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1.  Local control of hepatic phenotype with growth factor-encoded surfaces 
The goal of the present study was to modulate the phenotype expression of hepatocytes in vitro on surfaces imprinted with growth factors (GFs). Hepatocyte growth factor (HGF) or transforming-growth factor-β1 (TGF-β1) were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 300 μm or 500 μm diameter spots. Primary rat hepatocytes were seeded on top of the arrays, forming clusters corresponding in size to the underlying protein spots. The TGF-β1 spots appeared to downregulate markers of hepatic (epithelial) phenotype while upregulating expression of mesenchymal markers. Conversely, hepatocytes cultured on HGF spots maintained high level of epithelial markers. When hepatocytes were seeded onto alternating spots of HGF and TGF-β1, their phenotype was found to depend on center-to-center distance between the spots. At shorter distances cross-expression of epithelial and mesenchymal markers was observed while at distances exceeding 1.25 mm divergence of phenotypes, epithelial on HGF and mesenchymal on TGF-β was seen. Overall, our results demonstrate that GF-encoded surfaces can modulate phenotype within groups of cells cultured on the same surface. Given the importance of phenotype switching in development, fibrosis and cancer, this platform may be used to gain useful insights into the mechanisms of processes such as epithelial-to-mesenchymal transition or stem cell fate selections.
PMCID: PMC4331075  PMID: 24247788
2.  A cell–ECM screening method to predict breast cancer metastasis 
Breast cancer preferentially spreads to the bone, brain, liver, and lung. The clinical patterns of this tissue-specific spread (tropism) cannot be explained by blood flow alone, yet our understanding of what mediates tropism to these physically and chemically diverse tissues is limited. While the microenvironment has been recognized as a critical factor in governing metastatic colonization, the role of the extracellular matrix (ECM) in mediating tropism has not been thoroughly explored. We created a simple biomaterial platform with systematic control over the ECM protein density and composition to determine if integrin binding governs how metastatic cells differentiate between secondary tissue sites. Instead of examining individual behaviors, we compiled large patterns of phenotypes associated with adhesion to and migration on these controlled ECMs. In combining this novel analysis with a simple biomaterial platform, we created an in vitro fingerprint that is predictive of in vivo metastasis. This rapid biomaterial screen also provided information on how β1, α2, and α6 integrins might mediate metastasis in patients, providing insights beyond a purely genetic analysis. We propose that this approach of screening many cell–ECM interactions, across many different heterogeneous cell lines, is predictive of in vivo behavior, and is much simpler, faster, and more economical than complex 3D environments or mouse models. We also propose that when specifically applied toward the question of tissue tropism in breast cancer, it can be used to provide insight into certain integrin subunits as therapeutic targets.
Insight, innovation, integration
We developed a high-throughput method to rapidly screen cell adhesion, motility, and growth factor responses on biomaterial surfaces. This approach is analogous to systems biology, relying on cell phenotypes in lieu of genetics. We used this technique to reveal patterns of phenotypes associated with breast cancer metastasis to possible tissue sites (bone, brain, lung). By comparing the phenotypic patterns between cell lines that metastasize to only one tissue site with heterogeneous cell lines, we provide the first method to connect in vitro phenotype to in vivo fate. This method is successful without genetic analysis, yet it also predicts outcomes related to integrin gene expression, potentially identifying new targets for tissue-specific metastasis.
PMCID: PMC4323858  PMID: 25537447
3.  [No title available] 
PMCID: PMC3984951  PMID: 24343706
4.  [No title available] 
PMCID: PMC4000736  PMID: 24336811
5.  Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts 
This study tests the hypothesis that the cell cytoskeletal (CSK) network can rearrange from geodesic dome type structures to stress fibers in response to microenvironmental cues. The CSK geodesic domes are highly organized actin microarchitectures within the cell, consisting of ordered polygonal elements. We studied primary neonatal rat cardiac fibroblasts. The cues used to trigger the interconversion between the two CSK architectures (geodesic domes and stress fibers) included factors affecting spatial order and the degree of CSK tension in the cells. Microfabricated three-dimensional substrates with micrometre sized grooves and peaks were used to alter the spatial order of cell growth in culture. CSK tension was modified by 2,3-butanedione 2-monoxime (BDM), cytochalasin D and the hyphae of Candida albicans. CSK geodesic domes occurred spontaneously in about 20% of the neonatal rat cardiac fibroblasts used in this study. Microfabricated structured surfaces produced anisotropy in the cell CSK and effectively converted geodesic domes into stress fibers in a dose-dependent manner (dependence on the period of the features). Affectors of actin structure, inhibitors of CSK tension and cell motility, e.g. BDM, cytochalasin D and the hyphae of C. albicans, suppressed or eliminated the geodesic domes. Our data suggest that the geodesic domes, similar to actin stress fibers, require maintenance of CSK integrity and tension. However, microenvironments that promote structural anisotropy in tensed cells cause the transformation of the geodesic domes into stress fibers, consistent with topographic cell guidance and some previous CSK model predictions.
