The emerging role of exosomes in multiple myeloma

Blood Reviews 38 (2019) 100595

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Review

The emerging role of exosomes in multiple myeloma a

b,c

Milad Moloudizargari , Mohammad Abdollahi , Mohammad Hossein Asghari ⁎ Alina Andreea Zimtae, Ioana Berindan Neagoee,f, Seyed Mohammad Nabavig,

d,⁎

T

,

a

Department of Immunology, School of Medicine, Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran c Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran d Department of Pharmacology and Toxicology, School of Medicine, Babol University of Medical Sciences, Babol, Iran e MedFuture Research Center for Advanced Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, 400337 Cluj-Napoca, Romania f Research Center for Functional Genomics, Biomedicine and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, 400337 Cluj-Napoca, Romania g Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cancer Plasma cell Extracellular vesicle Stromal cell Exosome

Multiple myeloma (MM), one of the most prevalent hematological malignancies, accounts for approximately 10% of all blood cancers. In spite of the recent advancements in MM therapy, this malignancy of terminally differentiated plasma cells (PCs) continues to remain a hard-to-cure disease due to the emergence of drug resistance and frequent relapses. It is now well-established that the tumor-supportive involvement of the bone marrow microenvironment (BMM) including the cellular and non-cellular elements are the major causes behind treatment failures of MM as well as its main complications such as osteolytic bone loss. Exosomes (EXs) are membranous structures that carry signaling molecules and have recently received a great deal of attention as important mediators of inter-cellular communication in health and disease. EXs involve in the growth and drug resistance of many tumors via delivering their rich contents of bioactive molecules including miRNAs, growth factors, cytokines, signaling molecules, etc. With regard to MM, many studies have reported that EXs are among the main culprits playing key roles in the vicious network within the BMM of these patients. The main producers of EXs that largely contribute to MM pathogenesis are bone marrow stromal cells (BMSCs) as well as MM cells themselves. These cell types produce large amounts of EXs that affect a variety of target cells including natural killer (NK) cells, osteoclasts (OCs) and osteoblasts (OBs) to the advantage of tumor survival and progression. These EXs contain a different profile of proteins and miRNAs from that of EXs obtained from their counterparts in healthy individuals. MM patients exhibit distinguishable elevations in some of their contents such as miR-21, miR-146a, let-7b and miR-18a, while some molecules like miR-15a are markedly downregulated in EXs of MM patients compared to healthy individuals. These findings make EXs desirable biomarkers for early prediction of disease progression and drug resistance in the context of MM. On the other hand, due to the tumor-supportive role of EXs, targeting these structures in parallel to the conventional therapeutic regimens may be a promising approach to a successful anti-MM therapy. In the present work, an extensive review of the literature has been carried out to highlight the recent advances in the field.

1. Introduction Multiple myeloma (MM) is a cancer of plasma cells (PCs) within their late stages of differentiation mainly diagnosed by the presence of higher than 10% monoclonal long-lived PCs within the bone marrow (BM) population and bony/extra-medullary plasmacytoma in the biopsy plus evidence of end-organ damage [1–3]. MM patients demonstrate increased numbers of circulating PCs and higher proportions

⁎

of monoclonal free light chains (FLCs) in their sera, which is of diagnostic significance [4]. These patients frequently present with back pain and may have clinical signs including, anemia, dehydration, renal failure, proteinuria and osteolytic bone lesions in their bone scans [5–7] (Fig. 1). Almost all the clinically observable pathology of MM is due to the overproduction of aberrant monoclonal antibodies and FLCs. Beside the basic genetic elements known to be involved in the development of the founder transformed clone of PCs, the BM microenvironment

Corresponding authors. E-mail addresses: [email protected] (M.H. Asghari), [email protected] (S.M. Nabavi).

https://doi.org/10.1016/j.blre.2019.100595

0268-960X/ © 2019 Elsevier Ltd. All rights reserved.

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M. Moloudizargari, et al.

phenotype of a typical MM patient [13,14]. Albeit, the finding that mesenchymal stem cells (MSCs) from healthy individuals can acquire the tumor-promoting phenotype of MM patient-derived MSCs following co-culture with MM cells gives rise to the notion that MM cells are the first initiators of such a mutual communication within the BMM [15]. These findings may, at least partly, explain how EXs in the BMM including both MM-derived EXs and those of other cellular origins can complement the genetic and epigenetic predisposition of the founder MM clone to drive the cell towards the final stages of malignant transformation as well as its survival and proliferation. Moreover, it is now understood that despite the tremendous improvements in the overall survival that can be achieved using the current therapeutic choices, relapsed/refractory disease is still a major problem which has been mainly attributed to the role of BMM [16–18]. Therefore, the novel MM research has been skewed towards the development of medications that influence BMM such as proteasome inhibitors, immune checkpoint inhibitors against PD1 and PDL1 (Programmed cell death protein 1 and its ligand, respectively) [19], immunotherapies and inhibitors of harmful cell signaling pathways [4,20]. Acknowledging this and the recent prominent findings regarding the importance of EXs in the pathogenesis of MM and their potential to be exploited for therapeutic purposes, an extensive literature review was performed to present the latest information regarding the role of EXs in MM development and progression and their potential applications as diagnostic markers and therapeutic tools. 2. Biogenesis and trafficking of EXs Fig. 1. The clinical symptomatology of multiple myeloma (MM) includes: frequent infections, back pain, renal failure, proteinuria and frequent bone lesions due to osteolysis.

