The clonal expansion of plasma cells (PCs) in the bone marrow (BM) is the hallmark of multiple myeloma (MM), a hematological tumor that typically affects subjects over 60 years of age and represents about 0.9% of all cancers worldwide, contributing to about 1% of cancer-related deaths^[1]. Although the etiology of MM has not been fully elucidated, multiple cytogenetic alterations, such as chromosomal deletions and/or translocations, are present and correlated with disease progression and high-risk MM^[2]. Asymptomatic forms like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) represent premalignant stages that may advance to symptomatic MM. Disease progression is characterized by severe and irreversible impairment of vital organs, including hypercalcemia, renal failure, anemia, and lesions in the bones^[2].

The expression and function of adenosinergic pathways in MM have been previously described by our group and others^[3,4]. The canonical pathway for adenosine (ADO) production is initiated by the nucleoside triphosphate diphosphohydrolase/CD39, which converts adenosine triphosphate (ATP) into adenosine monophosphate (AMP)^[5]. Subsequently, the hydrolysis of AMP to ADO is mediated by ecto-5’-nucleotidase/CD73^[6,7]. In contrast, the non-canonical pathway, whose efficiency increases at low pH, starts with the conversion of NAD⁺ to adenosine diphosphate ribose (ADPR) mediated by the nicotinamide adenine dinucleotide (NAD⁺)-glycohydrolase/CD38. ADPR is subsequently converted to AMP by CD203a/ENPP1 (PC-1), which has been characterized in both tumor and immune cells^[8], although the conversion of ADPR to AMP was first demonstrated functionally by our group^[9]. AMP is finally metabolized to ADO by CD73^[7,10,11], which catalyzes the final step of both adenosinergic pathways. Functionally, adenosinergic ectoenzymes are key players in the establishment of an immune-suppressive BM microenvironment through a local production of ADO^[4,12]. These processes are facilitated by nicotinamide phosphoribosyltransferase/pre-B cell colony enhancing factor (NAMPT/PBEF1), a key enzyme involved in the conversion of nicotinamide and 5-phosphoribosyl-1-pyrophosphate into nicotinamide mononucleotide, which is subsequently transformed into NAD⁺. This pathway supports not only glycolysis but also the activity of NAD⁺-dependent enzymes and DNA repair mechanisms^[13-16]. NAMPT/PBEF1 expression is increased in both solid and hematological tumors^[17,18] as well as in inflammatory diseases^[19,20].

We have recently reported that many of these molecules are also expressed on extracellular vesicles (EVs) obtained from BM plasma samples of MM patients^[21]. Several studies have described these EVs as resembling the phenotypic and functional characteristics of their cells of origin. Furthermore, their role in multiple processes related to MM pathogenesis, including modulation of immune responses, progression of the disease, therapy resistance, osteolysis, angiogenesis, and formation of the metastatic niche has been addressed^[22-25]. However, to date, no information is available on tumor-derived EVs present in the peripheral blood (PB) of MM patients. Therefore, the aim of this study was to characterize these EVs in terms of size, number, and phenotype, and to assess whether they exert immune-regulatory functions in vitro. The data obtained in this study indicate that tumor-derived EVs can be also detected in the PB of newly diagnosed MM (NDMM) patients and may contribute to tumor-associated immune suppression.

In recent years, several studies have acknowledged the involvement of EVs in tumor progression and tumorigenesis both in solid and hematological malignancies^[26,27]. In addition, EVs are used as biomarkers for diagnostic purposes and for monitoring the course of different tumors. They have also been proposed as potential targets for novel therapeutic strategies^[28,29]. Indeed, tumor-derived EVs can modulate the tumor microenvironment by mediating intercellular communication, thereby promoting immune escape, metastatic dissemination and resistance to therapy^[24,27]. Importantly, EVs may provide insights into disease states and serve as diagnostic tools for early cancer detection, as they reflect gene mutations as well as phenotypic and functional features of their cells of origin, enabling minimally invasive tracking of disease progression^[30-32].

