Apoptotic Vesicles: Therapeutic Mechanisms and Critical Issues

Abstract

Apoptosis is the most prominent mode of programmed cell death and is necessary for the maintenance of tissue homeostasis. During cell apoptosis, a distinctive population of extracellular vesicles is generated, termed apoptotic vesicles (apoVs). ApoVs inherit a variety of biological molecules such as proteins, RNAs, nuclear components, lipids, and gasotransmitters from their parent cells. ApoVs have shown promising therapeutic potential for inflammation, tumors, immune disorders, and tissue regeneration. In addition, apoVs can be used as drug carriers, vaccine development, and disease diagnosis. Recently, apoVs have been used in clinical trials to treat a variety of diseases, such as temporomandibular joint osteoarthritis and the regeneration of functional alveolar bone. Here, we review the history of apoV research, current preclinical and clinical studies, and the potential issues of apoV application.

Introduction

Apoptosis is the dominant mode of programmed cell death (PCD) and plays an important role in the maintenance of tissue and organ homeostasis (Liu et al. 2018). It has been reported that more than 50 billion cells undergo apoptosis each day in an adult (Fu et al. 2023). Apoptotic disorders can cause a variety of diseases such as autoimmune disease, aging, and cancer (Ou et al. 2024). Apoptosis is a caspase-dependent process, which is regulated by the extrinsic pathway (death receptor–mediated pathway) and the intrinsic pathway (mitochondria-mediated pathway) (Xu et al. 2019). The end metabolite products of apoptosis are apoptotic vesicles (apoVs) (Ou et al. 2024).

ApoVs are a special type of extracellular vesicles, consisting of cellular materials in a spherical lipid bilayer (Zhang et al. 2022). ApoVs can inherit various components from parent apoptotic cells (Zhou et al. 2022). Compared with exosomes, apoVs have specific markers, such as integrin alpha-5 and calreticulin (Zhang et al. 2022). Recently, emerging evidence showed that apoVs contribute to a variety of physiological and pathophysiological events (Lin et al. 2023). In addition, apoVs are also used for drug carriers, vaccine development, and disease diagnosis (Zou et al. 2023). This review aims to introduce cutting-edge knowledge regarding the biological characteristics, therapeutic potential, and ongoing clinical trials of apoVs (Fig. 1).

Figure 1.

Timeline of major discoveries of apoptotic vesicles (apoVs) and current trends. In the past 5 decades, the important role of apoVs was recognized in both physiological and pathological contexts. Furthermore, the clinical application of apoVs was gradually carried out.

Biological Characteristics and Biogenesis of ApoVs

ApoVs are derived from apoptotic cells, initially named apoptosis bodies (apoBDs) (Kerr et al. 1972). Subsequently, apoptotic microvesicles (apoMVs) and apoptotic exosomes (apoExos) with smaller-sized vesicles derived from apoptotic cells were isolated and identified (Kakarla et al. 2020). Currently, apoVs can be classified into 3 subtypes: apoBDs, apoMVs, and apoExos (Table 1). Among apoV subtypes, the size and markers are different, and whether they play different physiological roles is also worthy of further exploration.

Table 1.

Properties and Differences among ApoV Subtypes.

Subtype (Size)	ApoBD b (1 to 5 μm)	ApoMV b (0.1 to 1 μm)	ApoExo b (30 to 150 nm)
Induction method	1. Serum starvation induction 2. Staurosporine treatment 3. Ultraviolet irradiation 4. Apoptosis receptor treatment (such as anti-Fas treatment) 5. 4-nitroquinoline 1-oxide treatment 6. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) treatment
Separation method	1. Sequential centrifugation with filtration: 300g for 10 min, supernatant is filtered with 5 µm and 1 µm filters, then 3,000g for 10 min 2. Fluorescence-activated cell sorting (FACS): 3,000g for 10 min, pellet is stained with Annexin V and TO-PRO-3 for 10 min. Then, the FSClowAnnexin Vintermediate/highTO-PRO-3intermediate/high vesicles are isolated via FACS	Sequential centrifugation with filtration: 300g for 10 min, 3,000g for 10 min, supernatant is filtered with 1 µm filters, then 20,000g for 30 min	Sequential centrifugation with filtration: 300g for 10 min, 3,000g for 10 min, 20,000g for 30 min, supernatant is filtered with 0.2 µm filters, then 120,000g for 60 min
Markers	Common markers: CD63, CD9, CD81, Annexin V
	Specific markers: Annexin V, C1q, TSP-1, GM130, TSP, C3b, calreticulin, calnexin, Bip/GRP78	Specific markers a : ARF6, VCAMP3, HSP70, integrin	Specific markers a : LAMP2, LG3, S1P1/3
Purity assessment	1. Transmission electron microscopy (TEM): structure (as described below) and size 2. Nano–flow cytometry and Western blot: specific markers 3. Nanoparticle tracking analysis: size, distribution, and Zeta potential
	Chromatin condensation and/or marginalization within the diameter range of 1 to 5 µm	Predominantly round and oval-shaped, membrane-bound structures of variable size and electron density within the diameter range of 0.1 to 1 µm	Cup-shaped or hemispherical with one side concave within the diameter range of 30 to 150 nm

