Efficient neutron capture therapy of glioblastoma with pteroyl-closo-dodecaborate-conjugated 4-(p-iodophenyl)butyric acid (PBC-IP).

Boron neutron capture therapy (BNCT) has been applied for clinical trials on glioblastoma patients since 1950s, however, the low survival rate under the treatments has hampered the widespread use of BNCT. In this study, we developed a novel boron agent, PBC-IP, which consists of three functional groups: FRα-targeting, 10B resource (twelve 10B atoms in the molecule), and albumin-binding moieties. PBC-IP was selectively taken up by glioma cell lines such as C6, F98, and U87MG cells and accumulated 10- to 20-fold higher than L-4­boronophenylalanine (BPA). PBC-IP administrated intravenously to the human glioblastoma (U87MG) xenograft model showed higher boron accumulation in tumors (29.8 µg [10B]/g at 6 h) than BPA (9.6 µg [10B]/g at 3 h) at a 25 mg [10B]/kg dose, effectively suppressing tumor growth after thermal neutron irradiation. PBC-IP administrated via convection-enhanced delivery (CED) accumulated in the F98 glioma orthotopic rat model, achieving 26.5 µg [10B]/g in tumors with tumor/normal (T/N) brain and tumor/blood (T/B) boron ratios of 37.8 and 94.6, respectively, 3 h after CED. Survival at 180 days after BNCT was 50% in the PBC-IP group and 70% in the combined BPA and PBC-IP groups, with no residual brain tumors.

Smart Nanofiber Mesh with Locally Sustained Drug Release Enabled Synergistic Combination Therapy for Glioblastoma.

This study aims to propose a new treatment model for glioblastoma (GBM). The combination of chemotherapy, molecular targeted therapy and radiotherapy has been achieved in a highly simultaneous manner through the application of a safe, non-toxic, locally sustained drug-releasing composite Nanofiber mesh (NFM). The NFM consisted of biodegradable poly(ε-caprolactone) with temozolomide (TMZ) and 17-allylamino-17-demethoxygeldanamycin (17AAG), which was used in radiation treatment. TMZ and 17AAG combination showed a synergistic cytotoxicity effect in the T98G cell model. TMZ and 17AAG induced a radiation-sensitization effect, respectively. The NFM containing 17AAG or TMZ, known as 17AAG-NFM and TMZ-NFM, enabled cumulative drug release of 34.1% and 39.7% within 35 days. Moreover, 17AAG+TMZ-NFM containing both drugs revealed a synergistic effect in relation to the NFM of a single agent. When combined with radiation, 17AAG+TMZ-NFM induced in an extremely powerful cytotoxic effect. These results confirmed the application of NFM can simultaneously allow multiple treatments to T98G cells. Each modality achieved a significant synergistic effect with the other, leading to a cascading amplification of the therapeutic effect. Due to the superior advantage of sustained drug release over a long period of time, NFM has the promise of clinically addressing the challenge of high recurrence of GBM post-operatively.

Strategies for Preclinical Studies Evaluating the Biological Effects of an Accelerator-Based Boron Neutron Capture Therapy System.

This review discusses the strategies of preclinical studies intended for accelerator-based (AB)-boron neutron capture therapy (BNCT) clinical trials, which were presented at the National Cancer Institute (NCI) Workshop on Neutron Capture Therapy held from April 20 to 22, 2022. Clinical studies of BNCT have been conducted worldwide using reactor neutron sources, with most targeting malignant brain tumors, melanoma, or head and neck cancer. Recently, small accelerator-based neutron sources that can be installed in hospitals have been developed. AB-BNCT clinical trials for recurrent malignant glioma, head and neck cancers, high-grade meningioma, melanoma, and angiosarcoma have all been conducted in Japan. The necessary methods, equipment, and facilities for preclinical studies to evaluate the biological effects of AB-BNCT systems in terms of safety and efficacy are described, with reference to two examples from Japan. The first is the National Cancer Center, which is equipped with a vertical downward neutron beam, and the other is the University of Tsukuba, which has a horizontal neutron beam. The preclinical studies discussed include cell-based assays to evaluate cytotoxicity and genotoxicity, in vivo cytotoxicity and efficacy of BNCT, and radioactivation measurements.

A Critical Review of Radiation Therapy: From Particle Beam Therapy (Proton, Carbon, and BNCT) to Beyond.

