1. To evaluate the effect of bevacizumab (BV) on local tumor response and lung metastatic potential in boron neutron capture therapy (BNCT), referring to the response of intratumor quiescent (Q) cells. To evaluate the effect of bevacizumab (BV) on local tumor response and lung metastatic potential in boron neutron capture therapy (BNCT), referring to the response of intratumor quiescent (Q) cells. B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2’- deoxyuridine (BrdU) to label all proliferating (P) tumor cells. The tumors received reactor thermal neutron beams following the administration of a 10 B-carrier (BPA or BSH), with or without the administration of bevacizumab (BV), and further in combination with an acute hypoxia-releasing agent (nicotinamide, NA)or mild temperature hyperthermia (MTH, 40°C for 60 min). The responses of the Q and total (= P+Q) cell populations were assessed based on the frequency of micronuclei using immuno- fluorescence staining for BrdU. In other tumor-bearing mice, 17 days after irradiation, lung metastases were enumerated. B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2’- deoxyuridine (BrdU) to label all proliferating (P) tumor cells. The tumors received reactor thermal neutron beams following the administration of a 10 B-carrier (BPA or BSH), with or without the administration of bevacizumab (BV), and further in combination with an acute hypoxia-releasing agent (nicotinamide, NA)or mild temperature hyperthermia (MTH, 40°C for 60 min). The responses of the Q and total (= P+Q) cell populations were assessed based on the frequency of micronuclei using immuno- fluorescence staining for BrdU. In other tumor-bearing mice, 17 days after irradiation, lung metastases were enumerated. Three days after BV administration, the sensitivity of the total tumor cell population after BPA-BNCT had increased more than after BSH-BNCT. The combination with MTH, but not with NA, further enhanced total tumor cell population. With or without a 10 B-carrier, MTH enhanced the sensitivity of the Q cell population. With or without irradiation, the administration of BV showed some potential to reduce the number of lung metastases, as well as NA treatment, especially in BPA-BNCT compared with BSH-BNCT. Three days after BV administration, the sensitivity of the total tumor cell population after BPA-BNCT had increased more than after BSH-BNCT. The combination with MTH, but not with NA, further enhanced total tumor cell population. With or without a 10 B-carrier, MTH enhanced the sensitivity of the Q cell population. With or without irradiation, the administration of BV showed some potential to reduce the number of lung metastases, as well as NA treatment, especially in BPA-BNCT compared with BSH-BNCT. In BNCT, BV has the potential to sensitize total tumor cell populations and cause a decrease in the number of lung metastases to a similar level to NA. In BNCT, BV has the potential to sensitize total tumor cell populations and cause a decrease in the number of lung metastases to a similar level to NA. Significance of Combined Treatment with Bevacizumab in Boron Neutron Capture Therapy in Terms of Local Tumor Response and Lung Metastasis Shin-ichiro Masunaga, Y. Sakurai, K. Tano, H. Tanaka, M. Suzuki, N. Kondo, M. Narabayashi, T. Watanabe, Y. Nakagawa, A. Maruhashi, K. Ono Abstract Materials/Methods Results Conclusions The use of BSH as a 10 B-carrier in combination with MTH is thought to be advantageous and promising in terms of local tumor response in BNCT because MTH reduces the difference in radiosensitivity between radiosensitive total (P+Q) and radioresistant Q cell populations. The use of BSH as a 10 B-carrier in combination with MTH is thought to be advantageous and promising in terms of local tumor response in