Potential Effect of Extracellular Vesicles in Clinical Settings of Lymphoma

Abstract

Liquid biopsy is gaining importance in oncology in the age of precision medicine. Extracellular vesicles (EVs), among other tumor-derived indicators, are isolated and analysed from bodily fluids. EVs are secreted by both healthy and cancerous cells and are lipid bilayer-enclosed particles that are diverse in size and molecular makeup. Since their quantity, phenotype, and molecular payload, which includes proteins, lipids, metabolites, and nucleic acids, mirror the nature and origin of parental cells, EVs are valuable transporters of cancer information in tumour context. This makes them interesting candidates for new biomarkers. Being closely linked to the parental cells in terms of composition, quantity, and roles is a crucial aspect of EVs. Multiple studies have shown the crucial part tumor-derived EVs plays in the development of cancer, and this subject is currently a hot one in the field of oncology. The clinical applications of EVs-based technology that are currently being tested in the areas of biomarkers, therapeutic targets, immune evasion tools, biologically designed immunotherapies, vaccines, neutralising approaches, targeting biogenesis, and extracorporeal removal were the main focus of this review. However, more bioengineering refinement is needed to address clinical and commercial limitations. The introduction of these new potential diagnostic tools into clinical practise has the potential to profoundly revolutionise the cancer field, primarily for solid tumours but also for haematological neoplasms. The development of EV-based therapies will be facilitated by improvements in EV engineering methodology and design, transforming the current pharmaceutical environment.

Introduction

Extracellular vesicles (EVs) are small membranous structures that are formed from cells and act as channels for the communication of important information between the cells [1, 2]. Signals that are dependent on and independent of the endosomal sorting complex required for transport (ESCRT) are essential for EV biogenesis [3]. EVs release a range of signals that are then received by recipient cells. Target cells take up EVs when they are released. Direct membrane fusion [4], ligand-receptor interactions [5], phagocytosis [6], as well as clathrin-dependent [7] and clathrin-independent [8] endocytosis are the major processes for EV absorption. Composition of Extracellular vesicles includes cytosol and membrane-associated components [9]. Gangliosides, transferrin receptors, cholesterol, ceramide, and sphingomyelin are all abundant in EV membranes [10, 11]. Non-coding RNA species that are carried by EVs include transference RNA (tRNA) fragments, vault RNA, short non-coding RNA (Y RNA), small interfering RNA, repetitive sequences, structural RNA, and other microscopic molecules [12, 13]. Classification of Extracellular Vesicles: [14] Microvesicles (MVs), which are large membrane vesicles (100–1000 nm diameter) branching away from plasma membrane, and exosomes (Exos), which are small membrane vesicles (30–100 nm in diameter) produced by endosome-multi-vesicular bodies (MVBs) pathway, are two different types of EVs that can be distinguished from one another. EVs contributes to NK cells' anticancer activity. Multiple pathways are used by NK cell-derived EVs (NKEVs) containing cytotoxic proteins, microRNAs, and cytokines to kill tumor cells, they also demonstrate immunomodulatory activity by boosting other immune cells. Diffuse large B cell lymphoma (DLBCL) exhibits variable behaviour and necessitates the development of more precise biological characterization, monitoring, and prognostic techniques. The presence of DLBCL has a significant impact on the EV proteome, with several proteins being specifically identified in DLBCL plasma. Plasma extracellular vesicle proteome identifies DLBCL cancer patients from healthy donors and contains potential EV protein markers for prediction of survival.

Applications of Extracellular Vesicles

Due to their intricate molecular cargo, which is dependent on parental cells, and their presence in all body fluids. EVs can be used as biomarkers, therapeutic targets, immune evasion tools, biologically designed immunotherapies, Vaccines, Neutralizing approaches, Targeting Biogenesis, Extra corporeal removal (Fig. 1).

Fig.1.

