Mapping immuno-vascular signatures to characterize blood brain barrier disruption and tumor colonization in triple negative breast cancer brain metastasis.

Introduction: The incidence of brain metastases in breast cancer patients is rising, with brain metastases affecting 25-40% of women with triple-negative breast cancer (TNBC), with a median survival of under six months. Disruption of the blood-brain barrier (BBB) leads to the formation of a blood-tumor barrier (BTB), which is a critical event in brain colonization. We hypothesize that metastatic tumor cells disrupt neurovascular integrity via specific molecular mechanisms, thereby enabling BTB formation. Elucidating the molecular mechanisms and key players underlying TNBC brain tropism-focusing on BBB disruption and immune evasion- will help identify and manipulate key players in this metastatic process, offering potential for preventing or reversing colonization. Methods: We performed bulk RNA sequencing to compare brain-tropic 4T1-Br5 cells with parental 4T1 cells and characterized the transcriptional programs supporting brain adaptation. We also established a dual-phased in vivo injection model (orthotopic mammary fat pad injection (MFP) followed by intracardiac injection (IC) 7 days later) to obtain BrM tissue for spatial analysis. Results: For the in vivo study, 10 mice got hybrid injections of 4T1-Br5 cells; however, only 5 mice survived to the endpoint. Robust, histologically confirmed brain metastases were detected in 1 of 5 mice (20%). This rate aligns with literature reports and also highlights modeling challenges in immunocompetent settings. RNA-seq showed upregulation of cell cycle drivers (MYC,E2F) and DNA repair pathways, indicating rapid proliferation and genomic resilience. altered BRCA1/TP53 balanced instability with survival often observed with aggressive metastasis. Adaptive pathways for brain colonization, including neuronal signaling (via NGFR-BDNF), cytoskeletal remodeling, and ESR1 activation, exhibited distinct upregulation. Immune-evasive signatures were characterized by the reduced expression of IL-1beta, IFN-gamma, as well as impaired antigen presentation (MHC I/II), and the activation of CCL5 and the complement cascade. As expected with brain vasculature remodeling, angiogenesis/vascular markers, such as VEGF, HCN, and BGN, were upregulated, while THBS2 and NOTCH signaling were downregulated. Conclusion: Overall, the brain-tropic 4T1-Br5 cells exhibit a coordinated reprogramming of proliferation, immune evasion, angiogenesis, epigenetic plasticity, and neuronal mimicry pathways, collectively empowering them cells to overcome the unique barriers of the brain, thus supporting their metastatic growth. This establishes a strong scientific foundation for identifying actionable targets for therapeutics. I n vivo model dose optimization will yield more consistent brain metastases, enabling a comparative spatial analysis of mouse tissue with patient-derived tissue samples.