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

Abstract 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<0.0001) along with decreased ATP (20%-40%, p<0.05), and pSTAT3 protein expression in spheroids derived from BC cell lines and rat BC organoids. Further, MitoSOX™ staining showed that STAT3 inhibitor treatment induced mitochondrial mediated ROS generation in BC spheroids. CA and EtBr staining showed that C188-9 and SH5-07 treatment induced cell death in BC spheroids that was also associated with caspase-3 cleavage. These findings indicate that C188-9 and SH5-07 could suppress the activation of the STAT3 pathway and inhibit the bladder cancer spheroid growth by inducing ROS production and thus warrants further evaluation in vivo. Furthermore, our study provided valuable spheroid and organoid models for evaluating therapeutic candidates in an in vivo-mimic microenvironment, thereby providing great potential for drug testing. (Partly supported by P30CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Targeting STAT3 for bladder cancer prevention – in vitro studies using spheroid and organoid models [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A018.