Three-dimensional Tissue Culture Research Articles

Third harmonic generation microscopy of cells and tissue organization.

The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra- and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in three-dimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice.

Visualization of three-dimensional oxygen concentration for three-dimensional tissue culture

Visualization of three-dimensional oxygen concentration for three-dimensional tissue culture

Alginate: Current Use and Future Perspectives in Pharmaceutical and Biomedical Applications

Over the last decades, alginates, natural multifunctional polymers, have increasingly drawn attention as attractive compounds in the biomedical and pharmaceutical fields due to their unique physicochemical properties and versatile biological activities. The focus of the paper is to describe biological and pharmacological activity of alginates and to discuss the present use and future possibilities of alginates as a tool in drug formulation. The recent technological advancements with using alginates, issues related to alginates suitability as matrix for three-dimensional tissue cultures, adjuvants of antibiotics, and antiviral agents in cell transplantation in diabetes or neurodegenerative diseases treatment, and an update on the antimicrobial and antiviral therapy of the alginate based drugs are also highlighted.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro.

The effect of simvastatin treatment on endothelial cell response to shear stress and tumor necrosis factor alpha stimulation.

BackgroundStatin drugs are one of the most commonly prescribed pharmaceuticals by physicians. By blocking the rate-limiting step in the cholesterol biosynthesis pathway, statins inhibit cholesterol synthesis, which benefits patient health. However, since many other important cellular processes are regulated within this pathway, they may also be influenced by statin therapy. These pleiotropic effects of statins have not been fully investigated, but are believed to positively influence endothelial cells (ECs), which line the vasculature in a confluent monolayer. Few studies have considered the effect of blood flow on ECs and how this may augment EC response to statins.MethodsIn this study, the effect of statin treatment on ECs is investigated for cells stimulated with tumor necrosis factor alpha (TNF-α), an inflammatory cytokine that promotes an atheroprone endothelium. Additionally, ECs are exposed to a physiologically relevant wall shear stress (WSS) of 12.5 dynes/cm2 using a three-dimensional tissue culture model to provide a realistic hemodynamic environment. ECs are analyzed for morphology using light microscopy as well as cytoskeletal structure and alignment using confocal microscopy. Statistical analysis is performed on the results using both the one-way analysis of variance with Bonferroni post-tests and the two-tailed t test.ResultsWe have shown that statin treatment caused cells to adapt to a rounded, atheroprone morphology, with a significantly higher shape index. Oppositely, TNF-α stimulation caused cells to elongate to an atheroprotective morphology, with a significantly lower shape index. WSS and TNF-α were unable to reverse any statin-induced cell rounding or F-actin disruption.ConclusionFurther work is therefore needed to determine why statin drugs cause cells to have an atheroprone morphology, but an atheroprotective genotype, and why TNF-α stimulation causes an atheroprotective morphology, but an atheroprone genotype. Despite the morphological changes due to statins or stimulation, ECs still respond to WSS. Understanding how statins influence ECs will allow for more targeted treatments for hypercholestemia and potentially other diseases.

Noninvasive Oxygen Monitoring in Three-Dimensional Tissue Cultures Under Static and Dynamic Culture Conditions.

We present a new method for noninvasive real-time oxygen measurement inside three-dimensional tissue-engineered cell constructs in static and dynamic culture settings in a laminar flow bioreactor. The OPAL system (optical oxygen measurement system) determines the oxygen-dependent phosphorescence lifetime of spherical microprobes and uses a two-frequency phase-modulation technique, which fades out the interference of background fluorescence from the cell carrier and culture medium. Higher cell densities in the centrum of the scaffolds correlated with lower values of oxygen concentration obtained with the OPAL system. When scaffolds were placed in the bioreactor, higher oxygen values were measured compared to statically cultured scaffolds in a Petri dish, which were significantly different at day 1–3 of culture. This technique allows the use of signal-weak microprobes in biological environments and monitors the culture process inside a bioreactor.

3D Bioprinted Muscle and Tendon Tissues for Drug Development.

