Boron neutron capture therapy for glioblastoma ...

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Feng-Yi Yang*1‡, Yi-Wei Chen2‡, Fong-In Chou3, Sang-Hue Yen1,2, Yi-Li Lin1 & Tai-Tong Wong4 1 Department of Biomedical Imaging & Radiological Sciences, National Yang-Ming University, No 155, Sec 2, Linong Street, Taipei, Taiwan 2 Cancer Center, Taipei Veterans General Hospital, Taipei, Taiwan 3 Nuclear Science & Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan 4 Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan *Author for correspondence: Tel.: +886 2 2826 7281 n Fax: +886 2 2820 1095 n [email protected] ‡ Authors contributed equally

Aim: This study investigated whether the efficacy of boron neutron capture thera py wa s en ha nced by mea n s of intravenou s a d m in i stration of boronophenylalanine (BPA) with blood–brain barrier disruption induced by focused ultrasound (FUS). Materials & methods: BPA was administered, followed by pulsed FUS, and the boron concentration in the treated brains was quantified by inductively coupled plasma mass spectroscopy. Growth of the firefly luciferaselabeled glioma cells was monitored through noninvasive biophotonic imaging. Finally, the brain tissue was histologically examined after sacrifice. Results: Compared with the nonsonicated tumor group, animals treated with an injection of 500 mg/kg of BPA followed by FUS exhibited not only significantly increased accumulation of the drug at the sonicated tumor site, but also a significantly elevated tumor-to-normal brain drug ratio (p 98%) BPA was converted to a more soluble fructose complex [18] . BNCT was initiated 8 days after stereotactic implantation of glioma cells into mice, randomized into two groups of eight animals each. Animals received an intravenous bolus injection of BPA (500 mg/kg) with or without FUS-induced BBB-D. All irradiations were performed at the 10

Future Oncol. (2012) 8(10)

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Boron neutron capture therapy for glioblastoma multiforme

Function generator

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Amplifier Power meter Transducer Cone Water PE membrane

Ear bar Rat

Stereotaxic apparatus

Figure 2. Schematic diagrams showing the sonication and radiation arrangement. (A) Pulsed focused ultrasound system for blood–brain barrier disruption. (B) Experimental setup for thermal neutron irradiation. PE: Polyurethane.

Tsing Hua Open-Pool Reactor (National Tsing Hua University, Taiwan) 1 h after administration of the compound. The head was adjusted perpendicular to the neutron beam, which was collimated using an acrylic holder as a template (Figure 2B) . The neutron fluence was determined by multiplying the reactor power level in megawatts by the duration of irradiation in Contralateral brain lpsilateral brain Tumor 70

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minutes to yield a dose in megawatt-minute. Animals were irradiated for 17 min at a reactor power of 1.2 MW to give a total exposure of 20.4 MW-min. The control group of glioma-bearing mice (n = 6) received no treatment. Upon completion of BNCT, the animals were returned to Yang-Ming University for observation.

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Figure 3. Concentration of boron for glioblastoma multiforme-bearing mice. (A) Boron concentration in tumor with or without FUS-induced blood–brain barrier disruption, for the ipsilateral brain and contralateral brain. (B) Tumor-to-ipsilateral brain ratio of boron concentration derived from (A). *p < 0.01; **p < 0.001. BPA: Boronophenylalanine; FUS: Focused ultrasound.


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Yang, Chen, Chou, Yen, Lin & Wong

Biophotonic tumor imaging

The mice were injected at the dose of 4.29 mg per mouse of freshly prepared luciferin substrate (D-lucferin, Biosynth International, Inc., AG, Staad, Switzerland) suspended in PBS. After anesthetic induction with isoflurane (1.5 l/min oxygen in 4% isoflurane), mice were imaged using the Xenogen IVIS imaging system (Xenogen, CA, USA) 10 min after the intraperitoneal injection of luciferin, with a 1 min acquisition time in small-bin mode. Luciferase activity was quantified using Living Image Software from Xenogen for a region of interest that encompassed the head of the mouse after anesthesia of the mouse and administration of luciferin substrate. Tumor size data acquired at each time point were normalized to the size obtained on day 4 after tumor implantation. MRI

