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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 c 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 pro- nounced for migration and invasion. mRNA expression of E- cadherin was significantly downregulated with low-dose photon exposures, but increased with dose or LET. Expres- sion 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 c 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.

The aim of this study was to measure the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for survival of cells within implanted solid tumors following exposure to 290 MeV/nucleon carbon-ion beams or X-rays. Squamous cell carcinoma cells (SCCVII) were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (SOBP, at 46 and 80 keV/micron) or X-rays was delivered to 5-mm or less diameter tumors. We defined three different oxygen statuses of the irradiated cells. Hypoxic and normoxic conditions in tumors were produced by clamping or not clamping the leg to avoid blood flow. Furthermore, single-cell suspensions were prepared from non-irradiated tumors and directly used to determine the radiation response of aerobic cells. Single-cell suspensions (aerobic condition) were fully air-saturated. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assays to obtain cell survival curves. The RBE and OER values for cell survival were evaluated by D10 values (the dose required to reduce surviving fraction to 10%). The RBE values increased with increasing LET in SOBP beams. The maximum RBE values in three different oxygen conditions; hypoxic tumor, normoxic tumor and aerobic cells, were 2.16, 1.76 and 1.66 at an LET of 80 keV/micron, respectively. After X-ray irradiation the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.87 +/- 0.13 and 2.52 +/- 0.11, respectively. The aerobic cell samples that were sensitive to X-rays under normoxic conditions may contain a hypoxic fraction. In fact we found that the normoxic tumors (< 5 mm diameter) contained approximately 10% hypoxic cells using the paired survival curve method with three or more independent experiments. The OER values of carbon-ion irradiated samples were small in comparison to that of X-ray irradiated samples. However, no significant changes of the OER at proximal and distal positions within the SOBP carbon-ion beams were observed in both pairs. To conclude, we found that the RBE values for cell survival increased with increasing LET and that the OER values changed little with increasing LET within the SOBP carbon-ion beams.

We studied DSB induction and rejoining in the clamped and non-clamped transplanted tumors in mice leg after 80 keV/µm carbon ions and X-rays. The yields of DSB in the tumors were analyzed by a static-field gel electrophoresis. The OER of DSB after X-rays was 1.68 +/- 0.31, and this value was not changed by one hour rejoining time (1.40 +/- 0.26). These damages in oxygenated conditions were rejoined 60 to 70% within one hour in situ. On the other hand, no difference between X-rays and carbon ions was found for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after carbon ions were similar to that after X-rays, and the calculated RBEs of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not the types of radiation quality.

Purpose: Respiration-gated irradiation for a moving target requires a longer time to deliver single fraction in proton radiotherapy (PRT). Ultrahigh dose rate (UDR) proton beam, which is 10-100 times higher than that is used in current clinical practice, has been investigated to deliver daily dose in single breath hold duration. The purpose of this study is to investigate the survival curve and relative biological effectiveness (RBE) of such an ultrahigh dose rate proton beam and their linear energy transfer (LET) dependence. Methods: HSG cells were irradiated by a spatially and temporally uniform proton beam at two different dose rates: 8 Gy/min (CDR, clinical dose rate) and 325 Gy/min (UDR, ultrahigh dose rate) at the Bragg peak and 1.75 (CDR) and 114 Gy/min (UDR) at the plateau. To study LET dependence, the cells were positioned at the Bragg peak, where the absorbed dose-averaged LET was 3.19 keV/um, and at the plateau, where it was 0.56 keV/um. After the cell exposure and colony assay, the measured data were fitted by the linear quadratic (LQ) model and the survival curves and RBE at 10% survival were compared. Results: No significant difference was observed in the survival curves between the two proton dose rates. The ratio of the RBE for CDR/UDR was 0.98+/-0.04 at the Bragg peak and 0.96+/-0.06 at the plateau. On the other hand, Bragg peak/plateau RBE ratio was 1.15+/-0.05 for UDR and 1.18+/-0.07 for CDR. Conclusions: Present RBE can be consistently used in treatment planning of PRT using ultrahigh dose rate radiation. Because a significant increase in RBE toward the Bragg peak was observed for both UDR and CDR, further evaluation of RBE enhancement toward the Bragg peak and beyond is required.

PURPOSE: The aim of this study was to clarify the effect of manipulating intratumor hypoxia on radiosensitivity under reduced dose-rate (RDR) irradiation. MATERIALS AND METHODS: Tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-rays or accelerated carbon-ion beams at high dose-rate (HDR) or RDR with or without tumor clamping to induce hypoxia. Some mice without clamping received nicotinamide, an acute hypoxia-releasing agent or misonidazole, a hypoxic cell radio-sensitizer before irradiation. The responses of quiescent (Q) and total (= P + Q) cells were assessed by the micronucleus frequency using immunofluorescence staining for BrdU. RESULTS: The clearer decrease in radiosensitivity in Q than total cells after RDR gamma-ray irradiation was suppressed with carbon-ion beams, especially with a higher linear energy transfer value. Repressing the decrease in the radiosensitivity under RDR irradiation through keeping tumors hypoxic during irradiation and enhancing the decrease in the radiosensitivity by nicotinamide were clearer with gamma-rays and in total cells than with carbon-ion beams and in Q cells, respectively. Inhibiting the decrease in the radiosensitivity by misonidazole was clearer with gamma-rays and in Q cells than with carbon-ion beams and in total cells, respectively. CONCLUSION: Manipulating hypoxia during RDR as well as HDR irradiation influences tumor radiosensitivity, especially with gamma-rays.

