Research review paperThree-dimensional in vitro tumor models for cancer research and drug evaluation
Introduction
Cancer is the major cause of death worldwide, and one in every four deaths in the United States is due to cancer-related diseases (Siegel et al., 2012). While cells in normal tissue reside in defined locations and maintain steady numbers, cancer cells remove these constraints through mutations in oncogenes and tumor suppressor genes (Esmaeilsabzali et al., 2013, Joyce and Pollard, 2009). Consequently, cells in the tumor tissues can sustain proliferative signaling, evade growth suppressors, resist cell death, enable replicative immortality, induce angiogenesis, and activate invasion and metastasis (Hanahan and Weinberg, 2011). During cancer progression and metastasis, malignant cells maintain their close interactions with surrounding cells and the stromal extracellular matrices (ECM) (Fig. 1) (DelNero et al., 2013, Hanahan and Weinberg, 2011, Infanger et al., 2013, Nyga et al., 2011, Seo et al., 2013). Numerous stromal cells, including endothelial cells of the blood and lymphatic circulation, stromal fibroblasts, and innate and adaptive infiltrating immune cells together comprise the complex tumor microenvironment (Hanahan and Weinberg, 2011, Joyce and Pollard, 2009, Koontongkaew, 2013). The stromal ECM is composed of complex assemblies of collagens, glycosaminoglycans and proteoglycans and the molecules that bind to them (Jain, 1999, Jain, 2012). Tumor cells interact with those stromal components dynamically through growth factor-mediated tumor-stromal cell crosstalk (Murata et al., 2011) and integrin-mediated tumor-ECM interactions (Desgrosellier and Cheresh, 2010). Moreover, these interactions evolve along with the progression of the disease (Tlsty and Coussens, 2006), where the stromal microenvironment can initially exert inhibitory effects on even aggressive malignant tumor cells (Bissell and Hines, 2011, Joyce and Pollard, 2009, Xu et al., 2012a). However, as the disease progresses, cancer cells exploit and modify their surroundings to facilitate the inappropriate growth, angiogenesis, invasion and ultimately metastasis in a secondary site (Chung et al., 2012, Joyce and Pollard, 2009, Psaila and Lyden, 2009). In general, tumor growth and progression require intricate interactions between cancer cells and their surrounding microenvironment.
In vitro studies aimed at gaining molecular understanding of cancer progression or the identification of effective anti-cancer therapeutics rely on the availability of a versatile platform that closely recapitulates pathophysiological features of the native tumor tissue and its surrounding microenvironment. Conventional two dimensional (2D) platforms (Hutmacher et al., 2010) are well established and straightforward to use. However, the absence of the third dimension can obscure the experimental observations, generating misleading and contradictory results (Hutmacher, 2010, Hutmacher et al., 2010). Additionally, screening in 2D may miss promising lead compounds whose actions are suppressed when cells are adhered to plastic. Often, promising results obtained from 2D cannot be translated similarly into in vivo settings (Goodman et al., 2008). Whereas cells on 2D are exposed to a uniform environment with sufficient oxygen and nutrients, cells in solid tumors are exposed to gradients of critical chemical and biological signals (Mehta et al., 2012), which can exert both stimulatory and inhibitory effects on tumor progression (Mehta et al., 2012). Intriguingly, certain tumor cells from cancer patients are intrinsically resistant to a broad spectrum of chemotherapeutic drugs without any previous exposure to those cytotoxic agents (Sanchez et al., 2009, Zhu et al., 2005, Zhu, 2012). This intrinsic drug resistance has been attributed to the overexpression of the multidrug resistance (MDR) proteins by tumor cells (Sanchez et al., 2009, Wartenberg et al., 1998, Zhu et al., 2005, Zhu, 2012). Characteristics of the tumor tissue, namely hypoxia (Milane et al., 2011, Zhu et al., 2005, Zhu, 2012), low nutrient supply (Zhu et al., 2012) and low pH (Webb et al., 2011, Wei and Roepe, 1994, Xu et al., 2014), all have been suggested to upregulate the expression of MDR proteins through specific cellular signaling pathways. Although one can partially recreate an MDR-conducive environment in 2D cultures, the lack of a three dimensional (3D) architectural context precludes the recapitulation and the maintenance of MDR behaviors (Correia and Bissell, 2012, Faute et al., 2002). Finally, the lack of the complex 3D ECM network structures in monolayer cultures can affect drug testing results. While anti-cancer agents applied to a monolayer cell culture typically reach cells without physical barriers, the same therapeutics delivered in vivo encounter an entirely different environment that significantly restricts the partition of the drugs throughout the entire tumor (Goodman et al., 2008). The 3D organization of the tumor mass, as well as the associated stroma, fundamentally alters the diffusion profile for drugs, through both cell–cell contacts and cell–matrix interactions (Chauhan et al., 2011). Detailed descriptions on physicochemical properties of native tumor microenvironments including cell–cell and cell–matrix interactions, tissue structure and mechanics, as well as juxtacrine and soluble factor signaling, can be found in recent reviews by Fischbach and coworkers (DelNero et al., 2013, Infanger et al., 2013, Seo et al., 2013).
