3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.     

Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments

Background

Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not.

Methods

We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures.

Results

3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures.

General significance

This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.     

Cultivation of avian malaria parasites in mammalian liver cells

The exoerythrocytic stages of Plasmodium lophurae and P. fallax were grown in cell cultures derived from embryonic mouse livers. Liver cell monolayers were maintained in continuous culture with frequent subculturing for extended periods of time. Morphologically, the main cell type was a large, flat cell which closely resembled in shape the mouse parenchymal cell, although small colonies of spindle-shaped cells could also be found. Mammalian cells were labelled with thymidine-³H prior to infection with avian merozoites so they could be positively distinguished from any avian cells which might be present in the merozoite inoculum as contaminants. Merozoites were observed inside the labelled mammalian cells within three hours and large forms were seen at 48 hr. The mammalian cells supported parasite growth comparable to that observed in avian cell cultures.     

Microporous cellulosic scaffold as a spheroid culture system modulates chemotherapeutic responses and stemness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) treatments are evaluated by two-dimensional (2D) in vitro culture systems, despite their limited ability to predict drug efficacy. The three-dimensional (3D) microporous scaffold provides the possibility of generating more reliable preclinical models to increase the efficacy of cancer treatments. The physical properties of a microporous cellulosic scaffold were evaluated. The cellulosic scaffold was biocompatible and had a highly porous network with appropriate pore size, swelling rate, and stiffness of cancer cell cultures. Cellulosic scaffolds were compared with 2D polystyrene for the culture of HepG2 and Huh7 human HCC cells. Cellulosic scaffolds promoted tumor spheroid formation. Cells cultured on scaffolds were more resistant to chemotherapy drugs and showed upregulation of EpCAM and Oct4. The migration ability of HCC cells cultured on scaffolds was significantly greater than that of cells grown in 2D cultures as evidenced by the downregulation of E-cadherin. In addition, the proportion of CD44+/CD133+ HCC cancer stem cells (CSCs) was significantly greater in cells cultured on scaffolds than in those grown in 2D cultures. These findings suggest that cellulosic scaffolds effectively mimic the in vivo tumor behavior and may serve as a platform for the study of anticancer therapeutics and liver CSCs.     

Fabrication of thin-layer matrigel-based constructs for three-dimensional cell culture

Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3-D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3-D culture systems suitable for analysis is difficult because of air–liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and nontreated 96-well plates to produce uniform, thin-layer constructs for 3-D culture of cells in Matrigel. The first technique, the floating 3-D cell culture method, uses phase-separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 μL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3-D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 μL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3-D culture platform for high-throughput production of disease models for evaluation of novel drug therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2733, 2019     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose–response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.     

Growth rate,labeling index,and radiation survival of cells grown in the matrigel threadin vitro tumor model

Summary Six rodent cell lines (36B10 rat glioma cells, 9L rat gliosarcoma cells, V79 Chinese hamster lung fibroblasts, EMT6/UW and EMT6/Ro mouse mammary sarcoma cells, and RIF-1 mouse fibrosarcoma cells) were tested for growth in cylindrical threads of Matrigel. These cells grew in the threads with doubling times of 17–23 h, reaching maximum cell densities on the order of 10⁸ cells/ml. Histological sections of these threads showed a heterogeneous cell distribution: cells grew to confluence at the thread surface and at somewhat lower cell densities in the thread core. [H-3]thymidine labeling index and radiation sensitivity were measured for 9L and EMT6/UW cells in Matrigel threads. For both cell types, the labeling index in Matrigel was lower than observed in cell monolayers, with higher labeling indexes at the thread periphery than in the thread core. When these threads were grown in stirred medium, lower thread diameters, higher cell yields per thread, and higher labeling indices were obtained. EMT6 cell monolayers coated with Matrigel were less radiosensitive than cells in uncoated monolayers. This protective effect was eliminated by irradiating in the presence of 1 mg/ml misonidazole. EMT6 cells consume nearly three times as much oxygen (mole/cm³-sec) as do 9L cells, which are equally radiosensitive in monolayers with or without a Matrigel coating. The radiation sensitivity of EMT6/UW cells in Matrigel threads was similar to that for monolayers of plateau phase cells, whereas for 9L cells, the response in threads was more similar to exponentially growing cells. We conclude that Matrigel threads provide an alternativein vitro model for studying the radiation response of cells in a three-dimensional geometry.     

Differential sensitivity to short-chain ceramide analogues of human intestinal carcinoma cells grown in tumor spheroids versus monolayer culture

The cytotoxic activity of short-chain (C(2)) ceramide was evaluated in human intestinal carcinoma cells grown as multicellular tumor spheroids versus the same cells cultured as monolayers under closely comparable conditions. A decrease in cell number was seen in monolayer cultures of HT-29, Caco-2, and HRT-18 cells, with an EC(50) (concentration for half-maximal toxicity) of between 13 and 23 microM. However, when the same cells were grown in the multicellular spheroid format, C(2) was markedly less potent in reducing cell number, with an EC(50) of between 44 and 63 microM, representing a 1.9- to 4.9-fold decrease in its potency. The chemotherapeutic agents 5-fluorouracil and cisplatin were equally potent against spheroids and monolayer cultures, indicating that although drug access is a problem in conventionally grown tumor spheroids it is not a problem for spheroids grown under the conditions used in this study. Our results suggest that although ceramide is capable of inducing cell death in intestinal carcinoma cells grown in spheroid culture, its cellular toxicity is constrained by influences that are independent of drug access and may be the consequence of the altered cellular relationships. Carcinoma cell populations show an intrinsically decreased responsiveness to the effects of ceramide when they are grown in a three-dimensional culture format.     

3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers

Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.     

High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy

Conventional two-dimensional cell monolayers do not provide the geometrical, biochemical and mechanical cues found in real tissues. Cells in real tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Such a highly interconnected communication network extends along all three dimensions. This architecture is lost in two-dimensional cultures. Therefore, at least in many cases, two-dimensional cell monolayers do not represent a suitable in vitro tool to characterize accurately the biology of real tissues. Many studies performed over the last few years have demonstrated that the differences between three-dimensional and two-dimensional cultured cells are striking at the morphological and molecular levels and that three-dimensional cell cultures can be employed in order to shrink the gap between real tissues and in vitro cell models. End-point and long-term imaging of cellular and sub-cellular processes with fluorescence microscopy provides direct insight into the physiological behavior of three-dimensional cell cultures and their response to chemical or mechanical stimulation. Fluorescence imaging of three-dimensional cell cultures sets new challenges and imposes specific requirements concerning the choice of a suitable microscopy technique. Deep penetration into the specimen, high imaging speed and ultra-low intensity of the excitation light are key requirements. Light-sheet-based fluorescence microscopy (LSFM) offers a favorable combination of these requirements and is therefore currently established as the technique of choice for the study of three-dimensional cell cultures. This review illustrates the benefits of cellular spheroids in the life sciences and suggests that LSFM is essential for investigations of cellular and sub-cellular dynamic processes in three-dimensions over time and space.     

Three-dimensional bioprinting of stem cell-derived central nervous system cells enables astrocyte growth,vasculogenesis, and enhances neural differentiation/function

Current research tools for preclinical drug development such as rodent models and two-dimensional immortalized monocultures have failed to serve as effective translational models for human central nervous system (CNS) disorders. Recent advancements in the development of induced pluripotent stem cells (iPSCs) and three-dimensional (3D) culturing can improve the in vivo-relevance of preclinical models, while generating 3D cultures though novel bioprinting technologies can offer increased scalability and replicability. As such, there is a need to develop platforms that combine iPSC-derived cells with 3D bioprinting to produce scalable, tunable, and biomimetic cultures for preclinical drug discovery applications. We report a biocompatible poly(ethylene glycol)-based matrix which incorporates Arg-Gly-Asp and Tyr-Ile-Gly-Ser-Arg peptide motifs and full-length collagen IV at a stiffness similar to the human brain (1.5 kPa). Using a high-throughput commercial bioprinter we report the viable culture and morphological development of monocultured iPSC-derived astrocytes, brain microvascular endothelial-like cells, neural progenitors, and neurons in our novel matrix. We also show that this system supports endothelial-like vasculogenesis and enhances neural differentiation and spontaneous activity. This platform forms a foundation for more complex, multicellular models to facilitate high-throughput translational drug discovery for CNS disorders.     

Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel

Objective:  Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel.
Materials and Methods:  We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves.
Results:  hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone.
Conclusion:  Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel.     

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.     

Ex vivo cultures of glioblastoma in three-dimensional hydrogel maintain the original tumor growth behavior and are suitable for preclinical drug and radiation sensitivity screening

Identification of new drugs and predicting drug response are major challenges in oncology, especially for brain tumors, because total surgical resection is difficult and radiation therapy or chemotherapy is often ineffective. With the aim of developing a culture system close to in vivo conditions for testing new drugs, we characterized an ex vivo three-dimensional culture system based on a hyaluronic acid-rich hydrogel and compared it with classical two-dimensional culture conditions. U87-MG glioblastoma cells and seven primary cell cultures of human glioblastomas were subjected to radiation therapy and chemotherapy drugs. It appears that 3D hydrogel preserves the original cancer growth behavior and enables assessment of the sensitivity of malignant gliomas to radiation and drugs with regard to inter-tumoral heterogeneity of therapeutic response. It could be used for preclinical assessment of new therapies.     

Permeation of Insulin,Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

Objectives

Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs.

Methods

The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer.

Results

Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures.

Conclusions

Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.     

Isolation,culture, and characterization of equine oviduct epithelial cells in vitro

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley-Liss, Inc.     

Tumorspheres but Not Adherent Cells Derived from Retinoblastoma Tumors Are of Malignant Origin

Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.     

Three-dimensional in vitro tumor models for cancer research and drug evaluation

Cancer occurs when cells acquire genomic instability and inflammation, produce abnormal levels of epigenetic factors/proteins and tumor suppressors, reprogram the energy metabolism and evade immune destruction, leading to the disruption of cell cycle/normal growth. An early event in carcinogenesis is loss of polarity and detachment from the natural basement membrane, allowing cells to form distinct three-dimensional (3D) structures that interact with each other and with the surrounding microenvironment. Although valuable information has been accumulated from traditional in vitro studies in which cells are grown on flat and hard plastic surfaces (2D culture), this culture condition does not reflect the essential features of tumor tissues. Further, fundamental understanding of cancer metastasis cannot be obtained readily from 2D studies because they lack the complex and dynamic cell–cell communications and cell–matrix interactions that occur during cancer metastasis. These shortcomings, along with lack of spatial depth and cell connectivity, limit the applicability of 2D cultures to accurate testing of pharmacologically active compounds, free or sequestered in nanoparticles. To recapitulate features of native tumor microenvironments, various biomimetic 3D tumor models have been developed to incorporate cancer and stromal cells, relevant matrix components, and biochemical and biophysical cues, into one spatially and temporally integrated system. In this article, we review recent advances in creating 3D tumor models employing tissue engineering principles. We then evaluate the utilities of these novel models for the testing of anticancer drugs and their delivery systems. We highlight the profound differences in responses from 3D in vitro tumors and conventional monolayer cultures. Overall, strategic integration of biological principles and engineering approaches will both improve understanding of tumor progression and invasion and support discovery of more personalized first line treatments for cancer patients.