We aimed to determine the effect of carbon ion irradiation on the uptake of N-benzyl-N-11C-methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([11C]DAC), a positron emission tomography (PET) ligand for the peripheral-type benzodiazepine receptor (PBR), in tumor cells and tumor-bearing mice. Spontaneous murine fibrosarcoma (NFSa) cells were implanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with 290 MeV/u carbon ions was delivered to the 7- to 8-mm tumors In vitro uptake of [11C]DAC was measured in single NFSa cells isolated from NFSa-bearing mice after irradiation. In vivo biodistribution of [11C]DAC in NFSa-bearing mice was determined by small animal PET scanning and dissection. In vitro autoradiography was performed using tumor sections prepared from mice after PET scanning. In vitro and in vivo uptake of [11C]DAC in single NFSa cells and NFSa-bearing mice was significantly reduced by carbon ion irradiation. The decrease in [11C]DAC uptake in the tumor sections was mainly due to the change in PBR expression. In conclusion, [11C]DAC PET responded to the change in PBR expression in tumors caused by carbon ion irradiation in this study. Thus, [11C]DAC is a promising predictor for evaluating the effect of carbon ion radiotherapy.

To compare the biological effectiveness of 290 MeV/amu carbon-ion beams in Chiba, Japan and in Darmstadt, Germany, given that different methods for beam delivery are used for each.

Purpose: To clarify the radiosensitivity of intratumor quiescent cells in vivo to accelerated carbon ion beams and reactor neutron beams. Methods and Materials: Squamous cell carcinoma VII tumor-bearing mice were continuously given 5-bromo-20-deoxyuridine to label all intratumor proliferating cells. Next, they received accelerated carbon ion or g-ray high-dose-rate (HDR) or reduced-dose-rate (RDR) irradiation. Other tumor-bearing mice received reactor thermal or epithermal neutrons with RDR irradiation. Immediately after HDR and RDR irradiation or 12 h after HDR irradiation, the response of quiescent cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for 5-bromo-20-deoxyuridine. The response of the total (proliferating plus quiescent) tumor cells was determined from the 5-bromo-20-deoxyuridine nontreated tumors. Results: The difference in radiosensitivity between the total and quiescent cell populations after g-ray irradiation was markedly reduced with reactor neutron beams or accelerated carbon ion beams, especially with a greater linear energy transfer (LET) value. Clearer repair in quiescent cells than in total cells through delayed assay or a decrease in the dose rate with g-ray irradiation was efficiently inhibited with carbon ion beams, especially with a greater LET. With RDR irradiation, the radiosensitivity to accelerated carbon ion beams with a greater LET was almost similar to that to reactor thermal and epithermal neutron beams. Conclusion: In terms of tumor cell-killing effect as a whole, including quiescent cells, accelerated carbon ion beams, especially with greater LET values, are very useful for suppressing the dependency on the heterogeneity within solid tumors, as well as depositing the radiation dose precisely.

Background: The present retrospective cohort study was performed to determine the efficacy of contact-mode 1064 nm neodymium-yttrium-aluminum-garnet (Nd:YAG) laser laser for keloids and hypertrophic scars. The indication and limitations of this modality are discussed. Methods: The cohort consisted of 102 consecutive Japanese patients (23 males and 79 females) with keloids and hypertrophic scars for more than 1 year. They were treated every 3–4 weeks for 1 year with a long-pulsed 1064 nm Nd:YAG laser (Cutera, Brisbane, Calif.) in contact mode. Thirty-eight patients had hypertrophic scars and 64 had keloids. The scars were evaluated before the treatment commenced and 1 month after the last session by using the Japan Scar Workshop Scar Scale 2011. Recurrence was assessed at 6 months after the termination of treatment. Results: The average total Japan Scar Workshop score of the keloid and hypertrophic scar region groups dropped significantly after 1 year of treatment compared with before treatment (all P < 0.05). None of the hypertrophic scars or keloids deteriorated. However, 3 of the 34 anterior chest keloids (8.8%) did not respond. The following recurrence rates were observed 6 months after stopping laser treatment: 1 of the abdomen hypertrophic scars (4%), 18 of the anterior chest keloids (52.9%), 5 of the upper arm keloids (35.7%), and 4 of the scapula keloids (25%). Conclusions: Hypertrophic scars responded significantly better to 1064 nm Nd:YAG laser treatment than keloids. However, keloid recurrence occurred when there was remaining redness and induration, even if only a small part of the scar was affected.

A radiobiological method was developed to measure the initial growth of clonogenic tumor cells metastasizing to lung. Thorax of mice was externally irradiated by gamma rays after an intravenous transplantation of syngeneic fibrosarcoma cells. Lung colonies developed 11 days after irradiation were counted and provided surviving fractions. Survival curves moved downward when time interval between transplantation and radiation delayed from 1 to 21 hr, but shifted upward at 48 hr or later. Survival ratios at given doses and the extrapolation number of survival curves fitted to multi-target model were calculated, and plotted against time after the intravenous transplantation. Doubling times of 13.3 and 13.1 hr were obtained by use of the survival ratio and of the extrapolation number, respectively. This method is useful to measure growth dynamics of clonogenic tumor cells at the site of metastasized organ.

The biological effectiveness of carbon ions relative to γ rays (RBE) was compared between the tumor growth delay and an early skin reaction of syngeneic mice. The RBE was larger for a tumor than skin when irradiated with large doses of high-LET (linear energy transfer) carbon ions. The intra-track damage (a term of a linear quadratic model) of a tumor and skin increased equally with an increase of the LET, while the inter-track damage (β term) of skin alone increased with the LET. These data provide evidence that high-LET radiotherapy could achieve therapeutic gain by minimizing the difference in response to fractionated irradiation between the tumor and normal tissue.

