Abstract
Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.
Highlights
Using a combination of cell culture, microscopic and live-cell imaging techniques, cell lines or primary cells from patients, grown in 3D matrices, can be used to investigate key mechanisms inherent to cancer biology. 3D models are increasingly considered more biologically relevant than 2D monolayer cultures on plastic
Immortalized, non-transformed epithelial cell lines EP156T and RWPE-1 cells were cultured in Keratinocyte Serum-Free Medium (KSFM; Invitrogen, Carlsbad, CA, USA), supplemented with 50 mg/l bovine pituitary extract, 5 mg/l EGF and 2% FBS for 3D conditions. 1 nM R1881 was added to LAPC-4 medium for growth support
To demonstrate the potential of the 3D platform combined with automated image analyses and statistics, we utilized smallmolecule inhibitors to modulate pathways involved in re-organization of the actin cytoskeleton, in particular transformation of differentiated ‘‘round/acinar’’ into invasive ‘‘stellate’’ spheroids [6,7,52]
Summary
Using a combination of cell culture, microscopic and live-cell imaging techniques, cell lines or primary cells from patients, grown in 3D matrices, can be used to investigate key mechanisms inherent to cancer biology. 3D models are increasingly considered more biologically relevant than 2D monolayer cultures on plastic. To warrant significant throughput, reduced cost and a high level of miniaturization and standardization, suitable 3D platforms and matching 3D image analysis tools must be fully compatible with each other and existing laboratory automation They must further comply with cell culture standards, HCS instrumentation and microscopes, robotics (liquid handling), plate readers etc. No satisfactory, fully integrated and sufficiently user-friendly solution that addresses all of these aspects simultaneously exists to date This manuscript describes a combined approach, illustrating how 3D cultures can be generated in miniaturized and standardized fashion, and subsequently analysed by a dedicated software package that and quantitatively addresses the complex phenotypes formed under these conditions
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