Label-Free Flow Cytometry For Cancer Cell Discrimination

Project Summary Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of diseaseStandard flow cytometers use fluorescent tagsoften conjugated to monoclonal antibodiesto give qualitative and quantitative information about specific molecules in the cell on a cell by cell basis at very high throughputMetabolic imaging of cancer cells relying on measurements of the inherent fluorescence of the ubiquitous metabolic co enzyme NADHthe reduced form of nicotinamide adenine dinucleotideparticularly the fluorescence lifetime of NADHyields a robustgeneralized indicator of carcinogenesisThis is based on the well establishedWarburg Effecta shift from oxidative phosphorylation to glycolysis as the preferred metabolic pathway in many cancer cellsand the involvement of NADH as a key player in both of these pathwaysHowevermost of this work has been done in the field of fluorescence lifetime imaging microscopyFLIMa low throughput method not amenable to development of a clinical diagnostic assayTranslating this method to high throughput flow cytometry would pave the way for such a clinical diagnostic testSince thesefforts to implement lifetime measurements in flow have been hampered by a variety of technical barriers associated with hardware cost and speedand computationally intense analysisThis work overcomes these obstaclesleveraging an existing fluorescence lifetime flow cytometry platform and modifying it to create a built for purpose tool for cancer cell analysis using NADH fluorescence lifetime that also employs an innovative method for rapid data analysis which would enable on line analysis and cell sortingFeasibility will be demonstrated by challenging the system with a comprehensive set of verification and validation testsThe method requires no exogenous cell labelingrelying instead on the inherent fluorescence of NADHThis instrument will provide a uniquehigh throughputlabel free method for cancer cell discrimination at the singlecell level compatible for use on both blood borne cancer cells and dissociated cells from solid tumorsSuch an instrument would speed cancer drug developmenthave utility in cancer diagnostics and monitoring cancer treatmenthelp identify and understand tumor heterogeneity and treatment resistant subpopulations of cellsand could enable development of personalized treatment regimens Project Narrative Flow cytometry is a widely used cell analysis technique in research and clinical diagnosticse gdrug developmentcancerAIDSand other diseasesin part due to its extremely high throughputNovellabel free cancer cell techniquesbased on fluorescence lifetimeexistbut are amenable only to low throughput and not suitable for clinical diagnostic testsThis proposal translates these techniques to the high throughput realm of flow cytometrypaving the way for better and more informative cancer diagnostics