Cancer is one of leading causes of death worldwide with 14 million new cases and 8.2 million deaths in 2012 (2014). Numerous efforts have been aimed at finding new and more effective ways to treat cancer. Among these strategies is screening of anticancer drugs. Standard screening has typically been evaluated in animal models. However, some results have shown that animal experiments do not always predict clinical outcome in humans, especially with regard to toxicity assessments Knight, 2008. Moreover, the use of animals for research is often restricted due to ethical concerns Festing, 2007. In light of these issues, an in vitro cell-based model is great alternative, minimizing the need for and number of animal experiments. 2D cell culture was the first procedure established for cell-based screening assays. Although 2D cell culture methods are simple, quick and cost-effective to set up, and have been widely investigated, there remain many disadvantages. The primary disadvantage of a 2D system is that it does not mimic an actual 3D tumor and is not biologically relevant Carrie J. Lovitt, 2014. Cells in the in vivoenvironment usually interact with neighboring cells and the extracellular matrix (ECM); however, 2D cell models cannot recapitulate those characteristics. Thus, a 2D culture model may be starkly different from an actual growing tumor with regards to cell morphology, cell proliferation, and gene and protein expression (Edmondson et al., 2014). As a result, only 10% of the drugs passed through in vitro testing have had a positive effect in the clinic, or led to drug approval. The percentage of anticancer drugs which have shown clinical efficacy is even lower, at about 5% (Westhouse, 2010). The high rate failure in the clinical testing phase is a waste of time and money. Therefore, it is important to identify promising in vitro culture models for evaluating drug efficacy in the early stages of drug discovery and development Wong et al., 2012. Given the advantages of 3D versus 2D cell culture models, 3D cell culture techniques garnered increasing attention. The number of publications related to 3D cell cultures have rapidly increased in the last decade- from 7 publications in 1992 to 421 in 2013 Ferro et al., 2014Ravi et al., 2015. The 3D cell culture systems allow cell-based assays to be more physiologically relevant, particularly since cell behavior in 3D culture is much more similar to that of cells in in vivo tissues. In 3D models, cell-cell and cell- ECM interactions are maintained, such that cell morphology, proliferation, differentiation, migration, apoptosis, gene expression and protein expression are comparable to those of cells in vivoEdmondson et al., 2014.

Cell-based assays play a critical role in anticancer drug screening. Traditionally, 2D cell culture was widely used in cancer drug discovery. However, a large number of drugs reported to have strong anticancer effect in 2D cell culture models failed in clinical tests Xu and Burg, 2007. In 2011, although approximately 900 antineoplastic agents had passed through cell-based assay testing, only 12 were approved by the FDA after clinical testing America, 2011Kantarjian et al., 2013.

In recent years, the potential and critical role of 3D cultures in cancer research have gained greater interest. Through the use of sophisticated 3D multicellular tumor spheroid (MCTS) systems, the microenvironment, phenotype and cellular heterogeneity of tumors are effectively represented Thoma et al., 2014. MCTS systems create a gradient of oxygen and nutrients from the outside of tumor spheroids to the core. Spheroids in MCTS systems are constructed with different zones of cells, including proliferating cells on the outside, quiescent viable cells in the middle, and necrotic cells at the inner core ( Figure 1 ), which realistically mimic in vivo tumors Ma et al., 2012. Many research studies have shown that the genotypic profile of cells in MCTS, versus cells grown in monolayer, are more similar to in vivo tumors Smith et al., 2012. Cells in 3D culture conditions were found to exhibit gene expression profiles different to those grown in monolayer Luca et al., 2013Myungjin Lee et al., 2013. This may be a primary reason as to why results of anticancer drug assessments using MCTS are more predictive of clinical efficacy than 2D cell assessments Carver et al., 2014.