EXs constitute a portion of extracellular vesicles with a diameter ranging between 30 and 100 nm. These membranous structures, produced by a wide range of cells, have been shown to be involved in various physiological functions such as maintenance of the cross-talk between the hematopoetic system and distant sites in the body [9,21], T cell stimulation and antigen transfer to dendritic cells (DCs) within the immune system [22], and maintenance of cell homeostasis via removal of unwanted molecules [23]. EXs are also involved in some pathological processes such as malignant transformation of cells and induction of pre-metastatic niche in the context of malignancies [21,24]. These structures constitute a conserved collection of molecules including tetraspanins [25], various other proteins, different RNA species and lipids. The modulation of the targeted cell is highly dependent on the specific profile of EX cargo [26]. Additionally, EXs reflect the surface properties of their cellular source, since they normally share common surface markers with their cells of origin [9]. The biogenesis of EXs can be simply summarized in the following steps: 1) initially, early endosomes give rise to the formation of small intraluminal vesicles (ILVs) [27]; 2) these ILV-containing structures then transform and become multivesicular bodies (MVBs), a process which is mainly regulated by a group of endosomal sorting complexes required for transport (ESCRT) each responsible for different events during the maturation process including the identification and sorting of ubiquitinated proteins into the vesicles by ESCRT-0, engulfment of the confined ubiquitinated proteins by ESCRT-I and ESCRT-II, and the detachment of the matured vesicle from the endosomal structure [28]; 3) finally, fusion of the EXcontaining MVBs with the plasma membrane of the cell via the action of soluble N-ethylene maleimide-sensitive factor attachment protein receptors (SNAREs) gives rise to the release of EXs into the extracellular space [29]. Although this sequence of events has been shown to be the main pathway of EX biogenesis in many cells, other ESCRT-independent pathways including a pathway involving the neutral sphingomyelinase 2 (nSMase) and one involving the fusion of small microdomains have been explained [21]. All together, these processes result in the formation of different populations of EXs [30–32]. Given that EXs are key players in maintaining the intercellular communications, their biogenesis, trafficking, and final uptake by target cells require to be finely regulated [33]. Recent studies have revealed that like many other

(BMM) has been demonstrated to play a prominent role not only in the pathogenesis of MM and its resistance to chemotherapeutic agents, but also in the malignant transformation of less pathogenic forms of PCrelated conditions including monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) into MM [1,4]. In addition to the genetic and epigenetic events that precede the later functional changes in the course of MM development, a variety of alterations in the BMM including the presence of specific soluble factors, enhanced state of angiogenesis and osteoclast (OC) production coupled with dampened immune surveillance and osteoblastogenesis give rise to the proliferation, migration and drug resistance of mutated PCs as well as their decreased apoptosis and DNA repair function [1]. Hence, the cross-talk of MM cells and other cellular/non-cellular elements of the BM is a major determinant of disease progression and response to treatment [8]. Such a cross-talk takes place via directs cellto-cell contact and production of soluble factors including cytokines, growth factors and extracellular vesicles mainly exosomes (EXs). EXs are critical components of the BMM secretome and carry a variety of biologically active molecules mainly nucleic acids and proteins [9]. These spherical structures are shown to play crucial roles in facilitating information transfer between the original EX-producing cell and the target cell(s) [10]. Previous studies revealed that the content of EXs released by BM mesenchymal cells and BM stromal cells (BMSCs) differ significantly between MM patients and healthy individuals [11]. MM cells, in turn, produce an altered profile of free molecules (including cytokines, growth factors) and EXs [5], which per se, further affect the BMM in favor of tumor progression giving rise to a vicious self-potentiating cycle. For instance, MM cells produce EXs which contain miRNAs that turn the normal stromal cells into tumor-supporting cells via producing a modified profile of tumor-promoting cytokines [12]. Whether the alterations in the BMM precedes the altered secretory profile of MM cells or vice versa is not completely understood; however, it is clear that a strong cross-talk between the MM cells and their microenvironment in the BM exists, which gives rise to the final 2

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intracellular mechanisms such as the apoptosis pathway within the MM cells [9]. The exosomal cargo can be mainly classified into non-coding RNAs, soluble proteins and EX-bound surface ligands [47]. These EXs exert their effects via delivering an altered profile of cargo compared to that of EXs secreted by healthy PCs. For instance, the downregulation of oncosuppressor non-coding RNAs like miR-15a and the upregulation of tumor-promoting cytokines, chemokines and proteins such as IL-6, CCL2, CD 146 are among some of the previously studied elements of such a MM-promoting conversion of EX contents [13]. There is also an example showing that MM-EXs negatively affect the NK-mediated immune response via their shedding of NKG2D ligands, which induces the downregulation of this activating receptors on the surface of NK cells [53]. Other molecules capable of promoting tumor progression such as matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) have also been shown to greatly improve tumor invasion in the context of other cancers [54,55], which remain to be also studied in MM-EXs.

physiological processes in the body, EX production is regulated by a negative feedback mechanism [34]. There are also a variety of tightly related molecules including syndecan, syntenin and (ALG-2)-interacting protein X (ALIX) (collectively known as the syndecan-syntenin-ALIX axis) that, in parallel with the ESCRT complexes regulate EX formation and release [35,36]. Heparanase triggers this axis by the cleavage of heparin sulphate residues and increases EX formation [37]. Rab proteins, which are known for their roles in regulating intracellular trafficking, are also involved in regulating EX biogenesis [38]. There are also studies that link autophagy, an important cellular regulatory mechanism, to EX biogenesis [39,40]. It has been shown that autophagymediated regulation of EX biogenesis is done via non-conventional autophagy pathways and by virtue of changing the pH of the endosomes in which ILVs are formed [39]. EX regulation mechanisms can also be found at the level of EX uptake by the target cells. For instance, in the context of MM, it has been reported that the fibronectin on the surface of EXs produced by resident cells of BMM can interact with the heparin sulphate of their target cells such as BMSCs and endothelial cells and induce several signaling pathways involved in MM progression [41]. EXs induce a variety of diverse biological activities in health and disease via their contents consisting of a great diversity of proteins, nucleic acid sequences, etc. [42]. Almost all the cells in human body use EXs as signal vehicles to selectively transfer their content to specific target cells [43]. miRNAs are among the most important signaling molecules which are sorted into EXs, making them desirable therapeutic targets and/or diagnostic biomarkers. An interesting finding is that EX-producing cells only sort a very limited set of their rich miRNA content into their EXs, which shows that this process is a highly selective and controlled phenomenon [44–46]. Malignant cells produce larger amounts of EXs, the contents of which differ widely from that of EXs produced by their normal counterparts [47]. Cancer cells use the ability of EXs as carriers of selected signaling molecules and proteins to the advantage of promoting their tumor niche as well as their progression [48].