In our aging societies, there is a huge medical need for treatments of degenerative muscle and tendon diseases, for which there are currently no approved pharmaceutical therapies. Furthermore, also devastating muscle diseases that affect children and younger patients such as Duchenne muscular dystrophy or amyothrophic lateral sclerosis (ALS) lack curative drug treatments. A major hurdle in new drug discovery and development is the nonexistence of normal functional human tissues and diseased tissues for compound screening and testing. Currently, most high-throughput drug screening campaigns are performedwith target-centeredbiochemicalor simplehumancellbased assays if the drug target is known, or with two-dimensional (2D) cell culture phenotypic screens, if the target is unknown. Identified hits and further optimized compounds (leads) are then usually analyzed in low-throughput rodent ex vivo and/or in vivo preclinical animal models for efficacy, potency, specificity and safety. Besides the obvious slowness of this step to assess the pharmacodynamic and physiological effects of new drug candidates the jump from human cell-based systems to animal preclinical models and to human clinical trials is very often too large and not reliable enough to master. Fortunately, recent years have seen an incredible progress in new approaches generating functional 3D human tissues from normal and disease donors. Three-dimensional (3D) human organotypic tissue cultures are generally more predictive for in vivo effects, because they model much more in vivo tissue physiology than conventional 2D cell cultures. Thus, 3D tissue culture has the potential to revolutionize drug discovery and development. The paradigm shift from 2D to 3D cell culture is already showing great benefit in basic research of tissues differentiation and homeostasis as well as in tissue engineering efforts for regenerative medicine applications.[1] Skeletal muscle and tendon tissues basically consist each of one single functional cell type: the multinucleated myotube and the tenocyte. Myotubes are the basic building block of skeletal muscle and are also called myofibers. They are formed by cell fusion of myoblasts. Myofibers are bundled into fascicles and an individual skeletal muscle can be made up of many fascicles surrounded by a connective tissue sheet. At each end, skeletal muscles are seamlessly and tightly connected to tendon through an interface called the myotendinous junction (MTJ). In contrast to skeletal muscle, which is mainly composed of cells with very little extracellularmatrix (ECM), tendon tissuemainly consists of collagen ECM with few embedded threads of aligned cells. Over the last years, different groups have generated 3D skeletal muscle and tendon tissues for basic research and screening purposes.[2–5] Tissues were generated between different kinds of attachment posts allowing mechanical tension and/or stimulation and functional readouts. However, although these systems allowed better in vitro investigations of skeletal muscle and tendon tissue generation and physiology as well as compound screenings they all suffer from a lack of reproducibility, robustness and cost effectiveness for high-throughput analyses and routine compound screenings.

Cartilage-derived extracellular matrix extract promotes chondrocytic phenotype in three-dimensional tissue culture

Cell transplantation is a promising regenerative therapy for cartilage degeneration. However, obtaining sufficient numbers of cells for this purpose is a challenge, due a lack of autologous donor tissue and the difficulty of culturing chondrocytes in vitro. Tissue engineering strategies that induce or maintain chondrocytic phenotype may solve these problems by (1) broadening the range of available donor tissue, and (2) facilitating the expansion of these cells while controlling phenotypic drift. In this study, bone marrow-derived mesenchymal stem cells (MSCs) and cartilage-derived cells (CDCs) were cultured on composite hydrogels containing agarose and homogenized cartilage extracellular matrix (ECM). MSCs cultured on agarose-ECM scaffolds did not show significant signs of chondrogenic differentiation in the absence of additional cues. However, CDCs cultured on agarose-ECM scaffolds proliferated more rapidly than their ECM-free counterparts and MSCs, while retaining chondrocytic morphology. These results were corroborated via expression of cartilage marker genes: in autologous constructs, SOX 9 expression was upregulated by 12.6 ± 5.3-fold, and COL II was upregulated by 2.0 ± 0.3-fold. Agarose-ECM composite hydrogels are therefore useful for expanding partially differentiated CDCs for applications in regenerative medicine.

