Abstract 5255: Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models
Authors: Singh Surya P. , Pathuri Gopal , Asch Adam , Cholewa Brian , Shoemaker Robert , Rao Chinthalapally V. , Madka Venkateshwar 
Published: April 2023

Abstract
Bladder cancer (BC) is a lethal genitourinary malignancy associated with frequent recurrence and poor survival due to metastatic potential. Identification of key cancer cell signaling networks and developing promising agents is critical for effectively inhibiting tumor growth and progression. In many cancers, including bladder cancer (BC), signal transducer and activator of transcription 3 (STAT3) has emerged as an important molecular pathway due to its role in promoting proliferation, invasion, and chemoresistance. Thus, developing STAT3 targeting, orally bioavailable small molecule inhibitors may be helpful for the prevention of BC progression and improving the survival rate of patients with muscle invasive BC. Monolayer culture has limitations for drug testing. Therefore, spheroid and organoid culture are used extensively as they may mimic in-vivo drug response more accurately. The aim of our study is to examine the preclinical anticancer efficacy of STAT3 inhibitors [TTI-101 (C188-9) and SH5-07] in 3D (spheroid and tumoroid) invitro models of BC. We optimized the spheroid growth using various BC cell lines [human (J82), rat (NBT-II), and mouse (MB49) BC cells]. Similarly, tumoroids from rat (BBN-induced bladder tumors) and transgenic mice (UPII-SV40T) bladder tumors were developed. These spheroids and tumoroids were treated with various concentrations (0 - 50 μM range) of STAT3 inhibitors and evaluated for their viability [Calcein AM (CA) and EtBr staining], ATP production (CellTiter-Glo™ 3D), and ROS production (MitoSOXTM). Effect of drug treatment on biomarkers of cell proliferation, apoptosis, stemness, STAT3 signaling, and immune modulation was determined using western blotting and immunofluorescence. Treatment with TTI-101 (0 - 50 μM or SH5-07 (0 - 50 μM) for 144 hrs resulted in significant reduction in the spheroids size (39-45% smaller Vs untreated; p&lt;0.0001), along with decreased ATP levels (20%-40%, p&lt;0.05). MitoSOXTM staining suggested that STAT3 inhibitors treatment increased ROS production in BC cells. CA and EtBr staining revealed that TTI-101 and SH5-07 treatment resulted in increased cell death in BC spheroids compared to control. Decreased spheroids and organoids size also correlated with increased apoptotic marker (cleaved caspase-3) along with decreased cyclin D1, PCNA, and pSTAT3 protein expression. Drug treated BC spheroids/tumoroids also showed reduction in CD44 (BC stemness and invasion marker) and induction of cGAS-STING pathway (cGAS, STING, TBK1, and IRF3) in comparison to the control. These findings indicate that STAT3 inhibitors, TTI-101 and SH5-07, could inhibit bladder cancer by suppressing STAT3 pathway activation and therefore warrant further study in vivo. (Supported by P30 CA225520 and Kerley-Cade Endowed Chair)
Citation Format: Surya P. Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5255.