Emerging role of exosome signalling in maintaining cancer stem cell dynamic equilibrium

Abstract Cancer stem cells (CSCs) are a small subset of heterogeneous cells existed in tumour tissues or cancer cell lines with self‐renewal and differentiation potentials. CSCs were considered to be responsible for the failure of conventional therapy and tumour recurrence. However, CSCs are not a static cell population, CSCs and non‐CSCs are maintained in dynamic interconversion state by their self‐differentiation and dedifferentiation. Therefore, targeting CSCs for cancer therapy is still not enough,exploring the mechanism of dynamic interconversion between CSCs and non‐CSCs and blocking the interconversion seems to be imperative. Exosomes are 30‐100 nm size in diameter extracellular vesicles (EVs) secreted by multiple living cells into the extracellular space. They contain cell‐state‐specific bioactive materials, including DNA, mRNA, ncRNA, proteins, lipids, etc. with their specific surface markers, such as, CD63, CD81, Alix, Tsg101, etc. Exosomes have been considered as information carriers in cell communication between cancer cells and non‐cancer cells, which affect gene expressions and cellular signalling pathways of recipient cells by delivering their contents. Now that exosomes acted as information carriers, whether they played role in maintaining dynamic equilibrium state between CSCs and non‐CSCs and their mechanism of activity are unknown. This review summarized the current research advance of exosomes’ role in maintaining CSC dynamic interconversion state and their possible mechanism of action, which will provide a better understanding the contribution of exosomes to dedifferentiation and stemness acquisition of non‐CSCs, and highlight that exosomes might be taken as the attractive target approaches for cancer therapeutics.

experimental investigations suggested that CSCs could be isolated from several types of cancers, including leukaemia 2-4 and solid tumours. [5][6][7][8][9] CSCs possess unlimited self-renewal and multilineage differentiation potentials, and played very important roles in tumour initiation, recurrence, metastasis and therapeutic resistance. [10][11][12] Due to their properties, CSCs were considered to be responsible for the failure of traditional (surgical operation, radiotherapy and/or chemotherapy) and target therapy as well as tumour relapse. CSCs can be isolated and characterized from tumour tissues and cell lines based on their surface markers (such as, CD133, CD24, CD90, ALDH1, etc.) and Hoechst 33342 exclusion by flow cytometry, and selectively cultured with SFM. [13][14][15] CSCs have their own specific surface markers and signalling pathways, which might offer an optimistic opportunity to target eliminating CSCs and/or induce them differentiation for preclinical anti-cancer therapy. However, recent studies indicated that CSCs are not a static cell population with the capacity of initiating tumour through asymmetric cell division, but a cell population in highly dynamic equilibrium state, which could be maintained through the dedifferentiation of matured cancer cells. [16][17][18][19] Until now, the CSC plasticity or dedifferentiation of matured cancer cells (or non-cancer stem cells, non-CSCs) with acquisition of stem-like properties was reported in several cancers. [20][21][22][23] Taken together, all the findings mentioned above suggest that CSCs and non-CSCs are not in a motionless but in a dynamic equilibrium state: CSCs differentiate into non-CSCs under some circumstances, and non-CSCs could dedifferentiate into CSCs. Therefore, targeting CSCs seems to be not enough, and blocking the process of non-CSC dedifferentiation to disturb the dynamic equilibrium between CSCs and non-CSCs appears to be particularly important for cancer therapeutics. However, the cellular and molecular mechanisms of interconversion between differentiated non-CSCs and CSCs are unclear. More recently, emerging evidence revealed that the molecular cross-talking between CSCs and non-CSCs in tumour microenvironment plays a critical role in this process. The intercellular communication between tumour cells and other cells is accomplished via cell-cell interactions. Tumour cell released growth factors, chemokines, proteins, mRNAs, microRNAs, etc., are carried and transferred by carriers. Exosomes may serve as important molecular information carriers to communicate with CSCs, non-CSCs and other cells in tumour microenvironment. 24 Exosomes, nanovesicles originated from the endosome, are 30-100 nm EVs released by all types of cells. 25 They modulate intercellular communication by transfer their molecular contents between different types of cells. 26 Cancer cell released exosomes that play a crucial role in the intracellular communications involved in cancer progress. 27 These tumour-cell-derived exosomes are found in all body fluids, upon contact with target cells, they can alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to tumour growth, metastasis and immunosuppression. [28][29][30] However, the role of exosomes in the reciprocal conversion between non-CSCs and CSCs was rarely investigated. As information carriers between cells, whether exosomes regulate non-CSC dedifferentiation in CSC dynamic equilibrium, and targeting exosome signalling could attenuate the production of CSCs and finally eradicate cancers is worthy of serious study.

