Growth inhibition in a brain metastasis model by antibody delivery using focused ultrasound-mediated blood-brain barrier disruption

Abstract

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 brain. We investigated whether we could improve the response by temporary disruption of the BBB using focused ultrasound in combination with microbubbles. To study this, we inoculated 30 nude rats with HER2-positive cells derived from a brain metastasis of a breast cancer patient (MDA-MB-361). The animals were divided into three groups: a control-group that received no treatment; an antibody-only group that received six weekly treatments of trastuzumab and pertuzumab; and an ultrasound+antibody group that received trastuzumab and pertuzumab in combination with six weekly sessions of BBB disruption using focused ultrasound. In two animals, the leakiness of the tumors before disruption was evaluated using contrast-enhanced T1-weighted magnetic resonance imaging and found that the tumors were not leaky. The same technique was used to evaluate the effectiveness of BBB disruption, which was successful in all sessions. The tumor in the control animals grew exponentially with a growth constant of 0.042±0.011mm(3)/day. None of the antibody-only animals responded to the treatment and the growth constant was 0.033±0.009mm(3)/day during the treatment period. Four of the ten animals in the ultrasound+antibody-group showed a response to the treatment with an average growth constant of 0.010±0.007mm(3)/day, compared to a growth constant 0.043±0.013mm(3)/day for the six non-responders. After the treatment period, the tumors in all groups grew at similar rates. As the tumors were not leaky before BBB disruption and there were no responders in the antibody-only group, these results show that at least in some cases disruption of the BBB is necessary for a response to the antibodies in these brain metastases. Interestingly, only some of the rats responded to the treatment. We did not observe a difference in tumor volume at the start of the treatment, nor in HER2 expression or in contrast-enhancement on MRI 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. In conclusion, we demonstrate that BBB disruption using focused ultrasound in combination with antibody therapy can inhibit growth of breast cancer brain metastasis.

Keywords: Blood-brain barrier; Brain metastasis; Focused ultrasound; HER2-targeting antibodies; Targeted drug delivery.

Figure 1

Study design.

Figure 2

(A–B) Growth curves (relative cell number to Day0 at days 3 and 6) for fluorescence expressing BT-474-HB2-GFP (A) and MDA-MB-361-H2B-GFP (B) cells treated with trastuzumab (red), pertuzumab (green) or both antibodies (magenta) at a concentration of 22 μg/ml. Controls received no treatment (blue). (C–D) Endpoint measurement of growth inhibition relative to cells without treatment for BT-474 cells (C) and MDA-MB-361 (D) after six days of exposure to trastuzumab, pertuzumab or the combination at 22 μg/ml.

Figure 3

A) T1w image before contrast administration. The tumor is indicated with the red arrow. B) Before the sonications, no difference in tumor enhancement is observed after contrast administration (ΔSI=0.4%). C) After focused ultrasound-mediated blood-brain barrier disruption, the tumor enhances after contrast administration (ΔSI=30.1%)

Figure 4

Tumor volume measurements for each group from the start of the treatment period (day 35) till sacrifice. The measurements were obtained every other week. Four rats in the FUS+antibody group were classified as responder during the treatment period. After treatment, their growth curves follow the same pattern as the other groups.

Figure 5

Kaplan-Meier survival curves for the three treatment groups.

Figure 6

MRI images and histology of a control animal who did not receive treatment. A) Tumor on T2w imaging 5 weeks after implantation. B) Tumor on MR imaging when the animal reached the study endpoint. The tumor shows a heterogeneous appearance with cystic cavities. C) The hematoxylin & eosin-stained section corresponds well with MR-imaging and cysts are present. D) The HER2-stained section demonstrates that the complete tumor is HER2-expressing.

Figure 7

MRI images and histology of a FUS+antibody animal who was classified as a responder. A) Tumor on MR imaging 5 weeks after implantation. B) Tumor on MR imaging when the animal reached the study endpoint. C) The hematoxylin & eosin-stained section has, like the MR image, a heterogeneous appearance with cystic areas, which was observed also in the other animals at the study endpoint. D) HER2-stained section confirms that the tumor is HER2-expressing. The box indicates the location of the enlargements in E (H&E) and F (HER2).