3.2. Bone marrow microenvironment bridging Unlike solid tumors in which the primary tumor site and the metastatic sites are readily distinguishable, MM involves different sites within an altered BMM [1]. Such a microenvironment constitutes a variety of cellular elements including BMSCs, MSCs, osteoblasts (OBs), OCs, fibroblasts, adipocytes, cells of innate and adaptive immunity, macrophages, and myeloid-derived suppressor cells (MDSCs) as well as non-cellular elements such as cytokines, chemokines, growth factors, EXs and the extracellular matrix (ECM) [1,5,56]. Although the BMM itself, due to its nature of hosting and nurturing highly proliferating hematopoietic cells, has a lot of survival advantages that make it a suitable place for the growth and maintenance of MM cells, the alteration of its checking mechanisms which normally control the growth of cells makes it an even more optimal environment for the MM cells to survive and grow [5,57]. EXs are among the major culprits through which these tumor-promoting BMM alterations take place [6]. Among the aforementioned BM components, the most highly influential interactions exist between MM cells and BMSCs/MSCs, which by altering the production of soluble factors from these cell types, contribute largely to the survival and progression of MM cells [45,58,59]. Almost all cells in the BMM of MM patients are functionally different from their healthy counterparts acting to the advantage of tumor survival [60–63]. It has been shown that BMSCs in MM patients are highly permissive for adherent MM cells providing the space where free or EX-loaded cytokines, growth factors and miRNAs induce MM cell proliferation and block apoptosis signals [5]. In one study in which human MSCs were treated with MM cell conditioned media, 19 miRNAs were found to be dysregulated among which miR-146a was strongly upregulated. This miRNA, via the Notch signaling pathway, along with other dysregulated miRNAs resulted in an altered profile of chemokines and cytokines including elevated levels of the chemokines C-X-C motif ligand 1 (CXCL1), C-C motif ligand 5(CCL5), monocyte chemoattractant protein-1(MCP-1), interleukin 8(IL-8), interleukin 6(IL-6) and interferon gamma-induced protein 10 (IP-10), which act in concert to improve MM cell survival and progression [12]. It was then shown that MM-derived EXs contained miR-146a, which could be transferred to MSCs to drive the aforementioned changes. Supporting these finding, Cheng et al. showed that EXs secreted by the MM cell line OPM2 in vitro are enriched in miR-146a and mi-R21 which could increase cell proliferation, IL-6 production and cancer-associated fibroblast (CAF) transformation of MSCs after co-culture of MSCs with OPM2 conditioned media [64]. Similarly in another study, MM-EXs were shown to induce the growth and proliferation of BMSCs, which in turn acted in favor of MM cell survival and expansion [65]. These findings are good examples of how MM cells affect the BMM and its resident cells (especially MSCs and BMSCs) to function in favor of tumor progression and survival via secreting soluble factors and differentiating them into

3. Roles in pathogenesis and disease progression In this section, the role of EXs in the pathogenesis of MM will be discussed in detail. Emerging evidence suggests that EXs are critically involved in the emergence and progression of tumors. In the context of malignancies, harmful EXs may be either produced by the malignant cells, which in that case may be referred to as tumor-derived exosomes (TEXs) or by other non-malignant cells surrounding the tumor or distant from the primary tumor site [9]. Whatever the case, EXs via their contents of bioactive molecules can affect different aspects of the tumors and alter the function of their target cells to the advantage of tumor survival and proliferation [45,49]. In hematological malignancies, TEXs were shown to be involved in the malignant transformation of the adjacent normal cells and promoting their survival by changing the BMM, dampening anti-leukemic immune responses, and inducing drug resistance [21]. MM as a malignancy of hematologic origin is not an exception. Both MM-derived EXs and those produced by other resident cells of the BM have been shown in various studies to be important non-cellular mediators of cell-to-cell contact within the BMM [50]. In addition, EXs contribute to many of the major complications of MM and treatment failures via promoting osteolysis, angiogenesis and drug resistance [51,52], which will be discussed in the following section. 3.1. Exosomal cargos and their role in MM progression EXs from MM cells and other cells, mainly BMSCs, within the deregulated BMM of patients have been shown to affect disease progression via their rich exosomal cargos affecting a variety of mechanisms ranging from the anti-MM immune response to other fate-determining 3

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Table 1 Alterations in the exosomal content of MM patients compared to healthy individuals. EX origin

Content

Mode of alteration

Reference

BMSCs

Primary MM cells MM cells MM cell lines: MM1 and U266 OPM2 cell line Peripheral blood of MM patients

miR-146a lncRNA RUNX2-AS1 Bone marrow stromal protein 2 (BST-2) miR-21 and miR-146a let-7b and miR-18a

Serum samples of MM patients underwent BM allograft

CD146 (MCAM-1)

Downregulated compared to healthy Upregulated Downregulated in bortezomib-resistant MM patients Upregulated in bortezomib-resistant MM patients Upregulated? Upregulated Exclusively sorted Upregulated Upregulated in patients with shorter OS and PFS Upregulated in patients with higher GVHD risk (by 60%) Upregulated in patients with lower GVHD risk (by 40% and 60%) Overexpressed

[66]

Peripheral blood of MM patients

Oncosuppressor miR-15a IL-6 CCL2 Fibronectin miR-16-5p, miR-15a-5p, miR-20a-5p, miR-17-5p, miR125b-5p, miR-19a-3p, and miR-21-5p miR-513a-5p, miR-20b-3p, let-7d-3p

