The avatar principle: exosomal dynamics guiding tumor adaptation and next-generation therapeutic strategies

Abstract

Exosomes are nanoscale extracellular vesicles that transfer proteins, nucleic acids, and lipids, reflecting the state of their parent cells. A persistent scientific challenge is that tumor-derived exosomes (TDEs) facilitate immune evasion, remodel the tumor microenvironment, and create premetastatic niches, intensifying tumor aggressiveness and undermining therapeutic efficacy, ultimately narrowing treatment options to palliative strategies in advanced settings. Yet their dual roles as suppressive agents and potential therapeutic tools remain poorly integrated within current cancer immunotherapy frameworks. This review examines the molecular mechanisms underlying TDE-mediated immune suppression and therapeutic resistance, while also highlighting engineering strategies to exploit or counteract exosome biology. Exosomes derived from chimeric antigen receptor (CAR) T cells preserve antigen specificity and cytotoxic components without the risks of uncontrolled proliferation or cytokine release, offering a safer class of cell free immunotherapies. Advances in genetic engineering, hybrid vesicle design, and nanotechnology have extended exosome applications to the delivery of CRISPR/Cas systems, chemotherapeutic agents, immunoregulatory RNAs, and vaccines, with liposome or nanoparticle integration enhancing targeting and efficacy. Remaining obstacles include the lack of standardized protocols, scalability issues in production, and unresolved regulatory frameworks. Drawing on The Art of War, exosomes can be envisioned as avatars of strategy, discreet messengers capable of undermining host defenses while simultaneously carrying the potential to redirect immunity against the tumor. By embodying both deception and counterattack, they illustrate the capacity to penetrate hidden barriers and redefine the therapeutic battlefield, opening new horizons for precision cancer immunotherapy.

Graphical abstract

Introduction

Exosomes are the smallest type of extracellular nanoscale vesicles (EVs) (30–150 nm) secreted by nearly all cell types and function as key mediators of intercellular communication through their ability to transport proteins, lipids, and nucleic acids between cells [1–5]. Their molecular content mirrors the physiological or pathological status of their cells of origin, enabling them to influence immune responses, gene expression, and cell behavior across diverse biological contexts [6, 7].

In cancer biology, tumor-derived exosomes (TDEs) have emerged as critical mediators of tumor immune evasion and microenvironment remodeling. These vesicles carry tumor-specific antigens, oncogenic proteins, and immunomodulatory molecules that systematically reprogram the tumor microenvironment (TME) [8, 9]. By delivering oncogenic proteins, immunosuppressive ligands, and regulatory RNAs, TDEs reprogram the TME to promote regulatory T cell expansion, polarization of tumor-associated macrophages (TAMs), suppression of cytotoxic lymphocytes, angiogenesis, extracellular matrix (ECM) remodeling, and formation of pre-metastatic niches (PMN), reflecting at a nanoscale all hallmarks of cancer [10]. These features position TDEs as both a therapeutic target and a platform, offering a potential use in cell-free immunotherapy or as delivery vehicles for precision oncology.

Recent developments in chimeric antigen receptor (CAR) T-cell therapy have extended to the exosomal domain. CAR T-derived exosomes preserve key effector functions, including cytotoxic proteins and CAR surface expression, while lacking replicative capacity and immune checkpoint molecules such as programmed death-1 (PD-1). This profile reduces the risk of cytokine release syndrome (CRS) and immune exhaustion, supporting their utility in engineered immunotherapies [11]. Synthetic strategies including CRISPR/Cas9 loading and hybrid constructs are being explored to enhance their specificity and potency [12]. Nevertheless, challenges remain in standardization, large-scale manufacturing, and clinical translation. This review explores the molecular basis of TDEs-mediated immune suppression and resistance and highlights engineering approaches aimed at leveraging or counteracting exosome biology in cancer therapy (Fig. 1). Although TDEs constitute the primary pathogenic focus of this review, we also examine exosomes released by T cells, including CAR T cells, as they represent one of the most clinically advanced and experimentally tractable class of therapeutic exosomes. Within the heterogeneous TME, multiple cell types secrete vesicles that modulate tumor progression; however, T cell–derived exosomes emerge as a key subset currently subjected to systematic engineering, good manufacturing practice (GMP) compatible production, and early clinical evaluation. Their inclusion provides a mechanistic and translational counterpoint to TDE-mediated immune suppression and therapeutic resistance, illustrating how exosomal pathways co-opted by tumors can be redirected to achieve antitumor efficacy.

Fig. 1.

Biological and therapeutic exosomes: a general outlook at their structure, manufacture, and uses

In this review, we define the “Avatar Principle” as the mechanistic notion that exosomes act as molecular surrogates of their cells of origin, faithfully transmitting their genomic, proteomic, lipidomic, and regulatory signatures. Through precisely regulated ESCRT-dependent and ceramide-mediated pathways, together with RNA-binding protein–guided sorting, exosomes encapsulate oncogenic drivers, immunomodulatory ligands, metabolic regulators, and non-coding RNAs that reproduce the functional phenotype and adaptive programs of the parental tumor cell. In this way, exosomes work as operational extensions of malignant cells nanoscale agents capable of executing immune suppression, stromal reprogramming, therapeutic resistance, and pre-metastatic niche conditioning across spatial distances.

Within this architecture, we used the strategic elements borrowed from The Art of War that serve not as literary embellishment but as an interpretive framework that parallels established biological evidence. Sun Tzu’s principle that the outcome of conflict is shaped long before direct confrontation resonates with the documented capacity of TDEs to precondition the microenvironment: they reorganize the extracellular matrix, manipulate cellular signaling networks, alter metabolic gradients, and modulate immune surveillance, thereby shaping the biological “terrain” to the tumor’s advantage. Similarly, their ability to disseminate regulatory information via miRNAs, surface ligands such as PD-L1, and stress-response proteins reflects a strategic distortion of host communication pathways, akin to controlling the flow of intelligence in warfare [13].

Accordingly, this review is structured into three major sections: (i) the molecular and immunological mechanisms by which TDEs promote tumor progression; (ii) strategies designed to halt exosomal spread, disrupt TDE biogenesis, or counteract their immunosuppressive functions; and (iii) engineering approaches that repurpose exosomes including CAR-T-derived vesicles and hybrid nanoplatforms as therapeutic carriers capable of overcoming current barriers in cancer treatment. This crucial perspective not only provides conceptual coherence but also frames exosomes as both agents of deception and instruments of counterattack within the evolving landscape of cancer therapy.

Exosomes are nanoscale EVs (30–200 nm) generated via the endosomal pathway through intraluminal vesicle formation within multivesicular bodies and subsequent fusion with the plasma membrane. They interact with recipient cells through receptor–ligand binding, membrane fusion, or endocytosis, delivering bioactive cargo that modulates intracellular signaling, gene expression, immune responses, angiogenesis, proliferation, and apoptosis. Structurally, exosomes are composed of a lipid bilayer enriched in tetraspanins (CD9, CD63, CD81), MHC molecules, adhesion proteins, heat shock proteins, and diverse nucleic acids and cytoskeletal components. TDEs promote immune evasion, therapy resistance, and premetastatic niche formation by reprogramming immune cells, whereas CAR-T–derived exosomes enhance targeted cytotoxicity, immune activation, and drug delivery while reducing CRS and improving central nervous system penetration. Despite their therapeutic promise, clinical translation is limited by challenges in large-scale production, vesicle heterogeneity, CAR-T exhaustion, TME adaptation, purification standardization, cost-effectiveness, and potential toxicities including neurotoxicity and GvHD.

Know your enemy, know yourself: Understanding exosome biology in health and disease

The traditional classification of EVs into exosomes (30–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (> 1 μm) remains useful, but recent work demonstrates substantial heterogeneity and overlap among these populations. Proteomic analyses show that EV identity is more accurately defined by biogenesis pathway, physical properties, molecular composition, and context dependent markers rather than size alone. The International Society for Extracellular Vesicles recommends the use of the general term extracellular vesicles with explicit reporting of size, density, biochemical markers, and, when possible, biogenetic origin. The MISEV2023 guidelines further advise against relying on size based categorization and emphasize transparent reporting of isolation procedures, marker selection, and functional assays to delineate EV subtypes. In addition, emerging nanoparticle classes such as exomeres and supermeres illustrate the complexity of the extracellular nanoparticle spectrum and reinforce the need for precise terminology and rigorous methodology ​ [2, 14–18]​.​ Exosomes are formed via inward budding of endosomal membranes, resulting in intraluminal vesicles (ILVs) that are secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane [19, 20]. Microvesicles originate through outward budding of the plasma membrane, while apoptotic bodies are released during the late stages of programmed cell death [21, 22].

In the human body, EVs contribute to essential physiological activities, such as metabolic regulation, gene expression, and immune signaling. However, they have also been linked to the development and progression of several diseases, including cancer, diabetes, autoimmune disorders, and neurodegenerative conditions [2, 23–26]. Their uptake by recipient cells occurs via receptor-ligand binding, direct membrane fusion, or endocytic pathways [21, 27]. Distribution and biological fate are influenced by physicochemical parameters such as size, surface composition, and environmental pH [28–30]​.

The cargo of exosomes includes transmembrane and cytosolic proteins such as tetraspanins, heat shock proteins, integrins as well as mRNAs, microRNAs, DNA fragments, and lipid species (Table 1). This composition determines their tropism, immunomodulatory capacity, and potential for therapeutic engineering. Exosomes circulate in biological fluids including blood, cerebrospinal fluid, and urine, and can cross barriers such as the blood brain barrier (BBB), supporting their application as non-invasive diagnostic tools [31]​. Compared to synthetic platforms like liposomes, exosomes exhibit superior biocompatibility, lower immunogenicity, and intrinsic targeting capability, reinforcing their relevance in both regenerative medicine and immuno-oncology [32, 33].

Table 1.

