Very Late Antigen-4 (VLA-4) cells exhibit a unique behavior. Unlike other cells in the cell adhesion family, VLA-4 exhibit a ?roll and firmly adhere? function through inside-out signaling where other cells in the same class arrest and adhere. Heterodimeric integrins ?4-?1 can undergo several conformational changes through rapid affinity modulation in the presence of ligands. There remains an unresolved issue with incomplete and inconsistent activation of all integrins on the surface of these cells. Tangential related, phenotyping immune cells requires use of many biomarkers to detect the presence of clusters of differentiated proteins as well as their normal and abnormal functions. Detection of all biomarkers is crucial when tracking the progression of diseases and cancers. This may introduce a high level degree of complexity, and may become cost-prohibitive to early investigators. We hypothesize 1) multiharmonic high-throughput flow cytometry will resolve multiple fluorescence lifetimes for each single cell analyzed and 2) phenotyping of human peripheral blood mononuclear cells using a minimum of five(5) biomarkers, excite with a singular excitation source and establish conceptual framework for the expansion of this ability to ten or more colors while use of a singular excitation source. Acquisition of LDV-FITC fluorescence lifetimes at harmonics where phase shift is at a maximum. Detection of multiple fluorescence lifetimes per cell will allow for investigation of regions of a single cell that contain activated integrins and whether this activation is an inherent region of the cell or if activation of regional integrins is random. Progression of diseases and cancer is well tracked through multiparametric phenotyping through use of multiple biomarkers. Newer cytometers allow investigators very sensitive detection of multiple biomarkers using extensive number of color channels. However this introduces complexity in instrumentation and very large data files and becomes cost-prohibitive to many. Under the fellowship training plan, we will address two(2) specific aims. We will 1) expand current flow cytometer to allow highly sensitive multiharmonic fluorescence lifetime measurements and quantify integrin behavior per cell and 2) use the multi-lifetime approach in phenotyping across multiple spectral channels to reduce bioinstrumentation complexity and cost and reduce complexity of data acquisition and analysis.
Cellular affinity, is mediated by the stimulation of integrin dimers, is diverse in nature, relevant to cell fate in diseases such as cancer, and such phenotypes can be exploited for drug development. Yet the heterogeneous nature of cell avidity is not fully understood because of inconsistencies related to the activation of integrins. The goal of this fellowship is to use a new multi-harmonic time-resolved flow cytometer to quantify the heterogeneity of integrin activation in cells at a high throughput and thereby establishing a reliable means for studying large populations of cells and set the stage for biomarker screening of cellular adhesion.
