Abstract
Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.
Highlights
Despite the wealth of data generated, and strong recommendations to upgrade cell culture from 2D to 3D models[4], few of these more complex model systems have been incorporated into the drug discovery funnel
The lung adenocarcinoma cell line H1437 is sensitive to the pan-PI3 kinase/mTOR inhibitor GSK1059615 as the positive control targeted agent selected for the lung pathology[6]
Fibroblasts may be defined by their functionality rather than by their origin[13], and human non-immortalized dermal fibroblasts (HDF) may functionally be re-programmed by tumor cells to become CAFs14
Summary
Despite the wealth of data generated, and strong recommendations to upgrade cell culture from 2D to 3D models[4], few of these more complex model systems have been incorporated into the drug discovery funnel. Fibroblasts may be defined by their functionality rather than by their origin[13], and HDFs may functionally be re-programmed by tumor cells to become CAFs14 Given their ready availability, greater robustness, and suggested functional adequacy, we decided to use HDFs as a surrogate for breast-derived CAFs. Starting from simple 2D monocultures, the complexity was increased stepwise to include stromal cells in 2D co-cultures, and growth of the cultures in 3D. ECM embedded cultures were established in, (1) Matrigel, a basal membrane extract that induces polarization of normal epithelial cells[15], and would reflect a localized tumor environment, (2) collagen I as an interstitial stroma matrix component, providing an invasive growth environment[16], and, (3) a 1:1 mix of both. The robust protocols established by this collaborative effort in combination with the cross-comparisons performed to characterize the models provide a toolbox that should help to better incorporate complex models in the drug discovery process
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