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The strong cell killing effect of high linear energy transfer (LET) carbon ions is dependent on lethal DNA damage. Our recent studies suggest that induction of clusters of double-strand breaks (DSBs) in close proximity is one of the potential mechanisms. However, the relationship between LET, the degree of DSB clustering and the cell killing effect of carbon ions remains unclear. Here, we used high-resolution imaging technology to analyze the volume of γH2AX foci induced by monoenergetic carbon ions with a clinically-relevant range of LET (13–100 keV/μm). We obtained data from 3317 γH2AX foci and used a gaussian function to approximate the probability (p) that 1 Gy-carbon ions induce γH2AX foci of a given volume (vth) or greater per nucleus. Cell killing effects were assessed in clonogenic assays. The cell killing effect showed high concordance with p at vth = 0.7 μm3 across various LET values; the difference between the two was 4.7% ± 2.2%. This relationship was also true for clinical carbon ion beams harboring a mixed LET profile throughout a spread-out Bragg peak width (30–120 mm), with the difference at vth = 0.7 μm3 being 1.6% ± 1.2% when a Monte Carlo simulation-derived dose-averaged LET was used to calculate p. These data indicate that the cell killing effect of carbon ions is predictable by the ability of carbon ions to induce γH2AX foci containing clustered DSBs, which is linked to LET, providing the biological basis for LET modulation in the planning of carbon ion radiotherapy.

Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

The promising results of the PACIFIC study led to the approval of consolidation durvalumab for coverage by the National Health Insurance (NHI) in 2018 for patients with locally-advanced unresectable non-small cell lung carcinoma (NSCLC) treated with definitive concurrent chemoradiotherapy (CCRT). However, the effect of NHI coverage on the patterns of care for this population remains unclear. Here, we conducted a questionnaire-based survey to determine the patterns of care for patients with stage II-III NSCLC treated with definitive radiotherapy in 2017 (pre-durvalumab era) or in 2019 (post-durvalumab era). Data were obtained from 11 radiotherapy facilities in Gunma prefecture, which has a population of 1.94 million. We identified 80 and 83 patients with stage II-III NSCLC who received definitive radiotherapy in Gunma in 2017 and 2019, respectively. At a given facility, CCRT was the treatment of choice in a significantly greater proportion of patients in 2019 than in 2017 (66% ± 20% vs 51% ± 29%, P = 0.041). Intensity-modulated radiotherapy (IMRT) was more frequent in 2019 than in 2017 (24% vs 1.2%). Carboplatin plus paclitaxel was used for CCRT at higher rate in 2019 than in 2017 (73% vs 44%). Consolidation durvalumab was performed in 73% (40/55) of CCRT-treated patients in 2019, and the treatment was performed for the planned 12 months in 45% (18/40) of patients. These data indicate that NHI coverage of durvalumab might be a possible reason for choosing CCRT in patients with stage II-III NSCLC in the real-world setting.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

Photons, such as X- or γ-rays, induce DNA damage (distributed throughout the nucleus) as a result of low-density energy deposition. In contrast, particle irradiation with high linear energy transfer (LET) deposits high-density energy along the particle track. High-LET heavy-ion irradiation generates a greater number and more complex critical chromosomal aberrations, such as dicentrics and translocations, compared with X-ray or γ irradiation. In addition, the formation of >1000 bp deletions, which is rarely observed after X-ray irradiation, has been identified following high-LET heavy-ion irradiation. Previously, these chromosomal aberrations have been thought to be the result of misrepair of complex DNA lesions, defined as DNA damage through DNA double-strand breaks (DSBs) and single-strand breaks as well as base damage within 1–2 helical turns (

