Analysis Of Cell Behavior Research Articles

Abstract A34: Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition

Abstract Drug chemotherapy resistance is the leading cause of death for cancer patients. Most of the studies involving chemotherapy resistance are based on experiments making use of cancer cell lines maintained in two-dimensional (2D) cultures. In these conditions, the influence of extracellular matrix (ECM) and the three-dimensional (3D) microenvironment are not considered, despite clear evidence of their role in genesis and progression of cancer. In the present work, we evaluated the microenvironment control in the MCF-7 cellular response to doxorubicin using the three-dimensional laminin-rich ECM (3D lrECM) cell culture model. Based on cell plating on top of growth factor-reduced Basement Membrane (BD MatrigelTM), the 3D lrECM culture consists of a more physiologically applicable approach for ex vivo analysis of cell behavior. When maintained in a 3D context, these human breast cancer cells showed morphological differences, as viewed by phase contrast microscopy, forming round colonies. The MCF-7 growth rate showed also to be dependent of the cellular culture model used. Despite cancerous, these cells undergo a significant growth arrest on top of laminin-rich ECM. Using the cell viability XTT assays for doxorubicin sensitivity analysis, we observed a drug resistance event in 2D cells treated with low chemotherapy doses (250 and 625 nM of doxorubicin). Thus, while the cell growth on lrECM led to decrease on doxorubicin survival at 60%, the survival rates were almost unchanged (higher than 95% of survival) in monolayer cultures at same treatment conditions. Autophagy, a lysosome-dependent catabolic degradation process, has been implicated in anticancer drug resistance. Moreover, recent reports demonstrated that this cellular phenomenon plays a crucial role as a pro-survival factor in MCF-7 cells treated with epirubicin, a doxorubicin analog. Thus, we investigated whether autophagy is involved in doxorubicin response changes promoted by maintenance in 3D culture. By co-treatment with chloroquine, a pharmacological inhibitor of autophagy, we observed a synergistic effect with doxorubicin only in 2D, with 30% reduction of survival. We also evaluated the chloroquine effect in breast cancer cell response to doxorubicin using specific fluorescent dyes for living, apoptotic and necrotic cells. Inhibition of autophagy increased the cell death percentage in monolayer, by not in 3D cell culture, confirming that ECM abolishes autophagy pro-survival action. Subsequently, the autophagy levels were measured by LC3-II accumulation in both cell culture conditions. Our results suggested that autophagy is significantly inhibited by cellular growth in 3D lrECM. While a clear synergic LC3-II accumulation was observed in 2D cultured cells both treated with doxorubicin and chloroquine, very low levels of LC3-II expression were detected in 3D treated cells. Altogether, our results suggest that autophagy is essential for the tissue microenvironment control of breast cancer cells response to anticancer drugs. Therefore, presence of ECM compounds might prevent the autophagy protective effect from doxorubicin-induced cell death. Financial Support: FAPESP and CNPq Citation Format: Luciana R. Gomes, Alexandre T. Vessoni, Carlos FM Menck. Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A34.

Magnetically attachable stencils and the non-destructive analysis of the contribution made by the underlying matrix to cell migration

Cell migration is controlled by the integration of numerous distinct components. Consequently, the analysis of cell migration is advancing towards comprehensive, multifaceted in vitro models. To accurately evaluate the contribution of an underlying substrate to cell motility in complex cellular environments we developed a migration assay using magnetically attachable stencils (MAts). When attached to a culture surface, MAts create a defined void in the cell monolayer without disrupting the cells or damaging the underlying substrate. Quantitative analysis of migration into this void reveals the substrate's contribution to migration. The magnetically-guided placement of a microfabricated stencil allows for full experimental control of the substrate on which migration is analyzed. MAts enable the evaluation of intact, defined matrix, and make it possible to analyze migration on unique surfaces such as micropatterned proteins, nano-textured surfaces, and pliable hydrogels. These studies also revealed that mechanical disruption, including the damage that occurs during scratch assays, diminishes migration and confounds the analysis of individual cell behavior. Analysis of migration on increasingly complex biomaterials reveals that the contribution of the underlying matrix depends not only on its molecular composition but also its organization and the context in which it is presented.

