Evaluation of spheroid head and neck squamous cell carcinoma cell models in comparison to monolayer cultures

The use of animal models in drug discovery studies presents issues with feasibility and ethical concerns. To address these limitations, in vitro tissue models have been developed to provide a means for systematic, repetitive, and quantitative investigation of drugs. By eliminating or reducing the need for animal subjects, these models can serve as platforms for more tightly controlled, high-throughput screening of drugs and for pharmacokinetic and pharmacodynamic analyses of drugs. The focus of this review is three-dimensional (3D) tissue models that can capture cell-cell and cell-matrix interactions. Compared to the 2D culture of cell monolayers, 3D models more closely mimic native tissues since the cellular microenvironment established in the 3D models often plays a significant role in disease progression and cellular responses to drugs. A growing body of research has been published in the literature, which highlights the benefits of the 3D in vitro models of various tissues. This review provides an overview of some successful 3D in vitro models that have been developed to mimic liver, breast, cardiac, muscle, bone, and corneal tissues as well as malignant tissues in solid tumors. Copyright © 2010 Wiley-Liss, Inc. and the American Pharmacists Association

The efficacy of potential anticancer drugs during preclinical development is generally tested in vitro using cancer cells grown in monolayer; however, a significant discrepancy in their efficacy is observed when these drugs are evaluated in vivo. This discrepancy, in part, could be due to the three-dimensional (3-D) nature of tumors as compared to the two-dimensional (2-D) nature of monolayer cultures. Therefore, there is a need for an in vitro model that would mimic the 3-D nature of tumors. With this objective, we have developed surface-engineered, large and porous biodegradable polymeric microparticles as a scaffold for 3-D growth of cancer cells. Using the MCF-7 cell line as model breast cancer cells, we evaluated the antiproliferative effect of three anticancer drugs: doxorubicin, paclitaxel and tamoxifen in 3-D model vs in 2-D monolayer. With optimized composition of microparticles and cell culture conditions, a density of 4.5 x 10 (6) MCF-7 cells/mg of microparticles, which is an 18-fold increase from the seeding density, was achieved in six days of culture. Cells were observed to have grown in clumps on the microparticle surface as well as in their interior matrix structure. The antiproliferative effect of the drugs in 3-D model was significantly lower than in 2-D monolayer, which was evident from the 12- to 23-fold differences in their IC 50 values. Using doxorubicin, the flow cytometry data demonstrated approximately 2.6-fold lower drug accumulation in the cells grown in 3-D model than in the cells grown as 2-D monolayer. Further, only 26% of the cells in 3-D model had the same concentration of drug as the cells in monolayer, thus explaining the reduced activity of the drugs in 3-D model. The collagen content of the cells grown in 3-D model was 2-fold greater than that of the cells grown in 2-D, suggesting greater synthesis of extracellular matrix in 3-D model, which acted as a barrier to drug diffusion. The microarray analysis showed changes in several genes in cells grown in 3-D, which could also influence the drug effect. In conclusion, the cells grown in 3-D are more resistant to chemotherapy than those grown in 2-D culture, suggesting the significant roles of cellular architecture, phenotypic variations, and extracellular matrix barrier to drug transport in drug efficacy. We propose that our model provides a better assessment of drug efficacy than the currently used 2-D monolayer as many of its characteristic features are similar to an actual tumor. A well-characterized 3-D model can particularly be useful for rapid screening of a large number of therapeutics for their efficacy during the drug discovery phase.

The MEK/ERK and PI3K/AKT pathways are often concurrently activated by separate genetic alterations in colorectal cancer (CRC), which is associated with CRC progression and poor survival. However, how activating both pathways is required for CRC metastatic progression remains unclear. Our recent study showed that both ERK and AKT signaling are required to activate eIF4E-initiated cap-dependent translation via convergent regulation of the translational repressor 4E-BP1 for maintaining CRC transformation. Here, we identified that the activation of cap-dependent translation by cooperative ERK and AKT signaling is critical for promotion of CRC motility and metastasis. In CRC cells with coexistent mutational activation of ERK and AKT pathways, inhibition of either MEK or AKT alone showed limited activity in inhibiting cell migration and invasion, but combined inhibition resulted in profound effects. Genetic blockade of the translation initiation complex by eIF4E knockdown or expression of a dominant active 4E-BP1 mutant effectively inhibited migration, invasion and metastasis of CRC cells, whereas overexpression of eIF4E or knockdown of 4E-BP1 had the opposite effect and markedly reduced their dependence on ERK and AKT signaling for cell motility. Mechanistically, we found that these effects were largely dependent on the increase in mTORC1-mediated survivin translation by ERK and AKT signaling. Despite the modest effect of survivin knockdown on tumor growth, reduction of the translationally-regulated survivin profoundly inhibited motility and metastasis of CRC. These findings reveal a critical mechanism underlying the translational regulation of CRC metastatic progression, and suggest that targeting cap-dependent translation may provide a promising treatment strategy for advanced CRC.