Abstract 5172: Advanced models of breast cancer brain metastasis and imaging protocols for preclinical efficacy studies

Abstract Introduction: Approximately 30% of breast cancer (BC) patients eventually develop metastases in distant organs and especially breast cancer brain metastases (BCBM) are associated with poor prognosis and high mortality. Translatable animal models mimicking and predicting clinical disease and outcomes are urgently needed to increase the success of novel anti-cancer therapies. In this study, we generated advanced orthotopic BCBM models established by three different inoculation methods. Furthermore, we optimized imaging protocols to monitor tumor growth, BBB permeability and efficacy in preclinical studies. Methods: BCBM was established by stereotactic inoculation of human BC cells lines (BT474 and MDA-MB-231.Luc2) and cells isolated from patient derived xenografts (PDX) (ST1339, ST3338, ST941, ST340, ST1616B and ST1360B). To mimic metastatic spread, BCBM was also established by intracardial (ICar) and intracarotid (ICt) inoculation (BT474 and MDA-MB-231.Luc2). Metastatic spread and tumor growth were monitored using T2-weigthed MRI and bioluminescence imaging while BBB permeability was evaluated in vivo by Gd-contrasted MRI. Transport of HER2 targeting trastuzumab across the BBB was confirmed by PET/MRI, resulting in a subsequent study evaluating efficacy of Trastuzumab emtansine (T-DM1) (ST1339 and ST3338). IHC and light sheet microscopy (LSM) were performed to validate in vivo findings of tumor characteristics including metastatic spread and BBB permeability. Results: BCBM were successfully established by stereotactic inoculation with high tumor take rates (>80%) and consistent tumor location and number (n=1 per animal). The BCBM presented with varying brain infiltration profiles and BBB permeability. 64Cu-trastuzumab uptake in animals bearing ST1339 tumors (HER2 3+) confirmed delivery across the BBB. Animals treated with T-DM1 experienced tumor growth delay with ST1339 having the greatest response. ICar inoculation gave rise to metastatic spread, but <25% of animals inoculated developed BCBM prompting the need for a more targeted delivery of BC cells to the brain. ICt inoculation resulted exclusively in the formation of BCBM with a take rate of >65%. MRI imaging revealed 1-7 metastases per animal with intrasubject and intersubject variation in BBB permeability. Ex vivo analysis validated the in vivo imaging of metastases and showed multiple metastases of varying sizes and BBB permeability. Conclusion: We’ve successfully developed multiple complex orthotopic models for BCBM mimicking different aspects of BCBM. ICar and ICt models especially resemble the pathophysiology of BCBM to a large extent with extravasation and micro seeding in the brain. Together with the developed imaging protocols allowing longitudinal evaluation of compound delivery, tumor growth and BBB permeability this serves as a great platform for preclinical studies with novel anti-cancer agents. Citation Format: Sigrid Cold, Maria Z. Alfsen, Mark U. Juul, Camilla Malec, Camilla Christensen, Michael J. Wick, Lene Jessen, Trine B. Engel, Lotte K. Kristensen, Andreas Kjaer, Carsten H. Nielsen. Advanced models of breast cancer brain metastasis and imaging protocols for preclinical efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5172.