CD140-α (PECAM-1 and PDGFR-α) Serum samples of MM patients and MM cell lines (MM.1R and RPMI-8226) MM serum samples and corticosteroid resistant MM.1R cells Plasma cells from BM samples of MM patients MM cell lines RPMI8226, KMS-11, and U266 MM patient urine

HLA-1 and the associated β2-microglobulin

[4]

[12] [45] [109] [64] [94] [95]

[110]

CD44 Ectoenzymes responsible for the production of adenosine (CD39, CD38, CD73, etc.) miR-135b Monomeric light chains

Upregulated

[68]

Upregulated under hypoxic conditions Upregulated

[82] [101]

MM, Multiple myeloma; IL-6, Interleukin 6; CCL2, C-C Motif Chemokine Ligand 2; MCAM-1, melanoma cell adhesion molecule; PECAM-1, Platelet endothelial cell adhesion molecule 1; PDGFR-α, platelet-derived growth factor receptor A; GVHD, Graft-versus-host disease; HLA-1, Human leukocyte antigen 1.

for their recognition by NK cells), but also they may constitute a proportion of the soluble activating ligands, involved in the downregulation of activating ligands on NK cells [72]. Furthermore, co-localization of immunosuppressive molecules (e.g. TGF-β) with the exosomal surface ligands, may worsen the situation and provide more suppressive signals for NK cells [73]. Another aspect of the immunosuppressive role of BMM in MM patients is the activation and stimulation of MDSCs, which negatively regulate the anti-myeloma immune response. The activated MDSCs facilitate tumor growth by suppressing the antigen recognition and activation of lymphocytes [56,74]. In parallel with these findings, Wang et al. showed that MMEXs are able to promote the immunosuppressive phenotype of MDSCs via activating the signal transducer and activator of transcription 3(STAT3) pathway in vivo, finally inducing their growth and upregulating nitric oxide (NO) syntheses within these cells [65].

populations that support the expansion of MM cells. BMSCs of MM patients also secrete EXs which exhibit an altered composition compared to those produced by BMSCs of healthy individuals (Table 1). Lower levels of the oncosuppressor miR-15a and high levels of IL-6 and C-C motif ligand 2 (CCL2) are among these changes which, when delivered to MM cells, render them into highly proliferative cells [66]. Although similar results were obtained in another study indicating that BMSC-EXs of MM patients promote survival and proliferation of MM cells, the same effects were observed with BMSCs from normal donors [7]. This indicates that the secretions of BMSCs may favor MM cell migration, growth and survival regardless of the diseased or normal BMM status [7]. Adenosine, a potent immunosuppressor molecule, produced by different cells in the BMM of MM patients plays a critical role in the tumor niche formation and MM progression [67]. It has been shown that MM-EXs are enriched in ectoenzymes (CD39, CD38, CD73, etc.), which convert adenosine precursors (ATP or NAD+) into adenosine and therefore are probably the main contributors of adenosine production in the BMM niche [68]. These findings, coupled with other findings reporting that BM levels of adenosine are higher in MM patients compared with MGUS/SMM patients, indicate that this molecule might be potentially used as a biomarker for the differentiation of these conditions as well as early stage and advanced MM patients [68].

3.4. Induction of drug resistance and survival promotion Although treatment of MM varies considerably depending on the institution in which the therapy is carried out, treatment costs, patient's eligibility for autologous stem cell transplantation and some other factors, the chemotherapeutic alkylating agent melphalan, proteasome inhibitors such as bortezomib and carfilzomib and immunomodulatory agents like thalidomide and lenalidomide constitute the main ingredients of the therapeutic regimens [3]. Among these agents, melaphalan induces direct cytotoxic effects, bortezomib induces its effects by inhibiting the CT-L (β5) subunit of the 26S proteasome, carfilzomib is also a proteasome inhibitor which irreversibly blocks CT-L activity and lenalidomide, a more potent analog of thalidomide, exerts its effects via angiogenesis inhibition [18]. Despite the existence of various effective drugs, the reason for which MM is still regarded to as an incurable disease is the rapid emergence of drug resistance [75]. It has been now well-established that the interactions within the BMM and its resident cells are crucially involved in resistance to therapy. EXs produced by BMSCs have been found to significantly increase the viability of MM cells following treatment with bortezomib in a 5 T33 murine MM model of and in human MM cells, indicating a role for the BMSC-EXs in drug resistance of MM cells [7]. Activation of several pathways

3.3. Governing escape mechanisms On one hand, it has been shown that genotoxic stress induced by chemotherapeutic drugs can alter the activity of proteases such as a disintegrin and metalloproteinase (ADAM) which can be engaged in the proteolytic cleavage of NK-activating ligands mainly MHC class I polypeptide-related sequence A and B (MIC-A and MIC-B, respectively) from the surface of MM cells [69,70]. On the other hand, it has been demonstrated that soluble forms of these activating ligands can induce the downregulation of the activating receptors, NKG2D and NKp30, on NK cells [53,70]. Keeping in mind that the EXs released by MM cells also express these activating ligands [71], there is a strong possibility that the MM-derived EXs are not only engaged in a non-proteolytic form of ligand shedding by MM cells (which renders them less immunogenic 4

5 Via unknown mediators [56] ↑IL6 and CCL2 and ↓ miR-15a [7,66]

–

MM cell

–

BMSC

↑Survival and growth [56] MM clone expansion [66] Resistance to bortezomib [7]

Via unknown mediators [65]

↑Growth and proliferation

Target cell + consequences

IL15/IL15RA Complex [86] Via HSP70 [87]

NK cell

↑Proliferation and activation (druginduced senescence) [86] ↑IFN-γ production [87]

Via unknown mediators [56]

Via unknown mediators [65]

MDSC

↑Survival STAT1, STAT3 activation and ↑Bcl-xL, Mcl-1 ↑ Expansion ↑NO release [56]

miR-135b [82] Via unknown mediators [65]