Abstract B54: Non-malignant cells from metastatic niches induce estrogen receptor-positive breast cancer cell resistance to therapy

Abstract Metastatic lesions remain the main cause of mortality in breast cancer (BrCa) and their resistance to systemic therapies has been mainly attributed to mutations in BrCa cells, while the non-mutually exclusive role of the metastatic microenvironment has been largely overlooked. To address the role of the local microenvironment of metastatic sites in conferring drug resistance to BrCa cells, we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant cells from organs frequently targeted by metastatic disease. BrCa cells (MCF7, T47D, and ZR75-1) expressing the firefly luciferase gene were grown in extracellular matrices (collagen type I and Matrigel) alone or with nonmalignant cells from the respective metastatic target tissues, and exposed to clinically achievable concentrations of FDA-approved antineoplastic agents widely used for the treatment of BrCa. Interestingly, BrCa cell response to antiestrogens (hydroxytamoxifen (4-OHT), fulvestrant or raloxifene) in 3D conditions was marked by acinar differentiation of tumor spheroids into structures that resemble normal breast epithelium. Co-culture of BrCa cells with immortalized accessory cells from the bone (BMSCs and osteoblasts); brain (astrocytes); liver (hepatocytes); and lung (fibroblasts) in 3D (but not 2D) conditions, inhibited the antiestrogen-induced acinar differentiation and conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. The results obtained with immortalized human BMSCs were further confirmed in co-cultures of BrCa cells with primary human BMSCs isolated from bone metastatic site. MCF7 cells injected s.c. in nude mice together with BMSCs (heterotypic xenografts) had decreased response to tamoxifen compared with monotypic xenografts (MCF7 alone). Furthermore, immortalized BMSCs or metastatic patient-derived BMSCs induced MCF7 cell resistance to several chemotherapeutic agents including vinca alkaloids (vincristine, vinblastin and vinorelbin) and taxanes (docetaxel, paclitaxel, cabazitaxel). Notably, primary BMSCs from healthy donors and brain astrocytes isolated from cancer-free neurosurgical specimens did not induce BrCa cell resistance to anti-estrogens or chemotherapeutic agents. BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including genes involved in IGF1R, IL6 and EGFR superfamily members; NF-kappaB and other antiapoptotic pathways; and elevated c-Myc protein levels. The co-cultures of MCF7 cells with BMSCs had increased sensitivity to inhibitors of c -myc, IGF1R, IL-6 and JAK/STAT pathways when compared to the respective mono-cultures. Our results indicate that local nonmalignant cells present in the metastatic niche can function as “accessories” to the tumor, conferring BrCa cell resistance to antiestrogens and chemotherapeutic agents in 3D tissue cultures and xenograft models. Preclinical modeling of the composition and 3D architecture of metastatic lesions can help elucidate the mechanisms of stroma-induced drug resistance and may reveal new therapeutic targets for refractory BrCa patients with metastatic disease. Citation Format: Eugen Dhimolea, Richard Groen, Culhane Aedin, Mitsiades Nicholas, Polyak Kornelia, Mitsiades Constantine. Non-malignant cells from metastatic niches induce estrogen receptor-positive breast cancer cell resistance to therapy. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B54. doi:10.1158/1538-7445.CHTME14-B54

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm−1 in the spiral direction induced a mean migratory speed of 1015 μm day−1. Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

Nondestructive evaluation of progressive neuronal changes in organotypic rat hippocampal slice cultures using ultrahigh-resolution optical coherence microscopy.

