Microanatomy of intracranial Human Thrombus by Single Nuclei Transcriptomics

Introduction: Microvascular dysfunction and inflammation are thought to underlie reperfusion failure following endovascular thrombectomy. Here, we present the first single nuclei RNA-sequencing (snRNA-seq) analysis of an intracranial human thrombus to demonstrate the feasibility of transcriptomic analysis of thrombi, define and contextualize its cellular and transcriptomic landscape, and identify molecular pathways contributing to reperfusion dynamics post-recanalization.

Methods: We performed snRNA-seq on an intracranial human thrombus harvested during revascularization. Unsupervised clustering, data visualization, differential expression and functional gene ontology/pathway analysis were utilized to deconvolve expression data into cell type-specific expression profiles. Thrombus snRNA-seq data was integrated with single cell RNA-seq (scRNA-seq) data from symptomatic and asymptomatic human carotid plaques and healthy brain vasculature to contextualize the thrombus in atherosclerotic disease and identify ligand-receptor interactions (GSE 155512; https://compbio.mit.edu/scBBB/).

Results: Unbiased clustering of 2027 nuclei identified five cellular populations in distinct effector states within the thrombus microenvironment. Functional gene ontology and pathway analysis revealed a monocyte subpopulation in a distinct endothelial-activation state, supporting the notion of cellular trans-differentiation and interconversion. Integration with carotid plaque data revealed shared transcriptional programs existing in a continuum, progressing from asymptomatic plaque to embolized thrombus. Ligand-receptor analysis identified macrophage mediated T-cell activation, pro-inflammatory response, immunological synapse stabilization, and monocyte trans-endothelial migration. The ability of snRNA-seq to identify temporally-resolved ligand-receptor interactions provides the opportunity to uncover distinct targetable therapeutic vulnerabilities.

Conclusion : Our study provides the first transcriptome-based cellular landscape of a human intracranial thrombus and highlights the importance of cellular plasticity and intercellular communication at the site of occlusion. Our novel application of snRNA-seq to thrombectomy specimens provides the highest resolution exploration of cellular differences and a best understanding of individual contextualized cell function to date. This detailed definition of the cell communities at play will facilitate mapping of novel interventional targets with direct functional relevance and allow for greater personalization of post-reperfusion therapy.