizes (100–1,000nm in diameter)and are generally known in the literature as microvesicles,ectosomes,or microparticles.Other types of vesicles,the exosomes,are generated inside multive-sicular endosomes or multivesicular bodies(MVBs)and are secreted when these compartments fuse with the plasma mem-brane.Exosomes are vesicles smaller than150nm in diameter and are enriched in endosome-derived components.All EVs bear surface molecules that allow them to be targeted to recip-ient cells.Once attached to a target cell,EVs can induce signaling via receptor-ligand interaction or can be internalized by endocytosis and/or phagocytosis or even fuse with the target cell’s membrane to deliver their content into its cytosol,thereby modifying the physiological state of the recipient cell.
In this Review,we highlight and discuss the more recent studies on cancer-derived EVs,with a special focus on the latest discoveries on the role of EVs in cancer metastasis.The term ‘‘exosomes’’is often used in these articles to designate the EVs analyzed.However,we now know that the most popular exosome puri?cation protocols used historically in the literature (differential ultracentrifugation,220nm?ltration[Thery et al., 2006])—and the recently released commercial kits—co-isolate different types of EVs.Thus,the term exosomes is generally used to refer to a mixed population of small EVs(sEVs)without further demonstration of their intracellular origin.In fact,func-tions assigned to exosomes may either re?ect generic EV activ-ities or truly exosome-speci?c ones;however,the published data cannot be used to determine the precise speci?city.We thus chose here to use the generic term EVs when vesicles are isolated without speci?c attention to their size or sEVs when the method used selects vesicles smaller than200nm,indepen-dent of the term used in the article referred to.
EV-Borne Proteins Promote Cancer Progression and Metastasis
EVs have been shown to participate in the dissemination of can-cer cells,and many groups have described how tumor-and stroma-derived EVs are involved in the different steps of the met-astatic cascade(Figure1).Tumor sEVs can directly modify tumor cells’intrinsic motility and invasiveness capacity.In particular, sEVs can promote directional cell motility through ECM compo-nents,such as?bronectin,which bind to integrins present on sEVs and thus provide a substrate favoring cell adhesion and enhancing cell speed(Sung et al.,2015).Moreover,sEVs partic-ipate in the biogenesis and activity of an invasive structure called invadopodia through the MVB-dependent delivery of metallo-proteinases such as MT1-MMP and other cargo molecules (Hoshino et al.,2013),thus promoting cell motility.EVs can directly contribute to extracellular matrix(ECM)degradation by spreading matrix metalloproteinases present either on sEVs (Yue et al.,2015)or in tumor-shed large EVs(Clancy et al., 2015).The latter also have been shown to facilitate amoeboid movement and facilitate invasion.Not only tumor-derived EVs, but also sEVs from cancer-associated?broblasts can stimulate invasiveness of recipient breast cancer cells,in this case by acti-vating the planar cell polarity signaling pathway(Luga et al., 2012).
Tumor sEVs can alter the cellular physiology of both surround-ing and distant non-tumor cells to allow dissemination and growth of cancer cells,i.e.,by triggering vascular
permeability
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(Peinado et al.,2012;Zhou et al.,2014)or by conditioning pre-metastatic sites in distant organs (Costa-Silva et al.,2015;Hoshino et al.,2015;Peinado et al.,2012).In particular,mela-noma tumor sEVs bearing a tyrosine-kinase receptor can pro-mote migration of bone marrow progenitor cells to future sites of metastasis,whereas sEVs secreted by a less-aggressive version of the same tumor,devoid of the relevant receptor,do not display this effect (Peinado et al.,2012).Alternatively,sEVs from pancreatic cancer cells themselves migrate to distant organs and promote the formation of a pre-metastatic niche by creating a ?brotic environment enriched in TGF b ,?bronectin,and a macrophage-attracting chemokine (Costa-Silva et al.,2015).Interestingly,sEVs from different tumor types bear integ-rins (ITGs)that target these sEVs to speci?c organs and trigger signaling pathways,thereby initiating pre-metastatic niche formation (Hoshino et al.,2015).For example,sEVs expressing ITG a v b 5bind speci?cally to Kupffer cells,mediating liver tropism,while ITG a 6b 4and ITG a 6b 1on sEVs bind to lung-resident ?bro-blasts and epithelial cells,leading to lung tropism (Hoshino et al.,2015).Modi?cations induced by sEVs in these distant organs then attract metastatic tumor cells.