Animals were anesthetized with a mixture of oxygen and vaporized isoflurane throughout the scanning process. Tumor progression was assessed by MRI with a 3T scanner (Trio® with Tim®, Siemens MAGNETOM, Germany) on days 12 and 20 after tumor implantation. A loop coil (Loop Flex Coil, Siemens MAGNETOM; 4 cm in diameter) was exploited for radiofrequency reception. The following parameters for T2-weighted images were used in this

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study: repetition time/echo time = 3500/75 ms; matrix = 125 × 256; field of view = 25 × 43 mm and section thickness = 1 mm. Histopathology

Two tumor-bearing mice from the BPA group with or without sonication were sacrificed 20 days after tumor implantation for histological observations. The mice were perfused with saline and 10% neutral buffered formalin. The brains were removed and embedded in paraffin and then serially sectioned into 30-µm-thick slices. The slices were stained with hematoxylin and eosin to confirm tumor progression. TUNEL analysis was performed on floating brain sections modified by an apoptosis detection system, the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Basel, Switzerland). Briefly, sections were rinsed with PBS, followed by a 2-min incubation of sections with 0.1% Triton X-100 on ice. Sections were then rinsed twice in PBS and reacted with 50 µl of the TUNEL reaction mixture (Roche) for 18 h at 4°C. The sections were washed three-times in PBS, mounted on glass slides using FluoreGuard® mounting medium (ScyTek Laboratories, UT, USA) and stored in the dark and cold until imaged. Photomicrographs of hematoxylin and eosinstained and TUNEL-labeled tissue sections were obtained with a Mirax Scan digital microscope

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Figure 4. Biophotonic imaging of longitudinal brain tumor monitoring from days 4 to 20 after implantation. Firefly luciferase-expressing GBM 8401 cells were implanted into the left forebrain of NOD-scid mice that had received no treatment (control) or treatment with boron neutron capture therapy by means of intravenous injection of BPA with or without sonication on day 8 after tumor cell implantation. BPA: Boronophenylalanine; FUS: Focused ultrasound.

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Figure 5. Therapeutic efficacy of the tumors with boron neutron capture therapy with or without sonication. (A) Analysis of increases in tumor size (relative to day 8) is based on data obtained from bioluminescence images in Figure 4. (B) Tumor size change (relative to control tumor) of tumor-bearing mice treated by BPA with or without sonication on day 20. Compared with the control tumor mice, ‘*’ and ‘‡’ denote a significant difference for BPA with and without sonication, respectively. *,‡p < 0.05; **p < 0.01; ‡ ‡ ‡p < 0.001. BPA: Boronophenylalanine; FUS: Focused ultrasound.

slide scanner (Carl Zeiss, Mirax 3D Histech, Kft., Hungary) with Plan-Apochromatic 20/0.8 objective. The serial histology images were illustrated by Panoramic Viewer software (Carl Zeiss, Mirax 3D Histech, Kft.). Statistical analysis

Results were typically expressed as means ± standard error of the mean. Statistical analysis was performed with a t test. Statistical significance was defined as a p-value ≤ 0.05. Results

The concentrations of boron per unit mass (in µg/g of tissue) in contralateral brain, ipsilateral brain and tumor with or without sonication on day 8 after tumor implantation is shown in Figur e 3A . Not only was the accumulation of boron in the control tumor significantly greater than that of the adjacent normal brain region, but we found that the boron concentration was clearly greater at the sonicated tumor (56.9 ± 3.2 µg/g) than in the control tumor (29.5 ± 2.4 µg/g). Pulsed FUS exposure administered after BPA introduction significantly increased the boron concentration in the tumor. F igur e 3B reveals that the tumor-to-ipsilateral brain ratio derived from the concentration of boron in the tumors caused an approximately 82% increase in the target tumors (5.40 ± 0.81) compared with the control tumors (2.96 ± 0.51). The results have shown that the intravenous injection with FUS-induced BBB-D attains future science group

the enhanced tumor uptake of boron and the tumor-to-normal tissues ratio. The control tumors and the effect of tumors treated on day 8 by BNCT with or without sonication on tumor progression were monitored by bioluminescence imaging over time (Figure 4) . Tumor cells spread rapidly in the untreated control mice ( Figure 4 , top). Tumor treatment by BNCT with or without sonication slowed the growth of the tumors. The pattern of tumor progression in tumor-bearing mice treated by BNCT with sonication showed a slight improvement in antitumor effect compared with that in the BNCT alone mice. Treatment of the tumors by BNCT with or without sonication significantly slowed the growth of the tumor on days 12 and 16 after implantation (Figure 5A) . A significant improvement was observed in the antitumor efficacy in mice treated with BNCT plus sonication compared with the control mice on day 20 after implantation. However, BNCT alone did not show significant difference in limiting tumor growth at this time point compared with the control. Figure 5B indicates that the mean relative reduction of tumor size in the BNCT group with sonication was higher than that of BNCT alone group on day 20 after implantation. Thus, FUS sonication enhanced the treatment of BNCT alone. MRI was carried out in a subset of animals to monitor tumor growth noninvasively as a function of time for the tumor-bearing mice treated with BNCT with or without sonication www.futuremedicine.com

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( F igur e 6 , top). Moreover, the corresponding hematoxylin and eosin- and TUNEL-stained sections were observed for histopathologic examination and apoptotic evaluation on day 20 after implantation (Figure 6) . Based on the histological analysis of the sonicated and nonsonicated tumors, local displacement and extravasation of red blood cells were seen in the sonicated tumor tissues in and around the focal region (Figure 7) . In addition, cell apoptosis was clearly found in the sonicated tumor compared with the nonsonicated tumor treated by BNCT alone and the control tumor. Discussion

TUNEL stain

H & E stain

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BNCT is a binary system for tumor treatment. A relatively high amount of boron must be delivered to individual brain tumor cells while maintaining low levels in the normal tissues and the tumor is then irradiated to a sufficient level of thermal neutron. In the present study, we have shown that the intravenous injection of BPA followed by pulsed FUS-induced BBB-D resulted in an increase in tumor boron uptake compared with values obtained from those with intravenous injection without BBB-D.

It is known that adequate delivery of therapeutic agents to brain tumors is impeded by the BBB and blood–tumor barrier [19–22] . Previous work demonstrated that convection-enhanced delivery resulted in higher tumor and lower normal tissue boron concentrations, and improved tumor-tobrain ratio [23] . Although convection-enhanced delivery is a minimally traumatic approach, catheters do cause tissue damage along the insertion track. Intracarotid administration of hypertonic mannitol has been used to disrupt the BBB in animals [19,24,25] to enhance drug delivery into the brain. A previous study has reported that intracarotid injection with mannitol-induced was the optimal delivery to attain the highest tumor uptake of boron and the tumor-to-normal tissue ratio [9] . However, it was recommended that BPA be administered intravenously as this route was safer than the intracarotid injection. It should be noted that BBB-D followed by intravenous injection of the drug did not result in any increase in tumor uptake compared to intravenous injection of the drug without BBB-D [18] . Another study has demonstrated that pulsed FUS-induced BBB-D after Evans blue administration resulted in almost a fivefold higher level

Figure 6. Histopathology. Histological evaluations in the brains of (A) control mice and cured mice by boronophenylalanine (C) with and (B) without sonication. At 20 days after tumor implantation, three mice from the control, boronophenylalanine alone and the boronophenylalanine with sonication group were imaged by T2-weighted MRI, and then sacrificed for H & E and TUNEL staining. Scale bars: 2000 μm. H & E: Hematoxylin and eosin.

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TUNEL stain

H & E stain


Figure 7. Histological observation. For high magnification from the square regions of Figure 6 in the brains of (A) control mice and cured mice by boronophenylalanine (B) without and (C) with sonication. Local displacement and extravasation of red blood cells were seen in the sonicated tumor tissues (C), the damaged vessel was indicated by the arrow in the hematoxylin and eosin staining. There were significant apoptotic cells in the sonicated tumor (C) compared with the nonsonicated tumor (B) and control tumor (A). Scale bars: 200 μm. H & E: Hematoxylin and eosin.

of Evans blue extravasation in target liver cancer compared with contralateral controls. However, this effect was significantly lower while Evans blue was administered after sonication [26] . These results are consistent with our published data [27] . Therefore, this study was performed using pulsed FUS exposure after BPA intravenous injection to elevate concentration of boron in the brain tumor tissue as much as possible. The success of BNCT is dependent upon the accumulation of sufficient concentrations of boron and the thermal neutrons in the tumor cells. In addition, prolonged infusion was found to be beneficial for the efficacy of BNCT. It is suggested that a 6-h infusion of BPA must be used in future clinical applications of BNCT for GBM [28] . The reason for the poor treatment efficacy shown in this study is explained by the therapeutic window of boron concentration limitation of bolus injections of BPA. Recently, it has been reported that the uptake of 4-borono-2-18F-fluorol-phenylalanine in tumors and derived tumorto-ipsilateral brain ratio reached a maximum at about 1 h after intravenous administration of 4-borono-2-18F-fluoro-l-phenylalanine [9] . Thus, irradiations were performed by thermal neutrons 1 h after administration of the boron. The time interval required to achieve an optimum tumorto-ipsilateral brain ratio provides a partial explanation for the failure to demonstrate that pulsed FUS-induced BBB-D significantly improves the future science group

antitumor effects of BNCT in our study. The pharmacokinetics of intravenous injection of BPA followed by FUS-induced BBB-D is needed to suggest the optimal window for effective BNCT. Histopathologic examination (Figures 6 & 7) of the brain tumors treated by BNCT with sonication revealed obvious damaged tumor tissues compared with the BNCT alone and the control tumor. The changes that were seen within the sonicated region of the tumor most likely represented a reactive response following destruction of the tumor by BNCT with sonication that led to hemorrhage, cavity formation and apoptotic cells (Figure 7C , bottom). Pulsed-FUS exposure produced acoustic radiation forces in a focal region, thereby inducing local displacements in the sonicated tumor tissues (Figure 7C , top) [29] . Another study has shown that sonication induces widening of the intercellular gaps [30] . Pulsed-FUS exposure also reduces a high interstitial fluid pressure in tumors. The opening up of the intercellular spaces in the tumors by sonication seemed to enhance boron transport in the interstitium and, simultaneously, lowered interstitial fluid pressure for improved extravasation. The results of Figure 3 show that the concentration of boron in the tumor was enhanced when the sonication was applied. One of the problems associated with the use of mannitol-induced for boron delivery enhancement is the increased normal cell death that results from disruption of critical regions within a normal

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brain by drugs. However, pulsed FUS-induced BBB-D provides a powerful approach to locally increase boron uptake in the tumor site while sparing the normal brain tissue. It is particularly useful for higher-molecular-weight delivery agents such as boron-containing liposomes or antibodies that normally do not traverse the BBB [31] . Furthermore, the ligand-conjugated liposomal technology assisted by pulsed FUS to concentrate high-dose boron in brain tumors improved the efficacy of BNCT for GBM. The integration of targeted nano-boron and pulsed FUS technology opens windows to achieve enhanced selective drug delivery and treatment efficacy in the BNCT of high-grade gliomas and other tumor treatments that will have minimal toxic side effects. Conclusion

This study has demonstrated for the first time, to our knowledge, that BPA administration assisted by pulsed-FUS is able to selectively increase the accumulation of boron in brain tumors and elevate the tumor-to-ipsilateral brain ratio. This finding will have considerable impact in future BNCT research. Further studies are currently in progress in our group to optimize the prolonged infusion of BPA and to enhance the efficacy of BNCT with BBB-D induced by sonication.

Financial & competing interests disclosure

This study was supported by grants from the National S cience C ouncil of Taiwan ( No NSC 101‑2320‑B‑010‑036‑MY3, NSC 100‑2321‑ B‑010‑010 and NSC 99‑2321‑B‑010‑017), Cheng Hsin General Hospital Foundation (no. 101F195CY18 and 100F117CY25), Veterans General Hospitals University System of Taiwan Joint Research Program (#VGHUST100‑G1‑3‑3), Yen Tjing Ling Medical Foundation (grant CI‑100‑17), Department of Health of Taiwan (DOH101‑TD‑PB‑111‑TM012 and DOH101‑TD‑C‑111‑007). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human sub‑ jects, informed consent has been obtained from the participants involved.

Executive summary For boron neutron capture therapy to be successful, a sufficient amount of 10B must be delivered to the tumor for thermal neutron radiation. Our data demonstrated that focused ultrasound (FUS) not only significantly increased accumulation of the drug at the sonicated tumor site, but also significantly elevated the tumor-to-normal brain drug ratio (p