Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro – Implication for Drug Development

Abstract

Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

“…Thus, these results might qualify spheroids as proper models to recapitulate specific subsets of cancer metastasis. Nevertheless, comprehensive systematic investigations on phenotypic changes, potentially altered signaling pathways and the response to drug treatment in 2D vs 3D systems have only occasionally been performed (Ekert et al, 2014;Luca et al, 2013;Pickl and Ries, 2009). We focused on colon carcinoma, being the third most deadly cancer in the world, to thoroughly evaluate the main signaling pathways in 2D vs 3D spheroid cultures under normal and treated conditions.…”

“…Thus far, only a few comprehensive and systematic studies have been found that compare distinct signaling pathways in 2D vs 3D culture, in the presence or absence of ECM and/or stromal cells, or upon treatment with inhibitors (Ekert et al, 2014;Luca et al, 2013;Pickl and Ries, 2009). Changes in signaling cascades in cancer cells or crosstalk with other cell types in 3D cultures are still poorly understood.…”

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…Heterotypic MSs more closely mimic the heterogeneity of tumors and the heterotypic inter-relations between cells, and, in addition to monotypic culture, they allow the investigation of important tumor endpoints such as angiogenesis, tumor cell migration and invasion, and drug responsiveness [101]. Examples of heterotypic spheroids may encompass tumor cells with fibroblasts, macrophages, and endothelial cells [102,103]. For example, heterotypic MSs of melanoma cell lines and endothelial cells allowed to demonstrate that invasion and angiogenesis processes may be potentiated by cadherins [104].…”

Advanced in vitro systems for efficacy and toxicity testing in nanomedicine

“…Importantly, the lack of a 3D environment, or even the absence of appropriate cell anchorage to a 2D surface, can cause abnormalities in 2D primary cell function that essentially eliminate the potential advantages of adopting a primary cell-culture model in the first place (77). There are now numerous examples of the use of a 3D microenvironment as the crucial element in improving in-vitro disease models for cancer research and nanotoxicology, and these increasingly offer a meaningful alternative to ethically-unattractive animal testing, as well as a physiologically-relevant advance over conventional 2D culture models (78)(79)(80)(81)(82)(83)(84)(85). As an example, in case of cancer cells, a key characteristic is their acquisition of resistance to a variety of anticancer drugs through a variety of mechanisms.…”