Cellular homeostasis and reactive changes depend on signal transduction between the cytoplasm and nucleoplasm. In cancer, aberrant signal transduction is commonly observed and novel therapeutics targeting these signaling cascades are currently evaluated in clinical trials or have already secured a position in the therapeutic arsenal. Aberrant signal transduction is often associated with abnormal intracellular distribution of specific signaling intermediaries thus their sub-cellular localization could potentially be used as a parameter of treatment response. Traditionally, intracellular localization of molecules have been studied by molecular techniques or by immunological labeling techniques studied with (confocal) microscopy. These approaches have the disadvantage of lacking specificity for target cell populations or the ability to evaluate large cell populations required for statistically robust analyses. The ImageStream 100 multispectral imaging flow cytometer (IS 100) has been developed to produce high resolution brightfield, darkfield, and fluorescence images of cells prepared in suspension at rates up to 100 cells per second. The IDEAS(tm) analysis software quantifies over 200 morphometric and photometric parameters for each cell based on its imagery, including parameters that measure sub-cellular location of probes. The aim of this application is to evaluate the potential of multispectral imaging flow cytometry to evaluate target cell-specific, therapy-induced changes in nuclear-cytoplasmic distribution of specific signaling pathway intermediaries as response parameters in clinical samples. The proposed studies will ultimately be conducted in conjunction with a phase I clinical trial in patients with acute myeloid leukemia (AML) undergoing therapy with the macrolide rapamycin (sirolimus) which is known to affect the function (and cellular localization) of nuclear factor-KB (NF-icB). During the R21 phase of this application, system hardware, software and sample preparation conditions will be optimized to efficiently and accurately determine NF-icB translocation events using cell line model systems. Applicability of detecting other translocations of proteins associated with NF-icB signal transduction and rapamycin action, will also be assessed and if successful will lead to testing of additional hypotheses in the R33 phase of this application. The R33 phase will apply this technique in the analysis of patient samples and will test the hypothesis that cellular distribution of NF-icB can be used as a determinant of response in AML patients treated with rapamycin and will also test additional hypotheses generated in theR21 phase. Lay Summary: By analyzing images of cancer cells at a high rate the ImageStream cytometer can provide specific information on the success or failure of specific cancer treatment responses. The aim of the proposed studies is to determine necessary adjustments to this instrument's hardware and analysis software to accurately and efficiently use it in a clinical research setting.