PROJECT SUMMARY The Digital Pathology and Mass Cytometry Core (Core D) will provide state-of-the-art technologies to enable high-parameter single-cell proteomic profiling for all the Research Projects in the proposed P01 Program Project Grant. The core directors are experts in multicolor immune profiling (multispectral immunoflurescence staining) and high-dimensional proteomic profiling (Cytometry by Time-of-Flight; CyTOF) technologies and will work with project investigators to assay human and mouse tissues generated over the five years of the Program Project. Dr. Anders (core co-director) is an Associate Professor of Pathology, is a practicing GI surgical pathologist who co-directs the Tumor Microenvironment (TME) Laboratory in the Bloomberg Kimmel Institute for Cancer Immunotherapy. The core houses approximately 6 million dollars of equipment aimed at multicolor tissue staining and analysis. The major equipment includes 2 Leica Bond automated immunostainers, 6 Akoya automated high capacity multichannel high-resolution scanning microscopes (Vectra and Polaris systems), and 8 data servers linked by 10G and 40G internet connections to the rest of the campus. The TME laboratory is a central resource for multispectral immunofluorescence staining and image analysis for the Johns Hopkins Oncology research community. Dr. Ho (core co-director) is an Assistant Professor of Oncology and Scientific Director of the newly established CyTOF core, housing the third-generation cytometer Helios™, in the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins. Dr. Ho completed his postdoctoral research in the laboratory of Dr. Elizabeth Jaffee with a special emphasis on developing mass cytometry pipelines to profile the immunologic responses. Dr. Ho also co-led the development of analytical pipelines for CyTOF datasets with Dr. Elana Fertig (Bioinformatics and leader for Core C). Both directors, Anders and Ho, have demonstrated a strong track record of productivity utilizing these technologies. The lab spaces and offices dedicated to this Core are located in close proximity within the Cancer Research Building Complex. The goal of the Digital Pathology and Mass Cytometry Core (Core D) is to provide established and validated assay platforms. The Core Directors have already set-up the assay systems and validated specific marker panels. This provides quality assurance and control for assays performed within and across projects. Once acquired, the data will be provided to Core C for analysis and integration, and to Core B for clinical correlations.

PROJECT NARRATIVE The Digital Pathology and Mass Cytometry Core (Core D) will serve as a centralized resource for each project by providing state-of-the-art proteomic technologies to assay the status of the tumor microenvironment. The Core will employ multiple color image analysis (multispectral immunofluorescence tissue staining) and high- parameter single-cell protein profiling (mass cytometry by Time-of-Flight; CyTOF) of human and animal pancreatic tumor tissue in order to construct a multi-dimensional map of the tumor microenvironment.