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Molecular Diagnostics

EphA2 on urinary extracellular vesicles as a novel biomarker for bladder cancer diagnosis and its effect on the invasiveness of bladder cancer



Urinary extracellular vesicles (uEVs) secreted from bladder cancer contain cancer-specific proteins that are potential diagnostic biomarkers. We identified and evaluated a uEV-based protein biomarker for bladder cancer diagnosis and analysed its functions.


Biomarker candidates, selected by shotgun proteomics, were validated using targeted proteomics of uEVs obtained from 49 patients with and 48 individuals without bladder cancer, including patients with non-malignant haematuria. We developed an enzyme-linked immunosorbent assay (ELISA) for quantifying the uEV protein biomarker without ultracentrifugation and evaluated urine samples from 36 patients with and 36 patients without bladder cancer.


Thirteen membrane proteins were significantly upregulated in the uEVs from patients with bladder cancer in shotgun proteomics. Among them, eight proteins were validated by target proteomics, and Ephrin type-A receptor 2 (EphA2) was the only protein significantly upregulated in the uEVs of patients with bladder cancer, compared with that of patients with non-malignant haematuria. The EV-EphA2-CD9 ELISA demonstrated good diagnostic performance (sensitivity: 61.1%, specificity: 97.2%). We showed that EphA2 promotes proliferation, invasion and migration and EV-EphA2 promotes the invasion and migration of bladder cancer cells.


We established EV-EphA2-CD9 ELISA for uEV-EphA2 detection for the non-invasive early clinical diagnosis of bladder cancer.

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Fig. 1: Identification and validation of urinary EV-EphA2 as a biomarker for bladder cancer diagnosis.
Fig. 2: Development of EV-EphA2-CD9 ELISA and evaluation of bladder cancer diagnostic performance.
Fig. 3: Functional analysis and immunohistochemical staining of EphA2 in bladder cancer cells.
Fig. 4: The effect of EV-EphA2 on the invasion and migration potential of bladder cancer cells.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.


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We thank Sadamu Ozaki, Takahiro Nishibu and Shigeaki Nakazawa for their help in constructing the ELISA and Mutsumi Tsuchiya and Atsuko Yasumoto for their technical support.


This study was supported by the Japan Society for the Promotion of Science under KAKENHI (grant numbers: 17K16788 and 20K18140) and the Japan Agency for Medical Research and Development under Translational Research (grant number: A102).

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Authors and Affiliations



Study conception and design: ET and KF; data acquisition: ET, KM and RN; data analysis: ET, KM, RN, AY, TU, GY, YK, MM and YH; provision of resources: ET, KM, YH, MH, EB, RU, TT and ST; drafting the manuscript and figures: ET and KF; reviewing the manuscript: TK, KH, AK, MU, HU, JA, TT and NN. All authors have read and approved the final draft for submission.

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Correspondence to Kazutoshi Fujita.

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The authors declare no competing interests.

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Approval was obtained from the Institutional Review Board (Osaka University Hospital Institutional Review Board, Protocol Number: 13397-11) before initiating the study, and all patients provided written informed consent. The study was performed in accordance with the Declaration of Helsinki.

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Tomiyama, E., Fujita, K., Matsuzaki, K. et al. EphA2 on urinary extracellular vesicles as a novel biomarker for bladder cancer diagnosis and its effect on the invasiveness of bladder cancer. Br J Cancer (2022).

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