Exosome molecular inventory

Cargo type	Components	Function
Membrane and membrane-associated proteins	Tetraspanins (CD81, CD63, CD9, CD82, CD37); integrins; ICAM-1; MHC-II; syndecan; flotillin 1/2; IL-6R; EGFR; TCR; CAR; GPCR; PD-L1; TGF-β; ADAM proteases	Intrinsic enzyme-linked receptors or catalytic activity; immune signaling; immune evasion (PD-L1); cell–cell interaction
	Lipid bilayer: Phosphatidylserine, phosphatidylcholine, sphingomyelin, ceramides, and cholesterol.	Enables direct fusion with target cells, Lipid-mediated signaling, protects exosomal cargo from enzymatic degradation and hostile extracellular conditions.
Anchored or post-translationally modified proteins	GPI-anchored proteins (proteoglycans, glypican-1, DAF, MAC-IP); prenylated small GTPases; BASP-1; Src; ubiquitinated, SUMOylated, or phosphorylated proteins)	Regulation of exosome biogenesis; intracellular signaling; vesicular trafficking
Biogenesis-related proteins and chaperones	ESCRT-associated proteins: ALIX, TSG101, syntenin; chaperones: HSP70, HSP90, HSP20	Exosome biogenesis; protein folding and stability
RNA	mRNA fragments (≤ 1 kb), miRNAs, snRNAs, tRNA fragments, snoRNAs, mtRNAs, piRNAs, vtRNAs, Y RNAs, circRNAs, rRNA fragments, lncRNAs	Intercellular communication; disease biomarkers; gene regulation
DNA	Genomic dsDNA; ssDNA; mtDNA; viral DNA; histone-associated DNA	Potential cytoprotective role (removal of damaged DNA); liquid biopsy biomarkers; reflection of genomic instability

An army is forged in its barracks before marching to the battlefield: exosome biogenesis and structure

Exosomes can be generated through at least two pathways. The endocytic pathway is the most extensively characterized route of vesicular trafficking, primarily responsible for directing internalized cargo to lysosomal degradation but also capable of generating exosomes. Their formation involves the following steps: (a) invagination of the plasma membrane into an early secretory endosome; (b) formation of ILVs, also known as MVBs, through membrane budding driven by the incorporation of cargo into endosomes; (c) maturation of endosomes via acidification; and (d) extracellular release of ILVs as exosomes following fusion with the plasma membrane [34]. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery orchestrates ILVs and MVBs biogenesis through four core complexes (ESCRT-0, -I, -II, and -III) and accessory proteins including ALIX and VPS4. ESCRT-0 recognizes ubiquitinated cargo, ESCRT-I and -II induce membrane deformation and cargo sequestration, and ESCRT-III drives vesicle scission followed by VPS4-mediated disassembly, with disruption of these components altering exosome size, number, and cargo. Additional regulation is provided by the syndecan–syntenin–ALIX axis, stress-responsive ESCRT-III proteins, and GPR143-mediated recruitment of HRS, which modulates exosomal proteomic composition and integrin-enriched vesicle secretion [5, 35, 36]​.

Exosome biogenesis can also proceed through ESCRT-independent, lipid-driven mechanisms dominated by ceramide generated by neutral sphingomyelinase 2, which promotes intraluminal vesicle budding and cargo sorting independently of ESCRT components. This pathway is supported by microtubule-associated protein 1 A/1B-light chain 3 and RAB31, enhancing exosome secretion and epidermal growth factor receptor enrichment, particularly in cancer cells. While both ESCRT-dependent and ceramide-mediated pathways converge on exosome release, they differ in cargo specificity: ESCRT preferentially sorts ubiquitinated membrane and immune-regulatory proteins, whereas lipid-driven mechanisms enrich tetraspanins, proteolipid protein, and lipid-associated components. Together with RNA-binding protein–mediated RNA loading, this molecular divergence shapes exosomal composition, intercellular signaling, stress adaptation, and disease progression [36].

Exosomal cargo is determined by the lineage and activation state of the parent cell, while structural variation reflects gene expression and cellular topology [28]. In this sense, exosomes can be viewed as molecular avatars of their tumors, faithfully carrying and projecting the biological identity and programmed functions. At early stages of biogenesis, exosomal diversity is shaped by both intracellular pathways and systemic factors. Dietary lipids modify vesicle membrane composition, circadian rhythm regulates secretion timing, and hormonal signals such as estrogen influence microRNA cargo. Infections increase the release of immunomodulatory vesicles, while physical activity enhances regenerative signaling [3, 37].

This diversity has functional consequences. On the one hand, TDEs carry oncogenic and immunosuppressive molecules that facilitate immune evasion and disease progression. On the other hand, exosomes released by effector T cells display immune activating properties; for example, CD8⁺ T cell-derived exosomes contain perforins, granzymes, co-stimulatory receptors, and surface proteins such as CD3, CD8, and Fas ligand (FasL) [38]. These exosomes enhance low-affinity cytotoxic T cell responses, modulate PD-1/PD-L1 interactions, and contribute to anti-tumor immunity [39]. They have been implicated in treatment response, with uPAR-positive CD8 + exosomes serving as potential biomarkers in melanoma [40]. Also, exosomes from CD4⁺ T cells, particularly regulatory T cells (Tregs), mediate immune suppression [41, 42] and express CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and CD73, and support B-cell activation, M2 macrophage polarization, and CD8⁺ T cell inhibition [43]. Their release is modulated by Rab GTPases, particularly Rab27, and they frequently carry immunoregulatory microRNAs such as miR-150-5p and miR-142-3p, which may serve as non-invasive biomarkers in immunotherapy settings [44–47].

Within disorder, seek harmony: identifying the features of exosomes

Characterization of exosomes primarily focuses on the identification of molecular markers that distinguish exosomes subtypes and reflect their cellular origins. Tetraspanins such as CD9, CD63, and CD81 are widely recognized as exosome markers due to their involvement in the endosomal biogenesis pathway [22, 48]​. In contrast, proteins like annexin A1, annexin A2, ARRDC1, and α-actinin 4 are more specifically associated with microvesicle formation​ [21, 22, 49]. Certain molecules, including TSG101, syntenin-1, ALIX, ARF6, and VPS4, contribute to both exosome and microvesicle biogenesis, making them shared markers [22].

However, the small size of extracellular vesicles contributes to substantial variability in their membrane marker composition. For example, a 50 nm vesicle is estimated to accommodate only about 600 surface proteins, inherently limiting the range of detectable exosomal markers. Even exosomes originating from the same cell type can differ in marker presence and spatial distribution [50]. As a result, not all small EVs, often classified as exosomes, necessarily express classical tetraspanins, posing challenges for their consistent identification. To overcome these constraints, recent methodological advances have focused on single-vesicle and single-cell EV analysis. High-sensitivity flow cytometry, super-resolution and total internal reflection fluorescence microscopy, droplet-based digital immunoassays, Raman-based platforms, and microfluidic single-vesicle assays now enable multiplexed phenotyping of thousands of individual vesicles and, in some setups, the vesicle secretion profiles of single cells. These approaches begin to bridge the gap between physical characterization and functional heterogeneity, revealing EV subtypes that cannot be distinguished by classical bulk methods. Notably, Guo et al. employed a proximity barcoding assay for single-exosome proteomic profiling of plasma from colorectal cancer patients, identifying ITGB3⁺ and ITGAM⁺ exosome subpopulations with opposing effects on tumor progression and promising diagnostic and therapeutic value [51]. This type of single-exosome profiling exemplifies how methodological innovation can refine our understanding of exosome identity beyond conventional marker panels.

Isolation methods further shape how exosomes are defined and studied. Ultracentrifugation remains the most widely used technique, involving sequential centrifugation steps to eliminate cells, debris, and larger vesicles, followed by high-speed spins for exosome recovery [52]​. Density media like sucrose or iodixanol gradients are often used to enhance purity, with iodixanol being superior for distinguishing exosomes from retroviral particles [53]​. Polymer-based precipitation with polyethylene glycol (PEG) offers a convenient and scalable alternative, especially for large volumes, and was initially developed for virus isolation [54]​. Size-based methods, including ultrafiltration and size-exclusion chromatography (SEC), separate vesicles according to molecular weight or hydrodynamic diameter [55, 56]​, while immunoaffinity capture utilizes antibodies or ligands specific to exosomal surface proteins, enabling selective isolation from smaller sample volumes [57]. Commercial kits and microfluidic platforms have also emerged as rapid and user-friendly solutions for exosome recovery.

Despite this broad toolkit, no single isolation strategy simultaneously guarantees high purity, yield, and preservation of vesicle integrity. Differential ultracentrifugation and PEG precipitation can co-isolate protein aggregates, lipoproteins, and other nanoparticles; SEC may dilute samples and enrich only specific size fractions; and immunoaffinity capture is intrinsically biased toward vesicles bearing known markers, under-representing poorly characterized subpopulations [58]. These methodological trade-offs complicate cross-study comparisons and can profoundly influence the apparent molecular composition and bioactivity of “exosome” preparations, underscoring the need to interpret functional data in light of the underlying isolation protocol.

Once isolated, exosomes must be characterized to confirm their identity, morphology, and bioactive content. Structural features can be visualized through electron microscopy techniques such as TEM, SEM, or Cryo-EM, the latter preserving native architecture and avoiding fixation-related artifacts [59]​. Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) provide information on hydrodynamic size distribution and concentration, with NTA enabling recovery of samples post-analysis [59, 60]​. Protein-based methods, including Western blot, ELISA, and flow cytometry, are employed to assess specific surface and cargo proteins [61, 62]​. For storage, protocols such as cryopreservation at –80 °C, lyophilization, or spray drying help preserve exosomal integrity depending on downstream applications [49, 58, 63]. According to International Society for Extracellular Vesicles (ISEV) recommendations, vesicles should be maintained in PBS at −80 °C for long-term preservation, avoiding freeze–thaw cycles to prevent structural degradation [7, 64, 65]. Exosomes retain functional activity for several months under these conditions, and lyophilization offers the additional advantage of enabling stable room-temperature storage [66].

The enemy’s stratagems: Tumor-derived exosomes as instruments of deception

TDEs are secreted by malignant cells into the extracellular space. Although structurally like other EVs, TDEs are distinguished by their enrichment in tumor-specific proteins, lipids, glycans, and nucleic acids [67]. These components reflect the genomic, transcriptomic and proteomic profile of the tumor of origin and confer potent immunomodulatory properties. TDEs suppress anti-tumor immunity, modulate stromal cell function, and contribute to the formation of PMNs by reprogramming the phenotype and behavior of immune and non-immune cells​ [68]. Some examples are shown in Table 2.

Table 2.

Examples of TDEs promoting cell proliferation across cancer types

Neoplasm	Role	Reference
Glioblastoma	TDEs from glioblastomas have been shown to induce proliferation of the human glioblastoma cell line U87.	[6]
Chronic myeloid leukemia (CML)	TDEs from CML cells stimulate autocrine cell proliferation.	[69]
Gastric cancer	TDEs from gastric cancer cells stimulate autocrine proliferation.	[70]
Bladder cancer	TDEs induce cell proliferation via activation of the Akt and ERK pathways.	[71]
Melanoma	TDEs promote in vivo tumor growth and inhibit apoptosis in murine melanoma models	[72]
Prostate cancer	TDEs from hypoxic prostate cancer cells increase invasion and motility in human prostate cancer cells	[73]

The finest siege begins inside: how exosomes remodel the tumor microenvironment

The TME consists of blood vessels, ECM, fibroblasts, bone marrow–derived inflammatory cells, signaling molecules, and diverse immune cell populations [74]. Fibroblasts, endothelial cells, and infiltrating immune cells, the predominant stromal components, can interact with tumor cells through exosome-mediated signaling [75, 76].