^aSpecific markers are still lacking to distinguish between apoMV and apoExo.

^bApoV, apoptotic vesicle; ApoBD, apoptotic body; ApoMV, apoptotic microvesicle; ApoExo, apoptotic exosome.

The formation of apoBDs is tightly controlled by caspase activities, and it can be divided into 3 stages (Fig. 2A): plasma membrane blebbing, formation of thin membrane protrusion, and distinct apoBD generation (Santavanond et al. 2021). During membrane protrusions, apoptopodia resembles a “beads-on-a-string” structure, and the “beads” are frequently sheared off the “string” to form apoBDs, which is a previously unknown mechanism and negatively regulated by caspase-activated pannexin 1 channel (Santavanond et al. 2021).

Figure 2.

Biological characteristics and biogenesis of apoptotic vesicles (apoVs). (A) The biogenesis of apoVs during cell apoptosis. Apoptotic exosomes (apoExos) through exocytosis of multivesicular body (MVB) and apoptotic microvesicles (apoMVs) through plasma membrane shedding. Apoptotic body (apoBD) generation contains membrane blebbing, membrane protrusion including apoptopodia, beaded apoptopodia, and distinctly formation. (B) ApoVs makers contain CD9, CD63, and CD81 and apoptotic-specific makers PS and C1q as well as origin-specific markers. ApoVs contain various cellular components including proteins, nucleic acids, and lipid profiles, and they depend on the parent cells.

As for apoMVs and apoExos, the formation mechanisms are similar to that of extracellular vesicles (EVs) produced by viable cells, which are first formed as intracellular multivesicular bodies through the invagination of the plasma membrane and endosomal system but are specifically secreted during apoptosis (Kakarla et al. 2020). Notably, the generation of apoExos may not be dependent on the endosomal-sorting complexes but on S1P-S1PR signaling (Park et al. 2018), and the activation of caspase-3 may be important for the release of apoExos (Dieude et al. 2015).

The membrane markers of apoVs are partly overlapped with exosomes, such as CD9, CD63, and CD81 (Fig. 2B). In addition, apoVs can inherit substances from their parental apoptotic cells and express some specific markers, which contributes to the heterogeneity of apoVs. For example, mesenchymal stem cell–derived apoVs (MSC-apoVs) inherit various parent cell proteins, including factor-related apoptosis (Fas), integrin alpha-5, syntaxin-4, caveolin-1, caveolae associated protein 1 (Cavin 1), Lamin B1, calnexin, and calreticulin (Zhang et al. 2022). Owing to the various biogenesis mechanisms, cell origins, and diverse contexts of apoVs, the molecular characteristics and biological functions remain to be further investigated.

Therapeutic Function of ApoVs

The finding of therapeutic function of apoVs can be traced back to 2006. ApoVs from Mycobacterium tuberculosis–infected macrophages stimulate CD8⁺ T cells to produce interferon-γ, which induces protection from M. tuberculosis infection (Winau et al. 2006). The immunomodulatory function of apoVs was then gradually realized (Kakarla et al. 2020). We demonstrate that MSC-apoVs can improve the osteopenia phenotype in mice with apoptosis deficiency via regulation of MSC function, suggesting the therapeutic function of MSC-apoVs in the mouse model (Liu et al. 2018). The therapeutic functions of apoVs is currently being further explored, including anti-inflammation, antitumor, and tissue regeneration functions (Fig. 3).

