The interaction between neoplastic cells and the microenvironment (ME) is one of the main drivers in the progression of several hematological malignancies (HMs). Malignant cells interact with the ME, impacting resistance to apoptosis, survival, proliferation, migration, and chemoresistance (1). Furthermore, this crosstalk allows malignant cells to manipulate the immune response by hindering immune surveillance and fostering an immunosuppressive ME, allowing the disease to progress (2, 3). This interaction occurs in different ways: direct cell-to-cell contact, secretion of soluble factors, or release of extracellular vesicles (EVs) (1). Recently, EVs have increasingly proven to be of interest in HM research. An introduction to the impact of EVs on the signaling networks of leukemias and multiple myeloma as well as their potential therapeutic applications will be discussed in this review.

First thought to be “platelet dusts”, cell debris or experimental artifacts in the second half of the 20^th century (4), little interest was originally given to EVs. In 1996, Raposo and colleagues demonstrated the ability of EVs to act as antigen-presenting entities, and their biological significance was highlighted for the first time (5). Currently, EVs have been acknowledged as one of the main intercellular communication drivers (6). Their relevance for the progression of HMs cannot be ignored, as they shape the ME responsible for nurturing neoplastic cells, thus driving malignancy (6). EVs commonly referred to as ‘exosomes’ (30-150 nm in diameter) and ‘microparticles’ (100-1,000 nm in diameter) will be referred to as small EVs (sEVs, <150 nm in diameter) and medium/large EVs (m/lEVs, >150 nm in diameter), respectively, when mentioned. The different types of EVs are distinguished by their biogenesis. sEVs mainly arise from the endosomal compartment, whereas m/lEVs arise from plasma membrane budding (7, 8). Additionally, ‘apoptotic bodies’ (>1,000 nm in diameter) emerge during cell apoptosis and exhibit typical characteristics of cell death (condensed chromatin, fragmented DNA and proteolytic cleavage of the cytoskeleton). We will mainly focus our attention on sEVs and m/lEVs.

Although the cell of origin plays an immense role in EV cargo composition, the selection of molecules to be packaged is a highly regulated process. Specific sorting machinery ensures that proteins, nucleic acids (DNA, RNA, non-coding RNA) and glycolipids carried by EVs drastically differ from the cell of origin (9). Selective protein packaging is dependent on posttranslational modifications, particularly ubiquitination and sumoylation, that target specific proteins to be sorted by the endosomal sorting complex required for transport (ESCRT) machinery (10, 11). In addition to the presence of major histocompatibility complex-II (MHC-II) molecules on the EV surface, tetraspanins are enriched on EVs compared to the lysosomal membrane and plasma membrane (5, 12). Notably, CD63, CD81 and CD9 have been observed to be enriched on sEVs originating from diverse cell types (13). However, the presence of these tetraspanins has been detected on m/lEVs as well (14). In a proteomic comparative study, Kowal et al. demonstrated, on one hand, that sEVs are also enriched in Annexin XI (but also Annexins V and VI), ACE (angiotensin-converting enzyme), syntenin-1 [involved in receptor targeting to intraluminal vesicles of multivesicular bodies (15)], TSG101 (Tumor Susceptibility 101, a ESCRT pathway protein), ADAM10 (ADAM Metallopeptidase Domain 10), and EHD4 (EH Domain Containing 4), syntenin-1 and TSG101 being specific of the tetraspanin-enriched sEVs representing exosomes (16). On the other hand, GP96 (Glycoprotein 96) and possibly other ER-associated proteins are mainly present in large EVs while actinin-4 and mitofilin, and possibly other mitochondrial proteins, are present in both large and medium-sized EVs but absent from the sEVs. In addition, proteins associated with the outward budding cause notable differences in the proteomic profile of m/lEVs compared to their sEV counterpart. For instance, m/lEVs containing gelatinases, ARF6, MHC-1, MT1MMP, VAMP3 and β1-integrin likely originate from a myosin light-chain kinase (MLCK) cascade dependent release (17). Alternatively, other pathways involved in m/lEV release such as RAB22A dependent secretion and ARRDC1/TSG101/VSP4 dependent shedding result in TGM2 containing (18) and TSG101/ARRDC1 containing m/lEVs respectively (19). Outside of proteins associated with the biogenesis of EVs, a 2021 report exploring available EV datasets describes the tendency of m/lEVs to carry conserved proteins across prokaryotes and eukaryotes and related to mitochondrial function or energy synthesis, in symphony with the conserved process of membrane budding (20). sEVs on the other hand contain proteins more akin to the recent emergence of the endosomal vesiculation process. These proteins were part of signaling pathways or involved in the synthesis of intracellular building blocks (20). sEVs and m/lEVs also carry various RNA species: messenger RNA (mRNA) (21), non-coding RNAs (ncRNAs) such as microRNAs (miRs) along with their precursors (pre-miRNAs) (22), Y-RNAs (23, 24), small nuclear RNAs (snRNAs) (25), long non-coding RNAs (25, 26), piwi-interacting RNAs (21, 25), small nucleolar RNAs (21, 25), ribosomal RNAs (21, 25), transfer RNAs (21, 25), mitochondrial RNAs (27), circular RNAs (circRNAs) (28, 29) and vault RNAs (30). Among the RNAs encapsulated within human plasma derived EVs, microRNAs are the most abundant and have been described to play a significant role in cancer by promoting tumor progression and ME immunosuppression (25, 31). DNA species such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA) and mitochondrial DNA (mDNA) have been observed in sEVs; however, whether DNA packaging into sEVs is selective remains unclear (32). An overview of EV biogenesis, cargo and the process of internalization are illustrated in Figures 1 , 2 .