Realizing the limitations of monolayer cultures, and inspired by the complexity of the native tumor microenvironment, researchers have developed various 3D models that recapitulate certain features of solid tumor tissues, such as tumor morphology (Gurski et al., 2012), gradient distribution of chemical and biological factors (Fracasso and Colombatti, 2000), expression of pro-angiogenic and MDR proteins (Fischbach et al., 2009, Xu et al., 2014), dynamic and reciprocal interactions between tumor and its stroma (Xu et al., 2012a). Moreover, compared to 2D monolayer cultures, cells in 3D generally exhibit a reduced sensitivity to some chemotherapeutic agents (Fong et al., 2013). This review adds to the existing literature by summarizing recent advances in ex vivo assembly of pathologically-relevant 3D tumor models using custom-designed culture devices and biologically-derived or biomimetic matrices. Critical assessments are provided to highlight the applications of these models toward a mechanistic understanding of cancer biology and in therapeutic evaluations of anticancer drugs, both free and nanoencapsulated. Possible mechanisms for the altered drug sensitivity observed in 3D culture conditions as compared to the corresponding 2D systems also are discussed.
Section snippets
Device-assisted assembly of tumor models
Three-dimensional multicellular tumoroids can be grown using engineered devices that maximize cell–cell interactions and solute transport without the undesirable interference from scaffolding materials. Outlined below are two types of devices employed in cancer research: tissue engineering bioreactors and microfluidic systems.
Matrix-assisted assembly of 3D tumor models
The intricate molecular network of tumor-associated stromal ECM is an important component of the tumor microenvironment, and plays crucial roles in cancer progression and invasion (Dutta and Dutta, 2009, Weaver et al., 1997). Studies have shown that the blockage of ECM-integrin interactions led to death of highly metastatic breast cancer cells, thereby restoring morphologically normal breast structures (Wang et al., 2002). In general, association of cancer cells with the ECM produces survival
3D tumor models for drug testing
Although rapid advances in drug design methodology have led to dramatic increases in the discovery of both candidate drug compounds and their screenable drug targets (Dobson, 2004, Tan, 2005), a commensurate increase in the number of approved drugs has not been seen (LaBarbera et al., 2012). The high failure rate of drug candidates can be attributed in part to the usage of 2D monolayer cultures as the initial screening method that frequently produces inaccurate and unreliable results and does
Summary and outlook
Engineered 3D systems provide a realistic and controllable environment for the incorporation of specific cells, ECM molecules, growth factors and other biochemical cues to better simulate the native tumor microenvironment that favors tumor growth and progression. These models will likely be of significant use in delineating the biological mechanisms that govern the pathological abnormalities observed in cancer. They also will serve as more reliable platforms for generating predictive results on
Acknowledgements
Work in the authors’ laboratories has been funded by grants from the National Institutes of Health (P20 RR016458, X.J.; P01 CA098912, M.C.F.C.; R01 DE022969, M.C.F.C. and X.J.), Delaware Health Science Alliance (DHSA) and the University of Delaware (Graduate Fellowship to X.X.). The authors would like to thank Genzyme for generously providing hyaluronic acid.
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Present address: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.