Figure 3.

The therapeutic roles of apoptotic vesicles (apoVs) in anti-inflammation, antitumor, and tissue regeneration. ApoVs show promising therapeutic applications, such as anti-inflammation, antitumor, and tissue regeneration. The mechanisms of apoVs in disease treatment include the regulation of cell proliferation, cell differentiation, and immune microenvironment. Neutrophils undergo a unique form of cell death (NETosis) that generates neutrophil extracellular traps (NETs), which consist of citrullinated histone H3 (H3Cit) and myeloperoxidase (MPO). ECs, endothelial cells; MM, multiple myeloma; PLGA/pDA, poly(lactic-co-glycolic acid) scaffolds modified with polydopamine (pDA); Treg cells, regulatory T cells.

Anti-inflammation

Inflammation can be classified into acute inflammatory disease and chronic inflammatory disease. Sepsis is defined as acute life-threatening immune dysregulation with no optimal therapeutics. The immune dysregulation involves a special neutrophil PCD called NETosis, which forms excessive neutrophil extracellular traps (NETs) to amplify the inflammatory response (Boufenzer et al. 2021). In the treatment of sepsis, apoVs derived from apoptotic MSCs can alleviate the inflammatory response and reduce mortality. Systemic injected MSC-apoVs accumulate in the bone marrow of septic mice via electrostatic charge interactions with positively charged NETs and then inhibit the overproduction of NETs, offering insight into the therapeutic potential of apoVs in acute inflammatory disease (Ou et al. 2022).

ApoVs also exert anti–chronic inflammatory effects by modulating the immune microenvironment. Macrophages are the central mediators of innate immune responses, and the balance between proinflammatory M1-type macrophages and anti-inflammatory M2-type macrophages plays an important role in the progress of inflammation (Huang et al. 2022). A previous study showed that MSC-apoVs inhibit macrophages from polarizing into the M1 type via the AMPK/SIRT1/NF-κB pathway and downregulate the secretion of tumor necrosis factor–α from M1 macrophages to alleviate the progress of chronic periodontitis (Ye et al. 2022). MSC-apoVs express calreticulin, one of the “eat-me” signals, which promotes the efferocytosis of apoVs by liver macrophages and facilitates the polarization to M2-type macrophages to alleviate type 2 diabetes (Zheng et al. 2021). In addition, MSC-apoVs can suppress effector T-cell activation by reducing the activity of T-cell receptors and then relieve the inflammatory response and ameliorate murine lupus (Wang et al. 2023). In addition to MSC-apoVs, apoVs derived from other cells also have anti-inflammatory functions. ApoVs derived from both macrophages and osteoclasts can inhibit synovial inflammation and bone damage by inducing macrophage polarization to the M2 type (Li et al. 2024).

Dendritic cells (DCs) play an antigen-presenting role in the immune system and express high levels of major histocompatibility complex (MHC) class II (Worbs et al. 2017). It has been reported that apoVs from ultraviolet (UV)–irradiated peripheral blood mononuclear cells weaken the antigen-presenting function of DCs by downregulating the expression of MHC II. The reduction of MHC II is related to the inflammatory response and disease progression of systemic lupus erythematosus (Fehr et al. 2013). In addition, apoVs extracted from the UV-irradiated β-cell line NIT-1 are phagocytosed by immature DCs, which reduces the secretion of proinflammatory cytokines in DCs to prevent type 1 diabetes (Marin-Gallen et al. 2010; Yu et al. 2023).

In summary, apoV-mediated immunoregulation and efferocytosis show promising efficacy for inflammatory diseases (Table 2). A recent study indicated that the impaired clearance of apoptotic cells is a relevant factor leading to inflammation (Santavanond et al. 2021). The intracellular contents released by apoptotic cells can trigger an inflammatory response. ApoVs derived from apoptotic cells can carry the intracellular contents without causing obvious inflammatory responses (Santavanond et al. 2021). Therefore, promoting the formation of apoVs may help to alleviate the inflammatory response caused by the impaired clearance of apoptotic cells.

Table 2.

Therapeutic Functions of ApoVs.