JAGGED Controls Growth Anisotropy and Coordination between Cell Size and Cell Cycle during Plant Organogenesis

SummaryBackgroundIn all multicellular organisms, the links between patterning genes, cell growth, cell cycle, cell size homeostasis, and organ growth are poorly understood, partly due to the difficulty of dynamic, 3D analysis of cell behavior in growing organs. A crucial step in plant organogenesis is the emergence of organ primordia from the apical meristems. Here, we combined quantitative, 3D analysis of cell geometry and DNA synthesis to study the role of the transcription factor JAGGED (JAG), which functions at the interface between patterning and primordium growth in Arabidopsis flowers.ResultsThe floral meristem showed isotropic growth and tight coordination between cell volume and DNA synthesis. Sepal primordia had accelerated cell division, cell enlargement, anisotropic growth, and decoupling of DNA synthesis from cell volume, with a concomitant increase in cell size heterogeneity. All these changes in growth parameters required JAG and were genetically separable from primordium emergence. Ectopic JAG activity in the meristem promoted entry into S phase at inappropriately small cell volumes, suggesting that JAG can override a cell size checkpoint that operates in the meristem. Consistent with a role in the transition from meristem to primordium identity, JAG directly repressed the meristem regulatory genes BREVIPEDICELLUS and BELL 1 in developing flowers.ConclusionsWe define the cellular basis for the transition from meristem to organ identity and identify JAG as a key regulator of this transition. JAG promotes anisotropic growth and is required for changes in cell size homeostasis associated with accelerated growth and the onset of differentiation in organ primordia.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies

Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour. A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies. The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Use of PCR Template-Derived Probes Prevents Off-Target Whole Mount In Situ Hybridization in Transgenic Zebrafish

Transgenic zebrafish have been utilized for in vivo analysis of cell behaviors using advanced imaging techniques, for analyzing spatiotemporal gene regulation, and for targeted mis-expression of transgenes. The Tg(fli1a:EGFP)y1 vascular reporter has been particularly useful for examining the development of blood and lymphatic vessels, but it has been suggested that whole-mount in situ hybridization may result high background staining in this line, potentially limiting its usefulness. Here, we show that off-target hybridization of plasmid vector-derived probes to tissues expressing transgenes occurs in a number of different commonly used transgenic lines as a result of multiple cloning site sequences present in the cloning vectors, suggesting this may be a more general problem. However, we also show that this problem is easily avoided by performing in situ hybridization using probes synthesized from PCR templates lacking vector sequences.

Molecular Imaging of Stem Cells: Tracking Survival, Biodistribution, Tumorigenicity, and Immunogenicity

Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.

The use of fluorescent proteins for intravital imaging of cancer cell invasion.

The analysis of cancer cell behavior in the primary tumor in living animals provides an opportunity to explore the process of invasion and intravasation in the complex microenvironment that is present in vivo. In this chapter, we describe the methods that we have developed for performing intravital imaging of mammary tumors. We provide procedures for generating tumors through injection of tumor cell lines, and multiphoton imaging using a skin-flap tumor dissection and a mammary imaging window.

1G1448 Analysis of cell behavior near silicon based micro-scale pore(Cell Biology I,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)

1G1448 Analysis of cell behavior near silicon based micro-scale pore(Cell Biology I,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)

Hybrid Scaffolds for Tissue Regeneration: Chemotaxis and Physical Confinement as Sources of Biomimesis

Biomineralization is a complex ensemble of concomitant phenomena, driving the development of vertebrate and invertebrate organisms, particularly the formation of human bone tissue. In such a process collagen molecules assemble and organize in a complex 3‐D structure and simultaneously mineralize with nearly amorphous apatite nanoparticles, whose heterogeneous nucleation, growth, and specific orientation are mediated by various chemical, physical, morphological, and structural control mechanisms, activated by the organic matrix at different size levels. The present work investigates on in‐lab biomineralization processes, performed to synthesize hybrid hydroxyapatite/collagen scaffolds for bone and osteochondral regeneration. The synthesis processes are carried out by soft‐chemistry procedures, with the purpose to activate all the different control mechanisms at the basis of new bone formation in vivo, so as to achieve scaffolds with high biomimesis, that is, physical, chemical, morphological, and ultrastructural properties very close to the newly formed human bone. Deep analysis of cell behaviour in contact with such hybrid scaffolds confirms their strong affinity with human bone, which in turn determines high regenerative properties in vivo.

In vivo calcium dynamics during neural crest cell migration and patterning using GCaMP3

Examining calcium dynamics within the neural crest (NC) has the potential to shed light on mechanisms that regulate complex cell migration and patterning events during embryogenesis. Unfortunately, typical calcium indicators are added to culture media or have low signal to noise after microinjection into tissue that severely limit analyses to cultured cells or superficial events. Here, we studied in vivo calcium dynamics during NC cell migration and patterning, using a genetically encoded calcium sensor, GCaMP3. We discovered that trunk NC cells displayed significantly more spontaneous calcium transients than cranial NC cells, and during cell aggregation versus cell migration events. Spontaneous calcium transients were more prevalent during NC cell aggregation into discrete sympathetic ganglia (SG). Blocking of N-cadherin activity in trunk NC cells near the presumptive SG led to a dramatic decrease in the frequency of spontaneous calcium transients. Detailed analysis and mathematical modeling of cell behaviors during SG formation showed NC cells aggregated into clusters after displaying a spontaneous calcium transient. This approach highlights the novel application of a genetically encoded calcium indicator to study subsets of cells during ventral events in embryogenesis.

