外泌体与膀胱癌：新的生物标志物和靶点

Exosomes in bladder cancer: novel biomarkers and targets 外泌体与膀胱癌：新的生物标志物和靶点 1 equal first-author 2 equal 3 4 1 1 1 1 corresp Dexin YU, dxyu@ahmu.edu.cn Dexin YU, dxyu@ahmu.edu.cn dxyu@ahmu.edu.cn 3 corresp Zhaofeng LIANG, liangzhaofeng@ujs.edu.cn Zhaofeng LIANG, liangzhaofeng@ujs.edu.cn liangzhaofeng@ujs.edu.cn https://orcid.org/0000-0001-9799-0837 Department of Urology, the Second Affiliated Hospital of Anhui Medical University, Hefei230032, China Department of Urology, the Second Affiliated Hospital of Anhui Medical University, Hefei230032, China Department of Urology, the First Affiliated Hospital of University of Science and Technology of China, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei230001, China Department of Urology, the First Affiliated Hospital of University of Science and Technology of China, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei230001, China Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang212013, China Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang212013, China Department of Child Healthcare, Women and Children Health Hospital of Zhenjiang, Zhenjiang212001, China Department of Child Healthcare, Women and Children Health Hospital of Zhenjiang, Zhenjiang212001, China Exosomes are nanometer-sized vesicles that contain various types of biologically active components, including proteins, nucleic acids, carbohydrates, and lipids, which vary with the type and physiological state of the cell. In recent years, several studies have showed that exosomes can provide new non-invasive diagnostic and prognostic biomarkers in patients affected by cancers, including bladder cancer (BC), and the lipid bilayer membrane structure makes exosomes as promising delivery vehicles for therapeutic applications. Exosomes have the characteristics of high abundance, high stability, tissue specificity, and wide distribution in body fluids, and are secreted as various types by cells in different states, thereby possessing great potential as biomarkers for BC. Herein, we briefly summarize the functions and roles of exosomes in the occurrence and development of BC and the current progress of research on exosomes in BC, while focusing on potential clinical applications of the diagnosis, treatment, and prognosis of BC. 外泌体是纳米大小的囊泡，包含蛋白质、核酸、碳水化合物和脂质等各种生物活性成分，这些成分会随细胞的类型和生理状态而变化。近年来，一些研究表明不但外泌体可以为包括膀胱癌在内的多种肿瘤提供新的无创性诊断和预后生物标志物，而且外泌体脂质双层膜结构可使其成为治疗应用的理想载体。外泌体具有丰度高、稳定性好、组织特异性强，以及在体液中广泛分布等特点，且可以由在不同状态下的多种细胞所分泌。因此，外泌体具有作为膀胱癌诊断和预后生物标志物的巨大潜力。本文就外泌体在膀胱癌发生发展中的作用及其研究现状进行综述，并聚焦于外泌体在膀胱癌诊断、治疗和预后中的临床应用。 Exosome Bladder cancer Function Clinical application Biomarker 外泌体 膀胱癌 功能 临床应用 生物标志物 1 Introduction 1 Bladder cancer (BC) is the second most malignant tumor of the urinary tract, and the fifth most common cancer worldwide ( Liang et al., 2017 Liang et al., 2017 ; Christensen et al., 2019 Christensen et al., 2019 ). It has a significant incidence and mortality rate with annually 80 470 new cases diagnosed and 17 670 deaths recorded worldwide ( Li et al., 2019 Li et al., 2019 ; Siegel et al., 2019 Siegel et al., 2019 ). The cancer in approximately 75% of newly diagnosed BC patients is of the non-muscle invasive type, of which about 70% of cases are confined to the mucosa, 20% involve the submucosa, and 10% are carcinoma in situ. Nevertheless, 1%–45% of non-muscle invasive patients will still develop muscle invasive BC ( Mearini et al., 2017 Mearini et al., 2017 ). In addition, BC has the characteristics of high relapse rate, rapid progress, and poor prognosis ( Wang et al., 2018 Wang et al., 2018 ). The risks of recurrence and malignant progression of BC were found to increase with tumor stage, degree of malignancy, pathological changes, and the presence of carcinoma in situ ( Mearini et al., 2017 Mearini et al., 2017 ). Early diagnosis and monitoring is the key to improve the therapeutic effect in BC. Current BC diagnosis and monitoring techniques usually involve a combination of cystoscopy, histological evaluation, and urine cytology ( Miyamoto et al., 2018 Miyamoto et al., 2018 ; Wang et al., 2018 Wang et al., 2018 ). These methods, however, are uncomfortable, invasive and costly, and have low patient acceptance. Also, due to the lack of sensitivity and specificity, the early diagnostic application value of BC is limited ( Li et al., 2019 Li et al., 2019 ; Zhang et al., 2019 Zhang et al., 2019 ). For example, urine cytology has a weakness of low sensitivity (about 30%–92%), which is even lower (only 30%–40%) for low-grade tumors. The other most common diagnostic method is