Applications of EVs in different areas

Extracellular Vesicles as Biomarkers

The fact that EVs are released by all the cells and can be retrieved from all bodily fluids, makes them an obvious candidate for biomarker sources [15, 16]. EVs represent a more trustworthy source of information and can be easily included in the diagnostic practise compared to the majority of popular biomarkers derived from liquid biopsies, such as circulating tumour DNA (ctDNA). Mainly, ctDNA only makes up a small portion of plasma-cell-free DNA [17, 18]; hence, despite its excellent specificity, the initial quantity is already challenging [19]. EVs are incredibly stable and may be kept for extended periods at a range of temperatures (for instance, several years in liquid nitrogen, several weeks at 4 °C, and several months at −80 °C), depending on when they are used [20]. Cancer cells produce much EV that closely resembles the characteristics of the parental cancer cells, almost like a fingerprint [21–23]. There are list of some target specific biomarkers used in Multiple myeloma and Hodgkin lymphoma haematological malignancy (Table 1). Several important microRNAs, such as miR-21, miR-155, and miR-146a, are highly enriched in the plasma of a patient with chronic lymphocytic leukaemia (CLL). These microRNAs can potentially affect various pathways involved in the pathogenesis and development of the disease. In order to provide more individualised care and follow-up for CLL patients, the microRNA expression profile can also be used to group patients [24]. Importantly, these microRNAs and several others (miR-148a and Let-7 g) were discovered in CLL-derived EV, highlighting how these vesicles might indirectly infer cellular status [25, 26].

Table 1.

List of some target specific biomarkers used in Multiple myeloma and Hodgkin lymphoma haematological malignancy

Disease	Biofluid	EV biomarker	EV analysis method	EV isolation method	Impact	References
MM	Serum	CD38	FC	Ultracentrifugation	A higher diagnostic rate in MM than Hodgkin’s Lymphoma Comparing with stage, stage III has a higher level of complexity	[22]
CLL	Serum	Number	FC	Ultracentrifugation	MM diagnosis rates are higher compared to Hodgkin's lymphoma/MGUS	[22]
MM	Plasma	CD38, CD203a	DLS, FC, HPLC	Differential centrifugation	Higher at diagnosis in MM compared to MGUS/SMM	[31]
MM	Plasma	CD138	FC, SEM	Centrifugation + immunolabelling	Higher at diagnosis for MM than for hodgkin's lymphoma	[32]
MM	Serum	CD44	Immunoblotting	Exclusion chromatography	MM diagnosis rates are higher than those for Hodgkin's lymphoma	[30]
MM	Plasma	CD38, [PC-1], CD157, CD73, CD203a, CD39	Differential centrifugation	DLS,FC,HPLC	Higher upon diagnosis in MGUS/SMM vs. MM	[31]
MM	Plasma	MiR-129-5p,	Ultracentrifugation	qRT-PCR	Higher upon diagnosis in MM compared to SMM	[35]
HL,NHL	Serum	CD 30	Ultracentrifugation	–	Higher upon diagnosis in HL compared to SMM	[22]

MGUS monoclonal gammopathy of undetermined significance, MM multiple myeloma, CLL chronic lymphocytic leukaemia, FC flow cytometer, SMM smoldering multiple myeloma, DLS dynamic light scattering analysis, HPLC High performance liquid chromatography, qRT-PCR quantitative real-time polymerase chain reaction and NHL non-hodgkin lymphoma

Van Eijndhoven and colleagues discovered higher levels of EV with miR-24-3p, miR-127-3p, miR-21-5p, miR155-5p, and Let7a-5p in plasma of Hodgkin lymphoma patients as compared to controls. Patients who were in remission after treatment reported a sustained decrease in these miRNAs as opposed to patients who were in relapse [27]. Plasma-derived EV RNA can be utilised to describe tumour mutation landscape, such as B Raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E for melanoma, to identify cancer in its early stages or select the best course of treatment for particular patients [28]. Multiple studies have demonstrated that Major histocompatibility complex (HLA-A, HLA-B, and HLA-C) molecules and CD19, CD20, CD5, and CD37 are present in CLL-derived EV isolated from a patient's plasma [29, 30]. In a study by Harshman and colleagues, Multiple myeloma (MM) derived EV isolated from a patient's blood had markers typical of MM cells (CD38, CD138, and CD147), as well as markers associated with treatment resistance (CD44) [31, 32]. In conclusion, EV is an accurate snapshot of the mother cell that gave rise to them. Identifying proteins and RNAs by circulating EV is a promising source of prognostic markers for people with B-cell malignancies. List of some common biomarkers used in B-cell malignancies [33, 34] (Table 2).