9E-08 血液中を流動する細胞挙動の数値解析(OS-1(1) 微小循環器系のバイオエンジニアリング(2))

9E-08 血液中を流動する細胞挙動の数値解析(OS-1(1) 微小循環器系のバイオエンジニアリング(2))

A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse.

BackgroundUnderstanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.ResultsWe have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.ConclusionBy combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.

Analysis and modelling of motility of cell populations with MotoCell

BackgroundCell motility plays a central role in development, wound-healing and tumour invasion. Cultures of eucariotic cells are a complex system where most cells move according to 'random' patterns, but may also be induced to a more coordinate migration by means of specific stimuli, such as the presence of chemical attractants or the introduction of a mechanical stimulus. Various tools have been developed that work by keeping track of the paths followed by specific objects and by performing statistical analysis on the recorded path data. The available tools include desktop applications or macros running within a commercial package, which address specific aspects of the process.ResultsAn online application, MotoCell, was developed to evaluate the motility of cell populations maintained in various experimental conditions. Statistical analysis of cell behaviour consists of the evaluation of descriptive parameters such as average speed and angle, directional persistence, path vector length, calculated for the whole population as well as for each cell and for each step of the migration; in this way the behaviour of a whole cell population may be assessed as a whole or as a sum of individual entities. The directional movement of objects may be studied by eliminating the modulo effect in circular statistics analysis, able to evaluate linear dispersion coefficient (R) and angular dispersion (S) values together with average angles. A case study is provided where the system is used to characterize motility of RasV12 transformed NIH3T3 fibroblasts.ConclusionHere we describe a comprehensive tool which takes care of all steps in cell motility analysis, including interactive cell tracking, path editing and statistical analysis of cell movement, all within a freely available online service. Although based on a standard web interface, the program is very fast and interactive and is immediately available to a large number of users, while exploiting the web approach in a very effective way. The ability to evaluate the behaviour of single cells allows to draw the attention on specific correlations, such as linearity of movement and deviation from the expected direction. In addition to population statistics, the analysis of single cells allows to group the cells into subpopulations, or even to evaluate the behaviour of each cell with respect to a variable reference, such as the direction of a wound or the position of the closest cell.

The migration of autonomic precursor cells in the embryo

The neural crest is an excellent model system to study cell fate and cell guidance signaling. Neural crest cells emerge from a common multipotent subpopulation and follow stereotypical migratory pathways to contribute to many diverse peripheral structures throughout the vertebrate embryo. The neural tube and diverse embryonic microenvironments from which the neural crest originate and migrate through are important sources of signals, yet it is still unclear how a common pool of neural crest stem and progenitor cells diversify and become distributed along specific stereotypical migratory paths. In the post-otic hindbrain and trunk, the neural crest emerge and contribute to the autonomic nervous system, and failure of proper cell navigation and differentiation often leads to congenital disorders that include dysautonomias, Hirschprung's disease, and neuroblastoma cancer. Recent exciting studies of neural crest cell behaviors have revealed the interplay of several molecular signaling pathways that guide and shape autonomic precursor cells to and into proper target structures, suggesting further work may help to better understand autonomic nervous system assembly, derived from a convergence of time-lapse imaging and molecular analyses. In this mini-review, we summarize recent fluorescent cell labeling strategies and cell behavior analyses that elucidate the role of molecular signals on the migration of autonomic precursor cells. We highlight advances in our understanding of the autonomic precursor cell behaviors and fate determination studied within the embryonic microenvironment.

Analysis of Human Embryonic Stem Cell Behavior in Control and Experimental Conditions Using Time-Lapse Video Microscopy Endpoints.

