No abstract

Here, we report on the development of homo- and heterotypic ovarian and pancreatic microtissue tumor models for a screening of up to 40 compounds selected from the NCATS Oncology focused library of small molecules. The goal of this study was to develop a high throughput compatible drug screening platform that could leverage the best of high throughput and high content screening assays to generate a pharmacological profile of activities that we hope will help better predict activity in vivo .This platform utilizes 3D multicellular spheroids from ovarian (HEY and SKOV) and pancreatic (Panc-1) cancer and enables the discrimination of tumor-specific efficacy and nonspecific cytotoxicity of a drug candidate with subsequent identification and validation of the molecular mechanism of action (MMOA). The biological responses measured over a 10-day drug exposure period included (i) growth kinetic (microtissue size), (ii) potency (IC50ATP_10_days) and efficacy (max. response ATP and size). The biological response of the compounds was compared between the different cell culture formats tested, 2D, 3D homotypic and 3D heterotypic. The comparison of IC50 values among the different ovarian cancer cell cultures showed that 21 out of the 38 compounds tested were more potent in 3D than in 2D. Within the pancreatic models, 13 out of 20 compounds tested were more potent in 3D than in 2D. Interestingly, most of the compounds which showed stronger potency in 3D were targeted small molecule agents. Comparing drug responses of homo vs heterotypic ovarian tumor model systems, 3 compounds were effective only in the heterotypic model including the WNT inhibitor PNU-74654 and GABA uptake inhibitor (Gat-1) SK&F-89976A. We also evaluated nonspecific cytotoxic effects on the incorporated NIH3T3 fibroblasts by quantifying a secreted reporter over time. The assay system allowed us to discriminate between acute cytotoxic effects (3-day exposure) and sub-chronic toxicity (7-day exposure). Whereas only a very limited number of compounds had acute cytotoxic effects, there were considerable more compounds with sub-chronic cytotoxicity. Based on the single endpoint classifications, we chose an mTOR inhibitor, Torin-2, to further assess molecular mechanism of drug action for all three tumor microtissue models used in this study, applying reverse phase protein array (RPPA) and transcriptome analysis for signaling pathway profiling. Preliminary results of RPPA analysis revealed a clear down-regulation in the cell cycle and PI3K/Akt/mTOR signaling pathway, especially for S6 ribosomal protein phosphorylated at Ser 235, 236, 240 and 244, indicating a strong inhibitory effect on translation and cell growth control. In summary, we present a 3D tumor microtissue-based phenotypic drug discovery testing scheme which allows a multi-parametric endpoint analysis paired with subsequent molecular pathway identification. Citation Format: Madhu A. Lal, Zoe Weydert, Jens Kelm, Marc Ferrer. Multi-parametric 3D tumor microtissue-based phenotypic compound classification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 771.