cystoscopy, which is invasive, painful, and potentially infectious ( Poli et al., 2015 Poli et al., 2015 , 2017 2017 ). All of these deficiencies of current techniques highlight the urgent need to develop novel non-invasive methods and more sensitive biomarkers for BC diagnosis and treatment. The concept of "liquid biopsy" was proposed to characterize the utility of circulating cancer cells as non-invasive cancer biomarker candidates ( Pantel and Alix-Panabières, 2010 Pantel and Alix-Panabières, 2010 ). The significance of this novel approach lies in the fact that it was also found to involve the analysis of circulating subcellular structures, such as exosomes, cell-free DNA, and other substances ( de Palma et al., 2019 de Palma et al., 2019 ). These exosomes, as extracellular vesicles that can be secreted by almost all cells, have attracted increased attention in tumor diagnosis and treatment in recent years. Exosomes are nanometer-sized vesicles sized 30–150 nm that contain a variety of active components, including proteins, nucleic acids, carbohydrates, and lipids, which vary with the type and the physiological state of the cell that secretes the exosome ( Chen et al., 2020 Chen et al., 2020 ). Exosomes have lipid bilayer membranes to protect these active components from RNases and proteases ( Pegtel and Gould, 2019 Pegtel and Gould, 2019 ). It has been reported that the proteins and lipids on the surface of exosomes may vary according to the type of cell of origin, which makes it possible to further isolate and recognize bladder cell-derived exosomes ( Street et al., 2014 Street et al., 2014 ). More importantly, exosomes have been shown to be involved in cellular communication and cancer progression by interacting with target cells and delivering exosomal cargo into target cells ( Franzen et al., 2016 Franzen et al., 2016 ). The possibility to isolate exosomes from body fluids, such as blood and urine, provides a new non-invasive biomarker diagnostic tool in cancer patients, and the lipid bilayer membrane makes exosomes as promising delivery vehicles for therapeutic applications ( Franzen et al., 2016 Franzen et al., 2016 ). In addition, exosomes carry certain substances such as specific microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and proteins, which increase the diagnostic sensitivity and specificity of tumor markers, including those of BC. 2 Role of exosomes in the occurrence and progression of BC 1 In recent years, exosomes have attracted much attention as important messengers of cancer-related intercellular communication. They carry proteins, lipids, nucleic acids, and other biomolecules, such as surface molecules that can induce signal transduction through a receptor‍‒‍ligand interaction. In addition, exosomes can be internalized through endocytosis and/or phagocytosis, or even fuse with the target cell membrane to transport their contents into the cytoplasm ( Lin et al., 2020 Lin et al., 2020 ). Exosomes play a key role in the transmission of proteomics and genetic information to target cells. Evidence has been accumulating to highlight that exosomes from donor cells can alter the state of recipient cells and may play a crucial role in the process of tumor formation and development ( el Andaloussi et al., 2013 el Andaloussi et al., 2013 ; Lin et al., 2020 Lin et al., 2020 ). Several reports have shown that exosomes are highly important in tumorigenesis, progression, and metastasis for their role of transferring carcinogenic molecules ( Zhan et al., 2018 Zhan et al., 2018 ; Yoshida et al., 2019 Yoshida et al., 2019 ). The prognosis of BC patients with clinical lymph node metastasis is extremely poor. Exosomal lncRNA LNMAT2 secreted by BC cells was internalized by human lymphatic endothelial cells, triggering the epigenetic upregulation of Prox1 expression through hnRNPA2B1 recruitment and the increase of H3K4 trimethylation in the Prox1 promoter, which eventually leads to lymphangiogenesis and lymphatic metastasis in BC cells ( Chen et al., 2020 Chen et al., 2020 ). Progressive and metastatic invasive BC generally has a poor prognosis. Exosomal cysteine-rich receptor-like kinase (CRK) receptors secreted by BC cells increase epidermal growth factor receptor 2/3 (ErbB2/3) expression in other BC cells. These kinases/adaptors transfer from host BC cells to recipient cells through exons, inducing vascular leakage and proliferation, and leading to distant metastasis ( Yoshida et al., 2019 Yoshida et al., 2019 ). Intratumoral hypoxia is a tumor microenvironment that is not conducive to the