We performed dynamic MRI to reveal the characteristic gadopentetate dimeglumine (Gd-DTPA) uptake in carbon-ion irradiated tumor and compare it with photon irradiation. Fibrosarcomas in C3H mice legs were irradiated with either 16 Gy of carbon ions (74 keV/mm) or an equivalent dose (30 Gy) of Cs-137 gamma-rays. Dynamic MRI was performed 1 or 6 days after irradiation when the tumors showed an initial growth delay or incipient regrowth, respectively. The enhancement pattern was visualized by mapping the maximum enhanced time (Tmax), relative signal intensity maximum (SImax), and time delay of starting enhancement (Td). Significantly larger Tmax and Td values were observed in the tumors 1 day after carbon-ion irradiation than in the nonradiated tumors (No-R) and tumors 1 day after gamma-ray irradiation. Among the selected pixels in the tumors 6 days after carbon irradiation, 77% had Tmax values of less than 120 sec, significantly more than in the No-R group. The Tmax maps for the tumors irradiated with gamma-rays showed a similar tendency to the carbon-irradiated ones, and only a significant difference was obtained between tumors 1 and 6 days after irradiation. Tmax and Td in the carbon-ion irradiated tumors were different from those in the gamma-ray-irradiated tumors. These treatment-specific kinetics may be useful in predicting the therapeutic efficacy of carbon-ion treatment.

It is well established that the RBE (relative biological effectiveness) for cell killing depends on LET (linear energy transfer), and that a maximum RBE is observed at ~150 keV/mm. However, the therapeutic gain depends on the ratio of the RBEs for the effects on the cancer cell population and the effects on normal tissues. The RBE of a given radiation quality depends on LET but also on dose, biological system and effect, and irradiation conditions. There is no data available to answer the question: which LET is suitable to improve the biological therapeutic gain of carbon ions? Here, we selected 3 different LET values of 290 MeV/u carbon ions, and compared the relative biological effectiveness between tumor-growth retardation and skin damage using a murine transplantable tumor. Larger RBE values for tumors after than the skin type were obtained when carbon-ions of intermediate LET were daily delivered for 2 through 5 fractions. The biological therapeutic gain would be high for the carbon-ion SOBP if the number of fractions were correctly selected in clinical trials.

In clinical use of heavy-ion beams, we cannot overlook skin response appearing in the acute period. We previously experimented effects of fractionated irradiation on the mouse skin with carbon beams of 290 MeV/u. Changing the number of fractions in therapy may alter the normal tissue damages; we have concluded that skin damage should be carefully studied when the number of fractions is changed in new clinical trials. It is still unclear whether any specific LETs cause such change in skin reaction. Here we irradiated mice feet with mono-energetic and SOBP carbon ions, to obtain dose response of early skin reaction at different LETs. With an increase of LET, repair of skin damage may be decreased. Required isoeffect total-dose increased linearly with the fraction numbers on a semi-logarithmic chart at low LET (20 and 40 keV/µm) SOBP beam. Similar behaviors were also found at high LET (60 and 80 keV/µm) beam when the fraction numbers were large. However, no increases of isoeffect dose were observed at few fractionations. The same tendency was observed for monoenergetic carbon beams. Douglas-Fowler s Fe-plot could not fit to the results for few fractionations at high-LET region. The a/b ratios obtained from the Fe-plot, the values were 71, 37, 11 and 2.9 for LETs at 20, 40, 60, and 80 keV/µm in the SOBP, respectively. We conclude that irradiation with few fractionation number at high-LET produced much stronger effect on skin. The LQ-model based Fe-plot could not fit skin reaction at few fractions at high-LET. This result may account for clinical carbon therapy of lung tumors that requires larger doses than doses predicted by extrapolating from fractionation date., Heavy Ion in Therapy and Space Radiation Symposium 2013 （HITSRS2013)

The aim of this study was to clarify the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for cell killing and micronucleus formation in clamped and non-clamped transplanted solid tumors in mice leg after 290 MeV/n carbon-ion beams and X-rays. Furthermore, single-cell suspensions were prepared by tumors and were used directly to determine the radiation response of oxic cells. Squamous cell carcinoma (SCC VII) cells were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (46 and 80 keV/µm) or X-rays was delivered to 5-mm diameter tumors. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assay to obtain cell-survival curves. Moreover, a large portion of single-cell suspensions were incubated with cytochalasin-B to examine the dose-response curves for micronucleus (MN) formation. The RBE and OER for cell survival were evaluated by D10 values (the dose required to reduce surviving fraction down to 10%), and the RBE and OER for MN formation were evaluated by D0 values (the dose corresponding to 37% retained binucleated cell without micronuclei. The RBEs for survival and MN formation increased with increasing LET. These maximum values were 1.7-2.4 at an LET of 80 keV/µm. The OERs for in vivo (clamped tumor / non-clamped tumor) were smaller than that for in vitro (clamped tumor / single-cell suspension) in both endpoints, and the dependence of OERs for in vivo toward LET was small in comparison to the OERs for in vitro., The 10th International Symposium on Chromosomal Aberrations

