Spatial Analysis of Neural Cell Proteomic Profiles following Ischemic Stroke in Mice using High-Plex Digital Spatial Profiling

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at three-days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN, an increase in the autophagy proteins BAG3 and CTSD, an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39, and an increase in the neurodegenerative proteins BACE1, APP, αβ 1-42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic immunoreactivity, apoptotic, and neurodegenerative proteomic profile, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. Overall, our data highlight the importance of identifying ischemic mechanisms in a spatial manner to understand the complex, dynamic interactions throughout ischemic progression and repair as well to introduce potential targets for successful therapeutic interventions.

Spatially-resolved proteomics and transcriptomics: An emerging digital spatial profiling approach for tumor microenvironment

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.