Challenges. Once metastatic, only 5 out of 100 patients are alive at five years. Immune checkpoint inhibitors have demonstrated increasing clinical traction yet conventional imaging such as CT scans struggle to accurately assess tumor response in this treatment context. Serially accessible sources of tumor and host biomarkers could add earlier insights into response this informing timely go / no-go decision making to render precision immunotherapy. Innovation: In light of needed pre-competitive nanotechnology tools for EV investigation to fill temporal and scientific gaps precluding accurate immunotherapy-based treatment monitoring, our group developed and validated a magneto- electrochemical platform without need for EV purification and capable of 96 parallel readouts within 45 minutes. This proposal exploits the bladder's intimate anatomical location within the genitourinary system; urine would thus provide us with rich repositories of bladder cancer EVs. We previously demonstrated feasibility of urine EV testing in kidney transplant rejection. Through increased excited charges generated by inducing plasmonic resonance in gold nanoparticles, we recently accelerated electrochemical reactions within our most current and scalable platform to achieve 12-fold signal increase from EV surface markers. We hypothesize that advancing our nanoplasmonic EV sensor for human urine and optimizing assay protocols to measure intra-EV and surface markers, could identify high value bladder cancer and host biomarkers (protein and mRNA) to better examine their interplay over time and under treatment pressures. We propose three specific aim: AIM 1: To optimize our nanoplasmonic sensor (NEXT) assay and instrumentation for high-throughput urine-based analyses and comprehensive profiling of both surface and intra-EV markers. AIM 2: To employ pre-clinical and banked biospecimens for NEXT analyses to examine profiling performance and inform optimal biomarker panel. AIM 3: Use NEXT to prospectively monitor and profile urinary EVs from patients undergoing immunotherapy-based therapies for bladder cancer. Impact: Our highly complementary group of accomplished investigators bring to bear longstanding expertise and translational experience in EV biology, bioengineering, systems biology, bioinformatics, and clinical oncology. If successful, our urinary nanoplasmonic EV platform would add critical actionable insights into immunotherapy-based treatments of advanced bladder cancers with promise in other prevalent genitourinary cancers such as kidney and prostate.