Neon ion (20 Ne10 + ) charged particle beams manipulate rapid tumor reoxygenation in syngeneic mouse models.

Charged particle beams induce various biological effects by creating high-density ionization through the deposition of energy along the beam's trajectory. Charged particle beams composed of neon ions (20 Ne10+ ) hold great potential for biomedical applications, but their physiological effects on living organs remain uncertain. In this study, we demonstrate that neon-ion beams expedite the process of reoxygenation in tumor models. We simulated mouse SCCVII syngeneic tumors and exposed them to either X-ray or neon-ion beams. Through an in vivo radiobiological assay, we observed a reduction in the hypoxic fraction in tumors irradiated with 8.2 Gy of neon-ion beams 30 h after irradiation compared to 6 h post-irradiation. Conversely, no significant changes in hypoxia were observed in tumors irradiated with 8.2 Gy of X-rays. To directly quantify hypoxia in the irradiated living tumors, we utilized dynamic contrast-enhanced magnetic resonance imaging (MRI) and diffusion-weighted imaging. These combined MRI techniques revealed that the non-hypoxic fraction in neon-irradiated tumors was significantly higher than that in X-irradiated tumors (69.53% vs. 47.67%). Simultaneously, the hypoxic fraction in neon-ion-irradiated tumors (2.77%) was lower than that in X-irradiated tumors (4.27%) and non-irradiated tumors (32.44%). These results support the notion that accelerated reoxygenation occurs more effectively with neon-ion beam irradiation compared to X-rays. These findings shed light on the physiological effects of neon-ion beams on tumors and their microenvironment, emphasizing the therapeutic advantage of using neon-ion charged particle beams to manipulate tumor reoxygenation.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

A Phase Ib Study of Durvalumab (MEDI4736) in Combination with Carbon-Ion Radiotherapy and Weekly Cisplatin for Patients with Locally Advanced Cervical Cancer (DECISION Study): The Early Safety and Efficacy Results.

We conducted a phase Ib study to examine the safety of a combination of carbon-ion RT (CIRT) with durvalumab (MEDI4736; AstraZeneca) in patients with locally advanced cervical cancer. This was an open-label, single-arm study with a modified 3 + 3 design. Patients with newly diagnosed histologically proven locally advanced cervical cancer were enrolled. All patients received 74.4 Gy of CIRT in 20 fractions and concurrent weekly cisplatin (chemo-CIRT) at a dose of 40 mg/m2. Durvalumab was administered (1500 mg/body) at weeks two and six. The primary endpoint was the incidence of adverse events (AEs) and serious AEs (SAEs), including dose-limiting toxicity (DLT). All three enrolled patients completed the treatment without interruption. One patient developed hypothyroidism after treatment and was determined to be an SAE. No other SAEs were observed. The patient recovered after levothyroxine sodium hydrate treatment. None of the AEs, including hypothyroidism, were associated with DLT in the present study. All three patients achieved complete responses within the CIRT region concerning treatment efficacy. This phase 1b trial demonstrates the safety of combining chemo-CIRT and durvalumab for locally advanced cervical cancer in the early phase. Further research is required as only three patients were included in this study.

α-particle therapy for synovial sarcoma in the mouse using an astatine-211-labeled antibody against frizzled homolog 10.

