Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

OBJECTIVE: Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. METHODS: Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. RESULTS: Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. CONCLUSION: Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.

Upright proton therapy for esthesioneuroblastoma: a single-institution experience.

Aim: This study presents an analysis (efficacy and toxicity) of outcomes in patients with esthesioneuroblastoma after pencil beam proton therapy with a fixed beamline in the upright position. Background: Esthesioneuroblastoma (ENB) is an extremely rare tumor of sinonasal area located in critical proximity to vital structures. Proton therapy (PT) is often considered the optimal radiation treatment for head-and-neck tumors, although of limited availability. Upright PT delivered using fixed pencil beamline and rotating chair is a fairly promising option. Methods: This is a single-center experience describing the outcomes of PT in 14 patients with ENB treated between January 2016 and October 2022; half of the cases had a history of previous irradiation. The therapy was applied using a fixed pencil beamline with 6D-chair for positioning. The median dose was 63 GyRBE (total range 48-70 GyRBE; based on 1.1 RBE multiplier for protons) with 2.0 GyRBE per fraction. The mean gross tumor volume was 109.5 cm3 (17.1-257.7 cm3). Patient demography, pathology, treatment parameters and toxicity data were analyzed. Radiation-induced reactions were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v 4.0. Results: The median follow-up time was 28 months. The 1- and 2-year locoregional control rates constituted 100% and 88.9%, respectively; the median duration of local control was 52 months. The 1- and 2-year progression-free survival (PFS) rates constituted 92.9% and 75.0%, respectively; the median PFS duration was 52 months. The 1- and 2-year overall survival (OS) rates constituted 92.9% and 84.4%, respectively. Two patients died of non-cancer-related causes (coronavirus-induced pneumonia) and 1 patient died of tumor progression. All patients tolerated PT well without any treatment gaps. Serious late toxicity reactions included glaucoma in 1 patient and cataract in 2 patients, in over half a year since irradiation. Conclusion: PT with upright design of the unit affords promising outcomes in terms of disease control and toxicity rates in ENB, a sinonasal tumor of complicated localization.

Immunohistochemical and histochemical analysis of the rat skin after local electron irradiation.

Background: Skin cancer is the most frequently diagnosed type of cancer among all malignant neoplasms. The decrease in mitotic activity and the death of intact keratinocytes arise due to the constantly renewing epithelium being highly sensitive to ionizing radiation. Aim: The aim of the study is immunohistochemical evaluation of the proliferative-apoptotic balance of keratinocytes, the fibrous component of the skin, and the expression of pro-inflammatory and anti-inflammatory cytokines after single or fractional local electron irradiation. Methods: Wistar rats (n = 80) were taken from the ITM&B Vivarium (Sechenov University) and divided into groups: I-control, which were injected with saline; and experimental groups, local electron irradiation at doses: II-8 Gy (single), III-40 Gy (single), IV-summary dose 78 Gy (fractional; 13 Gy per day for 6 days). We performed histological analysis, histochemical analysis using Masson, safranine, and picrosirius red staining, immunohistochemical (Ki-67, caspase-3, p53, types I and III collagens, IL-1, IL-6, IL-4, and IL-10) and morphometric analysis of skin fragments of the outer surface of the thigh, irradiated in accordance with the design of the experiment. The early and delayed effects of local electron irradiation at different doses were studied. Results: After local electron irradiation, dose-dependent morphological changes in the skin of the experimental groups were observed: violation of the histoarchitectonics of the skin confirmed by morphological and morphometric analysis, the proliferation of connective tissue according to the results of histochemical and immunohistochemical studies with signs of the radiation-induced skin fibrosis development, an increase in the levels of pro- and anti-inflammatory cytokines. We observed the most pronounced signs of radiation-induced skin damage in the group of fractional irradiation after 3 months. Conclusion: 8 Gy and 40 Gy single local electron irradiation leads to a shift in the proliferative-apoptotic balance of keratinocytes toward their apoptosis, which activity is directly correlated with the dose of ionizing radiation, and 78 Gy in fractions leads to partial desquamation of the epithelium and inflammatory infiltration. In addition, after 3 months a significant increase in the expression of type I and III collagen fibers and the development of radiation-induced skin fibrosis takes place against the background of 78 Gy fractional local electron irradiation.

