TGF-ß3 increases the severity of radiation-induced oral mucositis and salivary gland fibrosis in a mouse model.

PURPOSE: Toxicities from head and neck (H&N) radiotherapy (RT) may affect patient quality of life and can be dose-limiting. Proteins from the transforming growth factor beta (TGF-ß) family are key players in the fibrotic response. While TGF-ß1 is known to be pro-fibrotic, TGF-ß3 has mainly been considered anti-fibrotic. Moreover, TGF-ß3 has been shown to act protective against acute toxicities after radio- and chemotherapy. In the present study, we investigated the effect of TGF-ß3 treatment during fractionated H&N RT in a mouse model. MATERIALS AND METHODS: 30 C57BL/6J mice were assigned to three treatment groups. The RT + TGF-ß3 group received local fractionated H&N RT with 66 Gy over five days, combined with TGF-ß3-injections at 24-hour intervals. Animals in the RT reference group received identical RT without TGF-ß3 treatment. The non-irradiated control group was sham-irradiated according to the same RT schedule. In the follow-up period, body weight and symptoms of oral mucositis and lip dermatitis were monitored. Saliva was sampled at five time points. The experiment was terminated 105 d after the first RT fraction. Submandibular and sublingual glands were preserved, sectioned, and stained with Masson's trichrome to visualize collagen. RESULTS: A subset of mice in the RT + TGF-ß3 group displayed increased severity of oral mucositis and increased weight loss, resulting in a significant increase in mortality. Collagen content was significantly increased in the submandibular and sublingual glands for the surviving RT + TGF-ß3 mice, compared with non-irradiated controls. In the RT reference group, collagen content was significantly increased in the submandibular gland only. Both RT groups displayed lower saliva production after treatment compared to controls. TGF-ß3 treatment did not impact saliva production. CONCLUSIONS: When repeatedly administered during fractionated RT at the current dose, TGF-ß3 treatment increased acute H&N radiation toxicities and increased mortality. Furthermore, TGF-ß3 treatment may increase the severity of radiation-induced salivary gland fibrosis.

Radiation-induced long-term dysphagia in survivors of head and neck cancer and association with dose-volume parameters.

BACKGROUND: Although dysphagia is a common side effect after radiotherapy (RT) of head and neck cancer (HNC), data on long-term dysphagia is scarce. We aimed to 1) compare radiation dose parameters in HNC survivors with and without dysphagia, 2) investigate factors associated with long-term dysphagia and its possible impact on health-related quality of life (HRQoL), and 3) investigate how our data agree with existing NTCP models. METHODS: This cross-sectional study conducted in 2018-2020, included HNC survivors treated in 2007-2013. Participants attended a one-day examination in hospital and filled in patient questionnaires. Dysphagia was measured with the EORTC QLQ-H&N35 swallowing scale. Toxicity was scored with CTCAE v.4. We contoured swallowing organs at risk (SWOAR) on RT plans, calculated dose-volume histograms (DVHs), performed logistic regression analyses and tested our data in established NTCP models. RESULTS: Of the 239 participants, 75 (31%) reported dysphagia. Compared to survivors without dysphagia, this group had reduced HRQoL and the DVHs for infrahyoid SWOAR were significantly shifted to the right. Long-term dysphagia was associated with age (OR 1.07, 95% CI 1.03-1.10), female sex (OR 2.75, 95% CI 1.45-5.21), and mean dose to middle pharyngeal constrictor muscle (MD-MPCM) (OR 1.06, 95% CI 1.03-1.09). NTCP models overall underestimated the risk of long-term dysphagia. CONCLUSIONS: Long-term dysphagia was associated with higher age, being female, and high MD-MPCM. Doses to distally located SWOAR seemed to be risk factors. Existing NTCP models do not sufficiently predict long-term dysphagia. Further efforts are needed to reduce the prevalence and consequences of this late effect.

Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro.

OBJECTIVE: The influence of radiation backscatter from titanium on DNA damage and migration capacity of human osteoblasts (OBs) and mesenchymal stem cells (MSCs) may be critical for the osseointegration of dental implants placed prior to radiotherapy. In order to evaluate effects of radiation backscatter, the immediate DNA damage and migration capacity of OBs and MSCs cultured on titanium or plastic were compared after exposure to ionizing irradiation. MATERIALS AND METHODS: Human OBs and MSCs were seeded on machined titanium, moderately rough fluoride-modified titanium, or tissue culture polystyrene, and irradiated with nominal doses of 2, 6, 10, or 14 Gy. Comet assay was performed immediately after irradiation, while a scratch wound healing assay was initiated 24 h post-irradiation. Fluorescent live cell imaging documented the migration. RESULTS: DNA damage increased with higher dose and with backscatter from titanium, and MSCs were significantly more affected than OBs. All doses of radiation accelerated the cell migration on plastic, while only the highest dose of 10 Gy inhibited the migration of both cell types on titanium. CONCLUSIONS: High doses (10 Gy) of radiation inhibited the migration capacity of both cell types on titanium, whereas lower doses (2 and 6 Gy) did not affect the migration of either OBs or MSCs. CLINICAL RELEVANCE: Fractionated doses of 2 Gy/day, as distributed in conventional radiotherapy, appear not to cause severe DNA damage or disturb the migration of OBs or MSCs during osseointegration of dental implants.

The dose-dependent impact of γ-radiation reinforced with backscatter from titanium on primary human osteoblasts.