This observation has been recently used in an innovative way to redirect tumor cell dissemination in a non-deleterious location (de la Fuente et al.,2015).An arti?cial pre-metastatic niche generated by embedding tumor sEVs in a 3D scaffold and then implanted in mouse peritoneum was able to capture ovarian tu-mor cells present in the peritoneum and divert them from their normal organ target for dissemination,resulting in strikingly increased survival of the animal.The possible application of this device in human patients could represent a very promising approach to suppress metastasis.However,despite being extremely appealing,we must stress that the working model of circulating tumor-derived sEVs fostering pre-metastatic niche formation has not been demon-strated in a fully physiological in vivo context.In published arti-cles to date,animals were subjected to sustained injections of in-vitro-puri?ed tumor-derived sEVs,resulting in this enhanced metastasis.Whether sEV secretion in vivo by tumor cells is able to achieve this function is still not clear.One possible way to address this is by interfering in vivo with sEV biogenesis in cancer cells.Some studies have attempted to do this by inhibit-ing Ras-related RAB proteins.RAB27A or RAB35have been ?rst shown to be required for sEV secretion in HeLa cervical carci-noma (Ostrowski et al.,2010)and Oli-Neu oligodendroglial pre-cursor cell lines (Hsu et al.,2010),respectively.Consistently,knocking down RAB27A in melanoma (Peinado et al.,2012),breast (Bobrie et al.,2012),?brosarcoma (Sung et al.,2015),or prostate cancer cell lines (Webber et al.,2015)reduces the secretion of sEVs.Cells lacking RAB27A,when injected in vivo,displayed reduced local migration (Sung et al.,2015)or reduced growth due to impaired recruitment of bone-marrow-derived pro-tumoral immune cells (Bobrie et al.,2012),or impaired modi?cation of co-injected ?broblasts into pro-tumoral myo?broblasts (Webber et al.,2015)(Figure 1).Lower incidence of metastasis was also observed (Bobrie et al.,2012;Peinado et al.,2012).However,RAB27A does not exclusively regulate EV secretion.Loss of the protein also decreases EV-indepen-dent secretion of soluble factors,such as some growth factors and metalloproteinases that are also involved in tumor metas-tasis (Bobrie et al.,2012;Peinado et al.,2012).The same prob-lem has arisen with the other molecules proposed so far to regulate speci?cally sEV secretion,such as
sphingomyelinases
Figure 1.EV-Mediated Effects Promoting Tumor Growth,Invasiveness,and Metas-tasis
Tumor-derived EVs can have several effects on recipient cells.At the site of the primary tumor (left),EVs can enhance cancer cell motility by stabilizing cellular protrusions promoting an effective and directionally persistent migration via deposition of ECM cargoes,such as ?bronectin,into sEVs.The secretion of EVs containing metal-loproteinases also directly participates in ECM remodeling and promotes function of specialized cell protrusions endowed with degradative activ-ity,called the invadopodia.ECM remodeling supports tumor cell motility through the tissues.EVs can also promote differentiation or recruit-ment of pro-tumoral stromal cells (?broblasts and bone-marrow-derived cells).Reciprocally,tumor cell motility,but also acquisition of drug resis-tance,can be enhanced via a complex interplay with EVs secreted by surrounding ?broblasts.In addition,sEVs can enter the circulation and travel to distant sites from the primary tumor (right).Various sEV cargoes promote vascular perme-ability,and EVs can enter the distant tissue,where they may generate a pre-metastatic niche by inducing ECM remodeling and promoting the recruitment of bone-marrow-derived cells and eventually,tumor cells.This ?gure schematizes the effects of EVs demonstrated by mixed in-vivo-/in-vitro-based experiments.See the text for dis-cussion on the evidence for fully physiological in vivo occurrence of these functions.
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(SMases)(Trajkovic et al.,2008).Knockdown or inhibition of SMases by small-molecule inhibitors,which results in impaired ceramide formation,has often been used to inhibit exosome/sEV secretion but without demonstration of the speci?city of this effect for sEV secretion,as opposed to other secretions or other physiological features of the cells.Thus,to understand the functions of EVs in vivo,the development of complementary methodologies will be required.
Catching Communication in Action
A major challenge for the EV ?eld and,more broadly,for under-standing how EVs may support both physiological and patho-physiological processes is being able to demonstrate in vivo EV transfer between cells.To address this problem,a few groups have recently developed clever modi?cations of EVs,allowing tracking of their behavior and their target cells in vivo (Figure 2).Either proteins or mRNA cargoes of EVs have been thus modi?ed.For instance,fusing a ?uorescent protein to a palmi-toylation sequence induces its localization at the plasma mem-brane as well as in secreted EVs of all sizes.The EVs could then be visualized in the tumor microenvironment by intravital microscopy on animals bearing tumors expressing this fusion protein (Lai et al.,2015).Fusion of luciferase to a protein trans-membrane domain also allowed its secretion in EVs and subse-quent enzymatic measurement of luciferase activity in distant cells (Lai et al.,2014).These authors also designed an intracel-lular probe to ?uorescently label mRNA secreted in EVs and tools to measure EV-borne mRNA encoding luciferase signal (Lai et al.,2015).While these tools are promising,studies so far have only demonstrated EV-borne mRNA transfer between cultured cells in vitro.
Another elegant approach to address EV transfer has now demonstrated functional EV-mediated transfer in vivo of an mRNA into target cells in the absence of any ex vivo manipulation (Ridder et al.,2014).Transgenic mice expressing the CRE re-combinase speci?cally in immune cells and a LacZ reporter gene,expressed only upon excision of a STOP sequence,were used for this purpose (Ridder et al.,2014).Ridder et al.showed that EVs containing the CRE mRNA,but devoid of the CRE protein,are present in blood circulation of these transgenic mice.Strikingly,LacZ expression was observed in some neurons and other non-immune cells throughout the animals.Leakiness of the immune cell-speci?c promoter and fusion between CRE-expressing and reporter-expressing cells were carefully excluded,leading to the conclusion that the CRE mRNA carried by immune cell EVs was transferred in the recipient non-immune cells and translated into functional CRE protein.Although the number of recombined neural cells was very low,suggesting a limited ef?ciency of this transfer in normal conditions,induction of systemic in?ammation increased it,opening up the possibility that in vivo EV-mediated transfer may be particularly relevant to some pathological conditions.