TDEs suppress antitumor immunity through multiple mechanisms. In the case of CD8⁺ T lymphocytes, TDEs deliver inhibitory ligands that induce mitochondrial dysfunction, reduce expression of survival proteins including Bcl-2, Bcl-xL and cFLIP, and promote apoptosis [77]. In nasopharyngeal carcinoma, TDEs present PD-L1 to CD8⁺ T cells, reducing cytokine secretion, proliferation, and cytotoxicity, while in breast cancer, transforming growth factor-β (TGF-β) enhances PD-L1 loading, thereby suppressing T cell receptor (TCR) signaling [78]. In dendritic cells (DCs), TDEs downregulate antigen-processing machinery, major histocompatibility complex (MHC) class I and II molecules, and co-ligands receptors, resulting in defective maturation [79].

Exosomal microRNAs (miRNAs) and oncogenic proteins further induce DC anergy, impairing T and B lymphocyte activation and reducing natural killer (NK) cell cytotoxicity [80]; for example, exosomes carrying EGFR exon 19 deletion from Lewis lung carcinoma cells induce DC anergy [81]. In NK cells, TDEs decrease expression of the activating receptor NKG2D and reduce cytotoxicity, while bladder cancer exosomes enriched in miR-221-5p and miR-186-5p suppress perforin, granzyme B, and activating receptors [82]. In B lymphocytes, TDEs activate the adenosinergic pathway, increasing adenosine production, and inhibiting proliferation [83]. Within the TME, exosomal miR-1468-5p activates JAK2/STAT3 signaling in lymphatic endothelial cells by suppressing the SOCS1 promoter, thereby promoting immune tolerance [84].

TAMs, derived from bone marrow monocytes, infiltrate most tumors and undergo exosome-driven polarization toward either proinflammatory M1 or immunosuppressive, proangiogenic M2 phenotypes, characterized by anti-inflammatory cytokine secretion and ECM remodeling and induction of epithelial-to-mesenchymal transition (EMT) through TGF-beta secretion​ [85, 86]. Exosomal miRNAs contribute to this polarization through post-transcriptional regulation of target genes and activation of transcription factors such as peroxisome proliferator–activated receptor (PPAR), STAT3, and STAT6 [87]. Colorectal cancer–derived EMT exosomes carrying miR-106b downregulate programmed cell death protein 4 (PDCD4) and activate the PI3K/AKT mTOR pathway, favoring M2 differentiation [88]. Small cell lung cancer exosomes promote M2 conversion via NOD-like receptor family pyrin domain containing 6 (NLRP6)/NF-κB signaling [89]. Under hypoxic conditions, TDEs are enriched in immunomodulatory proteins and chemokines, which recruit macrophages and reinforce M2 polarization through oxidative phosphorylation and let-7a–mediated inhibition of insulin/AKT/mTOR signaling [90].

In parallel, TDEs expand Tregs and myeloid-derived suppressor cells (MDSCs), enhancing their suppressive functions through cytokine-mediated signaling and adenosine accumulation [91, 92]. TDEs also maintain CSC pluripotency and tumor heterogeneity by transferring stemness-associated signals [93]. Elevated TDEs concentrations in patient-derived fluids correlate with CD8⁺ T cell depletion, attenuated immunotherapy response, and disease progression, highlighting their relevance as prognostic and pharmacodynamic biomarkers [94].

Let distance be illusion, nearness your truth: exosomes in pre-metastatic niche formation

TDEs promote PMN formation through immune modulation, vascular remodeling, lymphangiogenesis, and organ-specific dissemination [95]. At the immune level, TDEs secrete miR-21 and miR-29a that bind to Toll-like receptor 7 (TLR7) in mice and Toll-like receptor 8 (TLR8) in humans, triggering proinflammatory cascades that facilitate metastasis [96]. In breast cancer, exosomal miR-200b-3p is internalized by alveolar epithelial type II cells, inducing CCL2, S100A8/9, MMP9, and CSF1, recruiting MDSCs and generating an inflammatory PMN [97].

To promote colonization, TDEs remodel vasculature by suppressing endothelial tight junction proteins, including zonula occludens-1 (ZO-1), occludin, and claudin-5. Glioblastoma stem-like cells exosomes enriched in vascular endothelial growth factor A (VEGF-A) enhance angiogenesis and permeability in brain endothelial cells (ECs) [98], while hepatocellular carcinoma (HCC) exosomal miRNAs (miR-638, miR-663a, miR-3648, miR-4258) downregulate ZO-1 and VE-cadherin, increasing intrahepatic permeability [99]. Melanoma exosomes containing urokinase plasminogen activator receptor (uPAR) are internalized by ECs, upregulating VE-cadherin, EGFR, and uPAR expression in ECs, thereby amplifying pro-angiogenic signaling [100]. Hypoxia intensifies these effects: In multiple myeloma, hypoxia-induced exosomal miR-135b targets hypoxia-inducible factor 1 (HIF-1) in ECs [101], while in lung cancer, exosomal miR-23a suppresses prolyl hydroxylases (PHD1/2), stabilizing HIF-1α and driving angiogenesis [102]. In the lymphatic niche, tumor-derived exosomes drive nodal metastasis by delivering oncogenic RNAs and chemokine receptors that activate ERK/AKT signaling, disrupt immune barriers, and promote lymphatic endothelial migration, tube formation, and invasion [103–106].

TDEs also dictate organotropism: colorectal cancer (CRC) commonly spreads to the liver and lung, whereas breast cancer preferentially metastasizes to the lung, bone, and brain. Exosomal α6β4 and α6β1 integrins promote lung metastasis, while αvβ5 integrin is associated with liver metastasis [107]. Clinically, circulating exosomal integrin β3 in lung cancer patients with brain metastases correlated with survival and intracranial control after whole-brain radiotherapy [108]. In gastric cancer, exosomal EGFR is transferred to liver stromal cells, activating the hepatocyte growth factor (HGF)/c-MET axis in cancer cells and fostering liver colonization [109]. In pancreatic ductal adenocarcinoma (PDAC), exosomal macrophage migration inhibitory factor (MIF) is internalized by Kupffer cells, initiating PMN formation and liver metastasis [110]. In clear cell renal cell carcinoma (ccRCC), CD103⁺ CSCs–derived exosomes drive lung-specific metastasis [111].

Importantly, in vivo models corroborate these molecular findings by defining the temporal dynamics of PMN formation. In breast cancer models, pulmonary PMN formation begins within two weeks of tumor inoculation and is established by week four; this process can be reproduced by intravenous exosome administration [112]. In pancreatic cancer, hepatic PMN forms following retro-orbital injection of TDEs every 48 h for three weeks, marked by fibronectin deposition and F4/80⁺ macrophage infiltration. In spontaneous pancreatic cancer models, Kupffer cell activation and TGF-β upregulation occur during pancreatic intraepithelial neoplasia stages, four to six weeks prior to malignant transformation [110]. Collectively, TDEs act as systemic vectors that reprogram distant tissues, integrating immune suppression, vascular and lymphatic remodeling, and integrin-mediated tropism to establish permissive PMNs.

Cut the flow, taint the source, and the spirit breaks: conventional methods to halt Exosomal spread

Targeting TDEs represents a promising approach to overcome immunosuppression and therapeutic resistance in cancer [113]. Effective strategies focus on disrupting exosome biogenesis, trafficking, and secretion through both ESCRT-dependent and ESCRT-independent pathways. In addition, bispecific antibodies such as CD73xEpCAM selectively block CD73-mediated adenosine production in exosomes from EpCAM-positive tumors, thereby restoring T cell function [114]. Some molecular strategies are shown in Table 3.

Table 3.

Pharmacologic and Molecular Strategies to inhibit TDEs. The targets involve multiple molecular pathways. The ESCRT mediates intraluminal vesicle formation within multivesicular bodies, while the syndecan–syntenin–ALIX axis provides an accessory route linking membrane proteoglycans to ESCRT machinery. Lipid handling is regulated by ABC transporters, including the ABCA3 subtype, which is essential for exosome export. Vacuolar H⁺-ATPase (V-ATPase) maintains acidic conditions required for vesicle release, and EGFR signaling enhances exosome biogenesis through oncogenic pathways. Additional regulators include farnesyltransferase, which anchors vesicular trafficking proteins to membranes; cholesterol synthesis, which sustains membrane curvature and rigidity; and syntenin, an adaptor protein that stabilizes protein– protein interactions during vesiclePharmacologic and Molecular Strategies to inhibit TDEs. The targets involve multiple molecular pathways. The ESCRT mediates intraluminal vesicle formation within multivesicular bodies, while the syndecan–syntenin–ALIX axis provides an accessory route linking membrane proteoglycans to ESCRT machinery. Lipid handling is regulated by ABC transporters, including the ABCA3 subtype, which is essential for exosome export. Vacuolar H⁺-ATPase (V-ATPase) maintains acidic conditions required for vesicle release, and EGFR signaling enhances exosome biogenesis through oncogenic pathways. Additional regulators include farnesyltransferase, which anchors vesicular trafficking proteins to membranes; cholesterol synthesis, which sustains membrane curvature and rigidity; and syntenin, an adaptor protein that stabilizes protein– protein interactions during vesicle formation

Target or Pathway	Mechanism of Action	Agents or Interventions	Notes	Reference
ESCRT-dependent pathway	Inhibits formation of intraluminal vesicles via disruption of ESCRT machinery	Sulfisoxazole; siRNA targeting HRS, STAM1, TSG101	Downregulates ALIX, VPS4B, Rab GTPases; reduces exosomal MHC class II release.	[115]
Syndecan-syntetin-ALIX axis	Disrupts ESCRT accesory signaling	Heparan sulfate analogs	Inhibits tumor cell proliferation and invasiveness.	[116]
ESCRT-independent (ceramide-dependent)	Inhibits neutral sphingomyelinase 2, blocking ceramide-mediated vesicle formation	GW4869; spiroepoxide; DPTIP	Reduces exosome release; DPTIP is more potent than GW4869; enhances anti–PD-L1 efficacy.	[117]
ABC transporter activity	Blocks lipid recycling essential for exosome formation and release	Glibenclamide	Inhibits ABC transporters involved in exosomal lipid handling.	[118]
ABCA3 transporter	Inhibits exosomal export; increases intracellular drug retention	Indomethacin	Enhances doxorubicin accumulation in tumor cells.	[119]
V-ATPase proton pump	Disrupts pH gradient necessary for TDE biogenesis and release	Lansoprazole, omeprazole	Targets pH regulation in the TME.	[115]
EGFR signaling	Inhibits EGFR-mediated pathways involved in exosome generation	Erlotinib	Blocks EGFR-dependent vesicle production.	[120]
Farnesyltransferase activity	Disrupts prenylation of small GTPases critical for vesicle trafficking	Manumycin A; tipifarnib	Tipifarnib modulates both ESCRT-dependent and independent pathways; downregulates PD-L1.	[121]
Cholesterol synthesis	Reduces membrane availability for vesicle formation	D-pantethine	Decreases cholesterol-dependent exosome production.	[122]
Cytoskeletal remodeling	Inhibits microvesicle release by targeting actin–myosin and membrane shedding pathways	Calpeptin (calpain inhibitor), Y27632 (ROCK inhibitor)	Enhances chemosensitivity; suppresses microvesicle formation.	[123]
Syntenin	Blocks protein-protein interactions essential for exosome release	RNA interference; C58 peptide	Inhibits syndecan–syntenin interaction; reduces migration and clonogenicity.	[124]
PD-L1 trafficking	Increases exosomal PD-L1 export	6J1 (triazine)	Alters PD-L1 localization.	[125]
	Decreases membrane-bound PD-L1	LSD1 knockdown; miR-16-5p overexpression	Restores T cell cytotoxicity	[126]
TDE secretion in drug-resistant tumors	Inhibits exosome production in drug-resistant cancer phenotypes	Ketoconazole	Effective in sunitinib- resistant renal carcinoma.	[127]

These strategies enhance immunotherapy by disrupting tumor-derived exosomal communication networks and increasingly integrate nanotechnology with cellular immunotherapy to overcome exosome-mediated immune suppression and resistance.

