Low Linear Energy Transfer X-rays Research Articles

A Novel Approach to Support Quality Assurance (QA) of Intensity Modulated Neutron Therapy (IMNT)

Neutron therapy is a form of high linear energy transfer (LET) radiation treatment shown to be beneficial for the treatment of locally advanced head and neck cancers (HNC) resistant to low LET x-ray and proton therapy treatments. The Clinical Neutron Therapy System (CNTS) at our institution has been in clinical operation for over 35 years, and over 3,400 patients have been treated using 3D conformal neutron radiation therapy. In October of 2022, the clinical commissioning of IMNT was completed and the first-ever patient was treated. A novel patient specific quality assurance (PSQA) program has been developed to support IMNT. We present an analysis of our early experiences and PSQA findings for the first 16+ patients treated with IMNT. Our pre-treatment IMNT PSQA program includes (1) ionization chamber measurements, (2) a log-file analysis of treatment delivery, and (3) a γ-analysis of the expected and measured doses (fluences) from a novel positron emission portal imaging system based on 12C(n,2n)11C reactions. Patient setup is confirmed on a daily basis with kV portal imaging. We use a modulation factor (MF), defined as the total number of monitor units (MU)/prescribed dose (cGy) per fraction, to identify and help anticipate IMNT plans that may fail our PSQA program. We have found that IMNT treatments for large, irregularly shaped treatment volumes (e.g., chest wall or head and neck cancers that require treatment of nodes in the lower neck) are more likely to fail PSQA when the MF exceeds 3 MU/cGy. For smaller, more spherical treatment volumes (e.g., base of tongue cancers), plans with a MF up to 3.5 MU/cGy pass our PSQA criteria. Re-optimized plans subsequently passed PSQA with insignificant or no change in tumor coverage and organ-at-risk (OAR) dose. Re-optimized plans also reduced the total number of MU and number of segments, which has the added benefit of decreasing the overall treatment time and patient time on the table. About 10% of early IMNT patients have required plan re-optimization. Our pre-treatment clinical PSQA program and workflow provides useful information to guide IMNT treatment planning and delivery, and helps ensure the safe and accurate delivery of IMNT. Our early experiences suggest IMNT plans with smaller MF values are more likely to pass PSQA than plans with larger values of the MF.

Preliminary study on radiosensitivity to carbon ions in human breast cancer.

The aim of the study was to investigate the various effects of high linear energy transfer (LET) carbon ion (12C6+) and low LET X-ray radiation on MDA-MB-231 and MCF-7 human breast cancer cells and to explore the underlying mechanisms of radiation sensitivity. Cell proliferation, cell colony formation, cell cycle distribution, cell apoptosis and protein expression levels [double-strand break marker γ-H2AX, cell cycle-related protein cyclin B1, apoptosis-related proteins Bax and Bcl-2, and the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase B1 (p70S6K) pathway] were detected after irradiation with carbon ions or X-rays at doses of 0, 2, 4 and 8 Gy. Our results showed that the inhibition of cell proliferation and cell colony formation and the induction of G2/M phase arrest, DNA lesions and cell apoptosis/necrosis elicited by carbon ion irradiation were more potent than the effects elicited by X-ray radiation at the same dose. Simultaneously, compared with X-ray radiation, carbon ion radiation induced a marked increase in Bax and prominent decreases in cyclin B1 and Bcl-2 in a dose-dependent manner. Furthermore, the Akt/mTOR/p70S6K pathway was significantly inhibited by carbon ion radiation in both breast cancer cell lines. These results indicate that carbon ion radiation kills MDA-MB-231 and MCF-7 breast cancer cells more effectively than X-ray radiation, which might result from the inhibition of the Akt/mTOR/p70S6K pathway.

Genomic Instability and Carcinogenesis of Heavy Charged Particles Radiation: Clinical and Environmental Implications.

One of the uses of ionizing radiation is in cancer treatment. The use of heavy charged particles for treatment has been introduced in recent decades because of their priority for deposition of radiation energy in the tumor, via the Bragg peak phenomenon. In addition to medical implications, exposure to heavy charged particles is a crucial issue for environmental and space radiobiology. Ionizing radiation is one of the most powerful clastogenic and carcinogenic agents. Studies have shown that although both low and high linear energy transfer (LET) radiations are carcinogenic, their risks are different. Molecular studies have also shown that although heavy charged particles mainly induce DNA damage directly, they may be more potent inducer of endogenous generation of free radicals compared to the low LET gamma or X-rays. It seems that the severity of genotoxicity for non-irradiated bystander cells is potentiated as the quality of radiation increases. However, this is not true in all situations. Evidence suggests the involvement of some mechanisms such as upregulation of pro-oxidant enzymes and change in the methylation of DNA in the development of genomic instability and carcinogenesis. This review aimed to report important issues for genotoxicity of carcinogenic effects of heavy charged particles. Furthermore, we tried to explain some mechanisms that may be involved in cancer development following exposure to heavy charged particles.