BNCT because MTH reduces the difference in radiosensitivity between radiosensitive total (P+Q) and radioresistant Q cell populations. BPA-BNCT in combination with NA and/or BV treatment may show a little greater potential to reduce the number of lung metastases from the primary tumor. BPA-BNCT in combination with NA and/or BV treatment may show a little greater potential to reduce the number of lung metastases from the primary tumor. In BNCT, BV has the potential to sensitize total (P+Q) tumor cell populations and cause a decrease in the number of lung metastases to a similar level to NA. In BNCT, BV has the potential to sensitize total (P+Q) tumor cell populations and cause a decrease in the number of lung metastases to a similar level to NA. Finally, it was elucidated that control of the chronic hypoxia-rich Q cell population in the primary solid tumor has the potential to impact the control of local tumors as a whole, and that control of the acute hypoxia-rich total (P+Q) tumor cell population in the primary solid tumor has the potential to impact the control of lung metastases from the primary tumor. Finally, it was elucidated that control of the chronic hypoxia-rich Q cell population in the primary solid tumor has the potential to impact the control of local tumors as a whole, and that control of the acute hypoxia-rich total (P+Q) tumor cell population in the primary solid tumor has the potential to impact the control of lung metastases from the primary tumor. Three days after BV administration, the sensitivity of the total tumor cell population after BPA-BNCT had increased more than after BSH-BNCT. The combination with MTH, but not with NA, further enhanced total tumor cell population. Three days after BV administration, the sensitivity of the total tumor cell population after BPA-BNCT had increased more than after BSH-BNCT. The combination with MTH, but not with NA, further enhanced total tumor cell population. With or without a 10 B-carrier, MTH enhanced the sensitivity of the Q cell population. With or without a 10 B-carrier, MTH enhanced the sensitivity of the Q cell population. With or without neutron beam irradiation, the administration of BV showed some potential to reduce the number of lung metastases, as well as NA treatment, especially in BPA-BNCT compared with BSH-BNCT. With or without neutron beam irradiation, the administration of BV showed some potential to reduce the number of lung metastases, as well as NA treatment, especially in BPA-BNCT compared with BSH-BNCT. Summary of Results B16-BL6 melanoma tumor-bearing C57 BL /6 mice were continuously given BrdU to label all proliferating (P) cells. The tumors received reactor neutron beam irradiation after i.p. administration of BPA (250 mg/kg) or BSH (125 mg/kg) with or without the administration of BV (3 days before irradiat., i.v.), and further in combination with an acute hypoxia-releasing agent (NA, 1 g/kg, 1 hour before irradiat., i.p.) or MTH (40°C for 60 min). Right after the irradiation, cells from some tumors 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. 17 days after neutron beam irradiation, lung metastases were enumerated. <Immunofluorescence staining for BrdU to detect BrdU-labeled cells> Assessment of the responses of Q and total (= P+Q) cell populations were based on the frequencies of micronucleation using immuno- fluorescence staining for BrdU. Arrows: Arrows: BrdU-unlabeled (Quiescent) binuclear tumor cell with a micronucleus (colored with nuclear staining (PI, red) only) BrdU-unlabeled (Quiescent) binuclear tumor cell with a micronucleus (colored with nuclear staining (PI, red) only) Arrowheads: Arrowheads: BrdU-labeled (Proliferating) binuclear tumor cell with a micronucleus (colored with immuno-fluorescence staining (FITC, green) and