MICROENVIRONMENT
Emerging evidence have been found that non-CSCs could be reprogrammed and transform into CSC-like cells, which indicated that CSCs and non-CSCs were in a dynamic equilibrium state ( Figure 1A), and the molecular crosstalk between cancer cells and CSCs in tumour microenvironment seems to play an important role in maintaining of this dynamic equilibrium. Recent study showed that M2 TAMs-derived prostaglandin E2 (PGE2) could endow colon cancer cells with stem-like qualities; this effect was abolished by celecoxib, the COX-2-selective inhibitor, which blocks PGE2 production. 36 Similarly, other cytokines such as EGF, TGF-b1 and IL-6 released by TMAs also promote transforming of non-CSCs into CSCs. [37][38][39] MDSCs played an important role in stemness phenotypic plasticity of non-CSCs through activation of notch signalling, EMT and up-regulation of stemness genes, such as, Nanog, Oct4 and Sox2, 40,41 previous study showed that MDSCs released IL-6 activated stat3/notch signalling pathway in breast cancer cells and endow these cells with stem-like properties. 42 In addition, CSCs could also dictate the characters of their surrounding stromal cells by secreting a variety of factors to promote tumour progression. The study showed that CSCs induced the transition of fibroblasts to CAFs by secreting TGF-b, breast CSCs can produce IL6, which attracts and activates MSCs to produce the CSC-supportive cytokine CXCL7. 43 Interestingly, recent studies reveal that a set of stemness and Previous studies showed that cancer cells can release and uptake small EVs containing a subset of the membrane and cytosolic proteins, RNA and lipids within the tumour microenvironment, which leads to reprogramming of recipient cells, including stemness phenotype. 44 Thus, it is reasonable to assume that EVs, including exosomes, might mediate the cell communication between non-CSCs and CSCs, and play important roles in maintaining the dynamic equilibrium state of CSCs.

| EXOSOME BIOLOGY
The term "exosome" was first used to describe the exfoliated microvesicles ranging from 40 to 1,000 nm released by various normal and neoplastic cells with ectoenzymes activity by Trams et al in the early 1980s. 45 In 1983, Stahl et al 46 50 During this process, the cytoplasmic DNA, RNA and proteins are specifically sorted into ILVs (pre-exosomes; Figure 2B). Interestingly, evidence also confirmed that the proportion of proteins and RNAs in exosomes was The role of exosomes in maintaining cancer stem cell dynamic equilibrium. A, The dynamic equilibrium between CSCs and non-CSCs: CSCs differentiated into cancer cells, and cancer cells dedifferentiated into CSCs. B, C, The role of exosomes in the reciprocal conversion between non-CSCs and CSCs: CSCs-derived exosomes may induce the dedifferentiation of cancer cells to acquire stemness phenotype through transfer their stemness-related molecules; cancer cellderived exosomes may also affect the surrounding cells within tumour microenvironment as well as these nontumour cells could promote tumour initiation and progression by releasing exosomes. Dotted line means the exosome-mediated cell-cell communication | 3721 different from that in the originating cells, which indicates that there are some specific mechanisms involved to control the sorting process of specific contents into exosomes. Actually, the specific sorting process of proteins cargo into exosomes is regulated by various pathways, including endosomal sorting complexes required for transport (ESCRT), 51 tetraspanins 52 and lipid-dependent mechanisms. 53 Moreover, researchers further found that a zip code in the 3 0 -UTR presented in exosomal mRNAs, which may guide their sorting to exosomes. 54 Other studies also found that a specific motif (GGAG) has been involved in the loading of specific miRNAs into exosomes through the interaction with specific chaperone proteins. 55

