Exosomes: Toward a potential application in bladder cancer diagnosis and treatment

Abstract Bladder cancer (BC) is a prevalent malignant tumor of the urinary system, known for its rapid progression and high likelihood of recurrence. Despite ongoing efforts, clinical diagnosis and treatment of BC remain limited. As such, there is an urgent need to investigate potential mechanisms underlying this disease. Exosomes, which contain a variety of bioactive molecules such as nucleic acids, proteins, and lipids, are regarded as extracellular messengers because they are implicated in facilitating intercellular communication in various diseases and are pivotal in tumor advancement, serving as a promising avenue for such researches. Nevertheless, the heterogeneous nature of BC necessitates further exploration of the potential involvement of exosomes in disease progression. This review comprehensively outlines the biological attributes of exosomes and their critical roles in tumorigenesis, while also discussing their potential applications in regulating the progression of BC involving clinical diagnosis, prognostication and treatment.


| INTRODUCTION
Bladder cancer (BC) is a prevalent malignancy of the genitourinary system, ranking as the ninth most common malignancy globally.BC can be broadly classified into urothelial BC and non-urothelial BC based on histopathological criteria.Urothelial carcinoma accounts for nearly 90% of BC cases, with 20% of cases being initially diagnosed as muscle-invasive bladder carcinoma (MIBC) and carrying a poor prognosis.However, even patients initially diagnosed with non-muscle-invasive bladder cancer (NMIBC) may experience disease progression to MIBC, with rates ranging from 10% to 30%. 1,2The survival rates for BC have exhibited no significant improvement over the last three decades, as evidenced by approximately 60%-70% of NMIBC patients experiencing disease recurrence post-surgery. 3The long-term survival rate of BC is intricately linked to the stage of diagnosis.For instance, the 5-year survival rate of bladder carcinoma in situ is as high as 95.8%, while that of metastatic BC is mere 4.6%.This underscores the criticality of timely and precise diagnosis in determining the prognosis of BC patients. 4urrently, the primary diagnostic methods for BC are cystoscopy and cytology. 5However, the sensitivity of cytological examination is notably low, particularly for low-grade tumors, with only a detection rate of 34%. 6,7lthough cystoscopy is the current gold standard for BC diagnosis, it is an invasive, costly, and poorly tolerated procedure, often resulting in urethral stricture, perforation, bleeding, and infection. 8In recent years, exosomes have emerged as a prominent tool in the field of oncology.Heterogeneous cells secrete membrane-bound vesicles that contain abundant nucleic acid, protein, and lipid contents. 9These exosomes, derived from tumor cells, act as mediators of bioactive molecules and play a role in inducing the tumor microenvironment (TME), including the formation of pre-metastasis niches. 10,11urthermore, exosomes offer a potential source of novel biomarkers for liquid tumor biopsy and may provide a safe and targeted alternative approach for tumor therapy due to their low immunogenicity and circulatory advantage. 12espite the development of exosome-based mechanisms and applications in various diseases, their utilization in BC remains in nascent stages.This review endeavors to elucidate the biological significance of exosomes in tumor progression and their prospective clinical applications in the diagnosis and treatment of BC.

| Biological characteristics of exosomes
Exosomes are extracellular vesicles (EVs) with diameter of 30-150 nm, which can mediate biological activity.They are composed of bilayer lipid coating and rich in biomolecules including nucleic acid, proteins, and lipids, which can reflect the characteristics of parent cells. 11,13ue to the lipid bilayer, exosomes are stable, resistant to the degradation of circulating RNases or other enzymes, and can maintain the integrity of their contents for a longer time.The biogenesis and release of exosomes are a complex multi-step process.Early endosomes are formed by endocytosis of the plasma membrane and further mature as multivesicular body (MVB).During this process, exosomes are secreted in the form of intracavitary vesicles after fusion of the MVB membrane with the plasma membrane and serve as the key mediators of intercellular communication [14][15][16] (Figure 1).
More and more studies have shown that exosomes are involved in many important physiological processes, including neutrophil chemotaxis, extracellular matrix remodeling, immunity, inflammation, and central nervous system physiology, etc. 17 Besides, it's reported that exosomes are related to a wide range of disease pathology, including different types of kidney disease, Parkinson's disease, Alzheimer's disease, and tumor, etc. 18 During tumorigenesis, exosomes play a key role in tumor progression by increasing the invasiveness and migration, promoting angiogenesis, activating cancer-related fibroblasts, and enhancing cell proliferation, thus participating in tumor formation, metastasis, immune escape and drug resistance, etc. [19][20][21][22][23] (Figure 2).