Assessment of Proliferation and Apoptosis in Testes of Rats after Experimental Localized Electron Irradiation.

BACKGROUND AND PURPOSE: With the emergence of linear accelerators in radiotherapy, it becomes necessary to accurately select new dosing regimens. The purpose of this study was to assess the morphological changes of spermatogenesis after radiation exposure. MATERIALS AND METHODS: Male Wistar rats (n = 40) were subjected to targeted ionizing radiation on a pulsed electron accelerator "NOVAC-11" with doses of 2, 8 and 12 Gy. Spermatogenesis was assessed a week later using light microscopy and immunohistochemical method (antibodies to Ki-67, Bcl-2, p53, Caspase 3). RESULTS: A decrease in the number of normal germ cells was seen in all experimental groups, while radioresistant Sertoli and Leydig cells were barely affected. The most serious damage to the tubules and germ cells was observed in 8 and 12 Gy irradiation groups. IHC analysis of testes after irradiation showed a shift in the proliferative-apoptotic balance toward apoptosis of germ cells: a decrease in the expression levels of Ki-67 and Bcl-2, an increase in p53-positive and caspase 3-positive cells by the end of the experiment. CONCLUSION: Dose-dependent progressive pathomorphological changes in histoarchitectonics of the testes are traced, and a decrease in the number of germ cells is seen on the seventh day after irradiation with a pulsed electron accelerator "NOVAC-11".

Fast and Furious: Fast Neutron Therapy in Cancer Treatment.

Fast neutron therapy has been used for decades. In conjunction with recent advances in photonic techniques, fast neutrons are no longer of much oncologic interest, which is not unequivocally positive, given their undoubted therapeutic value. This mini-review recalls the history of medical research on fast neutrons, considers their physical and radiobiological properties alongside their benefits for cancer treatment, and discusses their place in modern radiation oncology.

Hypersensitivity and Induced Radioresistance in Chinese Hamster Cells Exposed to Radiations with Different LET Values.

We study the impact of radiation LET on manifestation of HRS/IRR response in Chinese hamster cells ovary cells exposed to radiations used in radiotherapy. Earlier we have investigated this response to carbon ions (455 MeV/amu) in the pristine Bragg curve plateau and behind the Bragg peak, 60Co γ-rays, and 14.5 MeV neutrons. Now we present results of cytogenetic metaphase analysis in plateau-phase CHO-K1 cells irradiated with scanning beam protons (83 MeV) at doses < 1 Gy and additional data for 14.5 MeV neutrons. Dose curves for frequency of total chromosome aberrations (CA, protons), paired fragments (protons, neutrons), aberrant cells (neutrons) had typical HRS/IRR structure: HRS region (up to 0.1 and 0.15 Gy), IRR region (0.1−0.6 Gy and 0.15−0.35 Gy) for protons and neutrons, respectively, and regular dose dependence. Taken together with previous results, the data show that LET increase shifts the HRS upper border (from 0.08−0.1 Gy for γ-rays, protons and plateau carbons to 0.12−0.15 Gy for "tail" carbons and neutrons). The IRR regions shortens (0.52−0.4 γ-rays and protons, 0.25 plateau carbons, 0.2 Gy "tail" carbons and neutrons). CA level of IRR increases by 1.5−2.5 times for carbons as compared to γ-rays and protons. Outside HRS/IRR the yield of CA also enhanced with LET increase. The results obtained for different LET radiations suggest that CHO-K1 cells with G1-like CA manifested the general feature of the HRS/IRR phenomena.

1-Isobutanoil-2-isopropylisothiourea Phosphate, T1082: A Safe and Effective Prevention of Radiotherapy Complications in Oncology.