Table 2.

List of common biomarkers used in B-cell malignancies

B cell malignancy	Biomarker	References
CLL	miR-21, miR-148a, miR-146a, miR-155 and Let-7 g	[24–26]
HL	MiR-24-3p, miR-127-3p, miR-155-5p, miR21-5p and Let7a-5p	[27]
MM	MiR-18a and Let7b	[28]
CLL	CD37, CD20, CD5,CD19,CD52 and MHC	[29, 30]
MM	CD38, CD138, CD147 and CD44	[31, 32]

Extracellular Vesicles as Therapeutic Targets

Multiple targetable protein complexes are involved in the carefully regulated processes of EV biogenesis, EV release, and EV internalisation. Acting on two key axes can be used to reduce EV's pro-tumoral effect. One possibility is that EV could interfere with the autocrine signals that cause neoplastic cells to bind to and change themselves [36]. On the other hand, it might be possible to focus on the part that EV plays in the communication between cancer cells and the environment that supports them. This may significantly affect tumour survival, growth, and migration [37]. Koch and colleagues showed that the non-steroidal anti-inflammatory drug indomethacin inhibited EV release and markedly slowed tumour growth in a number of diffuse large B-cell lymphoma (DLBCL) cell lines. They also shown, both in vitro and in vivo, that cytostatic medicines such anthracyclines and anthracenediones have a stronger effect when EV release is reduced [38]. It has been proposed that HSPGs (heparan sulphate proteoglycans) serve as a receptor for EV internalisation. This is supported by our research, which showed that pre-treating EV with low molecular weight heparin, an HS analogue, significantly reduces the uptake of EV originating from CLL by target cells [25]. It is feasible to hypothesise that EV generated from B-cell malignancies may have a comparable effect, if not a greater one. According to Zhang et al. research's [39], it is possible to reduce the amount of EV produced by B cells that are CD73 and CD39-positive by downregulating the docking protein RAB27A. This has been done with the use of an inactivated Epstein-Barr virus-mediated siRNA, Still it is also feasible to create EV (for instance, derived from altered stroma cell lines) containing RAB27A siRNA and deliver it precisely to tumour cells. An intriguing approach to treating cancer is by focusing on B-cell malignancy EV. An intriguing approach to treating cancer is by focusing on B-cell malignancy EV. These methods involve obvious risks and limitations, such as the potential for drug resistance and off-targets, which can eventually result in reduced therapeutic efficacy.

Extracellular Vesicles in Immunotherapy

Immunotherapy has potential to effect EVs that ultimately affect immune suppression and the development of cancer. Immunotherapy works by stimulating the body's immune system to find and destroy cancer cells in the patient, Fig. 2 demonstrate EVs used as an Immunotherapy. Neoplastic cells secrete EVs as a significant source of selected antigens that educate immature immune cells [40, 41]. Leukemia-derived EV are loaded with antigens and many immunogenic chemicals, such as TGF-β and IL-6, which can hinder dendritic cells' (DCs') ability to mounting a targeted immune response against neoplastic cells [42–44]. Due to their unique structure and properties, EVs can uniquely drive DC-dependent immunisation; when DCs are stimulated with simply leukemic cell lysate, the proper immune response cannot be properly induced [45–48]. Additionally, immunotherapy can be utilised in conjunction with chemotherapy. Guo and colleagues mixed cyclophosphamide, polyinosinic polycytidylic acid sodium salt (poly I:C), and leukemia-specific DC-derived EV48. This combination is predicated on the fact that poly I:C affects DC maturation while DC-derived EV stimulates T lymphocyte proliferation and increases their cytotoxic effectiveness against leukaemia. Targeting EV surface chemicals produced from tumours is another effective tactic to significantly lessen their influence on immune cells.

