Establishment Of Organoids To Promote Personalized And Immunotherapeutic Research For Bladder Cancer

ObjectiveBladder cancer(BLCA)is a highly heterogeneous tumor with significant genetic and phenotypic differences between patients.Traditional treatment methods include surgery,chemotherapy,and radiation therapy,but these methods are not effective for all patients or may lead to drug resistance.Therefore,the development of personalized treatments and immunotherapy strategies based on individual differences and sensitivities of patients has become an important direction in bladder cancer treatment research.In this context,this study aims to establish an efficient three-dimensional(3D)in vitro organoid culture technique that can maintain homogeneity with the original patient while also preserving heterogeneity between different individuals.Using this technique,a patient-derived bladder cancer organoids(BCOs)model was successfully established,which can help more accurately characterize tumor biology and predict drug efficacy.This can be used to evaluate the individualized efficacy and tumor specificity of chemotherapy drugs and chimeric antigen receptor(CAR)T cells,thereby improving the precision of bladder cancer treatment.Methods(1)Obtaining fresh tissue samples from patients who have undergone surgery is crucial.The samples should be collected immediately after surgery and transported to the laboratory under refrigeration.(2)Establishment and characterization of BCOs models.Tissue mechanical and enzymatic dissociation into small fragments,then implanted in 3D matrix gel and cultured in a specialized medium.Immunohistochemistry and immunofluorescence techniques are then used to characterize the BCOs to confirm the presence of tumor cells and evaluate their morphology.(3)Using the CellTiter-Glo luminescent cell viability assay to test chemotherapy drugs at different concentrations and evaluate their effects on organoid survival and proliferation.(4)Determine the immunotherapy target B7H3 through literature collection and pan-cancer gene expression analysis of the UCSC database.(5)Validate B7H3 expression in cell lines and BCOs through immunohistochemistry(IHC)and immunofluorescence(IF).(6)Extract T cells from patients,construct B7H3-CAR,expand and culture B7H3-CAR T cells.(7)Detection of B7H3-CAR T cell killing specificity.B7H3-CAR T cells are co-cultured with T24-luc cells,and the killing efficiency is detected using the firefly luciferase method.(8)Detection of B7H3-CAR T cell killing function against BCOs.The secretion levels of cytokines IFN-y and IL-2 after co-culture of the two are used to evaluate efficacy and tumor specificity.Results(1)A total of 11 bladder cancer patients were included,and the success rate of BCOs culture was 82%.(2)BCOs preserved the cellular heterogeneity of the original tumor and showed key features of bladder cancer,such as Ki67 and cytokeratin 20(CK20).(3)BCOs from different patient sources showed differences in sensitivity to platinum drugs and olaparib,making them a preclinical model for drug screening.(4)B7H3 is widely expressed in bladder cancer tissues and cell lines,and B7H3-CAR T cells showed good tumor killing specificity and efficacy against BCOs.ConclusionThe establishment of patient-derived BCOs models and the development of B7H3-CAR T cells provide new ideas and methods for individualized and immunotherapy of bladder cancer.The use of BCOs for preclinical drug screening can also provide personalized treatment options for bladder cancer patients.