Abstract Background: Brain metastasis from triple-negative breast cancer (TNBC) is highly aggressive and remain difficult to treat, with a disproportionate burden among African American patients, especially in Texas. Chronic psychological stress, anxiety, or depression affecting more than 50% of cancer patients, disrupts neurotransmitter homeostasis and has been increasingly implicated in promoting cancer progression and metastasis. Among stress-associated neurotransmitters, serotonin (5-HT) levels are markedly elevated both under chronic stress and with selective serotonin reuptake inhibitor (SSRI) use, and can exacerbate brain metastasis. In this study, we investigate the mechanistic contribution of serotonin-HTR2B signaling to stress-induced brain metastatic progression in TNBC, using spatial transcriptomics and functional analysis. Methods: We conducted a comprehensive analysis of neurotransmitter-related gene expression using both single-cell RNA-seq (scRNA-seq) and bulk transcriptomic data from TNBC brain metastases and brain-tropic cell lines. Chronic restraint stress models were used to study metastasis development following inoculation with 4T1-BR or MDA-MB231-BR cells. Visium spatial transcriptomics was performed on brain tissue sections to spatially resolve transcriptional alterations within the metastatic niche under stress conditions. Functional validation included CRISPR -Cas9-mediated knockout (KO) of HTR2B in TNBC cell lines and in vivo bioluminescence imaging to monitor metastatic burden. Clinical datasets (GSE12276, n=204; TNBC cohort n=422) were analyzed to examine HTR2B expression levels and their association with patient survival outcomes. Results: TNBC brain metastases and brain-seeking cell lines showed widespread neurotransmitter-related gene upregulation. In vivo, chronic restraint stress significantly accelerated brain metastasis formation (p=0.022) and increased anxiogenic behaviors in mice. Spatial transcriptomic analysis revealed serotonin-HTR2B signaling enriched at the tumor-microenvironment interface in stressed mice, with activation of ERK/MAPK, PI3K/AKT, and STAT signaling pathways. CRISPR-mediated knockout of HTR2B resulted in a ∼60% reduction in brain metastatic burden under stress conditions (p 0.01), without affecting in vitro proliferation. Exogenous serotonin modestly increased in vitro growth but likely promotes metastasis via microenvironmental interactions. High tumor HTR2B expression was associated with shorter brain metastasis-free survival (p=0.012) and distant metastasis-free survival (p=0.013). Conclusions: Chronic stress promotes TNBC brain metastasis through activation of serotonin-HTR2B signaling, creating a pro-metastatic niche. Ongoing studies aim to identify specific stromal and immune cell populations responding to this signaling axis, using spatial transcriptomics-guided multiplex imaging. This work addresses a critical knowledge gap in understanding how stress reshapes the brain metastatic microenvironment. Collectively, these findings highlight serotonin-HTR2B as a promising therapeutic target to mitigate brain metastasis risk in stressed cancer patients. Citation Format: J. Ahn, J. Zambelas, D. Chen, M. Vasquez, W. Dong, H. Zhao, S. Wong. Chronic stress promotes brain metastasis via serotonin-HTR2B axis in triple-negative breast cancer: insights from spatial transcriptomic and functional analysis abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-02-21.
Ahn et al. (Tue,) studied this question.