rapid expansion of tumors. However, cancer cells can reshape the surrounding microenvironment, maintain survival and growth, and subsequently promote invasion and metastasis. Hypoxic BC cells were demonstrated to secrete lncRNA urothelial cancer associated 1 (UCA1)-enriched exosomes to reshape the tumor microenvironment and promote tumor growth and progress ( Xue et al., 2017 Xue et al., 2017 ). Exosomal lncRNA UCA1 in human serum can potentially be used as a diagnostic biomarker for BC. As a new type of non-coding RNAs, circRNAs have received a lot of attention in the pathogenesis of BC and other tumors in recent years. For example, Chenet al. (2018) Chen et al. (2018) found that circPRMT5 expression was increased in serum and urine exosomes of BC patients, and was significantly associated with BC metastasis. Exosomes secreted by cancer cells can also affect normal cell function, thereby promoting the occurrence of BC. Wuet al. (2019) Wu et al. (2019) showed that long-term exposure to cancer-derived exosomes induces the malignant transformation of urothelial cells. These transformed cells not only have a range of carcinogenic properties, such as increased genomic instability and invasiveness or loss of cell‍‒cell contact inhibition, but also show changes in morphology and cell structure, including cytoplasmic enlargement, small volume fusion of mitochondria, and others ( Wu et al., 2019 Wu et al., 2019 ). Franzen et al. (2015) Franzen et al. (2015) reported that the expression of mesenchymal markers (including α‍-smooth muscle actin (α‍-SMA), S100A4, and Snail) was increased and the expression of epithelial markers (such as E-cadherin and β‍-catenin) was decreased in urothelial cells treated by BC-derived exosomes, and further showed that BC-derived exosomes increased the migration and invasion characteristics of urothelial cells. Exosomes also can inhibit or delay tumorigenesis and progression by transporting tumor suppressor molecules. MiRNAs can be packaged into exosomes to mediate cell-to-cell communication, affecting cancer cell proliferation, metastasis, and apoptosis. Li et al. (2020) Li et al. (2020) demonstrated that exosomal miR-375-3p from BC cells inhibits cell proliferation and metastasis, and promotes cell apoptosis both in in vivo and in vitro models as a suppressor of BC, which can be used as a potential therapeutic approach for BC. The malignant proliferation and migration ability of BC cells plays a crucial role in the occurrence and development of BC. It was reported that miR-133b plays an important role in inhibiting the proliferation and promoting the apoptosis of BC cells by upregulating dual-specificity phosphatase 1 (DUSP1) expression, and this finding is expected to provide a new direction for BC treatment ( Cai et al., 2020 Cai et al., 2020 ). Exosomes secreted by other cell types have also been found to play a key role in BC occurrence and progression. Exosomes secreted by bone marrow mesenchymal stem cells, for example, significantly contribute to the occurrence and development of BC. Cai et al. (2019) Cai et al. (2019) found that exosomal miR-9-3p derived from bone marrow mesenchymal stem cells inhibits the progression of BC by downregulating endothelial cell-specific molecule 1 (ESM1), which may point to a new potential target for the treatment of BC. Fibroblast cells, as tumor microenvironment cells that respond to cancer cells, are activated and exhibit the characteristics of myofibroblasts, which is favorable for aggressive growth and metastasis. Ringuette Goulet et al. (2018) Ringuette Goulet et al. (2018) demonstrated that BC cells trigger the differentiation of fibroblasts to cancer-associated fibroblasts by exosome-mediated transforming growth factor-‍β (TGF-‍β) transfer and the activation of Sma- and Mad-related protein (SMAD) pathway, which plays a key role in the progression of BC. Exosomes play a highly important role in the occurrence and development of BC, as demonstrated by various studies ( Table 1 Table 1 ). An increasing number of studies are focused on the relationship between exosomes and BC, aiming to determine specific mechanisms of action ( Beckham et al., 2014 Beckham et al., 2014 ; Berrondo et al., 2016 Berrondo et al., 2016 ; Huang et al., 2020 Huang et al., 2020 ; Yin et al., 2020 Yin et al., 2020 ); relevant results will be beneficial for BC diagnosis, treatment, and prognostic judgment. BC: bladder cancer; LncRNA: long non-coding RNA; ErbB2: epidermal growth factor receptor 2; CRK: cysteine-rich receptor-like kinase; UCA1: urothelial cancer associated 