Fig. 2.

EVs as immunotherapy

Targeting EV surface chemicals originating by tumours is another effective approach that significantly decreases their influence on immune cells. TGF-1 is found in EV generated by a variety of tumours and it acts on different immune cells like DCs to trigger immunological escape [49]. High quantities of TGF-1 are present in the EV produced by the acute lymphocytic leukaemia (ALL) L1210 cell line. Huang and coworkers removed TGF-1 from their EV and restored DC maturation and activity in vivo by employing short hairpin RNA (shRNA) to knock down the ALL cell line. Furthermore, the same team showed that pulsed DCs could boost T cell development and cytotoxic activity against ALL cells [50]. It is possible to re-establish a correct cytotoxic activity against B-cell malignancies using immune cells other than dendritic cells. Natural killer (NK) cells are another worthwhile target, because chemicals like NKG2D ligands introduced to EV generated from haematological malignancy (HM) suppress their function [51, 52]. In one study, the NKp30 ligand BAG6 was shown to have two functions in CLL. First, CLL patients have high levels of soluble BAG6 in their plasma, which is one of the reasons why these individuals have a deficiency in NK cytotoxicity [52]. The use of EV-based vaccinations is still up for debate despite their obvious immune system activation effects, even though numerous studies have already shown how EV can be utilised to develop a powerful immune response before tumour cells manifest. In agreement with this, it has been demonstrated that DC cells pulsed with DLBCL-derived EV stimulate T lymphocyte growth, increasing anti-lymphoma immunity in mice as a result [53]. These outcomes are consistent with prior research showing that EV generated from leukaemia can be exploited to develop anti-leukemia immunity, with observable outcomes both in vitro and in vivo [54]. Together, these results position EV as powerful immune modulators that have the potential to be important tools for vaccine and immunotherapy development.

Engineered Extracellular Vesicles

Lunavat and colleagues demonstrated that, creating nanoparticles containing siRNA, effectively triggers poly (ADP-ribose) polymerase-dependent apoptotic pathways in treated l820 lymphoma cells [55]. TGF-b1 has recently been silenced in lymphoma cells using the shRNA technique, which causes the cells to secrete EV that is depleted of TGF-b1. Immune system’s response to leukemic cells improve by eliminating antitumor-immunological surveillance inhibitor [56]. In accordance with this, modified EV were also utilised to transfer particular antigens in an effort to boost or reactivate the immune system. The internalisation of EV in leukemic cells and a potent immunogenic impact were obtained by the researchers by treating the cells of CLL patients, which resulted in a dual activation of tumor-associated and EBV-specific T cells [57]. In B-cell malignancies, further methods based on nanostructures have been investigated. Several HM cell lines exposed to artificial lipid vesicles consistently exhibited a pro-apoptotic effect while preserving normal cells (such CD4C and CD8C T cells) in vitro and without any evidence of toxicity in vivo [58]. Using a variety of cell lines, engineered EV can be produced in large quantities in vitro. It has been demonstrated that mesenchymal stem cells (MSCs) are a useful tool for the creation of EV with strong effect in malignant cells. In accordance with this, MSCs may be changed to release engineered EVs with a variety of downstream uses and methods [59]. A new class of modified EVs has recently been put to the test in an effort to boost T cell activity against tumour cells. Monoclonal antibodies against CD3 and the epidermal growth factor receptor linked with cancer cells are included in synthetic multivalent antibodies retargeted (SMART) EV stimated glomerular filtration rate (EGFR) [60]. These qualities enable them to function as a bridge, guiding T cytotoxic cells in the direction of tumour cells, causing crosstalk, and increasing the antitumor response [61]. Because MM has a high amount of EGFR,

which ensures cell proliferation and resistance to traditional treatments, SMART EV may have a possible use in treating MM [62, 63]. EV have the capacity to quickly and fully alter the phenotypic of target cells by epigenetically reprogramming them. Inhibiting the molecules responsible for this profound alteration and engineering EVs to carry specific cargo components, such as siRNA and shRNA, that can change the epigenetic landscape and alleviate immune cell depletion are promising strategies for reactivating the immune system. General techniques used in production of Engineered EVs (Table 3).

