Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Background: Standard 2D monolayer cultures used for target validation and drug screening studies do not adequately address the complexity of in vivo tumor pathophysiology. Although 3D tumor spheroids better represent a tumor microenvironment (with gradients of proliferation, oxygenation and drug access) they have not been routinely used in high-throughput (HTS) mode. The aim of the present study was to develop a suite of in vitro 3D spheroid-based assays to rapidly and accurately quantify key aspects of the malignant phenotype: growth, motility, invasion and angiogenesis.</jats:p>
<jats:p>Methods: We established a novel, standardised method for the generation of tumor spheroids in suspension using ultra-low attachment (ULA) 96-well plates. Spheroids were characterized in terms of growth kinetics, haptotaxis on extracellular matrix (ECM) and endothelial cell (EC) monolayers, invasion into MatrigelTM and co-culture with embryoid bodies (EB) to model tissue invasion/angiogenesis. Quantitative data were obtained by image analysis using a Celigo cytometer or microscopy. Cell viability was determined by the CellTiter Glo assay for direct comparison of 2D and 3D cultures. Highly malignant human adult (U87MG) and pediatric (KNS42) gliomas and breast carcinoma (MDA MB 231) spheroids were treated with inhibitors of HSP90 (17-AAG) and PI3K (PI-103) to demonstrate the power of the techniques.</jats:p>
<jats:p>Results: Our standardized protocol generates single spheroids/well which are reproducible in size and easy to handle. Images are obtained at intervals over 14 days for growth kinetics or 48-96 hours for functional assays. The Celigo cytometer enables rapid, multiparametric real-time analyses, and samples can be collected to measure target protein expression, drug levels and pharmacodynamic effects. Dose-dependent inhibition of 3D spheroids was readily demonstrated using the targeted agents 17-AAG and PI-103. All models also showed extensive migration and invasion mirroring their in-vivo behaviour; interestingly U87MG and KNS42 showed different patterns of dissemination: widely dispersed or contiguous respectively. MDA MB 231 migration and invasion were inhibited with sub GI50 concentrations of 17-AAG. GFP-labeled U87MG and MDA MB 231 tumor spheroids co-cultured with EBs rapidly attached and coalesced, providing a model to study simultaneous tissue invasion and angiogenesis. Direct comparisons of cell viability in 2D and 3D showed that U87MG and MDA MB 231 are more resistant to 17-AAG in 3D, conversely U87MG was more sensitive to PI-103 in 3D.</jats:p>
<jats:p>Conclusions: We have developed a unique repertoire of novel 3D multiplate assays reflecting key aspects of malignant cell behaviour and able to deliver rapid and reproducible results. We provide evidence that our methods potentially enhance target selection and the triaging of drug candidates prior to in vivo studies.</jats:p>
<jats:p>Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2011-4277</jats:p>