1; EDIL-3: endothelial locus 3; SV-HUC: SV-40 immortalized human ureteral epithelial cells; EMT: epithelial-mesenchymal transition; TGF-β: transforming growth factor-β. Sample Role in BC Functional component Reference BC cell exosomes Induce lymphangiogenesis and lymphatic metastasis LncRNA LNMAT2 Chen et al., 2020 Chen et al., 2020 BC cell exosomes Induce vascular growth in premetastatic niches and promote metastasis ErbB2 and CRK Yoshida et al., 2019 Yoshida et al., 2019 BC cell exosomes; serum exosomes from BC patients Remodel tumor microenvironment; promote tumor growth and development LncRNA UCA1 Xue et al., 2017 Xue et al., 2017 Serum and urine exosomes from BC patients Be correlated with tumor metastasis CircPRMT5 Chen et al., 2018 Chen et al., 2018 Urine exosomes from BC patients Promote cancer progression EDIL-3 Beckham et al., 2014 Beckham et al., 2014 BC cell exosomes Induce malignant transformation of SV-HUC cells Wu et al., 2019 Wu et al., 2019 BC cell exosomes; urine exosomes from BC patients Induce EMT and promote invasion in urothelial cells Franzen et al., 2015 Franzen et al., 2015 BC cell exosomes Inhibit proliferation and metastasis; promote apoptosis miR-375-3p Li et al., 2020 Li et al., 2020 BC cell exosomes Inhibit proliferation and promote apoptosis miR-133b Cai et al., 2020 Cai et al., 2020 Bone marrow-derived mesenchymal stem cell exosomes Inhibit BC cell viability, migration, and invasion; induce apoptosis miR-9-3p Cai et al., 2019 Cai et al., 2019 BC cell exosomes Trigger the differentiation of fibroblasts to cancer-associated fibroblasts TGF-β Ringuette Gouletet al., 2018 Ringuette Gouletet al., 2018 3 Application of exosomes in the diagnosis and prognosis of BC 1 As discussed above, exosomes contain essential functional components, including proteins, lipids, nucleic acids, and transcription factors. These functional biomolecules are not randomly packaged in exosomes, but depend on characteristic motifs determining the carried active components under different conditions or in different cell types ( Zhan et al., 2018 Zhan et al., 2018 ; Chen et al., 2020 Chen et al., 2020 ). Studies have confirmed that exosomes secreted by BC cells can be found in the blood and the urine, and functional components, such as proteins and nucleic acids, are stably present in these exosomes ( Zhan et al., 2018 Zhan et al., 2018 ). Therefore, the analysis of exosomes and their components in body fluids is expected to provide valuable clues for BC diagnosis. Exosomes play a role in BC progression by encapsulating lncRNAs to mediate extracellular communication, and thus can be used as potential non-invasive biomarkers for BC detection. The diagnostic accuracy of lncRNA phosphatase and tensin homolog pseudogene 1 (PTENP1) was evaluated by the area under the curve (AUC) of the receiver operating characteristic curve. Specifically, Zheng et al. (2018) Zheng et al. (2018) found that exosomal lncRNA PTENP1 could relatively correctly distinguish BC patients from healthy controls with an AUC value of 0.743; it was further discovered in the study that exosomes secreted by normal cells can transfer lncRNA PTENP1 to BC cells, which inhibit the invasion and migration capabilities, increase the apoptosis, and reduce the malignant biological behavior of BC cells in vitro. Zhan et al. (2018) Zhan et al. (2018) established a group composed of three different expressed lncRNAs (metastasis-associated lung denocarcinoma transcript 1 (MALAT1), prostate cancer-associated non-coding RNA transcript-1 (PCAT-1), and SPRY4-intronic transcript 1 (SPRY4-IT1)) in urine for BC diagnosis, with an AUC value of 0.854, which was significantly higher than that of urine cytology, and in addition these results also demonstrated that exosomal lncRNA PCAT-1 may be an independent prognostic indicator of BC. Zhang et al. (2019) Zhang et al. (2019) identified a panel of three lncRNAs (PCAT-1, up-regulated in bladder cancer 1 (UBC1), and small nucleolar RNA host gene 16 (SNHG16)) providing high diagnostic accuracy for BC, with the AUC of training set and verification set at 0.857 and 0.826, respectively, which were significantly higher than those of urine cytology. The corresponding AUC values of Ta, T1, and T2‒T4 in the panel for BC patients were 0.760, 0.827, and 0.878, respectively. It was also found that recurrence free survival was significantly lower in non-muscle invasive BC patients with higher lncRNA UBC1 expression ( Zhang et al., 2019 Zhang et al., 2019 ). The clinical significance of urine exosomal antisense non-coding RNA