Research Interest
High throughput 3D tumour spheroid assays for target validation and drug evaluation
Sue Eccles, Maria Vinci, Sharon Gowan, Frances Boxall, Cara Lomas, Lisa Patterson, Will Court, Lou Chesler
(funded by NC3R)
Standard 2D monolayer cultures used for target validation and drug screening studies do not adequately address the complexity of in vivo tumour pathophysiology. Although 3D spheroids better represent the cancer microenvironment (with gradients of proliferation, oxygenation and drug access) they have not been routinely used in high-throughput (HTS) mode. The aim of our studies is 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 and to deploy them in routine drug discovery projects.
We have established a novel, standardised method for the generation of tumour spheroids which have been 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 (Fig 1). Quantitative data are obtained by image analysis cytometry or microscopy. The power of the techniques has been exemplified using highly malignant human adult (U87MG) and pediatric (KNS42) gliomas and triple negative breast carcinoma (MDA MB 231) spheroids treated with inhibitors of HSP90 and PI3K.
Our standardized protocol generates spheroids 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. 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 (Fig 2). MDA MB 231 migration and invasion were inhibited with sub GI50 concentrations of 17-AAG. GFP-labeled U87MG and MDA MB 231 tumour spheroids co-cultured with EBs rapidly attached and coalesced, which can be followed in real-time, providing a dynamic model to study simultaneous tissue invasion and angiogenesis.
Current work is extending the system to other tumour types including additional paediatric cancers and we will deploy the assays in the evaluation of novel targeted compounds from the Cancer Therapeutics Unit. In addition, we are rigorously comparing data from 2D, 3D and in vivo assays to assess the relative predictive value of the two in vitro systems for in vivo efficacy.
Fig 1: Mouse embryonic stem cells are differentiated into embryoid bodies (EBs). These represent a source of endothelial cells as confirmed by CD31 staining. The co-culture is initiated by transferring a single tumour spheroid (TS) and a single EB per well to a 96 well plate.
Time-lapse imaging shows the relatively rapid attachment and coalescence of the two tissues (representing tissue invasion and angiogenesis).
Fig 2: Patterns of migration of glioma cell lines on endothelial cell monolayers or matrix proteins.