The radioprotective effects of a new 1-isobutanoil-2-isopropylisothiourea derivative named T1082 are presented. Research methods included toxic characteristics, radioprotective activity (Till-McCulloch's test and 30-day survival test) in γ-ray total-body-irradiated mice, and a clinical and histological study of the effect of T1082 on acute radiation skin reactions (RSR) in rats after a single or fractionated ß-ray local irradiation. T1082 is more effective than its analogue, the NOS inhibitor T1023, at low concentrations and doses (1/12-1/8 LD10), both parenterally and intragastrically. In this case, its therapeutic index (LD50/ED50) reaches 30, and the optimal radioprotective doses (ED84-98-141-224 mg/kg) are an order less than the maximum tolerated doses-1/16-1/10 LD10. These properties allowed T1082, at a low intragastrical dose (160 mg/kg; 1/14 LD10), to significantly limit the severity of acute RSR after single (40 Gy) and fractionated (78 Gy) ß-ray irradiation. The results confirm T1082 as one of the safest emergency radioprotectors and indicate the prospects for its further development as a pharmacological agent for the prevention of RT complications.

An experimental approach to comprehend the influence of platelet rich growth factors on spermatogenesis.

MATERIALS AND METHODS: Experimental animals (n = 135) were divided into 5 groups: I - control (n = 10); II - 2IR (n = 35; 2 Gy); III - 2IR + LP-PRP + IGF-1 (n = 30); IV - 2IR + LP-PRP (n = 30); V - LP-PRP (n = 30). RESULTS: Electron irradiation reduces the number of germ cells in comparison with the control group. After injection of LP-PRP + rhIGF-1 significantly increased the number of germ cells, Sertoli and Leydig cells, the height of germinal epithelium, area and diameter of seminiferous tubules. CONCLUSION: LP-PRP + rhIGF-1 has a normalizing effect on structural and functional disorders of the testis caused by electron irradiation.

In vitro modified microdosimetric kinetic model-based predictions for B14-150 cells survival in 450 MeV/u carbon ion beam with aluminum ridge filter for biologically optimized spread-out Bragg peak.

The relative biological efficiency of particle irradiation could be predicted with a wide variety of radiobiological models for various end-points. We validate the forecast of modified Microdosimetric Kinetic Modelin vitrousing combined data of reference Co-60 radiation and carbon ion plateau data for specific cell line to optimize the survival function in spread-out Bragg Peak obtained with an especially designed ridge filter. We used Geant4 Monte-Carlo software to simulate the fragment contribution along Bragg curve inside water phantom, open-source toolkit Survival to predict the expected linear-quadratic model parameters for each fragment, and in-house software to form the total survival curve in spread-out Bragg Peak. The irradiation was performed at U-70 synchrotron with an especially designed Aluminum ridge filter under the control of PTW and in-house ionization chambers. The cell clonogenic assay was conducted with the B14-150 cell line. The data analysis was accomplished using scipy and CERN ROOT. The clonogenic assay represents the survival in spread-out Bragg Peak at different points and qualitatively follows the modeled survival curve very well. The quantitative difference is within 3σ, and the deviation might be explained by the uncertainties of physical modeling using Monte-Carlo methods. Overall, the obtained results are promising for further usage in radiobiological studies or carbon ion radiotherapy. Shaping the survival curve in the region of interest (i.e., spread-out Bragg Peak) is a comprehensive task that requires high-performance computing approaches. Nevertheless, the method's potential application is related to the development of next-generation treatment planning systems for ion beams. This can open a wide range of improvements in patient treatment outcome, provide new optimized fractionation regimes or optimized dose delivery schemes, and serve as an entrance point to the translational science approach.

Proton therapy with a fixed beamline for skull-base chordomas and chondrosarcomas: outcomes and toxicity.