[Background and Purpose] The fractionated irradiation is standard protocol for radiotherapy including carbon-ion beam (C-ions) therapy. There are big differences between single and fractionation, for example on DNA damage repair, reoxygenation of hypoxic region, etc. It is necessary to get the results using fractionated irradiation to know the effects in clinical. The purpose of this study is to examine the effects of fractionated irradiation for metastasis using C-ions and X-rays. [Materials and Methods] A mouse osteosarcoma cells, LM8 having highly metastatic potential were used. C-ions irradiation (290 MeV/u) was performed at the center position of 6cm-SOBP, and X-rays were used as reference beam. The interval among each fraction was 24 hours, and 1 to 5 fractions were used. [in vitro] The cytotoxic effects were examined using colony formation assay, and the anti-metastatic effects were examined by the inhibition of migration and invasion activities using Boyden chamber assay and Matrigel invasion assay, respectively. [in vivo] LM8 tumors inoculated into right hind leg of mice were irradiated at 12days after inoculation. Radiosensitivity for individual cell in a tumor was examined by in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. [Results] [in vitro] After X-rays fractionation, cell survival and metastatic potentials (migration and invasion activities) were increased depend on the fraction number, and the degree was significant at low dose region. On the other hand, the enhancement was not significant after C-ions fractionation. [in vivo] The survival of cells in local tumor and the number of lung metastatic nodules were increased with the increment of the fraction number after fractionated irradiation. The degree of increase was more remarkable by X-rays than C-ions. The RBE values of C-ions were increased with fraction number on both cell killing and suppression of metastasis, and the values were larger on metastasis suppression than on cell killing. [Conclusion] It is confirmed that the anti-metastatic effects showed the dependency of the fraction number as well as cell killing effects. The degree of the dependency was more significant for X-rays compared with C-ions with high LETs than X-rays with low LETs. It was suggested that C-ions therapy was superior to photon therapy not only on tumor cell killing but also on metastasis suppression even for clinical fractionated irradiation., 39th Annual Meeting of the European Radiation Research Society

PTCOG51

Purpose: Up to 2011, over 5500 patients have been treated in HIMAC. Malignant melanoma showed high local control about 75%, whereas the overall survival was about 36% at 5-years. It is an important subject to control tumor distant metastasis for heavy-ion radiotherapy. The aim of this study is to clarify the effect of carbon ion beams (C-ions) on metastatic potential of melanoma in vitro and in vivo. Materials and Methods: A highly metastatic mouse malignant melanoma cell line, B16/BL6 was maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics. [in vitro] Samples were prepared 1.5 days before and then irradiated with C-ions at different LETs or X-rays. Surviving fractions (SF) and metastatic potentials (migration, invasion and adhesion activity) of the cells were examined. [in vivo] Local tumors implanted into right leg of mice were also irradiated with C-ions at different LETs, or gamma-rays. The metastatic effects were analyzed with the spontaneous lung metastasis model. Radiosensitivity for individual cell in a tumor was obtained by an in vivo-in vitro assay. Results: [in vitro] C-ions showed higher cytotoxic and anti-metastatic effects compared with X-rays, and the effects were significant depend on dose and LET values. The enhancement ratios obtained from metastatic potential were higher than that from cell killing. [in vivo] After irradiation for local tumors, the numbers of lung metastatic nodules decreased with the dose and LET values. C-ions were more effective compared with gamma-rays. The number of lung metastasis was analyzed with equivalent survival dose at D10 calculated form the survival curves of individual cell in a tumor. In this case, C-ions also showed smaller number of metastasis than gamma-rays. Conclusion: It might suggest that C-ions inhibit metastasis at radiotherapy compared with photons., 14th International Congress of Radiation Research (ICRR2011)

Cancer therapy with carbon ions at the HIMAC has started in 1994, more than 5,000 lesions have been treated by 2010, and fruitful results have been demonstrated. Following those results, few heavy-ion treatment facilities are working, some are under construction and many new facilities are planning all over the world now. To start treatments with HIMAC clinical experiences such as treatment planning, the beam quality at those new facilities must be very similar to the HIMAC in physics and biology. Biological results are easy to affected by the biological conditions, techniques or so on. We tried to establish a standard biological protocol for this purpose. Both C3H/He mice bead at NIRS (N) and Charles River Co. (C) were employed, and the crypt cell survival assay 3.5 days after the irradiation was performed with whole body single irradiation of carbon 290 MeV/u beam having 6 cm SOBP at HIMAC. Crypt survival data was taken from (N) and (C) mice, by both trained and fresh observers. The radio-sensitivity of the mice crypt was different by the breeders. The Dq values were similar between them (C and N), but the Do for (N) was higher than that for (C). Recognition level of the existing crypts at counting was different by the observers, but this technical difference was conquerable by simple training. Change in the recognition level with elapsed time could not found for an observer within 6 years. Use of mice supplied by worldwide breeder with good quality control may be required for intercomparison of RBE. Short training could unify technique of new observer., 14th International Congress of Radiation Research (ICRR2011)

56th Annual Meeting of Radiation Research Society

Purpose: The aim of this study is to clarify the effect of carbon ion beams (C-ion) on metastatic potential of melanoma in vitro and in vivo. Materials and Methods: A highly metastatic mouse malignant melanoma cell line B16/BL6 were maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics. [in vitro] Samples were prepared 2 days before and then irradiated with C-ions or X-rays. Surviving fractions were obtained using colony formation assay. Migration and invasion activity as metastatic potentials of the cells were examined using the Boyden-chamber method and the Matrigel invasion assay. [in vivo] The cells were implanted in right-leg of C57BL/6J mice at 1x106 cells/mouse 9-10 days before irradiation. Tumors were irradiated at the center of 6cm-SOBP of C-ions, or gamma-rays. Radiosensitivity for whole tumor was obtained by the tumor growth delay method, and that for individual cell in a tumor was obtained by an in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. The dose averaged LET values of carbon beams were approximately 50 keV/um. Results: [in vitro] Survival curves showed higher cytotoxic effects of C-ions compared with X-rays, and the RBE values were 1.96. The potential of migration and invasion were suppressed by C-ions at all dose points (0.50 to 8.0 Gy) tested, however it was enhanced by X-rays at low dose points (0.50 and 1.0 Gy) than non-irradiated controls. The RBE values obtained from migration and invasion test were higher than that from cell killing. [in vivo] C-ions significantly suppressed the tumor growth, and the RBE was 2.64. The numbers of lung metastatic nodules after tumor-irradiations decreased with the dose, and C-ions were more effective compared with gamma-rays. The metastatic potentials of survived cells in a tumor after irradiation was analyzed with the number of metastatic lung colony from implanted tumors and survival of irradiated and explanted cells from a tumor. Smaller number of metastasis was found for C-ions than gamma-rays when the numbers were compared with biological equivalent dose. Conclusion: It might suggest that C-ion inhibit metastasis at radiotherapy compared with low-LET photons., NIRS-KI Joint Symposium on Carbon Ion Therapy