PMCID: PMC4299856  PMID: 20023805
Nanoparticles can be engineered with distinctive compositions, sizes, shapes, and surface chemistries to enable novel techniques in a wide range of biological applications. The unique properties of nanoparticles and their behavior in biological milieu also enable exciting and integrative approaches to studying fundamental biological questions. This review will provide an overview of various types of nanoparticles and concepts of targeting nanoparticles. We will also discuss the advantages and recent applications of using nanoparticles as tools for drug delivery, imaging, sensing, and for the understanding of basic biological processes.
PMCID: PMC3865110  PMID: 24104563
7.  Robust Pluripotent Stem Cell Expansion and Cardiomyocyte Differentiation via Geometric Patterning 
Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening.
PMCID: PMC3918460  PMID: 24141327
8.  Motility efficiency and spatiotemporal synchronization in non-metastatic vs. metastatic breast cancer cells 
Metastatic breast cancer cells move not only more rapidly and persistently than their non-metastatic variants but in doing so use the mechanical work of the cytoskeleton more efficiently. The efficiency of the cell motions is defined for entire cells (rather than parts of the cell membrane) and is related to the work expended in forming membrane protrusions and retractions. This work, in turn, is estimated by integrating the protruded and retracted areas along the entire cell perimeter and is standardized with respect to the net translocation of the cell. A combination of cross-correlation, Granger causality, and morphodynamic profiling analyses is then used to relate the efficiency to the cell membrane dynamics. In metastatic cells, the protrusions and retractions are highly “synchronized” both in space and in time and these cells move efficiently. In contrast, protrusions and retractions formed by non-metastatic cells are not “synchronized” corresponding to low motility efficiencies. Our work provides a link between the kinematics of cell motions and their energetics. It also suggests that spatiotemporal synchronization might be one of the hallmarks of invasiveness of cancerous cells.
PMCID: PMC4122865  PMID: 24136177
9.  Cancer Cell Glycocalyx Mediates Mechanostransduction and Flow-Regulated Invasion 
Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) were investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiologic levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP actvity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx – a potential target for therapeutics.
PMCID: PMC4249644  PMID: 24077103
10.  A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions 
Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.
PMCID: PMC3848881  PMID: 24061208
11.  Changes in Translational Efficiency is a Dominant Regulatory Mechanism in the Environmental Response of Bacteria† 
To understand how cell physiological state affects mRNA translation, we used Shewanella oneidensis MR-1 grown under steady state conditions at either 20% or 8.5% O2. Using a combination of quantitative proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein-mRNA ratios were primarily due to differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Transcription levels were significant altered for 26% of the protein changes; translational efficiency was significantly altered for 46% and a combination of both was responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part due to altered tRNA pools, is a major determinant of regulated alterations in protein expression levels in bacteria.
PMCID: PMC3968953  PMID: 24081429
12.  From swimming to swarming: Escherichia coli cell motility in two-dimensions† 
Escherichia coli swarmer cells coordinate their movement when confined in thin layers of fluid on agar surfaces. The motion and dynamics of cells, pairs of cells, and packs of cells can be recapitulated and studied in polymer microfluidic systems that are designed to constrain swarmer cell movement in thin layers of fluid between no-slip surfaces. The motion of elongated, smooth swimming E. coli cells in these environments reproduces the behavior of packs of cells observed at the leading edge of swarming communities and demonstrates the delicate balance between the physical dimensions of fluids and bacterial cell behavior.
PMCID: PMC4222179  PMID: 24145500
13.  Bioactive sphingolipid metabolites modulate ovarian cancer cell structural mechanics 
Cancer progression is associated with an increased deformability of cancer cells and reduced resistance to mechanical forces, enabling motility and invasion. This is important for metastases survival and outgrowth and as such could be a target for chemopreventive strategies. In this study, we determined the differential effects of exogenous sphingolipid metabolites on the elastic modulus of mouse ovarian surface epithelial cells as they transition to cancer. Treatment with ceramide or sphingosine-1-phosphate in non-toxic concentrations decreased the average elastic modulus by 21% (p ≤ 0.001) in transitional and 15% (p ≤ 0.02) in aggressive stages while exerting no appreciable effect on non-malignant cells. In contrast, sphingosine treatment on average increased the elastic modulus by 33% (p ≤ 0.0002) in aggressive cells while not affecting precursor cells. These results indicate that tumor-supporting sphingolipid metabolites act by making cells softer, while the anti-cancer metabolite sphingosine partially reverses the decreased elasticity associated with cancer progression. Thus, sphingosine may be a valid alternative to conventional chemotherapeutics in ovarian cancer prevention or treatment.