Synovial sarcoma (SS) is a rare yet refractory soft-tissue sarcoma that predominantly affects young adults. We show in a mouse model that radioimmunotherapy (RIT) with an α-particle emitting anti-Frizzled homolog 10 (FZD10) antibody, synthesized using the α-emitter radionuclide astatine-211 (211 At-OTSA101), suppresses the growth of SS xenografts more efficiently than the corresponding ß-particle emitting anti-FZD10 antibody conjugated with the ß-emitter yettrium-90 (90 Y-OTSA101). In biodistribution analysis, 211 At was increased in the SS xenografts but decreased in other tissues up to 1 day after injection as time proceeded, albeit with a relatively higher uptake in the stomach. Single 211 At-OTSA101 doses of 25 and 50 µCi significantly suppressed SS tumor growth in vivo, whereas a 50-µCi dose of 90 Y-OTSA101 was needed to achieve this. Importantly, 50 µCi of 211 At-OTSA101 suppressed tumor growth immediately after injection, whereas this effect required several days in the case of 90 Y-OTSA101. Both radiolabeled antibodies at the 50-µCi dosage level significantly prolonged survival. Histopathologically, severe cellular damage accompanied by massive cell death was evident in the SS xenografts at even 1 day after the 211 At-OTSA101 injection, but these effects were relatively milder with 90 Y-OTSA101 at the same timepoint, even though the absorbed doses were comparable (3.3 and 3.0 Gy, respectively). We conclude that α-particle RIT with 211 At-OTSA101 is a potential new therapeutic option for SS.

Targeted cancer cell ablation in mice by an α-particle-emitting astatine-211-labeled antibody against major histocompatibility complex class I chain-related protein A and B.

Major histocompatibility complex class I chain-related protein A and B (MICA/B) are ligands of the immune receptor, natural-killer group 2 member D. MICA/B expression is often found in several types of cancer but is restricted in normal tissues. Here, we show that an α-particle emitting astatine-211 (211At)-labeled antibody targeting MICA/B (211At-anti MICA/B Ab) efficiently ablates cancer cells in vitro and in vivo. We generated 211At-anti MICA/B Ab, an anti-MICA/B antibody conjugated with a highly cytotoxic α-particle emitting radionuclide 211At. 211At-anti MICA/B Ab binds to human osteosarcoma SaOS2 and U2OS cells that exhibit high levels of MICA/B expression and efficiently kills those cells in vitro. Biodistribution analysis using xenograft mouse models of HCT116 p53-/- positive for MICA/B expression, showed increased 211At in the xenografts for up to 22 h after injection as time proceeded. A single dose of 211At-anti MICA/B Ab (1 MBq) showed significant reduction in the tumor growth rate of HCT116 p53-/- xenografts compared to 211At-labeled mouse IgG (1 MBq) at 21 days after injection. No body weight loss and erythrocytopenia was evident in mice that received 211At-anti MICA/B. Leukocytopenia and thrombocytopenia were observed within a week after 211At-anti MICA/B injection, but counts of red blood cells and platelets were recovered to control levels at about 3-4 weeks after injection. Taken together, these data strongly demonstrate that targeted α-particle therapy using 211At-anti-MICA/B Ab emitting highly cytotoxic α-particles is a potential new therapeutic option for several types of cancer.

Locoregional therapy with α-emitting trastuzumab against peritoneal metastasis of human epidermal growth factor receptor 2-positive gastric cancer in mice.

Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found, however, that locoregionally administered trastuzumab armed with astatine-211 (211 At-trastuzumab) is effective against human epidermal growth factor receptor 2 (HER2)-positive PMGC in a xenograft mouse model. We first observed that 211 At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in s.c. tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test α-particle radioimmunotherapy with 211 At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the i.p. administration of 211 At-trastuzumab (1 MBq) was a more efficient means of delivery of 211 At into metastatic tumors than i.v. injection; the maximum tumor uptake with i.p. administration was over 60% injected dose per gram of tissue (%ID/g) compared to approximately 18%ID/g with i.v. injection. Surprisingly, a single i.p. injection of 211 At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA double-strand breaks, and to drastically reduce the tumor burden in another three mice. No bodyweight loss, leukocytopenia, or significant biochemical changes in liver or kidney function were observed in the treatment group. Accordingly, locoregionally administered 211 At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211 At-trastuzumab may offer a new treatment option for HER2-positive PMGC.

Restoration of thymic T-cell development by bone marrow transplantation in mouse radiation lymphomagenesis.