–

–

Endothelial cell

HIF-1 regulation and ↑Angiogenesis [82] ↑ Endothelial growth via activation of STAT3, c-Jun N-terminal kinase, and p53 [65]

lncRUNX2-AS1 [45] miR21 and miR-146a [64] mir146a [12]

MSC

Osteogenesis repression [45] ↑proliferation ↑IL-6 secretion ↑ CAF transformation ↑miR-146a and production of tumorpromoting cytokines and chemokines [12]

–

Via DKK-1 (76)

OB

↓Runx2, Osterix, and Collagen 1A1 Apoptosis induction Differentiation inhibition

–

Via unknown mediators [6,76]

OC

Pre-OC migration, survival and differentiation into OCs [6]↑Resorption capacity [76]

IFN-γ, Interferon gamma; STAT, Signal transducer and activator of transcription; Bcl-xL, B-cell lymphoma-extra-large; Mcl-1, Induced myeloid leukemia cell differentiation protein; NO, Nitric oxide; CAF, Cancerassociated fibroblast; IL-6, Interleukin 6; HIF-1, Hypoxia-inducible factor 1; Runx2, Runt-related transcription factor 2; BMSC, Bone marrow stromal cells; MM cell, Multiple myeloma cell; NK; Natural killer; MDSC, Myeloid-derived suppressor cell; MSC, Mesenchymal stem cell; OB, Osteoblast; OC, Osteoclast.

EX-producing cell

Cell of origin/Target cell

Table 2 Exosome-mediated exchange of bioactive molecules and the functional consequences in the context of MM.

M. Moloudizargari, et al.

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Fig. 2. The bone marrow microenvironment (BMM) modulation by multiple myeloma (MM) exosomes (EX). A. MM cells secrete exosomes with unique characteristics. B. MM exosomes (MM-EXs) determine the mesenchymal stem cells (MSCs) to differentiate into cancer associated fibroblasts (CAFs). C. The MM-EXs target CAFs and cause IL-6 production and D. in endothelial cells (ECs) they cause VEGF production and growth. E. The MM-EXs lead to OPG suppression. F. The MM-EXs cause IFN-γ release by natural killer cells. G. The CAF-EXs target myeloid-derived suppressor cells (MDSC), which leads to local immunosuppression of dendritic cells (DC) and NK cells. H. The MM-EXs trigger osteoblast survival and differentiation. I. Through RANK-RANK ligand (RANKL) binding, the osteoclasts (OCs) are transformed into osteoblast (OBs). J. The MM-EXs target the bone marrow mesenchymal stem cells (BMSCs) and enhance their survival, while the BMSCs also sustain MM viability. MM, Multiple myeloma; EX, Exosome; VEGF, Vascular endothelial growth factor; FLC, Free light chain; IL-6, Interleukin 6; OC, Osteoclast; OB, Osteoblast; BMSC, Bone marrow stromal cell; NO, Nitric oxide; MSC, Mesenchymal stem cell; CAF, Cancer-associated fibroblast; EC, Endothelial cell; IFN-γ, Interferon Gamma; OPG, Osteoprotegerin; DC, Dendritic cell; RANKL, Receptor activator of nuclear factor kappa-Β ligand.

proteins, triggers several pathways including STAT3, c-Jun N-terminal kinases, and p53 and consequently stimulate endothelial cell growth, proliferation and invasion. At the molecular level, IL-6 and VEGF secretion are increased, thus promoting angiogenesis [78]. The BM, a highly vascularized tissue, is a primary target site for the angiogenesispromoting properties of EXs and is naturally hypoxic in comparison to other vascular tissues [79,80]. Interestingly in the context of MM, the BM becomes more hypoxic due to the overproduction of PCs, a condition that causes MM cells to produce higher amounts of EXs compared to the normoxic conditions [81,82]. EXs produced by these hypoxic MM cells have been reported to be enriched in miR-135b, which promotes in vitro angiogenesis via the factor-inhibiting hypoxia inducible factor 1 (FIH-1) pathway. Although many other contents of EXs (in addition to miR-135b) may be involved in this process, this is an important finding emphasizing how parental cells can change their miRNA sorting under controlled conditions to make the BMM more supportive of their survival [44,82]. There is a difference in the BMM secretome between young individuals and older adults. The EXs from younger donors are less effective in inducing angiogenesis than those from older donors. The tumor pro-angiogenic activities of EXs can be inhibited through loading of miR-340 and miR-365 mimics [83].

including p38, p53, c-Jun N-terminal kinases and Akt by BMSC-EXs in the target MM cells were shown to be responsible for the increased viability and survival [7]. Another study also showed that the inhibition of EX secretion by GW4869 can significantly augment the sensitivity of murine MM cells to bortezomib in vitro, a finding that emphasizes the important role of MM-EXs in developing drug resistance [76]. It has been interestingly shown that the conventional chemotherapeutic agents including melphalan and anti-proteases such as bortezomib and carfilzomib can trigger a burst of EX production by MM cells especially those that survive chemotherapy. These EXs (referred to as chemoexosomes) are shown to be rich in surface heparanase enzyme which is involved in a variety of changes required for developing chemoresistance and the consequent relapse of the patient. Following EX uptake by MM cells, these changes include the activation of ERK pathway via delivery of their heparanase content, induction of TNF-α production by macrophages, matrix degradation and migration promotion [77]. 3.5. Angiogenesis promotion Among the effects through which MM-derived EXs maintain and reprogram the BMM is their direct and indirect angiogenesis promotion. It has been shown that MM-EXs, via their content of angiogenic 6

