Study Protocol for Evaluating the Efficacy of Postoperative Adjuvant Chemotherapy Drugs for Bladder Cancer Based on Organoid Technology.

1. Research Background (Presenting the Rationale and Significance of This Study Based on Current Domestic and International Research)

Bladder cancer is a malignant tumor originating from the bladder urothelium and ranks first in incidence among urogenital tumors in China. Among these, urothelial carcinoma is the most common, accounting for over 90% of bladder cancer cases [1]. Clinically, based on the depth of tumor invasion into the bladder wall and prognostic characteristics, bladder cancer is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). MIBC constitutes approximately 20% of newly diagnosed bladder cancer cases [2-3], but its prognosis is significantly worse than that of NMIBC, with a 5-year overall survival (OS) rate of about 60%-70% [4-5].

For patients with T2~T4a stage disease without distant metastasis, radical cystectomy (RC) is typically the standard treatment [2-3]. However, for high-risk patients with pT3-pT4 disease and/or pN+ M0 status, the 5-year survival rate following RC is only 25-35% [2, 6-8]. Therefore, combining systemic therapy with surgery plays a crucial role in reducing tumor progression rates. Multiple retrospective studies have shown that adjuvant chemotherapy after RC can delay recurrence and improve overall survival, providing clinical benefit. Skinner et al. [9] reported the first randomized trial, which demonstrated benefits for the adjuvant chemotherapy group in terms of disease-free survival (DFS) (51% vs. 34%) and overall survival (44% vs. 39%, n.s.) at 5 years. Another prospective randomized trial reported by Freiha et al. investigated the benefit of adjuvant CMV chemotherapy after RC in 50 patients with pT3b-T4, N0/+, M0 disease [10]. Consequently, guidelines recommend adjuvant chemotherapy for high-risk patients with pT3~pT4 disease or lymph node metastasis [11]. Currently, commonly used adjuvant chemotherapy drugs for bladder cancer include: cisplatin, gemcitabine, methotrexate, vinblastine, doxorubicin, paclitaxel... However, the variety of chemotherapy drugs and differing administration methods lead to treatment regimen selection often relying on physicians' clinical experience or specific research findings, lacking a unified standard, and being characterized by empiricism and randomness. Furthermore, some patients exhibit intrinsic resistance to certain chemotherapeutic agents. By the time clinical assessment confirms a patient's insensitivity to the applied drug, severe toxic side effects have often already occurred, potentially even leading to multi-drug resistance (MDR), thereby depriving the patient of the opportunity to choose alternative treatments. Therefore, how to evaluate chemotherapy response, avoid primarily resistant drugs, and directly select highly sensitive agents to achieve individualized chemotherapy has become a research hotspot.

The realization of precise cancer treatment heavily relies on drug sensitivity testing. Through precise and individualized chemotherapy drug screening experiments, the most effective and least toxic treatment regimen can be identified for each patient before therapy begins. Developing patient-specific chemotherapy plans represents a new research direction for achieving precision treatment in bladder cancer and improving chemotherapy efficacy. Previously, the more commonly used preclinical models were traditional tumor cell lines and patient-derived xenograft (PDX) models. Tumor cell line culture is simple but insufficient to simulate the growth state of tumor cells within the patient's body, and drugs screened using this system have low clinical application value. Although PDX models can simulate in vivo tumor characteristics and preserve the tumor microenvironment, they have significant limitations, such as relatively low stable engraftment rates, long modeling and evaluation cycles (six months to one year), being time-consuming and labor-intensive, making them difficult to generate and utilize for high-throughput drug screening [12-15]. Therefore, to develop more personalized treatment and prevention strategies tailored to an individual's unique genetic, environmental, and lifestyle characteristics, minimizing risks and optimizing medical intervention outcomes, it is imperative to develop drug sensitivity testing models that can simulate the heterogeneity and complexity of bladder cancer.

Organoids, specifically Patient-Derived Organoids (PDOs), are three-dimensional organotypic structures formed through the self-assembly of stem cells in vitro. They can differentiate into multiple organ-specific cell types and exhibit cell-cell interactions, cell-extracellular matrix interactions, and spatial organization, thereby recapitulating key functions and structures of real human organs in vitro while maintaining stable phenotypic and genetic characteristics. Compared to two-dimensional tumor cell lines and PDX models, tumor organoids can be cultured directly from a patient's own tissue. These organoids can effectively replicate key properties of the primary tumor, preserve the pathological morphology and biological mechanisms of the patient's tissue, retain tumor heterogeneity and a more authentic tumor microenvironment, and have the advantage of a short growth cycle [16]. This facilitates their use for sensitivity testing of chemotherapy drugs, molecularly targeted agents, anti-tumor antibodies, etc., in clinical cancer patients, predicting patient response to drugs, and holds potential for assisting clinical treatment decision-making.