Fractionated total body irradiation (TBI) with X-rays induces thymic lymphoma/leukemia (TL) in C57BL/6 mice. Radiation-induced mouse TL (RITL) can be prevented by bone marrow transplantation (BMT) of unirradiated BM cells. However, the mechanisms underlying the prevention of RITL with BMT remain unclear. Here, we show that BMT restores thymic T-cell differentiation in mice subjected to TBI. TBI (four times of 1.8 Gy X-rays weekly) was conducted with C57BL/6 mice. BMT was performed immediately after the last irradiation of TBI in mice by transplantation of BM cells isolated from enhanced green fluorescence protein (eGFP) transgenic mice. Thymic cell numbers were drastically decreased in TBI and TBI + BMT mice compared to those in non-irradiated mice. Flow cytometry showed a dramatic decrease in double negative (DN, CD4-CD8-) thymocytes, especially DN2 (CD25+CD44+) and DN3 (CD25+CD44-) subpopulations, in the TBI mice on Day 10 after the last irradiation. In contrast, the DN2 and DN3 populations were recovered in TBI + BMT mice. Interestingly, these restored DN2 and DN3 cells mainly differentiated from eGFP-negative recipient cells but not from eGFP-positive donor cells, suggesting that transplanted BM cells may interact with recipient cells to restore thymic T-cell development in the RITL model. Taken together, our findings highlight the significance of restoring thymic T-cell differentiation by BMT in RITL prevention.

Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene.

H-ferritin (HF) is a core subunit of the iron storage protein ferritin, and plays a central role in the regulation of cellular iron homeostasis. Recent studies revealed that ferritin and HF are involved in a wide variety of iron-independent functions, including regulating biological processes during physiological and pathological conditions, and can be overexpressed in some human diseases. To investigate the in vivo function of HF, we generated transgenic (tg) mice overexpressing the human HF gene (hHF-tg). We established two independent hHF-tg mouse lines. Although both lines of hHF-tg mice were viable, they showed reduced body size compared to wild-type (WT) mice at 4-12 weeks of age. Serum iron concentration and blood parameters of hHF-tg mice such as hemoglobin and red blood cell counts were comparable to those of WT mice. At 3-5 weeks of age, hHF-tg mice exhibited temporary loss of coat hair on the trunk, but not on the head or face. Histological analyses revealed that although initial hair development was normal, hHF-tg mice had epidermal hyperplasia with hyperkeratosis, dilated hair follicles, bended hair shafts and keratinous debris during the hairless period. In conclusion, we showed that hHF-tg mice exhibited mild growth retardation and temporary hairless phenotype. Our findings highlight the physiological roles of HF and demonstrate that hHF-tg mice are useful for understanding the in vivo functions of HF.

H-ferritin overexpression promotes radiation-induced leukemia/lymphoma in mice.

H-ferritin (HF) is a core subunit of the iron storage protein ferritin and is related to the pathogenesis of malignant diseases. HF overexpression is present in human hematologic malignancies, suggesting that HF overexpression may contribute to the development of hematologic cancers. However, in vivo evidence that HF is directly linked to hematologic tumorigenesis has not yet been shown. In this study, we show that transgenic (tg) mice overexpressing the human HF gene (hHF-tg) developed aggressive radiation-induced thymic lymphoma/leukemia (TL) compared with wild-type (WT) mice, providing evidence that HF overexpression promotes leukemia/lymphomagenesis. Fractionated X-irradiation of hHF-tg mice caused a higher incidence and earlier onset of TL compared with WT mice. Immunological and pathological features of TLs were similar in both groups. However, proliferative activity of hHF-tg lymphoma cells was higher than that of WT lymphoma cells, and microarray analyses revealed that some leukemia/lymphoma-related genes were differentially expressed in hHF-tg TLs compared with WT TLs. To investigate whether cell damage induced by irradiation is related to leukemia/lymphomagenesis, we evaluated apoptotic levels in the thymus and bone marrow (BM) of hHF-tg and WT groups after fractionated X-irradiation. Apoptosis was augmented in the hHF-tg BM, but not in the thymus, compared with the WT BM, suggesting a possible linkage between increased BM apoptosis by HF overexpression and accelerated radiation-induced TL development. Our findings indicate that HF overexpression is closely related to the development of leukemia/lymphoma, which could have implications for the prevention of malignant hematologic diseases.