High Linear Energy Transfer Particle Irradiation Leads to Increased Expression of In Vitro Markers of Immunogenic Cell Death

Radiotherapy (RT) fosters a form of tumor cell death that results in dendritic cell activation, effector T-cell cross priming/tumor trafficking, and the formation of a tumor cell/T-cell immune response. This type of immunogenicity is comprised of the release of immune modulating signals, which include the release of high mobility group box 1 (HMGB1), ATP, and the translocation of calreticulin to the cell surface. Previous studies have demonstrated a positive correlation between markers of immunogenic cell death (ICD) and radiation dose with X-rays. Less is known about the effect of higher energies using charged particle irradiation and ICD. In this study, we explore whether varying linear energy transfer (LET) greater than 60 keV/μm with helium ion irradiation would impact immunogenicity in tumor cells as measured by levels of calreticulin translocation, ATP release, and HMGB1 release. Reporter cell lines designed to detect ATP luminescence (RLUs), red fluorescence protein tagged HMGB1 (relative fluorescent units [RFUs]), and cell surface calreticulin (percentage of calreticulin positive cells [%CRT+]) were derived from TSA (murine mammary carcinoma; syngeneic to BALB/c mice) cells. Prior to irradiation, the cells were seeded on six micron thick Mylar attached to 1.5 cm metal rings. The cells were then irradiated at the Radiological Research Accelerator Facility (RARAF) through a Track Segment Charged-Particle Irradiator. ATP and HMGB1 levels were measured 72 hours after RT and calreticulin was measured 24 hours after RT. Various doses of 250kV X-rays and LETs of helium ions were delivered and levels of these tumor ICD markers were determined. For ATP measurements, when compared to non-irradiated controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in RLU fold-changes of 1.00 ± 0.05 (non-irradiated), 2.33 ± 0.08, 3.27 ± 0.19, 3.24 ± 0.10, 3.99 ± 0.33, 4.12 ± 0.32, and 2.91 ± 0.16 respectively. For HMGB1 measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in RFU fold-changes of 1.00 ± 0.02 (non-irradiated), 1.43 ± 0.03, 1.72 ± 0.03, 1.97 ± 0.06, 1.64 ± 0.03, 1.61 ± 0.04, and 1.51 ± 0.03, respectively. For calreticulin measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/μm helium ions resulted in %CRT+ fold-changes of 1.00 ± 0.12 (non-irradiated), 2.32 ± 0.15, 3.07 ± 0.32, 3.16 ± 0.17, 3.01 ± 0.12, 2.74 ± 0.39, and 1.61 ± 0.15, respectively. Immunogenicity of tumor cells as measured by immune cell death signals increases with high LET ion irradiation when compared to low LET X-ray irradiation at the same dose. These marker levels appear to decrease at LETs greater than 110 keV/μm. Further investigation is warranted on the effects of high LET charged particle irradiation on the tumor immune response.

Contribution of caspase-independent pathway to apoptosis in malignant glioma induced by carbon ion beams.

High linear energy transfer (LET) carbon ion beam (CIB) is becoming the best tool for external radiotherapy of inoperable tumors because of its greater cell killing than conventional low LET gamma or X-rays. In the present study, whether the caspase-independent pathway exerts the important contribution in CIB-induced cell apoptosis was explored. Herein we showed, despite the absence of caspase activity using a pan caspase inhibitor Z-VAD-FMK, that apoptosis induced by high LET CIB were clearly observed in the glioma cells. Simultaneously, the increased 8-OHdG level, PARP-1 activity and AIF translocation occurred in response to CIB irradiation. Moreover, it was distinctly higher in the nuclear translocation frequency along with PARP-1 activation when the caspase protease cascade was suppressed in the irradiated glioma cells. Nuclear colocalization between PARP-1 and AIF as well as a positive association of the PARP-1 mRNA expression with AIF translocation frequency indicated that PARP-1 activation controlled the translocation of AIF to the nucleus. Our findings strongly demonstrated that caspase-independent cell apoptosis provided a prominent compensation in the glioma cell death involving the PARP-1/AIF signaling pathway at 24h after CIB exposure, and likely triggered by oxidative damage to DNA. The knowledge on the molecular mechanism of AIF-mediated cell death may be very useful for the improvement of the therapeutic efficacy of malignant gliomas with heavy charged particles.