nuclear staining (PI, red))MT BrdU-labeled (Proliferating) binuclear tumor cell with a micronucleus (colored with immuno-fluorescence staining (FITC, green) and nuclear staining (PI, red))MT Cadnium ratio: 9.4, at the power of 1 MW Thermal: 1.0 x 10 9 n/cm 2 /s, 48.0 cGy/h Epithermal: 1.6 x 10 8 n/cm 2 /s, 4.6 cGy/h Fast neutrons: 9.4 x 10 6 n/cm 2 /s, 32.0 cGy/h Contaminating + prompt  -rays: 66.0 cGy/h Irradiation was started from 60 min and was finished by 180 min after the administration of the 10 B-carriers, because it took approximately 60 through 120 min to deliver a sufficient physical dose of radiation. Irradiation was started from 60 min and was finished by 180 min after the administration of the 10 B-carriers, because it took approximately 60 through 120 min to deliver a sufficient physical dose of radiation. PS2B 02 Q cell P cell 10 B concentration (ppm) in tumors and boron dose rate (cGy/h) Without NA or MTHWith NAWith MTH 10 B concentration (ppm) Without BV BPA6.97.38.3 BSH7.28.47.9 With BV BPA7.27.48.6 BSH8.0 8.68.7 Boron dose rate (cGy/h) Boron dose rate (cGy/h) Without BV BPA184.2194.9221.6 BSH192.2 224.3210.9 With BV BPA192.2197.6229.6 BSH212.7 229.6231.3 Enhancement ratios by employing a 10 B-carrier BPABSH (P + Q) tumor cells Without BV1.71.5 With BV2.01.65 (P + Q) tumor cells Without BV2.452.25 With BV2.62.35 Q tumor cells Without BV1.51.8 With BV1.61.85 Enhancement ratios by BV combination Without NA or MTHWith NAWith MTH (P + Q) tumor cells 10 B-carrier (-)1.251.051.3 10 B-carrier (-)1.251.051.3 With BPA1.551.051.7 With BPA1.551.051.7 With BSH1.351.051.35 With BSH1.351.051.35 (P + Q) tumor cells 10 B-carrier (-)1.11.051.2 10 B-carrier (-)1.11.051.2 With BPA1.21.051.3 With BPA1.21.051.3 With BSH1.151.051.25 With BSH1.151.051.25 Q tumor cells 10 B-carrier (-)1.051.051.05 10 B-carrier (-)1.051.051.05 With BPA1.051.051.1 With BPA1.051.051.1 With BSH1.11.051.2 With BSH1.11.051.2 Enhancement ratios by NA or MTH combination NAMTH (P + Q) tumor cells Without BV 10 B-carrier (-) 1.31.2 10 B-carrier (-) 1.31.2 With BPA1.251.4 With BPA1.251.4 With BSH1.51.2 With BSH1.51.2 With BV 10 B-carrier (-) 1.21.1 10 B-carrier (-) 1.21.1 With BPA1.151.35 With BPA1.151.35 With BSH1.3 1.15 With BSH1.3 1.15 (P + Q) tumor cells Without BV Without BV 10 B-carrier (-) 1.251.15 10 B-carrier (-) 1.251.15 With BPA1.151.3 With BPA1.151.3 With BSH1.41.2 With BSH1.41.2 With BV With BV 10 B-carrier (-) 1.11.15 10 B-carrier (-) 1.11.15 With BPA1.11.2 With BPA1.11.2 With BSH1.21.15 With BSH1.21.15 Q tumor cells Without BV Without BV 10 B-carrier (-) 1.11.2 10 B-carrier (-) 1.11.2 With BPA1.151.4 With BPA1.151.4 With BSH1.21.45 With BSH1.21.45 With BV With BV 10 B-carrier (-) 1.051.15 10 B-carrier (-) 1.051.15 With BPA1.11.35 With BPA1.11.35 With BSH1.151.4 With BSH1.151.4 Dose-modifying factors for Q cells relative to the (P + Q) cell population Without NA or MTHWith NAWith MTH Without BV Without BV 10 B-carrier (-)1.31.451.25 10 B-carrier (-)1.31.451.25 With BPA2.12.351.7 With BPA2.12.351.7 With BSH1.61.751.25 With BSH1.61.751.25 With BV With BV 10 B-carrier (-)1.41.51.4 10 B-carrier (-)1.41.51.4 With BPA2.32.452.1 With BPA2.32.452.1 With BSH1.651.751.45 With BSH1.651.751.45 The numbers of metastases from the irradiated tumors that received cytotoxic treatment producing a similar initial local effect Without NA or MTHWith NAWith MTH Without BV Without BV 10 B-carrier (-)17.916.617.9 10 B-carrier (-)17.916.617.9 With BPA15.314.515.1 With BPA15.314.515.1 With BSH15.915.215.5 With BSH15.915.215.5 With BV With BV 10 B-carrier (-)17.416.316.8 10 B-carrier (-)17.416.316.8 With BPA14.514.214.3 With BPA14.514.214.3 With BSH15.614.815.4 With BSH15.614.815.4 Department of Radiation Life and Medical Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan.