Harnessing the power of exosomes in leukemia: from molecular messengers to clinical applications

Abstract

Exosomes, nanosized extracellular vesicles (30–150 nm), have emerged as pivotal mediators in leukemia pathogenesis, fundamentally altering our understanding of the disease and therapeutic approaches. These membrane-bound carriers transport complex molecular cargos, including proteins, nucleic acids, and lipids, facilitating intercellular communication that drives malignant transformation, treatment resistance, and remodeling of the bone marrow microenvironment. In leukemia, exosomes orchestrate angiogenesis, immune evasion, and disruption of normal hematopoiesis while serving as repositories of disease-specific biomarkers. Characteristic molecular signatures have been identified across various leukemia subtypes, including microRNA patterns (miR-155, miR-150 in CLL; miR-26a-5p in AML) and protein markers (IFITM3, CD146 in CML), which enable minimally invasive liquid biopsy-based diagnostics and real-time disease monitoring with high sensitivity and specificity. Therapeutically, exosomes function as natural drug delivery vehicles with inherent biocompatibility and barrier-crossing capabilities, while engineered platforms, including CAR-T cell-derived exosomes, offer novel immunotherapeutic strategies. Paradoxically, exosomes also mediate treatment resistance through intercellular transfer of resistant phenotypes, particularly in imatinib-resistant CML and chemoresistant AML, revealing both challenges and therapeutic targets. Clinical translation faces significant hurdles, including standardization of isolation protocols, optimization of cargo loading, scalable manufacturing, and regulatory framework development. The convergence of enhanced biological understanding, technological innovation, and evolving regulatory landscapes positions exosome-based strategies to revolutionize leukemia management through precision diagnostics and targeted therapies with reduced systemic toxicity.

Background

Exosomes have evolved from the humble status of cellular waste to that of significant mediators of intercellular communication, particularly in cancer biology [1, 2]. These membrane-bound vesicles of 30–150 nm contain a heterogeneous array of bioactive molecules (proteins, lipids, and nucleic acids like microRNAs, mRNAs, lncRNAs, and circRNAs), which they transport all around the body. In leukemia, exosomes play unique and multifunctional roles. They reprogram the bone marrow microenvironment directly to form supportive niches that enhance leukemic cell proliferation and survival and also promote hallmark cancer activities such as immune evasion, angiogenesis, and pre-metastatic niche formation [3, 4]. Notably, leukemic cells release exosomes more abundantly and with a distinct molecular content than their normal counterparts, exhibiting a similarity to the pathological phenotype of their cells of origin [5].

The clinical applications of exosomes in leukemia are immense and far-reaching. Their presence in a variety of bodily fluids means that minimally invasive sampling is possible through liquid biopsies and positions them as credible biomarkers for cancer diagnosis, prognosis, and response to therapy [6]. Exosomal contents, particularly microRNAs, serve as molecular barcodes that reflect the pathophysiological condition of their parent cells and are therefore extremely useful in diagnosis [6–8]. Besides diagnostics, their inherent biocompatibility, low immunogenicity, and ability to cross biological barriers make exosomes excellent candidates for therapeutic applications, including delivery vehicles for proteins, nucleic acids, and chemotherapeutic drugs [9]. Exosomes engineered by researchers have been found to enhance or inhibit tumor growth in leukemia through paracrine signaling pathways, which makes them cell-free therapeutic agents [3].

However, translation of exosome-based strategies to the clinic is thwarted by substantial challenges. Technical hurdles, including inefficient isolation protocols, difficulties in comprehensive characterization, the inherent heterogeneity of exosomal populations, and the lack of universally agreed specific biomarkers, impede progress [10]. Microfluidic technologies have been recognized as promising strategies for exosome extraction due to their high throughput, portability, and ability to process small sample volumes; however, further optimization is still needed [10]. These developments are set against the backdrop of the rapidly evolving world of leukemia, where precision diagnostics, targeted treatments such as CAR T-cell immunotherapy, and nanotechnology offer promise but also underscore the complexity of relapse and treatment resistance [11].

The role of exosomes in mediating drug resistance is particularly concerning in leukemia. Leukemic cell exosomes can transfer resistant features to previously sensitive cells through intercellular molecular exchange, leading to therapy failure [4]. As an example, leukemic K562 cell-derived exosomes have been shown to transmit miR-711 into the bone marrow mesenchymal stem cell, causing cell adhesive impairment and risking eventual disease development [12]. Additionally, exosomes can recondition the bone marrow microenvironment to establish protective niches for cancer cells, thereby reducing their susceptibility to standard chemotherapeutic agents [4, 5]. Moreover, they can also regulate immune responses in the tumor microenvironment, which may conflict with immunotherapeutic strategies, such as CAR T-cell therapy, one of the most promising therapeutic breakthroughs for certain leukemias over the last decade [11].

This narrative review synthesizes current knowledge of exosome biology in leukemia, focusing on their multifaceted roles in disease development, their use as therapeutic and diagnostic tools, and the research efforts aimed at closing the gaps in their clinical applications. Through an analysis of these factors, we hope to enlighten the possibilities and limitations that exosomes present in advancing the boundaries of personalized medicine for leukemia patients.

Exosome content and analytical methods

Composition

Leukemic exosomes are membrane-bound nanovesicles (40–150 nm) that are highly sophisticated intercellular messengers in leukemia pathogenesis. Their molecular composition is distinct from that of exosomes from normal cells, offering both diagnostic value and therapeutic targets [13].

The protein makeup of leukemic exosomes comprises characteristic surface markers that function as identifying markers. These include CD19, CD20, CD24, CD37, and HLA-DR [14]. In drug-resistant chronic myeloid leukemia (CML), exosomes exhibit a high upregulation of proteins like interferon-induced transmembrane protein 3 (IFITM3), CD146, and CD36 [15]. In chronic lymphocytic leukemia (CLL), the S100-A9 protein in exosomes increases NF-κB activity, indicating disease progression [16, 17].

The nucleic acid cargo contains distinct microRNA signatures, namely the miR-29 family, miR-150, miR-155, and miR-223, in chronic lymphocytic leukemia [6, 17] and miR-26a-5p and miR-101-3p in acute myeloid leukemia [18]. Protected from RNase degradation by the exosomal membrane, these miRNAs are stable biomarkers for longitudinal disease monitoring [19].

Their lipid content, rich in sphingomyelin, phosphatidylserine, cholesterol, and ceramides, is responsible, essentially, for the structural integrity and signaling function of exosomes [20, 21]. This unique lipid profile provides circulatory stability while facilitating the transport of bioactive lipids and enzymes [22] (Fig. 1).

Fig. 1.

Biogenesis and molecular composition of exosomes in leukemia. The diagram illustrates exosome formation via the endosomal pathway and their release through exocytosis. Leukemic exosomes (40–160 nm) contain distinct molecular cargo including: surface proteins (CD19, CD20, CD24, CD37, CD63, and others) that serve as identifying markers; signaling proteins (TGF-β1, VEGF, S100-A9, DKK1) and cell survival mediators (FasL, PD-L1, Survivin, BCL-2); disease-specific microRNAs with roles in drug resistance, angiogenesis, cell migration, and stem cell maintenance; leukemia-specific transcripts (BCR-ABL in CML, NPM1 and FLT3-ITD in AML); and structural lipids (sphingomyelin, phosphatidylserine, cholesterol, ceramides) that contribute to exosome stability and function. CLL chronic lymphocytic leukemia, AML acute myeloid leukemia, CML chronic myeloid leukemia (Table 1)

Table 1.

