Phenotypic Pattern-Based Assay for Dynamically Monitoring Host Cellular Responses to Salmonella Infections

The interaction between mammalian host cells and bacteria is a dynamic process, and the underlying pathologic mechanisms are poorly characterized. Limited information describing the host-bacterial interaction is based mainly on studies using label-based endpoint assays that detect changes in cell behavior at a given time point, yielding incomplete information. In this paper, a novel, label-free, real-time cell-detection system based on electronic impedance sensor technology was adapted to dynamically monitor the entire process of intestinal epithelial cells response to Salmonella infection. Changes in cell morphology and attachment were quantitatively and continuously recorded following infection. The resulting impedance-based time-dependent cell response profiles (TCRPs) were compared to standard assays and showed good correlation and sensitivity. Biochemical assays further suggested that TCRPs were correlated with cytoskeleton-associated morphological dynamics, which can be largely attenuated by inhibitions of actin and microtubule polymerization. Collectively, our data indicate that cell-electrode impedance measurements not only provide a novel, real-time, label-free method for investigating bacterial infection but also help advance our understanding of host responses in a more physiological and continuous manner that is beyond the scope of current endpoint assays

A Cell Culture Chip with Transparent, Micropillar-Decorated Bottom for Live Cell Imaging and Screening of Breast Cancer Cells

In the recent years, microfabrication technologies have been widely used in cell biology, tissue engineering, and regenerative medicine studies. Today, the implementation of microfabricated devices in cancer research is frequent and advantageous because it enables the study of cancer cells in controlled microenvironments provided by the microchips. Breast cancer is one of the most common cancers in women, and the way breast cancer cells interact with their physical microenvironment is still under investigation. In this study, we developed a transparent cell culture chip (Ch-Pattern) with a micropillar-decorated bottom that makes live imaging and monitoring of the metabolic, proliferative, apoptotic, and morphological behavior of breast cancer cells possible. The reason for the use of micropatterned surfaces is because cancer cells deform and lose their shape and acto-myosin integrity on micropatterned substrates, and this allows the quantification of the changes in morphology and through that identification of the cancerous cells. In the last decade, cancer cells were studied on micropatterned substrates of varying sizes and with a variety of biomaterials. These studies were conducted using conventional cell culture plates carrying patterned films. In the present study, cell culture protocols were conducted in the clear-bottom micropatterned chip. This approach adds significantly to the current knowledge and applications by enabling low-volume and high-throughput processing of the cell behavior, especially the cell-micropattern interactions. In this study, two different breast cancer cell lines, MDA-MB-231 and MCF-7, were used. MDA-MB-231 cells are invasive and metastatic, while MCF-7 cells are not metastatic. The nuclei of these two cell types deformed to distinctly different levels on the micropatterns, had different metabolic and proliferation rates, and their cell cycles were affected. The Ch-Pattern chips developed in this study proved to have significant advantages when used in the biological analysis of live cells and highly beneficial in the study of screening breast cancer cell-substrate interactions in vitro

Normalizing for individual cell population context in the analysis of high-content cellular screens

<p>Abstract</p> <p>Background</p> <p>High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.</p> <p>Results</p> <p>We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.</p> <p>Conclusions</p> <p>Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.</p

Comparing the value of mono- vs coculture for high-throughput compound screening in hematological malignancies

Large-scale compound screens are a powerful model system for understanding variability of treatment response and discovering druggable tumor vulnerabilities of hematological malignancies. However, as mostly performed in a monoculture of tumor cells, these assays disregard modulatory effects of the in vivo microenvironment. It is an open question whether and to what extent coculture with bone marrow stromal cells could improve the biological relevance of drug testing assays over monoculture. Here, we established a high-throughput platform to measure ex vivo sensitivity of 108 primary blood cancer samples to 50 drugs in monoculture and coculture with bone marrow stromal cells. Stromal coculture conferred resistance to 52% of compounds in chronic lymphocytic leukemia (CLL) and 36% of compounds in acute myeloid leukemia (AML), including chemotherapeutics, B-cell receptor inhibitors, proteasome inhibitors, and Bromodomain and extraterminal domain inhibitors. Only the JAK inhibitors ruxolitinib and tofacitinib exhibited increased efficacy in AML and CLL stromal coculture. We further confirmed the importance of JAK-STAT signaling for stroma-mediated resistance by showing that stromal cells induce phosphorylation of STAT3 in CLL cells. We genetically characterized the 108 cancer samples and found that drug-gene associations strongly correlated between monoculture and coculture. However, effect sizes were lower in coculture, with more drug-gene associations detected in monoculture than in coculture. Our results justify a 2-step strategy for drug perturbation testing, with large-scale screening performed in monoculture, followed by focused evaluation of potential stroma-mediated resistances in coculture