In this paper, we discuss the role of particle therapy-a novel radiation therapy (RT) that has shown rapid progress and widespread use in recent years-in multidisciplinary treatment. Three types of particle therapies are currently used for cancer treatment: proton beam therapy (PBT), carbon-ion beam therapy (CIBT), and boron neutron capture therapy (BNCT). PBT and CIBT have been reported to have excellent therapeutic results owing to the physical characteristics of their Bragg peaks. Variable drug therapies, such as chemotherapy, hormone therapy, and immunotherapy, are combined in various treatment strategies, and treatment effects have been improved. BNCT has a high dose concentration for cancer in terms of nuclear reactions with boron. BNCT is a next-generation RT that can achieve cancer cell-selective therapeutic effects, and its effectiveness strongly depends on the selective 10B accumulation in cancer cells by concomitant boron preparation. Therefore, drug delivery research, including nanoparticles, is highly desirable. In this review, we introduce both clinical and basic aspects of particle beam therapy from the perspective of multidisciplinary treatment, which is expected to expand further in the future.

Development of an Imaging Technique for Boron Neutron Capture Therapy.

The development of 4-10B-borono-2-18F-fluoro-L-phenylalanine (18FBPA) for use in positron emission tomography (PET) has contributed to the progress of boron neutron capture therapy (BNCT). 18FBPA has shown similar pharmacokinetics and distribution to 4-10B-borono-L-phenylalanine (BPA) under various conditions in many animal studies. 18FBPA PET is useful for treatment indication. A higher 18FBPA accumulation ratio of the tumor to the surrounding normal tissue (T/N ratio) indicates that a superior treatment effect is expected. In clinical settings, a T/N ratio of higher than 2.5 or 3 is often used for patient selection. Moreover, 18FBPA PET is useful for predicting the 10B concentration delivered to the tumor and surrounding normal tissues, enabling high-precision treatment planning. Precise dose prediction using 18FBPA PET data has greatly improved the treatment accuracy of BNCT. However, the methodology used for the data analysis of 18FBPA PET findings varies; thus, data should be evaluated using a consistent methodology so as to be more reliable. In addition to PET applications, the development of 18FBPA as a contrast agent for magnetic resonance imaging that combines gadolinium and 10B is also in progress.

A Diels-Alder polymer platform for thermally enhanced drug release toward efficient local cancer chemotherapy.

We reports a novel thermally enhanced drug release system synthesized via a dynamic Diels-Alder (DA) reaction to develop chemotherapy for pancreatic cancer. The anticancer prodrug was designed by tethering gemcitabine (GEM) to poly(furfuryl methacrylate) (PFMA) via N-(3-maleimidopropionyloxy)succinimide as a linker by DA reaction (PFMA-L-GEM). The conversion rate of the DA reaction was found to be approximately 60% at room temperature for 120 h. The reversible deconstruction of the DA covalent bond in retro Diels-Alder (rDA) reaction was confirmed by proton nuclear magnetic resonance, and the reaction was significantly accelerated at 90 °C. A PFMA-LGEM film containing magnetic nanoparticles (MNPs) was prepared for thermally enhanced release of the drug via the rDA reaction. Drug release was initiated by heating MNPs by alternating magnetic field. This enables local heating within the film above the rDA reaction temperature while maintaining a constant surrounding medium temperature. The MNPs/PFMA-L-GEM film decreased the viability of pancreatic cancer cells by 49% over 24 h. Our results suggest that DA/rDA-based thermally enhanced drug release systems can serve as a local drug release platform and deliver the target drug within locally heated tissue, thereby improving the therapeutic efficiency and overcoming the side effects of conventional drugs used to treat pancreatic cancer.

Lethal DNA Lesions Caused by Direct and Indirect Actions of X rays are Repaired via Different DSB Repair Pathways under Aerobic and Anoxic Conditions.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5-7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6-9% was about 20 µg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

Non-isotope enriched phenylboronic acid-decorated dual-functional nano-assembles for an actively targeting BNCT drug.

