Traditional in vitro models used for the development of anti-cancer drugs are based on the monolayer culture of cells, which has a limited predictivity of in vivo efficacy. A number of cell culture platforms have been developed in recent years to improve predictivity and further to elucidate the mechanisms governing the differing responses observed in vitro versus in vivo. One detrimental aspect of current in vitro models is their inability to decouple the effect of different extrinsic factors on the responsiveness of the cells to drug treatment. Here, we have used an engineered poly(dimethylsiloxane) (PDMS) microwell array as a reductionist approach to study the effect of environmental parameters, independently of each other. It is observed for MCF-7 breast cancer cells, that culture within the three-dimensional (3D) environment of the microwells alone had an effect on the response to Taxol and results in a reduction of cell death in comparison to cells cultured on flat substrates. Additionally the microwells allowed the response of single versus multicell clusters to be differentiated. It was found that the formation of cell-cell contacts alters the drug response, depending on the type of adhesive protein present. Thus, with this microwell platform it is revealed that the presence of cell-cell contacts in addition to the dimensionality and the matrix composition of the environment are important mediators of altered drug responses. In conclusion the microwell array can not only serve as a platform to reveal which parameters of the extracellular environment affect drug response but further the interdependence of these parameters.

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 °C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.

BackgroundIncreasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters.Methodology/Principal FindingsIn this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect.Conclusions/SignificanceThe application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in vitro drug development.

Cancer is one of the most common causes of death worldwide. Consequently, important resources are directed towards bettering treatments and outcomes. Cancer is difficult to treat due to its heterogeneity, plasticity and frequent drug resistance. New treatment strategies should strive for personalized approaches. These should target neoplastic and/or activated microenvironmental heterogeneity and plasticity without triggering resistance and spare host cells. In this review, the putative use of increasingly physiologically relevant microfabricated cell-culturing systems intended for drug development is discussed. There are two main reasons for the use of miniaturized systems. First, scaling down model size allows for high control of microenvironmental cues enabling more predictive outcomes. Second, miniaturization reduces reagent consumption, thus facilitating combinatorial approaches with little effort and enables the application of scarce materials, such as patient-derived samples. This review aims to give an overview of the state-of-the-art of such systems while predicting their application in cancer drug development.