Turning the enemy’s messenger into a weapon: exosomes as therapeutic carriers

In parallel to efforts aimed at halting exosomal spread, a contrasting but highly innovative approach leverages exosomes themselves as delivery vehicles for cytostatic drugs, RNA molecules, or engineered protein cargo. TDEs and immune cell–derived exosomes are being repurposed for targeted drug transport, exploiting their intrinsic tropism, stability, and biocompatibility. There are several clinical studies using cytostatic drugs-loaded exosomes derived from tumor cells for targeted cancer therapy, as Table 4 shows:

Table 4.

Studies of engineered TDEs as carriers of cytostatic molecules

Feature	GlioblastomaExo‑PTX	ProstatePTX‑EVs	A549 Lungcancer cellsCisplatin-EVs
Source	Exosomes derived from tumor cells (U-87) were loaded witd PTX	Exosomes derived from Tumor cells (LNCaP/PC‑3) were loaded witd PTX	Exosomes derived from tde human breast cancer cell line MDA‑MB‑231 were loaded witd cisplatin to create a tderapeutic nanoplatform called CaCE
Loading methods	Incubation, sonication	Incubation and endocytic pathway	Endocytosis
Cellular uptake	Cell-targeting adhesion molecules like tetraspanins and integrins on the surface of exosomes	Endocytosis	The TDEs entered cancer cells mainly through membrane fusion and endocytic pathways
Efficacy	The results showed exosomes in combination with PTX killed approx. 40% of tumor cells versus control group (approx. 8%)	Enhanced prostate cell killing	This exosome delivery system improved the survival rate in LLC tumor-bearing mice compared to co-administration of cisplatin and blocking antibodies
Reference	[128]	[129]	[130]

CAR-derived exosome-based nanoparticles represent a synergistic chemoimmunotherapeutic platform, exemplified by the CAR-EDC system-CAR-macrophage exosomes enriched with CXCL10 and conjugated to SN-38 which enables targeted drug delivery while simultaneously activating CD8⁺ T cells and promoting M1 macrophage polarization for enhanced antitumor efficacy [131]. A wide range of exosomes are in trials for solid tumors: paclitaxel (PTX) loaded exosomes, CAR exosomes, and RNA loaded exosomes are being tested across lung, breast, pancreatic, and other cancers [132]. In essence, this strategy targets the tumor from within using chemical agents while simultaneously mobilizing the immune system from the outside through immune activation.

One of the most formidable biological barriers is the BBB that limits the uptake of many therapies to treat glioblastoma and other brain tumors. The implementation of nanotechnology in CAR-T cell therapy can enhance the effectiveness of these cancer treatments while reducing adverse side effects [133, 134]. The enhanced permeability and retention (EPR) effect simultaneously promotes the passive accumulation of nanoparticles within tumor tissues, as they extravasate through the leaky tumor vasculature into the interstitial space [135]. Engineered TDEs loaded with rapamycin efficiently cross the BBB, enhance intratumoral drug accumulation, suppress VEGF-driven angiogenesis, and significantly inhibit glioblastoma growth [136]. Beyond their role as drug carriers, exosomes derived from CAR T cells offer a complementary mechanism of action: rather than transporting external agents, they infiltrate tumors and deliver intrinsic cytotoxic molecules, directly disrupting tumor growth [137]. For example, exosomes from anti-HER2 CAR-T cells containing cytotoxic proteins like granzyme B and perforin, enabling direct antitumor effects within the brain TME [11].

Shadows triumph where light misleads: cellular armies versus Exosomal tactics

Exosome-based therapies are emerging as cell-free alternatives to adoptive cellular therapies (ACT), enabling the delivery of functional biomolecules while avoiding the severe toxicities of whole-cell approaches. Although ACTs such as CAR-T, TCR-T, and TIL therapies achieve high response rates—CD19 CAR-T reaching ~67% complete remission in relapsed acute lymphoblastic leukemia and 82% objective response in lymphoma [138, 139], and BCMA CAR-T achieving 73–84.6% overall response in multiple myeloma [140, 141], they are frequently limited by CRS, ICANS, GvHD, and poor penetration into the immunosuppressive TME [142, 143]. In this context, CAR-T cell–derived exosomes represent a promising cell-free strategy with potent antitumor activity mediated by receptor interaction, membrane fusion, and endocytic uptake [144–147]. These vesicles deliver a cytotoxic arsenal including perforin, granzymes (particularly granzyme B), FasL, Apo2L/TRAIL, and lysosomal enzymes, which trigger apoptosis through death receptor–mediated DISC formation, caspase-8 and caspase-3 activation, and Bid-dependent mitochondrial permeabilization [145, 148] (Fig. 2). Although their effector mechanisms are largely antigen-independent, CAR expression confers tumor-specific targeting, enabling effective cytotoxicity even in tumors with low or heterogeneous antigen expression and potentially supporting transient antitumor immunity with reduced recurrence risk [115].

Fig. 2.

Structural and Molecular Composition of Conventional T Lymphocyte-Derived Exosomes vs. CAR-T Cell-Derived Exosomes

However, unlike memory CAR T cells, exosome-based therapies lack long-term persistence, and current evidence does not demonstrate that CAR T exosomes can prevent tumor reintroduction in the context of autologous CAR T cell therapy, thus requiring repeated infusions [149, 150]. CAR T exosomes are generated after antigen stimulation and can be isolated at high yield using GMP compatible procedures, with recovery rates of approximately 63% ± 5.7% for CD19 CAR–positive vesicles [151]. CAR T exosomes persist in circulation for up to two years after infusion, indicating sustained activity of their parental cells. Their protein cargo is enriched for granzyme B, perforin, and surface CAR molecules, which are absent in conventional T cell exosomes. HER2 CAR T exosomes contain at least 20-fold higher granzyme B levels than exosomes from unstimulated T cells (p < 0.001), and each CD19 CAR–positive exosome carries approximately 0.0005 µg of protein [152]. CAR T exosomes mediate antigen-specific cytotoxicity in vitro, producing dose-dependent killing of CD19-positive leukemia cells and inducing apoptosis in HER2-positive targets with caspase-3/7 activation comparable to CAR T cells but slower kinetics. In vivo, CAR T exosomes inhibit tumor growth with lower toxicity than CAR T cells and without cytokine release syndrome. Quantitatively, circulating CD19 CAR–positive vesicles eliminated 20.3% ± 3.8% of Raji cells and 37.8% ± 7.0% of SUP-B15 cells at 0.015 µg of vesicular protein per target cell, and mesothelin-targeted CAR exosomes reduced 4T1-MSLN cell viability to below 60% after 12 h [153]. Modeling estimates that circulating CD19 CAR–positive vesicles could eliminate 2.7 × 10¹¹ to 1.67 × 10¹³ tumor cells per day. By contrast, exosomes from non-CAR T cells showed negligible cytotoxicity, with killing rates of 0.71% ± 2.19% in CD19-positive cell lines and 2.35% ± 1.91% in primary CD19-positive cells [151]. These quantitative differences position CAR T exosomes as a potent and specific cell-free platform for targeted cancer therapy, with substantially greater cytotoxic activity and a more favorable safety profile than conventional T cell exosomes.

The figure highlights the most significant differences in CAR-T cell–derived exosomes, which are as follows: First, the absence of the PD-1 receptor, which allows these exosomes to evade neutralization by tumor cells upon binding to PD-L1. Second, they exhibit a higher abundance of granzymes and perforins, enhancing their tumor-destructive potential. Third and finally, CAR-T–derived exosomes uniquely express the CAR receptor, enabling precise tumor targeting with high specificity. It also shows key differences in lipid composition (e.g., ceramides, cholesterol, LBPA), membrane-associated proteins (e.g., annexins, flottins, RABs, ARFs), and cargo molecules (e.g., MHC-II/TCR-BCR complexes, adhesion molecules like LFA-1 and ICAM) between conventional T lymphocyte-derived exosomes and CAR-T cell-derived exosomes. Notable markers include CD3, CD4/CD8, CD63, CD81, CD57 and immune checkpoint molecules (e.g., PD-1). Abbreviations: LBPA (Lysobisphosphatidic Acid), MHC-II (Major Histocompatibility Complex Class II), TCR (T-Cell Receptor), BCR (B-Cell Receptor), LFA-1 (Lymphocyte Function-Associated Antigen-1), ICAM (Intercellular Adhesion Molecule), MFGE8 (Milk Fat Globule-EGF Factor 8), LAMP-1 (Lysosomal-Associated Membrane Protein-1), TSPAN-6 (Tetraspanin-6), PD-1 (Programmed Death-1), ARF (ADP-Ribosylation Factor), RAB (Ras-Associated Binding Protein). Created with BioRender.com.

Cellular immunotherapies exhibit immunogenicity and toxicity profiles that depend on cell origin and in vivo expansion: while allogeneic products can induce host–versus–graft reactions, currently approved autologous CAR T cells trigger severe toxicities such as CRS and ICANS as a consequence of antigen-driven activation and clonal expansion rather than host rejection. Despite their high efficacy in hematologic malignancies, CAR T cells show limited activity in solid tumors due to antigen heterogeneity, immunosuppressive TME, and off-tumor toxicity risk, prompting strategies such as improved antigen selection, accelerated manufacturing, frontline deployment, lymphodepletion, and locoregional delivery [154–156]. In contrast, exosomes display intrinsically low immunogenicity and toxicity owing to their endogenous nanoscale nature, immune-evasive lipid bilayer, protected cargo, and ability to penetrate biological barriers [157–161]. Unlike cellular therapies, exosomes do not proliferate or differentiate in vivo, eliminating risks of uncontrolled expansion and cytokine surges [162–164]. Preclinical models demonstrate that CAR T cell–derived exosomes retain potent antileukemic cytotoxicity without inducing systemic cytokine elevations or weight loss, and early clinical studies of exosome-based therapies report favorable safety profiles without immune hyperactivation, positioning exosomes as a safer, cell-free immunotherapeutic alternative [11, 151, 165]. Exosomes are non-replicating, acellular particles that lack the machinery to expand or secrete pro-inflammatory cytokines, which account for their favorable safety profile compared to ACT. Although some exosomes express MHC-peptide complexes and costimulatory molecules, their antigen presentation is often indirect, relying on uptake by host antigen-presenting cells (APCs). Without prolonged immune synapses or autonomous activation, exosomes trigger limited T cell expansion and minimal systemic inflammation [166].

