Isabelle Roland

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.

The α1 subunit of the sodium pump could represent a novel target to combat non‐small cell lung cancers

With an overall 5 year survival rate as low as 15% for non‐small cell lung cancer (NSCLC), even with surgical intervention and the use of newer molecules in adjuvant chemotherapy, there is an urgent need for new biological targets and associated novel anti‐cancer agents. The present study was undertaken to evaluate the potential of the Na+/K+‐ATPase α1 subunit as a novel target in NSCLC and revealed that α1 expression is markedly higher in a significant proportion of NSCLC clinical samples compared to normal lung tissue. Furthermore, reduction in α1 expression in A549 NSCLC cells by anti‐α1 siRNA resulted in markedly impaired proliferation and migration of these cancer cells. Finally, of three cardenolides investigated, UNBS1450, which is known to bind to Na+/K+‐ATPase and displays potent anti‐tumour activity in vivo in experimental models of human NSCLCs, is the most potent inhibitor of Na+/K+‐ATPase isozymes (α1β1, α2β1 and α3β1), most strikingly of α1β1. This was reflected in the compounds more potent anti‐proliferative activity in all NSCLC cell lines evaluated (A549, Cal‐12T, NCI‐H727 and A427); the first three of which over‐express α1. The marked impairment in A549 NSCLC cell proliferation and migration, and resulting similar morphology following anti‐α1 siRNA or UNBS1450 treatment, was associated with features of abnormal cytokinesis, mediated in the case of UNBS1450 by disorganization of the actin cytoskeleton. Collectively these data strongly suggest that targeting the Na+/K+‐ATPase α1 using specific cardenolides could represent a novel means to combat certain NSCLCs. Copyright

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.

In vivo assessment of temozolomide local delivery for lung cancer inhalation therapy

The aim of this study was to compare the efficacy of local drug delivery by inhalation to intravenous delivery in a B16F10 melanoma metastatic lung model. Temozolomide was formulated as a suspension, which was elaborated and evaluated in terms of particle size, shape and agglomeration. An endotracheal administration device was used to aerosolise the suspension. This mode of delivery was evaluated at different temozolomide concentrations and was optimized for the uniformity of delivered dose, the droplet size distribution and the distribution of droplets in vivo. Of the particles in the stabilised suspension, 79% were compatible with the human respirable size range, and this formulation retained 100% in vitro anticancer activity as compared to temozolomide alone in three distinct cancer cell lines. The pulmonary delivery device provided good reproducibility in terms of both the dose delivered and the droplet size distribution. Most of the lung tissues that were exposed to aerosol droplets contained the particles, as revealed by fluorescent microscopy techniques. The global in vivo antitumour activity of the inhaled temozolomide provided a median survival period similar to that for intravenous temozolomide delivery, and three out of 27 mice (11%) survived with almost complete eradication of the lung tumours. The present study thus shows that inhalation of a simple liquid formulation is well tolerated and active against a very biologically aggressive mouse melanoma pulmonary pseudo-metastatic model. This inhalation delivery could be used to deliver other types of anticancer drugs.

In vitro and in vivo evaluation of a dry powder endotracheal insufflator device for use in dose-dependent preclinical studies in mice

The aim of this study was to evaluate the ability of the Penn-Century Dry Powder Insufflator for mice (DP-4M) to reproducibly, uniformly, and deeply deliver dry powders for inhalation in the mouse lung. Itraconazole-based dry powder formulations produced by spray-drying were different in terms of composition (different ratios of drug and mannitol, with or without phospholipids), but relatively similar in terms of particle size and mass median aerodynamic diameter. The ability of the dry powder insufflator to disaggregate each formulation was the same, indicated by the absence of a statistically significant difference between the particle size distribution parameters, as measured by laser scattering. The emitted fraction varied in vivo compared to the in vitro condition. Fluorescent particle distribution in the lungs was uniform and reached the alveolar spaces, as visualized by fluorescent microscopy. In terms of drug recovery in lung tissue, a minimum administered powder mass (in this case ∼1 mg) was necessary to recover at least 30% of the emitted dose in the lung and to obtain reproducible pulmonary concentrations. To reduce the dose administered in the lung, it was preferable to dilute the active ingredient within the carrier instead of reducing the dry powder mass inserted in the sampling chamber. Dry powder insufflators are devices usable in dose-dependent preclinical trials but have critical parameters to efficiently deliver reproducible doses depending on the type of formulation.

Clustering methods applied in the detection of Ki67 hot-spots in whole tumor slide images: An efficient way to characterize heterogeneous tissue-based biomarkers

Whole‐slide scanners allow the digitization of an entire histological slide at very high resolution. This new acquisition technique opens a wide range of possibilities for addressing challenging image analysis problems, including the identification of tissue‐based biomarkers. In this study, we use whole‐slide scanner technology for imaging the proliferating activity patterns in tumor slides based on Ki67 immunohistochemistry. Faced with large images, pathologists require tools that can help them identify tumor regions that exhibit high proliferating activity, called “hot‐spots” (HSs). Pathologists need tools that can quantitatively characterize these HS patterns. To respond to this clinical need, the present study investigates various clustering methods with the aim of identifying Ki67 HSs in whole tumor slide images. This task requires a method capable of identifying an unknown number of clusters, which may be highly variable in terms of shape, size, and density. We developed a hybrid clustering method, referred to as Seedlink. Compared to manual HS selections by three pathologists, we show that Seedlink provides an efficient way of detecting Ki67 HSs and improves the agreement among pathologists when identifying HSs.

TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas.

Based on the molecular profiling of astrocytomas, we previously identified a series of genes involved in astrocytoma invasion. Of these, tissue inhibitor of metalloproteinase-4 (TIMP-4) was found to be overexpressed in pilocytic astrocytomas relative to diffuse astrocytomas of any histological grade. Although some data suggest that TIMP-4 may be an anti-tumoral actor in astrocytomas, recent findings challenge this concept. The present study aims to investigate the diagnostic and prognostic values of TIMP-4 and its putative partner CD63 in human astrocytomas. Tissue microarray and image analysis were first carried out to quantitatively analyze the immunohistochemical expression of these proteins in 471 gliomas including 354 astrocytomas. Pathological semi-quantitative scores of both markers’ expression were then established and correlated to astrocytoma diagnosis and patient prognosis. TIMP-4 and CD63 expressions were both overexpressed in astrocytomas compared with oligodendrogliomas (P<0.001) and in pilocytic astrocytomas compared with grade II diffuse astrocytomas (P<0.001). In glioblastomas, high TIMP-4/CD63 co-expression scores were identified as independent prognostic factors associated with progression and shorter survival. In conclusion, this work provides the first evidence of a TIMP-4/CD63 association in astrocytoma tumor cells. It identifies TIMP-4 and CD63 as markers of the astrocytic phenotype in patients with gliomas. In addition, this work highlights the contribution of high TIMP-4/CD63 co-expression to the adverse outcomes of patients with glioblastomas.

KI-67 hot-spots detection on glioblastoma tissue sections

Glioblastomas are very aggressive cerebral tumors which are characterized by strong proliferative activity which is often assessed using the Ki-67 staining method. We propose a novel clustering method that exhibits high performance to detect Ki-67 hot-spots on immunohistochemical slides of glioblastomas.