AIM: This study presents an analysis (efficacy and toxicity) of outcomes in patients with skull-base chordomas or chondrosarcomas treated with a fixed horizontal pencil proton beam. BACKGROUND: Chordomas (CAs) and chondrosarcomas (CSAs) are rare tumours that are usually located near the base of the skull and very close to the brain's most critical structures. Proton therapy (PT) is often considered the best radiation treatment for these diseases, but it is still a limited resource. Active scanning PT delivered via a fixed pencil beamline might be a promising option. METHODS: This is a single-centre experience describing the results of proton therapy for 31 patients with CA (n = 23) or CSA (n = 8) located near the base of the skull. Proton therapy was utilized by a fixed pencil beamline with a chair to position the patient between May 2016 and November 2020. Ten patients underwent resection (32.2%), 15 patients (48.4%) underwent R2 resection, and 6 patients had unresectable tumours (19.4%). In 4 cases, the tumours had been previously irradiated. The median PT dose was 70 GyRBE (relative biological efficacy, 1.1) [range, 60 to 74] with 2.0 GyRBE per fraction. The mean GTV volume was 25.6 cm3 [range, 4.2-115.6]. Patient demographics, pathology, treatment parameters, and toxicity were collected and analysed. Radiation-induced reactions were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) v 4.0. RESULTS: The median follow-up time was 21 months [range, 4 to 52]. The median overall survival (OS) was 40 months. The 1- and 2-year OS was 100%, and the 3-year OS was 66.3%. Four patients died due to non-cancer-related reasons, 1 patient died due to tumour progression, and 1 patient died due to treatment-related injuries. The 1-year local control (LC) rate was 100%, the 2-year LC rate was 93.7%, and the 3-year LC rate was 85.3%. Two patients with CSA exhibited progression in the neck lymph nodes and lungs. All patients tolerated PT well without any treatment interruptions. We observed 2 cases of ≥ grade 3 toxicity, with 1 case of grade 3 myelitis and 1 case of grade 5 brainstem injury. CONCLUSION: Treatment with a fixed proton beam shows promising disease control and an acceptable toxicity rate, even the difficult-to-treat subpopulation of patients with skull-base chordomas or chondrosarcomas requiring dose escalation.

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)-1.6-1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5-1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4-1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors-T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

Proton re-irradiation of unresectable recurrent head and neck cancers.

BACKGROUND: This study presents a retrospective analysis (efficacy and toxicity) of outcomes in patients with unresectable recurrence of previously irradiated head and neck (H&N) cancers treated with proton therapy. Locoregional recurrence is the main pattern of failure in the treatment of H&N cancers. Proton re-irradiation in patients with relapse after prior radiotherapy might be valid as promising as a challenging treatment option. MATERIALS AND METHODS: From November 2015 to January 2020, 30 patients with in-field recurrence of head and neck cancer, who were not suitable for surgery due to medical contraindications, tumor localization, or extent, received re-irradiation with intensity-modulated proton therapy (IMPT). Sites of retreatment included the aerodigestive tract (60%) and the base of skull (40%). The median total dose of prior radiotherapy was 55.0 Gy. The median time to the second course was 38 months. The median re-irradiated tumor volume was 158.1 cm3. Patients were treated with 2.0, 2.4, and 3.0 GyRBE per fraction, with a median equivalent dose (EQD2) of 57.6 Gy (α/ß = 10). Radiation-induced toxicity was recorded according to the RTOG/EORTC criteria. RESULTS: The 1- and 2-year local control (LC), progression-free survival (PFS), and overall survival (OS) were 52.6/21.0, 21.9/10.9, and 73.4/8.4%, respectively, with a median follow-up time of 21 months. The median overall survival was 16 months. Acute grade 3 toxicity was observed in one patient (3.3%). There were five late severe side effects (16.6%), with one death associated with re-irradiation. CONCLUSION: Re-irradiation with a proton beam can be considered a safe and efficient treatment even for a group of patients with unresectable recurrent H&N cancers.

Proton re-irradiation of unresectable recurrent brain gliomas: clinical outcomes and toxicity.