| EXOSOME RELEASE
Once formed, these MVBs will be mobilized to the cell periphery, and subsequently fused with the plasma membrane to release the exosomes into extracellular space. 56,57 In this process, several cellular model systems, including cytoskeleton, Rab GTPase and the fusion machinery, were found to be involved in transporting MVBs to the sites of the plasma membrane and their docking. 58,59 After docking of two different intracellular compartments, the SNARE complexes drive the fusion of MVBs with the plasma membrane, and finally exosomes were secreted into outer-cellular milieu 50,60 ( Figure 2C).
Interestingly, the secretion of exosomal microRNA (miRNA) can be regulated by the neutral sphyngomyelinase 2 (nSMase2), the release of exosomes was reduced after inhibiting the activity of nSMase2 with GW4869, and overexpression of nSMase2 increased extracellular amounts of miRNAs, hypoxia promoted exosomes release along with exosomal miRNA increase, while some drugs could inhibit exosome secretion. [61][62][63] These findings indicate that the generation and secretion of exosomes are of modulation and selectivity, and whether this property could be used for cancer therapy needs deeper study.

| EXOSOME UPTAKE
The elicitation of exosomal functions mediating cell-cell communication happened after the uptake by recipient cells. The first step of uptake is the binding of exosomes to the surface of recipient cells, which is mediated by the specific receptors. 64,65 After initial binding, exosomes will be internalized by recipient cells through endocytic processes, or directly fusion with the cellular membrane and releasing their contents into the cytoplasm and finally regulate cellular pathways 66,67 ( Figure 2D). In addition, a recent study showed that treatment of exosomes with proteinase K significantly reduced their uptake by cancer cells. 68 This indicates that the uptake of exosomes by recipient cells might not be a random process, and specific molecules expressed on exosomes may serve as receptors for uptake. Therefore, blocking the binding between exosomes and recipient cell might be a promising strategy for inhibiting exosome uptake.  has shown to promote cell proliferation and tumorigenesis 84 as well as enhance stemness phenotype of glioblastoma cells 85 by targeting upstream or downstream genes. In addition, miR-200 was found to enriched in EVs from metastatic breast cancer cells which transferred to non-metastatic cells and then promoted mesenchymal-toepithelial transition of recipient cells. 86 Challagundla et al 87 found that neuroblastoma (NBL)-derived exosomes could transfer miR-21 into human monocytes, and then up-regulate miR-155 levels to enhance NBL drug resistance. Moreover, certain stemness and metastasis-related mRNA were found to be enriched in breast cancer stem-like cell-derived exosomes that could stimulate tumour progression. 88 Besides, several studies demonstrated that long non-coding RNA Fortunately, recent studies revealed the possible mechanisms involved in the exosome biogenesis, release and uptake; thus, many potential strategies to interrupt exosome-mediated signalling can be envisioned ( Figure 2). For example, the ESCRT machinery is known to be involved in the formation of MVBs and ILVs, and knockdown the components of ESCRT, such as HRS, 97 STAM1 or TSG101, 51 could inhibit the exosome biogenesis. Besides ESCRT machinery, several lipids and lipid metabolizing enzymes were suggested to also regulate this process in some cells. 52,98 Inhibiting nSMase activity by hydrochloride hydrate (GW4869) or RNAi could reduce exosome production and prion packaging. 99 Rab27 family is small GTPases to regulate exosome release. Knockdown Rab27 via RNAi reduced exosome release, tumour growth and the dissemination of metastatic colonies significantly, 100 Conversely, overexpression of EPI64, a candidate GAP that is specific for Rab27, could promote exosome secretion in lung cancer cells. 101 Exosomes could be internalized by recipient cells through endocytic processes, or directly fusion with the cellular membrane, heparan sulphate proteoglycans have been implicated in this process, and treatment with heparin significantly inhibit exosome uptake by cancer cells. 102 Another strategy might be involved in manipulation of the exosome cargo. For example, treatment with vemurafenib, the BRAF inhibitor, significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles in the vesicular secretome of malignant melanoma cells. 103 In addition, inhibition of ESCRT components or aSMase activity also modulates the nature and content of the vesicles. 104 Therefore, inhibition of exosome biogenesis, release, uptake or modification of exosome cargo in tumour microenvironment may have beneficial effects on blocking the interconversion between CSCs and non-CSCs.

