P18.25.B SPATIALLY CONFINED TRANSCRIPTIONAL RESPONSES CHARACTERIZE THE EARLY PRE-METASTATIC NICHE IN BRAIN METASTASIS

Abstract BACKGROUND Brain metastases often originate from circulating tumor cells (CTCs) that become arrested within cerebral microvessels. Recent findings indicate that this process induces localized hypoxia, which in turn upregulates angiogenic mediators such as Angiopoietin-2 (Ang-2), thereby promoting vascular remodeling and facilitating extravasation of tumor cells into the brain parenchyma. Nevertheless, the early transcriptional landscape of the adjacent brain tissue—the so-called pre-metastatic niche—remains poorly defined. MATERIAL AND METHODS To simulate early metastatic seeding, syngeneic murine lung and breast carcinoma cells were injected into the right carotid artery of immunocompetent mice. The contralateral, non-injected hemisphere served as an internal control to allow intra-animal comparisons, reduce biological variability, and minimize animal usage. Brain tissue was collected at 1 and 4 days post-injection, processed via formalin fixation and paraffin embedding, and subjected to histological evaluation. Comparative bulk RNA sequencing of tumor-bearing and control hemispheres was performed, complemented by high-plex spatially resolved single-cell transcriptomic profiling to detect localized gene expression patterns. RESULTS Bulk RNA sequencing revealed no significant transcriptional differences between hemispheres. In contrast, spatial transcriptomic analysis uncovered robust, spatially restricted gene expression changes in the tumor-bearing hemisphere. These alterations involved genes associated with vascular remodeling, hypoxia-responsive pathways, and neuronal connectivity. Notably, spatial mapping demonstrated that the expression of several key transcripts peaked near sites of arrested tumor cells and declined with increasing distance, indicative of a highly localized, tumor-induced host response. CONCLUSION This study reveals distinct, spatially confined transcriptional alterations that define the early pre-metastatic niche in the brain. By leveraging advanced spatial single-cell transcriptomics, we provide a powerful framework to dissect the molecular cues underlying early metastatic interactions within the native tissue microenvironment. These insights not only deepen our understanding of early brain metastasis biology but also inform the development of innovative strategies for therapeutic intervention at the pre-metastatic stage.