Ultrasound-mediated blood‐brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model
Graphical abstract
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.
Section snippets
Animals and cell culture
All experiments were done in accordance with procedures approved by the Harvard Medical School Institutional Animal Care and Use Committee. A total of 41 male nude (nu/nu) rats (Charles River Laboratories, Boston, MA) weighing 180–210 g (six-week old) were used. Animals were anesthetized during all procedures with either 2% isoflurane (for estrogen pellet implantation) administered through a nose cone, or with ketamine (90 mg/kg) and xylazine (10 mg/kg) administered via IP injection (all other
Results
BBB and BTB permeabilization induced by FUS and microbubbles was confirmed by a signal intensity increase measured in contrast enhanced T1-weighted images obtained after the sonications. Fig. 3 shows example contrast-enhanced images before (a) and after (b) the sonications. Due to the enhanced delivery of the MR contrast agent across these barriers, higher MR signal intensities were observed throughout the vascular portion of the tumor and a narrow rim of tissue surrounding it.
The tumor volume
Discussion
Since it was approved in 1998, trastuzumab, a monoclonal antibody that has high affinity to the growth-promoting protein HER2/neu, has been used in treating metastatic breast cancers [45], [46], [47]. It binds to the extracellular segment of the HER2/neu receptor and suppresses dimerization of the HER2/neu receptor, leading to a disruption in signaling for cell proliferation. In patients with HER2-positive metastatic breast cancer, trastuzumab is used as a first-line treatment in combination
Conclusions
This study demonstrates for the first time that combining weekly trastuzumab therapy with BBB/BTB permeabilization induced by FUS and microbubbles can improve outcomes in HER2-postive breast tumors inoculated in the brain. A significant reduction in mean tumor volume and survival were found after six weekly FUS + trastuzumab treatments, and no sequelae were found as the result of the procedure. Moreover, after treatment with ultrasound and trastuzumab, four of the ten tumors disappeared
Acknowledgment
This research was supported by NIH grants P41RR019703, P41EB015898, a grant from CIMIT (Center for Integration of Medicine and Innovative Technology), and a gift from Betty Brudnick.
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