Adrian Ozinsky Group

The goal of the research projects in the Ozinsky laboratory is to develop high-resolution mRNA and protein measurement and imaging tools appropriate for investigations on mammalian cells. These tools are being applied to purify and characterize human breast cancer stem cells, and murine macrophages and T cells. For examples, we have been measuring the mRNA expression of candidate genes by qRT-PCR in single mammary epithelial cells in order to identify the role for stem cells in cancer metastasis. Together with using promoter-reporter constructs that enable cell selection and time-lapse imaging, these data are revealing for the first time how gene expression patterns of ~40 transcriptional regulators and markers of cancer stem cells are coordinated within individual cells. In addition, two new analytical capabilities are under development that will enable proteins to be quantified within individual cells by highly multiplexed fluorescent ELISA, and global mRNA signatures to be determined with single cell resolution.

Technology Development:
Microfluidic platform for multiplexed protein measurements by single-cell ELISA
Dr. James Spotts, a senior scientist in the Ozinsky group, who has been collaborating with researchers at NIST, Caltech, and Tampere University of Technology, to develop a scalable microfluidics-based ELISA platform to measure multiple proteins from sets of single-cell samples in parallel. This new capability will improve the level of multiplexing of protein analysis in single cells by 10-100 fold, and is anticipated to be widely applicable in many areas of biology.

Global mRNA transcriptome profiling with single-cell resolution
We have devised an approach for processing single-cell samples so that their transcriptome profiles can be measured at the scale of quantifying >1000 genes from each of >100 individual cells in a single experiment, using Next-generation sequencing. The procedure barcodes the signal from the individual cells, pools the population, and captures the targets in a single purification step. Dr. Greg Zornetzer, a postdoctoral fellow in the Ozinsky group, is working to optimize the performance of this approach, and there is an ongoing collaboration with researchers at Caltech to miniaturize the procedure so that single-cell samples can be efficiently processed in parallel.

Automated microscopy, and bright-field image analysis of mammalian cells
In high-throughput microscopy, automated cell detection generally is enabled by fluorescence labeling of cells. This approach, however, has several limitations, including phototoxicity, photobleaching, the limited number of distinct fluorescent channels that can be multiplexed, and the blinkered view imposed by monitoring few molecular parameters. In collaboration with the Shmulevich group at ISB, and collaborators at Tampere University of Technology, we have found that by imaging mammalian cells within brightfield z-stacks, the in- and out-of-focus z-slices can be used to create artificial images of greatly increased contrast that are sufficient for automated analysis. Our approach is fully label-free, does not require any special optics or equipment, and is implemented on standard software tools. These enhanced images enable cells to be counted and segmented, which then serves as the basis for many forms of further computational analysis of their phenotypes.

Image-based selection and laser-mediated purification of individual mammalian cells for single-cell transcriptomic analysis
Together with researchers at Cyntellect, we have been developing protocols to use the laser of the LEAP instrument to target selected cells within a cell population in a well of a microtiter plate, and release their mRNA for qRT-PCR analysis. Noel Blake of the Imaging, Cytometry and Microfluidics Core Facility is helping to refine the procedure to enable single cell mRNA profiling.

Biology:
Insight into the immune response
How do immune cells coordinate defenses against infection? What are the mechanisms used by white blood cells, such as macrophages, to detect engulf and kill microbes and how are these events coupled to the initiation of inflammation. The Ozinsky group has been taking advantage of approaches that enable highly sensitive multiplexed RNA and protein measurements to gain insight into questions related to the immune system regulation. Macrophages are the immune cells responsible for detecting and invading pathogen and initiating the immune response. Individual macrophages produce different kinds and levels of cytokines, and understanding how differences in the cytokine production is regulated in macrophages is one key to understanding how the body mounts an immune response, and will lay the groundwork for developing more effective vaccines. Biomarkers that classify inhaled biothreat lung infections

We are measuring protein and mRNA biomarkers that distinguish between different types of inhaled biothreat agents, including Yersinnia pestis (plague), Francisella tularensis (tularemia), and pandemic influenza. These defense responses are being compared to those induced by matched less virulent pathogen strains, and other pathogenic though non-biothreat bacterial species. This project is being performed by Dr. Kathie Walters and Rolf Kuestner, who also aim to use this data to define mechanisms that underlie the extreme pathogenesis of biothreat agents. These studies are being performed in collaboration with Dr Shawn Skerrett at Harborview Medical Center and the University of Washington, and Dr Mark Wolcott of USAMRIID. In addition, Dr. Walters is working with Dr John Kash and Dr Jeffrey Taubenberger at the NIH to study how different immune responses are elicited by different pandemic stains of influenza, including 2009 H1N1 pandemic influenza, 1918 H1N1 pandemic influenza, and seasonal non-pandemic influenza.

Assessing the role for stem cells in human breast cancer metastasis
Dr. Anne Grosse-Wilde, a postdoctoral scientist in the Ozinsky group, has been studying whether stem cells contribute towards breast cancer metastasis. The hypothetical role for stem cells in cancer is inspired by observations that oncogenic cell differentiation mimics features of normal cellular development, with metastatic epithelial cells acquiring new capabilities for motility, invasion and survival in a manner that suggests that they have differentiated and acquired mesenchymal properties. It has been postulated that cancer stem cells within a tumor are responsible for the malignancy and radiotherapy resistance of breast tumors, suggesting that efforts to develop anti-tumor therapy should focus on this rare subset of tumor cells. Are all epithelial cells capable of differentiating into mesenchymal cells, or are a subset of stem cells capable of giving rise to both lineages? How similar is cell differentiation in breast tumors to normal epithelial/mesenchymal differentiation? This area of research is the perfect application for high-resolution time lapse single-cell imaging and mRNA and protein measurement assays that resolve the properties of individual cells.