A multidisciplinary team from GE Global Research (engineers, biologists, image analysts, statisticians) together with cancer/cell biologists from Vanderbilt University will validate in-situ single cell analysis of formalin fixed paraffin embedde (FFPE) normal and neoplastic colonic tissue. Multiplexed combinatorial analysis of more than four protein signatures within in situ tissue context has been, up till now, impossible to achieve. GE Global Research has developed over the past six years a unique method of iteratively staining individual FFPE tissue sections with 60 or more dye conjugated antibodies without loss of signal for these antigens. This has enabled markers needed for high-resolution single cell segmentation (four or more) to be combined with markers to re- fine cellular phenotype and signaling activities. Mapping software has been developed to correlate specific expression patterns within individual cells to produce single cell multidimensional analysis of individual cel phenotypes. The overall goal of the project is to foster translation of this technology from a research prototype into a system that is commonly used in multiple labs and hospitals around the world. This approach uniquely allows individual cell characterization to a high level within th native tissue context, thereby allowing associations between the cells to be determined. This project will examine important biological principles not possible without this high-resolution technique for analysis of multiple markers of individual cells. In the first study two stem cell populations with unique proliferative and signaling capacities that represent separate identifiable cell populations will be characterized in the mouse colon with markers previously established to work in this system. In the second, changes within these cell populations in colon cancer will be tested in mouse models that activate colon cancer specifically in the stem cells with genetic targeting. Finally, these studies will be extended to examine the nature of the stem cell population within human colon cancer and to correlate known pathway activities relevant to disease progression and recurrence to these cells. This project includes the addition of new technologies that expand the platform to include genomic characterization by adding RNA analysis (miRNA and mRNA FISH). Improvements will be made to an existing automated system to allow broader use of the technology and in- creased sample processing. The results of these studies will enable a paradigm shift in how complex cellular environments with diverse cellular elements are defined, without the loss of information that occurs in other single cell analysis methods that require dispersal of tissue elements.