Comprehensive overview of exosomal molecular markers across subtypes of leukemia

Disease subtype	Protein markers	microRNA markers	Nucleic acid cargo	Lipid components	Clinical significance	References
Chronic lymphocytic leukemia (CLL)	• CD19, CD20, CD24, CD37, HLA-DR • S100-A9 protein (↑NF-κB activity)	• miR-29 family • miR-150 • miR-155 • miR-223	• Disease-specific transcripts • Protected from RNase degradation	• Sphingomyelin •Phosphatidylserine •Cholesterol • Ceramides	• Disease progression monitoring • NF-κB pathway activation • Longitudinal disease monitoring	[6, 14, 16, 17, 19–22]
Chronic myeloid leukemia (CML)	• IFITM3 (highly upregulated in drug-resistant) • CD146 • CD36	• miR-365 (resistance transfer to sensitive cells)	• BCR-ABL transcripts • Pro-angiogenic miRNAs delivery	• Sphingomyelin • Phosphatidylserine • Cholesterol • Ceramides	• Imatinib resistance monitoring • Enhanced survival upon imatinib exposure • Drug resistance transfer mechanisms	[20–23]
Acute myeloid leukemia (AML)	• TGF-β1 • VEGF • Tumor-associated antigens	• miR-26a-5p • miR-101-3p • miR-484 (adriamycin resistance) • miR-1246, miR-34c-5p, miR-34a (stem cell maintenance)	• NPM1 mRNAs • FLT3-ITD mRNAs • VEGF/VEGFR mRNA delivery	• Sphingomyelin • Phosphatidylserine • Cholesterol • Ceramides	• Treatment outcome correlation • Chemoresistance mechanisms • Leukemic stem cell survival • Suppression of differentiation	[18, 20–27]
Acute lymphoblastic leukemia	• CD19, CD22 (B-ALL specific) • ADAM17 (Notch pathway) • ATG3 (autophagy pathway) • IL-15 (CNS invasion) • TSG101, CD81 (exosome markers) • Galectin-3 (stromal communication)	• miR-181a (proliferation marker) • miR-181b-5p (promotes invasion/migration) • miR-24-3p (immune suppression) • miR-451a (prognostic significance) • miR-92a (angiogenesis) • miR-210 (poor prognosis)	• DNMT1 mRNA (epigenetic regulation) • Disease-specific transcripts • lncRNAs (tumorigenesis) • Protected nucleic acids • mRNA regulatory networks	• Sphingomyelin • Phosphatidylserine • Cholesterol • Ceramides • Phosphatidylinositol • Gangliosides	• Early disease detection • CNS infiltration monitoring • Treatment response assessment • Notch/autophagy pathway activation • Immune surveillance evasion • Non-invasive diagnostic biomarkers	[28–32]

Isolation and analytical methods

Exosome recovery from body fluids remains a crucial step in accurately assessing biomarkers. The latest techniques include ultracentrifugation (UC), size exclusion chromatography (SEC), and immunoaffinity capture (IAC) [33, 34]. Each of these techniques has specific advantages and limitations. While UC is applied most frequently as an inexpensive method for bulk, it typically yields impurities, requires enormous processing time, and has comparatively low efficiency [35]. Critically, UC imposes high shear stress that has the potential to compromise exosome integrity and alter cargo content, potentially producing artifacts that complicate analysis [36]. Side-by-side comparisons between UC and other methods have identified dramatic differences in exosome yield, size distribution, and molecular content from identical samples [37], which challenges the validity of data between isolation protocols.

Density gradient centrifugation increases purity but introduces extraneous methodological variables, including gradient composition and centrifugation conditions that exert significant effects on results and complicate cross-study comparisons. The SEC provides higher purity than UC but is hindered by extreme sample dilution and can lose low-abundance biomarkers during processing [36]. The method cannot distinguish between exosomes and particles of comparable size, routinely co-isolating lipoprotein contaminants from plasma samples.

On the other hand, IAC yields high-purity isolation through attachment to a target surface marker (e.g., CD63), and recent microfluidic platforms enable efficient processing from minimal sample material with recovery yields of 80–90% [38, 39]. IAC imposes significant bias through the selection of vesicle subpopulations bearing the target marker and potentially overlooking diagnostically relevant exosome subgroups [40]. The region is not consensual on which markers truly define exosomes, with CD9, CD63, and CD81 being used extensively, despite variation in their expression between exosome subpopulations having been documented [41].

Sophisticated technologies, such as alternating current electrokinetic chips that can separate exosomes in just 20 min from a 25 µL sample, and tangential flow filtration systems that minimize clogging for the analysis of viscous biological samples, have emerged as promising advances [42]. Following isolation, transmission electron microscopy, Western blotting, and mass spectrometry are employed to thoroughly analyze the contents of exosomes, with extracted RNA having particular promise as a source of biomarkers, as demonstrated in prostate cancer research [43].

Critically, characterization methods themselves introduce variability. Nanoparticle tracking analysis (NTA) yields highly variable results, depending on equipment settings, sample preparation, and the operator’s proficiency [44]. Mass spectrometry proteomic analysis is hindered by variable sample preparation, as different lysis buffers release varying sets of proteins. RNA analysis is compromised by pre-analytical variables, including sample storage conditions and RNA extraction protocols, which have a profound impact on miRNA profiles [45].

These methodological discrepancies effectively undermine the validity of reported properties and functions of exosomes. Combined with incomplete reporting of methodological information within publications, they present serious obstacles to reproducing results or establishing diagnostic and therapeutic applications. The continued refinement of these methods is essential for advancing exosome research in leukemia and translating findings into clinical practice.

Method selection framework

The optimal isolation strategy depends critically on sample characteristics, intended application, and resource constraints. For diagnostic applications requiring minimal sample volumes (< 100 µL), immunoaffinity capture coupled with microfluidic platforms provides superior performance, achieving 80–90% recovery from fingerstick blood volumes while maintaining diagnostic-grade purity [38, 39]. However, the higher cost per sample limits routine clinical implementation.

For research-scale isolation (0.5-5 mL samples), size exclusion chromatography represents the optimal balance between purity and practicality, particularly when downstream molecular analysis requires minimal protein contamination. The method’s gentleness preserves exosome integrity, which is crucial for functional studies. However, the high sample dilution necessitates concentration steps that may introduce artifacts [36].

Large-scale therapeutic production demands ultracentrifugation as the foundational method due to its scalability and cost-effectiveness. However, clinical-grade purity requirements often necessitate sequential UC-SEC protocols, accepting the trade-off between yield loss and regulatory compliance. Emerging tangential flow filtration systems hold promise for bridging this gap, offering throughput comparable to that of SEC with purity levels equivalent to those of UC, although validation studies remain limited.

The critical consideration is that no single method suits all applications - diagnostic specificity, research reproducibility, and therapeutic scalability require distinct optimization strategies.

Following isolation, transmission electron microscopy, Western blotting, and mass spectrometry are employed to thoroughly analyze the contents of exosomes (Fig. 2).

Fig. 2.

Workflow for Exosomal Biomarker Analysis in Leukemia. The diagram illustrates the sequential steps taken from the sample collection to clinical applications. Step 1 involves blood collection (5–10 mL) using non-invasive sampling. Step 2 details various isolation methods, including ultracentrifugation (UC), size exclusion chromatography (SEC), immunoaffinity capture (IAC), and emerging microfluidic technologies. Step 3 illustrates various characterization techniques, including transmission electron microscopy, Western blotting, nanoparticle tracking analysis, and advanced methods such as EV Array and DNA-PAINT. Step 4 encompasses content analysis with miRNA profiling, mRNA analysis, proteomics, and next-generation sequencing. The workflow culminates in clinical applications for early disease detection and exosome-based treatment response monitoring, including the assessment of minimal residual disease (Table 2)

Table 2.