PURPOSE: To assess the efficacy and tolerance of proton re-irradiation in patients with unresectable recurrence of previously irradiated brain gliomas. METHODS: Between February 2016 and December 2019, 44 patients with in-field recurrence after prior irradiation of brain gliomas were irradiated with intensity-modulated proton therapy. Seven patients (15.9%) originally had low-grade (WHO grade I-II) gliomas, nine patients (20.4%) had anaplastic astrocytoma (WHO grade III), and 28 patients (63.7%) had glioblastoma (WHO grade IV). All tumors were unresectable due to their localization. After a median time from the prior irradiation of 28.0 months [range, 12 to 173], patients received PT with 2.0 and 3.0 GyRBE per fraction, with median proton EQD2 (/=10) to a tumor of 55.0 GyRBE [range, 46.0 to 61.75]. Adjuvant chemotherapy (Temozolomide, or Procarbazine, Lomustine and vincristine, or Bevacizumab with Irinotecan) received 86.9% of the patients (n=40). Treatment-related toxicity was reported following CTCAE. RESULTS: The median survival time was 12 months, with 1-year and 2-years overall survival (OS) amounting to 49.6% and 35.1%, respectively. The median progression-free survival (PFS) was 9 months, with 1- and 2-years PFS of 30.5% and 10.2%, respectively. Twenty-six patients died by the time of analysis; among them were 5 non-cancer deaths (19.2%), and 4 patients (15.4%) died of chemotherapy-associated severe toxicity. The incidence rate of radiation-induced necrosis was 6.8% (3 events). CONCLUSIONS: Based on our results, we suggest re-irradiation of recurrent brain gliomas with proton therapy is able to achieve reasonable tumor control. Low adverse events rate and promising outcomes make it a safe treatment option with curative intent, even in unresectable cases.

A Clinical Case of 5 Times Irradiated Recurrent Orbital Hemangiopericytoma.

Orbital hemangiopericytomas (HPCs) are rare mesenchymal tumors with a high tendency to recur. Treatment options are quite limited in case of a relapse, but re-irradiation can be useful. Most of the available data limit the possibility of re-irradiation, while novel techniques (e.g., pencil beam proton therapy [PT]) open new approaches for the safe repeating of treatment. To the best of our knowledge, this is the first well-documented case of multi-times (>3) irradiation of tumors located intracranially. The case reported here describes orbital HPCs with proton irradiation performed two times since 1999 in a 30-year-old woman with a medical history as well as surgery followed by conventional radiotherapy (RT) and chemotherapy, and two times stereotactic RT (in 2009 and 2013). In 2016 the patient came to our hospital with the 3rd relapse of the tumor, located in the left orbit, with an intracranial part, involving cavernous sinus, which was close to the temporal lobe. The 4th course of irradiation was done in May to June 2016 by pencil beam PT. Radiation necrosis occurred after 2 years and was treated with bevacizumab (BVZ). Three years later, another relapse was treated with PT and BVZ. The 9-month follow-up showed complete tumor response without signs of brain toxicity. The patient died due to a brain abscess 1 year after the 5th irradiation. This case shows a possibility of irradiation, applied 5 times to the same location, with promising results and manageable toxicity.

Formation of γH2AX and pATM Foci in Human Mesenchymal Stem Cells Exposed to Low Dose-Rate Gamma-Radiation.

DNA double-strand breaks (DSB) are among the most harmful DNA lesions induced by ionizing radiation (IR). Although the induction and repair of radiation-induced DSB is well studied for acute irradiation, responses to DSB produced by chronic IR exposures are poorly understood, especially in human stem cells. The aim of this study was to examine the formation of DSB markers (γH2AX and phosphorylated kinase ATM, pATM, foci) in human mesenchymal stem cells (MSCs) exposed to chronic gamma-radiation (0.1 mGy/min) in comparison with acute irradiation (30 mGy/min) at cumulative doses of 30, 100, 160, 240 and 300 mGy. A linear dose-dependent increase in the number of both γH2AX and pATM foci, as well as co-localized γH2AX/pATM foci ("true" DSB), were observed after an acute radiation exposure. In contrast, the response of MSCs to a chronic low dose-rate IR exposure deviated from linearity towards a threshold model, for γH2AX, pATM foci and γH2AX/pATM foci, with an indication of a "plateau". The state of equilibrium between newly formed DSB at a low rate during the protracted exposure time and the elimination of a fraction of DSB is proposed as a mechanistic explanation of the non-linear DSB responses following a low dose-rate irradiation. This notion is supported by the observation of the elimination of a substantial fraction of DSB 6 h after the cessation of the exposures. Our results demonstrate non-linear dose responses for γH2AX and pATM foci in human MSCs exposed to low dose-rate IR and showed the existence of a threshold, which may have implications for radiation protection in humans.