Table 3.

General techniques used in production of Engineered EVs

Modification/generation technique	Description	Advantages	Disadvantages	Pre-clinical applications	References
Surface functionalization	Use of chemical reactions to couple molecules of interest (i.e., peptides, proteins) to the EV surface (i.e., Click Chemistry) to enhance targeted delivery or half-life in circulation	Targeting to specific organs and areas of interest (i.e., brain, tumor), increase half-life and stability in circulation, binding of negatively charged molecules (i.e., siRNA, miRNA, mRNA, DNA)	(i) May increase overall size of EVs, (ii) reduces natural targeting and delivery capability of EVs, (iii) peptide used must be very specific to the target site	Targeted therapy	[64, 65]
Modification of parental cells	Genome engineering of parental cells to modify produced EVs (i.e., surface protein that influences targeting)	Highly modifiable depending on application, can target wide range of luminal molecules for functional modification of EVs, or surface molecules for targeting modification of EVs	Requires extensive validation of the efficiency and off-target errors of genome engineering strategies, may alter the properties and structure of the EV	Lentivirus transfected MSCs to overexpress miRNA-let7c, known to possess anti-fibrotic properties,generated exosomes capable of transferring this cargo to renal cells in vitro	[64, 66, 67]
Microfluidics	Fragmentation of cells/lipid micelles using microfluidic systems (pressure) to generate EV mimetics or liposomes of consistent size	Fast, efficient, scalable, consistent, less manual handling	Expensive, complex equipment	Mimetics were generated from embryonic stem cells and used to deliver drugs/RNA in vitro	[67]
Serial extrusion	Generation of EV-mimetics by fragmenting whole cells through micro-sized pore filters of descending size to obtain vesicles of similar size to EVs	Consistent, scalable generation of EVs from cells of interest, easily modifiable protocol to suit application	Requires extensive manual handling, additional purification steps, quality assurance of progenitor cells	Human MSCs treated with ion oxide nanoparticles (IONP) were extruded serially (five times through 10, 5, and 1 µm and 400 nm), and demonstrated their feasibility for spinal cord-injury treatment	[68]
Centrifugation	Use of centrifugal force to fragment cells through a membrane generating EV mimetics	Fast, efficient, scalable, less manual handling compared to extrusion	Relative scalability depending on equipment capacity	Murine embryonic stem cells were generated by this method and were able to transfer RNAs	[69]

The use of modified EV as a cytokine is a fascinating application for these organisms (e.g., proinflammatory cytokines). With this method, the impact of tumours on nearby cells is actively reduced (e.g., reducing inflammation). An illustration can be seen in a recent preprint in which stroma-derived EV were modified to carry the signalling molecules for the tumour necrosis factor receptor 1 and interleukin 6.