	Parental Cells	Induction Ways	Mechanism of Function	Recipient Cells	Disease	Reference
Anti-inflammation	BMMSCs	STS	ApoVs are phagocytosed by macrophages then inhibit osteoclast differentiation by reducing TNF-α secretion of proinflammatory macrophages	Macrophages	Periodontists	Ye et al. 2022
	BMMSCs	STS	Positively charged NETs attract with negatively charged apoVs via electrostatic charge interactions, which switches neutrophils NETosis to apoptosis via the apoV-Fas ligand (FasL)–activated Fas pathway	Neutrophil	Sepsis	Ou et al. 2022
	BMMSCs	STS	Calreticulin exposed on the surface of apoVs mediates efferocytosis of apoVs by macrophage, which alleviates macrophage infiltration and promotes macrophage polarization toward anti-inflammation	Macrophages	T2D	Zheng et al. 2021
	BMMSCs	STS	Phagocytosis of apoBDs by macrophages polarizes macrophages to M2 type; macrophages treated by apoBDs promote migration and proliferation of fibroblasts	Macrophages	Cutaneous wound	Liu et al. 2020
	Macrophages/osteoclasts	STS	ApoVs are uptaked by macrophages to induce macrophage repolarization to M2 type, promote chondrocyte functions and chondrogenesis, and inhibit osteoclast formation and maturation	Macrophages	Rheumatoid arthritis	Li et al. 2024
	PBMCs	UV-B irradiation	ApoVs are engulfed by monocyte-derived dendritic cells to induce DC maturation by downregulation of MHC II and do not induce a proliferative T-cell response	DCs	SLE	Fehr et al. 2013
	β-cell line NIT-1	UV-B irradiation	DCs pulsed with apoBDs inhibit the expression of CD40 and CD86 and reduce the secretion of proinflammatory cytokines in DCs	Immature DCs	T1D	Marin-Gallen et al. 2010
	BMMSCs	STS	PS exposed on the surface of apoVs mediates direct contact with T cells, which inhibit proximal TCR signaling to suppress activation of CD4+ T cells	T cells	Lupus	Wang et al. 2023
Antitumor	B16 melanoma cells	Doxorubicin	ApoVs express immunogenic molecules to inhibit the growth of tumors	None	Melanoma	Muhsin-Sharafaldine et al. 2016
	BMMSCs	STS	ApoVs directly contact MM cells to facilitate Fas trafficking from the cytoplasm to the cell membrane by evoking Ca2+ influx and elevation of cytosolic Ca2+ then use the Fas ligand on apoVs to activate the Fas in MM cells to undergo apoptosis	MM cells	MM	Wang, Cao, et al. 2021
Tissue regeneration	hDPSCs	STS	ApoVs transport mitochondrial Tu translation elongation factor to ECs and regulate the angiogenic activation of ECs via the transcription factor EB-autophagy pathway, leading to pulp revascularization and tissue regeneration	Endothelial cells	Pulp regeneration	Li, Wu, et al. 2022
	BMMSCs	STS	ApoVs promote the proliferation, migration, and osteogenic differentiation of recipient BMMSCs	BMMSCs	Calvarial defects	Li, Xing, et al. 2022
	RBCs	RBC lysis buffer	ApoVs are internalized by hBMSCs to improve the osteogenic differentiation of MSCs via the activation of P38/MAPK pathway.	BMMSCs	Calvarial defects	Shao et al. 2023
	HepG2 cells	UV-C irradiation	ApoVs are cleared by neutrophils and stimulate neutrophils to be primed to a pro-regenerative phenotype to secrete hepatocyte growth factors and support the regeneration of liver remnants	Neutrophils	Partial hepatectomy	Brandel et al. 2022
	BMMSCs	STS	ApoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery	Hepatocytes	Partial hepatectomy	Sui et al. 2024
	BMMSCs	STS	Exogenous apoVs is metabolized in the integumentary skin and hair follicles. ApoVs activate the Wnt/β-catenin pathway to facilitate their metabolism in a wavelike pattern, which promotes wound healing and hair growth.	Epidermal stem cells	Skin and hair disorders	Ma et al. 2023
	BMMSCs	STS	ApoVs attenuate bone loss and stimulate bone regeneration via inhibiting expression of the target gene Sorting Nexin 14 (SNX14) and activating the SMAD1/5 pathway in target cells	MSCs	Long bone regeneration	Zhu et al. 2023

apoBDs, apoptotic bodies; apoVs, apoptotic vesicles; BMMSCs, bone marrow mesenchymal stem cells; DCs, dendritic cells, ECs, endothelial cells; FasL, Fas legend; hDPSCs, human deciduous pulp stem cells; MHC II, major histocompatibility complex class II; MM, multiple myeloma; NETs, neutrophil extracellular traps; PBMC, peripheral blood mononuclear cell; PS, phosphatidylserine; RBC, red blood cell; SLE, systemic lupus erythematosus; STS, staurosporine; T1D, type 1 diabetes; T2D, type 2 diabetes; TCR, T cell receptor; TNF-α, tumor necrosis factor–α; UV, ultraviolet.

