3D-UHU-TU: A Three-Dimensional Bladder Cancer Model in a Healthy Urothelial Environment

Bladder cancer cases and fatalities continue to rise worldwide with treatment outcomes not improving in the last four decades. Poor translation of potential new therapies from pre- clinical studies to the clinic could be one reason behind this. The patient-derived xenograft (PDX) mouse is the gold-standard for testing new bladder cancer therapies, but there are key physiological and molecular differences between mouse and human bladders. Thus, more human cell-based models may improve translation of treatments. Here, we introduce a bladder cancer microtissue model called 3D Urine-tolerant Human Urothelium-Tumour (3D-UHU-TU), which incorporates spheroids derived from human bladder cancer cell lines RT112 (low grade) and T24 (high grade) into the previously published 3D-UHU healthy urothelial model in a 100% urine environment. Both low- and high-grade 3D- UHU-TU models were characterised using immunofluorescence and immunohistochemistry staining with diagnostic markers (CK7, CK20 and GATA3), cadherin markers (E- and N-Cadherin), invasion and migration markers (MMP-2 and MMP-9) and a proliferation marker (Ki-67). Both models expressed the correct markers in the correct spatial areas. We also investigated the utility of both 3D-UHU-TU models as a platform to test treatments, using the conventional chemotherapeutic Mitomycin C as proof of principle. After 2 hours of treatment and 24 hours of recovery, cell lysis and nuclear damage were observed in both low- and high- grade cancer spheroids, with minimal damage to the surrounding healthy urothelium. At higher doses, cancer spheroids either disintegrated or were reduced in size, with the healthy urothelium still intact. Taken together, 3D-UHU-TU is a novel, in vitro model for testing both the safety and efficacy of new treatments. Furthermore, our work lays the foundation for testing treatments on patient-derived tumour spheroids in a personalised medicine approach.