Standardized isolation protocols for clinical and research applications

Method	Step-by-step protocol	Best application
Ultracentrifugation	1. Pre-clear: 300×g, 10 min, 4 °C< 2. Remove debris: 2,000×g, 10 min, 4 °C 3. Remove large vesicles: 10,000×g, 30 min, 4 °C 4. Pellet exosomes: 100,000×g, 70 min, 4 °C 5. Wash pellet: resuspend in PBS, 100,000×g, 70 min< 6. Final resuspension in 100–500 µL PBS	Large-scale production, cost-sensitive research [35, 37]
Size exclusion chromatography	1. Pre-filter sample through 0.22 μm filter 2. Equilibrate Sepharose CL-2B column with PBS 3. Load 0.5 mL sample onto column 4. Elute with PBS, collect 0.5 mL fractions 5. Pool fractions 7–9 (typically exosome-rich) 6. Optional: concentrate using 100 kDa filters	High-purity research, molecular analysis [36]
Immunoaffinity capture	1. Incubate sample with anti-CD63/CD9/CD81 magnetic beads (1:10 ratio) 2. Rotate 2 h at 4 °C 3. Magnetic separation for 15 min 4. Wash 3× with PBS + 0.1% BSA 5. Elute with 100 mM glycine-HCl (pH 2.5) 6. Neutralize immediately with 1 M Tris (pH 8.0)	Diagnostic applications, biomarker studies [38–40]
Microfluidic isolation	1. Prime microfluidic chip with PBS 2. Load sample at 5 µL/min flow rate 3. Apply AC electrokinetic field (20 V, 100 kHz) 4. Collect exosome fraction from outlet 5. Optional: on-chip characterization	Point-of-care diagnostics, small sample volumes [42]
Hybrid UC-SEC	1. Perform UC protocol steps 1–4 2. Resuspend pellet in 500 µL PBS 3. Load onto SEC column 4. Collect and pool exosome fractions 5. Optional: concentrate final product	Therapeutic applications, clinical trials [35, 36]

Biological functions of exosomes in leukemia

Biological functions

Leukemic exosomes modulate several biological processes that effectively reshape the bone marrow microenvironment and promote disease progression through six general mechanisms:

Microenvironment remodeling

Leukemic exosomes extensively reshape the bone marrow microenvironment by proliferating mesenchymal progenitors while simultaneously inhibiting osteoblastogenesis and normal hematopoiesis [46]. They induce the expression of Dickkopf-1 (DKK1) in stromal cells, suppressing normal hematopoiesis and osteogenesis. In AML, leukemic stem cells reshape the endosteal niche by preferentially inducing osteogenesis over adipogenesis, secreting bone morphogenetic protein (BMP), DKK1, and chemokine ligand 3 (CCL3) that collectively worsen osteoblast function [47]. In CML, exosomes activate the epidermal growth factor receptor (EGFR) signaling of stromal cells and induce the secretion of interleukin-8 (IL-8) for the survival of leukemic cells [48]. They also induce lipolysis in adipocytes, supporting leukemia growth through metabolic reprogramming [49].

Angiogenesis promotion

Leukemic exosomes induce the transformation of stromal cells into cancer-associated fibroblasts and promote endothelial cell proliferation in a Src-dependent manner. In AML, leukemic stem cells bind to endothelial cells via adhesion molecules (E-selectin/CD44 and VCAM-1/VLA-4), inducing angiogenesis through the VEGF/VEGFR and Notch/Dll4 signaling pathways [50]. Exosomal microRNAs, such as miR-92a and miR-135b, also induce angiogenesis under normoxic and hypoxic conditions [51]. In CML, exosomes promote tumor growth through transforming growth factor beta-1 (TGF-β1) signaling pathways [52].

Immune modulation

Leukemic exosomes suppress immune cell functions through various mechanisms, including the inhibition of T cell and natural killer (NK) cell functions, the promotion of differentiation of immune suppressor cells, and the interference with antigen presentation [53]. They activate the PD-1/PD-L1 pathway, causing immune tolerance, and can lead to systemic immunosuppression upon chronic exposure [53]. They carry immunosuppressive molecules (e.g., TGF-β1, FasL, PD-L1) and decoy ligands (e.g., MICA/B), promoting the expansion of regulatory T cells and myeloid-derived suppressor cells, which further inhibit anti-tumor immunity [54]. This is a very sophisticated immune evasion mechanism that “deactivates” the immune system, making immunotherapy more challenging but also providing new possibilities for therapeutic targeting.

Cell migration and homing

Leukemic exosomes target E-selectin receptors for specific tissue delivery [55] and surface integrins govern organotropic metastasis by facilitating fusion with resident cells at target organs [56]. They remodel gene expression and molecular composition of homing niches, modulating both immune and cancer cell migration [57]. In acute myeloid leukemia, exosomes facilitate the maintenance of leukemic stem cells and chemotherapy resistance by delivering specific miRNAs, such as miR-1246, miR-34c-5p, and miR-34a, which enhance leukemic stem cell survival and suppress differentiation [24].

Disruption of normal hematopoiesis

Leukemic exosomes suppress hematopoietic stem cell function through downregulating stromal cell retention factors and suppressing hematopoietic transcription factors [58]. Although hematopoietic stem cells retain their intrinsic function, progenitor cells exhibit increased proliferation and subsequent exhaustion within the leukemic microenvironment [59]. These functions are mediated through key signaling pathways, including CXCR4, VLA4, CD44, and hypoxia-related proteins HIF-1α and VEGF [60].

Hypoxia interaction

Hypoxia of the bone marrow microenvironment has a significant impact on the content and function of exosomes. Oxygen deprivation alters the composition of exosomes (including miRNAs, lncRNAs, and proteins) in leukemia, promoting proliferation, angiogenesis, and immune evasion [61]. Hypoxia-inducible factors (HIFs) regulate exosomal cargo sorting, with a significant impact on disease pathways [62]. Exosomal miRNAs (e.g., miR-155, miR-21) and proteins (e.g., TGF-β1, BCR-ABL) are non-invasive diagnostic and prognostic biomarkers in this context [30].

Understanding these pathways and exosome-mediated interactions provides hope for the development of novel therapeutic approaches in the management of leukemia [3]. Potential approaches target exosome secretion or neutralize their downstream effects, such as the neutralization of IL-6 [63, 64].

Critical analysis of functional studies

Despite extensive research, significant discrepancies remain in the described functions of exosomes. Studies on AML-derived exosomes exhibit conflicting results in their effects on stromal cells, where some report pro-proliferative effects [46] while others demonstrate anti-proliferative properties [65]. Such discrepancies are likely because of heterogeneity in exosome isolation methods, donor cell state, and experimental conditions.

The opposing bone marrow microenvironment remodeling effects outlined in CML [48] versus AML [47] also suggest divergent mechanisms, implicating disease-specific differences that must be investigated rather than generalizable functions. Furthermore, in vitro findings of immune suppression mediated through leukemic exosomes [53, 54] have proven difficult to validate in vivo, highlighting the challenge of translating molecular findings to the clinic. Most functional studies are based on supraphysiological exosome concentrations, which may not accurately represent in vivo conditions and may overestimate biological effects. The community lacks standardization of cell culture conditions, including parameters such as serum starvation and hypoxia, which substantially modulate the content and function of exosomes, but are seldom controlled across studies (Fig. 3).

Fig. 3.

Multifaceted biological functions of leukemic exosomes. The diagram depicts the central role of leukemic exosomes in modulating six key pathological processes in leukemia: (1) Microenvironment Remodeling through DKK1 expression, IL-8 production, EGFR signaling, and BMP/CCL3 release in stromal cells; (2) Cell Migration and Homing via E-selectin binding and miRNA-mediated targeting; (3) Angiogenesis Promotion through VEGF/VEGFR signaling, miR-92a, miR-135b, Notch/Dll4 signaling, and TGF-β1 pathways; (4) Immune Modulation via PD-1/PD-L1 pathway activation, TGF-β1 and FasL delivery, MICA/B decoy ligands, and T & NK cell suppression; (5) Hypoxia Interaction through HIF-1α regulation, transfer of miR-155, miR-21, miR-210, BCR-ABL, and altered cargo sorting in bone marrow; and (6) Disruption of Normal Hematopoiesis by downregulating retention factors, suppressing HSC function, and modulating CXCR4 & VLA4 signaling. HSC hematopoietic stem cell, NK natural killer

Mechanisms of exosome-mediated resistance

Treatment-resistant leukemic cell-derived exosomes convey resistance by delivering biologically active cargo to target cells. These distinct vesicles enhance cell viability, clonogenic survival, and therapeutic resistance in both cancer stem cells (CSCs) and non-CSC populations by modulating critical pathways associated with DNA repair, apoptosis evasion, and epithelial-mesenchymal transition (EMT) [66].

Exosomes from resistant cells can measurably promote the migration of recipient cells and the formation of the tumor microenvironment, both of which are indicators of CSC growth, a crucial feature of therapy resistance. Mechanistically, molecular cargo in exosomes derived from resistant cells reprograms recipient cells to activate survival signaling, such as augmented DNA damage repair pathways or overexpressed efflux pump activity [66]. Intercellular transfer measurably transforms drug-sensitive cell phenotypes into resistant ones, with a marked impact on treatment effectiveness.