PMCID: PMC3908782  PMID: 24056950
14.  Network Analysis of Differential Ras Isoform Mutation Effects on Intestinal Epithelial Responses to TNF-α† 
Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that can elicit distinct cellular behaviors under different molecular contexts. Mitogen activated protein kinase (MAPK) pathways, especially the extracellular signal-regulated kinase (Erk) pathway, help to integrate influences from the environmental context, and therefore modulate the phenotypic effect of TNF-α exposure. To test how variations in flux through the Erk pathway modulate TNF-α-elicited phenotypes in a complex physiological environment, we exposed mice with different Ras mutations (K-Ras activation, N-Ras activation, and N-Ras ablation) to TNF-α and observed phenotypic and signaling changes in the intestinal epithelium. Hyperactivation of Mek1, an Erk kinase, was observed in the intestine of mice with K-Ras activation and, surprisingly, in N-Ras null mice. Nevertheless, these similar Mek1 outputs did not give rise to the same phenotype, as N-Ras null intestine was hypersensitive to TNF-α-induced intestinal cell death while K-Ras mutant intestine was not. A systems biology approach applied to sample the network state revealed that the signaling contexts presented by these two Ras isoform mutations were different. Consistent with our experimental data, N-Ras ablation induced a signaling network state that was mathematically predicted to be pro-death, while K-Ras activation did not. Further modeling by constrained Fuzzy Logic (cFL) revealed that N-Ras and K-Ras activate the signaling network with different downstream distributions and dynamics, with N-Ras effects being more transient and diverted more towards PI3K-Akt signaling and K-Ras effects being more sustained and broadly activating many pathways. Our study highlights the necessity to consider both environmental and genomic contexts of signaling pathway activation in dictating phenotypic responses, and demonstrates how modeling can provide insight into complex in vivo biological mechanisms, such as the complex interplay between K-Ras and N-Ras in their downstream effects
PMCID: PMC3966991  PMID: 24084984
15.  Transfection in the third dimension 
An understanding of parameters that modulate gene transfer in 3-D will assist in the formation of gene delivery systems and scaffolds, which can mediate efficient non-viral delivery for guiding in-vivo tissue regeneration and therapy. We have previously demonstrated the cell area and length, integrin expression, and RhoGTPases mediated signalling to be pivotal parameters that guide gene transfer to mouse mesenchymal stem cells (mMSCs) cultured in 2-D and are modulated by ECM proteins. In this study, we were interested in determining if cationic polymer mediated gene transfer to cells seeded in 3-D would occur through different mechanisms as compared to 2-D. In particular, we examined the endocytosis pathways used to internalize polyplexes, and the role of cytoskeletal dynamics and RhoGTPases on non-viral gene transfer for cells seeded in 2-D and 3-D. Inhibition of clathrin- and caveolae- mediated endocytosis resulted in more drastic decrease in overall transgene expression for cells seeded in 3-D than those in 2-D. In addition, polyplex internalization was only significantly decreased in 3-D when clathrin-mediated endocytosis was inhibited, while caveolae-mediated endocytosis inhibition for cells seeded in 2-D resulted in the strongest polyplex internalization inhibition. Actin and microtubule polymerization affected 2-D and 3-D transfection differently. Microtubule depolymerization enhanced transgene expression in 2-D, but inhibited transgene expression in 3-D. Last, inhibition of RhoGTPases also affected 2-D and 3-D transfection differently. The inhibition of ROCK effector resulted in a decrease of transgene expression and internalization for cells seeded in 3-D, but not 2-D and the inhibition of effector PAK1 resulted in an increase of transgene expression for both 2-D and 3-D. Overall, our study suggests that the process of gene transfer occurs through different mechanisms for cells seeded in 2-D compared to those seeded in 3-D.
