Olivier Debeir

Tracking of migrating cells under phase-contrast video microscopy with combined mean-shift processes

In this paper, we propose a combination of mean-shift-based tracking processes to establish migrating cell trajectories through in vitro phase-contrast video microscopy. After a recapitulation on how the mean-shift algorithm permits efficient object tracking we describe the proposed extension and apply it to the in vitro cell tracking problem. In this application, the cells are unmarked (i.e., no fluorescent probe is used) and are observed under classical phase-contrast microscopy. By introducing an adaptive combination of several kernels, we address several problems such as variations in size and shape of the tracked objects (e.g., those occurring in the case of cell membrane extensions), the presence of incomplete (or noncontrasted) object boundaries, partially overlapping objects and object splitting (in the case of cell divisions or mitoses). Comparing the tracking results automatically obtained to those generated manually by a human expert, we tested the stability of the different algorithm parameters and their effects on the tracking results. We also show how the method is resistant to a decrease in image resolution and accidental defocusing (which may occur during long experiments, e.g., dozens of hours). Finally, we applied our methodology on cancer cell tracking and showed that cytochalasin-D significantly inhibits cell motility.

Assessment of Very High Spatial Resolution Satellite Image Segmentations

Since 1999, very high spatial resolution satellite data represent the surface of the Earth with more detail. However, information extraction by per pixel multispectral classification techniques proves to be very complex owing to the internal variability increase in land-cover units and to the weakness of spectral resolution. Image segmentation before classification was proposed as an alternative approach, but a large variety of segmentation algorithms were developed during the last 20 years, and a comparison of their implementation on very high spatial resolution images is necessary. In this study, four algorithms from the two main groups of segmentation algorithms (boundarybased and region-based) were evaluated and compared. In order to compare the algorithms, an evaluation of each algorithm was carried out with empirical discrepancy evaluation methods. This evaluation is carried out with a visual segmentation of Ikonos panchromatic images. The results show that the choice of parameters is very important and has a great influence on the segmentation results. The selected boundary-based algorithms are sensitive to the noise or texture. Better results are obtained with regionbased algorithms, but a problem with the transition zones between the contrasted objects can be present.

Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration.

Cancer cell motility and invasion are critical targets for anticancer therapeutics. Whereas in vitro models could be designed for rapid screening with a view to investigate these targets, careful consideration must be given to the construction of appropriate model systems. Most investigations focus on two-dimensional (2-D) assays despite the fact that increasing evidence suggests that migration across rigid and planar substrates fails to recapitulate in vivo behavior. In contrast, few systems enable three-dimensional (3-D) cell migration to be quantitatively analyzed. We previously developed a digital holographic microscope (DHM) working in transmission with a partially spatial coherence source. This configuration avoids the noise artifacts of laser illumination and makes possible the direct recording of information on the 3-D structure of samples consisting of multiple objects embedded in scattering media, such as cell cultures in matrix gels. The software driving our DHM system is equipped with a time-lapse ability that enables the 3-D trajectories of living cells to be reconstituted and quantitatively analyzed.

Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells

OBJECTIVEIon transporters play pivotal roles in cancer cell migration in general and in glioblastomas (GBMs) in particular. However, the specific role of Na+/K+-ATPase (the sodium pump) and, in particular, its α1 subunit, has remained unexplored in GBMs. MATERIALS AND METHODSThe expression of Na+/K+-ATPase α1 in GBM clinical samples, normal brain tissue, and a human GBM cell line has been investigated. Using the novel cardenolide UNBS1450 (Unibioscreen, Brussels, Belgium), which is a ligand of the sodium pump, we have characterized the effects of inhibiting Na+/K+-ATPase α1 in human GBM cells with respect to cell proliferation; morphology; impact on intracellular Na+, Ca2+, and adenosine triphosphate; and changes in the actin cytoskeleton. We have investigated the mechanism by which UNBS1450 overcomes the apoptosis resistance of GBMs and determined its anti-tumor effects in comparative studies in vitro in GBM cell viability assays and in vivo using an orthotopic human GBM xenograft model. RESULTSOverall, the α1 subunit of Na+/K+-ATPase is highly expressed in a majority of glioblastomas compared with normal brain tissues, and by binding to this subunit in human U373-MG GBM cells, UNBS1450 impairs cell proliferation and migration via an intracellular adenosine triphosphate decrease-mediated disorganization of the actin cytoskeleton and cytotoxic proautophagic effects. UNBS1450 also significantly increases the in vivo survival of mice orthotopically grafted with U373-MG GBM cells. CONCLUSIONInhibition of the Na+/K+-ATPase α1 subunit in human GBM cells impairs both cell migration and cell proliferation.

Evidence of galectin-1 involvement in glioma chemoresistance.

