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.     

Engineering tumors with 3D scaffolds

Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.     

Recent and advanced therapy for oral cancer

Oral cancer is a common and deadly kind of tissue invasion, has a high death rate, and may induce metastasis that mostly affects adults over the age of 40. Most in vitro traditional methods for studying cancer have included the use of monolayer cell cultures and several animal models. There is a worldwide effort underway to reduce the excessive use of laboratory animals since, although being physiologically adequate, animal models rarely succeed in exactly mimicking human models. 3D culture models have gained great attention in the area of biomedicine because of their capacity to replicate parent tissue. There are many benefits to using a drug delivery approach based on nanoparticles in cancer treatment. Because of this, in vitro test methodologies are crucial for evaluating the efficacy of prospective novel nanoparticle drug delivery systems. This review discusses current advances in the utility of 3D cell culture models including multicellular spheroids, patient-derived explant cultures, organoids, xenografts, 3D bioprinting, and organoid-on-a-chip models. Aspects of nanoparticle-based drug discovery that have utilized 2D and 3D cultures for a better understanding of genes implicated in oral cancers are also included in this review.     

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.     

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.     

Sequential assembly of cell-laden hydrogel constructs to engineer vascular-like microchannels

Microscale technologies, such as microfluidic systems, provide powerful tools for building biomimetic vascular-like structures for tissue engineering or in vitro tissue models. Recently, modular approaches have emerged as attractive approaches in tissue engineering to achieve precisely controlled architectures by using microengineered components. Here, we sequentially assembled microengineered hydrogels (microgels) into hydrogel constructs with an embedded network of microchannels. Arrays of microgels with predefined internal microchannels were fabricated by photolithography and assembled into 3D tubular construct with multi-level interconnected lumens. In the current setting, the sequential assembly of microgels occurred in a biphasic reactor and was initiated by swiping a needle to generate physical forces and fluidic shear. We optimized the conditions for assembly and successfully perfused fluids through the interconnected constructs. The sequential assembly process does not significantly influence cell viability within the microgels indicating its promise as a biofabrication method. Finally, in an attempt to build a biomimetic 3D vasculature, we incorporated endothelial cells and smooth muscle cells into an assembled construct with a concentric microgel design. The sequential assembly is simple, rapid, cost-effective, and could be used for fabricating tissue constructs with biomimetic vasculature and other complex architectures.     

鉴定肿瘤驱动基因的实验模型

人体肿瘤的形成是一个复杂的过程.在这个过程中会发生许多基因突变,这些突变中只有较少一部分具有驱动肿瘤发生的作用,大部分突变作为伴随性变化对肿瘤的发生并无明确的贡献.要确定哪些变异具有驱动肿瘤发生的作用及其作用机制,需要通过实验验证.伴随着新的研究技术的出现,鉴定肿瘤驱动基因的手段也不断演变.从早期主要是从动物诱癌实验、...     

Advancements in melanoma cancer metastasis models

Metastatic melanoma is a complex and deadly disease. Due to its complexity, the development of novel therapeutic strategies to inhibit metastatic melanoma remains an outstanding challenge. Our ability to study metastasis is advanced with the development of in vitro and in vivo models that better mimic the different steps of the metastatic cascade beginning from primary tumor initiation to final metastatic seeding. In this review, we provide a comprehensive summary of in vitro models, in vivo models, and in silico platforms to study the individual steps of melanoma metastasis. Furthermore, we highlight the advantages and limitations of each model and discuss the challenges of how to improve current models to enhance translation for melanoma cancer patients and future therapies.     

Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200–500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.     

Modeling tissue morphogenesis and cancer in 3D

Three-dimensional (3D) in vitro models span the gap between two-dimensional cell cultures and whole-animal systems. By mimicking features of the in vivo environment and taking advantage of the same tools used to study cells in traditional cell culture, 3D models provide unique perspectives on the behavior of stem cells, developing tissues and organs, and tumors. These models may help to accelerate translational research in cancer biology and tissue engineering.     

Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.     

Development of a 3D human in vitro skin co‐culture model for detecting irritants in real‐time

Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant‐induced inflammatory reaction and cytotoxicy. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi®)] at sub‐toxic concentrations and studied interleukin‐1 alpha (IL‐1α) release from human keratinocytes and activation of NF‐κB in human fibroblasts. NF‐κB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi®. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF‐κB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi® was required for NF‐κB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF‐κB. A technical advantage of using transfected cells in 3D was an ability to detect NF‐κB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL‐1α was measured in keratinocyte‐conditioned medium after incubation with SDS or Formi®, which correlated with fibroblast NF‐κB activity. In summary, this work has value for developing 3D tissue engineered co‐culture models for the in vitro testing of irritant chemicals at sub‐toxic concentrations, as an alternative to in vivo models. Biotechnol. Bioeng. 2010;106: 794–803. © 2010 Wiley Periodicals, Inc.     

Mathematical modelling of skeletal repair

Tissue engineering offers significant promise as a viable alternative to current clinical strategies for replacement of damaged tissue as a consequence of disease or trauma. Since mathematical modelling is a valuable tool in the analysis of complex systems, appropriate use of mathematical models has tremendous potential for advancing the understanding of the physical processes involved in such tissue reconstruction. In this review, the potential benefits, and limitations, of theoretical modelling in tissue engineering applications are examined with specific emphasis on tissue engineering of bone. A central tissue engineering approach is the in vivo implantation of a biomimetic scaffold seeded with an appropriate population of stem or progenitor cells. This review will therefore consider the theory behind a number of key factors affecting the success of such a strategy including: stem cell or progenitor population expansion and differentiation ex vivo; cell adhesion and migration, and the effective design of scaffolds; and delivery of nutrient to avascular structures. The focus will be on current work in this area, as well as on highlighting limitations and suggesting possible directions for future work to advance health-care for all.     