Purpose: Up to 2009, over 5000 patients have been treated in HIMAC. Malignant melanoma showed high local control of about 75%, whereas the overall survival was about 36% at 5-years. It is an important subject to control tumor distant metastasis for heavy-ion radiotherapy. The aim of this study is to clarify the effect of carbon ion beams (C-ion) on metastatic potential of melanoma in vitro and in vivo. Materials and Methods: A highly metastatic mouse malignant melanoma cell line B16/BL6 were maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics. [in vitro] Samples were prepared 2 days before and then irradiated with C-ions at 13, 50 or 75 keV/um or X-rays. Surviving fractions were obtained using colony formation assay. Migration and invasion activity as metastatic potentials of the cells were examined using the Boyden-chamber method and the Matrigel invasion assay. [in vivo] The cells were implanted in right-leg of C57BL/6J mice at 1x106 cells/mouse 9-10 days before irradiation. Tumors were irradiated at the center of 6cm-SOBP of C-ions, or gamma-rays. Radiosensitivity for whole tumor was obtained by the tumor growth delay method, and that for individual cell in a tumor was obtained by an in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. Results: [in vitro] Survival curves showed higher cytotoxic effects of C-ions compared with X-rays, and the RBE values were 1.1, 1.7 or 2.5 at 13, 50, or 75 keV/um, respectively. The potential of migration and invasion were suppressed by C-ions at all dose points (0.25 to 8.0 Gy) tested, however it was enhanced by X-rays at low dose points (0.25 to 0.5Gy) than non-irradiated controls. The RBE values obtained from migration and invasion test were higher than that from cell killing. [in vivo] C-ions significantly suppressed the tumor growth, and the RBE was 2.6. The numbers of lung metastatic nodules after tumor-irradiations decreased with the dose, and C-ions were more effective compared with gamma-rays. The metastatic potentials of survived cells in a tumor after irradiation was analyzed with the number of metastatic lung colony from implanted tumors and survival of irradiated and explanted cells from a tumor. Smaller number of metastasis was found for C-ions than gamma-rays when the tumor cell survivals were 10%. Conclusion: It might suggest that C-ion inhibit metastasis at radiotherapy compared with low-LET photons., PTCOG 49

PTCOG49

陽子線がん治療ではブラッグピークの位置を病変部に合わせて照射し線量の集中性を良くして治療を行う。実際の治療においては、位置決めの誤差や病変部自体の移動を考慮して病変部の周囲にマージンを追加し、病変部より広い範囲に陽子線を照射しているが、陽子線の治療計画では病変部周辺での物理線量分布と生物線量分布差異が考慮されていない。そこで本研究ではRBEの評価実験を基に、陽子線治療ビームの後方端での物理線量分布と生物線量分布の比較評価を行った。比較評価を行った結果、SOBP後方端の生物線量分布はSOBP中心と比較して、実験した全てにおいて、SOBP中心効果比が最大で1.41（D10）及び1.97(D55)になっていた。生物線量分布は2.2mm（D10）及び3.7mm(D55)延長し、右側にシフトしたような形となった。よって我々は、陽子線のデザインや治療計画を行う時は、SOBP後方端での生物学的効果が高くなり、数mm程度延長する事を考慮した方が良いと提案する。, PTCOG49

Particle Therapy Co-Operative Group 49

We studied DSB induction and rejoining in the clamped and non-clamped tumors after low- and high-LET radiations. SCCVII cells were transplanted into the right hind legs of C3H male mice. Conditioning irradiation with either 80 keV/μm carbon ions or 200 kVp X rays was delivered to the tumors at 5 mm diameter. Hypoxic cells were produced by clamping tumor-bearing mice 15min before irradiation in situ. Immediately after and 1 hr after irradiation, tumors were excised and rapidly cooled on ice. DSB in the tumors was analyzed by a static-field gel electrophoresis, and assayed for DSB induction and rejoining. The DSB immediately after X rays under normoxic condition was higher than under hypoxic condition. These damages in both tumor conditions were rejoined 60 to 70% within 1 hr in situ. The OER of DSB after X rays was 1.68 ± 0.31, and this value was not changed by 1 hr rejoining incubation (1.40 ± 0.26). On the other hand, no difference between X rays and carbon ions was found for the induction and rejoining of DSB. The rejoined fraction and the OER of DSB after carbon ions were similar to that after X rays. Additionally, the RBE of DSB immediately after and 1 hr after irradiation, these values were 0.9 to 1.3, and were not dependent on gases conditions. The yields of DSB induced by X rays in vivo might be similar to that after carbon ions, because the OER and RBE were not found a major difference., MICROS 2009 15th International Symposium on Microdosimetry