| Exosomes and tumor progression
TME is a key element of tumor generation.The interaction between cells and the microenvironment can promote the occurrence and progress of tumors. 24Studies have shown that exosome-derived bioactive molecules secreted by tumor cells can generate the pre-metastasis niche by reshaping the TME.This complex process involves the binding of tumor-cell-derived exosomes to the target organ stromal cells, leading to cell reprogramming, activation of signaling pathways, and ultimately the establishment of a pre-metastatic microenvironment in the target organ, providing a prerequisite for promoting tumor growth and metastasis. 25,26Among them, tumorcell-derived exosomes are considered as the crucial mediators of cell-cell interactions in the TME, which are involved in variety of tumorigenic mechanisms.

| Angiogenesis promotion
Tumor occurrence and progression is a dynamic, multistep process that requires adequate blood conduction to provide the continuous nutrients and oxygen to maintain the rapid growth and reproduction of tumor cells.Tumor cells promote angiogenesis by activating endothelial

Key points
� This paper reviewed the biological characteristics of exosomes and their biological significance in tumor genesis and progression.� The application prospect of exosomes in bladder cancer (BC) diagnosis and treatment was systematically discussed.� The potential role of exosomes in the heterogeneity of BC was reviewed.� A novel idea of using exosomes as the natural nanoparticles in clinical diagnosis and treatment was introduced.cells. 27Exosome-associated mRNA encodes vascular endothelial growth factor, fibroblast growth factor, angiopoietin-1, ephrin A3, matrix metallopeptidase 2, matrix metallopeptidase 9 and azurocidin 1, etc.9][30][31] This is not only a necessary condition for the normal growth of tumor tissue, but also an important pathway for tumor invasion.It is the transformation of epithelial cells into mesenchymal cells, which involves in embryogenesis, wound healing and malignant progression of tumors.Intercellular and extracellular matrix interactions are reshaped, resulting in reduced adhesion between epithelial cells and the basement membrane, further activating the new transcriptional processes that facilitate their mesenchymal transformation.In tumor progression, epithelial-mesenchymal transition (EMT) promotes tumor formation and metastasis while increasing the tolerance to clinical intervention.Exosomes promote tumor invasion and migration by regulating extracellular matrix and EMT process (Figure 3).Tumor exosomes acting on the urothelial cells have been found to induce EMT. 33,342.2.3 | Premetastatic niches formation This is a key molecular event for tumor target organ/ tissue metastasis. 35Primary tumors release biomolecules that migrate to preferred metastatic targets and dynamically reshape these sites before metastasizing to distant organs.Premetastatic niches are defined as the molecular and cellular changes in specific organs/tissues.Distant metastasis of tumors is facilitated by the colonization of target organs/tissues by circulating tumor cells, 36 which involves the release of initial tumor-derived exosomes into the circulatory system and subsequent escape from the vascular bed to the distant secondary organs. 27,37In this process, the organ affinity exosomes exhibit is key to their targeting of organ tissues.By modifying the function of target cells through content biomolecules, exosomes play an important role in tumor invasion and metastasis.

