A neutron beam for boron neutron capture therapy (BNCT) of deep-seated tumours is designed to maintain a high flux of epithermal neutrons, while keeping the thermal and fast neutron component as low as possible. These neutrons (thermal and fast) have a high relative biological effectiveness in comparison with high energy photon beams used for conventional X-ray radiotherapy. In the past, neutrons for the purpose of BNCT were generated using nuclear reactors. However, there are various challenges that arise when installing a reactor in a hospital environment. From 2006, the Kyoto University Research Reactor Institute, in collaboration with Sumitomo Heavy Industries, began the development of an accelerator-based neutron source for clinical BNCT in a bid to overcome the shortcomings of a nuclear reactor-based neutron source. Following installation and beam performance testing, in vitro studies were performed to assess the biological effect of the neutron beam. Four different cell lines were prepared and irradiated using the accelerator-based neutron source. Following neutron and gamma ray irradiation, the survival curve for each cell line was calculated. The biological end point to determine the relative biological effectiveness (RBE) was set to 10% cell survival, and the D-10 for each cell line was determined. The RBE of the accelerator-based neutron beam was evaluated to be 2.62.

Previous studies have demonstrated that angiogenesis inhibitors can enhance tumor inhibitory effects of chemo- and radiotherapy via their action on tumor vessels. Here, we studied the effect of the angiogenesis inhibitor, bevacizumab (Avastin), on boron distribution in a murine tumor model. The human head and neck squamous cell carcinoma cell line was used for inoculation into mice. Boron-10 concentrations in tissues were measured by prompt γ-ray spectrometry (PGA). Hoechst 33342 perfusion and p-boronophenylalanine (BPA) distribution were determined by immunofluorescence staining. Our results revealed enhanced tumor blood perfusion and BPA accumulation in tumors after Avastin treatment, suggesting that combination of angiogenesis inhibition with treatment with boron compound administration may improve the efficacy of boron neutron capture therapy (BNCT) by modifying tumor vessels. In addition, our results also demonstrated the usefulness of immunofluorescence staining for investigating boron compound distribution at the cellular level.

Purpose: To evaluate the usefulness of combined treatment with continuous administration of a hypoxic cytotoxin, tirapazamine (TPZ), and mild temperature hyperthermia (MTH) in γ-ray irradiation in terms of local tumour response and lung metastatic potential, referring to the response of intratumour quiescent (Q) cells. Materials and methods: B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumour-bearing mice then received γ-ray irradiation after a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ, either with or without MTH. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (= P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Results: Continuous administration elevated the sensitivity of both the total and Q cells, especially the total cells. MTH raised the sensitivity of Q cells more remarkably in both single and continuous administrations, probably because of more exposure to TPZ in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. With or without irradiation, TPZ, especially administered continuously and combined with MTH, decreased the number of lung metastases. Conclusion: The combination of continuous long-term administration of TPZ and MTH in γ-ray irradiation was thought to be promising because of its potential to enhance local tumour response and repress lung metastatic potential.

Objective The efficiency of boron neutron capture therapy(BNCT)depends on the sufficient accumulation of boron-10 in cancer cells.Currently,the methods for valuating boron distribution are effective to determine the boron-10 average concentration in tumor tissues,but they cannot demonstrate boron distribution in detail.Previous studies demonstrated mild temperature hyperthermia(MTH)can contribute the response of radiotherapy.Here,we tried to clarify the boron-10 compound distribution in cell level and the effect of MTH on boron distribution in detail using immunofluorescence staining.Methods The human lung carcinoma cell line A549 and human head and neck squamous cell carcinoma cell line SAS were cultured at subconfluent density.The cells were incubated with BPA(p-boronophenylalanine)at different 10B concentrations(A549:1.625,3.25,6.5,13,26,52 μg/ml;SAS:0.65,1.3,2.6,3.9,7.8,11.7 μg/ml)for 1 hour.In addition,A549 cells were treated with heat shock at 40 ℃ for 1 hour.They were then processed for immunofluorescence staining with anti-BPA monoclonal antibody and Alexa Fluor labeling antibody.The fluorescence intensity of BPA staining was measured by Adobe Photoshop Software.Results The merge of BPA and DAPI showed that BPA was accumulated mostly in the surrounding of nucleus of cells.The intensity of BPA staining increased with the increasing concentration of 10B.In addition,the peak of intensity histogram of 26 μg/ml 10B staining shifted to the higher intensity area compared with that of 13 μg/ml 10B staining.After treatment of MTH,the BPA distribution was not increased significantly.Conclusions In this study,the BPA accumulation in cells was investigated by an imaging method in vitro.The present results suggested the effect of MTH on boron distribution may be induced by improving the extracellular microenvironment including tumor vessels normalization.