Disease-specific resistance pathways

Acute myeloid leukemia (AML)

Exosomes confer chemoresistance in AML by a variety of mechanisms, including the promotion of leukemic stemness properties:

Stemness Maintenance AML-derived exosomes deliver specific miRNAs (miR-1246, miR-34c-5p, miR-34a) that maintain cancer stem cell (CSC) characteristics by suppressing differentiation pathways and enhancing self-renewal capacity. These miRNAs target tumor suppressor genes and differentiation-promoting transcription factors, effectively preserving the undifferentiated state of leukemic cells [24]. Exosomal transfer of stemness-related transcription factors and epigenetic regulators further reinforces the CSC phenotype in recipient cells, creating a reservoir of treatment-resistant cells capable of driving disease relapse.

Vascular Remodeling VEGF/VEGFR mRNA is delivered to endothelial cells by AML exosomes, promoting VEGF signaling and glycolytic reprogramming. It enhances vascular remodeling to support leukemic cell survival in therapeutic stress [67].

Microenvironment Modification These exosomes induce the production of proangiogenic cytokines, such as IL-6 and VEGF, in endothelial cells, creating a hypoxic, nutrient-rich niche that protects leukemic blasts from chemotherapy. They also induce bone marrow stromal cells to produce IL-8, which counteracts the cytotoxic effect of drugs such as etoposide [67].

MicroRNA Transfer Exosomes from adriamycin-resistant HL-60/ADR leukemia cells contain increased levels of miR-484 compared with parental HL-60 cells. When transferred to drug-sensitive HL-60 cells, these exosomes enhance proliferation and confer resistance to doxorubicin-induced apoptosis through inhibiting caspase-3 activation and lowering intracellular drug levels [25].

Stromal Reprogramming Exosomes from bone marrow stromal cells in AML induce disease onset and therapeutic resistance by modulating the proliferation, angiogenesis, and migration patterns of stromal cells. Exosomes from AML cells transfer signaling molecules that activate pro-survival pathways, such as the PI3K/AKT pathway, in stromal cells, inducing them to secrete cytokines like CXCL8, which sustains leukemic cell growth [68].

Chronic myeloid leukemia (CML)

Several intricate mechanisms mediate resistance in CML:

Membrane Protein Transfer Exosomes secreted by imatinib-resistant K562IR cells transfer resistance traits to imatinib-sensitive K562 cells by transferring specific membrane proteins. Proteomics analysis found pronounced upregulation of interferon-induced transmembrane protein 3 (IFITM3), CD146, and CD36 in K562IR-derived exosomes. Such markers, particularly CD146, are internalized by target cells, leading to enhanced survival upon exposure to imatinib [15].

miRNA-Mediated Resistance Exosomes from imatinib-resistant CML cells transfer miR-365 to sensitive cells, enabling resistance to tyrosine kinase inhibitors. Upon delivery, miR-365 suppresses pro-apoptotic proteins BAX and cleaved caspase-3, disrupting the intrinsic apoptosis pathway and reducing sensitivity to imatinib-induced cell death [69].

Bone Marrow Stromal Interactions BM-MSC-derived exosomes are involved in the survival of CML cells and resistance to tyrosine kinase inhibitors by transferring specific miRNAs (e.g., miR-21, miR-155) and anti-apoptotic proteins (e.g., survivin, BCL-2). Exosomal products promote the proliferation of leukemic cells, inhibit drug-induced apoptosis, and stimulate autophagy or drug efflux mechanisms [23].

Microenvironmental contributions to resistance

Bone marrow microenvironment handles resistance to exosome-based therapy in leukemia through interrelated processes with a complex network of interacting steps:

Cytokine Signaling Exosomes secreted from leukemia induce the secretion of interleukin-8 (IL-8) from bone marrow stromal cells, including endothelial cells. Increased IL-8 signaling enhances the secretion of adhesion molecules, such as ICAM-1 and VCAM-1, promoting angiogenesis and creating a pro-survival, therapeutically insensitive niche [46].

Vascular Remodeling CML exosomes deliver BCR-ABL transcripts and pro-angiogenic miRNAs (e.g., miR-126, miR-92a) to stromal cells, which remodel the vascular niche by increasing microvessel density and caliber. These structural adaptations facilitate the proliferation and survival of leukemia cells in the face of chemotherapy-induced apoptosis [46].

Hematopoietic Suppression AML exosomes suppress normal hematopoietic stem cell functions by inhibiting CXCL12 and insulin-like growth factor-1 (IGF-1) within the stroma through Dickkopf-1 (DKK-1), thereby further facilitating skewing of the niche towards pro-leukemogenic conditions [46].

Such intricate balance between exosomal signaling molecules and microenvironmental remodeling provides a chemoresistant niche, enabling leukemia cells to become resistant to treatment and survive despite intervention (Fig. 4).

Fig. 4.

Exosome-mediated mechanisms of treatment resistance in leukemia. The diagram illustrates how resistant leukemic cells transfer resistance traits to sensitive cells through exosomes. Key mechanisms include: (1) Transfer of Resistance Traits through specific proteins (miR-365, miR-484) and membrane proteins (IFITM3, CD146, CD36); (2) Stromal Cell Reprogramming involving IL-8 production, PI3K/AKT pathway activation, and creation of protective niches; (3) Immune Evasion through PD-L1 transfer, TGF-β1 signaling, and suppression of T cells and NK cells; and (4) Drug Efflux Modulation via enhanced P-glycoprotein expression and ABC transporter activation. Disease-specific mechanisms are highlighted for AML (VEGF/VEGFR mRNA transfer and miR-484-mediated resistance) and CML (IFITM3, CD146, and CD36 transfer, as well as miR-365 regulation of BAX). The diagram also shows the involvement of bone marrow stromal cells and osteoblast/osteoclast interactions in creating a chemoresistant niche

Diagnostic and prognostic applications

Biomarkers

Exosomal microRNAs (miRNAs) are being increasingly recognized as useful diagnostic and prognostic biomarkers of leukemia due to their high stability, specificity, and accessibility in body fluids [70, 71]. The miRNA patterns specific to various types of leukemia have been identified.

These miRNA signatures are consistent with pathological changes, correlate with disease subtypes and progression, and show differential expression in cancer patients compared with normal subjects. Importantly, their level may be modifiable following therapeutic intervention such as chemotherapy [72]. A meta-analysis concluded that the pooled sensitivity and specificity for detecting cancer using exosomal miRNAs were 86% and 89%, respectively, highlighting their high diagnostic value [73].

However, a stringent critical analysis of exosomal miRNA biomarker studies reveals significant discrepancies. For instance, while some studies reveal miR-155 overexpression in CLL exosomes [74], others found the reverse effect [75], which is due to varying experimental protocols on sample preparation and analysis techniques. Many biomarker study investigations are undertaken on small homogeneous populations of patients, whose results cannot be extrapolated to heterogeneous populations. In addition, the exceptionally high reported specificities and sensitivities tend to decrease in validation sets or on other analytical platforms.

Outside of miRNAs, exosomal proteins significantly enhance the diagnostic and prognostic potential of extracellular vesicles in leukemia [76]. Elevated concentrations of extracellular vesicles that carry tumor-associated antigens (e.g., NPM1 and FLT3-ITD mRNAs in AML and BCR/ABL mRNA in CML) are correlated with typical clinical presentation and disease course [26]. Exosomal contents actively engage in diverse processes of tumor biology, including survival, angiogenesis, metastasis, drug resistance, and immune evasion, primarily by remodeling the bone marrow microenvironment. Leukemic cell-lineage-derived extracellular vesicles transfer pro-angiogenic microRNAs, such as VEGF and miR-210, into endothelial cells and remodel the extracellular matrix by utilizing matrix metalloproteinases to generate pre-metastatic niches [26, 77].

Specific analytical platforms, such as the EV Array and DNA-PAINT, combined with machine learning algorithms, enable the parallel examination of multiple exosomal surface biomarkers, thereby increasing diagnostic accuracy and clinical significance [78, 79].