The feasibility of boron neutron capture therapy (BNCT) greatly depends on the selective accumulation of 10B in tumors. The p-boronophenylalanine-fructose (BPA-f) complex has been established as a conventional BNCT agent due to its preferential uptake into tumors, which is driven by amino acid transporters. However, the retention of BPA-f in tumors is highly limited because of an antiport mechanism, which is regulated by a gradient of amino acid concentration across the cancer cell membrane. Thus, to preserve a high 10B concentration in tumors, patients are inevitably subjected to a constant intravenous infusion. To this end, we employed a phenylboronic acid (PBA)-decorated polymeric nanoparticle (NanoPBA) as a sialic acid-targeting BNCT agent. In this manner, the PBA can exhibit dual functionalities, i.e., exhibiting a neutron capture capacity and hypersialyated cancer cell targeting effect. Our developed NanoPBA possesses a supramolecular structure composed of a core and shell comprised of poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) segments, respectively. The PBA moiety is installed at the PEG end, providing an unusually strong targeting effect, supposedly via multivalent binding onto the cancer cell membrane. As in BNCT, we verified the feasibility of NanoPBA against a B16 melanoma-bearing mouse model. By virtue of efficient tumor targeting, even at a 100-fold lower dose than BPA-f, the NanoPBA achieved a potent antitumor effect.

Boron analysis and imaging of cells with 2-hr BPA exposure by using micro-proton particle-induced gamma-ray emission (PIGE).

Particle-induced gamma-ray emission (PIGE), which measures prompt gamma rays at 428 keV from 10B (p, p'γ) 7Be, was used to confirm the boron distribution within 2hr-BPA-exposed cells. Distribution images of potassium, phosphate, and boron and the whole spectrum showed the ratios of boron counts to total (%) as follows: control group: 1.35 ± 0.073%; 2hr boron exposure group: 2.33 ± 0.35%; and boron exposure/wash group: 1.58 ± 0.095%. Micro-beam PIXE/PIGE can be a promising tool for visualization of intracellular Boron.

Chronoradiation Therapy for Prostate Cancer: Morning Proton Beam Therapy Ameliorates Worsening Lower Urinary Tract Symptoms.

BACKGROUND AND PURPOSE: Worsening lower urinary tract symptoms (LUTS) are a frequent adverse event following proton beam therapy (PBT) for localized prostate cancer. We investigated the differences in worsening LUTS among patients who received PBT at different times of day. PARTICIPANTS AND METHODS: Among 173 patients who underwent PBT for prostate cancer, 168 patients (median age 68.5 years) completed international prostate symptom score (IPSS) questionnaires and were included. Changes in the IPSS from baseline to the end of PBT were assessed by multiple linear regression analysis for age, National Comprehensive Cancer Network risk classification, androgen deprivation therapy, fractional PBT dose, clinical target volume, severity of IPSS, diabetes, LUTS medication use before PBT, anti-coagulant therapy and radiation time of day (morning (08:30-10:30), around noon (10:31-14:30), and late afternoon (14:31-16:30)). RESULTS: IPSS total score and IPSS-Quality of Life (QoL) score (12 patients were excluded due to missing IPSS-QoL score) increased from eight to 14.9 (p < 0.0001) and from two to four (p < 0.0001), respectively. Time of day (morning) was the only determinant for worsening LUTS (ß = -0.24, p < 0.01), voiding subscore (ß = -0.22, p < 0.05) and IPSS-QoL (ß = -0.27, p < 0.005), and was a determinant in item four (urgency) (ß = -0.28, p < 0.005) with age (ß = 0.19, p < 0.05). CONCLUSIONS: Morning PBT for localized prostate cancer significantly ameliorated worsening LUTS and improved QoL compared with treatment around noon or late afternoon. Chronoradiation therapy for localized prostate cancer may be effective and further research to elucidate the underlying mechanism is warranted.

Evaluation of the characteristics of the neutron beam of a linac-based neutron source for boron neutron capture therapy.

The linac-base neutron source "iBNCT" developed by the Tsukuba team has begun to generate a large intensity of neutrons. To confirm the applicability of the device to BNCT, several characteristic measurements have been implemented. In a water phantom experiment, when the accelerator was operated with an average current of 1.4 mA, the maximum thermal neutron flux was approximately 7.8 × 108 (n/cm2/s). Results demonstrate the stability of the linac over time, showing its promising potential for future patient treatment.

Folate-appended cyclodextrin improves the intratumoral accumulation of existing boron compounds.

In this study, the tumor accumulation and antitumor effect of folate-modified cyclodextrin (ND201) purified with folate receptor (FR) connotated with BSH were examined. ND201 and BSH were stably bound in blood, and the mixing ratio 1:1 was most efficient. ND-BSH showed higher boron concentration (38.5 ppm) than BSH alone (11.25 ppm). The maximum ND-BSH tumor/blood ratio was also markedly higher (6.58) than that of BSH alone (1.04). ND-BSH showed a significant antitumor effect compared with BSH after neutron irradiation.