Unlike CAR-T cells, CAR-T cell–derived exosomes lack PD-1, rendering their cytotoxic activity resistant to PD-L1–mediated suppression within the TME. These properties enable exosomes to achieve effective intratumoral delivery with reduced off-target toxicity, supporting their emerging role as versatile drug carriers, immunotherapeutic agents, and even cancer vaccines, in contrast to CAR-T cells that rely on physical migration and are frequently constrained by stromal barriers [167–176]. This functional versatility has enabled the extension of exosome-based strategies beyond direct cytotoxicity toward antigen delivery and cancer vaccination. In this context, exosomes derived from induced pluripotent stem cells (iPSC) function as carriers of tumor-associated antigens, overcoming the tumorigenicity and logistical limitations of direct iPSC administration [177]. A leading strategy involves loading iPSC-derived exosomes onto dendritic cells to generate vaccines, which in preclinical melanoma models induces efficient DC maturation, enhances T cell expansion 3.3–3.5-fold over controls, increases CD8⁺ T-cell infiltration, activates NK cells, reduces regulatory T cells in the tumor microenvironment, and translates into both prophylactic and therapeutic benefits, with survival rates reaching 70% in preventive and 60% in established tumor settings [178].

Exosomes offer a strategic advantage in ACT by enhancing antigen presentation. DC loaded with TDEs represents a complementary immunotherapeutic strategy by presenting a broad repertoire of tumor-associated antigens and inducing cytotoxic T lymphocyte responses. Recent models in glioma, leukemia, and hepatocellular carcinoma have shown that DC and TDE vaccination not only reshapes the immune landscape enhancing IFN-γ and reducing IL-10/TGF- β but also promotes long lasting antitumor T cell responses. This immune activation may synergize with CAR-T cell therapies by improving persistence and functionality. Moreover, combining CAR-T cell derived exosomes with DC vaccination platforms, particularly those leveraging engineered TDEs or iPSC-derived exosomes, could address antigen heterogeneity and tumor relapse [179, 180]. Summary of key comparisons are shown in Table 5. From a strategic standpoint, early intervention is essential: exosome-based approaches are most effective when implemented before tumor escape programs become fully established. Initiating these therapies during the early phases of microenvironmental remodeling increases the likelihood of durable responses and long-term disease control, reflecting the broader principle that complex challenges are best addressed before they fully manifest. See Fig. 3.

Table 5.

Summary of key comparisons ACT versus exosomal therapy

Aspect	Cellular Therapy	Exosomal therapy	Exosome advantage
Active agent	Living cells	Cell-derived vesicles	No replicating cells; no risk of secondary T cell neoplasms, in contrast to the potential tumorigenic events reported with cellular CAR T products.
Mechanism of action	Direct cell killing via antigen recognition; in vivo cytokine release	Delivery of cytotoxic proteins/miRNAs; antigen presentation; drug/RNA delivery	Versatile cargo delivery; can combine mechanisms
Tumor targeting	Good antigen specificity; poor solid-tumor penetration	Can be engineered with targeting ligands; cross barriers	Better tissue penetration; engineerable tropism
Safety/Side effects	Risk of CRS, neurotoxicity, on-target/off-tumor effects	Generally safe; minimal systemic toxicity observed	Far lower toxicity; no CRS or GvHD
Regulatory status	Established	Nascent	Off-the-shelf potential once standardized
Scalability/production	Complex autologous culture	Can use cell lines/bioreactors; scalable purification (GMP processes emerging)	Potential for large-scale batch production
Cost	Extremely high per patient	Expected lower (mass-produced; no patient-specific labs)	Economies of scale; reuse of a standard product
Storage/Handling	Requires cryopreservation of live cells; limited shelf-life	Cryo- and lyophilization-stable; long shelf-life	Easier transport/storage; room-temp stability possible
Clinical evidence	Proven and approved in hematologic malignancies	Early-stage trials; some small studies of DC-exosomes show safety and disease stabilization; ongoing trials for drug delivery	Emerging but promising

Fig. 3.

A complex interplay between Sun Tzu’s “Art of War” and oncologic treatment applications of exosomes

War strategies employed by tumor-derived exosomes: (Upper side, from left to right) (1) Avatars acting as spies: Tumor-derived exosomes (TDEs) act as stealthy emissaries of malignancy, transferring oncogenic and immunosuppressive cargo that reprograms immune and stromal cells in distant niches, preparing the ground for immune evasion and metastasis. (2) False appearance: TDEs are camouflaged in a double lipid membrane, shielding their contents from immune detection and proteolytic degradation, while presenting a composition that mimics non-threatening or physiological signals. (3) Battlefield preparation: By reshaping the tumor microenvironment, inducing Treg and MDSC proliferation, and polarizing macrophages to the M2 phenotype, TDEs convert immune battlefields into sanctuaries of tolerance. (4) Weapon delivery: TDEs are equipped with immunosuppressive molecules, miRNAs, cytokines, and resistance-mediating proteins such as PD-L1 and ABC transporters, disabling effector cells and countering therapy. (5) Molecular avatar: Their proteomic and genomic content mirrors their tumor of origin, embodying a molecular extension of the malignant self, and creating decoys for immune responses

CAR-T derived exosomes as protective warriors: (Bottom side, from left to right): (1) Subdue without fighting: Synthetic or CAR-T-derived exosomes block tumor strategies such as angiogenesis and immune suppression while minimizing systemic toxicity, reshaping the immune landscape without provoking collateral damage. (2) Deception: By mimicking tumor-derived vesicles, therapeutic exosomes infiltrate malignant tissues undetected, delivering cytotoxic payloads such as perforin and granzyme B directly into the tumor core.

3. Know your enemy: Engineered CAR-T exosomes exploit molecular mimicry to engage tumoral receptors, enhancing selective targeting and cytotoxicity without PD-1-mediated inhibition.4. Balk the enemy’s plans: CAR-T exosomes are being designed to interfere with exosome biogenesis (e.g., via inhibition of the ESCRT pathway or ceramide synthesis), halting TDE production at its source. 5. Subtleness: Their nanoscale size (~80 nm) allows CAR T exosomes to evade immune surveillance and deeply penetrate solid tumors, unlike bulkier cellular therapies. 6. Using baits: By resembling TDEs or incorporating ligands for tumor receptors, therapeutic exosomes act as Trojan horses—delivering cytotoxic or gene-editing cargo (e.g., CRISPR/Cas9) to malignant cells or redirecting TDEs to safe elimination zones. Abbreviations: TDEs – Tumor-Derived Exosomes. CAR-T – Chimeric Antigen Receptor T cell. TME – Tumor Microenvironment. Treg – Regulatory T cell. MDSC – -Derived Suppressor Cell. PD-L1 – Programmed Death Ligand 1. PD-1 – Programmed Death 1. ABC transporters – ATP-Binding Cassette transporters. ESCRT – Endosomal Sorting Complex Required for Transport. CRISPR/Cas9 – Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9. miRNA – MicroRNA. Created with BioRender.com.

Fire devours; smoke deceives: engineering exosomes and nanotechnology in cancer therapy

Although CAR-T therapy is highly effective, its efficacy is limited by the TME, antigen heterogeneity and loss, and physical barriers such as hypoxia, acidosis, and extracellular matrix density. These constraints have driven the development of interdisciplinary strategies that integrate cellular therapies with exosomes and synthetic nanoparticles, which offer biocompatible, targeted platforms for delivering tumor antigens, co-stimulatory signals, and immunomodulatory cargos [133, 181, 182]. Together with polymeric and lipid-based nanocarriers, these engineered systems enhance CAR-based therapeutic efficacy while reducing systemic toxicity, establishing a convergent nanotechnology–immunotherapy framework for improved antitumor responses [183, 184]. (Fig. 4). A systematic comparison of the diverse strategies employed for engineering these and other therapeutic platforms is provided in Table 6.

Fig. 4.

Exosomes, Nanotechnology, and Therapeutic Strategies in Cancer Treatment

Table 6.

Systematic comparison of therapeutic engineering platforms

Strategy	Key Advantages	Main Disadvantages	Primary Applications	Technical Maturity	Example(s)	References
CAR-T derived exosomes	Reduced toxicity vs. cellular therapy; tumor targeting	Limited cargo capacity; scalability; loss of multifunctionality; batch variability	Overcoming antigen heterogeneity; reversing immunosuppression; gene delivery	Preclinical	RN7SL1-loaded CAR-T exosomes for CD19+/CD19 − tumors	[187]
Exosome-liposome hybrids	Enhanced drug loading; improved stability/circulation; dual targeting	Complex manufacturing; regulatory hurdles; higher cost; storage stability	Solid tumors with barriers; combination chemo-immunotherapy	Advanced Preclinical	LipCExo@PTX with anti-MSLN/PD-L1 scFv for lung cancer	[188]
Exosome-MSN hybrids	High payload; controlled/sustained release; imaging; tumor mimicry	Immunogenicity of inorganic components; rapid clearance; complex synthesis	Breast cancer; chemo-phototherapy; cytokine delivery	Preclinical	DOX-ICG loaded ID@EMSNs; IL-2/TGF-β co-loaded MSNs	[189]
Engineered TDEs for radiotherapy	Natural tumor homing; radiosensitizer delivery; amplify ROS	May promote resistance; cargo changes post-radiation; bystander effects	Radiosensitization; overcoming radioresistance	Early Preclinical	Manganese/FeS2 nanomaterial-loaded TDEs	[190]
MHC-engineered TDEs	Activation of CD8+/CD4 + T cells; broad immune response; antigen presentation	Requires genetic modification; MHC-II engineering complexity; autoimmunity risk	Therapeutic vaccination; personalized immunotherapy	Preclinical	MHC-I/II dual-presenting TDEs	[191, 192]

The figure illustrates a pyramid in the background that symbolizes the progressive layers of knowledge gained over time. At its foundation lies the well-documented mechanisms of action of various exosome types, including their unique characteristics and potential therapeutic targets, which are intrinsically linked to their parent cells or lymphocytes. Building upon this foundational understanding, the mid-section highlights the therapeutic techniques currently in use, which leverage these mechanistic insights. At the pinnacle, the pyramid points toward the future of exosome research, where scientific efforts must address persistent therapeutic barriers and challenges to unlock new clinical applications.

It also illustrates the role of exosomes (e.g., tumor-derived exosomes, TDEs; CAR-T cell-derived exosomes) and nanotechnology in overcoming challenges such as tumor heterogeneity, immune evasion (e.g., PD-L1/PD-1 axis), and drug delivery. Key strategies include exosome engineering (e.g., CRISPR/Cas9 gene editing), combination therapies (e.g., radiotherapy, dendritic cell vaccines), and hybrid systems (e.g., liposome-exosome hybrids, mesoporous silica nanoparticles). Abbreviations: CAR (Chimeric Antigen Receptor), PD-1 (Programmed Death-1), PD-L1 (Programmed Death-Ligand 1), TGF-β (Transforming Growth Factor-beta), MHC-I (Major Histocompatibility Complex Class I), VEGF-B (Vascular Endothelial Growth Factor-B), IFN-γ (Interferon-gamma), IL-10 (Interleukin-10), RN7SL1 (Immunostimulatory RNA), BBB (Blood-Brain Barrier), EPR (enhanced permeability and retention effect). Created with BioRender.com.

