PD53-02: Cytotoxicity caused by cisplatin, venetoclax, and S63845 to urothelial carcinoma cells expanded in 3D organoids differs clearly from cytotoxicity to autologous 2D adherent cells

Yi Wei, Bastian Amend, Wilhelm K. Aicher*, Arnulf Stenzl, Niklas Harland, Tuebingen, Germany

Introduction: Organoids are 3-dimensional (3D) constructs reflecting the micro-anatomy of the tissue of origin and some of the cell-cell and cell-matrix interactions observed in vivo. They serve as important tools for research, complementing normal 2-dimensional (2D) cell cultures. The objective of this study was to compare the response of urothelial carcinoma cells to three cancer chemotherapy agents, in a 3D organoid form vs. a conventional 2D culture form, in order to assess if the 3D organoid method is better able to assess the efficacy of chemotherapy agents

Methods: Bladder cancer tissue was obtained from surgery to produce organoids. Cells were isolated, resuspended in a hydrogel, and incubated in media complemented with ROCK inhibitor Y-27632. To characterize the cells, the expression of urothelial and mesenchymal markers was assessed. To assess cytotoxicity, cisplatin (CIS, 1 – 30 μM), venetoclax (VTX, 0.6 – 10 μM), and S63845 ( 0.6 – 10 μM) were added to the organoids and incubated for 24, 48, or 72 hours. Cell viability was quantified by CellTiter Glo 3D reagents in a GloMax reader. Untreated organoids and solvent-only samples served as controls. The effects of these components on the autologous cells in 2D cultures were measured using CellTiter Glo 2.0 chemistry.

Results: Organoids were established from upper tract urothelial carcinoma (BCO#56) and bladder cancer (BCO#140 and BCO#147) samples. Urothelial markers AE1/AE3, CK5, CK20, and FGFR3 and mesenchymal marker vimentin were expressed in organoids. In drug tests, BCO#56 and BCO#147 yielded different IC50s in organoids compared to corresponding 2D cells towards CIS, VTX, and S63. The IC50s of BCO#140 yielded very similar sensitivities in organoids vs 2D cultures (table).

Conclusions: Two of the three samples tested showed significantly different sensitivity to all three chemotherapy agents tested when assessed in organoids compared to conventional 2D cell cultures Further research is needed to determine if conventional 2D cell culture or newer 3D organoids are the more reliable method for assessing cytotoxicity. Until then, results from either method should not be relied upon, unless they are consistent with results from the other method.

Source of Funding: Leuze Stiftung, DFG: GRK2543We thank for proofreading & feedback by Dr. M. Hanna