The aim of this study is to clarify cell cycle distribution and cytotoxicity after carbon-ion beams (C-ions). One human malignant melanoma cell line, HMV-I was exposed to X-rays or 290 MeV/u C-ions at the center of SOBP. We applied flow cytometry technique and colony formation assay. C-ions induced G2/M arrest effectively more than X-rays at 24 h. The iso-effect dose, i.e. the 10% survival dose (5.0Gy for X-rays, 2.7Gy for C-ions) was used to examine the cell cycle kinetics. G2/M fraction increased immediately after irradiations, and showed a peak at 15 h. G2/M arrest cells were stayed long time after C-ions, whereas it released quickly after X-rays. In addition, the mRNA expression of molecules related with the arrest was suppressed with D10 dose and the effects were transitory after X-rays, whereas C-ions showed prolonged suppression. The arrest was released significantly by caffeine, and the cell killing by C-ions was enhanced with caffeine together. It is suggested that the release from G2/M arrest after C-ions can enhance the cytotoxic effects. Repair of complex DNA damage by C-ions takes long time. When it released intermediate of the process, incomplete repaired damage cause cell death., MICROS 2009 15th International Symposium on Microdosimetry

(Introduction) A dose escalation study of non-small cell lung carcinoma in patients has shown that shape of dose-tumor control probability of carbon-ion radiotherapy is steeper than that of photon therapy, and that relative biological effectiveness (RBE) of carbon ions is larger at higher doses than at lower doses. This contradicts with the biological knowledge; RBE decreases with an increase of dose. A possible explanation is that tumors could contain heterogeneous sub-populations with different photon sensitivities. (Purpose) The purpose of present study is to clarify experimentally significance of tumor heterogeneity. (Materials and Methods) Two sarcomas of # 6107 and #9037 were transplanted into syngeneic C3H male mice. Single cell suspensions for each tumor were mixed together at various ratios just prior to transplantation. When tumors grew to reach 7 mm in diameter, leg tumors were locally irradiated with 290 MeV/n carbon ions at an LET of 74 keV/m that were accelerated by HIMAC synchrotron in our institute. Tumor growth (TG) time was obtained by calculating days required for a tumor to reach 5 times initial volume. Both Tumor Growth Delay (TGD) time and Specific Tumor Growth Delay was used to obtain isoeffect doses. RBE values were calculated by comparing isoeffect doses between carbon ions and reference Cs-137 gamma rays. (Results and Discussion) TG time of unirradiated control was similar between #6107 and #9073, i.e., 5.6 and 6.1 days. Tumors with different ratios of mixture (RM) showed various TG time such that TG times of 8.3, 3.8 and 6.1 days were for tumors with ratios of 9:1, 5:5 and 1:9 (#6107: #9037), respectively. Isoeffect doses for 5 tumors with different RM were ranging from 18 to 48 Gy for gamma rays, and from 8 to 18 Gy for carbon ions. A tumor with RM of 9:1 showed largest isoeffect dose, and was more resistant than the either parental tumors of #6107 or #9037 tumors. RBE of the tumor with RM of 9:1 was largest, and larger than that of the parental tumors with RM of 10:0 and 0:10. This means any interaction between sub-populations could modify radiosensitivity of a tumor. Possible reasons for this interaction are either hypoxic ell fraction or G1 cell cycle phase. Factor(s) and/or signals responsible to the interaction are unknown. (Conclusion) Tumor heterogeneity is a significant determinant for tumor radiosensitivity and RBE of high LET radiation., 13th International Congres of Radiation Research

Introduction: Oxygen consumption rate and blood flow are important parameters for the physiological and pathological evaluation of brain, myocardium and tumors1,2, where 15O PET has been utilized. 17O MRI will be another tool for the direct observation of tumor oxygenation. Measurements of the blood flow and oxygen consumption rate by in vivo 17O NMR has been reported recently3,4. We have developed 17O imaging by FISP and succeeded in the visualization of natural abundance H217O distribution in the mouse with 10 min data acquisition5. In this study, we will report the dynamical study of 17O imaging using 17O enriched saline in the tumor bearing mice. Phantom experiments demonstrated the feature of 17O in vivo NMR signals. Methods: MRS/MRI was performed on 7T/400mm/SS system (NIRS/KOBELCO/Bruker) with 40 mm 1H/17O Litz coil (Doty Scientific Inc.). Water, ethanol or 25-100 % ethyleneglycol -water was used in phantom experiments. 17O images of healthy and tumor bearing C3H/He mice (20 . 25 g) were obtained under Ketamine:Xylazine anesthesia by true FISP with a TR/TE = 4.3/2.15 ms. A saline (0.5ml) containing 5% 17O prepared from 10% 17O water (Cambridge Isotope Laboratories, Inc) was i.v. injected to tumor bearing mice. After imaging experiment, organs were excised for 17O spectral measurements. Results: Phantom studies: The 17O signal of ethyleneglycol or water in 25% ethyleneglycol-water phantom was detected by fid acquisition mode but not by echo mode within our experimental condition. Animal studies: The S/N in the 17O images of a mouse obtained by FISP with 10 min data acquisition was dramatically improved from 3.8 to 13.4 after the injection of a saline containing 5% 17O. The result of 10 sec dynamical imaging of H217O with under the spatial resolution of 2.5 mm without slicing was shown in Fig.1. The process of initial accumulation of the injected H217O in the heart and re-distribution to whole body was demonstrated. Spatial resolution of 1.25 mm was attained with the 10 min data acquisition. Discussion: The dynamical study of H217O was achieved in the tumor bearing mice with a temporal resolution of 10 sec. The temporal and spatial resolutions attained in this study will lead this imaging method to the evaluation of blood flow or oxygen consumption rate using 17O gas and water. FISP is shown to be an effective method for imaging in vivo 17O signals from free water. The phantom experiments with ethyleneglycol-water strongly suggest that the in vivo 17O images obtained here is from the mobile H217O and not from other molecular species or immobilized water. The echo image of 17O detecting solely the signal from free water, not from the body constituents contributing as background should have an advantage over the other NMR method with FID detection in the 17O2 gas study. Reference: (1) Secomb TW. et al, 34 :313 (1995), (2) Ando K, et al, Int J Radiat Biol 75 :505 (1999), (3) Zhu XH. et al, MRM 45:543 (2001), (4) Fiat D. et al, Neurol Res 26:803 (2004), (5) Narazaki M. et al, 14th ISMRM 3113 (2006), Joint Annual Meeting ISMRM-ESMRMB 2007