A medaka model of cancer allowing direct observation of transplanted tumor cells in vivo at a cellular-level resolution.

The recent success with small fish as an animal model of cancer with the aid of fluorescence technique has attracted cancer modelers' attention because it would be possible to directly visualize tumor cells in vivo in real time. Here, we report a medaka model capable of allowing the observation of various cell behaviors of transplanted tumor cells, such as cell proliferation and metastasis, which were visualized easily in vivo. We established medaka melanoma (MM) cells stably expressing GFP and transplanted them into nonirradiated and irradiated medaka. The tumor cells were grown at the injection sites in medaka, and the spatiotemporal changes were visualized under a fluorescence stereoscopic microscope at a cellular-level resolution, and even at a single-cell level. Tumor dormancy and metastasis were also observed. Interestingly, in irradiated medaka, accelerated tumor growth and metastasis of the transplanted tumor cells were directly visualized. Our medaka model provides an opportunity to visualize in vivo tumor cells "as seen in a culture dish" and would be useful for in vivo tumor cell biology.

Favorable tumor uptake and nuclear transport of Auger electrons by nuclear targeting with 111In-trastuzumab in an intraperitoneal tumor mouse model.

OBJECTIVES: The 111In-labeled anti-HER2 antibody trastuzumab modified with a nuclear-localizing sequence (NLS) peptide (111In-trastuzumab-NLS) is a radiopharmaceutical candidate for Auger electron radioimmunotherapy (AE-RIT). However, in-vivo action of 111In-trastuzumab-NLS is poorly understood in intraperitoneal tumors. We aimed to elucidate the nuclear targeting activity of 111In-trastuzumab-NLS in a mouse model of intraperitoneal tumors. METHODS: Trastuzumab, trastuzumab-NLS-S with shorter NLS peptides, and trastuzumab-NLS-L with longer NLS peptides were tested in an intraperitoneal tumor xenograft. The AE-emitting radionuclide 111In was labeled with these antibodies. The cell-binding activity, nuclear importation, and cytotoxicity of those radiolabeled antibodies were examined in human cancer cell lines. Analyses of the biodistribution and in-vivo nuclear importation of 111In were conducted in a mouse model. RESULTS: The two111In-trastuzumab-NLS variants delivered the radionuclide into the nucleus more efficiently and had a comparable cytotoxicity to 111In-trastuzumab against human gastric cancer cells, although had a lower cell binding affinity. 111In-trastuzumab-NLS-L exhibited both a superior tumor uptake and in vivo nuclear transportation of the radionuclide than 111In-trastuzumab. CONCLUSION: Nuclear targeting using 111In-trastuzumab-NLS promotes a more efficient tumor cell uptake and subsequent nuclear translocation of the 111In AE-emitting radionuclide in vivo. This radio-immunoconjugate will likely be an effective agent for HER2-targeting by AE-RIT.

Estimation of biological effect of Cu-64 radiopharmaceuticals with Geant4-DNA simulation.

The aim of this work is to estimate the biological effect of targeted radionuclide therapy using Cu-64, which is a well-known Auger electron emitter. To do so, we evaluate the absorbed dose of emitted particles from Cu-64 using the Geant4-DNA Monte Carlo simulation toolkit. The contribution of beta particles to the absorbed dose is higher than that of Auger electrons. The simulation result agrees with experimental ones evaluated using coumarin-3-carboxylic acid chemical dosimeter. The simulation result is also in good agreement with previous ones obtained using fluorescent nuclear track detector. From the results of present simulation (i.e., absorbed dose estimation) and previous biological experiments using two cell lines (i.e., evaluation of survival curves), we have estimated the relative biological effectiveness (RBE) of Cu-64 emitted particles on CHO wild-type cells and xrs5 cells. The RBE of xrs5 cells exposed to Cu-64 is almost equivalent to that with gamma rays and protons and C ions. This result indicates that the radiosensitivity of xrs5 cells is independent of LET. In comparison to this, the RBE on CHO wild-type cells exposed to Cu-64 is significantly higher than gamma rays and almost equivalent to that irradiated with C ions with a linear energy transfer of 70 keV/µm.

