Nanoluciferase-CD63 labeling reveals extracellular vesicle kinetics in a mouse intraductal model of ductal carcinoma in situ.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a non-invasive precursor to breast cancer that increases lifetime risk for invasive disease and, in rare cases, causes distant metastases without local recurrence. Tumor-derived small extracellular vesicles (sEVs), or exosomes, mediate tumor progression, systemic signaling, and metastasis; however, the timing of release and biodistribution of sEVs from early preinvasive lesions remains unclear. METHODS: Here, we investigated the release kinetics and systemic dissemination of sEVs secreted by DCIS cells confined to the mammary ducts using an orthotopic mouse mammary intraductal xenograft model with MCF10DCIS cells engineered to express NanoLuc (NLuc)-tagged CD63, an sEV marker. Tumor growth was monitored using bioluminescent imaging (BLI) in vivo and ex vivo; DCIS outgrowth and focal invasion were confirmed by fluorescent immunohistochemistry; and NLuc activity was assayed in tissue lysates. Next-generation RNA sequencing of brain tissue was used to evaluate gene expression changes at 28 days post-implantation (dpi). RESULTS: Longitudinal BLI revealed CD63-NLuc signals in mammary glands (P < 0.05), and distant organs, including spleen (P < 0.05), liver (P < 0.05), lungs (P < 0.01) and brain (P < 0.01) by day 14 followed by detection in heart tissue (P < 0.001) and in the circulation by day 21, preceding histological evidence of invasion. Further, CD63-NLuc activity was significantly and positively correlated with BLI levels in mammary glands and plasma (P < 0.05). Immune, metabolic, and stress response pathways were downregulated in brain tissue at 28 dpi compared to 3 dpi. CONCLUSIONS: These findings demonstrate systemic dissemination of tumor-derived sEVs during DCIS progression, suggesting roles in early microenvironmental conditioning and metastatic preconditioning prior to overt invasion.