(INTRODUCTION) We previously reported tumor induction in mice legs after fractionated irradiation with carbon ions (J.Radiation Res., 46, 185-190, 2005), reporting that the RBE value of carbon ions was 2.9. What puzzles us is that dose responses of carbon ions as well as reference gamma rays are linear without decrease at large doses, which is contrary to other reports using whole body irradiation. (PURRPOSE) We here studied and reported tumor induction in the same radiation conditions/endpoints after single doses. (MATERIALS and METHODS) Right legs of female C3H mice were irradiated with either Cs-137 gamma rays or 290 MeV/u carbon ions of Spread-Out-Bragg peak (SOBP). Dose averaged LET of carbon ions used was 15 (entrance plateau), 45 (proximal SOBP) or 75 (distal SOBP) keV/microm. Number of mice used was 50 for each dose, and a total of 1650 mice were irradiated. Irradiated legs were once a week observed and palpated to detect tumor induction up to 800 days. Incidence of tumor induction was analyzed against doe and time after irradiation. (RESULTTS and DISCUSSION) Tumor induction frequency for any radiation qualities increased with an increase of dose up to 40 Gy. The frequency saturated to increase at higher doses, and rather decreased at 80 Gy of gamma rays and at 60 Gy of 75 keV/microm carbon ions. These results indicate that the irradiate site of leg is the primary target of tumor induction, and not its margin which receives lower doses than prescribed. RBE values of carbon ions were calculated by using a dose to induce 20 % tumor, and were 1.0, 1.2 and 1.9 for 15, 45 and 75 keV/microm, respectively. Maximum frequency of tumor induction by 15 keV/microm carbon ions was lower than that by gamma rays. Time between irradiation and tumor appearing depended on dose except 45 keV/microm carbon ions, and was longer for low dose groups than high dose groups. (Conclusion) Carcinogenic effects of carbon ions are quantitatively and qualitatively different from gamma rays. Low LET carbon ions could be less dangerous than gamma rays., COSPAR Colloquium : Mutagenic consequences of the space environment

Since the installation of 7T MRI system (NIRS/KOBELCO/Bruker) in October 2004, we have been working on the in vivo multinuclear imaging and MRS. The magnet was developed by NIRS and JASTEC (KOBELCO), 400mm bore, self shielded, and zero boil off freezing system for liquid helium. This report is on the multinuclear applications to the pharmacodynamical study of fluorine containing drug 5-FU with 19F, and 17O imaging towards CMRO2 in mice. In order to obtain images from low concentration or low sensitivity nuclear spins, fast sequences are compared for 19F at 282 MHz, and for 17O at 40MHz. Fast spin echo was better than true-FISP for 19F in giving stable image intensity for all the metabolites in various organs. FSE is also capable of giving the full time course of drug dynamics by simultaneous multi-spectral line selection: the distribution of effective anabolites sepecially at the tumor, the formation and excretion of catabolotes from the liver as well as the fate of administrated drug. The method is benefited by the wide chemical shift separation and high sensitivity of high field MR. For 17O, true-FISP was the best among FISP, FSE and Flash. Unlike the case of 19F, the smaller effect of magnetic susceptibility at the resonance frequency of 40MHz enabled us to use FISP for whole body MRI of 17O., The 6th Japan-France Workshop on Radiobiology and Isotopic imaging

Introduction: The application of 17O MRI method has been proposed in the measurements of regional cerebral blood flow and cerebral metabolic rate of oxygen consumption1-3. Since hypoxic tumor cells are resistant to the radiation, monitoring the tumor oxygenation and blood flow is important for the effective treatment. Irrespective of such expectation, low sensitivity and short T2 of 17O make the direct measurement difficult. In order to overcome these difficulties, proton-detected 17O MRI4, Chemical Shift Imaging5, gradient echo and projection reconstruction6 have been proposed. In this study, FISP was employed for H2 17O imaging at 7.0 T., International Society for Magnetic Resonance in Medicine(ISMRM) 14th Scientific Meeting and Exhibition

Introduction: The evaluation of drug efficacy and safety for individuals will become more reliable by performing direct dynamical study of drug itself. This is a study towards this purpose using 5-FU in animals. CSI is often used for the metabolite distribution. However, for the compounds with metabolites in wide range of chemical shift, generation of pulse covering sufficient band width is difficult for imaging instrument. One of the best methods for the dynamical study of 19F containing drug, e.g. 5-FU, has been found to be frequency selective imaging [1]. Here, we evaluate fast imaging sequences including FISP and Flash in comparison to FSE at 7T., International Society for Magnetic Resonance in Medicine(ISMRM) 14th Scientific Meeting and Exhibition

Introduction: Chemical shift imaging under minimal relaxation weight should be a robust method for in vivo dynamical study of drugs and labeled compounds. Fast imaging sequences are compared at 7T with 5FU administrated mice., The 8th International Conference on Magnetic Resonance Microscopy