<正>从1895年伦琴发现射线,1898年居里夫妇发现镭,1950年代开始钴-60治疗以来,到现在放射线的医疗应用已经有近100年的历史。其原理是尽可能在正常组织损伤小的情况下,利用射线的能量对肿瘤细胞进行杀伤,达到抑制肿瘤的目的。由于对患者造成的负担小,受年龄以及身体状况影响小等优点,放疗已发展成包括手术,抗癌药物治疗在内的三大肿瘤治疗手段之一,甚至放疗已经成为了一些肿瘤的首选治疗手段[1]。

The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, i.e., 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in CHO, but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action.

Secondary cerebral hypoxia has recently been shown to play an important role in the outcome of patients suffering from traumatic brain injury (TBI). However, the precise mechanisms underlying secondary cerebral hypoxia are complex and interrelated. In this study, we investigate the effect of hypoxia within a rat model of trauma-induced late cerebral cortex injury. Using the hypoxia marker pimonidazole, we verified and isolated areas of the cortex that had suffered hypoxic damage. Using subsequent reverse-transcriptase PCR analyses, we found that the expressions of both transforming growth factor beta1 (TGF-beta1) and hypoxia-inducible factor-1alpha (HIF-1alpha) increased significantly under hypoxic conditions induced by TBI compared with uninjured control animals. In addition, the maximum mRNA expression of TGF-beta1 and HIF-1alpha was found at 3 days and 12h after TBI, respectively. Our data suggest that secondary cerebral hypoxia injury involves various cytokines including TGF-beta1 and HIF-1alpha. Furthermore, upon immunohistochemical analysis, both TGF-beta1 and HIF-1alpha expression were almost localized in the same types of cells by using immunohistochemical study. These results may have important implications in the understanding of trauma-induced secondary cerebral hypoxia injury.

Labeling of all proliferating cells in C57BL/6J mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2'-deoxyuridine (BrdU). Tumors were irradiated with γ rays at a high dose rate or a reduced dose rate at 1 h after the administration of pimonidazole. Assessment of the responses of quiescent and total ( =  proliferating + quiescent) cell populations were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. The total cell fraction that was not labeled with pimonidazole showed significantly enhanced radiosensitivity. However, a significantly greater decrease in radiosensitivity, evaluated using a delayed assay or a decrease in radiation dose rate, was observed in the quiescent cell compared with the total cell population. Overall, the quiescent cell population showed significantly greater radioresistance and capacity to recover from radiation-induced damage than the total tumor cell population. Thus we believe that the subfraction of the quiescent tumor cell population that was not labeled with pimonidazole and that was probably oxygenated is a critical target in the control of solid tumors.

The usefulness of hexamethylenetetramine as an adjuvant to radiation and cisplatin in the treatment of solid tumors and its dependency on the p53 status of tumor cells were examined. Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53), or with neo vector as a control (SAS/neo), were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. The tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. Then, they received hexamethylenetetramine (HMTA), intraperitoneally or continuously, combined with or without gamma-ray irradiation or cisplatin treatment. Immediately after treatment following HMTA, the response of quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (= P + Q) tumor cells was determined from the BrdU non-treated tumors. A higher toxicity of HMTA to Q cells than total cells, especially in SAS/neo, was made less clear by continuous administration. There was no apparent difference in the radio- and cisplatin-sensitivity enhancing effects by HMTA combination between SAS/neo and SAS/mp53 tumors, with a slightly greater effect in SAS/mp53. In both SAS/neo and SAS/mp53 tumors, continuous HMTA administration produced higher radio- and cisplatin-sensitivity enhancing effects than intraperitoneal single administration. Therefore, the use of HMTA as an adjuvant to radiation or cisplatin might be promising in curing solid tumors with large fraction of hypoxic cells and also with frequent loss-of-function in p53.

The aim of this study was to clarify the effect of manipulating intratumor hypoxia on radiosensitivity under reduced dose-rate (RDR) irradiation.

To elucidate the effect of tumor oxygenation status on recovery from damage following γ-ray or accelerated carbon ion irradiation in vivo, including in quiescent (Q) cells.

The effective treatments of radiation-induced intestinal injury are currently unavailable. Developing new treatments for radiation-induced intestinal injury is thus important. The present study investigated whether transplantation of mesenchymal stem cells (MSCs) is able to prevent radiation-induced intestinal injury. Intestines of female nude mice (ICR nu/nu) were irradiated at a single dose of 30 Gy. Transplantation of male MSCs (C57BL/6) was then immediately performed into the walls of irradiated intestine by direct injection for the irradiation + MSCs group. Mice were weighed daily and survival was recorded for 13 days after irradiation. From 13 to 27 days after irradiation, intestines of mice were obtained in order to assay histological changes by staining with hematoxylin-eosin and Masson trichrome. Mean body weight of the irradiation + MSC group was significantly higher than that of the irradiation-only group from 8 days after irradiation. In addition, survival rates were significantly higher in the irradiation + MSC group than for the irradiation-only group from 5 days after irradiation. Histological observation revealed that intestines of irradiation + MSC-transplanted mice were thick in the submucosal and muscle layers, and had almost fully recovered from radiation-induced intestinal injury at day 27. Specifically, ulcerated areas in the intestines of the irradiation + MSC-transplanted mice were smaller by 13 days after irradiation and were fewer in numbers at 27 days when compared with the irradiation-only group. Our results suggest that transplanted MSCs may play an important role in preventing radiation-induced injury and may offer a novel method to treat radiation-induced intestinal injury.