[Two Cases of Penoscrotal Strangulation].

Case 1 (42-year-old man) : The patient was examined for penoscrotal swelling that had continued for 1 month. An annular erosive skin ulcer was observed at the penoscrotal base, with distal swelling. Asking the patient about the history of his condition was difficult due to a history of mental illness. We suspected his symptoms were due to an embedded foreign object. As computed tomography indicated the presence of a subcutaneous foreign object, surgery was performed to remove it. A rubber band was found wrapped twice around the area. After releasing the strangulation, penoscrotal swelling improved. Case 2 (72-year-old man) : Penoscrotal swelling appeared after having an automobile tow hook attached to the penoscrotal base for 2 weeks. The patient was examined at the emergency room because he could not remove it on his own. A rescue squad was called, and they cut the strangulating object with an electric saw. After releasing the strangulation, penoscrotal swelling improved. Although we experienced 2 cases of penoscrotal strangulation involving strangulating objects with different characteristics, improvement was achieved in both by releasing the strangulation. The cases of penoscrotal strangulation reported in Japan with known strangulation type are reviewed.

Functionalized mesoporous silica nanoparticles for innovative boron-neutron capture therapy of resistant cancers.

Treatment resistance, relapse and metastasis remain critical issues in some challenging cancers, such as chondrosarcomas. Boron-neutron capture therapy (BNCT) is a targeted radiation therapy modality that relies on the ability of boron atoms to capture low energy neutrons, yielding high linear energy transfer alpha particles. We have developed an innovative boron-delivery system for BNCT, composed of multifunctional fluorescent mesoporous silica nanoparticles (B-MSNs), grafted with an activatable cell penetrating peptide (ACPP) for improved penetration in tumors and with gadolinium for magnetic resonance imaging (MRI) in vivo. Chondrosarcoma cells were exposed in vitro to an epithermal neutron beam after B-MSNs administration. BNCT beam exposure successfully induced DNA damage and cell death, including in radio-resistant ALDH+ cancer stem cells (CSCs), suggesting that BNCT using this system might be a suitable treatment modality for chondrosarcoma or other hard-to-treat cancers.

Impact of RhoA overexpression on clinical outcomes in cervical squamous cell carcinoma treated with concurrent chemoradiotherapy.

The Rho-associated coiled-coil-containing protein kinase (ROCK) pathway is known to influence metastasis in several cancers; however, the impact of the pathway on clinical outcomes in patients undergoing radiotherapy remains unknown. In the present study, the expression of RhoA, RhoC, ROCK-1, ROCK-2 and p53 was immunohistochemically evaluated using biopsy specimens obtained from 49 patients with stage II-III cervical squamous cell carcinoma treated with concurrent chemoradiotherapy (CCRT). The relationship between the expression of these proteins and patient outcomes was investigated. RhoA overexpression was associated with significantly impaired disease-free survival and distant metastasis-free survival (P = 0.045 and P = 0.041, respectively) in stage III cancer patients. No differences in survival were observed based on the expression of the other proteins among stage III cancer patients. In stage II cancer patients, no differences in survival were noted based on the expression of any of the proteins. The expression of RhoA was able to successfully differentiate cervical cancer patients with distant metastasis after CCRT. This information may help stratify patients according to the risk of metastasis, thereby leading to the potential to provide individualized treatment.

ESTIMATION OF RBE VALUES FOR CARBON-ION BEAMS IN THE WIDE DOSE RANGE USING MULTICELLULAR SPHEROIDS.

Hypofractionated carbon-ion therapy has been applied to treatment of several tumours. In this case, relative biological effectiveness (RBE) at high dose region must be considered, however, the RBE calculated physically has been not verified biologically. In this study, spheroid technique was adopted to estimate RBE in wide dose range. Cells were irradiated with X-rays and heavy-ions with LET of 13, 35, 100 and 300 keV/µm with monolayer and spheroid condition. Surviving fractions in wide dose range (0-15 Gy) were obtained to combined monolayer with spheroid survival data. The linear-quadratic and multi-target single-hit equation fitted well in survival data at low dose, and high dose region, respectively. A multi-process equation showed best fitting for survival data in wide dose range. RBE values of heavy-ions could be estimated by combination of monolayer and spheroid data. The values converged at 1.1-1.4 and varied by LET values at high and low dose region, respectively.