Disease monitoring and clinical applications

Effective management of leukemia requires constant assessment of disease progress and treatment response. Exosomes have emerged as the most suitable non-invasive agents for monitoring using liquid biopsies, offering certain benefits over conventional strategies [80]. They are present in peripheral blood and can internalize disease-specific molecular loads, making them particularly well-suited for longitudinal disease monitoring of leukemia [81].

Exosome-mediated monitoring provides real-time data on disease kinetics via global profiling of their cargo, including proteins, nucleic acids, and metabolites. This has demonstrated exemplary sensitivity in detecting low levels of residual disease (MRD) and the initiation of relapse in leukemia [82]. In AML, protein profiles shift and levels of TGF-β1 correlate closely with treatment outcomes, providing useful indicators for monitoring therapeutic effectiveness [27].

The role of exosomes in remodeling the bone marrow microenvironment adds another layer to disease monitoring. The vesicles play a role in reprogramming the bone marrow niche, making it supportive of abnormal cell growth [83]. Elevated levels of extracellular vesicles carrying tumor-associated antigens have been reported in most leukemia types, with corresponding levels to clinical presentation and disease stages [84].

Exosomes are also explored for MRD detection, an essential aspect of disease surveillance [85]. Next-generation sequencing technologies with ultra-deep capacity applied to exosomal content are significantly more sensitive than routine tests.

Therapeutic applications

The therapeutic potential of exosomes in treating leukemia has garnered significant interest, offering a revolutionary approach to treatment. Exosomes are natural nanovesicles with unique properties that render them ideal carriers and platforms for immune modulation. The wide variety of applications encompasses drug delivery, immunotherapy, and strategies for treating treatment resistance.

Exosomes as drug delivery vehicles

Exosomes have been identified as promising vehicles for drug delivery due to their inherent biological properties. Their bilayer membrane, which is phospholipid-based, naturally shields the therapeutic load against degradation. However, their ability to permeate across biological barriers, fuse into target cell membranes, and escape endo/lysosomal routes enables direct cytoplasmic delivery [86]. Exosomes differ from synthetic nanoparticles in that they exhibit natural biocompatibility, circulation stability, and inherent targeting properties, which significantly reduce off-target effects [87].

Various cell types release exosomes with unique therapeutic functions:

Dendritic cell-derived exosomes (Dex) acquire immune-stimulatory components like MHC class I/II molecules, co-stimulation signals (CD80, CD86), and adhesion proteins (ICAM-1). Their lipid bilayer composition, rich in sphingomyelin and phosphatidylinositol, imparts stability and long storage half-life. Functionally, Dex can stimulate T cells, activate NK cells via NKG2D ligands, and enhance B-cell activity through Th1 polarization [88].

Mesenchymal stem cell-derived exosomes have low immunogenicity, tumor-homing properties, and excellent biocompatibility. Production can be enhanced through three-dimensional culture systems, bioglass stimulation, and targeted genetic manipulations (e.g., MYC transfection) [89].

Macrophage-derived exosomes (M1-Exo) have also been reported to reprogram immunosuppressive M2-polarized tumor-associated macrophages into pro-inflammatory M1-like macrophages, characterized by higher phagocytic activity and improved antigen cross-presentation. M1-Exo has been reported to induce delayed tumor growth in orthotopic models and exhibit synergistic effects when combined with anti-PD-L1 therapy [90].

Cancer cell-derived exosomes exhibit a preferential fusion with their parental cells, potentially enhancing targeted delivery to specific cancer types [91].

CAR-T cell-derived EVs T cell-budded EVs are membrane-bound vesicles that serve as active modulators of immunological events, both extrinsically through their surface receptors and intrinsically through their cargo. CAR-T cell-derived EVs have been highlighted for their roles as a new therapeutic agent for leukemia immunotherapy. CAR-T cell-derived EVs (CAR + EVs) are found to carry CARs on their surfaces and high concentrations of cytotoxic agents (i.e., perforin and granzyme B). Therefore, CAR-T cell derived EVs have high anti-tumor activity, targeting their target cells, where they deliver their cytotoxic cargo, optimizing the net antitumor immune response and augmenting the therapeutic efficacy of CAR-T cells in vivo [92].

Dual confocal and STORM analysis in CAR T cells showed an enormous richness of CAR + vesicles in the cytoplasm. In control and eGFP-expressing CAR T cells, most of the CD19.CAR protein co-localized with CD63. Notably, perforin also showed the same cytoplasmic localization. These findings indicate that the CD19.CAR protein, predominantly sequestered in CD63 + cytoplasmic vesicles, is released in large amounts into the supernatant as CAR + EVs, primarily after target engagement. This process describes how the CAR-T cells can exert their therapeutic effects without direct cellular interaction, and potentially propagate their primary action through EV-mediated communication [93].

Despite these developments, some obstacles still lie ahead for clinical translation. Standardization of isolation processes, optimization of drug loading efficiency, and reproducible characterization are significant technical challenges [94]. Scale-up production is demanding, requiring advanced bioreactor systems and standardized processes. Systemic clearance via the reticuloendothelial system and non-uniform accumulation within the tumor, because of variable vascular permeability, compromises the efficacy of therapy [95]. Solutions are proposed to include the deployment of trehalose as a cryoprotectant to prevent freezing and storage damage, as well as the use of PEGylation techniques to enhance stability and prolong circulation time [96].

CAR + EVs Some of the most recent advancements involve γδ-T cell-derived extracellular vesicles (γδ-T-EVs) that have been engineered as cancer vaccines. Such platforms can be loaded with tumor-associated antigens (TAAs) for enhancing tumor-specific T-cell immune responses while preserving direct antitumor cytotoxicity [97]. One of the most significant advantages of this approach is that allogeneic γδ-T-EVs-based vaccines are as potent as their autologous equivalents, potentially enabling more standardized and centralized manufacturing processes that could overcome the manufacturing challenges associated with personalized treatments. This twofold use, both as antigen-delivery vehicles and as local tumor-cytotoxic agents, is a thrilling direction for immunotherapy of leukemia [97].

Advantages of CAR + EVs over Conventional CAR-T Cell Therapy CAR + EVs offer several distinct advantages over traditional CAR-T cell therapy, addressing significant clinical limitations. Unlike CAR-T cells, CAR + EVs do not proliferate uncontrollably, significantly reducing the risk of cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), which are dose-limiting toxicities in CAR-T therapy [93, 98]. Their cell-free nature eliminates the risk of graft-versus-host disease and reduces immunogenicity compared to allogeneic CAR-T cells. CAR + EVs demonstrate superior tissue penetration due to their nanoscale size (30–150 nm versus ~ 10–15 μm for T cells), enabling better access to solid tumor microenvironments and anatomically restricted sites, such as the central nervous system [98, 99]. Manufacturing advantages include the potential for off-the-shelf production, standardized dosing, improved storage stability, and simplified logistics compared to patient-specific CAR-T cell production, which requires complex ex vivo expansion. Additionally, CAR + EVs enable repeated dosing without the need for lymphodepletion conditioning, providing more flexible therapeutic regimens while maintaining the targeted cytotoxic effects through the delivery of perforin and granzyme B [99, 100].

Engineering approaches have enhanced the therapeutic potential of exosomes. Surface engineering enhances targeting specificity through techniques such as exosome engineering, which targets specific cancer markers like EGFR [101] or αv integrin [102]. Therapeutic compounds are loaded using complementary approaches: passive incubation for lipophilic drugs and small molecules, electroporation for nucleic acids, and sonication for large cargos, such as proteins, but at the expense of compromising exosome membrane integrity [103]. Recent concepts involve the magnetic modification of exosomes to enable better targeting and efficient separation from the blood [104].

Immunotherapeutic applications

Apart from their use in drug delivery, exosomes also possess interesting immunotherapeutic applications in leukemia. They are endogenous adjuvants, antigen-presenting vehicles, and controllers of the tumor microenvironment.

Dendritic cell-derived exosomes trigger immune responses through several mechanisms. They can present antigen-MHC complexes to directly stimulate memory T cells or indirectly stimulate naïve T cells by “cross-dressing” bystander dendritic cells. Exosomes also stimulate NK cells via surface ligands, such as NKG2D ligands, which activate and lead to the secretion of IFN-γ, while enhancing B-cell responses through Th1 polarization [105]. Similarly, tumor-derived exosomes can present tumor-associated antigens to dendritic cells, which stimulate antigen-specific CD8 + cytotoxic T lymphocytes [106].