Antitumor

Evasion of apoptosis is a hallmark of tumors that is related to the mutation or inhibition of important molecules in the apoptosis pathway (Pfeffer and Singh 2018). A study reported that the upregulation of antiapoptotic proteins and the downregulation of proapoptotic proteins are the predominant ways for the apoptotic evasion of tumors (Lopez and Tait 2015). Apoptotic evasion promotes the abnormal proliferation, angiogenesis, and invasiveness of tumor cells (Pfeffer and Singh 2018). Therefore, targeting tumor cell apoptosis is an important option for tumor therapy.

Recent studies have verified the contribution of apoVs to the antitumor response. ApoVs derived from B16 melanoma can inhibit the growth of tumors and increase the survival rate of B16 tumor mice as compared with exosomes via the expression of immunogenic molecules (Muhsin-Sharafaldine et al. 2016). Multiple myeloma (MM) is the second most common hematologic cancer, with a highly aggressive property (Krishnan and Bebawy 2023). We found that MSC-apoVs can initiate MM cell apoptosis to prolong the lifespan of MM mice by promoting the Fas transport from the cytoplasm to the cell membrane (Wang, Cao, et al. 2021). These findings offer insights into the current understanding of the interaction of apoVs and tumors and present a new antitumor strategy (Table 2).

Paradoxically, some studies have suggested that apoptosis of tumor cells is an active behavior (Pfeffer and Singh 2018). Apoptotic tumor cells can promote proliferation and drug resistance in surviving tumor cells by releasing apoVs (Kakarla et al. 2020; Zhou et al. 2022). For example, apoVs secreted by apoptotic glioblastoma cells promote the aggressive phenotype of recipient tumor cells via spliceosomal proteins RBM11, which switches cyclinD1 to the more oncogenic isoforms in recipient tumor cells (Pavlyukov et al. 2018). Moreover, colorectal tumor cell–derived apoBDs were considered to promote tumor growth via horizontally transferring tumor DNA in vivo (Samos et al. 2006). This paradoxical fact suggests that the detailed role of apoVs in cancer therapy needs further investigation.

Tissue Regeneration

Regeneration is defined as the regrowth of lost or destroyed parts of tissues or organs (Xuan et al. 2018). In orofacial regeneration, it is an important issue to regenerate the functional pulp tissue. We verified in a randomized, controlled clinical trial (Xuan et al. 2018) that human deciduous pulp stem cells (hDPSCs) can regenerate the whole dental pulp. On the other hand, hDPSC-exosomes can regenerate only dental pulp–dentin-like tissue in an in vivo tooth root slice model (Zhang, Yang, et al. 2020). In addition, apoptotic cells can release metabolites and apoVs to promote tissue repair and regeneration (Liu et al. 2018). Indeed, hDPSC-apoVs can promote pulp regeneration in situ in the root canal of a Beagle model. Mechanistically, hDPSC-apoVs promote vessel regeneration by enhancing the angiogenic function of endothelial cells through the activation of autophagy (Li, Wu, et al. 2022). This strategy provides a promising approach to regenerating the functional pulp tissue and lays the foundation for EVs in the application of organ regeneration.

Orofacial bone defects caused by trauma or tumor removal remain a major health issue. For orofacial bone regeneration, exosomes can enhance vascularization and facilitate tissue regeneration (Hu et al. 2022). However, there are still some limitations in exosome therapy, such as the low yield (Ju et al. 2023). Local transplantation of MSC-apoVs can promote bone regeneration of calvarial defects by promoting the viability of endogenous bone marrow MSCs (Li, Xing, et al. 2022). In addition, red blood cells (RBCs) are the most numerous cells in the body that can be obtained from patients conveniently. Meanwhile, large numbers of RBCs undergo apoptosis every day; thus, it is unnecessary for cell expansion before vesicle isolation (Shao et al. 2023). RBC-apoVs can improve the osteogenic differentiation of MSCs via the activation of the P38/MAPK pathway and promote calvarial skull bone regeneration in vivo (Shao et al. 2023). These results show that apoV-mediated orofacial regeneration is promising and viable.

