Ultrasound-mediated blood‐brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model

Abstract

Trastuzumab has shown positive results in many patients with metastatic HER2-positive breast cancer, but it is less effective for controlling metastases in the CNS, which remains a site of relapse. The poor prognosis for patients with brain metastases is thought to be largely due to the presence of the blood‐brain barrier (BBB) that prevents delivery of most drugs to the CNS and to the heterogeneous and limited permeability of the blood-tumor barrier (BTB). Focused ultrasound (FUS) bursts combined with circulating microbubbles can temporarily permeabilize both the BBB and the BTB. This technique has been investigated as a potential noninvasive method for targeted drug delivery in the brain. Here, we investigated whether BBB/BTB permeabilization in the tumor and surrounding brain tissue induced by FUS and microbubbles can slow tumor growth and improve survival in a breast cancer brain metastases model. HER2/neu-positive human breast cancer cells (BT474) were inoculated in the brains of 41 nude (nu/nu) rats. Animals in the treatment group received six weekly treatments of BTB/BBB permeabilization under MRI guidance combined with IV administration of trastuzumab (2 mg/kg). Tumor growth and survival rates were monitored via MRI for seven weeks after sonication. Starting at week seven and continuing through the end of the study, the mean tumor volume of the FUS + trastuzumab group was significantly (P < 0.05) less than those of the three control groups (no treatment, FUS alone, trastuzumab alone). Furthermore, in four out of 10 rats treated with FUS + trastuzumab, the tumor appeared to be completely resolved in MRI, an outcome which was not observed in any of the 31 rats in three control groups. Trastuzumab improved median survival by 13% compared to the no treatment group, a difference which was significant (P = 0.044). Treatment with FUS + trastuzumab produced the most significant benefit compared to the no-treatment controls (P = 0.0084). More than half (6/10) animals survived at the study endpoint, leading to a median survival time greater than 83 days (at least 32% longer than the untreated control group). Overall, this work suggests that BBB/BTB permeabilization induced by FUS and microbubbles can improve outcomes in breast cancer brain metastases.

Introduction

Among patients with advanced metastatic breast cancer, 10–16% develop metastases in the central nervous system (CNS) [1], [2] with a true rate that may be higher based on autopsy data [3]. Breast cancers that overexpress human epidermal growth factor receptor 2 (HER2, HER2/neu, ErbB2, or c-erbB2) have been found to metastasize to the brain at higher frequencies than those that do not [2], [4], [5]. The rate for CNS metastases in breast cancer patients has appeared to increase in recent years, a change thought to reflect advances in detection and improved survival rates resulting from better management of systemic disease [1], [2], [6].

The prognosis for patients with advanced breast cancer who have CNS metastases is generally poor, with reported one- and five-year survival rates of 20% and 1.3%, respectively [1], [7]. The current standard of care for patients with CNS metastases is treatment with steroids and radiotherapy [8], [9], [10], [11], with surgery or stereotactic radiosurgery an option that can improve survival for patients with limited tumor burden [11], [12], [13], [14]. Chemotherapy has not generally been considered an effective option for patients with CNS metastases due to the presence of the blood‐brain barrier (BBB), a physical and functional barrier that restricts the delivery of most substances from the vasculature and hence to the tumor(s).

While the blood vessels in most brain tumors, including metastases, do not have an intact BBB and are somewhat permeable, infiltrating cancer cells at the tumor margins and small metastatic seeds may be protected by the BBB of the surrounding normal and intact tissue [15]. Furthermore, it is known that tumor vasculature permeability is heterogeneous, and there are additional barriers to drug delivery such as increased interstitial pressures [16]. Indeed, work in mice suggests that the blood-tumor barrier (BTB) is only partially compromised in breast cancer brain metastases, and that toxic concentrations of chemotherapy agents are only achieved in a small subset of metastases that are highly permeable [17].

These barriers, along with the increased aggressiveness of tumors that is thought to exist for breast cancers that metastasize to the CNS [18], pose a challenge for a growing number of patients. As systemic treatments have improved, the number of patients with CNS metastases has increased, and those who face recurrence after radiation therapy currently lack effective treatment options. Indeed, while patients with HER2-positive breast cancer with CNS metastases who receive trastuzumab do show a survival benefit [6], [19], this is thought to be largely due to control of systemic disease, with the brain remaining a sanctuary site [20]. Smaller molecule agents such as lapatinib may be more effectively delivered across the BTB and have shown promise in patients with breast cancer brain metastases [21]. However, with its molecular weight of 581 Da, lapatinib also will not effectively cross the intact BBB and may not reach all cancer cells.

To effectively treat metastases in the CNS, drugs will need to get past the BBB and overcome limitations imposed by the BTB. A number of strategies have been developed to get drugs past the BBB that range from invasive interventions such as convection-enhanced delivery [22] and minimally-invasive approaches such as transarterial injection of substances that transiently disrupt the BBB [23], [24], to the development of drugs or drug carriers that utilize endogenous transport mechanisms to ferry drugs across the BBB [25], [26].

Another approach to get drugs past the BBB that has been investigated in pre-clinical studies is the use of focused ultrasound (FUS), that when combined with circulating microbubbles (ultrasound contrast agents), can induce temporary BBB disruption and has been investigated as a method for targeted drug delivery in the brain [27]. Acoustic waves can be focused deeply into soft tissue, and the mechanical interaction between the ultrasound, the microbubbles, and the vasculature can then induce a transient disassembly of tight junction proteins [28], [29] and the stimulation of active transport [30] to allow for a temporary window for drug delivery.

This technique has several advantages over other approaches in that it is completely noninvasive, readily repeatable, targeted only to desired regions in the brain, and compatible with currently-approved drugs. Animal studies have shown that the disruption is not associated with significant tissue damage [31], [32], [33]. It has also been shown to enhance delivery of therapeutic agents [34], [35], including trastuzumab (Herceptin®) [36], and it can increase the permeability of the BTB [37], [38]. Recent work has shown that the method is capable of enhancing delivery of BCNU, improving survival a rat glioma model [39], and delivering antibodies that improved outcomes in a mouse Alzheimer's disease model [40]. Most studies have shown that the barrier is restored in a few hours [27], [28], [41].

In this study, we investigated whether temporary permeabilization induced by focused ultrasound (FUS) and microbubbles combined with trastuzumab can improve outcomes in a HER2/neu positive breast cancer brain metastases model. It was designed to demonstrate effective control of tumor progression and survival time with a course of multiple treatments that approximates the trastuzumab therapy regimen that a patient would receive. It was also chosen because, due to its large molecular weight (~ 150 kDa), we hypothesized that it would benefit significantly from enhanced local delivery in the brain.