Engineering techniques have significantly contributed to the immunotherapeutic capabilities of exosomes. Genetic modification using lentiviral vectors can transfect dendritic cells to express fusion proteins that facilitate efficient loading of antigens into exosomes, enhancing antigen presentation and immune responses [107]. Exosome surface engineering with HLA molecules, costimulatory ligands, and immunostimulatory agents, such as α-galactosylceramide, augments antigen presentation and invariant natural killer T cell activation in parallel [108, 109]. Bi-specific targeting approaches, such as exosome engineering with anti-CD3 and anti-EGFR antibodies, create bridges between T cells and cancer cells, enhancing targeted interactions and anti-tumor immunity [110].

The complex interplay between exosomes and the tumor microenvironment is both challenging and rewarding. Exosomes derived from leukemia can suppress NK cell-mediated immunity through TGF-β and Fas-L mechanisms, whereas under hypoxia, they load more immunosuppressive cargo [111]. Such mechanisms have been targeted by therapeutic attempts, such as CD19 CAR-expressing exosomes, which specifically induce death in malignant B cells and, potentially, restore anti-tumor immunity.

Clinical use faces serious challenges, particularly in immunocompromised leukemia patients. Serum exosomes from patients with acute myeloid leukemia may induce tolerance in dendritic cells, thereby suppressing antitumor immunity [112]. To counteract this, researchers are employing bioengineering strategies to enhance the immunogenicity of exosomes and targeting specificity [113]. Currently, several clinical trials are evaluating exosome-based immunotherapies, including a Phase I study (NCT03608631) that tests dendritic cell-derived exosomes loaded with MAGE antigens for the treatment of melanoma, and NCT05116813, which investigates mesenchymal stem cell-derived exosomes for the treatment of COVID-19-related acute respiratory distress syndrome. Additionally, NCT04202783 is evaluating plant-derived exosome-like nanoparticles for oral delivery in colon cancer [114–117]. While these trials have demonstrated feasibility and safety, improving therapeutic efficacy remains a focus of ongoing research.

Strategies to overcome exosome-mediated resistance

Exosome-based therapy offers novel approaches for overcoming treatment resistance in leukemia by targeting mechanisms of exosome-mediated resistance, thereby sensitizing resistant tumor cells to standard treatments. Various strategic strategies are under development:

1. Inhibition of Exosome Biogenesis: Pharmacologic agents that inhibit key proteins involved in exosome production and secretion can reduce exosome-mediated resistance [118]. Azole compounds such as neticonazole and ketoconazole, for instance, suppress exosome biogenesis by inhibiting Rab27A’s function, a GTPase required for exosome trafficking and secretion, reducing the secretion of immunosuppressive exosomes that export PD-L1; [119]

2. Extracorporeal hemofiltration employs semipermeable membranes or affinity adsorbents (e.g., heparin-coupled polyvinyl alcohol microspheres) to selectively remove tumor-derived exosomes from the blood, disrupting their immunosuppressive function and enhancing therapeutic effects by inhibiting exosome-mediated immune evasion and drug resistance [120];

3. Blockage of calcium signaling by agents like dimethyl amiloride or calcium channel blockers (e.g., amlodipine) disrupts calcium-dependent processes (e.g., Munc13-4/Rab11-mediated membrane fusion) necessary for exosome formation and PD-L1 loading [119]; and

4. Combination Therapies: A Combination of exosome inhibitors with existing anti-PD-1/PD-L1 therapies might enhance immunotherapy efficacy by simultaneously blocking both cell-surface and exosome-derived PD-L1, evading resistance mechanisms, and restoring T-cell cytotoxicity [119]

The convergence of emerging technologies in exosome engineering, combined with an increased understanding of their biological functions and mounting clinical evidence, positions exosomes as promising platforms for next-generation therapies for leukemia. With augmented standardization and expanded production scale, such exosome-based therapies may become an integral component of individualized medicine approaches, potentially improving treatment efficacy for patients with refractory diseases and reducing off-target toxicity compared to conventional therapies.

Future directions and challenges

Technological advances and innovations

Recent technological advancements have significantly enhanced the ability to monitor exosomes [121]. New techniques, such as microfluidic chips, nanowire arrays, and electrochemical biosensors, now enable the fast, sensitive, and high-throughput measurement and analysis of exosomes. Currently, multiple clinical trials are validating exosome-based liquid biopsies for clinical use. The FDA has granted breakthrough device designation to several exosome-based diagnostic platforms, including the ExoDx Prostate test (Exosome Diagnostics) for prostate cancer risk assessment, indicating regulatory acceptance of exosome-based diagnostics as they move toward clinical implementation [122, 123].

Challenges and standardization issues

Despite these breakthroughs, significant challenges remain in standardizing exosome-based monitoring for clinical applications. State-of-the-art methods of isolation yield inconsistent yields and purity, which undermines the reproducibility and reliability of the outcomes [124, 125]. Clinical translation of exosomes has been hindered by the lack of universally accepted high-quality separation and definitive analytical methods, although they hold promise as new liquid biopsy biomarkers.

These methodological restrictions have a significant impact on clinical translation. The field is hampered by a “methods crisis,” where breakthrough discoveries in basic research cannot be reliably reproduced from laboratory to laboratory using different methods of isolation [126, 127]. Comparative studies of clinically meaningful biomarkers suggest that different isolation procedures applied to the same samples yield distinct molecular profiles and biomarker expression levels, casting doubt on the significance of many published biomarkers [128, 129]. Furthermore, sample treatment before isolation (e.g., type of collection tube, duration of storage, temperature, and number of freeze-thaw cycles) profoundly alters exosome characteristics; however, these pre-analytical parameters remain insufficiently standardized [130].

Purification difficulties are particularly pronounced in clinical applications, as most techniques are unable to effectively remove co-isolated impurities, such as protein aggregates, lipoproteins, or cell debris. Such impurities may lead to false-positive signals in biomarker studies, particularly in proteomic studies [131]. Additionally, most isolation techniques exhibit significant batch-to-batch variation, making it challenging to generate reliable clinical assays that must function consistently over an extended period.

Methods for overcoming these shortcomings include the creation of standardized processes through global efforts, such as the MISEV guidelines and EV-TRACK [132, 133]. Most crucially, the MISEV2018 guidelines have encouraged the application of operational terms such as ‘small extracellular vesicles’ instead of ‘exosomes’ unless the biogenesis of vesicles can be positively verified. Such a shift in nomenclature acknowledges that most isolation methods result in heterogeneous vesicle populations, rather than purified exosomes, a crucial distinction that is frequently not conveyed in clinical research reports.

Therapeutic interventions also have the potential to influence exosome release patterns and content composition, which can affect their feasibility as monitoring tools in leukemia [133, 134].

Unless these intrinsic methodological shortcomings are addressed, the field may produce biomarkers and therapies based on isolation artifacts rather than biologically significant exosome traits. Progressing towards clinical use will require rigorous, multi-center validation studies by standardized protocols in various patient populations.

Integrated multi-omics approaches

Future developments are likely to arise from combined multi-omics platforms that simultaneously explore exosomal proteins, nucleic acids, and lipids. The combined analysis holds the potential to enhance diagnosis specificity and reveal novel therapeutic targets [135, 136]. Advanced machine learning approaches applied to multi-omics data can identify complex biomarker patterns that single-analyte approaches might not detect, and this has the potential to revolutionize early detection and monitoring of treatment.

Engineered exosome platforms

Engineered exosome platforms of a preprogrammed composition and enhanced functionality are an appealing option. Synthetic or semisynthetic mimetics of exosomes of a predetermined composition form a solution for heterogeneity-associated problems with the prospect of reproducible drug delivery [137]. These platforms would be modified to accommodate a disease’s unique features, enabling the development of personalized medicine practices tailored to each individual.