A total of 186,400 people are estimated to have been diagnosed with leukemia, lymphoma, or myeloma in the US in 2021; combined, these new cases represent 9.8% of cancer cases diagnosed in 2021 (33). Here we will review the role of EVs in different diseases: acute myeloblastic leukemias (AML), myeloproliferative neoplasms, acute lymphoblastic leukemias (ALL) and lymphoproliferative neoplasms.

EVs are undeniably of great importance in the crosstalk between the leukemic cells and their microenvironment. Their presence in blood and other bodily fluids allows for a less intrusive liquid biopsy as opposed to classical biopsies for many cancers. As such, several research groups propose the use of EVs as biomarkers for the prognosis of patients in a straightforward and non-invasive analysis. EV have distinct nanomechanical signatures in HMs (162) and as described above, the deregulation of EV cargo is commonplace in HMs (163). Diverging miR content can be revealing for predicting patient outcomes (163). Other cargo components were shown to have easily quantifiable differences such as ncRNA species (164), mRNA (165–167), proteins (168–171) and dsDNA (172) that could guide the treatment strategy, estimate disease progression (173), establishing a prognosis (174), indicate treatment response or even detect minimal residual disease (MRD) (174–181).

The ubiquitous BCR-ABL fusion found in CML has been described to be detected by analyzing CML-derived sEVs (167). Compared to controls, CML sEVs carry this transcript allowing for an easy assessment of BCR-ABL mutation presence (167). Proteome profiling of imatinib resistant CML patient plasma sEVs reveals 279 differentially expressed proteins compared to their imatinib sensitive counterpart (171). Following a bioinformatic analysis, 2 candidate proteins were identified, notably, Ribosomal protein L13 and Ribosomal protein L14, that may serve as imatinib resistance biomarkers in future applications (171).

The prognostic potential of circulating EVs in CLL has long been considered (182). Firstly, changes of miR profile in CLL patient serum is evident, Yeh and colleagues demonstrated a substantial shift in several miRs in CLL patient samples when compared to controls (183). High circulating miR-150 expression in the serum is associated with diminished treatment free survival (TFS) and overall survival (OS) in CLL patients compared to patients with lower amounts (177). Intriguingly, cellular miR-150 levels and circulating miR-150 display opposite prognostic indications, with increased TFS and OS when cellular miR-150 levels were high (177). Additionally, miR-155, shown to be deregulated not only in CLL but in several other HM may serve as a broad biomarker to identify HM occurrences (184). sEV miR quantification was suggested as a predictor of richter syndrome (RS) in CLL patients, a sudden switch to an aggressive form of the disease, with miR-19b serving as a potentially strong indicator of RS transformation and thus a less favorable course (173).