Glioblastomas (GBMs) are resistant to apoptosis but less so to autophagy; a fact that may at least partly explain the therapeutic benefits of the pro-autophagic drug temozolomide in the treatment of GBM patients. Galectin-1 (Gal1) whose expression is stimulated by hypoxia is a potent modulator of GBM cell migration and a pro-angiogenic molecule. Hypoxia is also known to confer cancer cells with resistance to chemotherapy and radiotherapy and to modulate the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. The present study investigates whether decreasing Gal1 expression (by means of a siRNA approach) in human Hs683 GBM cells increases their sensitivity to pro-autophagic or pro-apoptotic drugs. The data reveal that temozolomide, the standard treatment for glioma patients, increases Gal1 expression in Hs683 cells both in vitro and in vivo. However, reducing Gal1 expression in these cells by siRNA increases the anti-tumor effects of various chemotherapeutic agents, in particular temozolomide both in vitro and in vivo. This decrease in Gal1 expression in Hs683 cells does not induce apoptotic or autophagic features, but is found to modulate p53 transcriptional activity and decrease p53-targeted gene expression including DDIT3/GADD153/CHOP, DUSP5 ATF3 and GADD45A. The decrease in Gal1 expression also impairs the expression levels of seven other genes implicated in chemoresistance: ORP150, HERP, GRP78/Bip, TRA1, BNIP3L, GADD45B and CYR61, some of which are located in the ER and whose expression is also known to be modified by hypoxia. This novel facet of Gal1 involvement in glioblastoma biology may be amenable to therapeutic manipulation.

A model-based approach for automated in vitro cell tracking and chemotaxis analyses.

Chemotaxis may be studied in two main ways: 1) counting cells passing through an insert (e.g., using Boyden chambers), and 2) directly observing cell cultures (e.g., using Dunn chambers), both in response to stationary concentration gradients. This article promotes the use of Dunn chambers and in vitro cell‐tracking, achieved by video microscopy coupled with automatic image analysis software, in order to extract quantitative and qualitative measurements characterizing the response of cells to a diffusible chemical agent.

Videomicroscopic extraction of specific information on cell proliferation and migration in vitro

In vitro cell imaging is a useful exploratory tool for cell behavior monitoring with a wide range of applications in cell biology and pharmacology. Combined with appropriate image analysis techniques, this approach has been shown to provide useful information on the detection and dynamic analysis of cell events. In this context, numerous efforts have been focused on cell migration analysis. In contrast, the cell division process has been the subject of fewer investigations. The present work focuses on this latter aspect and shows that, in complement to cell migration data, interesting information related to cell division can be extracted from phase-contrast time-lapse image series, in particular cell division duration, which is not provided by standard cell assays using endpoint analyses. We illustrate our approach by analyzing the effects induced by two sigma-1 receptor ligands (haloperidol and 4-IBP) on the behavior of two glioma cell lines using two in vitro cell models, i.e., the low-density individual cell model and the high-density scratch wound model. This illustration also shows that the data provided by our approach are suggestive as to the mechanism of action of compounds, and are thus capable of informing the appropriate selection of further time-consuming and more expensive biological evaluations required to elucidate a mechanism.

Exploring the distinctive biological characteristics of pilocytic and low-grade diffuse astrocytomas using microarray gene expression profiles.

Abstract Although World Health Organization (WHO) grade I pilocytic astrocytomas and grade II diffuse astrocytomas have been classified for decades as different clinicopathologic entities, few, if any, data are available on the biologic features explaining these differences. Although more than 50 microarray-related studies have been carried out to characterize the molecular profiles of astrocytic tumors, we have identified only 11 that provide sound data on low-grade astrocytomas. We have incorporated these data into a comparative analysis for the purpose of identifying the most relevant molecular markers characterizing grade I pilocytic and grade II diffuse astrocytomas. Our analysis has identified various interesting genes that are differentially expressed in either grade I or grade II astrocytomas when compared with normal tissue and/or high-grade (WHO grade III and IV) astrocytomas. A large majority of these genes encode adhesion, extracellular matrix, and invasion-related proteins. Interestingly, a group of 6 genes (TIMP4, C1NH, CHAD, THBS4, IGFBP2, and TLE2) constitute an expression profile characteristic of grade I astrocytomas as compared with all other categories of tissue (normal brain, grade II, and high-grade astrocytomas). The end products (proteins) of these genes act as antimigratory compounds, a fact that could explain why pilocytic astrocytomas behave as compact (well-circumscribed) tumors as opposed to all the other astrocytic tumor types that diffusely invade the brain parenchyma. Having validated these molecular markers by means of real-time reverse transcriptase-polymerase chain reaction, an integrated model was proposed illustrating how and why pilocytic astrocytomas constitute a distinct biologic and pathologic entity when compared with diffuse astrocytomas.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.

Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis

Antibody‐based proteomics applied to tissue microarray (TMA) technology provides a very efficient means of visualizing and locating antigen expression in large collections of normal and pathological tissue samples. To characterize antigen expression on TMAs, the use of image analysis methods avoids the effects of human subjectivity evidenced in manual microscopical analysis. Thus, these methods have the potential to significantly enhance both precision and reproducibility. Although some commercial systems include tools for the quantitative evaluation of immunohistochemistry‐stained images, there exists no clear agreement on best practices to allow for correct and reproducible quantification results. Our study focuses on practical aspects regarding (i) image acquisition (ii) segmentation of staining and counterstaining areas and (iii) extraction of quantitative features. We illustrate our findings using a commercial system to quantify different immunohistochemistry markers targeting proteins with different expression patterns (cytoplasmic, nuclear or membranous) in colon cancer or brain tumor TMAs. Our investigations led us to identify several steps that we consider essential for standardizing computer‐assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to be able to compare measurements between studies involving different TMAs. In conclusion, we recommend certain critical prerequisites that commercial or in‐house systems should satisfy in order to permit valid immunostaining quantification.