Extracellular vesicles in multiple myeloma-bone marrow niche crosstalk: from cellular dialogue to clinical perspectives

Abstract

Multiple myeloma (MM) is a haematological neoplasia defined by the clonal expansion of malignant plasma cells within the intricate and dynamic ecosystem of the bone marrow (BM). Although genetic aberrations intrinsic to tumour cells play a critical role in MM initiation, the advancement and aggressiveness of the disease are heavily influenced by ongoing signalling exchanges and persistent interactions with the surrounding microenvironment. Extracellular vesicles (EVs) are increasingly recognized as essential messengers in this reciprocal communication, acting as membrane-encapsulated vehicles able to transfer biologically active molecules. Through both local and distant signaling, EVs coordinate complex molecular pathways that modify recipient cell behaviour and maintain a supportive microenvironment conducive to MM progression. The present review offers a critical and up-to-date synthesis of current knowledge on EVs derived from both tumour cells and non-malignant populations within the BM milieu, including stromal, immune, and endothelial cells, and their role in shaping the pathophysiology of MM. Specifically, we examined how EV-mediated communication contributes to enhanced tumor proliferation, immune surveillance escape and the onset of resistance to therapeutic agents. We further explore the translational relevance of EVs, with a focus on their emerging potential as non-invasive biomarkers for disease monitoring and as novel agents or delivery systems in therapeutic interventions. Further mechanistic researches, as well as the optimization of reliable isolation methodologies, in parallel with rigorous and well-designed clinical investigations, will be essential to enable the effective translation of EV-based approaches into clinical practice and to support the development of more effective and targeted treatment strategies in MM patients.

Background

Multiple myeloma (MM), a B-cell neoplasia resulting from the clonal proliferation of malignant plasma cells in the bone marrow (BM), accounts for 1% of all cancers and 10–15% of all haematological malignancies [1, 2]. A 40% increase in MM incidence has been recorded in the United States and a roughly 130% increase globally since 1990, while the mortality rate, currently estimated at 3.3 per 100,000 of the population, has declined by 18% from the peak value observed in 1994 (4.0 per 100,000) [3, 4]. Despite the introduction of novel therapeutic approaches, which have improved the 5-year overall survival rate to almost 54%, MM endures as a considerable cause of morbidity and mortality due to the development of drug resistance phenomena that make MM a difficult-to-treat haematological disease [3, 4].

MM originates from an asymptomatic condition known as Monoclonal Gammopathy of Unknown Significance (MGUS), which is characterized by the presence of abnormal plasma cells ( < 10%) involved in the production of monoclonal proteins ( < 3 g/dL) consisting of heavy chain (IgG, IgA, IgD, IgE, or IgM) and light chain kappa or lambda detectable in the blood or urine [5]. MGUS patients may evolve towards Smoldering Multiple Myeloma (SMM), in which serum monoclonal proteins reach expression levels ≥3 g/dL and clonal plasma cells may increase until 60%, concomitant with urinary monoclonal protein levels ≥500 mg per 24 h. However, the criteria for MM diagnosis established by the International Myeloma Working Group define an overt stage of MM in the presence of clonal BM plasma cells ≥10% or bone or extramedullary plasmacytoma after BM biopsy, serum Free Light Chain (FLC) ratio ≥100 and focal lesion > 1 on Magnetic Resonance Imaging. Furthermore, MM patients exhibit various end-organ complications, including hypercalcemia, kidney damage, anaemia and lytic bone lesions [6].

Accumulating evidence has proposed BM microenvironment (BMME) as a pathogenic entity in MM due to its ability to regulate the transition from the asymptomatic pre-malignant stages of the disease to the symptomatic condition [7]. The genetic and epigenetic profiles of the BM niche in MM patients show distinct characteristics when compared with those observed in the microenvironment of healthy subjects [8]. Of note, the clonal proliferation of malignant plasma cell populations within the BM is sustained by the crosstalk between MM cells and cellular constituents of tumor microenvironment (TME), encompassing endothelial cells (ECs), mesenchymal stromal cells (MSCs), osteoblasts (OBs), osteoclasts (OCs) and immune cells, as well as non-cellular components such as extracellular matrix, chemokines, cytokines and growth factors [7, 9–12]. More recently, Extracellular Vesicles (EVs) have emerged as key mediators of intercellular communication within BM [13]. EVs, nano-sized membrane-bound structures secreted by normal and tumour cells, are a source of short- and long-range transfer of genetic and molecular signals able to modulate the phenotypic profile of recipient cells [14], including cell proliferation, dissemination, immune response and drug resistance in several malignancies [15–23], including MM [24–28]. Beyond their established biological functions, EVs hold significant promise in clinical practice as minimally invasive biomarkers for disease detection and monitoring, as well as innovative delivery systems for targeted therapeutic interventions.

In the present review, we will critically discuss the latest findings regarding the biological functions of EVs in regulating MM biology, disease monitoring and treatment options. First, we will explore the mechanisms through which MM cell- and BMME-derived EVs influence MM development and progression. Then, the potential role of EVs as biomarkers in the dynamic monitoring of MM will be highlighted. Finally, the function of EVs as innovative therapeutic tools in the clinical management of MM will be addressed.

EVs: an overview

EVs are a heterogeneous population of lipid bilayer-enclosed nanoparticles, naturally released from all cell types and detectable in all biological fluids [29–31]. Rather than being mere cellular debris, EVs are now recognized as crucial mediators of intercellular communication, capable of transferring complex molecular cargo, including proteins, lipids, metabolites, and various classes of RNA, from donor to recipient cells. EV cargo is not passively packaged but it reflects dynamic, context-dependent sorting mechanisms that are tightly regulated by the cellular state and microenvironmental cues [32].

In homeostatic contexts, EVs contribute to processes such as tissue repair, immune modulation, and maintenance of cellular equilibrium [33, 34]. Moreover, EVs are now increasingly recognised as pivotal agents in the biology of pathological states as cancer. The role of these vesicles in facilitating tumour growth and survival is multifaceted, involving the remodelling of the TME, the modulation of immune responses, the promotion of angiogenesis, and the dissemination of pro-metastatic signals [35–37]. Furthermore, a mounting body of evidence has demonstrated that EVs can act as vectors for the transmission of drug-resistance mediators, thus contributing to treatment failure and disease relapse [38, 39].

EVs have traditionally been classified into exosomes, microvesicles (MVs), and apoptotic bodies (ApoBDs), reflecting differences in their biogenetic origin and morphological features [40–42].

Exosomes are typically derived from the endosomal compartment and released into the extracellular space through the fusion of multivesicular bodies with the plasma membrane [43, 44].

MVs, conversely, are released directly from cells into the extracellular space by outward budding and pinching of the plasma membrane [45, 46]. ApoBDs, meanwhile, are primarily produced by cells undergoing apoptosis, thereby encapsulating the residues of dying cells [47, 48]. The key features of each EV subtype are reported in Table 1.

Table 1.

Classification of extracellular vesicle subtypes

EV subtypes	Size (nm)	Origin	Biomarkers	Ref.
Exosomes	30–150	Endocytic pathway	CD9, CD63, CD81, Tsg101, Hsp70, Hsp90, Alix	[40]
Microvesicles	100–1000	Plasma membrane	Integrins, selectins, metalloproteinase	[41]
Apoptotic bodies	500–2000	Apoptotic cells	Phosphatidylserine	[42]

Alix, ALG-2-interacting protein X; CD, Cluster Differentiation; Hsp, Heat shock protein; Tsg101, Tumor Susceptibility Gene 101

Although this classification offers a useful conceptual framework, EV populations exhibit considerable overlap in terms of size, cargo composition, and surface marker expression, thus complicating their categorization. Given these complexities, we have decided not to delve into the intricacies of EVs in this review. Instead, we will direct the reader to the extensive existing literature on this issue. Thus, we will focus on the critical evaluation of EVs in the context of MM pathogenesis, as well as on the opportunities and limitations associated with their clinical translation into diagnostic and therapeutic applications.

Role of EVs in the crosstalk between MM and BM resident cells in BMME

Acting as molecular messengers, EVs navigate the intricate cellular landscape of the BMME, establishing a dynamic and reciprocal communication network between malignant plasma cells and surrounding stromal and immune populations. Through this continuous and finely regulated exchange of molecular signals, EV-driven communication contributes to the creation of a permissive niche that facilitates disease progression. Consequently, EVs should not be regarded as passive by-products of malignant activity, but rather as active drivers of disease evolution, offering valuable insight into the dynamic nature of MM-BMME interactions [24]. A mounting body of evidence underscores the capacity of EVs to modulate the pathogenesis of MM by regulating a variety of biological processes [24, 27, 49, 50]. The main mechanisms involved were summarized in Table 2.

Table 2.

Biological and clinical relevance of extracellular vesicles in multiple myeloma

Source	Type of vesicles	Molecules	Target Cells	Study model	Mechanisms	Ref.
MM cells	EVs	NOTCH2 receptor	ECs and OCs	In vitro/ in vivo	↑ Angiogenesis and OC differentiation	[51]
	Exosomes	Angiogenin, VEGF, bFGF	ECs	In vitro/ in vivo	↑ Angiogenesis	[52]
	EVs	piRNA-823	ECs	In vitro/ in vivo	↑ Angiogenesis	[53]
	Exosomes	tsRNA tRF-1003	ECs	In vitro/ in vivo	↑ Angiogenesis	[54]
	Exosomes	miR-135b	ECs	In vitro	↑ Angiogenesis	[55]
	Exosomes	miR-21, miR-146a	Mesenchymal stem cells	In vitro	↑ Mesenchymal stem cells proliferation	[56]
	Exosomes	miR-146a		In vitro	↑ MM cell proliferation and dissemination	[57]
	Exosomes	miR-433	Neighbouring MM cells	In vitro	↑ MM cell senescence	[58]
	Exosomes	lncRNA MALAT1	OCs	In vitro/in vivo	↑ OC differentiation	[59]
	Exosomes	lncRNA H19	OC and OB progenitor cells	In vitro/ in vivo	↑ OC and OB differentiation	[60]
	Exosomes	AREG	OC and OB progenitor cells	In vitro	↑ OC differentiation ↓ OB differentiation	[61]
	Exosomes	PNPO	OC progenitor cells	In vitro/in vivo	↑ OC differentiation	[62]
	sEVs and mEVs	MICA*008	NK cells	In vitro	↑ Immune escape	[63]
	EVs	MICA*008 and MICA *019	MM cells	In vitro	↓ Immune escape	[64]
	Exosomes	lncRNA NEAT1	NK cells	In vitro/ in vivo	↑ Immune escape	[65]
	EVs	Unknown	Lymphoid cells	In vivo	↑ Immune escape	[66]
	Exosomes	Unknown	Macrophages	In vitro	↑ M2 polarization	[67]
	sEVs	Unknown	Macrophages	In vitro	↑ Immune escape	[68]
	Exosomes	miR-16-5p, miR-146a-5p, miR-197-3p, miR-20b-5p, miR-21-5p	Monocytes/ macrophages	In vitro	↓ Innate antiviral immune response	[69]
	Exosomes	Unknown	MDSCs	In vivo	↑ Immune escape	[52]
	Exosomes	miR-106a-5p, miR-146a-5p	Monocytic MDSCs	In vitro	↑ Immune escape	[70]
	Chemoexosomes	Heparanase	MM cells	In vitro	↑ MM cell survival	[71]
	EVs	miRNA-1252-5p	MM cells	In vitro	↑ Bortezomib sensitivity	[72]
	Exosomes	ASM	MM cells	In vitro	↑ Drug resistance	[73]
	EVs	HSPA9	MM cells	In vitro/ in vivo	↑ Bortezomib resistance	[74]
	Exosomes	miR-16-5p, miR-15a-5p, miR-20a-5p, miR-17-5p	MM cells	In vitro	↑ Bortezomib resistance	[75]
	Exosomes	circMYC		In vitro	↑ Drug resistance	[76]
	EVs	TGFβ and RNAs	Mesenchymal stem cells	In vitro/ in vivo	↑ Drug resistance	[77]
	EVs	CD38		In vitro	↑ Daratumumab resistance	[78]
MSCs	Exosomes	miRNA-15a, oncogenic proteins, cytokines, adhesion molecules	MM cells	In vitro/ in vivo	↑ MM cell proliferation	[28]
	EVs	miR-10a	MM cells	In vitro/ in vivo	↑ MM cells proliferation	[79]
	Exosomes	lncRNA LINC00461	MM cells	In vitro	↑ MM cell proliferation ↓ apoptosis	[80]
	Exosomes	miR-340	ECs	In vivo	↓ Angiogenesis	[81]
	Exosomes	Unknown	MDSCs	In vitro/ in vivo	↑ Immune escape	[82]
	Exosomes	Unknown	MM cells	In vitro/ in vivo	↑ Bortezomib resistance	[83]
	sEVs	miR-140–5p, miR-28–3p	MM cells	In vitro/ in vivo	↑ Drug resistance	[84]
Mesenchymal stem cells	MVs	VLA4	MM cells	In vitro	↑ MM cell tumorigenicity	[85]
	MVs	Ribosomal proteins	MM cells	In vitro	↑ MM cell proliferation	[86]
	MVs	Unknown	MM cells	In vitro	↓ MM cell proliferation	[87]
	Exosomes	microRNA let-7c	Macrophages	In vitro	↑ M2 polarization	[88]
	Exosomes	PSMA3, lncPSMA3–AS1	MM cells	In vitro/ in vivo	↑ PI resistance	[89]
CAFs	Exosomes	miR-21	ECs	In vitro	↑ Angiogenesis	[90]
Fibroblasts	EVs	VEGF, HGF, ANG-1	ECs	In vitro	↑ Angiogenesis	[91]
	Exosomes	miR-214-3p, miR-5100	MM cells	In vitro	↑ Bortezomib resistance	[92]
Adipocytes	Exosomes	LOC606724 or SNHG1 LncRNAs	MM cells	In vitro/ in vivo	↑ Drug resistance	[93]
Osteocytes	Exosomes	miR-483-3p, miR-513a-5p	MM cells	In vitro/ in vivo	↑ Drug resistance	[94]

ANG-1, Angiopoietin-1; AREG, Amphiregulin; ASM, Acid Sphingomyelinase; bFGF, Basic Fibroblast Growth Factor; CAFs, Cancer Associated Fibroblasts; CD, Cluster Differentiation; circMYC, circular MYC RNA; ECs, Endothelial Cells; EVs, Extracellular Vesicles; HGF, Hepatocyte Growth Factor; HSPA9, Heat shock 70 kDa protein 9; lncRNA, long non coding RNA; M2, alternatively activated or anti-inflammatory macrophages; MALAT1, Metastasis-Associated Lung Adenocarcinoma Transcript 1; MDSCs, Myeloid Derived Suppressor Cells; mEVs, medium Extracellular Vesicles; MM, Multiple Myeloma; MSCs, Mesenchymal stromal cells; MVs, Microvesicles; NEAT1, Nuclear Enriched Abundant Transcript 1; NK, Natural Killer; OBs, Osteoblasts; OCs, Osteoclasts; PI, Proteasome Inhibitors; piRNA, Piwi-interacting RNA; PNPO, pyridoxine-5’-phosphate oxidase; PSMA3, Proteasome subunit alpha type-3; PSMA3-AS1, Proteasome subunit alpha type-3 antisense RNA1; sEVs, small Extracellular Vesicles; TGFβ, Transforming Growth Factor Beta; tsRNA, transfer RNA-derived small RNAs; VEGF, Vascular Endothelial Growth Factor; VLA4, Very Late Antigen-4

First, EVs activate pro-survival signaling pathways and deliver oncogenic cargo capable of suppressing apoptosis and interfering with DNA damage responses, thereby enhancing tumor cell survival under stress or therapeutic pressure [83]. The proliferative advantage conferred by EVs is not limited to direct effects on MM cells, but also relies on the EV contribution to BMME reprogramming by promoting angiogenesis and osteolytic remodelling, two processes that synergistically support tumor expansion [55, 95–97]. Secondly, EVs facilitate immune escape by modulating the phenotype and function of BM-resident immune cells, impairing cytotoxic responses and promoting immunosuppressive networks [66, 98]. Third, EVs have been implicated in the development of drug resistance by transferring protective molecules, modulating gene expression, and fostering a therapy-tolerant microenvironment [99]. It’s worth noting that the bidirectional nature of this EV-mediated communication reinforces the complexity of MM pathophysiology and underlines the need to dissect the distinct contributions of tumor-derived versus BMME-derived EVs. Thus, the following sections will provide a comprehensive analysis of how EVs from both malignant and non-malignant sources shape MM progression, focusing on their roles in promoting cell proliferation and survival, enabling immune evasion, and sustaining drug resistance, three interrelated processes that collectively drive disease evolution and therapeutic failure.

Tumor-derived EVs in MM cell growth

Among the oncogenic processes exploited by MM-derived EVs to support the growth and survival of MM plasma cells, the promotion of angiogenesis represents a particularly noteworthy phenomenon [53]. Recently, Giannandrea and colleagues demonstrated that EVs derived from MM cell lines and primary MM cells from high-risk patients express high levels of the oncogenic NOTCH2 receptor, whose activation is involved in modulating the behaviour of several BM cell populations. Findings evidenced that MM-EVs can transfer NOTCH2 receptor into ECs and OCs, thus promoting their protumorigenic capacity following the activation of NOTCH signaling [51].

Previous studies on MM murine models had highlighted the capacity of exosomes to promote pro-angiogenic effects through the modulation of specific signaling pathways, including Signal Transducer and Activator of Transcription 3 (STAT3), c-Jun N-terminal kinase (JNK), protein kinase B (Akt), p38, and p53 and by delivering angiogenic proteins like Angiogenin, Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) whitin their cargo [52]. MM development and progression can be also influenced by a small non-coding Piwi-interacting RNA (piRNA), piRNA-823, detected in EVs derived from the peripheral blood of MM patients and from MM cell lines. Indeed, piRNA-823-EV can be internalized in ECs, thereby favouring their proliferation, invasion and capillary structure formation. These events, occurring concomitantly with an increased expression of VEGF, IL-6 and ICAM-1, may create a permissive environment for MM cell growth and survival [53]. Similarly, another class of small non-coding RNAs, known as transfer RNA-derived small RNAs (tsRNAs), have emerged as novel regulatory elements in MM. Among them, tRF-1003, found enriched in exosomes from newly diagnosed and relapsed/refractory MM patients, was shown to promote angiogenesis by enhancing EC migration and tubulogenesis through hypoxia-inducible factor 1α (HIF-1α)/VEGF signaling, partly via Mitogen-Activated Protein Kinase 1 (MAPK1) suppression [54]. A strong correlation between MM-derived EV-miRNAs and various alterations in the BMME, including angiogenesis, was also reported [100]. Exosomal miR-135b derived from hypoxia-resistant MM cells improve the proliferation of ECs hindering the Factor-inhibiting hypoxia-inducible factor 1α (FIH). Thus, interfering with HIF-1α/FIH signaling pathway, miR-135b may lead to the remodelling of BMME by supporting angiogenesis under hypoxic condition [55].

Moreover, MM progression is regulated by the ability of MM-EVs to influence different functions of BM mesenchymal stem cells through the transport of miR-21 and miR-146a, which are involved in mesenchymal stem cell proliferation [56]. The transfer of miR-146a from MM cell-derived exosomes to MSCs resulted in an increased release of several cytokines and chemokines, encompassing IL-6, IL-8, CXCL1, IP-10, CCL5 and MCP-1, which in turn promote the survival and migration of MM cells [57]. Notably, EVs are profusely released by senescent cells and thus recognized as senescence-associated secretory phenotype (SASP) factors that may affect the growth and proliferation of recipient cells [101]. Takasugi et al. evidenced that exosomal miR-433 released from senescent Doxorubucin-induced MM cells promoted senescence in neighbouring MM cells. Mechanistically, the miR-433-induced senescent phenotype was not dependent to p53 and p21 up-regulation but rather associated with a reduced expression of cyclin-dependent kinase 6 (CDK-6). Thus, miR-433 derived from senescent MM-EVs may support and spread the senescence initiated by Doxorubicin to the surrounding MM cells [58].

Circulating EVs were also involved in the formation of distant bone lesions, as evidenced in de novo MM patients in which MM-EVs have been demonstrated to contribute in increasing OC lytic activity during MM progression [97]. Recent studies have identified novel EV-mediated mechanisms that reinforce the imbalance between OC and OB levels in MM [59–61, 97, 102]. For example, Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) has been identified as the highest expressed MM cell-derived exosomal lncRNA, which can be transferred to OCs and support their activation by promoting Receptor Activator of Nuclear Factor-κB Ligand (RANKL) expression and its downstream AKT and MAPK signaling pathways, known to be involved in MM cell viability and growth. Furthermore, it has been demonstrated that the increased expression of MALAT1 in MM cells may be correlated with NSUN2 and YBX1-mediated m5C modifications [59]. MM cells-derived exosomal lncRNA H19 is another contributing factor to osteolysis induction. Studies revealed that exosomal H19 enhanced osteolysis and counteracted osteogenesis by promoting the differentiation of OC progenitors and reducing the osteogenic differentiation of BM mesenchymal stem cells, resulting in decreased resorptive activity [60]. During MM progression, the functional balance between OCs and OBs is further perturbed by the activation of Epidermal Growth Factor Receptor (EGFR) signaling induced by Amphiregulin, EGFR ligand detected into exosomes of both MM cell lines and BM aspirates of MM patients. Particularly, Amphiregulin, once transferred into human mesenchymal stem cells, sustained osteoclastogenesis through the secretion of the pro-osteoclastogenic cytokine IL-8 and the inhibition of OB differentiation [61]. More recently, Deng et al. identified pyridoxamine 5’-phosphate oxidase (PNPO), a vitamin B6-metabolizing enzyme, as a driver of MM progression via EV-dependent mechanisms. PNPO overexpression in MM cells leads to the release of exosomes enriched in PNPO, which promote OC differentiation by oxidizing Dishevelled 3 (DVL3), a key cytoplasmic mediator of the Wnt signaling pathway. This activates Wnt/β-catenin signaling, enhancing osteoclastogenesis and bone resorption [62]. Figure 1Aprovides an overview of the molecular mechanisms through which MM cell-derived EVs contribute to MM cell growth.

Fig. 1.

Role of EVs released by MM cells in disease progression. MM cell-derived EVs transfer their cargo to various cellular components of BMME, thereby promoting MM cell growth (A), immune escape (B), and drug resistance (C). The figure illustrates the cell types involved in this intercellular communication. Solid arrows point from MM cells toward recipient cells, indicating the direction of EV-mediated signalings. MM-derived EVs are represented as small green spheres. EV cargo and the related EV-mediated effects on target cells (shown in the box) are reported. AREG, Amphiregulin; ASM, acid Sphingomyelinase; bFGF, Basic Fibroblast Growth Factor; CD38, Cluster of Differentiation 38; circMYC, circular MYC RNA; CTLA-4, cytotoxic T-Lymphocyte antigen 4; ECs, Endothelial cells; HSPA9, Heat shock 70 kDa protein 9; lncRNA, long non coding RNA; M2, alternatively activated or anti-inflammatory macrophages; MALAT1, Metastasis-Associated lung Adenocarcinoma Transcript 1; MDSCs, Myeloid-Derived Suppressor Cells; MSCs, Mesenchymal Stromal Cells; NEAT1, Nuclear Enriched Abundant Transcript 1; NK, Natural Killer; OB, Osteoblast; OC, Osteoclast; PD-1, Programmed Cell Death 1 protein; piRNA, Piwi-interacting rna; PNPO, pyridoxine-5’-phosphate oxidase; R-NOTCH2, NOTCH2 receptor; TGFβ, Transforming Growth Factor beta; tsRNA, transfer RNA-derived small RNAs; VEGF, vascular endothelial growth factor. This figure was generated using BioRender.com

BM-derived EVs in MM cell growth

The first clarifications on the ability of the BMME to transfer genetic and protein changes to MM clonal plasma cells via EVs was documented in 2013, when Roccaro et al. unveiled a novel oncogenic mechanism mediated by BM-resident MSCs to drive MM cell proliferation and dissemination [28]. It has been reported that MM BM-MSC exosome levels of microRNA-15a, a recognised tumour suppressor, were reduced, while the expression levels of oncogenic proteins, cytokines, and adhesion molecules were elevated compared to normal BM-MSC exosomes. Umezu et al. demonstrated that exosomes derived from BM MSCs of MM patients are significantly enriched in miR-10a compared to those from healthy donors. Exosomal transfer of miR-10a to MM cells was found to promote cell proliferation. Interestingly, blocking EV secretion induced intracellular accumulation of miR-10a in MSCs, resulting in impaired proliferative capacity and enhanced apoptotic activity of MSCs, as well as diminished MM cell growth [79]. MM progression is further supported by the lncRNA LINC00461, which is overexpressed in MSC exosomes of MM patients. Specifically, the transfer of MSC-derived exosomes carrying LINC00461 sustains proliferation and hinders apoptosis of MM cells through the modulation of miR-15a/16 and Bcl-2 [80]. Previous studies indicated that Histone Deacetylases (HDACs) represent important therapeutic targets in MM. It has been reported that HDAC3 knock-down in BM MSCs negatively influenced MM cell proliferation through several mechanisms [103]. Further investigations showed that HDAC3 inhibition also resulted in a reduced secretion of BM MSC-derived exosomes, dependent on the inhibition of Tsg101, a protein involved in exosome biogenesis. Moreover, the quantitative modulation of BM MSC-derived exosomes is concomitant to their qualitative alteration. Indeed, HDAC3 down-regulation has been demonstrated to affect exosomal miRNA content of BM MSCs, sustaining the reduced expression of miR380, miR382, miR15b, miR9986, and miR5191, recognized as pro-survival factors [104]. Cellular components of the BMME that can exert a pro-cancerous effect on MM cells via EVs also include mesenchymal stem cells. A proteomic analysis of MVs obtained from mesenchymal stem cells of normal donors and MM patients revealed discrepancies in their membrane protein expression. Interestingly, a strong correlation between high levels of integrin partners CD49d and CD29 and staging and treatment response of MM patients exists, suggesting that VLA4-enriched MVs, isolated from mesenchymal stem cells of MM patients, are involved in MM cells tumorigenicity [86]. Dabbah et al. demonstrated that the myelomatous condition also resulted in MV-derived mesenchymal stem cells enrichment of ribosomal proteins that facilitated translational initiation and enhanced MM cell proliferation [85, 86]. It has been reported that MVs derived from mesenchymal stem cells of healthy donors have the capacity to exert anti-tumor effects by promoting apoptosis in MM cell lines. Specifically, MM cells exposed to mesenchymal stem cell-MVs exhibited reduced expression of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic marker Bax, and decreased expression of Cyclin D genes. These results suggest that MVs isolated from healthy mesenchymal stem cells may partially reproduce the therapeutic functions of the cells from which they derive and show tumor-suppressive properties. This is particularly relevant given the growing interest in stem cell-based therapies as innovative approaches for the treatment of various malignancies. However, while encouraging, these findings require further in-depth in vitro and in vivo investigations to fully understand the mechanisms involved and to assess their translational potential in the clinical management of MM [87].

It has been proposed that the therapeutic potential of exosomes derived from BM MSCs of healthy subjects may be correlated with the donor’s age. An in vitro study demonstrated that the inhibition of MM cells angiogenesis by young BM MSCs was associated with a distinct miRNAs profile. Indeed, compared to older BM MSCs, the younger BM MSCs exhibited a detrimental effect on the angiogenic process through the transfer of exosomal miR-340 to ECs. The underlying mechanism of angiogenesis inhibition occurred through the Hepatocyte Growth Factor (HGF)/mesenchymal-epithelial transition factor receptor (c-MET) signaling pathway [81].

Recently, an interesting report investigated the relationship between exosomes derived from Cancer-associated Fibroblasts (CAFs) and angiogenesis in MM. The analysis of primary CAFs isolated from the BM of MM patients revealed high expression levels of exosomal miR-21 and its involvement in MM angiogenesis when transferred to MM ECs. Specifically, exosomal miR-21 has been demonstrated to regulate angiogenesis through an increased proliferation and migration of ECs and formation of their tubular structures. Furthermore, miR-21 induced the up-regulation of Alpha Smooth Muscle Actin (α-SMA) and Fibroblast Activation Protein-α (FAP) in Normal Fibroblasts (NFs), further supporting its role as a pro-cancer factor in MM by driving NF transformation into CAFs and promoting angiogenesis [90]. Moreover, fibroblast-derived EVs influence MM angiogenesis through both EV uptake-dependent and -independent mechanisms. These EVs are enriched in angiogenic cytokines [i.e. VEGF, HGF and Angiopoietin-1 (ANG-1)] that create a pro-angiogenic milieu without exploiting EV internalization. Additionally, prolonged exposure of MM ECs to fibroblast-derived EVs induced the activation of mTORC1, MAPK, SRC and STAT signalling pathways, which are crucial in modulating molecular processes driving angiogenesis, including migration, metalloprotease secretion, capillary-like structure formation and chemotaxis [91]. The effects of EVs released from cellular components of BMME on MM progression are summarized in Fig. 2A.

Fig. 2.

Role of EVs released by cellular components of BMME in MM progression. EVs derived from BMME cells transfer their cargo to target cells, thereby promoting MM cell growth (A), immune escape (B), and drug resistance (C). The figure highlights the cell types involved in this intercellular communication. Solid arrows indicate the direction of EV-mediated signaling from BMME cells to recipient cells. Dotted arrows represent indirect effects on MM cells resulting from EVs transfer between different BMME cell types. EVs are depicted as small spheres in different colors, reflecting their cell of origin. EV cargo and the related EV-mediated effects on target cells (shown in the box) are reported. ANG-1, angiopoietin-1; CCL2, chemokine C-C motif ligand 2; ECs, Endothelial Cells; HGF, hepatocyte growth Factor; IL-6, interleukin 6; lncRNA, long non coding RNA; M2, alternatively activated or anti-inflammatory macrophages; MDSCs, Myeloid-derived Suppressor Cells; MSCs, Mesenchymal Stromal Cells; PI, Proteasome Inhibitors; PSMA3, Proteasome Subunit Alpha type-3; PSMA3–AS1, Proteasome Subunit Alpha type-3 antisense RNA1; VEGF, Vascular Endothelial Growth Factor; VLA-4, Very Late Antigen-4. This figure was generated using BioRender.com

Tumor-derived EVs in MM immune escape

Dysfunction of immunological processes represents a hallmark of MM, with the potential to influence the evolution of the disease from its precursor stages [105]. Of note, compelling evidence provides novel insights into the influence of MM-derived EVs on both innate and adaptive immune responses. Indeed, recent works have attempted to clarify the molecular mechanisms through which MM-originated EVs can modulate the behaviour of BM-resident immune cells, including natural killer (NK), CD4⁺ or CD8⁺ T cells and macrophages, thereby promoting MM immune escape.

NK cells have been recognised as critical players in MM immunosurveillance due to their involvement in cancer cell death and cytokines and chemokines production [106]. Impaired NK cell activity and reduced expression of activating receptors, including Natural Killer Group 2, Member D (NKG2D), have been found in advanced MM patients [63, 107]. This dysfunction is correlated to the presence of MM-derived EVs enriched with NKG2D ligands, including MICA, which can be transferred to NK cells. In particular, it has been observed that short-term EV-stimulation transiently activates NK cells, thereby enhancing their cytotoxic responses. In contrast, prolonged exposure has been shown to affect NK cell functions by either down-regulating NKG2D or facilitating the killing of NK cells. Consequently, the extent of these effects on NK cell activity against tumor cells is closely related to the duration and intensity of EV exposure. From a clinical perspective, the abundance and composition of MM-EVs may be used as indicators of disease progression and immune system dysregulation. In this context, the hypothesis is that exploiting the immunostimulatory effects of EVs expressing NKG2D ligand through a temporally controlled release could represent a novel strategy to potentiate NK cell activity during chemotherapeutic treatment [63]. Furthermore, MM cells have been observed to internalise EVs carrying NKG2D ligands, resulting in their surface expression, a phenomenon referred to as ligand cross-dressing. This process has been shown to increase the susceptibility of tumour cells to NK cell-mediated recognition and killing, thereby enhancing anti-tumour immune responses. However, the dissemination of NKG2D ligands via EVs introduces a complex dynamic within the TME: while it may promote NK cell-mediated cytotoxicity against MM cells displaying these ligands, it can also contribute to immune escape by inducing NKG2D downregulation or facilitating NK cell fratricide. Thus, targeting EV-mediated ligand transfer, or modulating EV release and composition, may represent a novel therapeutic approach to improve clinical outcomes in MM patients [64]. Suppression of NK cell activity is also sustained by the transfer of MM cells-derived exosomal lncRNA NEAT1, which encourages MM immune escape through the modulation of Enhancer of Zeste Homolog 2 (EZH2)/Pre-B-cell leukemia transcription factor 1 (PBX1) axis [65].

Tumor-derived EVs were shown to create a permissive niche for MM progression also by affecting the other lymphoid populations within the BMME. Particularly, the characterization of BMME of mice exposed to EVs obtained from MM cells highlighted the modulation of lymphocytes phenotype, defined by high expression of Programmed Death 1 (PD-1) and the Cytotoxic T-Lymphocyte–associated Antigen 4 (CTLA-4) immune checkpoints. Consistent with these data, enhanced levels of CD27⁺, CD25⁺, and CD4⁺ T cells were detected, confirming the involvement of MM-EVs in immunosuppression [66].

Macrophages represent another class of immunoregulatory cells able to support tumour cells in inducing TME alterations. Exosomes derived from the MM cell line U266 have been shown to modulate the molecular profile and cellular characteristics of M0 macrophages, evidencing their pivotal role in immune suppression. Specifically, Bahman et al. demonstrated that MM-derived exosomes polarize M0 macrophages toward an M2-like phenotype with pro-tumoral and anti-inflammatory activity, expressing high levels of typical M2 markers, including CD206, IL-10 and Arginase 1 [67]. In addition, the immunosuppressive microenvironment is also correlated with the upregulation of Programmed Death-Ligand 1 (PD-L1) and IL-6, and the consequent STAT3 signaling pathway activation, in M0 macrophages due to the internalisation of EVs derived from MM cell line [68].

It is worth noting that MM-derived exosomes cells hinder innate antiviral immune response of MM patients by transferring specific miRNAs to the peripheral immune cells such as monocytes/macrophages. In particular, five miRNAs were identified in the MM exosome cargo, including miR-16-5p, miR-146a-5p, miR-197-3p, miR-20b-5p and miR-21-5p, which are responsible for inhibition of the Cyclic GMP-AMP synthase (cGAS)- Stimulator of interferon genes (STING) pathway and reduction of type-I Interferon (IFN-I) production. Thus, this study provided the discovery of an exosomal miRNA signature in MM patients with prognostic relevance for DNA virus infection [69]. Moreover, it has been demonstrated that MM-derived exosomes support the induction of Myeloid-Derived Suppressor Cells (MDSCs), which are immature myeloid cells that act as negative modulators of the immune response counteracting lymphocyte activation [108]. This occurs through the MM exosomes-mediated STAT3 signaling pathway activation and inducible Nitric Oxide Synthase up-regulation [25], as well as through the transfer of MM cells-derived exosomal miR-106a-5p and miR-146a-5p, which are involved in the upregulation of several molecules with immunosuppressive/inflammatory functions [70]. The collective involvement of tumor-derived EVs in immune escape mechanisms is illustrated in Fig. 1B.

BM-derived EVs in MM immune escape

Despite the existence of a number of reports providing substantial evidence on the relationship between MM cells-derived EVs and MM immune response, the current knowledge regarding the MM immunosuppressive functions of EVs released by BM cellular component remains limited. However, it has been demonstrated that BM MSC exosomes support immunosuppression by targeting MDSCs [108]. An in vitro study evidenced that exosomes obtained from BM MSCs contributed to an enhanced accumulation and activation of MDSCs through the induction of pSTAT1 and pSTAT3 signaling pathways along with an up-regulation of the MCL-1 (Myeloid Cell Leukemia 1) protein. Furthermore, the intravenous injection of BM MSC-exosomes into 5T33MM mice induces a greater production of Nitric Oxide in mice MDSCs, thereby favouring their capability to suppress T cell proliferation [82]. In addition to MDSCs, tumor-associated macrophages also cooperate with the other cells of the BMME to create a MM immunosuppressive milieu [109]. It is noteworthy that macrophages activation may be mediated by mesenchymal stem cell-derived exosomes, which have been observed to transport microRNA let-7c. This appears to contribute to the polarization and activation of macrophages into their M2 state [88]. In addition, a recent examination of Matchett and Kornbluth proposes the use of immortalised NK cell-derived EVs as a promising therapeutic option for the regulation of immune responses in MM, given their capacity to influence the survival of MM cells [110]. Figure 2 B illustrates the role of BM-derived EVs in promoting immune escape in MM.

Tumor-derived EVs in MM drug resistance

The advent of novel therapeutic agents has significantly improved the prognosis of MM by reducing the adverse effects associated with conventional chemotherapy. Current frontline treatment combines proteasome inhibitors (PIs), such as Bortezomib and Carfilzomib, with immunomodulatory drugs like Thalidomide and Lenalidomide, followed by monoclonal antibodies including Daratumumab, Elotuzumab, and Isatuximab [53]. Despite these advances, relapse and resistance to anti-MM therapies remain common, making drug resistance, whether intrinsic or acquired, a major clinical challenge [77]. This resistance stems from a complex interaction between MM cells and the BMME, in which EV-mediated communication plays a key role [111]. Consequently, growing attention has been directed toward analyzing EV content of MM cells to assess their potential as prognostic and predictive indicators of treatment response [83, 99, 112].

Notably, MM cells can autologously promote drug resistance by secreting EVs. Indeed, exposure of MM cells to commonly administered anti-MM drugs significantly raised the release of chemotherapy-induced exosomes, identified as “chemoexosomes” and characterised by an altered proteome profile [71]. In particular, it was shown that the surface of chemoexosomes is enriched with high levels of heparanase, which is responsible for the degradation of heparan sulphate in the extracellular matrix. Consistently, transfer of these exosomes to MM cells favours the degradation of heparan sulphate and drives the activation of p-ERK signalling [71]. Moreover, it has been highlighted a strong correlation between heparanase and miRNA-1252-5p expression in MM cells treated with Bortezomib. Heparanase expression levels and enzymatic activity were dramatically reduced in MM cells carrying EVs overexpressing miRNA-1252-5p, suggesting that miRNA-1252-5p presence in MM-derived EVs may be a promising factor to enhance MM cell sensitivity to Bortezomib treatment [72]. Faict et al. demonstrated that Melphalan- or Bortezomib-treated MM cells and their exosomes expressed high levels of Acid Sphingomyelinase (ASM) and that ASM-enriched exosomes were involved in the transfer of a drug-resistant phenotype to MM cells that were initially responsive to therapy [73]. Furthermore, Wang et al. reported that EVs containing HSPA9 promote Bortezomib resistance by activating the Thyroid Hormone Receptor Interacting Protein 13 (TRIP13)/Ubiquitin-Specific Peptidase 1 (USP1) pathway, thereby supporting cell survival and DNA repair in MM cells, further confirming the key role of EV cargo in mediating resistance [74]. A study conducted in 2016 compared exosomal miRNA profiles in Bortezomib-resistant and Bortezomib-responsive MM subjects, revealing the down-regulation of exosomal miR-16-5p, miR-15a-5p, miR-20a-5p, and miR-17-5p in Bortezomib-resistant patients [75]. In addition, a deeper investigation by Tang et al. showed that the exosome cargo of MM patients resistant to Bortezomib was enriched with 582 lncRNAs along with 2099 differentially expressed mRNAs. Particularly, the bioinformatics analysis evidenced that a total of 78 mRNAs were found to be enriched in various signaling pathway correlated with drug resistance, encompassing mTOR, platinum DR, cyclic Adenosine Monophosphate (cAMP) and Phosphoinositide 3-Kinase (PI3K)-Akt. Moreover, it has been demonstrated that Bortezomib resistance in MM patients is supported by the deregulated exosomal lncRNAs, which may alter miRNA-mediated regulation [113]. The exosomal circMYC has been identified as a potential biomarker for drug resistance in MM since its higher levels were detected in exosomes derived from therapy-resistant MM patients compared to treatment-responsive ones [76]. Moreover, MM EVs were shown to be involved in the induction of a mesenchymal stem cell inflammatory profile through the transfer of Transforming Growth Factor Beta (TGFβ) and various RNAs, which promote the up-regulation of different chemokines, leading to the establishment of drug resistance [77]. Interestingly, Sortilin 1 (SORT1) and Lysosome-Associated Membrane Protein 2 (LAMP2)-dependent EVs release and cell adhesion appear to be associated with Lenalidomide resistance in MM. Indeed, SORT1 and LAMP2 knock-down resulted in reduced EV release and cell adhesion in the Lenalidomide-resistant counterpart, thereby increasing drug sensitivity [111]. Exposure of MM cell lines to Daratumumab, an anti-CD38 monoclonal antibody, has been shown to induce redistribution of the CD38 receptor into released EVs [114]. Brennan et al. further characterize EVs derived from MM patients, revealing increased levels of CD38-expressing EVs in the peripheral blood of Daratumumab-treated MM patients compared to newly diagnosed individuals, suggesting that Daratumumab may influence CD38 levels. Moreover, mass spectrometry analysis confirmed the presence of Daratumumab-derived peptides on EV surface, providing evidence of direct antibody interaction with CD38 on EV membranes. These findings support the hypothesis that CD38⁺ EVs may act as molecular decoys, capturing Daratumumab in the extracellular milieu and thereby reducing its bioavailability for binding to CD38 on malignant plasma cells. This mechanism could contribute to decreased therapeutic efficacy and the development of drug resistance [78]. The pathways underlying the relationship between MM-derived EVs and drug resistance are summarized in Fig. 1C.

BM-derived EVs in MM drug resistance

Several reports have evaluated the impact of EVs derived from different cells of BMME on the efficacy of the PI therapeutic approaches used in MM patients. In 2014, Wang et al. demonstrated that exosomes derived from murine and human BM MSCs activated specific survival pathways and negatively modulated the expression levels of proteins regulating apoptosis process, thereby reversing Bortezomib effects in MM cells [83]. The reduced MM cell chemosensitivity to Bortezomib dependent on BM MSC-originated EVs was further corroborated by Tu et al., demonstrating that the blockade of EV uptake using specific chemical endocytosis inhibitors counteracted Bortezomib resistance in MM cells [115]. A crucial factor that may enhance the release of EVs from BM MSCs is the hypoxic state associated with BMME. It has been reported that the increased secretion of EVs derived from BM MSCs grown in a hypoxic environment mitigates Bortezomib sensitivity to a greater extent than EVs originated in normoxic condition. In particular, the characterization of hypoxic BM MSC-derived EV cargo evidenced an increased expression of miR-140–5p and miR-28–3p. Mechanistically, it was shown that these miRNAs, inhibiting Sprouty Related EVH1 Domain Containing 1 (SPREAD1), support the activation of MAPK-related pathways, leading to Bortezomib resistance [84]. Moreover, it was found that targeting the cystine/glutamate antiporter System Xc⁻ disrupts exosome-mediated crosstalk between BM MSCs and MM cells, reversing Bortezomib resistance [116]. Several studies have investigated the contribution of specific BM subpopulations in PI resistance. It has been demonstrated mesenchymal stem cell’s role in PI resistance due to their ability to transfer exosomal PSMA3 (Proteasome subunit alpha type-3) and lncPSMA3–AS1 (PSMA3 antisense RNA1) to MM cells. Increased levels of mesenchymal stem cell-derived exosomal lncPSMA3–AS1 have been shown to be a critical player in mediating PI resistance through its ability to stabilise PSMA3, which is responsible for the encoding of the constitutive proteasome component subunit α7. PSMA3 interacts with PSMA3–AS1 pre-mRNA to form an RNA duplex, leading to increased stability of PSMA3 and thereby promoting Chymotrypsin-Like proteasome activity [89]. BMME-induced MM drug resistance may be further explained by a vicious cycle existing between MM cells and marrow adipocytes. In particular, MM cells may regulate the content of adipocyte-derived exosomes, favouring the enrichment of specific lncRNAs, which in turn may preserve MM cell from the effects of chemotherapy. It was found that increased levels of LOC606724 or SNHG1 lncRNAs, detected in Bortezomib-resistant MM patients but not in Bortezomib-sensitive counterpart, prevented chemotherapy-mediated apoptosis in MM cells. Investigation of the molecular mechanism underlying MM cell-induced adipocyte exosomal packaging highlighted the involvement EZH2/METTL7A/lncRNA axis [93]. Furthermore, the cross-talk between osteocytes and MM cells, orchestrated by osteocyte-secreted exosomal miRNAs, affects MM therapeutic response. In this context, a pivotal role of exosomal miR-483-3p and miR-513a-5p has been suggested, which, once transferred from osteocytes to MM cells, support the acquisition of MM cell stem-like phenotype by activating the HIF-1α signalling [94]. In addition, the ability of MM cells to internalise MM fibroblast-derived exosomes and incorporate only specific miRNAs through a mechanism mediated by lncRNAs has been proposed. Although MM fibroblast-derived exosomes overexpress miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p and miR-5100, MM cells selectively express high levels of miR-214-3p and miR-5100. Specifically, it was shown that miR-214-3p and miR-5100 upregulation in MM cells induces the downregulation of their target genes, PTEN and DUSP16 respectively, thereby protecting MM cells from Bortezomib-induced apoptosis and ensuring their proliferation and survival [92]. Figure 2 C outlines the molecular processes connecting BM-derived EVs to drug resistance in MM.

Role of EVs in the dynamic monitoring of MM

The detection and quantification of monoclonal immunoglobulins, including FLCs, remain the cornerstone biomarkers for diagnosing and monitoring MM, reflecting the secretory nature of malignant plasma cells [117]. Over time, MM cells may reduce or completely lose their ability to secrete M-proteins due to cellular de-differentiation, a process known as light-chain escape. As a result, in oligo-secretory or non-secretory MM patients M-protein levels may be extremely low or undetectable, with FLCs serving as the only measurable marker, potentially leading to a misinterpretation of disease status [118]. Furthermore, renal dysfunction might reduce the clearance of FLCs, resulting in elevated serum levels that may not accurately reflect the tumour burden, thus complicating diagnostic interpretation and response assessment [119]. Beyond these limitations, MM spatial and subclonal heterogeneity is another challenge. The disease typically exhibits patchy neoplastic infiltration in marrow environment, with genetically distinct subclones often residing at different BM sites [120]. Consequently, standard single-site BM aspirates, although recognized as a gold standard for disease assessment, may fail to capture the full genomic complexity of the tumor, underestimating high-risk subclones or resistance-driving mutations. This issue becomes particularly critical during Minimal Residual Disease (MRD) assessment, as subclonal evolution under therapeutic pressure can drive relapse [121]. In this context, liquid biopsy by analyzing circulating tumor-derived components, including cell-free DNA (cfDNA), circulating tumor cells (CTCs), and EVs, has gained increasing attention as a novel strategy to overcome current diagnostic constraints. Among these circulating biomarkers, EVs are particularly encouraging due to their distinctive molecular cargo and remarkable structural stability. Unlike cfDNA, which is susceptible to fragmentation and rapid degradation, EVs preserve their bioactive cargo within a protective lipid bilayer, enabling more reliable downstream molecular studies. In addition, compared to CTCs, which are relatively rare and technically challenging to isolate and characterize, EVs are more abundant and stable in peripheral blood, making them a more accessible and reproducible source of tumor-derived material for molecular analysis [122]. Several quantitative analyses have demonstrated a significant increase in circulating EV levels in the peripheral blood of MM patients compared to healthy individuals or those with MGUS, suggesting a direct association between tumor burden and EV production [13, 27, 123]. Interestingly, the gradual increase in EV concentrations observed along the disease spectrum reinforces the concept that EV abundance may act as a surrogate marker of both disease stage and biological activity. For instance, exosomes expressing CD138, a canonical marker of differentiated plasma cells, have been identified in the peripheral blood of MM patients. Concentration of EV-CD138⁺ is markedly higher in individuals with active or advanced disease compared to those in remission, suggesting a direct link between EV-CD138⁺ levels and tumor burden [124]. Higher levels of circulating EV-CD138⁺ have also been associated with more severe clinical manifestations such as myeloma bone disease and renal impairment [97], as well as with the co-expression of additional surface markers, P-glycoprotein, phosphatidylserine, and CD34, which have been implicated in the development of multidrug resistance [125]. In addition, in-depth profiling of surface markers and molecular cargo enclosed within EVs has significantly broadened their utility as potential biomarkers in the context of MM MRD detection. Proteomic analysis of MM-derived exosomes has identified CD44 as a promising prognostic biomarker, with elevated levels of exosomal CD44 detected in patient serum demonstrating a significant inverse correlation with overall survival [126]. A recent real-world study on patient-derived EVs found that a higher protein-to-particle ratio is linked to poorer survival outcomes and immune dysfunction in MM. The study also identified several EV-associated proteins, including PDIA3, C4BPA, BTN1A1, and TNFSF13, as promising prognostic biomarkers [127]. Importantly, the significant proteomic overlap between circulating and BM-derived EVs highlights the potential of peripheral blood EVs as non-invasive alternatives for disease monitoring [127, 128]. A large amount of study shed light on the contribution of EV-miRNAs as valuable biomarkers in MM. For example, patients with lower exosomal levels of miRNA-18a and let-7b, were more likely to be at an advanced stage of disease and exhibited a worse prognosis [129]. Decreased exosomal levels of let-7c-5p, let-7d-5p, miR-140-3p, miR-185-5p, and miR-425-5p have been associated with key clinical features of MM, including renal impairment and elevated β2-microglobulin and IL-6 levels, highlighting their potential role in reflecting disease severity [130]. Zhang et al. identified miR-20a-5p, miR-103a-3p, and miR-4505 as differentially expressed among MM, SMM and healthy individuals, with additional alterations in let-7c-5p, miR-185-5p, and miR-4741 distinguishing MM from earlier disease stages [131]. More recently, exosomal miR-451 has been identified as a promising diagnostic and prognostic marker in MM, with diminished levels in circulation correlating with adverse clinical outcomes and reduced survival [132]. Interestingly, Tang et al. highlighted the translational relevance of circRNAs in MM, identifying exosome-secreted circHNRNPU_603aa as a driver of IgD MM progression through SKP2 splicing dysregulation and inhibition of c-Myc ubiquitination, suggesting its potential as a biomarker for high-risk, treatment-refractory cases [133]. Additionally, exosomal circMYC has been linked to disease relapse and Bortezomib resistance. Its levels are elevated in MM patients, particularly in resistant cases, and correlate with adverse features such as del(17p), t(4;14), advanced stage, and poor prognosis [76].

Collectively, these studies highlight the growing impact of EVs as powerful tools in the context of liquid biopsy, providing a minimally invasive window into the evolving molecular landscape of MM (Fig. 3). Such insights have the potential to improve disease monitoring, enhance risk stratification, and facilitate more precise, and timely therapeutic decision-making. However, to translate these advances into routine clinical practice, standardization of EV isolation and characterization protocols, validation across large patient cohorts, and seamless integration with current diagnostic approaches remain essential.

Fig. 3.

Bridging diagnostic gaps in MM through EV-based liquid biopsies. The figure illustrates current challenges and emerging solution in the monitoring strategies for MM. (A). The current challenges of the peripheral blood test and bone marrow biopsy, the cornerstone diagnostic tools in MM, are reported in red boxes. Briefly, for blood tests, limitations include low sensitivity in detecting M protein and free light chains (FLCs), as well as interference from abundant serum proteins such as Albumin. For bone marrow biopsy, the patchy nature of neoplastic infiltration can lead to sampling bias and underestimation of disease burden. (B). EV-based liquid biopsy is proposed as a minimally invasive strategy. The advantages of using this approach, as higher biomarker stability, potential for early detection, and a more comprehensive representation of tumor heterogeneity are reported in green box. This figure was generated using BioRender.com

EVs in liquid biopsy: methodological barriers and technological innovations

The successful clinical translation of EV-based liquid biopsy continues to be limited by unresolved EV isolation challenges, especially in the context of blood-derived samples, rich in interfering proteins like albumin [134]. In MM, the pathological accumulation of monoclonal immunoglobulins and FLCs further exacerbates this challenge by intensifying background noise and negatively affecting the yield, purity along with the biological fidelity of isolated EVs, which are critical for accurate molecular analyses such as miRNA profiling, transcriptomic studies, and surface marker characterization [134].

Conventional EV isolation methods, such as ultracentrifugation and size-exclusion chromatography, struggle to effectively discriminate tumor-derived EVs from non-vesicular particles that closely overlap in size and density, such as lipoproteins and protein aggregates [14]. Moreover, while immunoaffinity-based techniques offer higher specificity, their performance is constrained in MM by the pronounced phenotypic heterogeneity of EVs and the limited discriminatory power of canonical markers like CD63 and CD81, which are not exclusive to vesicles of plasma cell origin. To address this, emerging strategies are exploring the use of MM-associated surface antigens such as CD138 and CD38 to achieve more selective capture of disease-relevant EV subpopulations [128].

Several technological advances have recently emerged to overcome the limitations of conventional EV isolation methods from human plasma. For instance, a pioneering bubble-based platform has been developed, enabling wash-free and single-step profiling of EV surface markers directly from plasma. This platform employs ultrasensitive detection techniques, achieving an accuracy that approaches the single-EV level and reducing background noise, which is especially helpful in MM where monoclonal proteins can interfere with EV detection [135]. A recent comparative study evaluated nine EV isolation methods using just 100 µL of plasma and found that magnet-based systems, such as MagNet and MagCap, offered the best compromise between purity and proteome coverage. This makes them particularly well suited for downstream analyses in MRD settings, where the detection of low-abundance tumor-derived markers is strongly dependent on high EV purity [136]. An additional proposed strategy was to combine asymmetric flow field-flow fractionation (AF4) with density cushion ultracentrifugation, enabling high-purity EV isolation from plasma with minimal lipoprotein contamination. Unfortunately, the study highlights that commonly used EV markers from cell lines are not always applicable to clinical samples, underscoring the need for tailored strategies in liquid biopsy workflows [137]. Further microfluidic advancements include acoustofluidic chromatography, which achieves rapid EV enrichment from only a few microliters of plasma with minimal protein contamination [138], and a magnetic-levitation microfluidic (EV-Lev) system using antibody-functionalized beads to isolate sEVs with high purity in low-volume clinical specimens [139]. Furthermore, rapid and automatable microfluidic systems have been introduced, such as a viscoelastic microfluidic device capable of isolating sEVs from whole blood in a continuous and label-free manner with over 97% purity and 87% recovery [140], and cascaded pulsatile microfluidic circuits that enable clog-resistant, high-yield EV isolation from whole blood in just 30 minutes [141].

Role of EVs in the therapeutic management of MM

The therapeutic management of MM has evolved significantly over the past two decades, shifting from a context of limited treatment choices and poor survival outcomes to a more complex landscape shaped by the availability of numerous effective agents and combination regimens [142]. However, the growing complexity of treatment options now demands tailored strategies that account for diverse clinical presentations, cytogenetic profiles, and varying levels of frailty and comorbidity. This approach is particularly crucial in elderly and intermediate-fit patients, where aggressive regimens may lead to excessive toxicity and impaired quality of life [143]. Furthermore, resistance mechanisms and subclonal heterogeneity remain major barriers to achieving durable responses in MM, as therapy-resistant clones frequently emerge despite deep initial remission. Recent single-cell multi-omics studies have mapped the clonal architecture of relapsed/refractory MM, revealing how distinct tumor subclones adapt to treatment over time through coordinated or divergent transcriptional and epigenetic changes, and how their specific interactions with the BMME may contribute to resistance and disease progression [144]. Against this backdrop, EVs are emerging as potential therapeutic candidates capable of addressing several of these limitations, offering novel opportunities for targeted drug delivery and immunomodulation within the BMME [24, 145] (Fig. 4).

Fig. 4.

EV-based strategies currently explored in the therapeutic landscape of MM. The figure delineates the primary strategies for leveraging EVs in the treatment of MM. These include: (a) surface functionalization, aimed at enhancing targeting specificity through the modification of EV membrane proteins; (b) cargo engineering, which involves loading therapeutic agents (small molecules, RNA, or proteins) into the EV lumen; (c) inhibition of EV-mediated signaling, aimed at disrupting pro-tumor communication by blocking EV release or uptake; (d) immunomodulatory applications, highlighting the use of EVs in vaccine design to elicit anti-tumor immune responses. This figure was generated using BioRender.com

EVs as therapeutic target and drug delivery platforms in MM

Recently, a growing body of preclinical evidence has highlighted the therapeutic potential of targeting EVs in MM. Particularly, approaches that are currently under active investigation include the pharmacological or genetic inhibition of EV release, interference with EV internalization by recipient cells and reengineering of EV surface to disrupt specific MM-supporting pathways. In parallel, EVs are being explored as next-generation delivery systems in MM, taking advantage of their biocompatibility, intrinsic targeting capabilities, and ability to transport therapeutic molecules across biological barriers. These features make EVs more efficacious delivery system for therapeutic payloads, including small-molecule drugs, siRNAs, and CRISPR/Cas components, when directly administered to MM cells, compared to synthetic nanoparticles [146]. Tu et al. reported that targeting endocytic machinery through pharmacological inhibition or shRNA-mediated silencing of key regulators, such as clathrin, caveolin, and dynamin‑2, impairs the uptake of stromal-derived EVs by MM cells, resulting in enhanced Bortezomib-induced cytotoxicity [115]. Interestingly, it has been reported that RAB22A expression correlates with exosome release and immune infiltration in MM, and that knocking down RAB22A in mesenchymal stem cells reduces exosome secretion, thereby limiting their ability to promote MM cell proliferation [147]. Rivoltini et al. functionalized exosomes with membrane-bound TRAIL (TNF‑Related Apoptosis‑Inducing Ligand) to deliver pro-apoptotic signals directly to MM cells. When administered intratumorally in murine models, these TRAIL-armed EVs induced marked tumor regression and necrosis, highlighting the potential of EVs not only as passive carriers, but also as active therapeutic agents [148]. Notably, it has been introduced a novel BCMA (B cell maturation antigen)-based nanovesicle system designed to act as molecular decoys by sequestering APRIL and BAFF, cytokines that support MM cell survival in the BMME. Rather than delivering therapeutic cargo, these vesicles interfered with NF-κB signaling and pro-survival gene expression [149]. Furthermore, innovative therapeutic strategies have been proposed that exploit the natural tropism of EVs. Specifically, Soma et al. developed a novel delivery approach by linking siRNAs to anti-CD63 antibodies, creating a construct capable of capturing endogenous exosomes. This method facilitates the natural EV uptake pathways in delivering therapeutic siRNAs directly into MM cells, thereby effectively reducing in vitro the expression of key oncogenes such as MYC and CTNNB1. This strategy offers a potentially viable alternative to engineered EVs for gene silencing therapies in MM [150]. A notable approach involved the encapsulation of Bortezomib into apoptotic vesicles derived from mesenchymal stem cells, generating so-called nano Bortezomib-loaded EVs. This strategy resulted in a substantial enhancement of drug efficacy and a reduction in systemic toxicity in murine models of MM when compared to the free drug. Interestingly, the authors identified Rab7 as a key regulator of vesicle loading efficiency, suggesting that vesicle engineering can be optimized at the molecular level to improve therapeutic output [151]. A further advancement in this field involved the engineering of monocyte-derived exosomes functionalized with anti-BCMA antibodies to facilitate the targeted delivery of Bortezomib to MM cells. In addition to selective targeting, these EVs exhibited superior intracellular drug accumulation and toxicity in vitro, along with a prolonged anti-myeloma effect in vivo. Notably, the study also revealed that monocyte-derived EVs may exert immunomodulatory and osteogenic effects, highlighting their broader therapeutic potential beyond drug delivery [152]. A recent preclinical study has investigated the use of umbilical cord-derived mesenchymal stem cell EVs engineered with CD38-targeting peptides for the selective delivery of Doxorubicin in MM. When administered via transdermal microneedles, these CD38-directed EVs achieved greater tumor targeting, enhanced cytotoxic efficacy, and reduced systemic distribution compared to both non-targeted EV formulations and intravenous delivery of analogous constructs [153].

EVs as cancer vaccines in MM

In addition to their role in therapeutic target and drug delivery, EVs have emerged as versatile platforms in cancer immunotherapy, owing to their intrinsic ability to modulate immune responses and convey antigenic information. Tumor-derived EVs have been shown to present antigenic signatures capable of priming immune effector cells, while EVs originating from immune cells have been observed to actively reshape the TME. The dual function of EVs as both antigenic vectors and immunological modulators position them as promising candidates in the design of innovative therapeutic cancer vaccines, particularly within the immunologically complex context of MM [154]. Xie et al. reported that myeloma-derived exosomes engineered to express membrane-bound HSP70 functioned as effective immunotherapeutic agents, capable of enhancing dendritic cell activation via upregulation of co-stimulatory molecules (CD40, CD80) and pro-inflammatory cytokines (IL-1, IL-12, TNF-α). This immunostimulatory profile translated into a heightened CD8⁺ T cell-mediated cytotoxic response, supporting the potential of these exosome-based formulations as anti-MM vaccines [155]. Consistent with this data, exosomes derived from MM cells engineered to express membrane-bound TNF‑α induced more robust CD8⁺ T-cell responses and complete tumor protection in murine models, outperforming EV modified to carry IL‑2 or IFN‑γ. These findings underscore the promise of cytokine-engineered EVs as effective anticancer vaccine platforms in MM [156].

A very recent study introduced EVs engineered to transport a TP53-derived circular RNA (hsa_circp53_0041947), which encodes a functional peptide (circp53–209aa) with potent antitumor properties. These vesicles, selectively targeted to MM cells via E7‑Lamp2b surface modification, activated mitochondrial apoptosis by engaging the CypD/TRAP1/HSP90 axis. Although this platform does not constitute a traditional vaccine, it exemplifies how EVs can be exploited to deliver immunomodulatory RNA-based cargoes, expanding the paradigm of EV-based immunotherapy in MM. Such strategies may complement or even converge with antigen-based vaccination by integrating direct tumoricidal activity with immune modulation [157].

Conclusions

The development and progression of MM are critically shaped by the complex and dynamic interactions between malignant plasma cells and BMME, a specialised niche comprised of a heterogeneous network of cell populations, which collectively establish a permissive environment that supports tumor survival, immune escape, and drug resistance. A mounting body of evidence indicates that the bidirectional communication in BMME can occur through the transfer of EVs, which function as carriers for the delivery of bioactive molecules capable of affecting the behaviour and phenotype of recipient cells. In light of these findings, growing research efforts are now directed toward exploring the translational relevance of EVs in MM, with the aim of harnessing their biological properties for clinical applications. Recent advances underscore their promise as non-invasive biomarkers for disease monitoring and therapeutic response, as well as their applicability as targeted delivery platforms, active therapeutic agents and immunomodulatory drugs. Nevertheless, translating these promising insights into clinical applications remains challenging, largely due to unresolved issues in preclinical research.

A major limitation is the incomplete understanding of the functional heterogeneity of EV subpopulations and their distinct roles across different phases of tumorigenesis, including disease initiation, progression, and interactions with the BMME. Although a variety of in vitro and in vivo models are available to study EV-mediated mechanisms, they often fail to fully capturing the complexity of EV-driven intercellular communication and their dynamic behaviour within the BM niche. These limitations hinder the definitive elucidation of causal relationships and the identification of specific molecular pathways through which EVs influence MM pathophysiology, thereby slowing progress toward mechanistic insight and clinical translation. Building on these preclinical challenges, translational progress is also hampered by insufficient clinical characterization of EVs across distinct disease stages, including MGUS, SMM, newly diagnosed MM, and relapsed/refractory disease. Most available studies compare EV profiles between healthy controls and MM patients, often neglecting the molecular and clinical heterogeneity within disease stages. This oversimplified approach overlooks the dynamic changes in EV cargo that may accompany disease progression, thus limiting the identification of stage-specific biomarkers for early detection or risk stratification.