PMCID: PMC3798060  PMID: 23929354
16.  Chemical principles for a novel fluorescent probe with high cancer-targeting selectivity and sensitivity 
Understanding of principles governing selective and sensitive cancer targeting is critical for development of chemicals in cancer diagnostics and treatments. We determined the underlying mechanisms of how a novel fluorescent small organic molecule, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), selectively labels cancer cells but not normal cells. We show that BMVC is retained in the lysosomes of normal cells. In cancer cells, BMVC escapes lysosomal retention and localizes to the mitochondrial or to the nucleus, where DNA-binding dramatically increases BMVC fluorescence intensity, allowing it to light up only cancer cells. Structure-function analyses of BMVC derivatives show that hydrogen-bonding capacity is a key determinant of lysosomal retention in normal cells, whereas lipophilicity directs these derivatives to the mitochondria or the nucleus in cancer cells. In addition, drug-resistant cancer cells preferentially retain BMVC in their lysosomes than drug-sensitive cancer cells, and BMVC can be released from drug-resistant lysosomes with lysosomotropic agents. Our results further our understanding of how properties of cellular organelles differ between normal and cancer cells, which can be exploited for diagnostic and/or therapeutic use. We also provide physiochemical design principles for selective targeting of small molecules to different organelles. Moreover, our results suggest that agents which can increase lysosomal membrane permeability may re-sensitize drug-resistant cancer cells to chemotherapeutic agents.
PMCID: PMC3915044  PMID: 23970166
17.  Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines 
Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via either or both of the CXCR1 and CXCR2 receptors, exerting profound impacts on tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.
PMCID: PMC4034677  PMID: 23995780
18.  FRET Imaging of Calcium Signaling in Live Cells under Microenvironment 
Microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and reduced peak of a transient intracellular calcium rise comparing to control cells without constrains. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that micro-patterned surface can have profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new lights on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.
PMCID: PMC4165894  PMID: 23250282
Micro/nano-fabrication; Surface pattern; FRET; soft lithography; live cell imaging
19.  Established and Novel Methods of Interrogating Two-Dimensional Cell Migration 
The regulation of cell motility is central to living systems. Consequently, cell migration assays are some of the most frequently used in vitro assays. This article provides a comprehensive, detailed review of in vitro cell migration assays both currently in use and possible with existing technology. Emphasis is given to two-dimensional migration assays using densely organized cells such as the scratch assay. Assays are compared and categorized in an outline format according to their primary biological readout and physical parameters. The individual benefits of the various methods and quantification strategies are also discussed. This review provides an in-depth, structured overview of in vitro cell migration assays as a means of enabling the reader to make informed decisions among the growing number of options available for their specific cell migration application.
PMCID: PMC4165521  PMID: 23038152
20.  Evidence for distinct mechanisms of uptake and antitumor activity of secretory phospholipase A2 responsive liposome in prostate cancer 
Secretory phospholipase A2 (sPLA2) cleave phospholipids at sn-2 ester bonds, releasing lysophospholipids and fatty acids, and are over expressed in several pathologies, including inflammation, arthritis, sepsis and breast and prostate cancers. Herein we evaluated the therapeutic activity of liposomes engineered to be responsive to different sPLA2 isoforms compared to clinically used long-circulating (pegylated) sterically stabilized liposomes (SSL) in vitro and in vivo, and assess differences in role of sPLA2 in the mechanism of uptake and delivery of these nanoparticles. Exposing sPLA2 responsive liposomes (SPRL) to sPLA2 increased the release of intraluminal entrapped contents in a time-dependent manner that was inhibited by the sPLA2 inhibitor LY3117272. Treatment of prostate cancer cells with doxorubicin encapsulated in SSL and SPRL resulted in cytotoxicity in LNCaP, DU-145 and PC-3 cells lines comparable to free drug. Interestingly, cytotoxicity was not altered by sPLA2 inhibition. Tracking of drug and liposome delivery using fluorescence microscopy and flow cytometry, we demonstrated that drug uptake was liposome-dependent, as encapsulation of doxorubicin in SPRL resulted in 1.5 to 2-fold greater intracellular drug levels compared to SSL. Liposome uptake was cell-dependent and did not correlate to doxorubicin uptake; however, doxorubicin uptake was generally greatest in PC-3 cells, followed by DU-145 cells and then LNCaP cells. In almost all cases, uptake of one of our formulations, SPRL-E, was greater than SSL. The therapeutic activity of SPRL in vivo was demonstrated using a mouse xenograft model of human prostate cancer, which showed that doxorubicin entrapped within SPRL decreased tumor growth compared to SSL, suggesting that SPRL are more effective at slowing tumor growth than a SSL formulation similar to the FDA approved DOXIL™. Collectively, these data show the therapeutic activity of SPRL compared to SSL, yield insights into the mechanisms of action of these nanoparticles and suggest that SPRL could be useful for treatment of other pathologies that over express sPLA2.
PMCID: PMC4164335  PMID: 22890797
Secretory phospholipase A2; drug delivery; nanoparticle; liposome; targeting
21.  CD19-antigen specific nanoscale liposomal formulation of a SYK P-site inhibitor causes apoptotic destruction of human B-precursor leukemia cells 
We report the anti-leukemic potency of a unique biotargeted nanoscale liposomal nanoparticle (LNP) formulation of the spleen tyrosine kinase (SYK) P-site inhibitor C61. C61-loaded LNP were decorated with a murine CD19-specific monoclonal antibody directed against radiation-resistant CD19-receptor positive aggressive B-precursor acute lymphoblastic leukemia (ALL) cells. The biotargeted C61-LNP were more potent than untargeted C61-LNP and consistently caused apoptosis in B-precursor ALL cells. The CD19-directed C61-LNP also destroyed B-precursor ALL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. This unique nanostructural therapeutic modality targeting the SYK-dependent anti-apoptotic blast cell survival machinery shows promise for overcoming the clinical radiochemotherapy resistance of B-precursor ALL cells.
PMCID: PMC4158964  PMID: 24910947
22.  Building risk-on-a-chip models to improve breast cancer risk assessment and prevention 
Preventive actions for chronic diseases hold the promise of improving lives and reducing healthcare costs. For several diseases, including breast cancer, multiple risk and protective factors have been identified by epidemiologists. The impact of most of these factors has yet to be fully understood at the organism, tissue, cellular and molecular levels. Importantly, combinations of external and internal risk and protective factors involve cooperativity thus, synergizing or antagonizing disease onset. Models are needed to mechanistically decipher cancer risks under defined cellular and microenvironmental conditions. Here, we briefly review breast cancer risk models based on 3D cell culture and propose to improve risk modeling with lab-on-a-chip approaches. We suggest epithelial tissue polarity, DNA repair and epigenetic profiles as endpoints in risk assessment models and discuss the development of ‘risks-on-chips’ integrating biosensors of these endpoints and of general tissue homeostasis. Risks-on-chips will help identify biomarkers of risk, serve as screening platforms for cancer preventive agents, and provide a better understanding of risk mechanisms, hence resulting in novel developments in disease prevention.
PMCID: PMC3748262  PMID: 23681255
23.  Microfluidic Technique to Measure Intratumoral Transport and Calculate Drug Efficacy Shows that Binding is Essential for Doxorubicin and Release Hampers Doxil 
Intratumoral transport and binding are important mechanisms that determine the efficacy of cancer drugs. Current drug screening methods rely heavily on monolayers of cancer cells, which overlook the contribution of tissue-level transport and binding. To quantify these factors, we developed a method that couples an in vitro, drug-delivery device containing a three-dimensional cell mass and a mathematical model of drug diffusion, binding to DNA, release from carriers, and clearance. Spheroids derived from LS174T human colon carcinoma cells were inserted into rectangular chambers to form rectangular cell masses (tissue) and subjected to continuous medium perfusion. To simulate drug delivery and clearance, the tissues were treated with doxorubicin followed by drug-free medium. To evaluate the effect of liposome encapsulation, tissues were treated with liposome-encapsulated doxorubicin (Doxil). Spatiotemporal dynamics of drug distribution and apoptosis was measured by fluorescence microscopy. The diffusivity and DNA binding constant of doxorubicin were determined by fitting experimental data to the mathematical model. Results show that an ideal combination of diffusivity, binding constant, clearance rate, and cytotoxicity contribute to the high therapeutic efficacy of doxorubicin. There was no detectable release of doxorubicin from Doxil in the tissues. The rate of doxorubicin release, evaluated by fitting experimental data to the mathematical model, was below therapeutically effective levels. These results show that despite enhanced systemic circulation obtained by liposome encapsulation, the therapeutic effect of Doxil is limited by slow intratumoral drug release. The experimental and computational methods developed here to calculate drug efficacy provide mechanisms to explain poor performance of drug candidates, and enable design of more successful cancer drugs.
PMCID: PMC3760710  PMID: 23860772
Mathematical modeling; microfluidics; in vitro models; stealth liposomes; drug binding; drug delivery
24.  Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation 
During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals in these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences of osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction, correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration.
PMCID: PMC3816390  PMID: 23925497
mesenchymal stem cells (MSCs); collagen; osteogenesis; morphogenesis
25.  Let's push things forward: disruptive technologies and the mechanics of tissue assembly 
Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems – embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly.
PMCID: PMC3869098  PMID: 23907401
mechanobiology; morphogenesis; patterning

Results 1-25 (178)