Multiple reports describing the potential of circulating EVs and their content in MM, in particular proteins and sncRNAs as biomarkers have been published. In a 2021 report, Laurenzana et al. demonstrate that MM patients have significantly higher amounts of circulating EVs in their serum compared to healthy controls (185). CD44, a protein involved in the adhesion of MM cells to BM-MSCs, was observed is highly expressed in the EV fraction of corticosteroid resistant MM cell lines and was found to be significantly overexpressed in newly diagnosed MM patients (168). Additionally, high CD44 content detected in patient serum EVs was associated with shorter OS (168). More recently, a high protein/particle ratio, i.e. high protein content within circulating peripheral blood EVs, was associated to immune dysfunction but also significantly lower OS compared to patients with lower protein/particle ratio (169). Following mass spectrometry analysis of peripheral blood EVs of MM patients, the authors uncovered a set of proteins (PDIA3, C4BPA, BTN1A1 and TNFSF13) able to be of use as biomarkers for patient outcomes such as survival, immune dysfunction and treatment response (169). As is the case in other HMs, the level of several microRNAs within serum EVs of MM patients were found to be greatly altered compared to healthy donors and could be linked to patient outcomes. Firstly, seven sEV microRNAs were shown to be decreased throughout the disease progression into MM (healthy controls vs monoclonal gammopathy of undetermined significance vs MM) leading the authors to believe that this expression-decrease may relate to MM progression and could therefore serve as biomarkers for this phenomenon (186). Moreover, Manier and colleagues observed an association between heightened levels of peripheral blood circulating let-7b and miR-18a in sEVs with PFS and OS of MM patients (187). miR-let-7c, a miR secreted by BM-MSCs was shown to be able to promote the M2 polarization of macrophages, contributing to angiogenesis in the bone marrow ME (146). The authors proposed sEV derived miR-let-7c as a biomarker for MM disease progression (146). Lastly, the profile of sEV-derived circRNA in MM patients was linked to peripheral neuropathy, a complication of MM. Of which, a particular circRNA, namely, chr2:2744228–2,744,407+ was overexpressed in the serum sEVs MM patients and positively correlated with peripheral neuropathy in MM patients (164). MRD monitoring may also be facilitated by the quantification of MM markers on EVs. Recent reports uncovered the potential of liquid biopsies in monitoring MRD by screening EVs isolated in MM remission patients for MM biomarkers such as CD56, CD117, CD27, CD55, CD59, PD-L1 and CD38 (188, 189).

In summary, various HM EVs have been proposed as a powerful non-invasive tool that would enable physicians to generate an early prognosis prediction in a rapid manner. Whether this prognosis relies on the presence of surface markers, proteomic content, dsDNA or sncRNAs, their accuracy remains to be verified. Indeed, a systematic review by Lopez-Santillan et al. disputes the etched in stone viability of EV miR content quantification as non-invasive biomarkers in diffuse large B-cell lymphoma due to high variability between publications (176). Standardization and repeatability of such findings will be crucial for EVs and contents to be of use as biomarkers in the various HMs. This prospect is currently being studied in a clinical trial examining miR and protein deregulation in circulating EVs as tools to predict bone (190). However, no results have yet been reported as per the time of writing to draw any conclusions. A non-exhaustive list of EVs as biomarkers is provided in Table 1 .

EVs can be shed by most cell types; however, in the context of HMs, malignant cells and crucial cells composing the ME often drive disease progression and interfere with therapy. Exploiting this aspect of the ME may provide targeted therapeutic prospects to counter disease progression. The active use of EVs as carriers inducing apoptosis or repressing the expression of certain genes has been described in several studies in recent years, with promising effects. Induced expression of certain miRs targeting key oncogenes has been adapted to EV delivery to MM cells (181, 193). Targeting of heparinase by miR-1252-5p mimics and elevated sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) by miR-335 targeted overexpression resulted in decreased BZ chemoresistance and drastically increased apoptosis, respectively. This demonstrates that key oncogene-targeted regulation through EV transfer lies within the realm of possibility (181, 193). Tumor-derived EVs have been described to carry tumor-associated antigens (TAAs) able to promote tumor-specific immunity by stimulating anticancer responses (194). By downregulating sEV TGF-β1 with lentiviral shRNA, tumor-derived sEVs internalized by dendritic cells (DCs) induced increased expression of surface costimulatory factors and MHC-II complexes in addition to increased inflammatory cytokine secretion, enhancing tumor-specific immunity (195). Similarly, Hu et al. demonstrated lentiviral-mediated induced expression of the costimulatory molecules CD80 and CD86 in a leukemic cell line that led to the release of leukemia-derived EVs with high CD80 and CD86 level that could drastically increase the DC response following internalization (196). DBA/2 mice immunized with leukemia-derived EVs rich in CD80 and CD86 demonstrated dramatically better survival when exposed to L1210 cells than when the mice were immunized with unaltered leukemia-derived EVs (196). Directly blocking the effects of leukemia derived EVs on disease progression and chemoresistance may become a promising avenue to alleviate patient burden. Indeed, by blocking the angiogenic effect of CML-derived EVs with the use of gold nanoparticles, de novo vessel formation was steeply reduced compared to the untreated control devoid of CML-derived EVs (197). By inhibiting the endocytic pathway, sEV internalization by MM cells is greatly diminished (157). MM BM-MSC-derived EVs can induce chemoresistance to BZ; however, when the endocytic pathway was inhibited, the apoptotic effect of BZ was enhanced, indicating that targeting sEV internalization by MM cells can be extremely beneficial to improving BZ therapy (157). Finally, the use of EVs as drug delivery systems for improved efficiency in therapy or to reach difficult-to-access areas is key for some treatments. KrasG12D siRNA-loaded exosomes are presently being studied in a clinical trial for pancreatic cancer patients (198). The outcome of this study will lead the way for similar applications in other malignancies. For instance, gp350, an envelope glycoprotein of the Epstein barr virus, is known to bind with great affinity to the CD21 protein on the surface of CLL B-lymphocytes (60). Therefore, Xiu et al. engineered gp350-anchored red blood cell (RBC)-derived EVs that were proven to efficiently bind CLL B lymphocytes (60). They continued by loading fludarabine and doxorubicin into RBC-EVs that were later anchored with gp350, and the EVs had substantial cytotoxic effects on malignant cells (199). Gärtner et al. applied a similar approach by transfecting gp350, CD40L and/or pp65 [an antigen of human cytomegalovirus (CMV)] expression plasmids into HEK293 cells that would later shed gp350+/CD40L+/pp65+ EVs (200).