Purpose: Evidence from ill Vivo Studies Suggests there are enhanced radiation effects in abscopal regions after local head gamma ray irradiation. Splenocyte apoptosis and T lymphocyte micronuclei were induced at higher rates than what would be estimated given the dose at a shielded, distant position. In addition, we evaluated the radio-protective effects of ascorbic acid, acting as a radical scavenger on enhanced radiation effects in the shielded spleen following local head irradiation.Methods and materials: The heads of C3H mice were exposed to gamma-rays (10-20 Gy), while the other parts of the body were shielded with a 5 cm-thick lead block. The effective dose for the spleen was calculated at 1.0-2.0 Gy. Splenocytes were isolated 24 h after cranial irradiation and their apoptosis was measured with an Elisa kit (Roche). The induction of T lymphocyte micronuclei was studied using the cytokinesis-block micronucleus assay. The ascorbic acid glucoside, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), was orally administered to mice I h before whole body irradiation. The radio protective effects of AA-2G were estimated by comparing the induction of splenocyte damage (by apoptosis) and micronucleus induction.Results: The splenocyte damage, as measured by the above two methods, was more excessive than what would be expected given exposure to 1.0-2.0Gy of radiation. Our results suggest that the effects were enhanced in a distant, non-irradiated organ after localized irradiation. plasma ascorbic acid concentrations were increased 8-10x over control. Treatment with ascorbic acid slightly protected mouse splenocytes from the induction of apoptosis by the enhanced effects of radiation in the abscopal region. However, ascorbic acid significantly inhibited micronucleus induction in splenic T lymphocytes following local head irradiation.Conclusions: Our results suggest that ascorbic acid effectively scavenged radiation-induced radicals and protected against the enhanced effects of radiation in an abscopal region after local head gamma ray irradiation. (C) 2009 Elsevier B.V. All rights reserved.

Previous studies have demonstrated that X-ray irradiation affects angiogenesis in tumors. Here, we studied the effects of gamma-ray irradiation on boron-10 compound accumulation in a murine tumor model. The mouse squamous cell carcinoma was irradiated with gamma-ray before BSH ((10)B-enriched borocaptate sodium) administration. Then, the boron-10 concentrations in tumor and normal muscle tissues were measured by prompt gamma-ray spectrometry (PGA). A tumor blood flow assay was performed, and cell killing effects of neutron irradiation with various combinations of BSH and gamma-rays were also examined. BSH concentrations of tumor tissues were 16.1 +/- 0.6 microg/g, 16.7 +/- 0.5 microg/g and 17.8 +/- 0.5 microg/g at 72 hours after gamma-ray irradiation at doses of 5, 10, and 20 Gy, compared with 13.1 +/- 0.5 microg/g in unirradiated tumor tissues. The enhancing inhibition of colony formation by neutron irradiation with BSH was also found after gamma-ray irradiation. In addition, increasing Hoechst 33342 perfusion was also observed. In this study, we demonstrated that gamma-ray irradiation enhances BSH accumulation in tumors. The present results suggest that the enhancement of (10)B concentration that occurs after gamma-ray irradiation may be due to the changes in the extracellular microenvironment, including in tumor vessels, induced by gamma-ray irradiation.

Purpose: To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects.Methods and Materials: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity A phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells.Results: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BIP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells.Conclusions: Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation. (C) 2009 Elsevier Inc.

PURPOSE:To evaluate the effect of manipulating intratumor hypoxia during radiotherapy on lung metastasis, referring to its effect on the sensitivity of quiescent tumor cells.MATERIALS AND METHODS:B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-ray irradiation following loading with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The sensitivity of quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. That of the total (=P + Q) tumor cell population was determined from BrdU non-treated tumors. In other tumor-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated.RESULTS:In the total cells, a more marked enhancement in sensitivity was observed with nicotinamide than MTH. In Q cells, MTH combination induced a more marked enhancement than nicotinamide. Both nicotinamide and MTH reduced the size of the hypoxic fraction in the two cell populations, especially nicotinamide in the total cells and MTH in Q cells. Without gamma-ray irradiation, nicotinamide loading tended to decrease the number of lung metastases. With gamma-ray irradiation, nicotinamide loading and MTH, especially the former, reduced the number of metastases more than gamma-ray irradiation only.CONCLUSION:Hypoxia manipulation in solid tumors has the potential to influence lung metastases. Notably, acute hypoxia-releasing nicotinamide may be promising for reducing the number of lung metastases.

