Administrative Supplement to U54 AG079754: Midwest Murine-Tissue Mapping Center (MM-TMC)

University Of Minnesota, Minneapolis MN

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Abstract

ADMINISTRATIVE SUPPLEMENT GRANT SUMMARY The Midwest Murine Tissue Mapping Center (MM-TMC) is focused on identifying, characterizing and spatially mapping senescent cells (SnCs) in 5 different murine tissues – adipose, brain, liver, lung and muscle– from two different mouse strains (C57BL/6J and FVB;C57BL/6J F1 mice) with age. To accomplish this, the MM-TMC proposed in the parent application to use sc/snRNAseq analysis for identification and characterization of SnCs at the transcriptional level and IHC/IF at the protein level and 10X Visium Spatial Gene Expression (SGE) and Nanostring GeoMx Digital Spatial Profiler (DSP) for the initial spatial transcriptomic analysis along with a ~600 antibodies for analysis of the proteome. However, neither of these spatial transcriptomics platforms provides single cell resolution. Low-abundance transcripts (~1.8 transcripts/cell) can be identified when a minimum of ~300 cells are spatially profiled, and high abundance transcripts can be identified in as few as ~50 cells. Thus, we recently expanded our spatial mapping efforts to our new CosMx Spatial Molecular Imager with the currently available CosMx Universal Cell Characterization RNA and Neuroscience panels that allow for the profiling of the expression of 1000 curated RNA targets with subcellular resolution. CosMx, like GeoMx, is compatible with a wide range of tissues, including FFPE. However, even though the 1000-plex CosMx panel can be customized by adding up to 50 RNA targets to the panel, the transcriptional profiling is still incomplete, especially when using the panel for analysis of SnCs in a wide spectrum of tissues including brain, adipose, liver, muscle and lung. Thus, as part of this Administrative Supplement, we propose to apply the CosMx Whole Transcriptome Mouse Panel to enable the detection and quantification of over 18,000 RNA transcripts at single-cell and subcellular resolution. Importantly, the MM-TMC has early access to the Mouse Whole Transcriptome Panel in the late Fall of this year that can be pre-ordered at a reduced cost. This unique early access from Bruker will allow for completion of the proposed whole transcriptome spatial analysis in the 1 year time frame of the supplement. The use of the CosMx Whole Transcriptome Mouse Panel will allow for greater identification and characterization of SnCs as well as their paracrine effects on surrounding cells. The analysis also will allow for determination of changes in cell identities or cell population alterations that correlate with age. Finally, the whole transcriptome analysis will allow for a more detailed characterization of the paracrine signaling in regard to ligands and their receptors between SnC and their SASP with neighboring cells. It is also important to note that the results from the CosMx Whole Transcriptome Mouse Panel also will be compared to other platforms being used by the MM-TMC (e.g., Visium, Visium HD, Xenium, GeoMx, and the 1000 RNA CosMx panel) in regard to not only identifying, characterizing and spatially mapping of SnCs, but also for the characterization of the paracrine effects of SnCs on neighboring cells.