This methodology has also been recently used to visualize cancer-derived EV transfer to other cancer cells (Zomer et al.,2015)and to immune cells (Ridder et al.,2015)in living mice and to study the effect of this transfer.In Zomer et al.,the authors designed a reporter system based on the conversion of DsRed+tumor cells to eGFP+tumor cells upon uptake of tumor EVs con-taining CRE mRNA and analyzed the behavior of these latter cells without any manipulation of the EV-releasing https://www.wendangku.net/doc/ff9873430.html,ing this system,intravital imaging revealed the EV transfer of functional mRNA from a malignant human tumor cell to a less
malignant
Figure 2.Approaches Used to Analyze EV-Mediated Transfer In Vivo
Recent novel approaches allowing the visualiza-tion of EV transfer to recipient cells in vivo involve genetic modi?cation of the secreting cells,which then secrete EVs containing labeled components.(I)Genetic fusion of ?uorescent proteins to a consensus palmitoylation sequence,enabling whole-cell membrane labeling,and to an mRNA allows tracking of cells with bound or internalized EVs by their newly acquired ?uorescence.(II)Expression of a membrane-bound luciferase al-lows analysis of distant bioluminescent cells in vivo,resulting from EV-bound luciferase protein capture and/or luciferase mRNA neo-expression.(III)To demonstrate speci?cally neo-expression of an EV-associated mRNA,the CRE recombinase can be expressed in EV-secreting cells.Endoge-nous nuclear localization of CRE results in absence of the protein but presence of the mRNA in the secreted EVs.Transfer of the CRE mRNA contained within an EV to a cell carrying a ?uo-rescent or enzymatic reporter gene expressed only after DNA recombination and excision of a STOP signal is detected by ?uorescence or colorimetric changes.
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one and demonstrated that the uptake of these EVs can alter the migratory behavior and metastatic capacity of the recipient cell (Zomer et al.,2015).It will be interesting to study this transfer of EVs among tumor cells in immunocompetent mice.In Ridder et al.,a similar strategy highlighted the transfer of vesicle-enclosed CRE mRNA to non-tumor cells and showed that myeloid-derived suppressor cells that took up tumor EVs dis-played increased immunosupressive functions(Ridder et al., 2015).In these two studies,the actual nature of the EVs involved in mRNA transfer was not investigated,nor was the transfer mechanism.It is therefore not clear whether transfer involves a direct fusion of EVs with the recipient cells or phagocytosis of live or apoptotic cell-derived EVs by the recipient cell.To more solidly support the pathway for EV-mediated transfer of CRE mRNA,inhibition of EV biogenesis in vivo would be ideal;how-ever,as mentioned before,these experiments are quite dif?cult to accomplish and are not completely speci?c.Other transfer mechanisms,like formation of gap junctions or of nanotubes connecting two adjacent cells,were also not formally excluded. However,it was also observed that transfer occurred at a dis-tance between two tumors localized in different parts of the an-imal,excluding the possibility of local communication between cells and supporting the idea of mRNA being transferred through a long-range extracellular carrier.Overall,this visualization sys-tem allows identi?cation and isolation of cells that are targets of EVs in vivo and will be very important for understanding how tumor-and stromal-derived EVs affect their environment.
A Role for EV-Mediated Small RNA Transfer?
In addition to proteins and mRNAs,miRNAs and other non-cod-ing RNAs are also possible active EV cargoes.The idea that miRNA secreted in sEVs can be functionally delivered to target cells,resulting in direct modulation of their mRNA targets,has become one of the most actively explored hypotheses in the EV?eld,especially in cancer.This idea was initially demon-strated for Epstein-Barr virus-infected cells,where secreted sEVs transferred viral miRNAs into neighboring non-infected cells,leading to repression of virus-target genes(Pegtel et al., 2010).Following this path,several groups have recently reported that EV-mediated secretion of a given miRNA in the tumor micro-environment is responsible for tumor metastasis.Various mech-anisms have been proposed,involving either an effect on the local or distant tumor stroma(including modulating endothelial cell permeability,metabolism,or the pre-metastatic niche)or on the tumor cells themselves(by increasing oncogenic proper-ties and/or invasiveness).
However,even if involvement of the described miRNA is generally well supported,direct demonstration that functional EV-mediated miRNA transfer is the relevant mechanism is still dif?cult to achieve.Importantly,carriers other than EVs could mediate miRNA transfer.All EV isolation techniques potentially co-isolate other RNA-binding structures,such as large protein complexes(Palma et al.,2012)and lipoproteins(Vickers et al., 2011).Lipoprotein-associated RNAs have been shown to be resistant to RNase treatment and to deliver miRNAs into host cells,and there is currently no reason for excluding that transfer of naked protein-miRNA complexes into the cytosol of host cells can occur.Therefore,as the International Society for Extra-cellular Vesicles recently highlighted(Lo¨tvall et al.,2014),addi-tional steps of separation of EVs from other structures,e.g., by?oatation into density gradients or by immuno-isolation via speci?c antibodies,are necessary before claiming speci?c EV-mediated miRNA transfer.
