Tomiyama Eisuke, Fujita Kazutoshi, Matsuzaki Kyosuke, Narumi Ryohei, Matsushita Makoto, Hayashi Yujiro, Hashimoto Mamoru, Kato Taigo, Hatano Koji, Kawashima Atsunari, Minami Takafumi, Takao Tetsuya, Takada Shingo, Uemura Hirotsugu, Adachi Jun, Tomonaga Takeshi, Nonomura Norio
Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Department of Urology, Kindai University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
Cancer Med. 2025 May;14(10):e70964. doi: 10.1002/cam4.70964.
Urinary extracellular vesicles (uEVs), directly secreted from bladder cancer (BCa) cells, harbor potential for biomarker discovery.
We performed proteomic analysis to explore and validate uEV-based diagnostic markers for BCa, with a focus on cytoplasmic EV proteins. Among the 1960 proteins identified by shotgun proteomics (tandem mass tag-labeled liquid chromatography-tandem mass spectrometry [LC-MS/MS]) of uEVs from seven patients with BCa and four healthy individuals, 17 cytoplasmic EV proteins were significantly elevated in the patients' urine (fold change > 1.5; p < 0.05). These 17 proteins were subsequently validated using targeted proteomics (selected reaction monitoring/multiple reaction monitoring) using urine samples from 49 and 48 patients with and without BCa, respectively, including those with non-BCa hematuria.
Ten measurable EV proteins remained significantly elevated in the urine of patients with BCa, with EV-EIF2S1 demonstrating the best diagnostic performance (area under the receiver operating characteristic [ROC] curve [AUC] [ROCAUC]: 0.83). Additionally, EV-EIF2S1 distinguished patients with BCa from those without BCa and hematuria in a suitable manner (ROCAUC: 0.92). Functional analysis of EIF2S1 in the BCa cell lines (T24 and 5637) showed that EIF2S1 knockdown markedly inhibited cell proliferation and induced cell cycle arrest and apoptosis, suggesting its essentiality for BCa cell growth and survival.
This study identified EV-EIF2S1 as a novel, uEV-based BCa diagnostic marker and demonstrated its functional significance in BCa cell growth and survival.
膀胱癌（BCa）细胞直接分泌的尿液细胞外囊泡（uEVs）具有发现生物标志物的潜力。
我们进行了蛋白质组学分析，以探索和验证基于uEVs的BCa诊断标志物，重点关注细胞质EV蛋白。在通过鸟枪法蛋白质组学（串联质量标签标记液相色谱-串联质谱[LC-MS/MS]）对7例BCa患者和4例健康个体的uEVs鉴定出的1960种蛋白质中，17种细胞质EV蛋白在患者尿液中显著升高（倍数变化>1.5；p<0.05）。随后，使用靶向蛋白质组学（选择反应监测/多反应监测）分别对49例和48例BCa患者及非BCa患者（包括非BCa血尿患者）的尿液样本对这17种蛋白质进行了验证。
10种可测量的EV蛋白在BCa患者尿液中仍显著升高，其中EV-EIF2S1表现出最佳诊断性能（受试者操作特征[ROC]曲线下面积[AUC][ROCAUC]：0.83）。此外，EV-EIF2S1能以合适的方式区分BCa患者与无BCa和血尿的患者（ROCAUC：0.92）。对BCa细胞系（T24和5637）中EIF2S1的功能分析表明，EIF2S1敲低显著抑制细胞增殖并诱导细胞周期停滞和凋亡，提示其对BCa细胞生长和存活至关重要。
本研究确定EV-EIF2S1为一种基于uEVs的新型BCa诊断标志物，并证明了其在BCa细胞生长和存活中的功能意义。
