3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

Abstract

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment

“…migration and toxicity assays) to characterize the metastatic property and drug response of cancer cells, two-dimensional cultures cannot recapitulate the three-dimensional architecture of tissue's complexity, biophysical and biochemical property of extracellular matrix (ECM), and cell-cell interactions of human tumours [22][23][24]. Furthermore, cell cycle, cellular signalling and drug sensitivity can be different if cell culture is performed in a threedimensional instead of a two-dimensional microenvironment [25][26][27]. In vivo three-dimensional models using animal xenografts are also popular but suffer from ethical concerns and are unable to mimic human-specific biology and physiology.…”

Concise Review: 3D cell culture systems for anticancer drug screening

“…Gene expression of cells cultured in 3D systems differs from that of cells in 2D monolayer; for instance, expression of genes related to chemoresistance has been found to vary from 3D versus 2D systems (Lin and Chang, 2008). Studies in breast cancer (Howes et al, 2014a) and colon cancer (Luca et al, 2013) have demonstrated decreased epidermal growth factor (EGFR) and human epidermal growth factor (HER) activation in cells cultured in 3D versus 2D. This could cause decreased sensitivity to anticancer drugs targeting EGFR and HE, and has been observed in 3D cell systems.…”

Modeling nasopharyngeal carcinoma in three dimensions

“…This model is based upon the physiological manner of culturing cells in their natural 3D state, where the cells aggregate to form multicellular or sphere-like structures, aided by artificial extracellular matrix (ECM) (11). 3D spheroids maintain their structural integrity, which is a key component for mimicking the conditions of a tumor in vivo, in addition to the presence of the ECM (33,34). Ideally, the 3D spheroid model serves to provide a balance between 2D culture and in vivo animal approaches.…”