The miRNA-dependent effect observed could,in fact,be mediated by induction of endogenous miRNA expression in the target cell by other EV components,rather than by the EV-en-closed miRNA.Such a mechanism can be excluded if the recip-ient cell is incapable of expressing the studied miRNA—for instance,when the transferred miRNA is encoded only by a foreign genome, e.g.,viral(Pegtel et al.,2010)or parasitic (Buck et al.,2014).Similarly,if the recipient cell comes from a mouse engineered to lack the miRNA(Alexander et al.,2015), the dependence on EV-delivered miRNAs is more certain.How-ever,this control has not yet been used to our knowledge in can-cer studies.For example,recently,Zhang et al.have proposed that sEV-mediated transfer of astrocyte-derived miRNAs target-ing PTEN leads to the loss of expression of this tumor suppressor in brain metastatic tumor cells,enhancing metastasis outgrowth (Zhang et al.,2015).Even though the hypothesis proposed in this work is very appealing,conclusive demonstration that miRNAs are being transferred through sEVs and that this is the mecha-nism responsible for PTEN downregulation in brain tumor metas-tasis is lacking.While the effects appear dependent on expres-sion of the miRNAs in astrocytes and are lost upon global deletion of Rab27a in the brain,both controls potentially alter many other aspects than miRNA-containing sEV secretion by as-trocytes;thus,speci?c EV-dependent transport of endogenous miRNA remains a model to be tested.
Other studies have explored similar questions,and it is clear that the?eld is working toward a suite of more de?nitive controls. For example,studies showing that co-treatment of recipient cells with an anti-miRNA together with miRNA-carrying EVs estab-lished that the analyzed miRNA is necessary for the functional effect observed(Le et al.,2014;Zhou et al.,2014).But since the anti-miRNA can inhibit expression of both the endogenous and the EV-transported molecule,this control does not actually demonstrate EV-mediated miRNA transfer.In this experimental setting,absence of upregulation of the endogenous pre-or pri-miRNA while the mature miRNA is increased in EV-recipient cells (Basu and Bhattacharyya,2014;Le et al.,2014;Zhou et al.,2014) may be the strongest observation arguing for acquisition of the mature miRNA.
An important mechanistic aspect remains,however,myste-rious.To achieve silencing of their mRNA targets,miRNAs must be associated with the RNA-induced silencing complex (RISC)containing the argonaute2(AGO2)endonuclease(Wilson and Doudna,2013).For endogenous miRNAs,this association is formed following processing of the double-stranded pre-miRNA into miRNA/miRNA*duplex by DICER and subsequent incorpo-ration into the AGO2-RISC-loading complex.Thus,how naked mature miRNAs brought in by EVs can associate with endoge-nous AGO2and compete with an overwhelming amount of endogenous miRNA will remain unclear unless new molecular mechanisms of miRNA transfer into the RISC-loading complex are discovered.The form of miRNA present in EVs(i.e.,as mature single-strand,miRNA/miRNA*duplex or other form, Cell164,March10,2016a2016Elsevier Inc.1229
naked or bound with AGO or other proteins)is generally not re-ported,and it will be important to answer this question in different cell types(e.g.,tumoral or non-tumoral)and physiolog-ical contexts to elucidate the relevance of EVs as ef?cient miRNA carriers.
In that line,a recent study has brought an unexpected and possibly controversial turn to this?eld(Melo et al.,2014).Melo et al.have observed that pre-miRNAs loaded into the RISC ma-chinery are secreted by tumor(but not by non-tumor)cell lines in sEVs and that miRNA maturation takes place extracellularly when sEVs are incubated at37 C.The implication is that trans-ferred AGO2-associated miRNA could thus be directly functional in a recipient cell.It will be interesting to see whether these re-sults are borne out in other types of tumors.Furthermore,the actual nature of the RISC/miRNA carrier will have to be deter-mined more precisely since another study reported that AGO2 secreted in tumor sEVs is not recovered in the same fractions as classical sEV markers(Van Deun et al.,2014).In conclusion, whether EV-mediated miRNA transfer is a functionally relevant communication mechanism in cancer,especially in vivo in the absence of arti?cial overexpression of a miRNA,is still an exciting but not yet fully demonstrated hypothesis.
In addition to miRNAs,EVs are now known to contain several other species of small non-coding RNA or RNA fragments (Nolte-’t Hoen et al.,2012).A recent report shows that miRNA may even be a minor form of RNA in all types of EVs,whereas tRNA fragments and Y-RNAs are speci?cally secreted in EVs (Tosar et al.,2015).It will be very interesting to determine now whether these comparatively poorly studied non-coding RNAs display some of the gene-regulatory functions so far attributed to miRNAs in EVs.Indeed,a recent report showed that tRNA fragments present in sperm regulate gene expression in the embryo and that the levels of these short tRNAs can be altered in response to paternal diet(Sharma et al.,2016).Interestingly, the tRNA fragments are not generated in spermatozoa them-selves but are acquired during their transit through the epidid-ymis,possibly via EVs named epididymosomes,which are secreted by cells forming the epithelium of this canal.The trans-fer of EVs to sperm had been previously documented in Drosophila in vivo(Corrigan et al.,2014)and in large mammals in vitro(Caballero et al.,2013).The novel idea that parental exposure can affect progeny through mechanisms involving transfer of information within EVs is a very promising and exciting hypothesis.
Ties to Immunity
A different consequence of EV-associated RNA transfer in the tu-mor microenvironment has been recently described(Boelens et al.,2014).EV-borne RNAs bearing50-triphosphate ends are recognized in the recipient cell cytosol by the RIG-I sensor, which induces development of an interferon response(including expression of genes like STAT1)similar to that induced upon viral infection.This response was shown to participate in cancer cell resistance to radiation or chemotherapy.
This observation provides one example of a growing appreci-ation of the similarities between sEVs and enveloped viruses (Assil et al.,2015).Indeed,enveloped viruses have recently been shown to carry a nucleotide-based compound,cGAMP,which can reach the cytosol of infected cells and induce an inter-feron response(Bridgeman et al.,2015;Gentili et al.,2015).In Gentili et al.,we observed that cGAMP was present in non-viral sEVs,which could also transfer this signal to recipient cells,but only if they bore a fusogenic viral protein.This observation raises questions about the molecular mechanism involved in fusion of tumor sEVs with recipient cells:what surface molecules allow fusion with the membrane of recipient cells for delivery of the RNA or small-molecule content into the cytosol?Or can other mechanisms contribute,such as formation of channels between the apposed membranes of an EV and the recipient cell?These questions will be important to address.
Conclusions
With the functional implications proposed for EVs,it is now vital to understand these vesicles themselves.As mentioned above, most studies published so far analyze mixed EV populations, and we think that some of the most important steps the?eld must take are to comprehensively compare the different sub-types of EVs and to determine whether some of their functions are speci?c or prominent in a given subtype,e.g.,exosomes, but not other EVs.This knowledge is necessary to identify which EVs should be targeted for any therapeutic approach.Indeed,EV research is now at the stage where the immunology?eld was in the1950s.At that time,researchers could only claim that circu-lating white blood cells were capable of very different functions, such as killing other cells or making antibodies,simply because there were no means to distinguish what we know now as B versus T lymphocytes!For EVs,a recent article reported a calcu-lation that the number of copies of a given miRNA present per EV,in a mixed sEV preparation,is below one(Chevillet et al., 2014),suggesting that either very few miRNA molecules are pre-sent within each sEV or,more likely,that only a restricted sub-type of EVs contain signi?cant amounts of miRNA molecules and thus are capable of transferring miRNA-based information. This situation may apply to other transfer functions of EVs, whether they involve mRNA or proteins:if only a minor subtype of EVs carries the relevant activity,its actual ef?ciency will be dif?cult to detect,as it will be undermined by an abundance of non-functional EVs present in the same preparation.One of the challenges is therefore to re-de?ne methods that allow discrimination between sEVs,exosomes,and other EVs.It is impossible to distinguish them on the basis of a single property, such as size,structure,buoyant density,or presence of a given protein,and the community is seeking novel methods of isolation leading to better enrichment of a speci?c subtype.For this,however,better knowledge of speci?c markers of EV sub-types is required.We have recently performed a quantitative comparison of the protein composition of several subtypes of EVs secreted simultaneously by human primary dendritic cells,which were separated by a combination of differential ultracentrifugation,?oatation in a density gradient,and im-muno-isolation(Kowal et al.,2016).Although a large majority of the proteins were shared between all isolated EV subtypes, including some generally used as‘‘exosome markers’’(e.g., heat shock proteins,?otillins,major histocompatibility complex molecules),we are able to propose a few new speci?c markers of medium and large EVs(e.g.,actinins),of endosome-derived
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exosomes(co-expressing three tetraspanins CD9/CD63/CD81 and including TSG101and syntenin-1),and of non-endosomal sEVs(some ITGs),whose validity as speci?c markers can be tested in all EV sources,which will hopefully enable further functional studies.Indeed,knowing which EV markers to follow will eventually allow identi?cation of molecular tools to specif-ically affect secretion of a given subtype of EVs and thus under-stand the patho/physiological function of a particular subtype. We hope that the near future will thus provide the necessary technical advances and subsequent understanding of the various and fundamental roles of each type of EV.The potential development of these delivery packets for ef?cient therapeutic strategies in cancer and in many other diseases depends on these next steps.
ACKNOWLEDGMENTS
Because of space restriction,only a few recent publications were discussed in this Review;we apologize to all our colleagues whose work has not been cited here.We thank Joanna Kowal,Claire Hivroz,and Lionel Navarro for critical reading of the manuscript.This work has received support under the program ‘‘Investissements d’Avenir’’launched by the French Government and imple-mented by ANR with the references ANR-11-LABX-0043and ANR-10-IDEX-0001-02PSL and by Fondation ARC pour la Recherche sur le Cancer through SL220120605293grant to C.T.and post-doctoral grant to M.T.
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Nolte-’t Hoen,E.N.,Buermans,H.P.,Waasdorp,M.,Stoorvogel,W.,Wauben, M.H.,and’t Hoen,P.A.(2012).Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.Nucleic Acids Res.40, 9272–9285.
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阳光心态演讲稿
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文明守纪中学生演讲稿3篇
文明守纪中学生演讲稿3篇 Speech document of civilized and discipline abiding mid dle school students 编订：JinTai College
文明守纪中学生演讲稿3篇 小泰温馨提示：讲话稿是为了在会议或重要活动上表达自己意见、看法或汇报思想工作情况而事先准备好的文稿，用来交流思想、感情，表达主张、见解，是演讲上一个重要的准备工作。本文档根据讲话稿内容要求和特点展开说明，具有实践指导意义，便于学习和使用，本文下载后内容可随意修改调整及打印。 本文简要目录如下：【下载该文档后使用Word打开，按住键盘Ctrl键且鼠标单击目录内容即可跳转到对应篇章】 1、篇章1：文明守纪中学生演讲稿 2、篇章2：文明守纪中学生演讲稿 3、篇章3：文明守纪中学生演讲稿 文明是什么呢?文明是路上相遇时的微笑，是同学有难时的热情帮助，是不小心撞到对方时的一声“对不起”，是自觉将垃圾放入垃圾箱的举动，是不讲脏话、粗话，是在安静无声的自修课上自觉遵守纪律……文明是一种品质，文明是一种修养，一种受人尊敬并被大家广泛推崇的行为。本文是小泰为大家整理的文明守纪的中学生演讲稿，仅供参考。 篇章1:文明守纪中学生演讲稿
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一个学生问智者：什么是地狱？什么是天堂？ 智者回答：地狱就好像一群人围坐在一口锅周围，每个人手里都有一双很长的筷子，都想把肉夹到自己嘴里，结果谁也无法办到，最后大家都骨瘦如柴，彼此诅咒谩骂。那么天堂呢？ 天堂也是同样的锅与筷子，不同的是大家夹起肉彼此喂食，所有人都可以吃饱，最后大家都满面红光，谈笑风生。 随时随地的愿意为别人付出，任何地方都会是幸福的天堂。 在我们生活的周围，形形色色的人都有，自私心胸狭隘的人往往对别人心存嫉妒，任何利益都想沾边，而从来不愿意帮助别人一点点忙，那么当他遇到困难的时候也很少有人愿意帮他＿就好像地狱里的食客拼命往嘴里夹肉，最后却一无所得。 而另外一些人对别人的点滴帮助都铭记在心，在对待别人的时候也总是愿意多付出一点，在碰到困难的时候也会获得更多的帮助＿像天堂里的食客，总是有更多的人愿意把食物夹到他的嘴里。 付出汗水，才能收获麦穗；付出真诚，才能收获友谊；付出微笑，才能得到友善！我们知道行业给我们策划了一个美好的未来，可以让我们在实现自己的梦想。但罗马不是一天建成的，我们不可能一步登天，这个世界上没有白吃的午餐。不经历风雨，怎会见彩虹？人生不去经历，不去拼搏，不去奋斗，再美好的事业都不可能实现！没有从天而降的幸福，也没有不劳而获的收获！ 斤斤计较的人一生只能得到两斤。我们付出的理由是荣耀和财富，但更多的是对美好人生的追求，对幸福生活的向往，促使我们不断付出努力。一个“以人为本，人人互助”的行业，主动去帮助别人其实也就没是帮助了自己，因为“人”字的结构就是相互的支撑，而“众”人的事业需要每个人的参与和付出。没有哪一个人富得可以不需要别人的帮助，也没有人穷得不能在某方面帮助别人，其关键就在于自身，在心理上的拒绝就表现了行动上的自私自利，我们可以没有金钱没有权势，但是不能没有互相帮助互相关怀的爱心。 独在异乡结识了许许多多的朋友，感谢他们的热情、微笑、鼓励，大家齐心协力为一个哪怕只有1%的希望付出100%努力的精神，让我深受感动！有这样一个强大的团队做后盾，还有什么理由不成功！ 生活就象一面镜子，你对生活微笑，生活也会还你微笑；你给别人机会，别人也会给你机会；你帮助了别人，别人也会在需要的时候帮你一把。感谢并快乐的付出，不仅仅是一种美德，更是一种幸福，也是另外一种形式的收获。在帮助别人的同时也收获了高尚，收获了自我心灵的满足与幸福，收获了别人的友善与信赖。付出就好象在黑暗中为别人点亮了蜡烛，在照亮别人的同时也照亮了自己。