Integration with cutting-edge technologies

CRISPR-exosome systems

The combination of CRISPR-Cas gene editing with exosome delivery systems provides a highly potent approach to address genetic aberrations in leukemia. Exosomes transfer CRISPR-Cas reagents with potentially reduced off-target effects compared to viral vectors efficiently [138]. Recent studies have also demonstrated the efficient transfer of CRISPR-Cas9 ribonucleoproteins via engineered exosomes to leukemia cells, resulting in targeted gene knockout with minimal toxicity [139].

Theranostic applications

Emerging exosome theranostic platforms integrate diagnostic and therapeutic capabilities into a single system. Platforms typically comprise imaging agents along with therapeutic cargo, enabling real-time monitoring of exosome biodistribution and therapeutic effect [140]. Integrated approaches can streamline the clinical process from diagnosis to treatment monitoring, particularly for rapidly evolving types of leukemia.

Evolving regulatory frameworks

Regulatory infrastructures are evolving to address the new challenges presented by exosome-based technologies. The FDA’s regenerative medicine advanced therapies (RMAT) framework may be extendable to engineered exosomes, and the European Medicines Agency has published targeted guidelines for cell-derived medicinal products that may encompass exosome therapeutics [141, 142]. Global harmonization of these infrastructures will be critical to enable global clinical translation.

Novel disease applications

Despite concerted efforts aimed at prevalent leukemia types, new evidence suggests a possible application to less common disorders and disease subtypes with distinct molecular profiles. Exosome therapy can offer treatments for disorders that have limited therapeutic options available to them, such as rare leukemia subtypes [143]. The exosome’s action of high specificity can offer good treatment options for such disadvantaged patient groups.

Pharmacoeconomic innovations

High manufacturing and development costs

Exosomal therapy faces substantial economic barriers that significantly limit clinical implementation and patient accessibility. The endogenous method for exosome production is associated with low production efficiency and high cost, while manufacturing processes require specialized equipment, reagents, and expertise [144]. Clinical applications face challenges including product heterogeneity, rapid systemic clearance, and long-term stability, necessitating standardization of doses, administration routes, and parent MSC sources to overcome manufacturing complexities. GMP-grade exosome production involves the highest quality materials, cells, culture environment, manufacturing technologies, and a skilled workforce, all operating under highly controlled and strictly monitored conditions. Purification and quality control release assays are essential to ensure that the final products meet the highest standards before administration [145]. Despite their potential advantages, including decreased immune response and reduced toxicity, the complex manufacturing requirements and regulatory compliance costs present significant barriers to widespread clinical adoption.

The availability of affordable manufacturing technologies will be crucial for widespread clinical adoption. Economic evaluations specific to exosome therapies remain limited but will remain critical as these technologies become clinically available. Cost-effective strategies include developing scalable manufacturing processes, optimizing dosing regimens through pharmacokinetic modeling, and selecting patient subpopulations with the most significant probability of benefit from exosome therapy [143]. New models of business, such as centralized manufacturing facilities or point-of-care production platforms, may help overcome economic hurdles to adoption.

However, improvements in production methods, scalability, and regulatory frameworks are expected to help reduce the costs associated with exosome-based treatments, making them more accessible to broader patient populations as the field continues to advance [144].

Priority areas for improving exosome-based platforms in leukemia

To advance exosome-based approaches in leukemia management, five critical areas require prioritized attention:

1. Standardization and Manufacturing: Development of standardized isolation protocols, scalable GMP-compliant production methods, and consistent quality control measures to ensure reproducible therapeutic outcomes across different leukemia subtypes [35–37].

   Current methodological discrepancies effectively undermine the validity of reported exosome properties and present serious obstacles to clinical translation, with standardization of isolation and characterization methods remaining essential for advancing exosome research [94, 95].

2. Leukemia-Specific Biomarker Validation: Large-scale multicenter validation of disease-specific exosomal biomarkers (miR-155, miR-150 for CLL; [6, 17] miR-26a-5p for AML; [18] IFITM3, CD146 for CML [15]) through heterogeneous patient populations to establish clinical utility and overcome current validation limitations affecting biomarker reproducibility.

3. Targeted Drug Delivery Optimization: Engineering exosomes with enhanced leukemia cell-specific targeting capabilities, improved cargo loading efficiency for chemotherapeutics and nucleic acids, and strategies to overcome drug resistance mechanisms [101–103]. Surface engineering approaches and therapeutic compound loading methods require optimization to enhance targeting specificity and therapeutic efficacy.

4. Combination Therapy Integration: Investigation of synergistic effects between exosome-based treatments and existing leukemia therapies, including chemotherapy, targeted drugs, CAR-T cell therapy [11], and immunotherapies to maximize therapeutic potential and overcome treatment resistance.

5. Patient Stratification and Personalized Approaches: Development of criteria for identifying patients most suitable for exosome-based interventions, creation of personalized exosome formulations based on individual disease characteristics and genetic signatures, and establishment of optimal dosing regimens and treatment protocols [143]. Patient-tailored exosome platforms represent the ultimate goal for precision medicine in leukemia management.

Current clinical implementation and translation challenges

Despite extensive preclinical research demonstrating considerable promise for exosome applications in leukemia, a significant gap exists between research and clinical practice in current implementation. While our review has highlighted substantial therapeutic and diagnostic potential across multiple leukemia subtypes, the clinical reality reveals that no FDA-approved exosome-based tests or treatments exist specifically for leukemia, and clinical implementation remains essentially nonexistent.

Active clinical investigations

Current clinical trials specifically investigating exosome applications in hematological malignancies remain limited but targeted. Several key studies provide important insights into the clinical potential we have discussed throughout this review. NCT01589302 [146], a completed Phase 2 trial sponsored by Ohio State University, investigated exosomes as biomarkers in chronic lymphocytic leukemia patients receiving ibrutinib therapy. This study successfully demonstrated that plasma exosome concentrations significantly decreased following ibrutinib treatment, establishing that BCR signaling regulates exosome secretion in CLL patients and validating the biomarker potential described in Sect. 3.1.

NCT00900809 [147] represents another completed clinical investigation, in which researchers at the University of Pittsburgh Medical Center examined exosomes as immunosuppressive factors in acute myeloid leukemia. This Phase 1 adoptive cell therapy trial with NK-92 cells in seven relapsed/refractory AML patients revealed that pre-therapy plasma contained elevated levels of immunosuppressive exosomes, which interfered with the anti-leukemia functions of NK-92 cells. These findings support the discussion in Sect. 2.1.3 regarding immune modulation mechanisms and identify exosomes as potential barriers to the efficacy of immunotherapy, rather than solely as therapeutic tools.

International clinical trial registries show limited specific entries for hematological malignancy exosome applications, with most exosome trials still in early developmental phases.

Regulatory environment and approval status

The regulatory landscape presents significant challenges to clinical translation of the promising approaches we have reviewed. No FDA-approved exosome-based tests or treatments exist specifically for leukemia or any hematological malignancies. The FDA has explicitly stated that “there are currently no FDA-approved exosome products.” [148] for any indication beyond the single exception of the ExoDx Prostate IntelliScore (EPI) Test [149], which received FDA Breakthrough Device Designation on June 17, 2019, but applies only to prostate cancer risk assessment.

Exosome products fall under the FDA’s Center for Biologics Evaluation and Research (CBER) authority, governed by Public Health Service Act Sect. 351 as biological products requiring premarket review and approval [150]. As of October 2023, the FDA has issued six warning letters to exosome companies, demonstrating its aggressive enforcement against unauthorized clinical applications [150]. This regulatory vigilance has a significant impact on the clinical translation of the therapeutic delivery vehicles and immunotherapeutic applications discussed in Sect. 4.1 and 4.2.

No breakthrough therapy designations, 510(k) clearances, or PMA approvals have been granted for exosome applications in leukemia. The FDA’s approved companion diagnostic devices list for hematological malignancies contains only cfDNA-based liquid biopsies, with no exosome-based technologies included, despite the biomarker potential demonstrated in Sect. 3.

Clinical practice integration

Current clinical implementation reveals a complete absence of exosome-based testing in leukemia patients across major healthcare systems. Professional society guidelines from the NCCN, American Society of Hematology (ASH), and European LeukemiaNet (ELN) do not contain recommendations for exosome testing in leukemia, with current guidelines focusing on traditional molecular diagnostics, including cytogenetics, flow cytometry, and PCR-based monitoring.

