Core 2. Imaging and Spatial Profiling Core Project Summary: Central technologies of the ST-Analytics U54 program are tissue imaging and profiling platforms, including two sets of tools for highly multiplex, multi-omic analysis of tumor tissue specimens including clinical archival FFPE and lightly preserved frozen tissues, and associated image analysis & informatics software. These Core resource technologies are used extensively by both ST Analytics Projects. The first spatial profiling tool is the well-established commercial Nanostring Digital Spatial Profiling (DSP) system, which permits analysis of regions of interest (ROIs) containing as few as 10-20 cells for detecting around 1000 genes or around 50 proteins (Lee 2017, Lu 2021). Adjacent slices from an FFPE block are prepared. The first is subjected to H&E staining for pathological analysis, while the second is stained with fluorescent antibodies to identify ROIs (for example, immune cell rich or cancer cell rich regions). Those ROIs are then interrogated at the transcriptome or proteome level. We have extensive experience with this tool for analysis of FFPE preserved patient tumor tissues (Lu 2021). The second profiling tools are those recently developed and reported by the Fan group (Deng 2021, 2021, Liu 2020). These powerful tools permit highly multiplex (up to 300-plex) proteomics and genome--wide transcriptome mapping at a resolution of 10 micrometers in tissue slices prepared from FFPE blocks. They also permit spatially resolved ATAC-seq and ChIP-seq type epigenetic profiling (Chen 2017; Chen 2018) at a similar spatial resolution in lightly fixed tissue slices. In particular, this epigenetic profiling capacity is unique to these tools. Similar to DSP, adjacent tissue slices can be H&E stained, and low-plex fluorescent imaging can be employed on the same slice for identification of nuclei as well as immune rich and immune poor regions for alignment validation. These methods can enable high throughput and cost-effective profiling of tissues, and some technology development to that end is described. The Core will continue to develop new spatial profiling tools to meet the emergent needs in the two projects as they advance in the next 5 years, thus serving as a Technoloy Innovation Hub for the U54 center and for the CSBC consortium as well.