Apoptosis and injuries of heavy ion beam and x-ray radiation on malignant melanoma cell.

This study aims to investigate the influence of high linear energy transfer (LET) heavy ion (12C6+) and low LET X-ray radiation on apoptosis and related proteins of malignant melanoma on tumor-bearing mice under the same physical dosage. C57BL/6 J mice were burdened by tumors and randomized into three groups. These mice received heavy ion (12C6+) and X-ray radiation under the same physical dosage, respectively; their weight and tumor volumes were measured every three days post-radiation. After 30 days, these mice were sacrificed. Then, median survival time was calculated and tumors on mice were proliferated. In addition, immunohistochemistry was carried out for apoptosis-related proteins to reflect the expression level. After tumor-bearing mice were radiated to heavy ion, median survival time improved and tumor volume significantly decreased in conjunction with the upregulated expression of pro-apoptosis factors, Bax and cytochrome C, and the downregulated expression of apoptosis-profilin (Bcl-2, Survivin) and proliferation-related proteins (proliferating cell nuclear antigen). The results indicated that radiation can promote the apoptosis of malignant melanoma cells and inhibit their proliferation. This case was more suitable for heavy ion (12C6+). High LET heavy ion (12C6+) radiation could significantly improve the killing ability for malignant melanoma cells by inducing apoptosis in tumor cells and inhibiting their proliferation. These results demonstrated that heavy ion (12C6+) presented special advantages in terms of treating malignant melanoma. Impact statement Malignant melanoma is a malignant skin tumor derived from melanin cells, which has a high malignant degree and high fatality rate. In this study, proliferating cell nuclear antigen (PCNA) can induce the apoptosis of malignant melanoma cells and inhibit its proliferation, and its induction effect on apoptosis is significantly higher than low LET X-ray; hence, it is expected to overcome its lower sensitivity to radiation. This study can provide theoretical basis for clinical trials, in which malignant melanoma is treated by heavy ion (12C6+), in order to accurately determine the clinical efficacy of heavy ion therapy. Clinical applications has revealed that local tumor control rate is high when heavy ion is used to treat malignant melanoma, indicating that heavy ion is an important direction in treating melanoma in the future.

Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition

ObjectiveAdipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition. MethodsASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture. ResultsThe 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs' potential of cellular transformation was lower after irradiation by soft agar assay. ConclusionThese findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37% fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

Adaptive Response of Low Linear Energy Transfer X-rays for Protection Against High Linear Energy Transfer Accelerated Heavy Ion-Induced Teratogenesis

Adaptive response (AR) of low linear energy transfer (LET) irradiations for protection against teratogenesis induced by high LET irradiations is not well documented. In this study, induction of AR by X-rays against teratogenesis induced by accelerated heavy ions was examined in fetal mice. Irradiations of pregnant C57BL/6J mice were performed by delivering a priming low dose from X-rays at 0.05 or 0.30 Gy on gestation day 11 followed one day later by a challenge high dose from either X-rays or accelerated heavy ions. Monoenergetic beams of carbon, neon, silicon, and iron with the LET values of about 15, 30, 55, and 200keV/μm, respectively, were examined. Significant suppression of teratogenic effects (fetal death, malformation of live fetuses, or low body weight) was used as the endpoint for judgment of a successful AR induction. Existence of AR induced by low-LET X-rays against teratogenic effect induced by high-LET accelerated heavy ions was demonstrated. The priming low dose of X-rays significantly reduced the occurrence of prenatal fetal death, malformation, and/or low body weight induced by the challenge high dose from either X-rays or accelerated heavy ions of carbon, neon or silicon but not iron particles. Successful AR induction appears to be a radiation quality event, depending on the LET value and/or the particle species of the challenge irradiations. These findings would provide a new insight into the study on radiation-induced AR in utero.

Early effects of low dose 12C 6+ ion or X-ray irradiation on human peripheral blood lymphocytes

The aim of this study was to estimate the acute effects of low dose 12C 6+ ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy 12C 6+ ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supernatant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-γ and TNF-α in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy 12C 6+ ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) 12C 6+ radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDI.

