HER2-targeting antibodies (i.e. trastuzumab and pertuzumab) prolong survival in HER2-positive breast cancer patients with extracranial metastases. However, the response of brain metastases to these drugs is poor and it is hypothesized that the blood-brain barrier (BBB) limits drug delivery to the brain¹. We aim to improve delivery by temporary disruption of the BBB using MR-guided focused ultrasound (MRgFUS) in combination with microbubbles. MR imaging is used to evaluate the treatment benefit of combining HER2-targeting antibodies with focused ultrasound-mediated BBB disruption in a breast cancer brain metastasis model.

Three groups of 10 nude rats were included: the control-group received no treatment; the antibody-only group was treated with trastuzumab and pertuzumab; and the FUS+antibody-group received trastuzumab and pertuzumab in combination with BBB disruption using MRgFUS. At week 0, HER2-positive human cancer cells (MDA-MB-361) were injected in the right brain hemisphere. Six weekly treatments started at week 5. The ultrasound treatments took place in a 7T MR-system (Biospec, Bruker) using a single-element, spherically-focused 690 kHz-transducer. Before and after the sonications, T2-weighted (T2w), T1w and T2*w images were obtained for tumor localization and targeting, confirmation of BBB disruption and evaluation of hemorrhages (see table). At the start of each sonication (duration 60s, 10-ms bursts, burst repetition frequency 1 Hz), the ultrasound contrast agent Optison (100 µl/kg) was injected. The complete tumor was treated in 4 to 14 sonications using peak negative pressures between 0.46 and 0.62 MPa.

After the sonications gadolinium (Magnevist) was injected to confirm BBB disruption. The difference in signal intensity change in pre- and post-contrast T1w images was determined between the tumor and contralateral brain region (=ΔSI%). In two animals tumor leakiness was studied before the tumors were sonicated and quantified in the same manner. Pre- and post-sonication T2*w images were qualitatively inspected for hypo-intense regions, which can indicate extravasated erythrocytes.

Every other week, high-resolution T2w imaging was performed to determine tumor volume. The volumes were fitted with: volume(t)=a*exp(r*t), in which r is the growth rate and t is the time in days. r was determined for the treatment period (week 5 to 11) and the follow-up period (week 11 till sacrifice). An animal was classified as ‘responder’ if r was lower than the mean r of the control animals minus two standard deviations. The animal was euthanized if its condition was poor or the tumor diameter exceeded 13 mm. Brains were stained for hematoxylin and eosin (H&E) and HER2.

BBB disruption was successful in all sessions with an average ΔSI% of 21.2% (range 4.5–77.6%). The mean ΔSI% of two tumors before BBB disruption during the six treatment weeks were 0.4% and 0.6%, indicating that the tumors were not leaky before disruption (Figure 1). In 20/60 FUS-sessions, regions were present that were clearly more hypo-intense on post- than on pre-sonication T2*w images, suggesting hemorrhages. In the remaining 67% of the sessions, no or a small difference in hypo-intensity was observed.

In the FUS+antibody-group, 4/10 animals were classified as responders during the treatment period with an average growth rate of 0.010±0.007, compared to 0.043±0.013 for the non-responders. There was no difference in the average ΔSI% of the responding rats (20.9%±16.1) and the non-responding rats (21.3%±10.4). None of the control or antibody-only animals were classified as responder. For the follow-up period, no differences in growth rates were present between the groups.

High-resolution T2w imaging showed that the tumor was homogenous in most animals till week 13-15, when cystic and necrotic areas started to develop. The tumors showed also a heterogeneous appearance on H&E-stained sections and the complete tumor was HER2-expressing in the examined brains.

In this study, we demonstrate that BBB disruption using MRgFUS in combination with antibody therapy can slow down the growth of breast cancer brain metastasis. As the tumors were not leaky before BBB disruption and there were no responders in the antibody-only group, the disruption of the BBB is necessary for drug delivery to these brain metastasis. Interestingly, only part of the rats responded to the treatment, the other animals had the same growth rate as the control-group. This is in line with a previous study², where antibody therapy was combined with FUS in a different model and only in part of the animals a response was observed. We did not observe a difference in tumor volume at the start of the treatment, in HER2 expression, or in contrast-enhancement on T1w images between the responders and non-responders to explain this. Better understanding of why certain animals respond is needed and will help in translating this technique to the clinic.