Although no phase III trials of exosomes implementation in the treatment of solid tumors have been publicly reported, exosome‑related technologies have already achieved meaningful clinical translation in diagnostics across multiple malignancies, including lung, pancreatic, colorectal, ovarian, glioma, and hepatocellular cancers, as Table 6 illustrates the different strategies. A Phase II clinical program (iEXPLORE, NCT03608631) has provided clinical evidence supporting engineered exosomes as delivery systems for KRASG12D-specific siRNA in metastatic pancreatic ductal adenocarcinoma, demonstrating excellent tolerability and safety in Phase Ia/Ib following systemic administration of iExoKrasG12D derived from allogeneic bone marrow mesenchymal stromal cells [9, 185]. Preclinical studies further show synergistic antitumor activity when iExoKrasG12D is combined with anti–CTLA-4 therapy, supporting its ongoing evaluation in combinatorial immunotherapy strategies.

Additionally, these technologies have already demonstrated real-world effectiveness in diagnostics. A notable example is the ExoDxTM Prostate (EPI) liquid biopsy for prostate cancer, which improves the detection of disease and reduces unnecessary biopsies compared with conventional approaches. This success highlights the translational potential of exosome-based platforms and their future applications, as ongoing research continues to establish their relevance as a promising therapeutic strategy [186].

This table summarizes the major exosome-based strategies, highlighting their key advantages, disadvantages, primary applications, technical maturity, and representative examples. Most approaches remain in preclinical or early translational stages, with scalability, regulatory, and safety challenges as common barriers to clinical adoption.

Where thought does not reach, let your form emerge: CAR-T exosomes as specialized warriors

Although CAR-T–derived exosomes retain intrinsic tumor-targeting specificity, they can be further engineered to enhance safety and efficacy [183], including the delivery of CRISPR/Cas9 systems against oncogenic drivers such as MYC. In Raji xenograft models, CAR-T exosomes loaded with MYC-targeting sgRNA/Cas9 plasmids effectively suppressed tumor growth, highlighting their therapeutic potential. Compared with viral vectors, exosomes offer lower immunogenicity, reduced carcinogenic risk, and improved cargo encapsulation efficiency [193]. Given that MYC is dysregulated in approximately 30% of human cancers and is strongly associated with poor prognosis, exosome-mediated gene editing represents a promising strategy for targeting this critical oncogenic pathway [12].

mRNA-engineered exosomes offer a virus-free alternative to traditional lentiviral CAR-T manufacturing, allowing fast, temporary, and well-controlled CAR expression. The CAR-encoding mRNA can be loaded into exosomes from inside the producing cells using plasmid transfection or RNA-binding proteins (such as MS2P, HuR, and L7Ae), or from outside using techniques like electroporation, nanoporational loading, cationic lipids, or liposome fusion, which can increase loading efficiency up to 1000-fold. These methods enable efficient transfer of CAR mRNA into T cells, reducing dependence on viral vectors and even allowing CAR-T generation directly in vivo [194, 195]. In addition, CAR-T–derived exosomes can be combined with RNA vaccines such as RN7SL1 (Ova-19-7SL), an endogenous RNA that activates innate immune sensors (RIG-I and MDA5) to boost CAR-T expansion and function. In mouse models with mixed CD19⁺ and CD19⁻ lung tumors, RN7SL1-loaded CAR-T exosomes enhanced CAR-T activity, activated other immune cells in the TME, reversed immunosuppression, and significantly reduced tumor growth [187]. Together, these results show that multifunctional CAR-T exosome engineering can help overcome antigen heterogeneity, strengthen both innate and adaptive immunity, and improve CAR-T therapy in solid tumors while potentially counteracting tumor-driven immune resistance [196].

The humble Reed sways to the unseen wind: Exosome–liposome and exosome–nanoparticle hybrids as engineered weapons in cancer therapy

Inorganic nanoparticles can be engineered for high drug payload, controlled release, and imaging capabilities, but may have issues with immunogenicity and rapid clearance. Hybridizing exosomes with inorganic nanoparticles can improve drug encapsulation efficiency, stability, and targeted delivery, while reducing off-target effects and systemic toxicity [189].

Hybrid nanovesicles generated by fusing CAR-T–derived exosomes with lung-targeted liposomes represent a highly effective multifunctional therapeutic platform. In murine lung cancer models, bispecific CAR-T exosomes loaded with paclitaxel (Lip-CExo@PTX) achieved > 95% lung accumulation and induced rapid tumor regression. Incorporation of an anti-mesothelin (MSLN) scFv enabled selective delivery of PTX together with cytotoxic effectors such as granzyme B and perforin to MSLN-positive tumors, while a PD-L1–blocking scFv prevented immune checkpoint–mediated inhibition, thereby enhancing immunogenic cell death and antitumor immunity. In metastatic CT-26 lung cancer, Lip-CExo@PTX significantly prolonged survival, demonstrating that engineered exosome–liposome hybrids can function as precisely coordinated, high-efficacy chemoimmunotherapeutic systems [188, 193].

Also, mesoporous silica nanoparticles (MSNs) have emerged as versatile drug-delivery platforms. Tian et al. developed an exosome-coated MSN system (ID@E-MSNs) using 4T1-derived exosomes to mimic tumor cells, enhancing tumor accumulation and enabling synergistic chemo-photothermal therapy through co-delivery of doxorubicin and indocyanine green. In parallel, Liu et al. designed MSNs for controlled co-release of IL-2 and TGF-β, which strengthened antitumor immunity by promoting terminal effector T-cell differentiation and reshaping the TME through vascular normalization and increased T-cell infiltration [197].

Different methods regarding drug delivery with EV have been explored. Engineering approaches generally fall into two categories: surface modification to enhance targeting specificity and cargo modification to increase biological activity. By combining the intrinsic targeting capacity, immunomodulatory properties, and biocompatibility of exosomes with the tunable functionalities of liposomes or other nanoparticles, hybrid systems can achieve therapeutic effects that surpass those of natural vesicles alone. These exosome nanoparticle hybrids not only enhance cargo delivery and cellular uptake but also enable multifunctional platforms combining imaging, controlled release, and immunomodulation in a single vehicle. To generate such hybrid systems, several surface-engineering strategies have been developed, including lipid–PEG–lipid insertion, glycan modification, covalent bioconjugation, and click chemistry-based conjugation [198].

Lipid–PEG–lipid insertion has enabled the formation of hybrid nanocarriers that fuse long-circulating liposomes with exosomes, creating platforms with improved pharmacokinetics for cancer therapy [199]. Incorporation of PEGylated lipids into exosomal membranes extends circulation time while preserving exosome biochemistry and biophysical integrity, and PEG-mediated fusion has become a widely used strategy to enhance exosome drug-delivery capacity [200, 201]. Because PEG-driven fusion remains limited, newer approaches employ Tat-PEG-lipids whose membrane-associating peptide (YGRKKRRQRRR) and lipid tail particularly myristoyl (C14) and dodecanoyl (C12) promote more efficient exosome–liposome fusion [202]. Although concerns persist regarding potential toxicity or immunogenicity of PEG and related polymers, these issues are increasingly viewed as engineering challenges, prompting systematic investigation into how membrane modifications influence exosomal “self” identity and surface protein composition [203]. Collectively, growing in vitro and in vivo evidence supports the refinement of hybrid exosomes as complex, tunable nanocarriers with expanding therapeutic promise.

Surface insertion and lipid anchoring provide modular strategies to functionalize exosomal membranes while preserving their native architecture. In lipid-PEG–ligand post-insertion, functionalized PEG-lipids carrying maleimide, biotin, or azide groups are introduced into liposomes and then transferred to exosomes, enabling the addition of targeting ligands, stealth coatings, or stimuli-responsive elements with minimal structural disruption. Hydrophobic tail-anchored ligands such as cholesterol- or long-alkyl–conjugated peptides integrate spontaneously into the exosomal bilayer, offering rapid functionalization although they may influence membrane packing and protein mobility [198].

Covalent bioconjugation and click chemistry enable precise, stable, and site-specific exosome surface modification, typically by attaching targeting ligands to surface protein amines through copper-catalyzed or strain-promoted azide–alkyne cycloadditions [204]; using this strategy, Jia et al. enhanced glioma targeting via CuAAC [205]. More traditional chemistries, including maleimide–thiol and EDC/NHS coupling, offer scalable routes for modifying abundant amine and thiol groups, though they risk heterogeneous conjugation. Enzymatic ligation methods such as sortase a provide true site specificity while preserving membrane integrity, but at the cost of greater technical complexity [198].

Combining methods such as extrusion-based fusion, PEG-lipid insertion, and final covalent ligand attachment can significantly enhance hybrid exosome performance, as long as the steps are applied in the correct order to preserve membrane integrity and previously added components. These engineered exosome–nanoparticle hybrids surpass natural exosomes by offering adjustable pharmacokinetics, multimodal cargo delivery, and controlled therapeutic activation, making them a flexible platform for personalized therapies that integrate targeted cytotoxicity, immune modulation, and real-time imaging.

Like water, shaping nothing, becoming formless: tumor exosomes in radiotherapy

Recent studies highlight the dual role of TDE in radiotherapy: Not only do they mediate radioresistance and contribute to radiation induced bystander effects, but they also present a promising therapeutic target for improving treatment efficacy. Yang et al. demonstrated that irradiation significantly increases exosome biogenesis and release, and modulates their cargo such as DNA fragments and miRNAs, that can shuttle DNA damage signals and survival promoting messages to both irradiated and adjacent non-irradiated cells. These insights suggest that disrupting exosomes-mediated communication could enhance radiosensitivity or selectively target tumor cells. Building on this, engineered exosomes can be loaded with radiosensitizers or immune adjuvants to counteract TDE-mediated survival pathways, directly linking radiotherapy strategies to exosome-based therapeutic engineering. The TDEs can be engineered to carry radiosensitizer, such as manganese-based nanomaterials, which amplify reactive oxygen species (ROS) production upon irradiation, thereby increasing DNA damage in tumor cells. Other strategies include loading exosomes with sodium iodide symporters to improve uptake or combining exosomes with immune adjuvants to reverse local immunosuppression [115, 190].