Herein, we investigate the long-term clinical outcomes for cervical cancer patients treated with in-room computed tomography–based brachytherapy. Eighty patients with Stage IB1–IVA cervical cancer, who had undergone treatment with combined 3D high-dose rate brachytherapy and conformal radiotherapy between October 2008 and May 2011, were retrospectively analyzed. External beam radiotherapy (50 Gy) with central shielding after 20–40 Gy was performed for each patient. Cisplatin-based chemotherapy was administered concurrently to advanced-stage patients aged ≤75 years. Brachytherapy was delivered in four fractions of 6 Gy per week. In-room computed tomography imaging with applicator insertion was performed for treatment planning. Information from physical examinations at diagnosis, and brachytherapy and magnetic resonance imaging at diagnosis and just before the first brachytherapy session, were referred to for contouring of the high-risk clinical target volume. The median follow-up duration was 60 months. The 5-year local control, pelvic progression-free survival and overall survival rates were 94%, 90% and 86%, respectively. No significant differences in 5-year local control rates were observed between Stage I, Stage II and Stage III–IVA patients. Conversely, a significant difference in the 5-year overall survival rate was observed between Stage II and III–IVA patients (97% vs 72%; P = 0.006). One patient developed Grade 3 late bladder toxicity. No other Grade 3 or higher late toxicities were reported in the rectum or bladder. In conclusion, excellent local control rates were achieved with minimal late toxicities in the rectum or bladder, irrespective of clinical stage.

Anti-tumor immunity modulates the local effects of radiation therapy. High mobility group box 1 (HMGB1) plays a pivotal role in activating antigen-specific T-cell responses. Here, we examined the relationship between linear energy transfer (LET) and HMGB1 release. We assessed the proportions of KYSE-70, HeLa and SiHa cells surviving after carbon-ion (C-ion) beam irradiation with different LET values, using a clonogenic assay. The D10, the dose at which 10% of cells survived, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants of C-ion beam-irradiated tumor cells were assessed by enzyme-linked immunosorbent assay. The D10 doses for 13 keV/μm of C-ion irradiation in KYSE-70, HeLa and SiHa cells were 2.8, 3.9 and 4.1 Gy, respectively, whereas those for 70 keV/μm C-ion irradiation were 1.4, 1.9 and 2.3 Gy, respectively. We found that 70 keV/μm of C-ion irradiation significantly increased HMGB1 levels in the culture supernatants of all cell lines 72 h after irradiation compared with non-irradiated controls. Furthermore, 70 keV/μm of C-ion irradiation significantly increased HMGB1 levels in the culture supernatants of all cell lines 72 h after irradiation compared with 13 keV/μm. The results suggest that HMGB1 release from several cancer cell lines increases with increased LET.

Carbon-ion radiotherapy (CIRT) holds promise in the treatment of glioblastoma, an aggressive X-ray-resistant brain tumor. However, since glioblastoma cells show a highly invasive nature, carbon-ion (C-ion) irradiation of normal tissues surrounding the tumor is inevitable. Recent studies have revealed the existence of neural stem cells in the adult brain. Therefore, the damaging effect of C-ion beams on the neural stem cells has to be carefully considered in the treatment planning of CIRT. Here, we investigated the growth and death mode of human neural stem cells (hNSCs) and glioblastoma A172 cells after X-ray or C-ion beam irradiation. The X-ray dose resulting in a 50% growth rate (D(50)) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D(50) for C-ion beams was 0.4 Gy in hNSCs and 1.6 Gy in A172 cells; the relative biological effectiveness value of C-ion beams was 2.0 in hNSCs and 1.9 in A172 cells. Importantly, both X-rays and C-ion beams preferentially induced apoptosis, not necrosis, in hNSCs; however, radiation-induced apoptosis was less evident in A172 cells. The apoptosis-susceptible nature of the irradiated hNSCs was associated with prolonged upregulation of phosphorylated p53, whereas the apoptosis-resistant nature of A172 cells was associated with a high basal level of nuclear factor kappa B expression. Taken together, these data indicate that apoptosis is the major cell death pathway in hNSCs after irradiation. The high sensitivity of hNSCs to C-ion beams underscores the importance of careful target volume delineation in the treatment planning of CIRT for glioblastoma.

X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear-quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams.