For this reason, the characterization of circulating tumor-derived EVs in the PB as a form of liquid biopsy has attracted considerable interest for diagnostic and therapeutic purposes in recent years^[30,33-35]. Accordingly, several studies have examined EVs in the PB of patients with solid and hematological malignancies, characterizing their role in tumor progression as well as their diagnostic and prognostic value^[33,36-41]. More recently, Reale and coworkers described different methods for EVs isolation and characterization from the PB of patients with MM, highlighting their relevance for diagnostic and monitoring purposes^[42]. However, detailed phenotypic and functional characterization of these EVs remains limited. The present study addresses this gap.

Here we report that PB samples from MM patients, compared with those from HD, contained an increased concentration of EVs, while displaying a similar size distribution. Increased levels of circulating EVs compared with healthy subjects have already been reported in patients with lung cancer, where they correlate with disease stage^[43]. We also found that EVs obtained from MM patients’ PB samples displayed higher expression levels of CD138, NAMPT/PBEF1, CD38, and CD73 than those from HDs. The increased expression of CD138 on EVs from MM patients confirms that tumor-derived, CD138-expressing EVs are detectable in the PB of MM patients, and not only in the BM niche, as previously reported^[21]. Nonetheless, the detection of CD138 on EVs from HDs, although at lower levels, was not entirely unexpected. Indeed, CD138 can be detected on normal epithelial cells, normal PCs, and other healthy tissues^[44,45], and its expression can be increased during inflammation^[46] or in autoimmune and inflammatory diseases^[47,48].

The finding that NAMPT/PBEF1 is expressed at higher levels in EVs from MM patients than in those from HDs is novel and of particular interest. NAMPT/PBEF1 is a rate-limiting enzyme involved in NAD⁺ biosynthesis, and its expression has been reported in MM, where is crucial for tumor cell growth and osteoclast activity^[18]. More importantly, NAMPT/PBEF1 expression has been shown to correlate with disease progression^[49]. In addition, it has been proposed both as a therapeutic target^[50] and as a biomarker of chemotherapy efficacy in patients with NDMM, particularly in combination with nicotinate phosphoribosyltransferase (NAPRT)^[51]. Among the adenosinergic pathways, we observed significantly higher expression of CD38, which belongs to the non-canonical pathway, and of CD73, which represents a functional bridge between the canonical and non-canonical pathways. These data are consistent with our previous observations on EVs obtained from BM samples of MM patients^[21]. Accordingly, EVs potentially capable of producing the immunosuppressive molecule ADO from different substrates, including ATP and NAD⁺, are detectable in the PB of MM patients and not only within the BM microenvironment. Furthermore, we demonstrated that CD138⁺ EVs, which are mostly derived from malignant PCs (although the contribution of CD138⁺ healthy cells cannot be excluded), expressed significantly higher levels of NAMPT/PBEF1, CD38, CD39, and CD73. These data suggested that those EVs display an increased capacity for NAD⁺ and ATP metabolism leading to ADO production, as compared with EVs derived from healthy cells. Again, this feature mirrors what has been observed in EVs isolated from BM samples of MM patients^[21]. During MM progression, the BM niche becomes enriched in extracellular nucleotides (e.g., ATP and NAD⁺), as well as dinucleotides such as cyclic Guanosine monophosphate (GMP)-AMP (cGAMP). It has been reported that both mono- and di-nucleotides may contribute to the establishment of immune tolerance^[52], through their metabolization by ectoenzymes expressed by BM resident cells, such as CD38, CD203a, and CD73. The resulting enzymatic reactions trigger intracellular calcium flux signaling and drive the production of extracellular ADO, thereby linking purinergic metabolism to immunosuppressive processes^[4].

In this scenario, the interplay between the DNA damage response and purinergic metabolism remodeling is increasingly recognized as a critical determinant of MM adaptation to therapy. Genotoxic stress induces the release of ATP, NAD⁺, and cGAMP, which are rapidly processed by ectonucleotidases in the MM microenvironment. Among these ectonucelotidases, CD203a acts as a central regulator by converting ATP and NAD⁺ to AMP^[53]. Thus, CD203a activity fuels the non-canonical adenosinergic pathway independently of the enzymatic function of CD39^[9]. Furthermore, CD203a can suppress cGAS-Stimulator of Interferon Genes (STING)-mediated innate immune activation by degrading extracellular cGAMP^[53]. In MM, PCs and BM stromal cells frequently upregulate CD203a, enabling efficient conversion of therapy-induced nucleotides into AMP and sustaining CD73 dependent ADO accumulation^[54]. The combined enhancement of ADO signaling and the attenuation of STING responses promotes an immunosuppressive microenvironment that supports MM cell survival and contributes to therapeutic resistance.

Data from the functional studies reported here indicate that EVs obtained from MM patients’ PB samples may exert immune-suppressive activities, as evidenced by their ability to dampen T-cell proliferation in vitro in both helper and cytotoxic T lymphocytes. Previous studies on EVs derived from patients with MM have reported inhibitory effects on osteogenesis and angiogenesis in vitro^[55,56].

Several studies support the concept that EVs in MM act as key mediators that reprogram the BM microenvironment into an immunosuppressive niche, thus facilitating tumor progression through multiple, parallel mechanisms. One of the primary mechanisms involves the polarization of macrophages toward a pro-tumorigenic M2 phenotype. Exosomal miR-let-7c derived from mesenchymal stem cells drives this M2 shift, while MM-derived exosomes specifically increase the expression of M2-associated markers such as Arginase (Arg)-1, interleukin (IL)-10, and CD206 in resting macrophages^[57,58]. Furthermore, tumor-derived small EVs induce the expression of Programmed Death-Ligand (PD-L)-1 and pro-inflammatory cytokines such as IL-6 in macrophages by activating the Signal Transducer and Activator of Transcription (STAT) 3 and Toll-like receptor (TLR) 4/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways^[59]. Beyond macrophages, EVs are instrumental in the expansion and activation of myeloid-derived suppressor cells (MDSCs). In this context, Bone Marrow Stromal Cells (BMSC)-derived exosomes are internalized by MDSCs, directly promoting their survival through activation of STAT1 and STAT3 signaling and upregulation of the anti-apoptotic proteins B-cell lymphoma-extra large (Bcl-xL) and Myeloid Cell Leukemia-1(Mcl-1)^[60]. T-cell activity is likewise severely compromised through multiple EV-mediated pathways. Hagiwara et al. reported that EVs derived from MM promote T-cell exhaustion by upregulating immune checkpoint markers such as PD-1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and Lymphocyte-activation gene (LAG) 3 on CD8⁺ T cells, a process largely driven by Sphingosine kinase (SPHK) 1/Sphingosine-1-phosphate (S1P) signaling^[61]. Moreover, conditioning of the BM microenvironment with these vesicles increases PD-1 expression specifically on CD4⁺ T cells^[62].

Here, we also report that circulating EVs in PB are equipped with complete adenosinergic pathways (CD38, CD39, CD203a, and CD73). This feature enables them to produce the immunosuppressive molecule ADO. Thus, circulating EVs are capable of dampening both CD4⁺ and CD8⁺ T-cell proliferation at sites far from the primary tumor. Natural killer (NK) cells are also targeted by EV-mediated mechanisms, since chronic exposure to EVs carrying the MHC class I polypeptide-related sequence A (MICA) ligand leads to downmodulation and degradation of the Natural Killer Group (NKG) 2D receptor and can even trigger “fratricide” when MICA is transferred directly from vesicles to the NK-cell surface^[63]. In addition, exosomal lncRNA Nuclear Paraspeckle Assembly Transcript (NEAT) 1 inhibits NK-cell activity and cytokine production by downregulating Pre-B-cell leukemia transcription factor (PBX) 1 through an Enhancer of zeste homolog (EZH) 2-mediated axis^[49]. Finally, MM EVs are enriched in Complement Component 4 Binding Protein Alpha (C4BPA), which helps tumor cells evade the host complement system^[64], and phosphatidylserine-positive vesicles contribute to a highly immunosuppressive state^[65].

We have further recently demonstrated that EVs obtained from BM samples of MM patients regulate multiple immune cell functions, including T-cell proliferation and cytokine release. Those effects are partly mediated by the immune checkpoints Human Leukocyte Antigen (HLA)-G and PD-L1 expressed on their surface^[66]. Moreover, EVs from BM samples of MM patients express functional adenosinergic ectoenzymes, paralleled by the production of ADO from ATP and NAD^+[21]. Based on these observations, we previously hypothesized that EVs released within the BM microenvironment may modulate local anti-tumor immune responses. Furthermore, those EVs may arrive in the PB and, from there, they reach other BM niches or other organs (see Graphical Abstract). Here we provide the first evidence that EVs detected in MM patients’ PB samples have potential immunomodulatory effects. It is conceivable that these EVs may recirculate through the blood stream to perform these modulatory activities on immune cells at distant sites, rather than in the PB. Downregulation of T-cell proliferation in vitro positively and significantly correlated with the expression of CD138 and CD203a/PC-1 on EVs, suggesting that these effects can be attributed, at least in part, to the presence of tumor-derived EVs expressing a functional non-canonical adenosinergic pathway. Nonetheless, these findings require validation using specific ectoenzyme inhibitors, and we cannot exclude the contribution of additional immune-suppressive mechanisms. Further studies will therefore be necessary to validate the data here presented. Nonetheless, we provide the first evidence that immune-regulatory EVs derived from MM cells are clearly present in the PB of MM patients. Those EVs may be involved in tumor-derived immune-suppression in the periphery and, presumably, in tumor progression.

This study has limitations, including the relatively small number of MM patients analyzed. Although the use of primary patient samples inevitably introduces a degree of clinical heterogeneity, the application of strict inclusion criteria, uniform pre-analytical processing, and standardized functional assays mitigates this limitation. Nevertheless, future studies based on larger and clinically stratified cohorts will be essential to dissect the relative contribution of disease stage, comorbidities, and prior treatments to EV-mediated immune modulation in MM. Another limitation is the lack of experiments employing specific inhibitors of adenosinergic ectoenzymes to directly assess their role in the immune-regulatory properties of MM-derived EVs. Despite these limitations, our findings, together with results from other groups, underscore the relevance of EVs found in the PB of MM patients. Circulating CD138⁺ microparticle levels accurately reflect tumor burden, often signaling early relapse weeks before symptoms or standard tests show any change^[65]. The proteomic content of EVs, specifically the enrichment of CD44, is a key indicator of corticosteroid resistance and a predictor of shorter overall survival^[67]. Beyond tracking the tumor itself, MM-EVs inhibit bone formation through transfer of molecules like miR-103a-3p, providing a precise biomarker for the diagnosis of bone lesions^[68]. EVs may also enable real-time monitoring of treatment failure, as the presence of P-glycoprotein and phosphatidylserine on microparticles correlates with multidrug resistance and aggressive disease progression^[65]. Finally, it has been established that a high EV cargo ratio (> 0.6 × 10⁸ particles) is a significant prognostic parameter associated with immune dysfunction and lower survival rates, supporting a more personalized approach to long-term clinical management^[64]. In conclusion, PB EVs may represent a valuable tool for the diagnosis and monitoring of MM, potentially overcoming some limitations of invasive BM procedures; however, additional studies are required to confirm these hypotheses.