A Research Protocol for Evaluating the Efficacy of Perfused Chemotherapeutic Agents for Bladder Cancer Based on Organoid technologyKYLL-202407-046-1

1. Background Bladder cancer is a malignant tumor originating from the uroepithelium of the bladder, accounting for the first place in the incidence of genitourinary tumors in China. Among them, bladder uroepithelial cancer is the most common, accounting for more than 90% of bladder cancers. According to the depth of tumor invasion into the bladder and the prognostic characteristics, bladder cancer is clinically classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), with NMIBC as the main type, accounting for about 75% of all bladder cancer patients .

NMIBC is usually treated with Transurethral Resection of Bladder Tumor (TURBt). Despite being in the early stages of the disease, NMIBC has a high recurrence rate, with studies reporting a 5-year recurrence rate of approximately 70% . Therefore, postoperative bladder perfusion chemotherapy has become a key therapeutic measure to prevent recurrence of bladder cancer.For more than 60 years, people have been exploring more effective bladder perfusion drugs and methods.In 1961, Thiotepa was used for bladder perfusion therapy, and gained a certain degree of efficacy in preventing the recurrence of superficial tumors of the bladder.In 1976, Morales believed that bladder cancer was related to immune deficiencies, and accordingly BCG bladder perfusion therapy has been extremely successful, creating a new way of immunotherapy for bladder tumors, which has been widely valued and is still considered the most effective bladder perfusion drug. However, BCG instillation can cause serious side effects, including urethral and prostatic granulomas, bladder irritation, fever, hematuria and other local manifestations and systemic influenza-like symptoms, resulting in intolerance and interruption of treatment in some patients, which has limited the wide clinical application of BCG. Studies have shown that intravesical instillation of chemotherapeutic agents in the bladder can reduce the near-term recurrence rate of superficial bladder cancer by about 15%-20% and the long-term recurrence rate by about 6% . Currently there are many choices of chemotherapeutic agents in bladder instillation, including (1) pirenzolubicin; (2) gemcitabine; (3) mitomycin; (4) epirubicin; (5) doxorubicin; (6) alternating bladder-infusion chemotherapy with gemcitabine and pirenzolubicin; and (7) other combined-infusion chemotherapies. However, due to the wide variety of chemotherapeutic drugs, drug selection mostly relies on clinical experience or certain research results, there is no uniform standard, and it is blind, empirical, and randomized. Chinese urological disease diagnosis and treatment guidelines also did not provide the selection of perfusion drugs, and individual patients for some chemotherapeutic drugs show natural resistance, when the clinic determines that the patient is not sensitive to the application of the drug, the drug has produced serious toxic side effects, and even lead to the phenomenon of multi-drug resistance (multi-drug resistance, MDR) produced, so that the clinical The recurrence rate is still as high as 36%-44%, and at the same time, the tumor of this group of patients progresses rapidly, losing the opportunity to re-select the treatment method. Therefore, how to avoid the selection of primary drug-resistant drugs and directly choose drugs with high sensitivity to achieve individualized chemotherapy has become a hot spot in the research of bladder perfusion chemotherapy.

The realization of precision tumor therapy greatly depends on the detection of drug sensitivity. Through precise individualized chemotherapeutic drug screening experiments, the most effective and least toxic therapeutic regimen can be judged for each patient before the start of treatment, so as to propose a chemotherapeutic regimen for a single patient is a new direction of research to realize the precision treatment of bladder cancer and to improve the efficiency of bladder cancer chemotherapy. Previously, the more commonly used preclinical models are traditional tumor cell lines and human-derived tumor tissue xenografts (PDX). Tumor cell line culture method is simple but insufficient to simulate the growth state of tumor cells in patients, and the drugs screened by its drug screening system have low value for clinical application.PDX, although it can simulate in vivo tumor characteristics and preserve the tumor microenvironment, has obvious limitations such as low stable tumorigenicity, long modeling and evaluation period (half a year to one year), time-consuming and laborious, and it is difficult to generate and use for high-throughput drug screening. Therefore, the development of drug-sensitivity assay models that can mimic the heterogeneity and complexity of bladder cancer has become necessary in order to develop more personalized therapeutic and preventive strategies to minimize risk and optimize the effectiveness of medical interventions by targeting the unique genetic, environmental and lifestyle characteristics of individuals.