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studies that have recently shown the effects of MM-EXs on the immune response and more importantly how the routine therapeutic interventions can change the quality and quantity of such effects. It has been shown that both MM cell lines and primary malignant PCs from the BM of MM patients secrete EXs that carry the IL-15/IL-15RA complex, which is required for the proliferation and activation of NK cells [86]. Moreover, the treatment of the cells with very low doses of doxorubicin and melphalan can not only increase EX production by MM cells but it can also boost the expression of IL-15/IL-15RA complex on the surface of MM cells and their EXs via induction of senescence [86]. A similar study reported that treatment of MM cells with doxorubicin and melphalan can dramatically boost MM cell secretion of EXs with the potential to induce IFN-γ production in NK cells, probably through mechanisms involving TLR2 and HSP70-dependent activation of the NF-ƙB pathway [87]. This shows that, besides the immunosuppressive and tumor-promoting properties that have been previously defined, the immunomodulatory effects of MM-EXs can work to the advantage of boosting NK cell responses which act at the frontline of anti-myeloma immune response, especially following chemotherapeutic interventions. The same research group showed that although genotoxic stress induced by chemotherapeutic agents (doxorubicin and melphalan) can induce the expression of senescence-associated NK activating ligands on the surface of MM cells, it can also play an important role in sheddase and metalloproteinase (ADAM)-mediated shedding of such ligands rendering them into soluble ligands which have been previously shown to act differently from membrane-associated ligands, inducing the downregulation of activating receptors on NK cells [88,89]. The authors then concluded that targeting metalloproteinases in parallel to chemotherapy may be useful in boosting NK-based immune response. However, as stated above, it can be postulated that the increased state of EX release (four times the normal levels) from MM cells compared to normal PCs [90] could be involved in decreasing the surface concentration of NK-activating ligands needed for NK cell activation on MM cells and this hypotheses, if true, may provide the basis for pharmacological interventions targeting EX release from MM cells in combination with conventional routine treatments. This gives rise to the notion that, by choosing a suitable chemotherapeutic regimen, one could not only directly target MM cells, but also can change the MM-EXmediated transfer of information to the advantage of an enhanced antitumor immunity. This notion has been referred to as “outsmarting tumor EX to steal the founder cancer clone its niche” which can be defined as manipulating the tumor-mediated interactions within its microenvironment in hopes that the manipulation blocks its progression or even helps in its regression [91]. It has been shown that fibronectin-heparan sulphate interactions play important roles in the

3.6. Osteolysis promotion A characteristic feature of MM is the disruption of the existing balance between bone formation and bone absorption to the advantage of osteolysis, which gives rise to many of the clinically signs and symptoms of the disease [84,85]. There is an increasing bulk of evidence suggesting that EXs within the BMM play critical roles in relaying the information responsible for such an impairment of bone formation through their small non-coding RNA contents. For instance, MM cells have been shown to have high levels of the lncRNA RUNX2-AS1, a gene product of RUNX2 antisense, which can be transferred to MSCs where they hamper the normal osteogenic-differentiation activity of MSCs via regulating RUNX2 gene expression [45]. MM-derived EXs also contribute to bone lysis via targeting OCs. These EXs can induce differentiation, survival and migration of both primary OCs and the murine Raw264.7 cell line as evidenced by the increased expression of C-X-C chemokine receptor type 4 (CXCR4) and OC markers including MMP9, cathepsin K, and tartrate-resistant acid phosphatase [6]. Although the responsible molecules and mechanisms are poorly understood, there is some information which provide evidence for the involvement of the AKT pathway in the survival-inducing effects of MM-EXs [6]. It was shown in a recent study that EXs secreted from the murine MM cell line 5TGM1 can increase OC resorption capacity and inhibit OB differentiation, a vicious disbalance that finally results in osteolysis promotion (Table 2). This is mainly done via DKK-1 transfer from EXs to OBs and it confirms the results from other studies that have previously indicated the role of MM-EXs in osteolytic bone disease [76]. Another finding regarding MM patients is the downregulated ratio of receptor activator of nuclear factor kappa-Β ligand to osteoprotegerin (RANKL/ OPG), which further contributes to the osteolysis observed in these patients [6]. MM-EXs modulate pro-tumoral BMM through constant delivery of messages between various cell types (Fig. 2). 4. EX-targeting therapies It is now evident that the communication within the BMM of MM patients through EXs is an important determinant of disease progression, drug resistance development and osteolysis; therefore, targeting EX secretion could be useful in reversing all the above-mentioned effects [76]. On such a basis, recent studies have been making efforts to combine niche-modifying agents with the routine cytotoxic regimens that act directly on the malignant cells with the anticipation of stronger responses and overcoming relapses and residual disease [5,82]. In one of these studies, the use of an EX release inhibitor, GW4869, prevented MM-associated bone loss and improved bone formation and anti-catalytic activity; see Table 3 [44]. However, there are some interesting Table 3 EX-targeting interventions and their effects on EX-mediated outcomes. Steady state

EX-targeting intervention

Outcome following the intervention

EX-mediated bone loss

Treatment with the EX release inhibitor GW4869

MM EXs containing IL-15/IL-15RA activate NK response

Treatment with low dose doxorubicin and melphalan

→ → → → →

MM cells (primary and cell lines)

Treatment with doxorubicin and melphalan

→

MM cells

Genotoxic stress by doxorubicin and melphalan treatment

→ →

MM patients (Phase I trial)

Inhibition of fibronectin-heparan sulphate interactions by Roneparstat

Tumor-bearing models

Injection of TRAIL-decorated EXs

→ → → →

Prevention of bone loss Improvement of bone formation Anti-catalytic activity Increased EX production by MM cells Senescence induction and increase in IL-15/IL-15RA expression on EXs Boosting EX secretion from MM cells capable of inducing IFN-γ production in NK cells Increasing the expression of NK-activating ligands on MM cells Increasing EX-mediated shedding of these ligands capable of downregulating NK receptors Suppression of MM-EX interactions with target cells High safety profile Therapeutic efficacy? Induction of tumor-specific apoptosis

Ref [44]

[86]

[87] [88]

[92]

[93]

MM, Multiple myeloma; EX, Exosome,?, Not determined; TRAIL, TNF-related apoptosis-inducing ligand; NK, Naturak killer; IL-15, Interleukin 15; IL-15RA, Interleukin 15 receptor, alpha subunit. 7