Integrated Single Cell Imaging and RNA-Sequencing in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor and is comprised of transcriptionally heterogeneous cells and a complex microenvironment. Despite decades of research effort, few treatments significantly benefit clinical outcomes. This may be, in part, due to the lack of tools to directly measure functional responses of these heterogeneous cell types under therapy. This thesis aims to advance the understanding of cell type-specific therapeutic response by the development and application of integrated single cell imaging and RNA sequencing technology. Chapter 1 provides an overview of GBM and its heterogeneity, how investigation of cell type-specific phenotypes would benefit the development of GBM treatments, and current sequencing and imaging technologies to examine cell phenotypes with single-cell resolution. Chapter 2 presents a new microfluidic technology for joint single cell imaging and RNA sequencing that can link imaging-based phenotypes and transcriptional identity of the same individual cells with high throughput, molecular capture efficiency, linking accuracy, and user-friendliness. Chapters 3 and 4 present applications of this technology in investigating cell type-specificities of GBM treatments. Chapter 3 focuses on the specificities of 5-aminolevulinic acid (5-ALA), an FDA approved fluorogenic agent, used in fluorescence guided surgery and reveals 5-ALA labeling is not specific to transformed glioma cells, which encourages further studies to systematically compare its performance with potential alternatives. Chapter 4 focuses on the specificities of drug responses by presenting a functional drug screening approach that directly links cell states measured by apoptosis indicators with transcriptional states, which greatly enhances the interpretability of single cell-resolved drug perturbation assays

Investigating gene function for neuronal survival after metabolic stress using semi-automated fluorescence microscopy and automated image analysis

Overexpression approaches and fluorescence microscopy techniques allow investigating important spatiotemporal aspects of gene regulation as well as quantifying gene function. Consequently, fluorescence microscopy techniques help answer important questions on gene regulation such as addressing the role of a specific gene product for neuronal survival under different treatments. Here, we describe a versatile tool to measure effects of a transfected gene of interest on neuronal survival upon metabolic stress. We focus on nutrient starvation of cultured rodent primary neurons as a model of metabolic stress but our approach can easily be generalized and adapted to other cell types or to investigate single gene function in regulating neuronal survival under various conditions

Development and application of a high throughput cell based assay to identify apoptosis inducing proteins, and functional characterization of the candidate Vacuole Membrane Protein 1 (Vmp1)

The aim of the project was to identify and functionally characterize novel human proteins that dominantly induce apoptosis upon overexpression. To achieve this, a cell-based high throughput assay was developed. The assay is based on the detection of activated caspase-3 in cells overexpressing proteins tagged C- and N-terminally with YFP. Apoptotic cells were detected by staining with a specific antibody directed against the activated form of caspase-3. The assay was automated and data acquisition was done using a flow cytometer with an integrated 96-well plate reader. A total of 200 proteins have been screened in the assay, out of which five were identified to be significant activators of apoptosis. One of the candidates, Vacuole Membrane Protein 1 (Vmp1), which forms vacuoles in cells and subsequently induces apoptosis when overexpressed, has been functionally characterized in detail. It has been reported that VMP1 mRNA is differentially expressed in cancer, acute pancreatitis and kidney ischemia and that the overexpressed protein is localized to the Endoplasmic reticulum. But the function of this protein and its role in cancer and other diseases was previously unknown. In this study I show that the vacuoles are formed by the Endoplasmic reticulum due to accumulation of overexpressed Vmp1, and that Vmp1 is actually a plasma membrane protein involved in the formation of initial cell-cell contacts. Its function as a cell-cell adhesion protein was confirmed by identifying that Vmp1 interacts with the tight junction protein ZO-1, and that down regulation of Vmp1 induces cell detachment. Further, down regulation of Vmp1 resulted in a massive increase in the invasion potential of kidney cancer cells, which is consistent with the findings that VMP1 mRNA level is significantly lower in kidney metastases compared to primary tumours. Thus, these results are the first to show that Vmp1 is a cell adhesion protein, and that its expression level is a critical determinant of cancer cell invasiveness, metastasis formation and induction of apoptosis. In summary, I have established and applied a high throughput cell-based assay to screen for inducers of apoptosis. Functional characterization of one of the candidates from this screen revealed it to be a disease relevant regulator of cell-cell adhesion. This demonstrates the strength of this high throughput approach in the identification of proteins involved in diseases

Development of high content image-based cell viability assays

Aim and applications: 1. Optimize fluorescence-based cellular assays for getting insights about mechanisms leading to cell death 2. Informative high content assays development with the aim to implement them in order to obtain preliminary toxicity information of compounds 3. Toxicity profiling of chemicals or biologicals used in screening, in particular hits behavior relative to the characterization of cell viability. Methods: Different fluorescent probes for cell viability were tested and optimized in HeLa cells by varying parameters of staining, controls and incubation times. Automated fluorescence microscopy was used to image cells in 96-well plates and automated image analysis provided tool for quantification of the response to the different assays. Image quality was estimated in each condition and ability to discriminate between positive and negative populations was statistically determined after image segmentation and features extraction. Results: Live/dead assay using calcein-AM and ethidium homodimer-1 showed significant difference in signal between living and dead cells but high variability was observed within the same population. Apoptosis probes FLICA and annexin V conjugate were tested on cells treated with staurosporine. FLICA staining gave low signal-to-background ratio and apoptotic population was not significantly discernible. Living, early apoptotic and necrotic/late apoptotic cells populations were segregated using annexin V assay. Autophagy detection using LC3B immunostaining was observable in cells treated with chloroquine. Developed image analysis method reliably segmented autophagosomes in autophagic cells but failed with dead cells. Assays were tested on a set of toxic compounds and results showed differences with information found on these substances. Conclusion: Fluorescent probes are a powerful tool to assess cell death mechanism in high content screening assay as they can report multiple biological activities at the same time. As information in this type of assay are gathered at a single cell level, high variability is expected and must be reduced as possible to successfully characterize the phenotypes encountered and reach screening requirements