Three-dimensional tissue cultures have been used as effective models for studying different diseases, including epilepsy. High-throughput, nondestructive techniques are essential for rapid assessment of disease-related processes, such as progressive cell death. An ultrahigh-resolution optical coherence microscopy (UHR-OCM) system with [Formula: see text] axial resolution and [Formula: see text] transverse resolution was developed to evaluate seizure-induced neuronal injury in organotypic rat hippocampal cultures. The capability of UHR-OCM to visualize cells in neural tissue was confirmed by comparison of UHR-OCM images with confocal immunostained images of the same cultures. In order to evaluate the progression of neuronal injury, UHR-OCM images were obtained from cultures on 7, 14, 21, and 28 days in vitro (DIVs). In comparison to DIV 7, statistically significant reductions in three-dimensional cell count and culture thickness from UHR-OCM images were observed on subsequent time points. In cultures treated with kynurenic acid, significantly less reduction in cell count and culture thickness was observed compared to the control specimens. These results demonstrate the capability of UHR-OCM to perform rapid, label-free, and nondestructive evaluation of neuronal death in organotypic hippocampal cultures. UHR-OCM, in combination with three-dimensional tissue cultures, can potentially prove to be a promising tool for high-throughput screening of drugs targeting various disorders.

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting.

Three-dimensional (3D) tissue culture models may recapitulate aspects of the tumorigenic microenvironment in vivo, enabling the study of cancer progression in vitro. Both hypoxia and matrix stiffness are known to regulate tumor growth. Using a modular culture system employing an acrylated hyaluronic acid (AHA) hydrogel, three hydrogel matrices with distinctive degrees of viscoelasticity - soft (78±16 Pa), medium (309± 57 Pa), and stiff (596± 73 Pa) - were generated using the same concentration of adhesion ligands. Oxygen levels within the hydrogel in atmospheric (21 %), hypoxic (5 %), and severely hypoxic (1 %) conditions were assessed with a mathematical model. HT1080 fibrosarcoma cells, encapsulated within the AHA hydrogels in high densities, generated nonuniform oxygen distributions, while lower cell densities resulted in more uniform oxygen distributions in the atmospheric and hypoxic environments. When we examined how varying viscoelasticity in atmospheric and hypoxic environments affects cell cycles and the expression of BNIP3 and BNIP3L (autophagy and apoptosis genes), and GLUT-1 (a glucose transport gene), we observed that HT1080 cells in 3D hydrogel adapted better to hypoxic conditions than those in a Petri dish, with no obvious correlation to matrix viscoelasticity, by recovering rapidly from possible autophagy/apoptotic events and alternating metabolism mechanisms. Further, we examined how HT1080 cells cultured in varying viscoelasticity and oxygen tension conditions affected endothelial sprouting and invasion. We observed that increased matrix stiffness reduced endothelial sprouting and invasion in atmospheric conditions; however, we observed increased endothelial sprouting and invasion under hypoxia at all levels of matrix stiffness with the upregulation of vascular endothelial growth factor (VEGF) and angiopoeitin-1 (ANG-1). Overall, HT1080 cells encapsulated in the AHA hydrogels under hypoxic stress recovered better from apoptosis and demonstrated greater angiogenic induction. Thus, we propose that oxygen tension more profoundly influences cell fate and the angiogenic potential of 3D cultured HT1080 fibrosarcoma cells than does matrix stiffness.

Rapid uptake of glucose and lactate, and not hypoxia, induces apoptosis in three-dimensional tumor tissue culture

The spatial arrangement of cellular metabolism in tumor tissue critically affects the treatment of cancer. However, little is known about how diffusion and cellular uptake relate to intracellular metabolism and cell death in three dimensions. To quantify these mechanisms, fluorescent microscopy and multicellular tumor cylindroids were used to measure pH and oxygen profiles, and quantify the distribution of viable, apoptotic and necrotic cells. Spheroid dissociation, enzymatic analysis, and mass spectrometry were used to measure concentration profiles of glucose, lactate and glutamine. A mathematical model was used to integrate these measurements and calculate metabolic rate parameters. It was found that large cylindroids, >500 μm in diameter, contained apoptotic and necrotic cells, whereas small cylindroids contained apoptotic but not necrotic cells. The center of cylindroids was found to be acidic but not hypoxic. From the edge to the center, concentrations of glucose, lactate and glutamine decreased rapidly. Throughout the cell masses lactate was consumed and not produced. These measurements indicate that apoptosis was the primary mechanism of cell death; acidity was not caused by lactic acid; and cell death was caused by depletion of carbon sources and not hypoxia. The mathematical model showed that the transporter enzymes for glucose and lactate were not saturated; oxygen uptake was limited by intracellular metabolism; and oxygen uptake was not limited by membrane-transport or diffusion. Unsaturated transmembrane uptake may be the cause of both proliferative and apoptotic regimes in cancer. These results suggest that transporter enzymes are excellent targets for treating well oxygenated tumors.

Application of Porous Materials to Three-dimensional Tissue Culture in Capillary Formation in Vitro

Application of Porous Materials to Three-dimensional Tissue Culture in Capillary Formation in Vitro

Development of a novel 3D culture system for screening features of a complex implantable device for CNS repair.

Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI.

Modeling inflammation and oxidative stress in gastrointestinal disease development using novel organotypic culture systems.

Gastroesophageal reflux disease (GERD), Barrett's esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions.

Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs – clearing mucus and pathogens from the airway – we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3–7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial–mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.

Abstract C283: Non-malignant cells from tissues targeted by metastatic breast cancer induce tumor cell resistance to antiestrogens and conventional chemotherapeutic agents.

Abstract The role of the microenvironment of the metastatic niche on breast cancer (BrCa) de novo or secondary resistance to currently available treatments is poorly understood. To study the tumor-stroma interactions and explore potential therapeutic targets in the context of the local metastatic microenvironment of disseminated BrCa we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant accessory cells from organs frequently targeted by metastatic disease, which model the composition and architecture of metastatic lesions. MCF7, T47D and ZR75-1 cells expressing luciferase were immersed in extracellular matrices (collagen type I and Matrigel) alone or with accessory cells, and exposed to clinically achievable concentrations of FDA-approved antineoplastic agents widely used for the treatment of BrCa. The presence of HS-5 bone marrow stromal cells (BMSCs) induced BrCa cell resistance to vinca alkaloids (vincristine, vinblastin and vinorelbin) and taxanes (docetaxel, paclitaxel, cabazitaxel) both in 2D and 3D co-cultures. BMSCs sensitized the MCF7 to antifolates (methotrexate, pralatrexate) in 2D (but not in 3D) cultures. Importantly, BrCa spheroid response to antiestrogens (hydroxytamoxifen (4-OHT), fulvestrant or raloxifene) in 3D conditions was marked by acinar differentiation of tumor spheroids that resembles normal breast epithelium. Co-culture of BrCa cell and immortalized accessory cells from the bone (BMSCs; HOBIT and hFOB osteoblasts), brain (SVGp12 astrocytes), liver (THLE3 hepatocytes), and lung (MRC9 fibroblasts) in 3D conditions, conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. MCF7 cells injected s.c. in nude mice together with BMSCs (heterotypic xenografts) had reduced response to tamoxifen compared with monotypic xenografts (MCF7 alone). BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including members of the JAK/STAT pathway, interferon signal gene signature, signaling through EGFR superfamily members, NFkB and other antiapoptotic pathways; and elevated c-Myc protein levels. The growth of MCF7 cells in co-culture with BMSCs or hepatocytes was inhibited by exogenous TGFβ1/2 or neutralizing antibodies against IL-6, INF-gamma, and TNF-alpha. Inhibition of JAK1/2 using ruxolitinib also inhibited MCF7 cell growth in co-culture conditions. Our results indicate that accessory cells representing the microenvironment of metastatic sites confer BrCa cell resistance to antiestrogens and chemotherapeutic agents in conventional and/or 3D tissue cultures and xenograft models. The mechanisms of stroma-induced drug resistance reveal candidate therapeutic targets for refractory BrCa patients with metastatic disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C283. Citation Format: Eugen Dhimolea, Richard W.J. Groen, Kornelia Polyak, Constantine S. Mitsiades. Non-malignant cells from tissues targeted by metastatic breast cancer induce tumor cell resistance to antiestrogens and conventional chemotherapeutic agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C283.