Native-mimicking in vitro microenvironment: an elusive and seductive future for tumor modeling and tissue engineering

Human connective tissues are complex physiological microenvironments favorable for optimal survival, function, growth, proliferation, differentiation, migration, and death of tissue cells. Mimicking native tissue microenvironment using various three-dimensional (3D) tissue culture systems in vitro has been explored for decades, with great advances being achieved recently at material, design and application levels. These achievements are based on improved understandings about the functionalities of various tissue cells, the biocompatibility and biodegradability of scaffolding materials, the biologically functional factors within native tissues, and the pathophysiological conditions of native tissue microenvironments. Here we discuss these continuously evolving physical aspects of tissue microenvironment important for human disease modeling, with a focus on tumors, as well as for tissue repair and regeneration. The combined information about human tissue spaces reflects the necessities of considerations when configuring spatial microenvironments in vitro with native fidelity to culture cells and regenerate tissues that are beyond the formats of 2D and 3D cultures. It is important to associate tissue-specific cells with specific tissues and microenvironments therein for a better understanding of human biology and disease conditions and for the development of novel approaches to treat human diseases.     

Prenatal exposure to environmental factors and congenital limb defects

Limb congenital defects afflict approximately 0.6:1000 live births. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants, represents a major contributing factor to limb defects. Examples of well‐recognized limb teratogenic agents include thalidomide, warfarin, valproic acid, misoprostol, and phenytoin. While the mechanism by which these agents cause dymorphogenesis is increasingly clear, prediction of the limb teratogenicity of many thousands of as yet uncharacterized environmental factors (pollutants) remains inexact. This is limited by the insufficiencies of currently available models. Specifically, in vivo approaches using guideline animal models have inherently deficient predictive power due to genomic and anatomic differences that complicate mechanistic comparisons. On the other hand, in vitro two‐dimensional (2D) cell cultures, while accessible for cellular and molecular experimentation, do not reflect the three‐dimensional (3D) morphogenetic events in vivo nor systemic influences. More robust and accessible models based on human cells that accurately replicate specific processes of embryonic limb development are needed to enhance limb teratogenesis prediction and to permit mechanistic analysis of the adverse outcome pathways. Recent advances in elucidating mechanisms of normal development will aid in the development of process‐specific 3D cell cultures within specialized bioreactors to support multicellular microtissues or organoid constructs that will lead to increased understanding of cell functions, cell‐to‐cell signaling, pathway networks, and mechanisms of toxicity. The promise is prompting researchers to look to such 3D microphysiological systems to help sort out complex and often subtle interactions relevant to developmental malformations that would not be evident by standard 2D cell culture testing. Birth Defects Research (Part C) 108:243–273, 2016. © 2016 Wiley Periodicals, Inc.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

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.     

Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma

An in vitro 3D model was developed utilizing a synthetic microgravity environment to facilitate studying the cell interactions. 2D monolayer cell culture models have been successfully used to understand various cellular reactions that occur in vivo. There are some limitations to the 2D model that are apparent when compared to cells grown in a 3D matrix. For example, some proteins that are not expressed in a 2D model are found up-regulated in the 3D matrix. In this paper, we discuss techniques used to develop the first known large, free-floating 3D tissue model used to establish tumor spheroids. The bioreactor system known as the High Aspect Ratio Vessel (HARVs) was used to provide a microgravity environment. The HARVs promoted aggregation of keratinocytes (HaCaT) that formed a construct that served as scaffolding for the growth of mouse melanoma. Although there is an emphasis on building a 3D model with the proper extracellular matrix and stroma, we were able to develop a model that excluded the use of matrigel. Immunohistochemistry and apoptosis assays provided evidence that this 3D model supports B16.F10 cell growth, proliferation, and synthesis of extracellular matrix. Immunofluorescence showed that melanoma cells interact with one another displaying observable cellular morphological changes. The goal of engineering a 3D tissue model is to collect new information about cancer development and develop new potential treatment regimens that can be translated to in vivo models while reducing the use of laboratory animals.     

Modelling species distributions without using species distributions: the cane toad in Australia under current and future climates

Accurate predictions of the potential distribution of range-shifting species are required for effective management of invasive species, and for assessments of the impact of climate change on native species. Range-shifting species pose a challenge for traditional correlative approaches to range prediction, often requiring the extrapolation of complex statistical associations into novel environmental space. Here we take an alternative approach that does not use species occurrence data, but instead captures the fundamental niche of a species by mechanistically linking key organismal traits with spatial data using biophysical models. We demonstrate this approach with a major invasive species, the cane toad Bufo marinus in Australia, assessing the direct climatic constraints on its ability to move, survive, and reproduce. We show that the current range can be explained by thermal constraints on the locomotor potential of the adult stage together with limitations on the availability of water for the larval stage. Our analysis provides a framework for biologically grounded predictions of the potential for cane toads to expand their range under current and future climate scenarios. More generally, by quantifying spatial variation in physiological constraints on an organism, trait-based approaches can be used to investigate the range-limits of any species. Assessments of spatial variation in the physiological constraints on an organism may also provide a mechanistic basis for forecasting the rate of range expansion and for understanding a species' potential to evolve at range-edges. Mechanistic approaches thus have broad application to process-based ecological and evolutionary models of range-shift.