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Background: Incidence of patients having brain metastasis of breast cancer has increased due to enhanced treatment for peripheral disease. Breast cancer brain metastasis is often fatal and occurs in 15-20% of patients diagnosed with stage IV breast cancer. Traditional chemotherapy delivery to the brain is limited by the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Low intensity focused ultrasound (LIFU) is a unique technique recently investigated to non-invasively disrupt these barriers and aid neuro-modulation. Herein, we demonstrate the use of a clinical LIFU device to modulate blood-tumor permeability and deliver combined chemotherapy in a novel preclinical model of brain metastases of breast cancer. Methods: Brain colonizing MDA-MB-231Br cells transfected with luciferase were injected into athymic Nu/Nu female mice by intracardiac injection into the left ventricle (per mouse 175,000 cells). Brain metastasis was allowed to develop, and tumor burden was monitored by bioluminescent imaging IVIS Spectrum CT (Perkin Elmer). Mice were randomized into sham or single treatment or combined treatment groups on Day 21 (Doxil nanoparticles 5.6 mg/kg or Paclitaxel 10mg/kg). Mice receiving LIFU were placed on a specially designed animal restraint with the skull partially immersed in degassed water. T1 weighted MRI was used to identify and localize sonication spots within the cortex, and mice were sonicated using 0.4uL/kg Definity© at 0.3 cavitation dose for 60sec. Treatment with LIFU was continued once weekly for three consecutive weeks. Survival and tumor burden was recorded until mice developed neurological symptoms. LIFU induced blood-brain barrier breakdown in healthy mice was also analyzed by in-situ brain perfusion with 14CSucrose, 3HGlucose, and 3HIvermectin to monitor BBB integrity and transporter function. Results: Mice receiving LIFU+paclitaxel+Doxil-NP showed significantly lower tumor burden (peak area 635.9) and higher median survival of 60 days as compared to vehicle (29d) and LIFU+vehicle (32d) groups. Analysis of BBB permeability in healthy mice demonstrated significantly higher passive permeability marker accumulation than non-sonicated contralateral hemisphere. Conclusion: Timing the combined chemotherapy with LIFU induced windows of maximal BBB opening resulted in slower tumor progression and increased survival in a preclinical model of breast cancer brain metastasis. Future directions aim to combine these results and evaluate underlying mechanisms of enhanced permeability post-LIFU. Citation Format: Tasneem A. Arsiwala, Kathryn E. Blethen, Samuel A. Sprowls, Ross A. Fladeland, Brooke Keilkowski, Morgan Glass, Trenton Pritt, Peng Wang, Ali Rezai, Paul R. Lockman. Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3461.

Background Brain metastasis is a devastating stage of cancer progression, occurring in ~30% of metastatic breast cancer patients. Two-year survival rates for these patients is low, and most typically survive less than one year. Treatments for these women are limited by the blood-brain barrier, but include cytotoxic chemotherapy, surgical resection, and radiation therapy (whole-brain radiotherapy or stereotactic radiosurgery). Radiotherapy is considered to be capable of inducing disruption of the blood-brain barrier and eliciting an abscopal response to extracranial tumors. Methods A combination of ionization chamber and Gafchromic film dosimetry was used to commission and determine dose outputs for our experimental design. Dose deposition in-vivo was verified by immunohistochemistry. To evaluate the effects of ionizing radiation at the normal blood-brain barrier and the blood-tumor barrier, athymic nude and FVB mice were used. Athymic nude mice were injected with MDA-MB-231Br cells. Lesions were allowed to develop for ~28 days. Mice were then irradiated at the prescribed dose. Prior to tissue collection, mice were injected with Texas red, followed by a vascular washout with physiological buffer. Fluorescence in normal and diseased brain was quantified by fluorescent microscopy. Results Using a 10mmx10mm collimator, determined to have adequate field homogeneity as determined by Gafchromic film analysis, we were able to successfully treat a single hemisphere in mice. The blood-brain-barrier remained undisrupted in athymic nude mice at doses up to 12Gy compared to untreated brain and radiation naïve controls. Immune competent FVB mice treated with radiation showed significant blood-brain barrier disruption at a dose of 12Gy only. The blood-tumor barrier showed significant disruption at 24hrs following radiation treatment (6 or 12Gy). Conclusion Our study demonstrated that radiation therapy disrupts the blood-tumor barrier, but fails to disrupt the normal blood-brain barrier in athymic nude mice. However, in FVB immune competent mice, the blood-brain barrier was disrupted at a dose of 12Gy, suggesting an abscopal-like response impacts extent of barrier leakage.