Besides the effect on orofacial regeneration, apoVs also exert promising potential in nonorofacial regeneration (Fu et al. 2023). ApoVs from residual liver tissue stimulate circulating immune cells to secrete hepatocyte growth factors and support the regeneration of liver remnants (Brandel et al. 2022). In addition, internalization of MSC-apoVs by hepatocytes can safeguard diploid hepatocytes and rescue the injured liver (Sui et al. 2024). In skin regeneration, MSC-apoVs are metabolized in the integumentary skin and hair follicles, which promotes cutaneous wound healing and hair regeneration (Ma et al. 2023). As for long bone regeneration, MSC-apoVs can stimulate bone regeneration via the miR1324/SNX14 signaling axis (Zhu et al. 2023).

Taken together, apoVs exhibit an excellent ability to promote tissue regeneration, which provides a new approach to a cell-free strategy for tissue regeneration (Table 2).

Other Functions of ApoVs

In addition to their therapeutic function, apoVs have shown prospects as nanodrug carriers, vaccine components, and disease diagnosis. The abundant surface proteins of apoVs qualify them as an available drug delivery system to carry drugs/nucleic acids to target cells. Meanwhile, the inheritance of proteins and antigens from their parental cells makes apoVs a promising medium for vaccine development. However, these strategies are still in their infancy, and further investigation is needed to test the clinical feasibility, safety, and long-term effects.

Engineering ApoVs as Carriers

The application of EVs as carriers has several advantages, such as high drug bioavailability, low toxicity, and excellent therapeutic effects. ApoVs have shown great promise as carriers for high production efficiency, which is necessary for clinical application (Wang, Pang, et al. 2021). In addition to abundant surface protein inherited from parental cells, apoVs have the specific “find me” and “eat me” signals on their surface, which shapes their excellent targeting abilities.

Currently, there are 2 common ways to load nanodrugs into apoVs. One way is to co-incubate nanodrugs with living cells, by which cells endocytose nanodrugs and subsequently release nanodrug-loaded apoVs under apoptotic induction. However, it is difficult to produce large-scale nanodrug-loaded apoVs due to the low loading efficiency (Yong et al. 2019). Another way is to load the nanodrug into preprepared apoVs by different technologies, such as electroporation, extrusion, ultrasound, and repeated freeze-thaw cycles (Gui et al. 2024). These approaches can directly load nanoparticles, proteins, or even nucleic acids into apoVs (Zhang, Huang, et al. 2020; Zhou et al. 2022). However, this method may affect the electric potential and colloidal stability (Wu et al. 2021), damage membrane integrity and membrane protein composition (Wu et al. 2021), and disrupt the stability of apoVs (Jhan et al. 2020). Recently, Cao et al. (2023) explored a new way by adding nanoparticles into apoptotic MSCs to form nano-bortezomib-apoVs with a high loading efficiency of 22.14%. This method utilized the innate highly organized vesicle assembly system of apoptotic cells to form intact apoVs, displaying vesicle structure stability and considerable loading efficiency.

Constructing ApoVs as Vaccines

As apoVs can inherit tumor-related antigens from their parental cells, they can be used as vaccine development for tumor immunotherapy. There are 2 main approaches to using apoVs as vaccine components. One is to pulse the tumor’s apoVs into DCs to use them as a source of antigens in DC vaccines (apoV-DCs). The other is to directly inject them in vivo to stimulate T-cell responses by DCs (Fu et al. 2023). Compared with tumor lysate, tumor-apoVs are a preferred source of tumor-associated antigen for DCs-based vaccines, because apoVs can induce higher secretion of proinflammatory cytokines and costimulatory molecules and lower secretion of anti-inflammatory cytokines in DCs (Ruben et al. 2014). The treatment of elderly acute myeloid leukemia by apoV-DCs has entered phase I/II clinical study, which shows that 5 vaccinated patients survived longer than the control group (Chevallier et al. 2021). However, in another clinical trial, apoV-DCs showed limited inhibitory effects on melanoma, indicating the necessity to develop more potent vaccine strategies (Palma et al. 2012).