| TME regulation
An increasing number of studies have demonstrated that tumor progression is the result of autocrine and paracrine communication between tumor cells in the microenvironment, which tend to secrete large amounts of exosomes. 38,39For example, cancer-associated fibroblasts (CAFs) induced by exosome secretion of miR-9 and telomerase reverse transcriptase (hTERT) support tumor cell proliferation and increase their drug resistance.Signaling pathways activation is also one of the pathways that exosomes participate in the regulation of TME.For example, transforming growth factor-β (TGF-β)1 promotes the differentiation of human umbilical cord mesenchymal stem cells into CAFs and activates the TGF-β/Smad pathway. 40In the communication between normal stromal cells and tumor cells, exosomes may stimulate another signaling pathway, such as nuclear factor κB and epidermal growth factor receptor signaling pathway, which also play a critical role in tumor proliferation and migration.After exposure to tumor exosomes, endothelial cells showed enhanced proliferation, migration, and tubular formation.In addition, tumor cell exosomes have been proved to promote cancer progression by reducing the immune response.Notably, some specific exosomes may even reduce the cytotoxicity of natural killer cells and T cells.Tumor-derived exosomes can also influence autologous cells through autocrine, stimulating tumor cell invasion and migration while reducing the adhesion by enhancing MMP 9 or chemokine receptor 4. 41

| Exosomes and tumor diagnosis
Exosomes are secreted by various types of parent cells and are produced by endosomal budding.The lipid bilayer membranes protect bioactive molecules from enzymatic degradation in the extracellular environment through membrane fusion and internalization.Bioactive molecules, such as nucleic acids (non-coding RNA, messenger RNA, etc.) and proteins, are selectively classified, packaged and delivered to cells to participate in immune response, antigen presentation, and intercellular communication, and have become novel mediators for tumor progression in the past decade. 42The abundance of exosomes in blood and urine makes them a potential source of effective biomarkers for tumor diagnosis and detection. 43n recent years, exosome-derived biomarkers as potential candidates for primary disease diagnosis and prognostic monitoring have attracted extensive attention in laboratory diagnosis.As an important liquid biopsy technique, the detection of blood-derived and urinalderived exosomes has an ideal clinical application prospect in the in vitro diagnosis of tumors due to the unique advantages of non-invasive and simple operation, as well as the comprehensive display of complete genetic information of tumor patients.Compared to the traditional tissue biopsies, liquid biopsies allow easy and quick specimen access with less patient trauma.In the diagnosis and monitoring of tumors, liquid biopsy makes it easier to ensure the standardization and accuracy of the procedures, so that the analysis results have a better repeatability.Exosomes present different levels of enrichment in blood and urine based on the different tumor characteristics.Therefore, selective sampling can be used to detect the target exosome-derived biomarkers to improve the clinical diagnostic efficacy.Urinary exosome-related biomarkers have better applications in the diagnosis and monitoring of urinary tumors, while blood-derived exosome markers may be a better choice for the non-urinary tumors.Exosome-related biomarkers show promising clinical application prospects in improving the diagnostic sensitivity and accuracy, especially for the low-grade tumors.

| Exosomes and tumor treatment
In recent years, many studies have reported the applications of exosomes in tumor therapy.By exploring the effective biological effects and the reduction of toxicity of traditional drugs, exosomes can provide an important laboratory foundation for tumor research.

| Exosomes and tumor immunity
Tumor cells and their TME-derived exosomes directly or indirectly influence the tumor progression through cellular action and TME regulation, respectively.Therefore, blocking the production of exosomes and the mediated tumor-TME cellular signaling pathway, and eliminating the exosome-specific active contents, have been proposed as the therapeutic strategies to inhibit the tumor progression.In addition, the efficacy of exosomes in immunotherapy may hold promise for tumor treatment.Recent studies have shown that immunotherapy, especially the emergence of immune checkpoint blocking (i.e., PD-1/PD-L1), has become a new breakthrough in cancer treatment.Tumor-derived exosomes deliver functional PD-L1 and inhibit the immune response. 44herefore, systemic anti-tumor immunity and memory can be induced by the inhibition of exosome PD-L1. 45To date, studies based on exosomes promoting anti-tumor immunity have begun to explore their safety and efficacy in humans.

