Abstract B70: High-throughput analysis of 3D tumor colony formation of primary cell suspensions derived from xenografts to identify efficacy of anti-tumor agents in single agent or combination therapy

Abstract

Abstract Recent experience in anti-cancer research has demonstrated that three-dimensional (3D) tumor cell culture models represent a biological assay system, being more relevant than two-dimensional (2D) monolayer cell cultures. Anchorage-independent 3D growth is one of the hallmarks of cell transformation and performance of 3D colony formation assays in semi-solid media is considered as an accurate and stringent measure for detecting growth of malignant, transformed cells. Here, we describe the high-throughput application of a 3D soft-agar clonogenic assay using primary cell suspensions of patient-derived (PDX) and cell line-derived (CDX) xenografts in a 96 well microplate format, using targeted anti-tumor agents, such as Braf, Wnt pathway, and telomerase inhibitors. Colony formation was determined by automated image analysis. Our data show that this is a more appropriate read-out compared to indirect measurements, such as determination of metabolic activity by measuring intracellular ATP. Specificity of the assay was demonstrated by testing Braf inhibitors vemurafenib and dabrafenib, which potently inhibited colony formation of V600E mutated tumors of melanoma and colon carcinoma (MEXF 1732, MEXF 672, MEXF 989, HT-144, and Colo 205), whereas Braf wildtype tumors of the same histotypes were less sensitive (MEXF 1792, MEXF 1870, MEXF 535, MEXF 622, and HCT-116). Moreover, anti-tumor activity was determined for several standard of care agents. Erlotinib, for example, exhibited differential in vitro activity, which closely matched results obtained for xenografts when tested in vivo, and EGFR was overexpressed in sensitive tumor models. The 3D soft-agar clonogenic assay is customizable for different treatment layouts, which makes it a valuable tool for efficacy determinations of single drug treatments in broad screens or drug combinations. By using a 5×5 matrix combination approach, we identified synergistic interactions between Wnt pathway (XAV-939) and telomerase (RHPS4) inhibitors using Bliss independence analysis. These results are in line with a postulated molecular link between Wnt/β-catenin signalling and telomerase expression (Hoffmeyer et al. Science 336, 1549, 2012). In conclusion, the 3D assay platform provides a fast and robust method for the assessment of anti-tumor agents using primary cell suspensions in vitro without needing to establish permanent cell lines. Screening approaches with single agents and combinations in broad tumor panels are of high value for investigating cytotoxic and new targeted anti-tumor agents, as well as for hypothesis generation and selection of development or treatment strategies. Citation Format: Armin Maier, Sumeer Dhar, Andreas Maunz, Bruno Zeitouni, Anne-Lise Peille, Torsten Giesemann, Heinz-Herbert Fiebig. High-throughput analysis of 3D tumor colony formation of primary cell suspensions derived from xenografts to identify efficacy of anti-tumor agents in single agent or combination therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B70.