High Throughput Ratio Imaging to Profile Caspase Activity: Potential Application in Multiparameter High Content Apoptosis Analysis and Drug Screening

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based

VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis

Quantitative RT-PCR profiling of the Rabbit Immune Response: Assessment of Acute Shigella flexneri Infection

Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions

A Concerted HIF-1α/MT1-MMP Signalling Axis Regulates the Expression of the 3BP2 Adaptor Protein in Hypoxic Mesenchymal Stromal Cells

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour

Quorum Sensing Influences Vibrio harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence

The light-emitting Vibrios provide excellent material for studying the interaction of cellular communication with growth rate because bioluminescence is a convenient marker for quorum sensing. However, the use of bioluminescence as a marker is complicated because bioluminescence itself may affect growth rate, e.g. by diverting energy. quorum mutants. growth rate can be either positive or negative and includes both bioluminescence-dependent and independent components. Bioluminescence tends to slow growth rate but not enough to account for the effects of quorum sensing on growth rate