Extracellular Vesicles Based Vaccine

Cancer vaccines are also known as Biologic response modifiers (BRMs), which are regarded as a class of treatments [70]. To promote immune protection, single or several tumour antigens are administered [71]. Numerous cancer vaccination trials have been designed as a result of the discovery of specific antigens are highly expressed on cancer cells [72]. However, there are not many data to support the viability of tracking the immune response, therefore vaccination isn't yet thought to be a viable treatment for lymphoma [73]. The most significant drawback of vaccination-based cancer therapy is thought to be heterogeneity of molecular profile of tumour cells both during tumour growth and after the treatment. Additionally, variations in antigen-specific T-cell repertoires and the presence or absence of pre-existing immunological tolerance have also been linked to poor outcomes from vaccine-based therapy [74, 75]. According to studies, lymphoma cell derived extracellular vesicles (LCEVs) can cause CD8 + T cells to launch a powerful and targeted cytotoxic response. This result depends on the elevation of MHC, costimulatory, and cytokine molecules by DC functional maturation caused by LCEV [53]. The most effective APCs are DCs, which may acquire tumour antigens and, in response to environmental cues, deliver antigens at tumour sites and lymphoid organs to either prime, maintain, or abort EV-based active immunisation [76, 77]. Menay et al. have shown that LCEVs produced from T-cell lymphoma can effectively elicit an immune response and memory against lymphoma. T helper 1 mediated responses were linked to this impact. But Chen et al. [53] have more recently shown that a dual LCEV-mediated impact exists. They demonstrated that LCEVs may exhibit immunosuppressive behaviour that can foster the formation of tumours in vivo. On the other hand, they also showed that T cells with higher anti-lymphoma activity were retrieved from tumor-bearing mice treated with LCEVs. This result depends on the stimulation of T cells by DCs and the suppression of Th2 immunosuppressive activity [75]. These findings imply that a novel DC-based immunotherapy may involve the training of DCs employing LCEVs as tumor-associated antigens [76].

More recently, the idea has been advanced to modify immunity in solid tumours by combining standard-of-care chemotherapy with EV-based vaccines. For instance, when coupled with DC-derived EVs, cyclophosphamide has been found to enhance the cytotoxic T-cell response against malignancies [78, 79]. The effectiveness of this combination strategy as a complementary treatment for lymphomas should also be assessed in the future.

Neutralizing EV-Based Approaches

Despite the fact that rituximab has been shown to improve overall survival in individuals with aggressive or indolent B-cell NHL, only 50% of these patients achieve remission [79]. This suggests that more specific and extra antibodies are needed. Thanks to lymphoma molecular profiling [80, 81] CD22, CD19, CD37, and CD40 have been identified, and they are currently being researched in order to provide new antibody-based treatments. Alternative antibody-based therapies for lymphomas are currently being employed with a number of new targeted antibodies, such as the anti-CD22 [82], anti-CD40 [82], anti-CD19 [83], anti-CD19/CD3 [84], and anti-CD37 antibodies [85]. Since LCEVs also express these markers, if these methods are proven effective, they could potentially be utilised to eliminate LCEVs from circulation. As a result, a dual impact can be desired.

Targeting EV Biogenesis

An alternative EV-mediated treatment strategy for cancer has been suggested: blocking the release of EV from tumour cells [86, 87]. For this reason, it would be essential to identify important cellular pathways that involved in EV biogenesis. EBV infection has been demonstrated to affect EV biogenesis and miRNA profile in Burkit lymphoma cells [88]. According to a recent literature, ABCA3 expression is absolutely necessary for the creation of LCEVs, and genetic deletion or pharmacological interference with ABCA3 expression increases drug accumulation in nuclei of B-cell lymphomas [89]. Additionally, it has been demonstrated that EV production is hampered by the prevention of sphingolipid ceramide formation by a variety of methods 88. Manumycin A20 (MA) and tipifarnib, two farnesyl transferase inhibitors (FTIs), as well as many imidazoles (neticonazole, ketoconazole, and climbazole), have been discovered to disrupt the particular processes involved in EV formation in cancer cells [88]. The Rab family has a well-established history of controlling vesicular trafficking. This regulation encompasses the fusion of several vesicular transport intermediates, docking, budding, and vesicular motility [90]. More significantly, it has been demonstrated that the enrichment of Rab35 on EVs is essential for controlling vesicle density [91]. Ostrowsky and others, [92] have demonstrated that many Rab family members participate in EV synthesis and have detectable effects, supporting the use of Rab27 inhibitors to prevent EV creation. It has also been shown that intracellular Ca2 + concentration affects EV biogenesis in erythroleukemia cell lines.

Extracorporeal Removal of LCEVs

Novel strategy for elimination of EVs in the cancer and in lymphoma patients has been proposed: EV dialysis [93]. However, there are currently no reliable statistics on EV elimination using hemofiltration. However, The Aethlon ADAPTTM (adaptive dialysis-like affinity platform technology) system, developed by Aethlon Medical, is a therapeutic hemofiltration technique that combines either a continuous renal replacement therapy device or a conventional dialysis unit with immobilised affinity agents in outer-capillary space of hollow-fiber plasma separator cartridges (CRRT) [93]. This technology and dialysis have so far been combined to treat hepatitis C patients who needed viral clearance or decrease. A decrease in number of infected cells provided indirect support for the feasibility [93]. Despite being a beneficial creative solution, its prospective applicability in LCEV removal still has to be validated.

Ongoing Clinical Trials on Extracellular Vesicles

There are presently several EV clinical trials running. The bulk of treatments for cancer are used on solid tumours. The objectives range from developing EV based vaccines (NCT01159288), developing new sources and standardised procedures to isolate the EV from patients (NCT03821909), deepening characterization of the EV as predictive biomarkers (NCT03830619) to ultimately furthering EV-based treatment (NCT03608631), List of Ongoing and completed clinical trials on Extracellular based therapy (Table 4). The ExoReBly research (NCT03985696) aims to analyse DLBCL derived EV from patient samples with regard to B-cell malignancies. This clinical trial's justification is based on observation that immunotherapy fails to provide any effect in 50% of patients who receive it, often with the goal of inducing immune activity against CD20. The main theory is that the high levels of PD-L1 and CD20 found on DLBCL-derived EV function, respectively, as a strong immunosuppressive signal and a decoy target for the rituximab antibody, resulting in treatment resistance. The initiative intends to characterise DLBCL-derived EV and use them as a measure of therapeutic responsiveness and disease outcome. We found that CLL-derived EV displayed significant amounts of CD20 on their surface and had the ability to fool rituximab, which is intriguing and may point to a shared mechanism among B-cell malignancies. EV have been the subject of more than 70 clinical trials, and their potential for therapeutic and practical use is increasing recognition. Only one of these clinical trials is being developed for B-cell malignancies, and only a small portion of them consider HM. Nevertheless, fresh knowledge about the impact of EV on the development of cancer is produced annually, which strongly suggests that HM will find more uses in the future.

Table 4.