Many antineoplastic agents have been reported to be less effective for cancer cells cultured in 3D than 2D Frankel et al., 2000Imamura et al., 2015Karlsson et al., 2012. The architectural structure of MCTS is the main reason for this difference. Firstly, the 3D structure of MCTS reduces the number of cancer cells exposed to anticancer agents; these drugs have more accessibility to cells in monolayer culture Carrie J. Lovitt, 2014. Secondly, the tightly adhered cells and ECM in MCTS can limit drug penetration Frankel et al., 2000. Moreover, the hypoxic core generates a G0- dormant cell population which is highly resistant to chemotherapy Imamura et al., 2015. 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. On the other hand, some drugs show equal, or even greater, therapeutic effect in 3D models compared to 2D Hongisto et al., 2013Howes et al., 2007Pickl and Ries, 2009. The absence of a hypoxic, necrotic core in 2D culture models makes cells more resistant to antineoplastic agents, which are effectively activated by hypoxic conditions of 3D tumors; tirapazamine (TPZ) is an example of this kind of drug Tung et al., 2011. Given that 3D models not only mimic tumor architecture but mimic similar environmental challenges, these models are great and conservative systems to study candidate drug.

Although MCTS is still an in vitro model, its similarity to an in vivo tumor environment allows for a more accurate model to study drug efficacy while minimizing the cast of failed clinical trials.

Due to the advantages of 3D culture systems, there have been many studies focused on the development and optimization of 3D cell culture technologies. Up until now, there have been several types of 3D culture modeis, same of which have been used for anticancer drug screening.

Cell culture systems have long been a foundation for testing and comparing the cytotoxicity and pharmacodynamics of anticancer drug candidates. Even now, many results from 3D cell culture have consistently stressed the importance of these models in drug screening. Research by Jayme L. Horning et al., published in 2008, indicated that 3D MCF7 cells were more resistant to many popular anticancer drugs (e.g. doxorubicin, paclitaxel and tamoxifen) compared with MCF7 cells cultured in monolayer. Using polymeric microparticle surfaces to create 3D tumors, they found that 2D MCF7 cells were significantly more sensitive to these drugs than 3D MCF7 cells, with a 12- to 23- fold disparity in the IC50 values. The study also showed that the sum of collagen in the 3D model was 2 times greater than that of 2D condition and the expression of many genes were different, possibly accounting for the difference in responses to the drugs Horning et al., 2008. Vesa Hongisto et al. suggested in their 2013 studies that 3D cell models can effectively replace traditional 2D cell monolayers and that with regard to screening of drug compounds, 3D models provide better comparability to clinical results. In their study, 102 compounds were tested on JIMT1 breast cancer cells. Results showed that JIMT1 cells were significantly more sensitive to 63 compounds when cultured on Matrigel as compared to 2D condition Hongisto et al., 2013. Using 96-well roundbottom ultra-low attachment plates to create 3D cancer tumors, Amy L. Howes et al. showed, from their studies in 2014, that 3D BT-474 cells were more sensitive to lapatinib, gefitinib, vinblastine and vinorelbine than 3D MCF-10A cells. The authors also found that microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors are two classes of compounds to have selective effects on cancer cells in 3D culture Howes et al., 2014b. Work by Yukie Yoshii et al., published in 2016, on human colon cancer HCT116 cell line demonstrated that regorafenib was most effective on 3D HCT116- RFP cells among 8 drugs tested (capecitabine, bevacizumab, irinotecan, cetuximab, 5-fluorouracil (5- FU), panitumumab, oxaliplatin and regorafenib). Based on their 3D culture studies, the authors were able to demonstrate effective and non-effective drugs for colon cancer treatment Yoshii et al., 2016.

Anticancer drug screening is an important component in the fight against cancer. Several 3D cell culture systems have been developed as suitable platforms for drug screening and are serve as more reliable models for in vitro testing, compared to 2D, given that MCTS have greater structural similarity and cellular zone components to in vivo tumors. The 3D model systems should provide more accurate results for prediction of clinical outcome. Tremendous efforts have been made to establish various 3D cell culture systems. It is important for researchers to look carefully at the advantages and disadvantages of each to find the most suitable system for their studies. However, all the 3D systems can be utilized for cancer research, particularly for testing of new anticancer agents.