DIFFERENCE IN DEGREE OF SUB-LETHAL DAMAGE RECOVERY BETWEEN CLINICAL PROTON BEAMS AND X-RAYS.

Fractionated proton beam radiotherapy is spreading worldwide these days. However, biological data of sub-lethal damage recovery (SLDR) after proton irradiation is not known yet. We here conducted split-dose experiments (20-360 min intervals) to clarify SLDR kinetics, and also compared the kinetics between cells with different repairability of DNA double-strand breaks. CHO and 51D1 cell lines but not V3 cell line showed significant SLDR, which reached plateau in 4-6 h. The recovery rates and recovery halftime of SLDR after X-rays were significantly higher and shorter than proton beams for CHO and 51D1 cells, respectively. Additionally, the frequency of remaining gamma-H2AX foci after two fractions was remarkably higher for X-rays than proton beams. These data suggest that there is a difference between proton beam and X-rays in SLDR and the retained DNA double-strand breaks after split-dose irradiation.

Antimetastatic Effects of Carbon-Ion Beams on Malignant Melanomas.

The goal of this work was to clarify the effect of carbon-ion beams on reduction of the metastatic potential of malignant melanoma using in vitro and in vivo techniques. We utilized a 290 MeV/u carbon beam with a 6-cm spread-out Bragg-peak (SOBP), 137Cs γ rays or 200 kVp X rays for irradiation, and in vitro murine melanoma B16/BL6 cells that were implanted into C57BL/6J mice. The metastatic abilities (migration, invasion and adhesion) were suppressed by carbon ion treatment at all doses that were tested, whereas invasion and migration tended to increase after X-ray irradiation at low dose. Biological effects of carbon ions increased with linear energy transfer (LET) for both cell killing and metastatic abilities, although the effects were more pronounced for migration and invasion. mRNA expression of E-cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expression of Mel-CAM and L1-CAM was upregulated after low-dose photon exposure, but decreased with dose, especially after carbon-ion treatment. Conversely, these molecules showed a reversal in expression changes, especially after low-dose photon exposure. Cell-cell adhesion may be an important contributor to the antimetastatic effect of carbon ion treatment. The number of lung metastases after local tumor irradiation significantly decreased with increased dose and LET, with carbon ions being more effective than γ rays. Integrating dose-response curves to examine the relationship between cell killing and lung metastasis clearly showed that carbon ions inhibit lung metastasis more efficiently than photons at the iso-effective level of cell killing. Thus, carbon ions were more effective than photon beams, not only at killing tumor cells, but also at inhibiting metastatic spread caused by tumor cells that survived irradiation.

[Sarcomatoid Urothelial Carcinoma of the Bladder Including an Osteosarcoma Element].

A 68-year-old Japanese man was referred to Tsukuba University Hospital for bladder cancer treatment. He had undergone a transurethral resection of a bladder tumor (TURBT) at a local hospital, but the pathological specimen did not contain muscle layer. Abdominal computed tomography (CT) and magnetic resonance imaging revealed a 3 cm non-papillary bladder tumor with muscle invasion, but there was no apparent calcification. The patient underwent re-TURBT at our hospital for diagnosis and staging. A non-papillary pedunculated tumor was identified in the bladder dome, and it contained a small papillary part. The non-papillary part was stony hard and difficult to cut with electrocautery, whereas the small papillary part was easily cut. Histologically, the non-papillary part was composed of sarcomatous elements including osteosarcoma, chondrosarcoma, and spindle cell sarcoma. The papillary part was composed of high-grade urothelial carcinoma and spindle cell sarcoma. Muscularis propria was not present in the specimen. Since the staging study with CT was negative for metastases, we performed a total cystectomy with an ileal conduit under the clinical diagnosis of muscle-invasive sarcomatoid urothelial carcinoma. The pathological findings were identical to those of the re-TURBT specimens, and our diagnosis was pTxN0 sarcomatoid urothelial carcinoma. The patient received adjuvant chemotherapy with two courses of gemcitabine and cisplatin. There was neither recurrence nor metastases during the 20-month follow-up. Reports of sarcomatoid urothelial carcinoma of the bladder with an osteosarcoma element are rare, and only eight other cases hane been reported in the Japanese literature.