Biological effectiveness of 12C and 20Ne ions with very high LET

Purpose: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of 12C and 20Ne ions.Materials and methods: Chinese hamster ovary (CHO-K1) cells were exposed to high LET 12C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and 20Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up.Results: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440–830 keV/μm for 12C and for 1020–1600 keV/μm for 20Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET.Conclusions: The RBE decreases with increasing LET in the range between 440 and 1600 keV/μm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.

Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice.

Mutation rates at two expanded simple tandem repeat loci were studied in the germ line of first- and second-generation offspring of inbred male CBA/H, C57BL/6, and BALB/c mice exposed to either high linear energy transfer fission neutrons or low linear energy transfer x-rays. Paternal CBA/H exposure to either x-rays or fission neutrons resulted in increased mutation rates in the germ line of two subsequent generations. Comparable transgenerational effects were observed also in neutron-irradiated C57BL/6 and x-irradiated BALB/c mice. The levels of spontaneous mutation rates and radiation-induced transgenerational instability varied between strains (BALB/c>CBA/H>C57BL/6). Pre- and postmeiotic paternal exposure resulted in similar increases in mutation rate in the germ line of both generations of CBA/H mice, which together with our previous results suggests that radiation-induced expanded simple tandem repeat instability is manifested in diploid cells after fertilization. The remarkable finding that radiation-induced germ-line instability persists for at least two generations raises important issues of risk evaluation in humans.

High linear energy transfer carbon radiation effectively kills cultured glioma cells with either mutant or wild-type p53

Purpose: A mutation in the p53 gene is believed to play an important role in the radioresistance of many cancer cell lines. We studied cytotoxic effects of high linear energy transfer (LET) carbon beams on glioma cell lines with either mutant or wild-type p53. Methods and Materials: Cell lines U-87 and U-138 expressing wild-type p53 and U-251 and U-373 expressing mutant p53 were used. These cells were irradiated with 290 MeV/u carbon beams generated by the Heavy Ion Medical Accelerator in the National Institute of Radiologic Science or X-rays. A standard colony-forming assay and flow cytometric detection of apoptosis were performed. Cell cycle progression and the expression of p53, p21, and bax proteins were examined. Results: High LET carbon radiation was more cytotoxic than low LET X-ray treatment against glioma cells. The effects of the carbon beams were not dependent on the p53 gene status but were reduced by G 1 arrest, which was independent of p21 expression. The expression of bax remained unchanged in all four cell lines. Conclusion: These results indicate that high LET charged particle radiation can induce cell death in glioma cells more effectively than X-rays and that cell death other than p53-dependent apoptosis may participate in the cytotoxicity of heavy charged particles. Thus, it might prove to be an effective alternative radiotherapy for patients with gliomas harboring mutated p53 gene.

Hyperthermic enhancement of tumour growth inhibition by accelerated carbon-ions in transplantable human esophageal cancer

The study examined the effects of combination of hyperthermia (42d`C) and 290MeV/u carbon-ion (C—) beams or 200 kVp X-rays on tumour regrowth delay of transplantable human esophageal cancer as an in vivo model for radiotherapy of cancer. The C-beams were more effective in the tumour growth inhibition than X-rays. The relative biological effectiveness (RBE) of C-beams against X-rays was 2.00. It was observed that the interactive hyperthermic (42d`C, for 30 min) enhancement of tumour regrowth delay by high—linear energy transfer (LET) C-beams was similar to that of combination of low—LET X-rays with hyperthermia. The thermal enhancement ratios (TER) were 6.10 and 5.57 for X-rays and C-beams, respectively. These results suggest that hyperthermic treatment is effective in radiotherapy not only by low-LET radiation but also by high—LET radiation such as C-beams. In conclusion, the depression of the tumour growth by the combined treatment of hyperthermia (42d`C) and the C-beams strongly suggests the available possible application of interdisciplinary cancer therapy for refractory tumours.

Results of heavy ion radiotherapy.

The potential of heavy ion therapy for clinical use in cancer therapy stems from the biological parameters of heavy charged particles and their precise dose localization. Biologically, carbon, neon, and other heavy ion beams (up to about silicon) are clinically useful in overcoming the radioresistance of hypoxic tumors, thus increasing the biological effectiveness relative to low linear energy transfer x-ray or electron beams. Cells irradiated by heavy ions show less variation in cell-cycle-related radiosensitivity and decreased repair of radiation injury. The physical parameters of these heavy charged particles allow precise delivery of high doses to tumors while minimizing irradiation of normal tissues. Clinical use requires a close interaction between radiation oncologists, medical physicists, accelerator physicists, engineers, computer scientists, and radiation biologists.