Commercial laboratories offer no exosome-based leukemia tests, focusing instead on established molecular methods. ExosomeDx (Bio-Techne), the only company with FDA-approved exosome diagnostics, currently offers only a prostate cancer test [151], with no leukemia-specific applications in development.

Translation barriers

Several critical barriers impede the clinical translation of the promising exosome applications we have reviewed. Insurance coverage and reimbursement mechanisms do not exist for exosome-based leukemia testing, creating significant financial barriers to clinical implementation even if tests were available. No CPT codes, medical necessity criteria, or cost-effectiveness data support the clinical use of this product.

Manufacturing and quality control challenges persist as significant barriers, including a lack of standardized isolation and characterization methods, as discussed in Sect. 1.2, concerns about batch-to-batch variability, and manufacturing scalability issues. The FDA requires demonstration of safety and efficacy through controlled studies with proper GMP manufacturing [152], creating high development costs and technical challenges that must be overcome before the therapeutic platforms described in Sect. 4 can reach clinical implementation.

The standardization issues highlighted throughout this review remain critical obstacles. As noted in Sect. 5.2, methodological discrepancies effectively undermine the validity of reported properties and functions of exosomes, presenting serious challenges to reproducing results or establishing diagnostic and therapeutic applications.

Evidence from completed clinical studies

Completed clinical trials provide preliminary evidence for the utility of exosomes as biomarkers rather than therapeutic agents. The CLL ibrutinib study successfully demonstrated changes in exosome concentration correlating with treatment response, establishing proof of concept for the treatment monitoring applications discussed [146]. The AML immunotherapy trial identified exosomes as therapeutic barriers, showing that elevated immunosuppressive exosomes interfered with the anti-leukemia functions of NK-92 cells [79], which aligns with the discussion of immune modulation mechanisms in Sect. 2.1.3.

Future implementation pathway

The pathway to clinical implementation requires addressing the multifaceted challenges we have identified throughout this review. No breakthrough designations have been explicitly granted for leukemia-related exosome applications, in contrast to the single breakthrough designation for prostate cancer, which led to clinical implementation. The regulatory pathway for exosome-based leukemia applications remains undefined, necessitating early engagement with the FDA through pre-IND meetings to clarify development strategies.

The integration of exosome-based approaches into clinical practice will require addressing the technological advances, standardization issues, and regulatory frameworks outlined in Sect. 5. The convergence of enhanced biological understanding, technological innovation, and evolving regulatory landscapes, as described, positions exosome-based strategies for eventual clinical translation; however, significant work remains to bridge the current research-to-practice gap.

This clinical reality provides essential context for the promising research developments reviewed, emphasizing that while the scientific foundation is strong, substantial translational work is required to realize the clinical potential of exosome-based approaches in leukemia management (Table 3).

Table 3.

Clinical applications of exosomes in leukemia

Application	Details	Challenges	References
Biomarkers	• Elevated levels of tumor-specific antigen-expressing exosomes • Exosomal phosphatidylserine for early detection	• Standardization of isolation protocols • Validation across diverse populations	[70–73, 153–156]
Disease monitoring	• Detection of minimal residual disease • Differentiation between disease stages (e.g., WM) • Correlation with treatment responses (e.g., AML)	• Variable yield and purity with current isolation methods • Influence of treatments on exosome release	[27, 80–84, 124, 132, 134]
Drug delivery	• Sources: dendritic cells, MSCs, macrophages, cancer cells • Enhancement strategies: surface modification, engineering for targeting • Loading methods: incubation, electroporation, sonication	• Standardization of production • Optimization of drug loading efficiency • Large-scale production hurdles	[86–91, 94, 95, 101–104, 157]
Immunotherapy	• Dexosomes for activating immune responses • Engineering strategies: fusion proteins, HLA expression • Applications: CD19 CAR exosomes for B-cell leukemia	• Safety concerns in immunocompromised patients • Potential induction of dendritic cell tolerance (AML)	[105, 114, 143]
Overcoming treatment resistance	• Targeting exosome machinery • Extracorporeal hemofiltration • Inhibition of exosome biogenesis (e.g., azole drugs) • Targeting calcium signaling to reduce exosomal PD-L1	• Complex interactions between exosomes and conventional treatments • Therapeutic window considerations	[23, 25, 66–68, 118–120, 158]

Conclusion

Exosomes have evolved from cellular waste products to sophisticated intercellular messengers with transformative potential in the management of leukemia. This review highlights their multifaceted roles in disease progression through microenvironment remodeling, immune modulation, and mechanisms of treatment resistance, while also revealing their promise as diagnostic biomarkers and therapeutic vehicles. Disease-specific molecular signatures enable liquid biopsy applications with high sensitivity and specificity, while engineered exosome platforms offer novel drug delivery and immunotherapeutic strategies, including CAR-T derived approaches. However, significant challenges persist, including methodological standardization, scalable manufacturing, and regulatory hurdles that have prevented clinical implementation despite extensive preclinical evidence. The current research-to-practice gap highlights the need for harmonized protocols, large-scale validation studies, and the development of a regulatory framework. Future success will require integration with artificial intelligence, multi-omics platforms, and personalized medicine approaches, alongside continued collaboration between researchers, clinicians, and regulatory bodies. As these challenges are addressed, exosomes are positioned to revolutionize leukemia care through precision diagnostics, targeted therapies with reduced toxicity, and novel strategies for overcoming treatment resistance, ultimately transforming patient outcomes in hematological malignancies.

Abbreviations

AML

Acute myeloid leukemia

BCR-ABL

Breakpoint Cluster Region-Abelson Murine Leukemia

BM-MSC

Bone marrow mesenchymal stem cell

BMP

Bone morphogenetic protein

CAR

Chimeric Antigen Receptor

CCL3

Chemokine ligand 3

CD

Cluster of Differentiation

CLL

Chronic lymphocytic leukemia

CML

Chronic myeloid leukemia

CSC

Cancer stem cell

CXCL

C-X-C Motif Chemokine Ligand

CXCR4

C-X-C Chemokine Receptor Type 4

Dex

Dendritic Cell-derived Exosomes

DKK1

Dickkopf-1

EGFR

Epidermal growth factor receptor

EMT

Epithelial-mesenchymal transition

EV

Extracellular vesicle

FDA

Food and Drug Administration

HIFs

Hypoxia-inducible factors

HLA

Human Leukocyte Antigen

IAC

Immunoaffinity capture

ICAM

Intercellular adhesion molecule

IFITM3

Interferon-induced transmembrane protein 3

IGF-1

Insulin-like growth factor-1

IL

Interleukin

lncRNAs

Long non-coding RNAs

M1-Exo

M1 macrophage-derived exosomes

MICA/B

MHC Class I Chain-related Protein A/B

miRNA

MicroRNA

MISEV

Minimal information for studies of extracellular vesicles

MRD

Minimal residual disease

MSC

Mesenchymal stem cell

NK

Natural killer

NKG2D

Natural Killer Group 2D

NTA

Nanoparticle tracking analysis

PD-1

Programmed Death-1

PD-L1

Programmed Death-Ligand 1

RMAT

Regenerative medicine advanced therapies

SEC

Size exclusion chromatography

TAAs

Tumor-associated antigens

TGF-β

Transforming growth factor beta

UC

Ultracentrifugation

VCAM

Vascular Cell Adhesion Molecule

VEGF

Vascular Endothelial Growth Factor

VEGFR

Vascular Endothelial Growth Factor Receptor

VLA4

Very Late Antigen-4

WM

Waldenström’s Macroglobulinemia

Author contributions

Mohammad Amin Ansarian and Mahsa Fatahichegeni were responsible for literature analysis, figure design, and manuscript writing; Yuqi Wang contributed to manuscript preparation with focus on diagnostic applications; Juan Ren analyzed therapeutic applications and evaluated clinical translation challenges; Xiaoning Wang provided conception and design and reviewed and revised the manuscript; all authors contributed to the article and approved the submitted version.

Funding

The authors thank the following financial support for the research, authorship, and publication of this article: National Key R&D Program, Grant Number: 2022YFC2502700. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.