Diagnostic Applications of ApoVs

As products of dying cells, apoVs have the potential to be the diagnostic biomarkers for malignant tumors. In oral squamous cell carcinoma (OSCC) lesion biopsies, the number of apoVs increases gradually from normal to dysplasia to carcinomas (Viswanathan et al. 2015). The ratio of apoVs to non-apoVs in saliva is significantly decreased in patients with poorly differentiated OSCC (Zhong et al. 2019). OSCC patients with a higher ratio of apoVs to non-apoVs are predicted to have a better outcome, suggesting the prognostic value and biomarker potential of apoVs in OSCC disease (Zhong et al. 2019). In addition, the diagnostic application of apoVs in other malignant tumors is reported, such as ovarian carcinomas and carcinoma of prostate (Zou et al. 2023).

In addition, apoVs are also used in the diagnosis of graft versus host disease (GVHD) and autoimmune diseases. ApoVs in oral mucosa along with epithelial atrophy, hydropic degeneration of the basal cells, interface mucositis, and a subepithelial lymphocyte infiltrate are important diagnostic criteria of GVHD (Lin et al. 2023). ApoVs, along with nuclear proteins in the lupus band of patients with cutaneous lupus erythematosus, can potentially be used in the diagnosis of cutaneous lupus erythematosus (Koopman et al. 2017). The count of portal apoVs can be used for the diagnosis of active autoimmune hepatitis, particularly in early biopsies when no other conventional characteristics can be found (Franceschini et al. 2021).

Clinical Application and Critical Issues of ApoVs

In the past decade, exosomes and microvesicles have been used in many clinical trials (Mori et al. 2019). However, the complicated isolation process and low product yield largely limited their clinical application (Mori et al. 2019). Compared with these 2 vesicles, apoVs can be easily isolated with relatively high yields, suggesting that apoVs are a kind of promising EV for clinical application (Liu et al. 2018). Recently, apoVs have begun to be used in clinical treatment. In the year of 2022, Zhang et al. evaluated the therapeutic effect of apoVs on temporomandibular joint osteoarthritis (ChiCTR ID: 2200063153), which isolated apoVs from autogenous peripheral blood mononuclear cells. Mao et al. 2023, conducted a single-center, prospective, 2-arm study to evaluate the efficacy and safety of alveolar bone regeneration induced by gelatin sponge–loaded apoVs complex in patients with mandibular third molar extraction (NCT No. 05971342; currently unpublished). In this study, apoVs derived from allogeneic umbilical cord mesenchymal stem cells were loaded in a gelatin sponge for the regeneration of functional alveolar bone.

However, there are still some critical issues in apoV’s characterization and application. Since many methods can induce the generation of apoVs, the components and functions of apoVs induced in different ways may be different. It is necessary to establish a standard protocol for the induction, isolation, and characterization of apoVs. In addition, the different types of apoVs may have different therapeutic effects and mechanisms. Therefore, it is important to clarify the heterogeneity of apoVs to improve the potential clinical application. Recent studies showed that other types of cell death, such as pyroptosis and necroptosis, also generate death-related vesicles that may contribute to specific physiological and pathological activities. Thus, elucidating the biological roles of death-related vesicles will broaden the understanding of the PCD field. Finally, with the advancement in EV labeling and tracer imaging technique, in vivo tracing apoVs will help elucidate the distribution and mechanism of apoVs in clinical application.

Conclusion

With the advancement of research on and application of apoVs, previous studies have demonstrated that apoVs are more than just cell debris left behind from dying cells and have an important role in the physiological process, including bone homeostasis and immunomodulation. In addition, apoVs also show promising potential in clinical applications, such as in treatment, as nanodrug carriers, and in vaccine development. ApoVs are gradually being used in clinical trials. As critical issues of apoVs are gradually solved, apoVs are expected to be used in disease treatment and become the bioactive treasures left behind by dying cells.

Author Contributions

Q. Ou, W. Huang, contributed to data conception and design, drafted the manuscript; B. Wang, L. Niu, Z. Li, X. Mao, contributed to data acquisition, drafted the manuscript; S. Shi, contributed to conception and design, critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.