| Exosomes as tumor vaccines
Tumor-derived exosomes follow the antigen presentation pathway and can be used as cell-free vaccines. 46As key regulators in biological systems, non-coding RNAs influence the differentiation, maturation and function of immune cells. 47For example, non-coding RNA-modified exosomes target interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-1B (IL-1B), TGF-β, interferon-γ, and Tolllike receptor 4, inducing dendritic cells to mature and enhance their immunostimulatory capacity. 48Therefore, tumor-derived exosomes modified with specific noncoding RNA are expected to play an important role in the treatment of BC.

| Exosomes as drug delivery vectors
Studies have shown that therapeutic nanoparticles can be loaded into liposomes to increase the drug concentration in target tissues while reducing the toxic effects on normal tissues. 49,50Numerous studies have extensively endeavored to ascertain the most suitable exosome donor cell type, while also focusing on the efficient exosome isolation, engineered exosome production, and storage to facilitate the abundant accumulation of exosomes from non-specific locations toward the targeted tissues.Further major challenges include unsatisfactory preservation and delivery efficiency of exosome structural integrity during drug loading.Clarification of these issues will enhance the application prospect of exosomes as drug delivery vectors.

| Exosomes in BC progression
A number of studies have demonstrated that BC-derived exosomes are similar to exosomes from other tumors, participating in the regulation of biological processes such as cell proliferation, migration, invasion, apoptosis and angiogenesis (Table 1),  playing an important role in the tumorigenesis and progression at different stages of BC 80 (Figure 4). Yang t al. showed that BC exosomes induced the proliferation of moderately differentiated 5637 BC cells and poorly differentiated T24 bladder transitional cells in a dose and time dependent manner.They found that after the BC exosomes treatment, the expressions of anti-apoptotic genes B-cell lymphoma/ leukemia-2 (BCL-2) and Cyclin D1 were increased, while pro-apoptotic genes Bax and Caspase3 decreased.81 Xu et al. reported that exosomes down-regulate the expression of BCL-2 and MCL-1 by transporting miR-29c in BC, inducing BIU-87 cell apoptosis.82 Beckham et al. found that exosomes isolated from the urine of patients with high-grade radical BC could promote the migration and invasion of 5637 cells cultured in vitro and stimulate the angiogenesis of human umbilical vein endothelial cells.83 Franzen et al. showed that BC exosomes induced EMT in urothelial cells. Copared with controls, urothelial cells exposed to the invasive BC cell lines or bladder patientderived exosomes showed the decreased expression of epithelial markers and increased expression of mesenchymal markers.33 This could explain the interaction between urinary exosomes and bladder urothelial cells, as well as the fact that the former induces the latter to undergo transformation, making them at a higher tumorigenic risk.Ostenfeld et al. demonstrated that silencing Rab27a and Rab27b members of the Rab family could down-regulate the expression of miR-23b and miR-921 in BC cells to reduce cell invasion, indicating that exosomemediated tumor-suppressor miRNA can be coordinated to inhibit the progression of tumor metastasis.82

| Non-coding RNAs
Exosomes are found to be rich in content molecules, among which a large number of different types of noncoding RNAs are involved in the regulation of cell activity and intercellular communication. 104The types and amounts of non-coding RNAs are influenced by the surrounding microenvironment and parent cells, and are delivered and utilized as the recipient cells (Figure 5).Current reviews on non-coding RNAs from BC-derived exosomes mainly focus on miRNAs, long-chain noncoding RNAs and circular RNAs, which are involved in the regulation of tumor cell proliferation, invasion, apoptosis, cell cycle arrest, angiogenesis and lymphangion genesis.Due to the protective bilayer membrane, bioactive substances in exosomes achieve the resistance to degradation and stability, which are important advantages of exosomes as biomarkers 105 (Figure 6).By reviewing the previous studies, the relationship between exosomal noncoding RNAs and BC can be more clearly identified.Therefore, exosome derived non-coding RNAs are expected to be the potential diagnostic biomarkers and therapeutic targets.Moreover, exosomal non-coding RNAs can also be used to predict the patient survival parameters such as cancer-specific mortality, relapse-free survival, overall survival, and disease-free survival.Tong et al. collected information from studies reporting survival data and evaluated 9 exosome non-coding RNAs that were identified as the prognostic biomarkers. 23