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uptake of EXs by BMM resident cells and that the recognition of fibronectin by target cells can trigger signaling pathways like p38 and pERK, in addition to the consequent downstream expression of Dickkopf-related protein 1 (DKK-1) and MMP-9, two molecules with wellknown roles in MM progression. It was then suggested that the inhibition of such fibronectin-mediated interactions of EXs with their target cells by different means such as anti-fibronectin antibody, heparanase, heparin mimetic, and the Hep II fragment of fibronectin, can negatively affect MM progression [41]. Based on these findings, a phase 1 clinical trial (NCT01764880) used heparin and its mimetic Roneparstat to suppress the interactions between MM-EXs and the target cells [92]. The results showed that Roneparstat has a high safety profile with no systemic reactions of clinical relevance. The drug was shown to be tolerable at doses of 300–400 mg/day, however its efficacy remained to be studied in other trials. An important way to intervene into EX uptake by malignant cells is by targeting B-cell lymphoma-extra-large (Bcl-xL). Caspase 3 is activated on BMSC-EXs and tumor EXs, which cleaves the anti-apoptotic protein Bcl-xL. Such a cleavage is required for EX uptake by cells and it can inhibit EX-mediated tumor-supporting effects of the BMM [10]. It has been shown that TNF-related apoptosisinducing ligand (TRAIL)-decorated EXs, when injected to the patients either directly at the tumors site or in a systemic manner, can induce detectable tumor-specific apoptosis in tumor cells, which might be augmented by the parallel use of TRAIL-sensitizing drugs in clinics to hinder tumor growth [93]. Moreover, these TRAIL-EXs can also be loaded with genetic material with the purpose of hijacking the tumorpromoting signaling pathways in the MM cells [93]. The results of the aforementioned studies suggest that intervening in the EX-mediated BMM interactions can be effectively done at different levels of EX biogenesis from its formation and content sorting to EX trafficking its uptake by target cells. All of these therapeutic EX modulations are to be done with the aim of improving treatment outcomes and the implementation of more long-lasting therapies [6,9,32].

allograft transplantation in MM patients. An exploratory study of 41 MM patients reported that the expression of 3 exosomal surface antigens correlates with the onset of acute GVHD. The expression of CD146 (melanoma cell adhesion molecule 1; MCAM-1) correlated positively with an increased risk (by 60%) of GVHD, while CD31 (Platelet endothelial cell adhesion molecule; PECAM-1) and CD140-α (plateletderived growth factor receptor Alpha; PDGFR-α) were indicative of a lower risk of developing GVHD (by 40% and 60%, respectively) [95]. Considering the fact that a high proportion of MM patients have innate resistance to bortezomib or develop multi-drug resistance in the course of therapy, MM relapse is a common event [97,98]. Early prediction of drug resistance in MM patients is one of the hottest goals of the researchers in the field, which remains to be achieved. In a previous study, EXs from the peripheral blood of 204 MM patients, stratified based on the presence/absence of resistance to conventional drugs, were obtained and analyzed for their miRNA content. It was shown that specific miRNAs were either upregulated or downregulated in the bortezomib-resistant patients compared to the non-resistant. MM-EXs contain oncogenic long non-coding RNAs (lncRNAs), which by interacting with tumor suppressor miRNAs and releasing the oncogenic mRNAs from miRNA control, sustain malignant development in their targeted cells. In MM-EXs, the lncRNA LINC00461 was found to be overexpressed. This lncRNA further targets miR-15a and miR-16, thus releasing the anti-apoptotic gene B-cell lymphoma 2 (BCL-2) [99]. The plasma EXs contain an increased level of the psoriasis susceptibilityrelated non-coding RNA induced by stress (l ncRNA-PRINS), which has a high sensitivity and specificity of differentiating between healthy donors and either MM or MGUS. This lncRNAs is also correlated with patients' survival, however is unable to differentiate between MGUS and MM for diagnosis [100]. More than 10 miRNAs with the greatest changes overlapped in the literature-based findings and were deemed to be potential candidates to be used as predictive panels for drug resistance [4]. Such panels to predict the possibility of developing drug resistance are of great importance since they can largely help in deciding which therapeutic regime might best suit each patient and that the routine workups undertaken for MM patients do not reveal any information in this regard. Detection of light chains associated with urinary EXs has proven promising as a diagnostic technique of higher specificity compared to other methods of protein detection used in the diagnosis of nephrotoxic monoclonal gamopathies with absent symptomatic clonal mass. It is also of note that these EXs can also distinguish between amyloidosis and MM, since the associated light chains are oligomeric in amyloidosis and only monomeric in MM. This makes urinary EXs great biomarkers for renal response in gammopathy-associated nephropathies [101].

5. Role of EXs as prognostic and diagnostic tools Determination of the stage of the disease as well as the probable prognosis in MM patients is a crucial requirement for the selection of the most appropriate therapy. This requires careful monitoring of MM progression. Due to the lack of adequate monitoring tools, there is an urgent need to develop novel non-invasive approaches for the diagnosis of MM, its differentiation from other less severe forms of PC dyscrasia and prediction of treatment response [75]. There is compelling evidence showing that EXs isolated from the peripheral blood of MM patients can serve as useful markers for the prediction of disease progression. This has been proven by the findings of several studies indicating that certain miRNAs are either downregulated or upregulated in the peripheral blood EXs isolated from MM patients compared to healthy donors and that the levels of these miRNAs vary significantly among patients with different risk factors [4,12,94,95]. Such differences can result from mutations in the compartments of the EX sorting machinery in different cancers. For instance, DIS3, an EX protein complex with exoribonuclease activity is involved in controlling the translocation of certain RNAs from the nucleus to the cytoplasm where they can be packaged into the future EXs. DIS3 has two isoforms (isoform 1 and 2) the amounts of which, in comparison to healthy individuals, have been shown to be different in many hematological malignancies including MM. It has been suggested that the altered ratio of these two isoforms may have symptomatic value in prediction of MM and other hematological cancers [96]. Two exosomal miRNAs including let-7b and miR18a were found to predict patient prognosis based on overall and progression free survivals (OS and PFS, respectively). This is of significant importance since, using exosomal miRNA panels, the riskfactor-based stratification of patients can be done soon after the MM diagnosis is made [94]. EXs have also been shown to be useful in predicting the risk of graft-versus-host disease (GVHD) following BM

6. EXs as therapeutic tools EXs are emerging as novel tools of cancer immunotherapy in combination with the existing therapies including surgery, radiotherapy or chemotherapy. Unique migration properties as well as the immuneneutral nature of EXs makes them good options for the selective targeting of either cancer or immune cells with the aim of eliciting a potent anti-tumor immune response [102]. The co-modulation between MM-EXs and NK cells is controversial. Although there is mounting evidence that EXs carrying the NK-activating ligands on their surfaces can cause deleterious effects on NK-mediated anti-tumor immune response, some studies showed that DC EXs expressing UL16 binding protein 1 (ULBP1) can stimulate NK cells [103]. These findings may give rise to the idea that by designing EXs decorated with ligands and surface molecules of interest, one may restore the dampened NKmediated response, which is present in MM patients and even turn it into a more effective anti-tumor effect [70]. In line with this, Rivoltini et al. exploited the modifiable surface properties of EXs to attach molecules of interest on their surfaces. They also knew that cancer cells are selectively sensitive to TRAIL, a ligand of TNF family receptors. 8

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Fig. 3. Exosome applications in clinical practice are divided in three directions. First, exosomes are extensively studied as diagnostic tools for early detection of multiple myeloma (MM) based on their cargo. Secondly, exosomes are studied from the point of view of their prognostic potential due to their capacity of differentiating between different disease outcomes. Finally, exosomes are proposed to be engineered in order to deliver a therapeutic “message” either by increasing the local immune response or via inducing MM apoptosis. There are very few studies regarding the use of exosomes as therapeutic options in MM. MCAM-1, melanoma cell adhesion molecule; PECAM-1, Platelet endothelial cell adhesion molecule 1; PDGFR-α, platelet-derived growth factor receptor A; Bz, Bortezomib; ULBP1, UL16 binding protein 1; IL-6, Interleukin 6; GVHD, Graft-versus-host disease; TRAIL, TNF-related apoptosis-inducing ligand.

the tumor niche can enhance the therapeutic efficacy of routine chemotherapeutic agents, overcome drug resistance and directly improve the fate of common MM-associated complications such as osteolytic bone disease. Moreover, the augmented production of EXs by MM patients as well as their specific miRNA contents has shown promising to be recruited as early biomarkers for the determination of patient prognosis, prediction of GVHD risk following HSC transplantation and early stratification of patients based on their probability of developing drug resistance. This can be of great importance for the selection of the best therapeutic regimen that best fits the requirements of patients and can be a step forward towards personalized precision medicine for MM patients. However, the large-scale EX production is still highly dependent on the generating cells. In addition, the great impairment on the way of translating EX research into clinical practice is the lack of standardized methods, high would give a content value of purity and specificity. Significant progress has been made in this regard over the last few years due to the use of nanoparticle-mediated EX isolation [107,108]. These findings call for more research in the field to uncover the usefulness of EXs and their contents as biomarkers and to develop new treatment strategies combining niche-targeting agents with the routine anti-myeloma drugs.

Therefore, they designed EXs decorated with TRAIL to target K562 cells and they could successfully induce apoptosis signals in these chronic myelogenous leukemia cells [93]. Keeping in mind all the deleterious effects of EXs in the context of cancers, it can be easily concluded that these effects may be effectively interrupted by outsmarting TEXs via designing tailored EXs that could compete the TEXs or by designing surface-modified EXs and EXs carrying our contents of interest to achieve therapeutic goals [91]. The general characteristics of EXs based on their translational potential are illustrated in Fig. 3. 7. Summary and future directions In spite of the recent progress in MM therapy and the development of effective drugs such as bortezomib and melphalan, this malignancy of PCs is still regarded as a fatal disease due to the tumor-promoting role of BMM, which results in drug resistance, treatment failures and frequent relapses [7,104]. The disease is characterized by loss of antimyeloma immunity and an immunosuppressive environment that induces immune escape of the malignant cells and their progression [105]. The BMM, possessing an intricate network of cellular communication, contains different cells of hematopoietic lineage that work in favor of tumor niche formation [106]. There is compiling evidence indicating that targeting this inter-cellular network can enhance therapeutic outcome [41]. EXs are among the main role players of the BMM network, which due to their direct interaction with the target cells can serve as desirable therapeutic targets. A great deal of evidence has shown that interventions with the EX-mediated communication within

Practice points

• Targeting the EX-mediated interactions solely or in conjunction with

the routine anti-MM agents can largely improve the treatment outcomes and may also prevent the frequent relapses associated with

9

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MM.

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• The cells of MM patients produce larger amounts of EXs which differ in terms of their contents between different strata of patients such as the drug-resistant and the none-drug-resistant, making them useful options to be used as biomarkers in diagnostics.

Research agenda

• EXs are key role players in maintaining BMM and the tumor niche • • •

required for the malignant transformation of the founder clone of MM, its survival and progression. These EXs are mainly produced by MM cells and BMSCs and can target a vast variety of cells present in the BMM. Clinical studies determining the precise effects of EX-targeting interventions in MM patients are highly warranted to eliminate ambiguities regarding the tumor supportive role of EXs and to set up standard treatment protocols. Studies with high numbers of patients stratifying MM patients based on their disease stage, drug-resistance and occurrence of relapses with long follow up periods are required to determine the exosomal miRNA profiles of these patients to be used as biomarkers.

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