in the INK4 locus (ANRIL) and PCAT-1 as biomarkers was evaluated in BC patients classified as T1 or T2 by Abbastabar et al. (2020) Abbastabar et al. (2020) . Compared with normal subjects, the expression of ANRIL and PCAT-1 in BC patient urine exosomes was significantly higher, and the diagnostic efficacies of ANRIL and PCAT-1 detected by AUC were 0.7229 and 0.7292, respectively. The above results suggest that body fluid exosomes may be used as potential important biomarkers for the diagnosis and prognosis of BC. Exosomes are regarded as emerging tools for cancer diagnosis; more and more research is conducted on the application of exosomes in the diagnosis, prognosis, and risk assessment of BC ( Yazarlou et al., 2018a Yazarlou et al., 2018a , 2018b 2018b ; Elsharkawi et al., 2019 Elsharkawi et al., 2019 ; Poli et al., 2020 Poli et al., 2020 ). These studies elaborate on cancer-testis antigens, the secretion of exosomes at different stages of BC, and the active molecules in exosomes such as nucleic acids and proteins, all providing new strategies for BC diagnosis and treatment. 4 Current challenges and future prospects 1 Exosomes provide the possibility of non-invasive observation of bladder cells, enhancing the potential to establish the occurrence and development stage of BC ( Fig. 1 Fig. 1 ). At present, however, the vast majority of exosomal biomarkers are only used as research tools. In order to reach the level of their successful clinical application, it is necessary to utilize the experience of establishing mature biomarkers, focus on supporting decision making in the nursing of BC patients, and eliminate the obstacles of clinical application ( Street et al., 2014 Street et al., 2014 ). Despite the prospect of exosomes being utilized in fields of BC diagnosis, prognosis, and risk assessment research, many unresolved questions remain in experimental and clinical research. Firstly, there is a shortage of efficient and standardized exosome extraction and identification methods. Secondly, the packaging mechanism of active molecules carried by BC-related exosomes is still unclear. Thirdly, the number of functional components carried in exosomes with known biological function and mechanism of action is limited in BC. Furthermore, the conditions under which exosomes play a cancer‍‒‍promoting effect or a cancer‍-suppressing effect are yet to be investigated. It is also unclear whether exosomes can influence the BC cell microenvironment and thus play a role in BC progression. Nevertheless, the successful clinical application of exosomes as targets or drug delivery vehicles in BC requires more detailed and complete experimental data, such as that on safety, targeting ability, efficacy, and many other aspects; a systematic assessment including the cost, accuracy, and repeatability of exosomal biomarker analysis in a large number of BC samples, is also lacking. 5 Conclusions 1 Exosomes contain various types of biologically active components, including proteins, nucleic acids, carbohydrates, and lipids, and have been regarded as emerging tools for cancer diagnosis. The analysis of exosomes and their components from body fluids has a great potential to provide valuable clues for the diagnosis and prognosis of BC. In the current stage, however, knowledge on exosomes regarding the precise mechanisms of secretion, selective transport of functional components, and biological function in BC, is still limited. Nevertheless, we consider that these constraints will eventually be resolved, and novel diagnosis and treatment strategies based on exosomes carrying active molecules will be established to provide efficient tools for BC clinical diagnosis, prognosis, and treatment tasks. References Abbastabar M , Sarfi M , Golestani A , et al. , 2020 . Tumor-derived urinary exosomal long non-coding RNAs as diagnostic biomarkers for bladder cancer . EXCLI J , 19 : 301 - 310 . https://doi.org/10.17179/excli2019-1683 https://doi.org/10.17179/excli2019-1683 Tumor-derived urinary exosomal long non-coding RNAs as diagnostic biomarkers for bladder cancer Beckham CJ , Olsen J , Yin PN , et al. , 2014 . Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression . J Urol , 192 ( 2 ): 583 - 592 . https://doi.org/10.1016/j.juro.2014.02.035 https://doi.org/10.1016/j.juro.2014.02.035 Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression Berrondo C , Flax J , Kucherov V , et al. , 2016 . Expression of the long non-coding RNA hotair correlates with disease progression in bladder cancer and is contained in bladder cancer patient urinary exosomes . PLoS ONE , 11 ( 1 ): e0147236 . https://doi.org/10.1371/journal.pone.0147236 https://doi.org/10.1371/journal.pone.0147236 Expression of the long non-coding RNA hotair correlates with disease progression in bladder cancer and is contained in bladder cancer patient urinary exosomes Cai HZ , Yang XJ , Gao Y , et al. , 2019 . Exosomal microRNA-9-3p secreted from BMSCs downregulates ESM1 to suppress the development of bladder cancer . Mol Ther Nucleic Acids , 18 : 787 - 800 . https://doi.org/10.1016/j.omtn.2019.09.023 https://doi.org/10.1016/j.omtn.2019.09.023 Exosomal microRNA-9-3p secreted from BMSCs downregulates ESM1 to suppress the development of bladder cancer Cai XX , Qu LL , Yang J , et al. , 2020 . Exosome-transmitted microRNA-133b inhibited bladder cancer proliferation by upregulating dual-specificity protein phosphatase 1 . Cancer Med , 9 ( 16 ): 6009 - 6019 . https://doi.org/10.1002/cam4.3263 https://doi.org/10.1002/cam4.3263 Exosome-transmitted microRNA-133b inhibited bladder cancer proliferation by upregulating dual-specificity protein phosphatase 1 Chen CH , Luo YM , He W , et al. , 2020 . Exosomal long noncoding RNA LNMAT2 promotes lymphatic metastasis in bladder cancer . J Clin Invest , 130 ( 1 ): 404 - 421 . https://doi.org/10.1172/JCI130892 https://doi.org/10.1172/JCI130892 Exosomal long noncoding RNA LNMAT2 promotes lymphatic metastasis in bladder cancer Chen X , Chen RX , Wei WS , et al. , 2018 . PRMT5 circular RNA promotes metastasis of urothelial carcinoma of the bladder through sponging miR-30c to induce epithelial-mesenchymal transition . Clin Cancer Res , 24 ( 24 ): 6319 - 6330 . https://doi.org/10.1158/1078-0432.CCR-18-1270 https://doi.org/10.1158/1078-0432.CCR-18-1270 PRMT5 circular RNA promotes metastasis of urothelial carcinoma of the bladder through sponging miR-30c to induce epithelial-mesenchymal transition Christensen E , Birkenkamp-Demtröder K , Sethi H , et al. , 2019 . Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma . J Clin Oncol , 37 ( 18 ): 1547 - 1557 . https://doi.org/10.1200/JCO.18.02052 https://doi.org/10.1200/JCO.18.02052 Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma de Palma G , di Lorenzo VF , Krol S , et al. , 2019 . Urinary exosomal shuttle RNA: promising cancer diagnosis biomarkers of lower urinary tract . Int J Biol Markers , 34 ( 2 ): 101 - 107 . https://doi.org/10.1177/1724600819827023 https://doi.org/10.1177/1724600819827023 Urinary exosomal shuttle RNA: promising cancer diagnosis biomarkers of lower urinary tract el Andaloussi S , Mäger I , Breakefield XO , et al. , 2013 . Extracellular vesicles: biology and emerging therapeutic opportunities . Nat Rev Drug Discov , 12 ( 5 ): 347 - 357 . https://doi.org/10.1038/nrd3978 https://doi.org/10.1038/nrd3978 Extracellular vesicles: biology and emerging therapeutic opportunities Elsharkawi F , Elsabah M , Shabayek M , et al. , 2019 . Urine and serum exosomes as novel biomarkers in detection of bladder cancer . Asian Pac J Cancer Prev , 20 ( 7 ): 2219 - 2224 . https://doi.org/10.31557/APJCP.2019.20.7.2219 https://doi.org/10.31557/APJCP.2019.20.7.2219 Urine and serum exosomes as novel biomarkers in detection of bladder cancer Franzen CA , Blackwell RH , Todorovic V , et al. , 2015 . Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes . Oncogenesis , 4 ( 8 ): e163 . https://doi.org/10.1038/oncsis.2015.21 https://doi.org/10.1038/oncsis.2015.21 Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes Franzen CA , Blackwell RH , Foreman KE , et al. , 2016 . Urinary exosomes: the potential for biomarker utility, intercellular signaling and therapeutics in urological malignancy . J Urol , 195 ( 5 ): 1331 - 1339 . https://doi.org/10.1016/j.juro.2015.08.115 https://doi.org/10.1016/j.juro.2015.08.115 Urinary exosomes: the potential for biomarker utility, intercellular signaling and therapeutics in urological malignancy Huang CS , Ho JY , Chiang JH , et al. , 2020 . Exosome-derived LINC00960 and LINC02470 promote the epithelial-mesenchymal transition and aggressiveness of bladder cancer cells . Cells , 9 ( 6 ): 1419 . https://doi.org/10.3390/cells9061419 https://doi.org/10.3390/cells9061419 Exosome-derived LINC00960 and LINC02470 promote the epithelial-mesenchymal transition and aggressiveness of bladder cancer cells Li Q , Wang HL , Peng HR , et al. , 2019 . MicroRNAs: key players in bladder cancer . Mol Diagn Ther , 23 ( 5 ): 579 - 601 . https://doi.org/10.1007/s40291-019-00410-4 https://doi.org/10.1007/s40291-019-00410-4 MicroRNAs: key players in bladder cancer Li Q , Huyan T , Cai SN , et