VEHICLES FOR TARGETED CANCER THE RAPY
Unlike synthetic nanoparticles, the lipid bilayer membrane of exosomes can pass through the blood-brain barrier and have low toxicity and immunogenicity. 105 Anti-inflammatory or chemotherapeutic drugs delivered by exosomes exhibited much stronger stability, bioavailability and effectiveness, and no toxicity compared with conventional therapy. 106 Perhaps, exosomes have great potential for targeting CSCs. For example, in a human lung tumour xenografts model, exosomes-delivered paclitaxel showed significantly inhibit tumour growth in vivo with remarkably lower systemic and immunologic toxicities as compared with i.v. injection of paclitaxel. 107 Moreover, exosomes can be used as natural nanovesicles to deliver exogenous small RNAs, including siRNA and microRNAs, to target tissues and/or cells for gene therapy, the targeting effect of exosomes on cancer cells can be enhanced by modifying their surface molecules, such as, coated with CSC marker antibodies and anti-cancer drugs. Exosomes designed to express iRGD-Lamp2b showed highly efficient targeting potential and Dox delivery capacity to av integrin-positive breast cancer cells in vitro and in vivo. Besides anti-cancer therapeutic drugs, exosomes can also deliver various tumour antigens. Thus, exosomes could present CSC-specific antigens to T cells and activate T cells for anti-CSC immunization. 105 In addition, exosomes can be used as natural nanovesicles to deliver exogenous small RNAs, including siRNA and microRNAs, which target CSC-specific signal pathways, such as Wnt, Notch, Hippo, and Hedgehog, etc. for CSC targeting therapy. Therefore, developing more specific CSC targeting exosomes will be a promising cancer therapy strategy in the future.

| CONCLUDING REMARKS
Originally, CSCs were thought to be a fixed subset of cancer cells Based on that exosomes-mediated transformation between non-CSCs and CSCs in tumour microenvironment mentioned above, targeting exosomes would be a promising strategy for cancer therapy.
On the one hand, more and more molecular targets in exosome regulating non-CSCs dedifferentiating were explored and corresponding molecular inhibitors usher in the dawn such as GW4869, heparin, etc.; on the other hand, controlling exosome biogenesis, release or uptake through regulating donor or recipient cells could also block non-CSCs dedifferentiating. Moreover, exosomes as drug-delivery vehicles attracted a wide spread attention; through structure modification, such as surface marker enhancing or replacing, the designed exosomes are more suitable for delivery vehicles, and possess high specificity and stability.
Taken together, exosomes existed in the tumour microenvironment, acted as information carriers, played important and essential roles in maintaining the dynamic equilibrium state between non-CSCs and CSCs. Facing the difficulties and challenges in effectively targeting CSCs for cancer eradication, future studies will be focused on comprehensive understanding cell-specific biogenesis, content sorting of exosomes, and exosomes-recipient cell affinity 108

ACKNOWLEDG EMENT
This work was supported by National Natural Science Foundation of China (Grant number 31371148).

CONFLI CT OF INTEREST
The authors declare that they have no conflicts of interest.