奋进强军新时代 演讲稿
奋进强军新时代 “惟希望也，故进取；惟进取也，故日新。”党的十九大作出了“中国特色社会主义进入了新时代”的重大判断，这是新时代坚持和发展中国特色社会主义的政治宣言和行动纲领，向全党全军全国各族人民发出了奋进新时代、开启新征程的进军号令。时代的面貌日新月异，唯有紧跟潮流、砥砺奋进、奋勇拼搏，奋进强军新时代，方能开创美好未来。 为什么要奋进强军 强国必先强军，强军才能安邦。 历史经验教育我们牢记强军。从1860年第一次鸦片战争以来近一百年的屈辱的近代史深深的展示了一个民族如果只有富裕的经济而没有强大的军队，那么只会沦为“人为刀俎，我为鱼肉”。中华民族作为有悠久历史的四大文明古国之一,历史上长期居于世界经济发展的前列,但是到了近代,西方列强用坚船利炮,打开了中国的大门,疯狂地瓜分抢劫中国。近百年的屈辱,使我们明白了一个道理——民族复兴必须有强有力的国防力量作保障。 当前局势促使我们加紧强军。我们不是出生在一个和平的年代，而是出生在一个和平的国家。进入新时代，中国周边地区形势发生着深刻而复杂的变化。有利因素和不利因素的存在与发展使周边地区形势呈现出总体稳定与局部动荡并存的局面。与此同时，周边安全环境在地缘方向上存在较大的差异性，表现为“陆稳海动、陆缓海紧”和“北稳、南和、东紧、西动”的不同态势。在外部因素中，台海问题，南海争端，中印边界争端，朝鲜半岛问题等，一直都是威胁着我国的安全，其中，美国是影响中国周边安全环境的最主要因素。由此可见，如果不加紧强军备战，一旦国际形势有变，将对我国安全及发展产生重大威胁。 实现复兴需要我们继续强军。今天，我国正处于由大向强的关键阶段，正处于近两百年来最好的发展时期，国家的命运、复兴的征程与军队的现代化建设紧密地联系在一起。我军要为实现中国梦提供安全保障和战略支撑，困难不比战争年代少。当前，我国仍然面临严峻复杂的挑战，对我军履行新时代使命任务是一种严峻考验。只有登高望远、居安思危，深入推进练兵备战，扎实开展实战化训练，提升部队战斗力，才能掌握主动，有效化解风险，应对挑战，实现发展强大、复兴民族的重任。 奋进强军的意义 是我们党建军治军理念的凝练升华 在不同的历史阶段，我党根据形势任务的变化，及时提出明确的目标要求，引领我军建设不断向前发展。在国防和军队建设的实践中，我们党始终强调必须坚持党对军队绝对领导的根本原则，始终强调军队必须履行战斗队的根本职能，始终强调军队必须牢记全心全意为人民服务的根本宗旨，使军队建设发展与党的历史任务相一致、与国家发展战略相协调、与国家地位相适，充分彰显了我党建军的主张，展示了我党治军的理念，体现了我党伟大思想的发展进步。 是实现民族复兴中国梦的战略抉择 伟大的中国梦，承载着全体中华儿女的共同向往，体现了中华民族和中国人民的整体利益，昭示着国家富强、民族振兴、人民幸福的美好前景。强国梦蕴含强军梦，强军梦支撑强国梦，富国和强军，是实现中华民族伟大复兴的两大基石，两者相辅相融，互为支撑。中华民族伟大复兴绝不是轻轻松松、顺顺当当就能实现的，我国由大向强发展进程中无法回避会出现各种挑战，这是实现中华民族伟大复兴绕不过的门槛。建设一支强大的人民军队，实现国家富强、民族振兴、人民幸福才能有更好的保证。 是加快推进国防和军队现代化的根本遵循 经过多年的发展，我军建设站在了新的历史起点上，虽然我军建设有了大的发展进步，但现代化水平与国家安全需求相比差距还很大，与世界先进军事水平相比差距还很大，打现代化战争能力不够。定好强军目标体现了对军队建设发展现状的清醒认识，能使广大官兵清醒起来、警醒起来、发奋起来，正确看待存在的差距不足，找到解决矛盾问题、应对挑战考验的思路办法，进一步激励广大官兵攻坚克难、勇于超越，焕发投身强军兴军实践的高昂斗志。 怎样去奋进强军 铸牢强军之魂，坚定理想信念
文明建设演讲稿：文明守纪,团结进取,争创先进班集体
文明建设演讲稿：文明守纪，团结进取，争创先进班集体 老师们，同学们大家上午好： 新的学期已经拉开了帷幕，全校xx个班级xx多名同学今天齐聚在国旗下，意味着新的开始，新的征程，也蕴含着新的奋斗和新的辉煌。李校长在开学工作中明确指出：学生行为规范不仅是守好学生学习与成长的底线，更是营造良好的学习氛围、育人文化和提升学生做人做事的核心素养。我们要以完善文明班集体、优秀班主任考核评价为导向，以大数据管理为依托，使常规管理推陈出新。那么我今天国旗下讲话的题目是“文明守纪，团结进取，争创先进班集体”。 上学期末，学校启动了最新的班级常规考核机制，对于各班级各方面的表现以大数据的方式进行量化统计。负责提供考核数据的不仅有学生会的同学，医务室的老师，更有学校行政和值班老师。同学们可能已经看到，在学校文化书廊内我们升起了班旗，获得升旗资格的都是在文明班集体考核中表现优秀的班级。本学期，学校加强了值班工作，尤其是加强了班主任值班，值班行政和老师将加大对同学们日常行为规范的督查力度，这就要求同学们时刻牢记规范，用文明、勤奋书写省xx的求学生涯。在此我提出几点要求： 一、珍惜时间，做勤奋的xx学生。本学期是考试年，高一将面临新高考方案公布后的选科分班，高二迎接学业水平测试同时将做好准高三的准备，高三全力冲刺高考。这都要求同学们有良好的学习习惯。我虽然很感动我们的同学在宿舍打手电熬夜背书做作业，但绝不赞同，如果长期熬夜让你白天课堂效率低下，那是得不偿失的。所以我们要合理安排学习时间，不能成为刷作业的机器，要真正成为自主学习的主人。 二、文明礼貌，做有素养的xx学生。高一军训的时候，教官指导我们要懂礼貌，路上见到师长大声问候，同学有难时热情帮助。这些最起码的文明素养，我们有的同学慢慢淡忘了。于是同学们的宿舍不再象军训时整洁干净，教室与清洁区的卫生也需要时常提醒。我曾经拍过一段我们学生励志广场扫地的视频，“轻轻的我扫过了，未带走一片落叶”，还有的同学将落叶清扫后，不愿多走几步路，直接将落叶倒在周边的草坪中，美其名曰“回归大自然，生态系统的分解者会自动分解”。看到这些场景，试问省xx学生的文明素养何在？我们要做文明太仓人，我们不仅要创建文明校园，更要成为太仓创建文明城市的生力军。“社会主义核
付出心态演讲稿
付出心态演讲稿 Revised as of 23 November 2020
付出心态演讲稿 核心提示：提起付出心态，我立刻想起了“受得苦中苦，方为人上人”，这名格言，这句格言虽然是激励人艰苦奋斗，奋发向上的诗意，之间也包含了付出的意思，“受得苦中苦”实际就是一种不平凡的付出，你看那位古代练武的，为了练一身好武艺，冬练三九，夏练三伏，练的一身伤痕，两手老茧，吃尽了苦头，才有了成就，再看古代习文的书生，... 一个学生问智者：什么是地狱什么是天堂 智者回答：地狱就好像一群人围坐在一口锅周围，每个人手里都有一双很长筷子，都想把肉夹到自己嘴里，结果谁也无法办到，最后大家都骨瘦如柴，彼此诅咒谩骂。 那么天堂呢 天堂也是同样的锅与筷子，不同的是大家夹起肉彼此喂食，所有人都可以吃饱，最后大家都满面红光，谈笑风生。 随时随地的愿意为别人付出，任何地方都会是幸福的天堂。 在我们生活的周围，形形色色的人都有，自私心胸狭隘的人往往对别人心存嫉妒，任何利益都想沾边，而从来不愿意帮助别人一点点忙，那么当他遇到困难的时候也很少有人愿意帮他＿就好像地狱里的食客拼命往嘴里夹肉，最后却一无所得。 而另外一些人对别人的点滴帮助都铭记在心，在对待别人的时候也总是愿意多付出一点，在碰到困难的时候也会获得更多的帮助＿像天堂里的食客，总是有更多的人愿意把食物夹到他的嘴里。
付出汗水，才能收获麦穗；付出真诚，才能收获友谊；付出微笑，才能得到友善！我们知道行业给我们策划了一个美好的未来，可以让我们在实现自己的梦想。但罗马不是一天建成的，我们不可能一步登天，这个世界上没有白吃的午餐。不经历风雨，怎会见彩虹人生不去经历，不去拼搏，不去奋斗，再美好的事业都不可能实现！没有从天而降的幸福，也没有不劳而获的收获！斤斤计较的人一生只能得到两斤。我们付出的理由是荣耀和财富，但更多的是对美好人生的追求，对幸福生活的向往，促使我们不断付出努力。一个“以人为本，人人互助”的行业，主动去帮助别人其实也就没是帮助了自己，因为“人”字的结构就是相互的支撑，而“众”人的事业需要每个人的参与和付出。没有哪一个人富得可以不需要别人的帮助，也没有人穷得不能在某方面帮助别人，其关键就在于自身，在心理上的拒绝就表现了行动上的自私自利，我们可以没有金钱没有权势，但是不能没有互相帮助互相关怀的爱心。独在异乡结识了许许多多的朋友，感谢他们的热情、微笑、鼓励，大家齐心协力为一个哪怕只有1%的希望付出100%努力的精神，让我深受感动！有这样一个强大的团队做后盾，还有什么理由不成功！生活就象一面镜子，你对生活微笑，生活也会还你微笑；你给别人机会，别人也会给你机会；你帮助了别人，别人也会在需要的时候帮你一把。感谢并快乐的付出，不仅仅是一种美德，更是一种幸福，也是另外一种形式的收获。在帮助别人的同时也收获了高尚，收获了自我心灵的满足与幸福，收获了别人的友善与信赖。付出就好象在黑暗中为别人点亮了蜡烛，在照亮别人的同时也照亮了自己。如果你此时正取得成功，而你一直没有做出牺牲，那是走在前面的人已经为你付出，做过一些惠及于你的事情。如果你此时正付出着，但你又见不到任何成功，不用担心，总会有人在日后享受这些付出带来的果实，无论是你自己还是别人。赠人玫瑰，手留余