Philippe Van Ham

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.

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.

Gastrin inhibits motility, decreases cell death levels and increases proliferation in human glioblastoma cell lines

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

Characterization of Gastrin-Induced Proangiogenic Effects In vivo in Orthotopic U373 Experimental Human Glioblastomas and In vitro in Human Umbilical Vein Endothelial Cells

Purpose: This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo. Experimental Design: The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3′-kinase, protein kinase C, and nuclear factor-κB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-κB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers. Results: Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor α (CXCR1) and IL-8 receptor β (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel. Conclusions: The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.

Computer-assisted analysis of epiluminescence microscopy images of pigmented skin lesions.

BACKGROUND Epiluminescence microscopy (ELM) is a noninvasive clinical tool recently developed for the diagnosis of pigmented skin lesions (PSLs), with the aim of improving melanoma screening strategies. However, the complexity of the ELM grading protocol means that considerable expertise is required for differential diagnosis. In this paper we propose a computer-based tool able to screen ELM images of PSLs in order to aid clinicians in the detection of lesion patterns useful for differential diagnosis. METHODS The method proposed is based on the supervised classification of pixels of digitized ELM images, and leads to the construction of classes of pixels used for image segmentation. This process has two major phases, i.e., a learning phase, where several hundred pixels are used in order to train and validate a classification model, and an application step, which consists of a massive classification of billions of pixels (i.e., the full image) by means of the rules obtained in the first phase. RESULTS Our results show that the proposed method is suitable for lesion-from-background extraction, for complete image segmentation into several typical diagnostic patterns, and for artifact rejection. Hence, our prototype has the potential to assist in distinguishing lesion patterns which are associated with diagnostic information such as diffuse pigmentation, dark globules (black dots and brown globules), and the gray-blue veil. CONCLUSIONS The system proposed in this paper can be considered as a tool to assist in PSL diagnosis.

IN VITRO MOTILITY EVALUATION OF AGGREGATED CANCER CELLS BY MEANS OF AUTOMATIC IMAGE PROCESSING

BACKGROUND Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. METHODS Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. RESULTS The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. CONCLUSIONS The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Identification of High Versus Lower Risk Clinical Subgroups in a Group of Adult Patients with Supratentorial Anaplastic Astrocytomas

The present work investigates whether computer-assisted techniques can contribute any significant information to the characterization of astrocylic tumor aggressiveness. Two complementary computer-assisted methods were used. The first method made use of the digital image analysis of Feulgen-stained nuclei, making it possible to compute 15 morphonuclear and 8 nuclear DNA content-related (ploidy level) parameters. The second method enabled the most discriminatory parameters to be determined. This second method is the Decision Tree technique, which forms part of the Supervised Learning Algorithms. These two techniques were applied to a series of 250 supratentorial astrocytic tumors of the adult. This series included 39 low-grade (astrocytomas, AST) and 211 high-grade (47 anaplastic astrocytomas, ANA, and 164 glioblastomas, GBM) astrocytic tumors. The results show that some AST, ANA and GBM did not fit within simple logical rules. These “complex” cases were labeled NC-AST, NC-ANA and NC-GBM because they were “non-classical” (NC) with respect to their cytological features. An analysis of survival data revealed that the patients with NC-GBM had the same survival period as patients with GBM. In sharp contrast, patients with ANA survived significantly longer than patients with NC-ANA. In fact, the patients with ANA had the same survival period as patients who died from AST, while the patients with NC-ANA had a survival period similar to those with GBM. All these data show that the computer-assisted techniques used in this study can actually provide the pathologist with significant information on the characterization of astrocytic tumor aggressiveness.

Classification strategies for the grading of renal cell carcinomas, based on nuclear morphometry and densitometry.

The various grading systems proposed for renal cell carcinomas all suffer from problems related to inter‐observer variability. Some of these grading systems are based, either partially or wholly, on morphonuclear criteria, such as nuclear size and shape, anisonucleosis, and chromatin pattern. These criteria can be quantitatively (and thus objectively) evaluated by means of the computer‐assisted microscopic analysis of Feulgen‐stained nuclei. In the present work, 39 quantitative variables, including two morphometric, 28 chromatin pattern‐related, and nine DNA ploidy level‐related, were computed for 65 renal cell carcinomas. The actual diagnostic information contributed by each variable was determined by means of multifactorial statistical analysis (discriminant analysis) and two artificial intelligence‐related methods of data classification (the decision tree and production rule methods). The results show that quantitative information, as provided by the computer‐assisted microscopy of Feulgen‐stained nuclei and analysed by means of artificial intelligence‐related methods of data classification, contributes significant diagnostic information for the grading of renal cell carcinoma, thus reducing the problem of inter‐observer reproducibility.

The use of the decision tree technique and image cytometry to characterize aggressiveness in World Health Organization (WHO) grade II superficial transitional cell carcinomas of the bladder

The aggressiveness of human bladder tumours can be assessed by means of various classification systems, including the one proposed by the World Health Organization (WHO). According to the WHO classification, three levels of malignancy are identified as grades I (low), II (intermediate), and III (high). This classification system operates satisfactorily for two of the three grades in forecasting clinical progression, most grade I tumours being associated with good prognoses and most grade III with bad. In contrast, the grade II group is very heterogeneous in terms of their clinical behaviour. The present study used two computer‐assisted methods to investigate whether it is possible to sub‐classify grade II tumours: computer‐assisted microscope analysis (image cytometry) of Feulgen‐stained nuclei and the Decision Tree Technique. This latter technique belongs to the Supervised Learning Algorithm and enables an objective assessment to be made of the diagnostic value associated with a given parameter. The combined use of these two methods in a series of 292 superficial transitional cell carcinomas shows that it is possible to identify one subgroup of grade II tumours which behave clinically like grade I tumours and a second subgroup which behaves clinically like grade III tumours. Of the nine ploidy‐related parameters computed by means of image cytometry [the DNA index (DI), DNA histogram type (DHT), and the percentages of diploid, hyperdiploid, triploid, hypertriploid, tetraploid, hypertetraploid, and polyploid cell nuclei], it was the percentage of hyperdiploid and hypertetraploid cell nuclei which enabled identification, rather than conventional parameters such as the DI or the DHT.

Dynamic behavior analysis of in-vitro cancerous cells by means of an automatic image-processing device

The study of in vitro cell motility has received a great deal of attention for many years. All former studies typically imply the use of a microscope equipped with a camera, a digitizer board and a computer that process the frames to evaluate cell motility. The present paper reports an improved device incorporating the typical components of in vitro cell motility analyzers, which also makes possible the evaluation of clustered cell motility, an important variable for assessing aggressiveness in cancer models. Two methods have been developed. The first avoids the segmentation step and thus provides a measurement of the motility of the whole cluster considered as a single entity. The second consists of a clustered cell tracking software developed in our laboratory. The segmentation process is performed by means of an algorithm based on watershed transformation applied to a modified gradient image. This gradient modification consists of a new algorithm using the correlation between the frames. As a result, the cells are extracted from the background and can be tracked through the sequence. A set of five experiments was performed on cell colonies from the PC-3 human prostate cancer in vitro model colonies in order to illustrate the features of the device.