The present study investigated the ability of carboplatin and paclitaxel to sensitize human non-small-cell lung cancer (NSCLC) cells to carbon-ion beam irradiation. NSCLC H460 cells treated with carboplatin or paclitaxel were irradiated with X-rays or carbon-ion beams, and radiosensitivity was evaluated by clonogenic survival assay. Cell proliferation was determined by counting the number of viable cells using Trypan blue. Apoptosis and senescence were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and senescence-associated β-galactosidase (SA-β-gal) staining, respectively. The expression of cleaved caspase-3, Bax, p53 and p21 was analyzed by western blotting. Clonogenic survival assays demonstrated a synergistic radiosensitizing effect of carboplatin and paclitaxel with carbon-ion beams; the sensitizer enhancement ratios (SERs) at the dose giving a 10% survival fraction (D10) were 1.21 and 1.22, respectively. Similarly, carboplatin and paclitaxel showed a radiosensitizing effect with X-rays; the SERs were 1.41 and 1.29, respectively. Cell proliferation assays validated the radiosensitizing effect of carboplatin and paclitaxel with both carbon-ion beam and X-ray irradiation. Carboplatin and paclitaxel treatment combined with carbon-ion beams increased TUNEL-positive cells and the expression of cleaved caspase-3 and Bax, indicating the enhancement of apoptosis. The combined treatment also increased SA-β-gal-positive cells and the expression of p53 and p21, indicating the enhancement of senescence. In summary, carboplatin and paclitaxel radiosensitized H460 cells to carbon-ion beam irradiation by enhancing irradiation-induced apoptosis and senescence.

This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.

Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy.

To compare the incidence and degree of hematological toxicity between innovator and generic cisplatin formulations, decreases in white blood cell (WBC) count (leukopenia) and platelet counts (thrombocytopenia) were retrospectively examined, using the Common Toxicity Criteria for Adverse Events ver. 4.0, in patients with uterine cervical cancer treated with concurrent chemoradiotherapy using innovator (innovator group, n = 22) or generic (generic group, n = 22) cisplatin formulations. There were no significant differences in patient characteristics except in the technique of external irradiation; larger numbers of patients in the innovator and generic groups were irradiated using the parallel-opposed two-field technique and the four-field box technique, respectively (P = 0.00012), which is in line with the historical progress of external beam radiation therapy. The numbers of patients showing Grade 1, 2, 3 and 4 leukopenia were 1 (4.5%), 14 (64%), 7 (32%) and 0 (0.0%) in the innovator group, and 1 (4.5%), 6 (27%), 13 (59%) and 2 (9.0%) in the generic group, respectively. The number of patients showing Grade 3–4 leukopenia was significantly greater in the generic group than in the innovator group (P = 0.034). There was no significant relationship between the incidence of Grade 3–4 leukopenia and the technique of external irradiation. There were no significant differences in the incidence and degree of thrombocytopenia between the two groups. These results indicate the possibility that the generic cisplatin formulation may have a different toxicity profile compared to the innovator formulation in terms of the incidence of leukopenia.

Uterine cervical cancer/Radiotherapy/Small bowel/Pelvic insufficiency fracture/Common iliac lymph node region. The purpose of this study was to compare dose red uction to the small bowel and sacral bone by twofield and four-field techniques when the common iliac lymph node region is excluded from the radiation field in external beam radiotherapy of uterine cervical cancer. Thirteen patients with cervical cancer were entered into the study. Conventional treatment plans based on bony landmarks were made with parallelopposed two-field technique (C2F) and four-field box technique (C4F). Modified C2F (M2F) and C4F (M4F) plans of excluding the common iliac lymph node region from the conventional radiation fields were created in reference to the bifurcations of pelvic arteries in computed tomography images. For each patient, the dose volume histograms for the small bowel and sacral bone resulting from the C2F, C4F, M2F, and M4F plans were compared. The volumes were obtained at 10 levels of prescribed dose, at increments of 10%, from 5 Gy to 50 Gy. By sparing both small bowel and sacral bone, the M2F and M4F plans were s ignificantly better than the C2F and C4F plans at any dose level (p < 0.05), respectively. In addition, the M4F plan was significantly better than the M2F plan in sparing both small bowel at 10–50% of the prescribed dose (p < 0.05) and sacral bone at 40–100% of the prescribed dose (p < 0.05). The present study suggests that modified treatment planning could be useful for selected patients for reducing small bowel complications and insufficiency fracture after radiotherapy.

The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D(10) values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D(10) values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D(10) values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer.