Eisuke Tomiyama1, Kazutoshi Fujita2,3, Kyosuke Matsuzaki1, Ryohei Narumi4, Akinaru Yamamoto1, Toshihiro Uemura1, Gaku Yamamichi1, Yoko Koh1, Makoto Matsushita1, Yujiro Hayashi1, Mamoru Hashimoto5, Eri Banno5, Taigo Kato1, Koji Hatano1, Atsunari Kawashima1, Motohide Uemura1, Ryo Ukekawa6, Tetsuya Takao7, Shingo Takada8, Hirotsugu Uemura5, Jun Adachi4, Takeshi Tomonaga4, Norio Nonomura1. 1. Department of Urology, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Osaka, 565-0871, Japan. 2. Department of Urology, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Osaka, 565-0871, Japan. kfujita@med.kindai.ac.jp. 3. Department of Urology, Kindai University Faculty of Medicine, Ohno-Higashi, Osaka-Sayama, Osaka, 589-8511, Japan. kfujita@med.kindai.ac.jp. 4. Laboratory of Proteome Research, National Institutes of Biomedical Innovation, Health and Nutrition, Saito-Asagi, Ibaraki, Osaka, 567-0085, Japan. 5. Department of Urology, Kindai University Faculty of Medicine, Ohno-Higashi, Osaka-Sayama, Osaka, 589-8511, Japan. 6. FUJIFILM Wako Pure Chemical Corporation, Takata-cho, Amagasaki, Hyogo, 661-0963, Japan. 7. Department of Urology, Osaka General Medical Center, Bandai-higashi, Sumiyoshi-ku, Osaka, 558-8558, Japan. 8. Department of Urology, Osaka Police Hospital, Kitayama-cho, Tennoji-ku, Osaka, 543-0035, Japan.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: We established EV-EphA2-CD9 ELISA for uEV-EphA2 detection for the non-invasive early clinical diagnosis of bladder cancer.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: We established EV-EphA2-CD9 ELISA for uEV-EphA2 detection for the non-invasive early clinical diagnosis of bladder cancer.
Authors: Bernd J Schmitz-Dräger; Michael Droller; Vinata B Lokeshwar; Yair Lotan; M'Liss A Hudson; Bas W van Rhijn; Michael J Marberger; Yves Fradet; George P Hemstreet; Per-Uno Malmstrom; Osamu Ogawa; Pierre I Karakiewicz; Shahrokh F Shariat Journal: Urol Int Date: 2014-12-10 Impact factor: 2.089