At the same time, more recent findings have revealed specific molecular mechanisms by which exosomes contribute to radioresistance and how these can be therapeutically reversed. For instance, in breast cancer, irradiation has been shown to co-induce CD47 and HER2 expression, promoting immune evasion and tumor proliferation [206]. The blockade of both targets results in reduced clonogenic survival and enhanced macrophage-mediated clearance of tumor cells. In parallel, nanotechnology-based interventions have utilized exosome membranes to deliver FeS2-based oxidative stress amplifiers, which selectively target tumors and enhance low-dose radiotherapy by increasing hydroxyl radical generation. These advanced strategies further underscore the dual potential of TDE as mediators of resistance and as tools to sensitize tumors to radiotherapy through targeted delivery systems [207].

Radiation therapy can induce profound alterations in the TME, largely through epigenetic remodeling. Off-target radiation effects may promote aberrant DNA methylation, while exosomes released from irradiated cells often contain modified molecular cargo that can be horizontally transferred to non-irradiated cells. Several exosomal microRNAs have been shown to dampen the activity of immune effector populations such as NK cells thereby facilitating tumor progression, adaptive responses to radiation, and late recurrences long after the initial therapeutic exposure. These vesicles act as molecular proxies, extending the influence of irradiated cells beyond their physical boundaries, subtly shaping the biological terrain [208].

Radiation exposure also triggers extensive histone modifications, including ubiquitination, phosphorylation, acetylation, and methylation, ultimately altering chromatin structure and gene expression. Such epigenetic disruptions contribute to ionizing-radiation resistance, and exosomes can disseminate these regulatory cues to neighboring, non-irradiated cells, allowing the adaptive phenotype to propagate through the TME much like a silent strategic maneuver [209].

Non-coding RNAs within exosomes released from irradiated cells can further promote EMT through activation of β-catenin, Notch, and Smad2/3 signaling pathways, enhancing the malignant behavior of recipient cells. These exosomal long non-coding RNAs also hold potential as biomarkers for prognostic surveillance and post-irradiation therapeutic monitoring [210, 211].

Hypoxic tumor cells—intrinsically more radioresistant than oxygenated cells—exhibit an increased release of exosomes and significant alterations in their cargo. Hypoxia-derived exosomes can stimulate angiogenesis through shuttling of molecules such as miR-135b, which modulates components of the HIF-1 axis [212]. Furthermore, these vesicles can reduce radiation-induced apoptosis and enhance DNA-damage repair, conferring radioresistance via transfer of miR-340-5p to recipient normoxic cells. In this way, hypoxic cells deploy exosomal signals that reinforce survival pathways and recalibrate the microenvironment, enabling the tumor to endure therapeutic pressures with a quiet but deliberate precision [213].

TDEs promote radioresistance through defined mechanisms, including increased DNA repair signaling, transfer of survival-associated microRNAs, metabolic reprogramming, and immune evasion driven by CD47 or HER2. These pathways constitute actionable engineering targets for exosome and nanotechnology platforms. Exosomes derived from CAR T cells can be engineered to deliver CRISPR Cas9 cargos that disrupt genes associated with radiation tolerance, such as MYC or CD47, or to transport immunostimulatory RNAs, including RN7SL1, to counteract the immunosuppressive environment intensified by radiotherapy. Hybrid systems that combine exosomes with liposomes or inorganic nanoparticles can be designed to co-deliver radiosensitizers, ROS amplifiers, or scFv that block immune checkpoints within irradiated tumors, this approach involves the dual action of inhibiting TDE-mediated pro-survival signaling while exploiting the enhanced tumor permeability resulting from radiation exposure. In this framework, engineered exosomes and synthetic hybrids function as targeted countermeasures against the molecular circuits underlying TDE-driven radioresistance.

The purest honey yields but drops: limitations of exosomes as therapeutic agents

Several cellular therapies are FDA-approved, such as CAR-T for leukemia/lymphoma or TIL for melanoma, with defined regulatory pathways [214, 215]. In contrast, exosome therapies are largely experimental. Currently there is an increasing number of clinical trials investigating exosome-based therapies in solid tumors, but most are still in Phase I/II [216]. Exosome-based products do not yet have established regulatory guidelines, and agencies are still discussing how to classify and standardize them [161, 217]. Challenges include heterogeneity of vesicle populations and lack of standardized isolation. Recent work is addressing these technical gaps by introducing tangential flow filtration and developing stable, well-characterized producer cell lines for clinical-grade exosome production [218, 219].

Although some manufacturing steps may become more cost-efficient, regulatory barriers remain substantial. Because exosome therapies are acellular and non-replicative, they fall outside the ATMP classification yet are often regulated under frameworks designed for live-cell products in the United States and European Union. This mismatch imposes disproportionate requirements for characterization, GMP manufacturing, and preclinical testing. In the United States, for example, the FDA regulates exosome products as biologics under the Public Health Service Act and has issued safety warnings against unapproved exosome-based interventions marketed directly to patients [220].

In Europe, the EMA’s Committee for Advanced Therapies (CAT) assesses exosome products individually, and they are not classified as ATMPs unless they contain genetic modifications [221]. Nonetheless, manufacturers are often required to meet ATMP-level GMP, biodistribution, immunogenicity, and sterility standards, increasing cost and slowing clinical development. This regulatory ambiguity has also encouraged a gray market of unregulated exosome products marketed as cosmetics or wellness supplements, prompting safety warnings from agencies such as the FDA.

Accordingly, regulators and scientific societies are advocating for exosome-specific guidelines that reflect their acellular nature and establish standardized manufacturing and quality criteria. Initiatives toward such frameworks are underway in the United States, Japan, and international consortia, but exosome therapies still remain in an early and evolving regulatory stage compared with the more established pathways governing cellular therapies (see Table 8) [7, 222, 223].

Table 8.

Comparative overview of regulatory landscapes for EV-based therapeutics

Region/Agency	Regulatory Classification	Existing Documents	Key Gaps/Challenges
United States (FDA/CBER)	No EV-specific classification. EV-based products are currently assessed by analogy to biologics, cell- and gene-based therapies.	-General CBER regulatory science initiatives for complex and cell-derived products -No EV-specific guidance.	-Absence of EV-specific quality, potency, identity, and manufacturing guidance. -Developers must extrapolate from cell/gene therapy rules, which do not fully address EV heterogeneity. -Lack of validated potency assays.
European Union (EMA/CAT)	Case-by-case assessment. Some EV-based products may fall under “biological medicinal products” per CAT criteria.	-EMA/CAT Reflection Paper on EV-based Medicinal Products. -General ATMP guidance.	-Reflection paper provides considerations but not prescriptive requirements. -Need for consensus on minimal characterization, CQAs, and release criteria.
Japan (PMDA)	Actively evaluating EV-based therapies; some products classified under regenerative medicine products.	-PMDA considerations for cell-derived and EV-derived products.	-Early-stage framework; limited detail on manufacturing controls or potency assays.
Global scientific standardization (ISEV – MISEV2023/24)	Not a regulatory classification.	- MISEV guidelines for reporting and characterization	- Reporting-only framework; does not define manufacturing or regulatory pathways.

Personalized cell therapies are costly and operationally complex: autologous CAR-T products require individualized cell collection, 2–3 weeks of expansion, strict sterility control, and extensive coordination, resulting in 17–22 days manufacturing times and costs of $300,000–$500,000 USD per patient [224, 225]. Allogeneic platforms aim to reduce this burden but still face rejection risks and demanding bioprocessing requirements.

In contrast, exosomes can be generated from standardized producer cell lines, enabling an off-the-shelf model [219]. Scalable GMP production using bioreactors, tangential flow filtration, and chromatography has already been demonstrated, and exosomes remain stable for months even after lyophilization greatly simplifying storage, distribution, and overall logistics compared with living cell therapies [66].

Despite their remarkable potential in drug delivery and immunomodulation, exosomes face significant challenges that hinder their clinical translation [226]. One of the foremost limitations lies in the standardization of isolation and purification techniques. Conventional methods such as ultracentrifugation, size exclusion chromatography, and precipitation kits often result in heterogeneous populations with varying purity and yield, leading to inconsistencies in downstream applications [7]. Moreover, cross-contamination with other EVs or soluble proteins can alter their therapeutic efficacy and safety profile [227, 228]. Without robust, reproducible, and scalable isolation methods, the reliability of exosome-based products remains questionable.

Another critical barrier is the lack of control over exosomal cargo loading and retention. Both passive and active loading methods like electroporation, sonication, or chemical transfection exhibit low efficiency, potential structural damage to the vesicles, and often result in suboptimal therapeutic payloads [228]. Furthermore, the cargo naturally enclosed during biogenesis is highly variable and depends on the physiological state and source of the parent cell, raising concerns about batch-to-batch variability [227, 229]. This unpredictability not only compromises therapeutic consistency but also introduces safety concerns, especially if the exosomes inadvertently carry oncogenic or pro-inflammatory molecules [229].

Biodistribution and targeting specificity remain unresolved issues in exosome therapy [230]. Although engineering approaches like ligand modification or surface protein fusion have been explored to enhance targeting, these methods are still in early development and lack validation in clinical settings [227]. Additionally, pharmacokinetics and biodistribution data are scarce, which complicates the design of optimal dosing regimens and safety evaluations [228].

To overcome these limitations and enable clinical scalability, future research must prioritize a deeper understanding of exosome biogenesis and cargo-sorting mechanisms, (see Table 7). Elucidating the molecular machinery that governs the selective packaging, retention, and release of bioactive molecules into EVs would significantly reduce cargo variability and improve therapeutic consistency [227, 228]. Additionally, advances in bioengineering strategies, including genetic or chemical surface modifications, could enhance tissue-specific targeting and mitigate off-target accumulation [227]. Efforts should also focus on developing GMP-compliant, scalable manufacturing platforms and robust in vivo tracking tools to ensure reproducibility and safety [228, 229]. Advances in bioreactor-based systems, artificial exosome synthesis, and emerging isolation technologies such as microfluidics and tangential flow filtration (TFF) are addressing issues of scalability and reproducibility [231]. Artificial intelligence (AI), including machine learning and deep learning, has demonstrated value in exosome biomarker discovery, diagnostic precision, and therapeutic optimization [232, 233]. Algorithms trained on diverse datasets can reduce bias and improve clinical generalizability, while AI model exosome TME interactions and in silico simulations of hybrid exosome design enabling precise ligand selection and optimized therapeutic cargo loading [234, 235].

Table 7.