CAR-T cell derived EVs are a recent avenue being explored in solid cancers (201, 202). Naturally, similar approaches are being explored in hematological malignancies. Haque et al. showed that the treatment of CD19 B-lineage leukemia cell lines with exosomes expressing CD19 Chimeric Antigen Receptor (CAR) induced cytotoxicity and elevated pro-apoptotic genes in CD19-positive leukemia B-cells without inducing cell death in CD19-negative cells (203). Interestingly, the selective characteristic of CAR-T cells is maintained by the EVs they shed, pointing towards their potential for targeted therapy (203).

By either acting as carriers inducing malignant cell apoptosis or downregulating oncogene expression, EVs have the potential to become invaluable tools to treat HM. However, many challenges remain, and future studies elucidating the applicability of these proposed therapies remain to be performed. Although the limited available studies are promising, much is yet to be studied before the widespread therapeutic use of EVs. The practical applications of EVs in the context of HM have been illustrated in Figure 6 .

Studies exploring the effects and reach of EVs often encounter problems related to the methods, nomenclature, and reproducibility. For instance, the lack of homogenous protocols throughout the studies referenced in this review, different isolation methods and variations thereof, as well as different media for culturing EV-producing cells, were used. A summary of the methods used in the referenced studies is provided in Table 2 . Previous studies have demonstrated a lack of consistency, particularly with regards to microRNAs (176, 204). The actual influence that EV-carried miRs have on the microenvironment in hematological malignancies may be overstated as EVs do not all contain microRNAs in a significant manner, neither are these EVs directly fused to the target cell membrane to release their cargo in the cytosol (204). Therefore, more attention should be brought to the other elements within the extracellular vesicles that may participate in the perceived effects often only attributed to microRNAs. Additionally, an argument can be made regarding the use of physiologically relevant doses of EVs in most studies. Compared to the actual microenvironment in these HMs most reports inflate the number of EVs that would meet the cell type of interest. That in turn could lead to observations that would be less likely to occur in physiological conditions. On the other hand, having an insight into the exaggerated effects provided by these EVs can guide us to a better understanding of the inner workings of EV shedding and internalization within the HM microenvironment. Furthermore, inconsistent nomenclature is rife and should be avoided, as the definition of EVs and their subcategories often varies. Therefore, the use of a universally accepted nomenclature, such as MISEV 2018 guidelines (205), will be crucial to avoid misunderstandings. Moving forward, these factors will hamper progress in the field, and efforts should be made to establish proven protocols for consistent and replicable results.

Key interactions between hematological malignancies and their microenvironment have been proven to be of utmost importance in disease progression. Direct cell-to-cell contact and the release of cyto- and chemokines are known to possess great modular capacity to shape the microenvironment toward a tumorigenic niche. Extracellular vesicles recently became a subject of great interest when their paracrine and autocrine effects in myeloid and lymphoid neoplasms were unraveled. Circulating EVs released by neoplastic cells reach cells within the local ME, enhancing the tumorigenic niche while simultaneously suppressing the immune system and allowing for unhampered proliferation and migration of neoplastic cells. In contrast, cells impacted by the tumor microenvironment that underwent phenotypic changes favoring the disease may shed EVs that can further promote growth and proliferation and, in some cases, enhance chemoresistance, reducing therapeutic efficiency. Naturally, the potency and accessibility of EVs have sparked great interest in academia as tools to improve the detection and treatment of HMs. The non-invasive approach to collect and analyze vesicles compared to more invasive approaches demonstrates the potential of EVs in the diagnosis of HMs and other cancers through routine liquid biopsies. Furthermore, the ability to load therapeutic agents in EVs and targeting malignant cells may prove to be of great aid in cancer that are nonresponsive to therapy. Although many roadblocks lie ahead of EV research, their potential in therapy and diagnosis is limitless.

DV: Conceptualization, Writing – review & editing, Visualization, Writing – original draft. ND: Conceptualization, Writing – review & editing, Writing – original draft. DB: Writing – review & editing. NM: Writing – review & editing. LL: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. BS: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.