Since their first derivation in 1998, researchers have used human embryonic stem cells (hESC) for various purposes. However, the current methods for understanding hESC behavior have been problematic, and better technology is needed for efficient evaluation of hESC behavior in vitro. The purpose of this study was to evaluate and use time-lapse video microscopy (TLVM) to analyze hESC dynamics in vitro using various experimental conditions. The endpoints that were evaluated by observing cellular dynamics included pre-attachment "dancing", attachment/spreading, proliferation, and cell death (apoptosis or necrosis). For all experiments, WiCell H9 hESCs were cultured on Matrigel or gelatin-coated Petri dishes, and images were recorded with a Nikon BioStation IM at 1 or 2 minutes intervals for 3 hours using different experimental conditions. Quantification of the endpoints was done by different individuals counting multiple frames from each experiment. Different individuals also evaluated the same data repeatedly, and the coefficients of variation for both an individual's multiple counts as well as counts by different individuals were low (<1.0%), demonstrating the reliability of the method. One phenomenon observed in the control was pre-attachment "dancing" or rapid movements of paddle-like protrusions on cell surfaces in control culture environments. Similar behavior was observed in prostate stromal cells and mouse embryonic fibroblasts, but the movements were less vivacious and were short-lived compared to hESC. A number of protocols have been used to passage hESCs. Here, we utilized TLVM to compare the attachment/spreading ability of hESCS on Matrigel versus gelatin coated dishes after trypsin or Accutase treatment. The time lapse data showed that hESC survived best when removed from plates using trypsinization followed by subsequent plating on Matrigel-coated dishes. Changes in cellular dynamics also enabled analysis of apoptosis and necrosis. The surface of apoptotic cells underwent rapid blebbing, while necrotic cells became swollen. The toxicity of cigarette smoke and alcohol on hESC survival and growth was evaluated with TLVM endpoints. Treatment of hESC with sidestream (SS) smoke (0.1PE, PE=puff equivalent = smoke in 1 puff that dissolves in 1 ml of solution) from traditional (Marlboro Red) and harm reduction (Advance Lights, Marlboro Lights, and Quest) cigarettes decreased the percentage of cell attachment to Matrigel when compared to untreated controls. hESCs treated with 0.5% alcohol were undergoing apoptosis by 2 hours of treatment, and attached colonies treated with 0.1% alcohol (a level found in the blood of human drinkers) also showed apoptotic blebbing. hESC treated with 0.1% alcohol also had activated caspases that were demonstrated using a poly-caspase FLICA inhibitor. In summary, we have demonstrated that TLVM is a valuable and effective method to evaluate hESC behavior allowing close observation and detailed analysis of cellular dynamics under various experimental conditions. (poster)

Large-Scale Clonal Analysis Reveals Unexpected Complexity in Surface Ectoderm Morphogenesis

BackgroundUnderstanding the series of morphogenetic processes that underlie the making of embryo structures is a highly topical issue in developmental biology, essential for interpreting the massive molecular data currently available. In mouse embryo, long-term in vivo analysis of cell behaviours and movements is difficult because of the development in utero and the impossibility of long-term culture.Methodology/Principal FindingsWe improved and combined two genetic methods of clonal analysis that together make practicable large-scale production of labelled clones. Using these methods we performed a clonal analysis of surface ectoderm (SE), a poorly understood structure, for a period that includes gastrulation and the establishment of the body plan. We show that SE formation starts with the definition at early gastrulation of a pool of founder cells that is already dorso-ventrally organized. This pool is then regionalized antero-posteriorly into three pools giving rise to head, trunk and tail. Each pool uses its own combination of cell rearrangements and mode of proliferation for elongation, despite a common clonal strategy that consists in disposing along the antero-posterior axis precursors of dorso-ventrally-oriented stripes of cells.Conclusions/SignificanceWe propose that these series of morphogenetic processes are organized temporally and spatially in a posterior zone of the embryo crucial for elongation. The variety of cell behaviours used by SE precursor cells indicates that these precursors are not equivalent, regardless of a common clonal origin and a common clonal strategy. Another major result is the finding that there are founder cells that contribute only to the head and tail. This surprising observation together with others can be integrated with ideas about the origin of axial tissues in bilaterians.

Development of EIS cell chips and their application for cell analysis

We report the development of EIS cell chips able to monitor cell growth and adhesion. They are made of transparent or semitransparent materials to allow complementary analysis of cell behaviour during the measurements through optical microscopy. Our approach is cheap both in fabrication and usage, it is not invasive for cells and it does not require any additional reagent. Our devices are particular suitable to count cells or to evaluate cell morphology and changes as a consequence of different treatments.

Cell behavior analysis to evaluate proliferative potentials of human lymphocytes expanded and activated for therapeutic use

The cluster designation 3-lymphokine activated killer (CD3-LAK) culture was conducted using human lymphocytes obtained from different donors. It was found that donor-dependent variances existed in terms of lag time and minimum doubling time, which were process parameters for comprehending the proliferative potentials of cells in an early phase with weak growth and in a subsequent phase with active growth, respectively. To correlate these variances with culture performances, cellular behaviors were estimated by constructing a custom-made observation tool that can capture and process images of culture surfaces. The tool enabled us to determine time-lapse changes in an average projected cell area and the frequency of cell aggregates during the culture in a noninvasive manner. It was found that linear relationships were obtained both between lag time and average projected cell area, and between minimum doubling time and formation rate of cell aggregates, irrespective of culture performance fluctuation depending on each donor state. It is concluded that the developed tool is helpful for operating CD3-LAK culture while monitoring the state of human lymphocytes.

Study on analysis of cell behavior on scaffolds

Study on analysis of cell behavior on scaffolds