Critical bottlenecks in exosome therapeutics and emerging solutions

Bottleneck	Description	Emerging Solutions
Cargo Sorting and Loading	Inefficient, poorly understood mechanisms for selective cargo incorporation	Genetic engineering, advanced loading technologies [236]
In Vivo Tropism and Biodistribution	Unpredictable targeting, off target effects	Surface functionalization, ligand engineering [237]
Isolation and Purification	Low yield, heterogeneity, lack of standardization	Microfluidics, improved protocols, GMP compliance [238]
Regulatory Ambiguity	Fragmented global standards, unclear safety/efficacy requirements	Harmonization efforts, collaborative frameworks [239]
Preclinical vs. Clinical Potency	Discrepancy between animals and human outcomes	Enhanced targeting, robust clinical trial design [240]
Large-Scale Production and Stability	Scalability, storage, and reproducibility issues	Bioprocess optimization, AI-driven manufacturing [241]
Safety and Immunogenicity	Risk of immune response, infectious transmission	Advanced characterization, rigorous safety testing [242]

Although the U.S. FDA has not yet issued EV-specific regulatory guidance, recent scientific and technological advances offer practical paths to reduce current gaps [243]. A deeper mechanistic understanding and increasingly standardized engineering methods help define Critical Quality Attributes (CQAs) by extrapolating from existing biologic and cell therapy frameworks, while harmonized characterization standards such as ISEV-MISEV2023/2024 improve comparability across studies [67, 244]. In parallel, scalable manufacturing platforms including tangential flow filtration, microfluidics, and improved potency and release assays are becoming aligned with expectations outlined by EMA/CAT and CBER for complex cell-derived biologics, even in the absence of dedicated EV pathways. Together, these developments illustrate how the integration of nanotechnology, bioengineering, and AI is accelerating the clinical translation of exosome-based therapies and advancing their potential in next-generation cancer treatment (See Table 8). Our conceptual findings align with the principal research domains identified through bibliometric analysis of recent publications (Fig. 5), which highlight exosomes as central nodes connecting tumor biology, immunotherapy, and nanotherapeutics. This convergence underscores the relevance of the strategies discussed, as they parallel the most active directions currently pursued by the scientific community.

Fig. 5.

Co-occurrence network of author keywords related to exosome research in oncology

The network was generated from n = 456 indexed publications (2020–2025) using VOSviewer (version 1.6.20). Each node represents an author keyword, with node size proportional to its occurrence frequency and edges reflecting co-occurrence strength (association strength normalization). Colors denote clusters identified by modularity-based clustering, revealing four major thematic domains: (i) exosome biology and tumor microenvironment (red), (ii) drug delivery and nanotherapeutics (green), (iii) immunotherapy and immune modulation (blue), and (iv) experimental models and translational studies (yellow/purple). The figure highlights the centrality of “exosome(s)” and “tumor microenvironment” as research hubs, with growing convergence toward therapeutic applications and translational models.

Conclusion

Exosomes have emerged as both adversaries and allies in cancer biology and immunotherapy. TDEs, acting as molecular proxies of malignancy, orchestrate immune suppression, metastasis, and therapeutic resistance. Yet the same architecture that enables them to broadcast disruptive signals can be redirected toward therapeutic purpose. CAR-T–derived exosomes embody this turning point. As cell-free vectors, they bypass several constraints of conventional CAR-T therapy: their nanoscale dimensions allow efficient navigation of the dense tumor stroma, the absence of checkpoint receptors limits susceptibility to immunosuppressive cues, and their acellular nature minimizes risks of uncontrolled expansion or cytokine storm, enabling safer and more predictable dosing.

Advances in bioengineering and nanotechnology further refine these vesicles into deliberate therapeutic instruments. Engineered exosomes capable of delivering CRISPR/Cas9 systems, chemotherapeutics, or immunomodulatory RNAs illustrate the breadth of this platform. Hybrid constructs that integrate exosomes with liposomes or inorganic nanoparticles enhance tumor selectivity, reshape the TME, and amplify anti-tumor responses. In this sense, engineered exosomes operate as disciplined extensions of their cellular origin avatars crafted to act with the stealth, precision, and adaptability required to navigate cancer’s shifting terrain.

Challenges, however, remain substantial. Isolation and characterization methods lack standardization, often producing heterogeneous preparations that limit reproducibility and scalability. Cargo loading remains inconsistent, and control over biodistribution and pharmacokinetics is still incomplete. Regulatory frameworks provide no dedicated pathway for exosome-based products, forcing developers to adapt guidelines built for fundamentally different therapeutics. Addressing these gaps will require tightly integrated progress across molecular biology, bioprocess engineering, analytics, and regulatory science.

Despite these obstacles, the direction of the field is unmistakable. Exosomes are emerging as modular, programmable, and scalable therapeutic agents capable of adapting to the complexities of cancer. If TDEs represent the quiet advance of malignancy, CAR-T exosomes and engineered exosomes stand as their deliberate counterparts—subtle, strategic, and designed to restore equilibrium within the host. As mechanistic insight deepens and technological capability expands, these cell-free vectors may transition from passive biomarkers to decisive actors in cancer therapy. Echoing Sun Tzu’s principle that victory begins long before the battlefield is visible, the groundwork for outmaneuvering cancer is already being laid.

Abbreviations

ABC transporters

ATP-Binding Cassette transporters

ACT

Adoptive Cell Therapy

ADC

Antibody–Drug Conjugate

ADAM

A Disintegrin and Metalloprotease

ALIX

Apoptosis-Linked Gene 2-Interacting Protein X

APC

Antigen-Presenting Cell

ARF

ADP-Ribosylation Factor

ATMP

Advanced Therapy Medicinal Product

ATPase

Adenosine Triphosphatase

BBB

Blood–Brain Barrier

BCR

B Cell Receptor

BCMA

B-Cell Maturation Antigen

BiTE

Bispecific T-cell Engager

BMSC

Bone Marrow–Derived Mesenchymal Stem Cell

CAR

Chimeric Antigen Receptor

CAR-T

Chimeric Antigen Receptor T cell

CAR-M

Chimeric Antigen Receptor Macrophage

CAT (EMA)

Committee for Advanced Therapies

circRNA

Circular RNA

CNS

Central Nervous System

CQAs

Critical Quality Attributes

CRISPR/Cas9

Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9

CRS

Cytokine Release Syndrome

CuAAC

Copper-Catalyzed Azide–Alkyne Cycloaddition

CXCL10

C-X-C Motif Chemokine Ligand 10

DAF

Decay Accelerating Factor

DC

Dendritic Cell

DISC

Death-Inducing Signaling Complex

DLS

Dynamic Light Scattering

DNA

Deoxyribonucleic Acid

DOX

Doxorubicin

DR4/DR5

Death Receptor 4 / Death Receptor 5

DPTIP

Diperodon Tip Inhibitor of Neutral Sphingomyelinase 2

dsDNA/ssDNA – Double-Stranded DNA / Single-Stranded DNA

ECM – Extracellular Matrix

EDC/NHS

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide / NHydroxysuccinimide

EGFR

Epidermal Growth Factor Receptor

ELISA

Enzyme-Linked Immunosorbent Assay

EMA

European Medicines Agency

ESCRT

Endosomal Sorting Complex Required for Transport

EV/EVs

Extracellular Vesicle(s)

FasL

Fas Ligand

FDA

U.S. Food and Drug Administration

GPI

Glycosylphosphatidylinositol

GPCR

G Protein-Coupled Receptor

GvHD

Graft-versus-Host Disease

GZMB

Granzyme B

HLA/MHC

Human Leukocyte Antigen / Major Histocompatibility Complex

HRS

Hepatocyte Growth Factor–Regulated Tyrosine Kinase Substrate

HSPs

Heat Shock Proteins (Hsp60, Hsp70, Hsp90)

HuR/MS2P/L7Ae

RNA-binding proteins used for mRNA loading

ICAM-1

Intercellular Adhesion Molecule 1

ICD

Immunogenic Cell Death

ICG

Indocyanine Green

ICANS

Immune Effector Cell–Associated Neurotoxicity Syndrome

IFN-γ

Interferon Gamma

IL-2

Interleukin 2

ILVs

Intraluminal Vesicles

iPSC

Induced Pluripotent Stem Cell

iExoKrasG12D

Exosomes Loaded with siRNA Against KRAS G12D

LBPA

Lysobisphosphatidic Acid

LFA-1

Lymphocyte Function–Associated Antigen 1

lncRNA

Long Noncoding RNA

Lip-CExo@PTX

Liposome–CAR-T Exosome Hybrid Nanoparticles with

Paclitaxel

LOX

Lysyl Oxidase

MAC-IP

Membrane Attack Complex–Inhibitory Protein

MDA5

Melanoma Differentiation–Associated Protein 5

MDSC

Myeloid-Derived Suppressor Cell

miRNA

MicroRNA

mRNA

Messenger RNA

MSLN

Mesothelin

MSNs

Mesoporous Silica Nanoparticles

MSC

Mesenchymal Stem Cell

mtDNA/mtRNA

Mitochondrial DNA / Mitochondrial RNA

MVBs

Multivesicular Bodies

MYC

v-myc Avian Myelocytomatosis Viral Oncogene

NK

Natural Killer Cell

NTA

Nanoparticle Tracking Analysis

ORR

Overall Response Rate

PBS

Phosphate-Buffered Saline

PD-1/PD-L1

Programmed Death 1 Programmed Death-Ligand 1

PEG

Polyethylene Glycol

PiRNA

PIWI-Interacting RNA

PTX

Paclitaxel

RAB

Ras-Related Proteins in Vesicle Trafficking

RIG-I

Retinoic Acid–Inducible Gene I

RNA-seq

RNA Sequencing

RN7SL1

7SL1 Noncoding RNA Used as Immune Adjuvant

rRNA

Ribosomal RNA

scFv

Single-Chain Variable Fragment

SEC

Size-Exclusion Chromatography

sgRNA

Single Guide RNA

siRNA

Small Interfering RNA

snRNA/snoRNA

Small Nuclear RNA / Small Nucleolar RNA

SPAAC

Strain-Promoted Alkyne–Azide Cycloaddition

SRC

Proto-Oncogene Tyrosine-Protein Kinase Src

SUMOylation

Small Ubiquitin-like Modifier Conjugation

TAA

Tumor-Associated Antigen

Tat-PEG-Lipids

Tat Peptide–Conjugated PEGylated Lipids

TCR

T Cell Receptor

TDE/TDEs

Tumor-Derived Exosome(s)

TFF

Tangential Flow Filtration

TGF-β

Transforming Growth Factor Beta

TIL

Tumor-Infiltrating Lymphocyte

TLRs

Toll-Like Receptors

TME

Tumor Microenvironment

Treg

Regulatory T cell

TSG101

Tumor Susceptibility Gene 101

tRNA/vtRNA/YRNA

Transfer RNA / Vault RNA / Y RNA

V-ATPase

Vacuolar-Type H+ ATPase

VEGF

Vascular Endothelial Growth Factor

VLP

Virus-Like Particle

VPS4

Vacuolar Protein Sorting 4

Author contributions

JB: Visualization, Writing– original draft, Conceptualization, Methodology, Supervision, Project administration, Investigation, Validation, Writing– review & editing, Data curation. SC-S: Conceptualization, Validation, Supervision, Methodology, Project administration, Investigation, Writing– review & editing, Resources, Visualization, Formal analysis, Writing– original draft. YC: Investigation, Writing- review & editing. JB-V: Investigation, Writing- review & editing. LR-S: Investigation, Writing- review & editing. MG-M: Visualization, review & editing. ME-S: Visualization, review & editing. JV: Writing- review & editing. AT-P: Review & editing. JG-R: Review & editing. CC: Review & editing. JO-G: Review & editing. AL: Revise & editing.

Funding

Not applicable.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.