Development of a Glymphatic Pathway-based Rat Model for Cancer Metastasis from Brain to Lung

Background/Aim: Brain-to-lung metastasis is clinically rare but represents an underexplored route of cancer dissemination. Understanding this process may reveal novel mechanisms involving cerebrospinal fluid (CSF) and glymphatic clearance pathways. Existing preclinical models fail to replicate this directional spread or allow controlled investigation of central nervous system (CNS)-driven metastasis. This study aimed to develop a reproducible rat model to examine whether tumor cells introduced into the CNS can disseminate to peripheral organs, specifically the lungs. Materials and Methods: We established a brain-to-lung metastatic model in immunocompromised nude rats using intrathecal injection of A549-LUC human lung adenocarcinoma cells. This approach enabled precise tumor placement within the subarachnoid space for controlled modeling of metastatic progression. In vitro bioluminescence assays confirmed robust luciferase activity, peaking at 4 minutes post D-luciferin addition. In vivo imaging using the In Vivo Imaging System (IVIS) was employed to track tumor localization and dissemination over time. Results: IVIS imaging revealed early tumor localization in the CNS, followed by progressive and asymmetric spread to the lungs, with higher radiance in the right lung. Intracranial tumor detection was limited by poor signal penetration through the skull. Post-mortem hematoxylin and eosin staining confirmed tumor lesions in brain and lung tissues. Physiological monitoring showed initial weight gain followed by decline, and survival analysis indicated a median survival of 36 days, with complete mortality by day 40. Conclusion: This intrathecal model overcomes limitations of systemic injection techniques by enabling stepwise investigation of brain-to-lung metastasis. While immune interactions are restricted, this reproducible platform supports therapeutic testing – including ultrasound-enhanced glymphatic drug delivery – and provides a foundation for studying rare but clinically significant metastatic routes.