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They received hexamethylenetetramine (HMTA) either once intraperitoneally or continuously subcutaneously together with chemotherapy using intraperitoneally administered free doxorubicin (DXR) or intravenously injected pegylated liposomal doxorubicin (PLD). One hour after the free DXR loading or 24 h after the PLD loading, the response of intratumor quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cell population was determined from the tumors not treated with BrdU. Encapsulation of DXR into pegylated liposomes significantly enhanced cytotoxicity, especially in Q cells. HMTA, especially when administered continuously, efficiently increased the sensitivity to DXR, particularly in Q cells. The increase in sensitivity on the continuous rather than single administration of HMTA was a little clearer in the total cell population than in Q cells. DXR's encapsulation into pegylated liposomes and combination with HMTA, particularly when administered continuously, apparently reduced the difference in sensitivity to free DXR between the total and Q cell populations. In terms of the tumor cell-killing effect as a whole, including Q cells, the encapsulation of DXR into pegylated liposomes and combination with HMTA, particularly through continuous administration, are very promising, taking into account that HMTA has been used clinically.

We have been developed an epithermal neutron source for boron neutron capture therapy(BNCT), consisting of a cyclotron accelerator that can provide a ∼ 1 mA, 30 MeV proton beam, a neutron production beryllium target and the moderator that can reduce the energy of fast neutrons to an effective energy range. In order to validate the simulations, we measured the depth distribution of the thermal neutron flux in water phantom located at the treatment position. The measured results were compared with the simulations using the MCNPX Monte Carlo code. The good agreement between the simulations and measurements was shown. The thermal neutron flux with the proton current of 430 μA was 7.4 × 108 (neutrons cm−2 s−1) at the depth of around 20 mm in the water phantom. This intensity corresponds to the neutron source of Kyoto University Research Reactor (KUR), at which 275 clinical trials of BNCT have been performed. We experimentally confirmed that our cyclotron based neutron source can use for clinical trials of BNCT.

Background and Purpose; (10)B deriving from (10)B-para-boronophenylalanine (BPA) and (10)B-borocaptate sodium (BSH) have been detected in blood samples of patients undergoing boron neutron capture therapy (BNCT) using prompt gamma ray spectrometer or Inductively Coupled Plasma (ICP) method, respectively. However, the concentration of each compound cannot be ascertained because boron atoms in both molecules are the target in these assays. Here, we propose a simple and rapid method to measure only BPA by detecting fluorescence based on the characteristics of phenylalanine. Material and Methods; (10)B concentrations of blood samples from human or mice were estimated by the fluorescence intensities at 275 nm of a BPA excited by light of wavelength 257 nm using a fluorescence spectrophotometer. Results; The relationship between fluorescence to increased BPA concentration showed a positive linear correlation. Moreover, we established an adequate condition for BPA measurement in blood samples containing BPA, and the estimated (10)B concentrations of blood samples derived from BPA treated mice were similar between the values obtained by our method and those by ICP method. Conclusion; This new assay will be useful to estimate BPA concentration in blood samples obtained from patients undergoing BNCT especially in a combination use of BSH and BPA.

Background and purpose To confirm the feasibility of accelerator-based BNCT (AB-BNCT) for treatment of multiple liver tumors and malignant pleural mesothelioma (MPM), we compared dose distribution and irradiation time between AB-BNCT and reactor-based BNCT (RB-BNCT). Material and methods We constructed treatment plans for AB-BNCT and RB-BNCT of four multiple liver tumors and six MPM. The neutron beam data on RB-BNCT were those from the research reactor at Kyoto University Research Reactor Institute (KURRI). The irradiation time and dose–volume histogram data were assessed for each BNCT system. Results In BNCT for multiple liver tumors, when the 5 Gy-Eq dose was delivered as the mean dose to the healthy liver tissues, the mean dose delivered to the liver tumors by AB-BNCT and RB-BNCT was 68.1 and 65.1 Gy-Eq, respectively. In BNCT for MPM, when the mean lung dose to the normal ipsilateral lung was 5 Gy-Eq, the mean dose delivered to the MPM tumor by AB-BNCT and RB-BNCT was 20.2 and 19.9 Gy-Eq, respectively. Dose distribution analysis revealed that AB-BNCT is superior to RB-BNCT for treatment of deep-seated tumors. Conclusions The feasibility of the AB-BNCT system constructed at our institute was confirmed from a clinical viewpoint in BNCT for multiple liver tumors and MPM.