Phase Ib study of durvalumab (MEDI4736) in combination with carbon-ion radiotherapy and weekly cisplatin for patients with locally advanced cervical cancer (DECISION study): study protocol for a prospective open-label single-arm study.

INTRODUCTION: Concurrent chemoradiotherapy is considered the standard treatment strategy for locally advanced cervical cancer. Most recent reports indicate that patients with bulky tumours or adenocarcinoma subtypes have poorer local control. Carbon-ion radiotherapy (CIRT) with the concurrent use of chemotherapy has shown promising results in such cases of difficult-to-treat uterine cervical cancer. Programmed death-ligand 1 (PD-L1) upregulation was observed in tumour tissue samples from patients who had undergone CIRT. Thus, a combination of CIRT and anti-PD-L1 antibody may suppress metastasis by activating antitumour immune response, in addition to exhibiting strong local effects. OBJECTIVE: We will assess the safety and tolerability (primary endpoint) of the concomitant use of durvalumab, an anti-PD-L1 antibody, with CIRT and weekly cisplatin for locally advanced cervical cancer. METHODS AND ANALYSIS: This study is a non-randomised, open-label, prospective phase 1b study. Up to 10 patients with histologically proven uterine cervical cancer at stage IIB, IIIA, IIIB, IIIC1 or IVA as per International Federation of Gynecology and Obstetrics (2018) staging will be enrolled. All patients will receive CIRT of 74.4 Gy relative biological effectiveness in 20 fractions over 5 weeks (four fractions per week). Weekly cisplatin at a dose of 40 mg/m2 will be administrated up to five times. Durvalumab at a dose of 1500 mg/body will be administrated at weeks 2 and 6. Safety and tolerability will be evaluated based on the frequency of dose-limiting toxicities until 92 days after CIRT starts. Patients will be followed-up strictly as per the scheduled protocol for 1 year after CIRT initiation. ETHICS AND DISSEMINATION: The Human Research Ethics Committees of QST Hospital (#C21-002) and Chiba University (#2021006) have approved this study protocol. The findings will be published in peer-reviewed journals and presented at scientific conferences. TRIAL REGISTRATION NUMBER: Japan Registry of Clinical Trials (jRCT2031210083), registered on 12 May 2021.

Molecular imaging of mesothelioma by detection of manganese-superoxide dismutase activity using manganese-enhanced magnetic resonance imaging.

Malignant mesothelioma (MM) is a fatal malignancy with a rapidly increasing incidence in industrialized countries because of the widespread use of asbestos in the past centuries. Early diagnosis of MM is critical for a better prognosis, but this is often difficult because of the lack of disease-specific diagnostic imaging. Here, we report that manganese-enhanced magnetic resonance imaging (MEMRI) represents a promising approach for a more selective mesothelioma imaging by monitoring a high-level expression of manganese-superoxide dismutase (Mn-SOD), which is observed in many MM. We found that most human MM cells overexpressed Mn-SOD protein compared with human mesothelial cells and that NCI-H226 human MM cells highly expressed Mn-SOD and augmented Mn accumulation when loaded with manganese chloride (MnCl(2)). The cells showed marked T(1)-signal enhancement on in vitro MRI after incubation with MnCl(2) because of the T(1) shortening effect of Mn(2+). H226 subcutaneous tumor was preferentially enhanced compared with a lung adenocarcinoma cell tumor and another human MM cell tumor in MnCl(2)-enhanced T(1)-weighted MR image (T(1)WI), correlating with their respective Mn-SOD expression levels. Moreover, in a more clinically relevant setting, H226 xenografted pleural tumor was markedly enhanced and readily detected by MEMRI using manganese dipyridoxyl diphosphate (MnDPDP), a clinically used contrast agent, as well as MnCl(2). Therefore, we propose that MEMRI can be a potentially powerful method for noninvasive detection of MM, with high spatial resolution and marked signal enhancement, by targeting Mn-SOD.

Radiation-Induced Autophagy in Human Pancreatic Cancer Cells is Critically Dependent on G2 Checkpoint Activation: A Mechanism of Radioresistance in Pancreatic Cancer.

PURPOSE: Autophagy and cell-cycle checkpoints act in concert to confer cellular radioresistance. We investigated the functional interaction between radiation-induced autophagy and G2 checkpoint activation in highly radioresistant human pancreatic ductal adenocarcinoma (PDAC) cells. METHODS AND MATERIALS: Four human PDAC cell lines (MIA PaCa-2, KP-4, Panc-1, and SUIT-2) were analyzed. These cells were first irradiated using x-rays, and their cell cycle status, autophagy, and cell cycle checkpoint marker expression and ATP production levels were evaluated. Autophagic flux assays and siRNA knockdown were used to evaluate autophagy activity. Double thymidine block experiments were performed to synchronize the cells. Two inhibitors (MK-1775 and SCH 900776) were used to attenuate G2 checkpoint activation. Cell survival assays and animal experiments were performed to evaluate the radiosensitizing effects of the G2 checkpoint inhibitors. RESULTS: Autophagy and G2/M accumulation were synchronously induced in human PDAC cells with an activated G2 checkpoint at 12 hours after x-ray irradiation of 6 Gy. Radiation-induced autophagy produced the ATP levels required for cell survival. Double thymidine block experiments revealed that no autophagy occurred in cells that were solely in G2 phase. MK-1775 or SCH 900776 exposure attenuated not only G2 checkpoint activation but also postirradiation autophagy, indicating the dependence of radiation-induced autophagy on an activated G2 checkpoint. The inhibitors demonstrated a higher radiosensitizing effect in the PDAC cells than the autophagy inhibitor chloroquine. MK-1775 in combination with x-rays significantly suppressed the tumor growth of MIA PaCa-2 xenografts compared with other treatment groups, including radiation or drug exposure alone, to enhance the radiosensitivity of PDAC cells in vivo. CONCLUSIONS: Biological crosstalk exists between the G2 checkpoint activation and radiation-induced autophagy processes that are believed to independently contribute to the radioresistance of human PDAC cells. These findings have important implications for the development of future radiation therapy strategies for PDAC.

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.

Utility of 211At-Trastuzumab for the Treatment of Metastatic Gastric Cancer in the Liver: Evaluation of a Preclinical α-Radioimmunotherapy Approach in a Clinically Relevant Mouse Model.

A liver metastasis from a primary gastric cancer (LMGC) is relatively common and results in an extremely poor prognosis due to a lack of effective therapeutics. We here demonstrate in a clinically relevant mouse model that an α-particle radioimmunotherapy approach with 211At-labeled trastuzumab has efficacy against LMGCs that are positive for human epidermal growth factor receptor 2 (HER2). Methods:211At was produced in a cyclotron via a 209Bi (α,2n) 211At reaction. 211At-trastuzumab was subsequently generated using a single-step labeling method. NCI-N87 cells (HER2-positive human gastric cancer cells) carrying a luciferase gene were intrasplenically transplanted into severe combined immunodeficiency mice to generate an HER2-positive LMGC model. A biodistribution study was then conducted through the intravenous injection of 211At-trastuzumab (1 MBq) into these LMGC xenograft mice. In parallel with this experimental therapy, phosphate-buffered saline, intact trastuzumab, or 211At-nonspecific human IgG (1 MBq) was injected into control groups. The therapeutic efficacy was evaluated by monitoring tumor changes by chemiluminescence imaging. Body weights, white blood cell counts, and serum markers of tissue damage were monitored at regular intervals. Microdosimetry using a CR-39 plastic detector was also performed. Results: The biodistribution analysis revealed an increased uptake of 211At-trastuzumab in the metastasized tumors that reached approximately 12% of the injected dose per gram of tissue (%ID/g) at 24 h. In contrast, its uptake to the surrounding liver was about 4 %ID/g. The LMGCs in the mouse model reduced dramatically at 1 wk after the single systemic injection of 211At-trastuzumab. No recurrences were observed in 6 of 8 mice treated with this single injection, and their survival time was significantly prolonged compared with the control groups, including the animals treated with 211At-nonspecific antibodies. No severe toxicities or abnormalities in terms of body weight, white blood cell number, liver function, or kidney parameters were observed in the 211At-trastuzumab group. Microdosimetric studies further revealed that 211At-trastuzumab had been delivered at an 11.5-fold higher dose to the LMGC lesions than to the normal liver. Conclusion: α-radioimmunotherapy with 211At-trastuzumab has considerable potential as an effective and safe therapeutic option for LMGC.

131I-IITM and 211At-AITM: Two Novel Small-Molecule Radiopharmaceuticals Targeting Oncoprotein Metabotropic Glutamate Receptor 1.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed 2 new radiopharmaceuticals, 4-131I-iodo- and 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (131I-IITM and 211At-AITM), targeting the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanomas for TRT studies. Methods:131I-IITM and 211At-AITM were synthesized by reacting a stannyl precursor with 131I-NaI and 211At in the presence of an oxidizing agent. The therapeutic efficacy and safety of the 2 radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results:131I-IITM and 211At-AITM were obtained with a radiochemical purity of greater than 99% and radiochemical yields of 42.7% ± 10.4% and 45.7% ± 6.5%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited a maximum uptake of 4.66% ± 0.70 and 7.68% ± 0.71 percentage injected dose per gram (%ID/g) in the targeted melanomas, respectively, and were rapidly cleared from nontarget organs after intravenous injection. Both agents markedly inhibited melanoma growth compared with the controls (61.00% and 95.68%, respectively). In the melanoma model, considerably greater therapeutic efficacy with negligible toxicity was observed using 211At-AITM. Conclusion: The nontoxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells.

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

Significance of PD-L1 expression in carbon-ion radiotherapy for uterine cervical adeno/adenosquamous carcinoma.

OBJECTIVE: Programmed cell death-ligand 1 (PD-L1) is expressed in tumor cells and has been shown to predict clinical outcomes of several types of malignancies. The aim of this study was to investigate the effects of carbon-ion (C-ion) beam irradiation on PD-L1 expression in human uterine cervical adeno/adenosquamous carcinoma (UCAA) cells and clinical samples and to identify the prognostic factors for outcomes after C-ion radiotherapy (CIRT). METHODS: The effects of C-ion irradiation on PD-L1 expression in human UCAA and cervical squamous cell carcinoma cells were examined by flow cytometry. We examined PD-L1 expression in UCAA biopsy specimens from 33 patients before CIRT started (pre-CIRT) and after 12 Gy (relative biological effectiveness [RBE]) irradiation (post-12Gy-C) in 4 fractions of CIRT to investigate the correlation between PD-L1 status and clinical outcomes. RESULTS: The PD-L1 expression was upregulated by C-ion beam in a dose-dependent manner in HeLa and SiHa cells through phosphorylated Chk1. The overall frequencies of pre-CIRT and post-12Gy-C PD-L1 positivity were 45% (15/33) and 67% (22/33), respectively. The post-12Gy-C PD-L1 expression was significantly elevated compared to the pre-CIRT PD-L1 expression. There was no significant relationship between the pre-CIRT PD-L1 status and clinical outcomes, such as local control (LC), progression-free survival (PFS), and overall survival (OS). However, the post-12Gy-C PD-L1 expression had better correlation with PFS, but not with LC and OS. CONCLUSION: CIRT can induce PD-L1 expression in UCAA and we propose that PD-L1 expression after starting CIRT may become as a predictive prognostic marker in CIRT for UCAA.