Patient-Derived Organoids (PDO) are 3D organoid structures formed by stem cells self-assembled in vitro, which can be differentiated into multiple organ-specific cell types and can exhibit cell-cell and cell-surrounding matrix interactions and spatial location patterns, recreating in vitro some of the key functions and structures of real organs, and having a stable phenotype. structures with stable phenotypic and genetic characteristics. Compared with two-dimensional tumor cell lines and PDX, tumor organoids can be cultured directly using the patient's own tissues, and at the same time, these organoids can well replicate some of the key characteristics of the primary tumors, retain the pathomorphology and biological mechanisms of the patient's tissues, and preserve the heterogeneity of the tumor tissues and a more realistic tumor microenvironment, as well as having a short growth cycle. It is helpful for its use in clinical cancer patients for drug sensitivity testing of radiotherapy drugs, molecular targeting drugs, anti-tumor antibodies and other drugs, to predict the patient's responsiveness to drugs, with the potential to assist in clinical treatment decisions.

In 2018, Science reported a study on the use of metastatic gastrointestinal tumor-like organs for drug sensitivity testing, which comparatively analyzed the differences in sensitivity between 21 clinical patients and their corresponding PDOs to a series of targeted and chemotherapeutic drugs, and the results showed strong consistency between the two. In comparison with the actual patient outcomes, the PDOs were well predicted (sensitivity 100%, specificity 93%, positive predictive value 88%, and negative predictive value 100%).20 In 2020, Yao Y et al constructed 96 rectal cancer organoids using biopsies from 112 cases of locally advanced rectal cancer, and selected 80 of them to test their response to radiotherapy, and the results showed that rectal cancer organoids were sensitive to radiotherapy and the patient's clinical response. The results showed that the sensitivity of rectal cancer organoids to radiotherapy was highly consistent with the clinical response of patients (sensitivity 78%, specificity 92%, accuracy 84%). Subsequently, in tumors such as gastric cancer and breast cancer, the concordance between PDO and tumor patients' response to drugs was also found. Yan HHN et al constructed a gastric cancer organoid library using tumor tissues, paracancerous tissues and lymph node metastases from 34 gastric cancer patients. Two of them developed tumor metastases and underwent a combination of cisplatin and 5-FU after surgery, both of which responded well. The other case received chemotherapy before surgery and did not respond to capecitabine after surgery. Examination of the sensitivity of the corresponding compounds of PDO in these three cases showed that the drug sensitivity of PDO was in perfect agreement with the clinical response of each patient.Guillen KP et al constructed PDX and PDO using tumor samples from endocrine therapy-resistant, relapsed, and metastatic breast cancer patients, and these samples were examined histomorphologically, genomically, and drug sensitivity. The results showed that both breast cancer PDX and PDO were highly reductive of the histobiological and genomic properties of their source tumors, and both responded consistently to anti-tumor drugs. A patient with triple-negative breast cancer in stage IIA in this study developed liver metastases about 1 year after undergoing preoperative chemotherapy and surgical treatment. The investigators subjected the constructs PDO and PDX to ex vivo drug sensitivity testing and found that the microtubule inhibitor eribulin had the best therapeutic effect. Based on this result, patients were instructed to undergo treatment with eribulin, which resulted in complete remission of liver metastases for nearly 5 months after dosing. These studies confirm to some extent the possibility of PDO to guide the medication of patients with clinical tumors. It has also been shown that by comparing the difference in response to drugs between normal-like organs and PDO, it has been found that drugs with high selectivity help to reduce toxic side effects in clinical patients. Thus, it is clear that drug sensitivity testing by PDO to discover the most appropriate drug regimen will help to improve the clinical efficacy of tumor patients, reduce toxic side effects, risk of drug resistance, and chances of tumor recurrence, and maximize the benefits to patients.