List of Ongoing and completed clinical trials on extracellular based therapy

CT No	Mechanism of action	Target diseases	Clinical phase	Primary outcome	Secondary outcome	Number of patients
NCT04164134	EVs	Retinoblastoma	Recruiting	Adult RB1 mutation carriers (Rb-survivors) and retinoblastoma patients' blood showed platelet RNA expression and an allelic DNA balance of EVs (children)	Platelet RNA expression, blood EV allelic DNA balance, and tumour tissue genomic analyses in RB1-mutation carriers who have been diagnosed with a second primary malignancy	396
NCT04727593	EVs	Cancer	Recruiting	Change on extracellular vesicles and distress	Change on Cancer antigen, Prostate-specific antigen (PSA), Carcinoembryonic antigen (CEA), adrenocorticotropic hormone (ACTH), extracellular vesicles, distres	111
NCT04529915	Exosomes	Lung cancer	Active, not recruiting	Evaluation of the difference between healthy controls and patients with lung cancer using deep learning to analyse exosomes	Through quantitative study of lung cancer-specific exosomal proteins and deep learning analysis of exosomes, it is possible to identify the early pathological phases in patients with lung cancer	470
NCT04852653	Exosomes	Adenocarcinoma of rectum	Recruiting	response to neoadjuvant chemotherapy alone or in combination with radiochemotherapy for rectal cancer and the presence of onco-exosomes and/or exoDNA	to determine whether the kinetics of oncoexosome and exoDNA detection may be used to forecast how rectal cancer would respond to neoadjuvant therapy determining whether the tumor's early mutational profile is a reliable indicator of rectal cancer's responsiveness to neoadjuvant chemotherapy and radiochemotherapy	40
NCT03102268	EVs	Cholangiocarcinoma	Unknown	Characterization of ncRNAs in tumor-derived exosomes from patients with cholangiocarcinoma before receiving anti-cancer treatments and patients with benign biliary strictures	Time-to-event endpoints and exosomes-derived ncRNAs (at baseline and monthly during treatment till death) Time-to-event endpoints and exosomes-derived ncRNAs (at baseline and monthly during treatment till tumour progression)	80
NCT05587114	Exosomes	Lung cancer	Recruiting	Comparative analysis of CEA and lung cancer-specific exosome marker concentrations in peripheral blood and pulmonary blood using ELISA assay, Characterization of exosomes using western blot, NTA, and TEM analysis and Evaluation of the clinical usefulness of lung cancer diagnosis using lung cancer-specific exosome markers in peripheral vein blood and primary lung cancer outflow pulmonary vein-derived blood	Clinical evaluation of recurrence or cancer metastasis after treatment in patients who underwent lung cancer surgery using lung cancer-specific exosome biomarkers according to blood sample location (peripheral vein blood and primary lung cancer outflow pulmonary vein derived blood)	150
NCT05469022	EVs	Non small cell lung cancer	Recruiting	using RECIST version 1.1, the objective response rate (ORR) was assessed. It is determined by the percentage of patients who experienced a full or partial recovery after receiving lazertinib for nine weeks	The ratio of pathology stage downstages to clinical stage downstages the percentage of patients in the surgical sample with less than 10% cancer cells The period of time following surgical resection during which the patient doesn't experience recurrence, progression, or death from any reason The BALF EGFR mutation concordance rate compared to the surgically removed tissue's EGFR mutation status	40

Conclusion

In the past ten years, significant progress has been made in understanding the intricate features of EV in B-cell malignancies and biological function that they perform in tumour activity. The community has continued to be interested in investigating EV, in part because of their promise as a tool to enhance cancer diagnosis and its therapy, as seen by the number of studies recently published. It is now well known that EV can mimic the tumour cells from which they originate and transport important targetable chemicals closely associated with cancer biology, such as immunological checkpoints among many others.

However, translational research stage not been successfully applied in clinical settings for patients having B-cell malignancies. While advanced clinical trial phases of treatment of solid malignancies based on an EV-derived reasoning are already underway, this is not the case for diagnosis, follow-up, or any form of treatment for B-cell-originated neoplastic disorders. Use of LCEVs as innovative "multiomic shells" for disease detection, drug resistance, immunological evasion and therapeutic response has also been studied. According to a recent publication, treatment-related changes in circulating EVs that express the programmed death ligand 1 (PD-L1) are brought on by certain immunological responses [94]. As a result, it is possible LCEVs could serve as real-time indicators for therapy efficacy [38]. The discovery of mechanisms that control LCEV biogenesis has also been a focus of research. This would make it possible to create novel therapeutic strategies where targeting LCEVs might prevent tumour growth. Additionally, International Prognostic Index (IPI) only have survival time rather than treatment success, the utilisation of LCEVs as indicators of treatment response would be especially pertinent for patient management throughout therapy [95]. If enhanced therapy options are to be successfully created, a precise and in-depth understanding of LCEV activities is still necessary. In addition, EV-based strategies are effective in treating new chronic neoplastic and non-neoplastic disorders serves as a driving force for future HM endeavours to fully develop this reasoning.

We have outlined the uses of EVs and their functions in the development and spread of tumours in this review. This review examines previous and ongoing studies on the uses of EVs, their conclusions, and how to apply this knowledge to repurpose EVs as a medicinal tool. The clinical utility of EV as a potent tool for patients with B-cell neoplastic disorders is thus expected to be clarified over the course of the next 10 years.

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