Abstract This proposal requests a one-year extension to the Meldrum CEGS Microscale Life Sciences Center (MLSC). We propose to apply single cell analysis technology recently developed and integrated in our Center for Biosignatures Discovery Automation to two biological questions of immediate clinical relevance. 1) We will conduct metabolic and genetic analyses of premalignant Barretts esophagus cell lines that we have selected for resistance to hypoxia. There is substantial evidence that hypoxic stress is a potent mediator of progression to adenocarcinoma in Barretts esophagus, as well as in development of other types of cancer. However, the details of this process are largely unknown. We will conduct bulk and single cell experiments that correlate metabolic indicators and gene expression profiles in order to gain mechanistic insight into the role of hypoxia in the development of a cancerous metabolic phenotype. 2) We will also use our single cell analysis platform along with a set of newly developed intracellular sensors to investigate molecular regulation of pyroptosis, a pro-inflammatory cell death pathway. Inflammation signaling plays an important role in autoimmune diseases, sepsis and cancer. Potassium fluxes and alterations in cytosolic ATP concentration correlate with initiation of pyroptosis. However, the mechanisms involved remain unexplored. We propose to clarify the role of intracellular potassium dynamics as well as metabolic regulation of ATP in the activation of pro-inflammatory cell death. The supplemental funds requested will allow us to demonstrate the utility of our single cell analysis technology by answering pressing biological questions in carcinogenesis and inflammation. Our recent advances in and integration of novel technology have positioned us to meet the original goal of our CEGS of making a significant clinical impact by means of dynamic biological measurements having single cell resolution.
In order to understand fundamental pathways involved in disease states, it is necessary to link preexisting cell state to cell fate in the disease process at the individual cell level. The Microscale Life Sciences Center in the Center for Ecogenomics at Arizona State Universitys Biodesign Institute is focused on solving this problem, by developing cutting-edge microscale technology for high throughput genomic-level and multi-parameter single-cell analysis, and applying that technology to fundamental problems in biology and health. Our vision is to address pathways to disease states directly at the individual cell level, at increasing levels of complexity that progressively move to an in vivo understanding of disease. Cancer, heart disease and stroke all involve an imbalance in this cellular decision making process. Because of intrinsic cellular heterogeneity in the live/die decision, this fundamental cellular biology problem is an example of one for which analysis of individuals cells is essential to elucidate relationships among genomics, cell function, and disease. The specific systems to be studied are proinflammatory cell death (pyroptosis) in a mouse macrophage model, and neoplastic progression in the Barretts Esophagus (BE) precancerous model. In each case, diagnostic signatures for specific cell states will be determined by measuring both physiological and genomic parameters. These will then be correlated with cell fate via the same sets of measurements after a challenge (acid reflux for BE). Ultimately, time series will be taken to map out the pathways that underlie the live/die decision. Finally, this information will be used as a platform to define cell-cell interactions at the single-cell level, to move information on disease pathways towards greater in vivo relevance.
We propose to accelerate our studies of cell]cell interactions using the breakthrough cell analysis technologies developed, validated and currently exploited in our laboratory for single]cell physiological and transcriptomic studies. Cell]cell interactions play a central role in cell proliferation, differentiation and the live/die decision. They constitute a biological core for physiological homeostasis, development and response to stimuli in multicellular organisms. Enhancing our understanding of intercellular communications is indispensable for improving our approaches to the prevention, diagnosis and management of serious diseases including cancer. The major goal of the parent CEGS MLSC is to provide deeper understanding of cell heterogeneity and cell]cell signaling associated with cells' fate at the single]cell, multiple cell, tissue and in vivo scales. Based on our accummulated expertise working with single]cell techniques, we have elaborated a detailed experimental plan that will facilitate accelerated completion of the originally]approved project goals. We propose a study of intercellular interactions with a panel of four different cell lines obtained from the same tissue type and representing the progression from normal]to]dysplastic]to]adenocarcinoma in human esophageal epithelial cells. We will combine our single]cell loading, metabolism phenotype measurement, and single]cell harvesting platforms with single]cell whole genome transcriptomics and qRT]PCR analysis. This will enable us to correlate alterations in transcription profiles resulting from cell]cell interactions with changes in cellular metabolic rates. We will apply statistical analysis and data mining approaches to reduce the number of variability factors in order to extract the most significant information. Our expectations from this proposal are: 1.. deeper understanding of how and to what extent intercellular interactions among normal, pre]cancerous and cancerous cells affect cellular metabolism; and 2.. determination of the genes most affected by the presence of cell]cell interactions. A major hurdle hindering us from pursuing this research goal in a more timely fashion has been a lack of manpower. The funds requested include augmenting our personnel resources to accelerate the scientific activities required to achieve this key objective of the parent CEGS grant.
We propose to involve undergraduate students in our research by employing them to work in our lab in the summers of 2009 and 2010. The funding under this requested supplement will provide two benefits to public health: Our scientific progress will be accelerated by the students' contributions to the work, and the students will be encouraged to seriously pursue research careers in the health related sciences.
|Effective start/end date||1/1/07 → 7/31/13|
- HHS: National Institutes of Health (NIH): $20,407,298.00