放射線治療後において，MRIやCTでは治療効果を判定するには数週間を要するため，治療後早期の効果判定は腫瘍増殖能や照射計画を推測する上で重要であると考えられる．そこで今回我々はThymidineおよびFDGを用いたダブルトレーサ法により，炭素線照射後の腫瘍体積の変化が認められない早期(12時間後)と再増殖時期（14日後）におけるDNA合成能と糖代謝について検討を行った． 【方法】C3H雄性マウスの下肢にNFSa腫瘍を移植し，腫瘍直径が7.5mmのマウスに線量およびLET(14keV/μm，74keV/μm)の異る炭素線(6cm-SOBP)を照射して腫瘍体積を経日的に計測した．またDNA合成能と糖代謝を計測するため，照射12時間後と14日後に炭素線を照射したマウスに，18F-DGおよび14C-Thymidineを尾静脈投与した．投与30分後に屠殺して速やかに下肢腫瘍と筋肉および血液(Plasma)を採取し，液体シンチレーションカウンターで計測した． 【結果・考察】腫瘍体積は照射後2日まで，照射群（3-60Gy）は非照射群と比べ変化は認められなかった．糖代謝は照射12時間後では，照射群において軽度の減少がみられるもののLETおよび線量依存性(22-40Gy)は認められなかった．また腫瘍再増殖がみられる照射14日後においても変化は認められなかった．一方で，14C-Thymidine取込能は照射12時間後に，線量およびLETに依存した有意な取込能の減少が認められ，照射12時間後の腫瘍取込能と照射14日後の腫瘍体積との間に高い相関性が認められた．さらに照射14日後においては，腫瘍増殖速度に応じた取込能の増大が認められた．以上の結果から，14C-Thymidineは照射後早期において放射線治療効果を判定すると共に，再発する腫瘍の増殖速度を推定出来ることが示唆された．, 第48回日本放射線影響学会

High LET radiation minimizes any difference in the radiosensitivity of different types of cells to which photon irradiation produces large variation. It is anticipated that side effects of high LET particle radiotherapy would not bring a biological therapeutic gain. The purpose of this paper is to answer a question whether high LET radiation could discriminate tumor and normal tissues so that biological advantage could be achieved. We here selected and measured the growth delay of an experimental tumor and the skin reaction in C3H mice, as a model for biological gain. The tumor was a syngeneic NFSa fibrosarcoma, and were transplanted intramuscularly into the right hind legs of male mice 7 days before the first irradiation. Hairs on the right hind leg of female mice were removed by applying a depilatory agent 7 to 8 days before the first irradiation. Carbon-12 ions were accelerated by the HIMAC synchrotron up to 290 MeV/u. Cs-137 gamma-rays were used as a reference beam for determining the RBE. Daily fractionation was given with equal daily doses using an interfractional interval of 24 hours. Several graded doses were used to determine an isoeffect dose, and animals assigned to a given dose group received equal daily doses. The tumor growth (TG) time obtained for all animals were averaged per each dose group. Irradiated legs were observed for skin reaction scoring every other day up to 5 weeks. The skin scores in an individual mouse were averaged, and was used for obtaining dose-response. Carbon ions of LET 42 and 77 keV/micro.m, but not 14 or 20 keV/ micro.m, showed larger RBE values in retarding tumor growth than causing skin reaction after daily-fractionated irradiation. Using Fe-plot, we analyzed the isoeffect dose to evaluate dependence of intra-track damages (alpha term) and of inter-track damages (beta terms) on LET. The alpha term of tumor growth delay and skin reaction equally increased with an increase in LET, and no difference was observed between the two tissues. Beta term was, however, different between the two tissues such that the beta term of skin reaction apparently increased with LET while that of tumor growth delay was independent of LET. It is clear that beta term is a determinant of tissue specific response to high LET radiation, while alpha term solely depends on LET. It is implied that high-LET radiotherapy could achieve a large therapeutic gain when a large dose per fraction is used., 9th workshop on Heavy Charged Particles in Biology and Medicine and 3rd ENLIGHT co-ordination meeting

The purpose of this paper is to report that gut crypt cells become radioresistant after single or fractionated irradiation with intermediate LET carbon ions. C3H female mice received whole body irradiation with 20 keV/mm carbon ions, and served the jejunum for crypt survival assay 3.5 days after irradiation. Dose-crypt survival relationship showed that the D0 value increased from 1.06 Gy to 1.94 Gy when the first dose of 9.0 Gy was followed by single graded doses of carbon ions with an interval time of 4 hr. The Increased D0 gradually decreased with a half decay time of 50 hr, and returned to 1.08 Gy, a similar value of single irradiation. When mice received 1 Gy carbon ions for 9 fractions with an interval time of 4 hr each, the D0 value of crypt cells surviving the preceding total dose of 9 Gy also increased to 1.89 Gy. The D0 increase was less prominent when the size of first or preceding dose was small. It is concluded that charged particles with intermediate LET produce a unique biological response, and would be beneficial for radiotherapy of visceral tumors., ICRR

HCPBM and ENLIGHT-2002

HCPBM and ENLIGHT-2002

HCPBM and ENLIGHT-2002

HCPBM and ENLIGHT-2002

Europian Radiation Research 2001

13th Symposium on Microdosimetry

The 2nd International Workshop on Space Radiation Research

The evaluation of drug efficacy and safety for individuals will become more reliable by performing direct dynamical study of drug itself. This is a study towards this purpose using 5-FU in animals. CSI is often used for the metabolite distribution. However, for the compounds with metabolites in wide range of chemical shift, generation of pulse covering sufficient band width is difficult for imaging instrument. One of the best methods for the dynamical study of 19F containing drug, e.g. 5-FU, has been found to be frequency selective imaging [1]. Here, we evaluate fast imaging sequences including FISP and Flash in comparison to FSE at 7T.