Reelin-LRP8 signaling mediates brain metastasis of breast cancer cells via abluminal migration

Brain metastasis (BM) remains a significant challenge in breast cancer (BC) management. While conventional metastatic routes involve hematogenous dissemination, emerging evidence suggests that BC cells can also migrate along the abluminal surface of blood vessels, bypassing the blood-brain barrier (BBB). To investigate this phenomenon, we established a zebrafish xenograft model utilizing GFP-labeled MDA-MB-231 cells, allowing real-time observation of BC cell migration along the posterior cerebral veins. Our findings revealed that LRP8, a apolipoprotein E receptor, is upregulated in BC patients with brain metastases. Functional studies demonstrated that LRP8 knockdown significantly inhibited proliferation, migration, and invasion of triple-negative breast cancer (TNBC) cells both in vitro and in vivo. Mechanistically, LRP8 promotes the activation of CDC42, enhancing filopodia formation and cell motility, a process influenced by the neuronal extracellular matrix protein, Reelin. Furthermore, we demonstrate the therapeutic potential of MEN 10207, a neurokinin-2 receptor antagonist, in inhibiting TNBC cell migration and suppressing BM formation in both zebrafish and mouse models. These findings provide novel insights into the mechanisms underlying extravascular brain metastasis in BC, highlighting the Reelin-LRP8-CDC42 axis as a potential therapeutic target for this devastating complication. Following the transplantation of MDA-MB-231 (BM0) cells into zebrafish, tumor progression was assessed through daily fluorescence imaging. Brain metastatic lesions were subsequently examined, excised and minced using the M-shot fluorescence stereomicroscope (Guangzhou, China) under sterile conditions. Then the GFP-labeled tumor cells were isolated and transferred into DMEM supplemented with antibiotic-antimycotic solution (Beyotime, C0224) and 15% fetal bovine serum, denoting this cell line as first-generation of brain metastatic (BM1) cells. Following dissociation and subsequent expansion in culture, BM1 underwent additional five rounds of transplantation and isolation, denoting this cell line as BM6 cells. RNA from MDA-MB-231 cells transfected with sh-ctrl and sh-LRP8-2# plasmids, BM0 cells and BM6 cells were isolated using TRIZOL. RNA-Seq was performed in the Gene Denovo Biotechnology (Guangzhou, China). Differentially expressed genes (DEGs) were detected using DESeq2. Reactome enrichment analysis and GO enrichment analysis were performed to elucidate the functional implications of DEGs. DEGs with a false discovery rate (FDR) of less than 0.05 were selected for enrichment analysis. The protein-protein interaction (PPI) network involving CDC42 and various guanine nucleotide exchange factors (GEFs) was constructed using the STRING database (https://cn.string-db.org/).