Monolayered 2D cell culture remains the most used cell model in anticancer drug discovery. Immortalized cultured cell lines and more recently primary human tumor cell lines derived from patients have been a useful model to understand molecular pathways leading to cell proliferation. However, in vitro growth conditions in 2D cell culture are lacking important components of the tumor microenvironment (TME) [1]. The tumor microenvironment (TME) is a complex biochemical network of multiple factors surrounding tumor cells, including vascular networks, the extracellular matrix (ECM), and an heterogenous cell population composed by multiple cell types.

Complementary to 2D cell culture, in vivo xenograft mouse models (PDTX: patient-derived tumor xenograft) are widely used for anticancer drug testing. Despite being an in vivo model, the evolution of tumor in mice cannot fully recapitulate the human tumor microenvironment. Additionally, PDTX is a time and resource consuming model which doesn’t allow for high throughput screening [3].

One main cause of tumor chemoresistance to anticancer drugs is the dynamic behavior of cancer cells, with genetic and epigenetic modifications occurring over time and allowing cancer cells to escape the targeted effect. Tumor microenvironment (TME) can affect gene expression, cell metabolism, chromatin structure, cell proliferation and cell migration of both, cancer and stromal cells. Therefore, cytotoxicity of anticancer drugs is highly dependent on the TME. Because of this, results of drug testing are very sensitive to the cell culture model used. Following are some examples showing how TME can affect drug delivery and/or drug efficacity (reviewed in [1]):

– Cell density at solid tumors is not comparable to cell density in monolayered cultures. Cell density is a physical barrier which affects drug delivery.

– Changes in interstitial fluid pressure due to tumor mass growth and due the modification of composition in the extracellular matrix (ECM). Composition and stiffness of the ECM are dynamic and can influence drug intake [2]

– Cell contacts between cancer cells and the extracellular matrix (ECM) can activate signaling pathways which can induce drug resistance in cancer cells, in a process termed CAM-DR (cell adhesion mediated drug resistance). Cell-cell interactions between cancer cells and stromal cells in the ECM also trigger anti-apoptotic signaling. As an example, in vitro co-culture of cancer cells with stromal cells can generate chemoresistance to drugs which are effective in mono-cultured cancer cells [1].

– Changes in vascularization in the TME can induce resistance to apoptosis. High vascularization is induced in cancer cells in a process of abnormal angiogenesis (see Scientific note Hypoxia and cancer). Angiogenesis is induced by secretion of VEGF (Vascular Endothelial Growth Factor), synthesized by tumor and endothelial cells. In addition to angiogenesis, VEGF has a role in anti-apoptotic signaling. Cancer cells can express VEGF receptors, which provide them resistance to chemotherapy [2].

– Shear stress in the TME can activate signaling pathways inducing chemoresistance.

– Hypoxic conditions: While oxygen and nutrients are unlimited in 2D cultured cells, there is a gradient of oxygen accessibility across the tumor mass in solid tumors. Hypoxic growth increases survival rates of cancer cells. Thus, mimicking oxygen conditions in in vitro assays is critical to asses drug efficacity and tumor growth.