Full Research Project 4

Partnership for the Advancement of Cancer Research Project

Full Research Project 4

Time-resolved flow cytometric study of cell signaling

Project Leaders:
Roger Brent, Ph.D. Full Member, Basic Sciences - Fred Hutch
Jessica Houston, Ph.D. Assistant Professor, Department of Chemical Engineering - NMSU

Jessica Houston at New Mexico State University (NMSU) and Roger Brent at Fred Hutch plan to continue a now well-established collaboration to develop fluorescent lifetime methods to quantify signaling in living cells. Dysfunctions in cell signaling leading to inappropriate cell proliferation contribute to most cancers. Since 2010, during the pilot phase, we have overcome technical challenges to develop time-resolved flow cytometry (Houston) and fluorescent lifetime confocal microscopy (Brent), and have engineered reporter proteins to facilitate measurements of changed fluorescence lifetimes when signaling systems operate. Here, we will continue to develop these complementary methods and use them to measure signaling in living cells. We will isolate cells with proteins expressing useful fluorescence lifetime variants: establish quantitative assays that are less impacted by signal-to-noise-related complications than time independent studies: and facilitate quantification of signaling at a high throughput using time-resolved flow cytometry. Our hypothesis is that these experiments will lead to greater understanding of normal and aberrant cell signaling, and, eventually, to ability to identify and isolate subpopulations in which signaling is altered. Such basic information and abilities would be valuable in a number of applications including cancer diagnosis and, possibly in the future, in therapy (see below). 

Since 2010, Drs. Houston and Brent have worked closely to develop approaches to rapidly quantify signaling in the model organism Saccharomyces cerevisiae. Use of the yeast model has been invaluable, enabling rapid construction and testing of reporters of different signaling events. Over the next three years, we will continue this use of yeast as a test bed to develop better means to study signaling, and we will extend this collaborative work to study signaling in immortalized "normal" and cancerous mammalian cell lines. We look forward to optimizing these methods so that they can be applied to clinical applications, which we envision could occur after this 3-year project is completed. This project will provide training opportunities in New Mexico and Washington state for underrepresented minority students and research fellows, connecting them to a highly active area of international scientific research.

To these ends, during the three years of the full project, we will:

Specific Aim 1.  Develop temporally distinguishable and high-signal reporter protein moieties and measurement methods to allow sensitive quantification of yeast and mammalian signaling in flow cytometry.  We will refine microscopic and flow cytometry instrumentation and algorithms to allow more precise detection of shifts in fluorescence lifetimes. We will use yeast genetics with microscopic methods to generate fluorescent proteins with different lifetimes and test these in time resolved flow cytometry. We will use microscopic and cytometric methods to isolate high signal-to-noise fluorescent protein derivatives that give robust readouts for cell signaling events. We will use these with our instrument technologies to quantify key classes of signaling events in mammalian cells.

Successful work on this project will allow us to address now un-addressable scientific questions such as rates of formation of signaling complexes in specific subcellular locations. Successful work might also eventually, in the long term, have clinical impact. For example, these methods might impact cell-based therapy for cancer with tumor infiltrating lymphocytes. In order for such therapies to become routine, critical issues now include best means to immunize ex vivo and to identify and expand populations of T cells that engage tumors productively and consistently. In the future, we envision that these methods might be used to define immunization procedures specific for each patient and to identify and effector cell populations that mediate tumor regression might be transformative for these cancer treatments. This vision and related ideas animate our long-term interest in developing robust, high-throughput means to quantify and sort on phenotypes, genotypes and similarities in subpopulations of single cells.

For More Information:

Click here to learm more about the research taking place in Dr. Brent's lab.

Click here to learn more about Dr. Houston's research.