| Messenger RNAs
Perez et al. analyzed exosomal RNAs from BC patients and controls using a whole-transcriptome array and found that there was 55% overlap between BC patients and controls among 4102 transcripts.Based on the differential expression of patient and the control samples, 15 genes were selected for further PCR analysis at eight sites.They found that two genes, GALNT1 and LASS2, were only present in BC patients, while two genes, ARGH-GEF39 and FOXO3 only present in the control samples.   of 15

| Protein biomarkers
Exosome membranes are rich in transmembrane proteins and cytoplasmic proteins, some of which are considered as universal exosome markers regardless of cell origin, such as CD9, CD63, CD81, members of the quaternary transmembrane protein family, and endosomal sorting complex required for transport complex associated with biogenesis.Welton et al. conducted proteomic studies on exosomes of HT1376 bladder carcinoma cells by mass spectrometry and selected 18 proteins for western blot and flow cytometry verification.Flow cytometry analysis of protein expression in three patients with BC and four healthy controls showed that the increased expression in BC included MUC1, integrin β1, integrin α6, CD36, CD44, CD10, 5T4, basigin, and CD73. 76 Abbreviations: EMT, epithelial-mesenchymal transition; HLEC, human lymphatic endothelial cell.

F I G U R E 4
Role of urinary and blood exosome-derived biomarkers in tumorigenesis at different stages of bladder cancer.Reproduced under terms of the CC-BY license. 80Copyright 2021, The Authors, published by MDPI.
growth factor receptor pathway, the alpha subunit of GsGTP binding protein, Resistin, and retinoic acidinduced protein. 99Chen et al. evaluated urinary exosomal protein expression in BC patients and inguinal hernia using mass spectrometry.They analyzed 2964 urinary exosomal proteins, of which 168 proteins were differentially expressed in patients with BC and inguinal hernia, and 17 proteins were also upregulated in the patients with BC in Welton et al.Furthermore, this work identified 7 proteins (Apo A1, CD5L, FGA, FGB, FGG, HPR, and HP) that were differentially enriched in low and high grades of BC. 73  -9 of 15

| Exosomes and BC treatment
Due to the small size and histocompatibility of exosomes, the biomolecules inside the exosomes are enveloped by bilayer lipid membranes to avoid degradation by RNases in the circulation. 106Therefore, they can be used as delivery vectors targeting tumor cells 107 (Figure 7).Franzen et al. demonstrated that BC cells interacted with exosomes and internalized through receptor-mediated endocytosis. 108There is precedent for untargeted delivery of drugs through the bladder.However, the adverse effect of untargeted drug delivery on normal epithelial cells surrounding tumor cells is a possible concern.It was found that BC cells absorbed exosomes more readily (≥50 times) than normal urothelial cells. 109Polo-like kinase 1 siRNA was electro-perforated into human embryonic kidney cell-derived exosomes and delivered to BC cells in vitro.The expression of PLK1 mRNA in BC cells was significantly reduced after treatment with the PLK1 siRNA exosomes.This work supported the idea that exosomes could deliver siRNA to the target cells.However, the current research progress in this area is still limited, and more exploration is needed to advance the treatment of exosomes in BC.