Furthermore, the potential of EVs as predictive biomarkers of treatment response or relapse risk remains largely unproven. While preliminary studies have suggested correlations between EV-associated signatures and therapeutic outcomes, these findings are predominantly derived from small, cross-sectional cohorts with limited clinical annotation. The absence of longitudinal datasets and well-defined clinical endpoints, such as MRD status, depth of response, or progression-free survival, hampers a robust assessment of their prognostic or predictive value. Thus, without expanding and improving clinical studies, the integration of EV-based markers into real-time disease monitoring or personalized treatment strategies remains aspirational. Moreover, the clinical validation of EV-based biomarkers in MM is further impeded by their limited integration into prospective clinical trial designs. Currently, the bulk of EV-related evidence derives from retrospective or exploratory studies, which often lack the robustness and reproducibility necessary for clinical translation. Furthermore, the rapidly evolving therapeutic landscape of MM, characterized by the introduction of novel agents and combination regimens, demands dynamic biomarker tools to guide personalized treatment decisions. Accordingly, the systematic incorporation of EV analyses into ongoing and future clinical trials, particularly those evaluating innovative therapies, is essential to clarify their predictive and prognostic potential. Additionally, the role of EVs derived from metabolically or immunologically dysregulated peripheral tissues, such as adipose tissue in the context of obesity, remains a largely neglected aspect of MM pathogenesis. Systemic perturbations may alter the molecular composition and functional properties of circulating EVs, enabling them to influence the BMME and potentially promote tumor-supportive changes. Elucidating how peripheral tissue dysfunction shapes the EV landscape could uncover additional layers of systemic signaling and contribute to a deeper understanding of disease heterogeneity and progression in MM.

A relevant critical barrier to clinical translational is the lack of methodological standardization, essential to improve the reliability and comparability of EV studies. Updated Minimal Information for Studies of Extracellular vesicles (MISEV) 2023 guidelines recommend employing orthogonal characterization methods to provide a comprehensive EV profile. Furthermore, the use of standardized reference materials and stringent controls, along with careful documentation of pre-analytical variables including sample handling, anticoagulant type, and storage conditions, is crucial. Despite these recommendations, many MM studies still rely predominantly on a single isolation method without adequate validation of EV purity or contamination, limiting the interpretability and reproducibility of the results. Current research trend is increasingly directed toward integrating next-generation EV isolation and detection technologies with multi-omic methodologies and single-vesicle analysis, in order to dissect the complexity of EV populations at high resolution. Overall, this convergence of technologies, coupled with their systematic integration into clinical studies, may offer a powerful means to identify disease-specific signatures associated with MM stage, therapeutic response, and MRD, moving the field closer to clinically actionable applications.

Abbreviations

AF4

Asymmetric Flow field-flow fractionation

ALIX

ALG-2 interacting protein X

AKT

protein kinase B

ANG-1

Angiopoietin-1

ApoBDs

Apoptotic Bodies

APRIL

A PRoliferation-Inducing Ligand

ASM

Acid Sphingomyelinase

BAFF

B cell-Activating Factor

Bcl-2

B Cell Lymphoma-2

BCMA

B Cell Maturation Antigen

bFGF

Basic Fibroblast Growth Factor

BM

Bone Marrow

BMME

Bone Marrow Microenvironment

BTN1A1

Butyrophilin 1A1

CAFs

Cancer-Associated Fibroblasts

cAMP

Cyclic Adenosine Monophosphate

CCL5

C-C motif chemokine Ligand 5

CD

Cluster differentiation

CDK-6

Cyclin-Dependent Kinase 6

C4BPA

Complement Component 4-Binding Protein Alpha

cfDNA

Cell-free DNA

cGAS

Cyclic GMP-AMP Synthase

circMYC

Circular MYC RNA

c-MET

Mesenchymal-Epithelial transition Factor receptor

CTCs

Circulating Tumor Cells

CTLA-4

Cytotoxic T-Lymphocyte–associated Antigen 4

CXCL1

Chemokine (C-X-C motif) Ligand 1

DVL3

Dishevelled 3

ECs

Endothelial Cells

EGFR

Epidermal Growth Factor Receptor

EV

Extracellular Vesicle

EVLev

Magnetic-Levitation microfluidic

EZH2

Enhancer of Zeste Homolog 2

FAP

Fibroblast Activation Protein-α

FIH

Factor Inhibithing Hypoxia-inducible factor 1α

FLCs

Free Light Chains

HDACs

Histone Deacetylases

HGF

Hepatocyte Growth Factor

HIF-1α

Hypoxia-Inducible Factor 1α

HSP

Heat Shock Proteins

ICAM-1

Intercellular Adhesion Molecule-1

IFN-I

Type-I Interferons

IL-10

Interleukin-10

IL-6

Interleukin-6

IL-8

Interleukin-8

IP-10

Interferon-gamma-induced protein 10/C-X-C motif chemokine ligand 10

JNK

c-Jun N-terminal Kinase

LAMP2

Lysosome-Associated Membrane Protein 2

lncRNA

Long non coding RNA

M2

Alternatively activated or anti-inflammatory macrophages

MALAT1

Metastasis-Associated Lung Adenocarcinoma Transcript 1

MAPKs

Mitogen-activated Protein Kinases

MCL-1

Myeloid Cell Leukemia 1 protein

MCP-1

Monocyte Chemoattractant Protein-1/Chemokine (C-C motif) ligand 2

MDSCs

Myeloid-Derived Suppressor Cells

METTL7A

Methyltransferase-like protein 7A

MGUS

Monoclonal Gammopathy of Unknown Significance

MICA

Major Histocompatibility Complex Class I-Related Chain A

miRNAs

microRNAs

MISEV

Minimal Information for Studies of Extracellular Vesicles

MM

Multiple Myeloma

MRD

Minimal Residual Disease

mRNAs

messenger RNAs

MSCs

Mesenchymal Stromal Cells

mTORC1

Mechanistic Target of Rapamycin Complex 1

MVs

Microvesicles

NEAT1

Nuclear Enriched Abundant Transcript 1

NFs

Normal Fibroblasts

NK

Natural Killer

NKG2D

Natural Killer Group 2, Member D

NSUN2

NOP2/Sun RNA Methyltransferase 2

OBs

Osteoblasts

OCs

Osteoclasts

PBX1

Pre-B-cell leukemia transcription factor 1

PD-1

Programmed Death 1

PDIA3

Protein Disulfide-Isomerase A3

PD-L1

Programmed Death-Ligand 1

p-ERK

phosphorylated Extracellular signal-Regulated Kinase

PI

Proteasome Inhibitors

PI3K

Phosphoinositide 3-Kinase

piRNA

Piwi-interacting RNA

PNPO

Pyridoxamine 5’-Phosphate Oxidase

PSMA3

Proteasome subunit alpha type-3

PSMA3-AS1

Proteasome subunit alpha type-3 antisense RNA1

RANKL

Receptor Activator of Nuclear Factor-κB Ligand

SASP

Senescence-Associated Secretory Phenotype

SMM

Smoldering Multiple Myeloma

SORT1

Sortilin 1

SPREAD1

Sprouty Related EVH1 Domain Containing 1

STAT3

Signal Transducer and Activator of Transcription 3

STING

Stimulator of interferon genes

TGFβ

Transforming Growth Factor Beta

TME

Tumor Microenvironment

TNFSF13

Tumor Necrosis Factor Superfamily Member 13

TRAIL

TNFRelated ApoptosisInducing Ligand

TRIP13

Thyroid Hormone Receptor Interacting Protein 13

Tsg101

Tumor Susceptibility Gene 101

tsRNA

transfer RNA-derived small RNAs

USP1

Ubiquitin-Specific Peptidase 1

VEGF

Vascular Endothelial Growth Factor

VLA-4

Very Late Antigen-4

YBX1

Y box-binding protein 1

α-SMA

Alpha Smooth Muscle Actin.

Author contributions

M. F. contributed to this research by drafting the manuscript, reviewing the literature, and writing the main sections of the article. A.M.Z., G.G. and M.P. contributed to the literature review and drafting the manuscript. E.A.M., E.V., M.G. and D.B. assisted with the structural design and revisions of the manuscript. R.M. and L.G. contributed to the design and preparation of figures and tables. I. B., S.C., C.G. supervised the project, provided critical revisions, and coordinated the overall research efforts. All authors have read and approved the final manuscript.

Funding

This work was supported by PRIN 2022, European Union’s NextGenerationEU initiative under the Italian Ministry of University and Research—M4 C2-I1.1 (#2022 AA4 FTJ, CUP H53D23006420006) to Giordano C; National Plan for NRRP Complementary Investments-AdvaNced Technologies for Human-centrEd Medicine (ANTHEM) (PNC0000003); POS RADIOAMICA project funded by the Italian Minister of Health (H53C22000650006); POS CAL.HUB.RIA project funded by the Italian Minister of Health (H53C22000800006); AIRC Investigator Grant (IG) (#30782) to Catalano S. PNRR-MAD-2023-12378037 (Next Generation EU, M6/C2_CALL 2023) and PNRR-MAD-2022-12375673 (Next Generation EU, M6/C2_CALL 2022), Italian Ministry of Health to Gentile M.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.