Abstract A018: Targeting STAT3 for bladder cancer prevention – in vitro studies using spheroid and organoid models

Signal Transducer and Activator of Transcription 3 (STAT3) is tightly regulated in normal cells to maintain a transiently active state. In contrast, persistent STAT3 activation is frequently observed in bladder cancer (BC) and is associated with poor prognosis and chemoresistance. Hence, developing small molecule inhibitors targeting STAT3 may be helpful for preventing BC progression and improving the survival rate of patients with metastatic BC. Recently, the use of three-dimensional in vitro models in drug development has gained popularity as they closely resemble, to an extent, the in vivo environment in heterogeneity and physiological conditions. Here we established spheroid and organoid models for bladder cancer and evaluated STAT3 inhibitors (C188-9 and SH5-07) for their anticancer activity in vitro. Initially, we optimized the spheroid growth from human, rat, and mouse BC cell lines (J82, NBT-II, MB49, respectively) and tumoroid growth from the BBN-rat bladder cancer model. The anticancer efficacy of C188-9 and SH5-07 was evaluated in vitro at various doses (0-50 µM) in the 3D models of BC. Assays were performed to determine spheroid viability (calcein AM (CA) and EtBr staining), ATP and ROS production (MitoSOX™). Protein isolated from control and drug treated spheroids/tumoroids was used to evaluate pharmacodynamic biomarkers of cell proliferation, apoptosis, and STAT3 signaling. We demonstrate that treatment with C188-9 and SH5-07 significantly decreased the spheroids size (39-45% smaller compared to untreated, p