| Exosomes as natural nanoparticles in BC
Exosomes are the tiny endosome derived membrane nanovesicles that play an important role in cell communication. 42Currently, nanotechnology has been widely used in tumor-related research, including in vitro diagnosis, in vivo imaging enhancement and drug loading.
As the natural nanoparticles, exosomes exist in most biological fluids.Combined with the advanced biomedical engineering technologies, exosomes show important application potential in the tumor diagnosis and treatment [110][111][112][113] (Figure 8A).Liang et al. constructed an optimized dual-filter microfluidic system, which isolated and purified EVs from urine and quantified them by microchip ELISA.Receiver operating characteristic curve analysis of 16 BC patients and 8 healthy controls showed that the integrated system had a sensitivity of 81.3% and a specificity of 90%, and had the expected potential to improve the clinical diagnosis of BC in the combination with urocytology and cystoscopy 114 (Figure 8B).Inspired by the unique structure of pollen, Li et al. adopted hydrothermal synthesis method to propose a photonic crystal barcode with prickly surface for the capture and screening of multiple exosomes.These pollen-inspired PhC barcodes had extremely high specific surface area and excellent prickly surface nanostructure, which could improve the exosome capture rate and detection sensitivity 115 (Figure 8C).In another work, Wei et al. proposed a multiplexed analysis of urinary exosomes from BC based on Janus magnetic microspheres as barcodes.They were composed of colloidal silica nanoparticles and magnetic nanoparticles co-assembled through a droplet template with both structural color encoding and magnetic responsiveness [116][117][118] (Figure 8D).These schemes not only effectively enrich BC-related exosomes, but also support multiple screening of exosomes with high sensitivity, providing new strategies for laboratory diagnosis based on exosomes.

| CONCLUSION
Exosomes, which contain a plethora of active molecular components, serve as crucial mediators for intercellular communication.Their ubiquitous presence in blood and urine renders them efficacious and auspicious biomarkers for clinical diagnosis and prognostic monitoring of various diseases.Furthermore, the bioactive molecules encapsulated within exosomes hold potential as drug carriers and therapeutic targets.Prior researches have indicated that cystoscopy utilizing tissue sampling is the fundamental method for diagnosing BC, while exosome biomarkers possess a heightened sensitivity and can serve as a valuable and encouraging supplementary diagnostic tool in clinical settings.Notably, the isolation of exosomes derived from tumors in patients with lowgrade tumors highlights the potential importance of exosomes as tumor markers in the absence of conventional clinical symptoms, cystoscopy, or imaging results.Furthermore, the utilization of urinary exosome-derived biomarkers in the clinical detection of BC is advantageous due to the accessibility of urine as a bodily fluid specimen.Although exosome-based non-invasive tools and potential therapies have been employed for clinical diagnosis and prognostic monitoring in certain tumors, the current researches on exosomes in BC is still in its preliminary phases.Despite exosomes being a promising candidate for the diagnosis and prognosis of BC, there remain numerous challenges in the areas of mechanism research and technical advancements.Thus, a comprehensive exploration of the biological characteristics and potential clinical applications of BC exosomes is necessary.

AUTHOR CONTRIBUTIONS
Yefei Zhu and Dagan Zhang conceived the topic of this review; Xiaowei Wei wrote the manuscript and revised the content.

F I G U R E 5 F I G U R E 6
Biogenesis and potential roles of exosome-derived noncoding RNAs in bladder cancer.Reproduced under terms of the CC-BY license.23Copyright 2021, The Authors, published by Frontiers Media S. A. Biological properties of non-coding RNAs.Reproduced under terms of the CC-BY license.23Copyright 2021, The Authors, published by Frontiers Media S. A.

F I G U R E 7
Application prospect of exosomes in urological cancer.Reproduced under terms of the CC-BY license.107Copyright 2019, The Authors, published by John Wiley and Sons. 52 Potential role and mechanism of BC-derived exosomes.
T A B L E 1 Potential of exosomal biomarkers as diagnostic and prognostic tool in BC.
WEI ET AL.