al. , 2020 . The role of exosomal miR-375-3p: a potential suppressor in bladder cancer via the Wnt/β-catenin pathway . FASEB J , 34 ( 9 ): 12177 - 12196 . https://doi.org/10.1096/fj.202000347R https://doi.org/10.1096/fj.202000347R The role of exosomal miR-375-3p: a potential suppressor in bladder cancer via the Wnt/β-catenin pathway Liang ZF , Lu L , Mao JH , et al. , 2017 . Curcumin reversed chronic tobacco smoke exposure induced urocystic EMT and acquisition of cancer stem cells properties via Wnt/β-catenin . Cell Death Discov , 8 ( 10 ): e3066 . https://doi.org/10.1038/cddis.2017.452 https://doi.org/10.1038/cddis.2017.452 Curcumin reversed chronic tobacco smoke exposure induced urocystic EMT and acquisition of cancer stem cells properties via Wnt/β-catenin Lin F , Yin HB , Li XY , et al. , 2020 . Bladder cancer cell-secreted exosomal miR-21 activates the PI3K/AKT pathway in macrophages to promote cancer progression . Int J Oncol , 56 ( 1 ): 151 - 164 . https://doi.org/10.3892/ijo.2019.4933 https://doi.org/10.3892/ijo.2019.4933 Bladder cancer cell-secreted exosomal miR-21 activates the PI3K/AKT pathway in macrophages to promote cancer progression Mearini E , Poli G , Cochetti G , et al. , 2017 . Expression of urinary miRNAs targeting NLRs inflammasomes in bladder cancer . Onco Targets Ther , 10 : 2665 - 2673 . https://doi.org/10.2147/OTT.S132680 https://doi.org/10.2147/OTT.S132680 Expression of urinary miRNAs targeting NLRs inflammasomes in bladder cancer Miyamoto DT , Mouw KW , Feng FY , et al. , 2018 . Molecular biomarkers in bladder preservation therapy for muscle-invasive bladder cancer . Lancet Oncol , 19 ( 12 ): e683 - e695 . https://doi.org/10.1016/S1470-2045(18)30693-4 https://doi.org/10.1016/S1470-2045(18)30693-4 Molecular biomarkers in bladder preservation therapy for muscle-invasive bladder cancer Pantel K , Alix-Panabières C , 2010 . Circulating tumour cells in cancer patients: challenges and perspectives . Trends Mol Med , 16 ( 9 ): 398 - 406 . https://doi.org/10.1016/j.molmed.2010.07.001 https://doi.org/10.1016/j.molmed.2010.07.001 Circulating tumour cells in cancer patients: challenges and perspectives Pegtel DM , Gould SJ , 2019 . Exosomes . Annu Rev Biochem , 88 : 487 - 514 . https://doi.org/10.1146/annurev-biochem-013118-111902 https://doi.org/10.1146/annurev-biochem-013118-111902 Exosomes Poli G , Brancorsini S , Cochetti G , et al. , 2015 . Expression of inflammasome-related genes in bladder cancer and their association with cytokeratin 20 messenger RNA . Urol Oncol Semin Orig Invest , 33 ( 12 ): 505.e1 - 505.e7 . https://doi.org/10.1016/j.urolonc.2015.07.012 https://doi.org/10.1016/j.urolonc.2015.07.012 Expression of inflammasome-related genes in bladder cancer and their association with cytokeratin 20 messenger RNA Poli G , Cochetti G , Boni A , et al. , 2017 . Characterization of inflammasome-related genes in urine sediments of patients receiving intravesical BCG therapy . Urol Oncol Semin Orig Invest , 35 ( 12 ): 674.e19 - 674.e24 . https://doi.org/10.1016/j.urolonc.2017.08.004 https://doi.org/10.1016/j.urolonc.2017.08.004 Characterization of inflammasome-related genes in urine sediments of patients receiving intravesical BCG therapy Poli G , Egidi MG , Cochetti G , et al. , 2020 . Relationship between cellular and exosomal miRNAs targeting NOD-like receptors in bladder cancer: preliminary results . Minerva Urol Nefrol , 72 ( 2 ): 207 - 213 . https://doi.org/10.23736/S0393-2249.19.03297-1 https://doi.org/10.23736/S0393-2249.19.03297-1 Relationship between cellular and exosomal miRNAs targeting NOD-like receptors in bladder cancer: preliminary results Ringuette Goulet C , Bernard G , Tremblay S , et al. , 2018 . Exosomes induce fibroblast differentiation into cancer-associated fibroblasts through TGFβ signaling . Mol Cancer Res , 16 ( 7 ): 1196 - 1204 . https://doi.org/10.1158/1541-7786.MCR-17-0784 https://doi.org/10.1158/1541-7786.MCR-17-0784 Exosomes induce fibroblast differentiation into cancer-associated fibroblasts through TGFβ signaling Siegel RL , Miller KD , Jemal A , 2019 . Cancer statistics, 2019 . CA Cancer J Clin , 69 ( 1 ): 7 - 34 . https://doi.org/10.3322/caac.21551 https://doi.org/10.3322/caac.21551 Cancer statistics, 2019 Street JM , Yuen PST , Star RA , 2014 . Bioactive exosomes: possibilities for diagnosis and management of bladder cancer . J Urol , 192 ( 2 ): 297 - 298 . https://doi.org/10.1016/j.juro.2014.05.050 https://doi.org/10.1016/j.juro.2014.05.050 Bioactive exosomes: possibilities for diagnosis and management of bladder cancer Wang JS , Yang K , Yuan WX , et al. , 2018 . Determination of serum exosomal H19 as a noninvasive biomarker for bladder cancer diagnosis and prognosis . Med Sci Monit , 24 : 9307 - 9316 . https://doi.org/10.12659/MSM.912018 https://doi.org/10.12659/MSM.912018 Determination of serum exosomal H19 as a noninvasive biomarker for bladder cancer diagnosis and prognosis Wu CH , Silvers CR , Messing EM , et al. , 2019 . Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells . J Biol Chem , 294 ( 9 ): 3207 - 3218 . https://doi.org/10.1074/jbc.RA118.006682 https://doi.org/10.1074/jbc.RA118.006682 Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells Xue M , Chen W , Xiang A , et al. , 2017 . Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1 . Mol Cancer , 16 : 143 . https://doi.org/10.1186/s12943-017-0714-8 https://doi.org/10.1186/s12943-017-0714-8 Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1 Yazarlou F , Mowla SJ , Kholghi Oskooei V , et al. , 2018a . Urine exosome gene expression of cancer-testis antigens for prediction of bladder carcinoma . Cancer Manage Res , 10 : 5373 - 5381 . https://doi.org/10.2147/CMAR.S180389 https://doi.org/10.2147/CMAR.S180389 Urine exosome gene expression of cancer-testis antigens for prediction of bladder carcinoma Yazarlou F , Modarressi MH , Mowla SJ , et al. , 2018b . Urinary exosomal expression of long non-coding RNAs as diagnostic marker in bladder cancer . Cancer Manage Res , 10 : 6357 - 6365 . https://doi.org/10.2147/CMAR.S186108 https://doi.org/10.2147/CMAR.S186108 Urinary exosomal expression of long non-coding RNAs as diagnostic marker in bladder cancer Yin XB , Zheng XP , Liu M , et al. , 2020 . Exosomal miR-663b targets Ets2-repressor factor to promote proliferation and the epithelial-mesenchymal transition of bladder cancer cells . Cell Biol Int , 44 ( 4 ): 958 - 965 . https://doi.org/10.1002/cbin.11292 https://doi.org/10.1002/cbin.11292 Exosomal miR-663b targets Ets2-repressor factor to promote proliferation and the epithelial-mesenchymal transition of bladder cancer cells Yoshida K , Tsuda M , Matsumoto R , et al. , 2019 . Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer . Cancer Sci , 110 ( 7 ): 2119 - 2132 . https://doi.org/10.1111/cas.14080 https://doi.org/10.1111/cas.14080 Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer Zhan Y , Du LT , Wang LS , et al. , 2018 . Expression signatures of exosomal long non-coding RNAs in urine serve as novel non-invasive biomarkers for diagnosis and recurrence prediction of bladder cancer . Mol Cancer , 17 : 142 . https://doi.org/10.1186/s12943-018-0893-y https://doi.org/10.1186/s12943-018-0893-y Expression signatures of exosomal long non-coding RNAs in urine serve as novel non-invasive biomarkers for diagnosis and recurrence prediction of bladder cancer Zhang SJ , Du LT , Wang LS , et al. , 2019 . Evaluation of serum exosomal lncRNA-based biomarker panel for diagnosis and recurrence prediction of bladder cancer . J Cell Mol Med , 23 ( 2 ): 1396 - 1405 . https://doi.org/10.1111/jcmm.14042 https://doi.org/10.1111/jcmm.14042 Evaluation of serum exosomal lncRNA-based biomarker panel for diagnosis and recurrence prediction of bladder cancer Zheng R , Du ML , Wang XW , et al. , 2018 . Exosome-transmitted long non-coding RNA PTENP 1 suppresses bladder cancer progression . Mol Cancer , 17 ( 1 ): 143 . https://doi.org/10.1186/s12943-018-0880-3 https://doi.org/10.1186/s12943-018-0880-3 Exosome-transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression Hao GENG, Qingchen ZHOU, and Zhaofeng LIANG designed research and wrote the paper. Wenhao GUO, Ling LU, Liangkuan BI, Yi WANG, and Jie MIN participated in data collection and analysis. Dexin YU and Zhaofeng LIANG participated in the writing and revisions. All authors have read and approved the final manuscript. The present work was supported by the National Natural Science Foundation of China (No. 8157100782), the Natural Science Research in Universities of Anhui Province (No. KJ2018A0209), the Foundation for Excellent Young Teachers of Jiangsu University, Zhenjiang Social Development Guidance Project (No. FZ2019038), the Research and Practice Innovation Program for Graduate Students of Jiangsu Province (No. KYCX20_3089), and the Experimental Animal Center of Jiangsu University, China. 10.1631/jzus.B2000711 1673-1581（2021）05-0341-07 2020-11-03 2020-12-30 2021-05-15 2022-05-09 2021-04-12 © Zhejiang University Press 2021 Hao GENG, Qingchen ZHOU, Wenhao GUO, Ling LU, Liangkuan BI, Yi WANG, Jie MIN, Dexin YU, and Zhaofeng LIANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 22