In head and neck cancer patients receiving dental implants prior to radiotherapy, backscatter from titanium increases the radiation dose close to the surface, and may affect the osseointegration. The dose-dependent effects of ionizing radiation on human osteoblasts (hOBs) were investigated. The hOBs were seeded on machined titanium, moderately rough fluoride-modified titanium, and tissue culture polystyrene, and cultured in growth- or osteoblastic differentiation medium (DM). The hOBs were exposed to ionizing γ-irradiation in single doses of 2, 6 or 10 Gy. Twenty-one days post-irradiation, cell nuclei and collagen production were quantified. Cytotoxicity and indicators of differentiation were measured and compared to unirradiated controls. Radiation with backscatter from titanium significantly reduced the number of hOBs but increased the alkaline phosphatase activity in both types of medium when adjusted to the relative cell number on day 21. Irradiated hOBs on the TiF-surface produced similar amounts of collagen as unirradiated controls when cultured in DM. The majority of osteogenic biomarkers significantly increased on day 21 when the hOBs had been exposed to 10 Gy, while the opposite or no effect was observed after lower doses. High doses reinforced with backscatter from titanium resulted in smaller but seemingly more differentiated subpopulations of osteoblasts.

2D mapping of radiation dose and clonogenic survival for accurate assessment ofin vitroX-ray GRID irradiation effects.

Spatially fractionated radiation therapy (SFRT or GRID) is an approach to deliver high local radiation doses in an 'on-off' pattern. To better appraise the radiobiological effects from GRID, a framework to link local radiation dose to clonogenic survival needs to be developed. A549 lung cancer cells were irradiated in T25 cm2flasks using 220 kV x-rays with an open field or through a tungsten GRID collimator with periodical 5 mm openings and 10 mm blockings. Delivered nominal doses were 2, 5, and 10 Gy. A novel approach for image segmentation was used to locate the centroid of surviving colonies in scanned images of the cell flasks. GafchromicTMfilm dosimetry (GFD) and FLUKA Monte Carlo (MC) simulations were employed to map the dose at each surviving colony centroid. Fitting the linear-quadratic (LQ) function to clonogenic survival data for open field irradiation, the expected survival level at a given dose level was calculated. The expected survival levels were then mapped together with the observed levels in the GRID-irradiated flasks. GFD and FLUKA MC gave similar dose distributions, with a mean peak-to-valley dose ratio of about 5. LQ-parameters for open field irradiation gaveα=0.24±0.02Gy-1andß=0.019±0.002Gy-2. The mean relative percentage deviation between observed and predicted survival in the (peak; valley) dose regions was (4.6; 3.1) %, (26.6; -1.0) %, and (129.8; -2.3) % for 2, 5 and 10 Gy, respectively. In conclusion, a framework for mapping of surviving colonies following GRID irradiation together with predicted survival levels from homogeneous irradiation was presented. For the given cell line, our findings indicate that GRID irradiation causes reduced survival in the peak regions compared to an open field configuration.

Sharp dose profiles for high precision proton therapy using strongly focused proton beams.

The main objective of radiotherapy is to exploit the curative potential of ionizing radiation while inflicting minimal radiation-induced damage to healthy tissue and sensitive organs. Proton beam therapy has been developed to irradiate the tumor with higher precision and dose conformity compared to conventional X-ray irradiation. The dose conformity of this treatment modality may be further improved if narrower proton beams are used. Still, this is limited by multiple Coulomb scattering of protons through tissue. The primary aim of this work was to develop techniques to produce narrow proton beams and investigate the resulting dose profiles. We introduced and assessed three different proton beam shaping techniques: (1) metal collimators (100/150 MeV), (2) focusing of conventional- (100/150 MeV), and (3) focusing of high-energy (350 MeV, shoot-through) proton beams. Focusing was governed by the initial value of the Twiss parameter [Formula: see text] ([Formula: see text]), and can be implemented with magnetic particle accelerator optics. The dose distributions in water were calculated by Monte Carlo simulations using Geant4, and evaluated by target to surface dose ratio (TSDR) in addition to the transverse beam size ([Formula: see text]) at the target. The target was defined as the location of the Bragg peak or the focal point. The different techniques showed greatly differing dose profiles, where focusing gave pronouncedly higher relative target dose and efficient use of primary protons. Metal collimators with radii [Formula: see text] gave low TSDRs ([Formula: see text]) and large [Formula: see text]([Formula: see text]). In contrast, a focused beam of conventional ([Formula: see text]) energy produced a very high TSDR ([Formula: see text]) with similar [Formula: see text] as a collimated beam. High-energy focused beams were able to produce TSDRs [Formula: see text] and [Formula: see text] around 1.5 mm. From this study, it appears very attractive to implement magnetically focused proton beams in radiotherapy of small lesions or tumors in close vicinity to healthy organs at risk. This can also lead to a paradigm change in spatially fractionated radiotherapy. Magnetic focusing would facilitate FLASH irradiation due to low losses of primary protons.

Spatially fractionated radiotherapy: tumor response modelling including immunomodulation.

A mathematical tumor response model has been developed, encompassing the interplay between immune cells and cancer cells initiated by either partial or full tumor irradiation. The iterative four-compartment model employs the linear-quadratic radiation response theory for four cell types: active and inactive cytotoxic T lymphocytes (immune cells, CD8+T cells in particular), viable cancer cells (undamaged and reparable cells) and doomed cells (irreparably damaged cells). The cell compartment interactions are calculated per day, with total tumor volume (TV) as the main quantity of interest. The model was fitted to previously published data on syngeneic xenografts (67NR breast carcinoma and Lewis lung carcinoma; (Markovskyet al2019Int. J. Radiat. Oncol. Biol. Phys.103697-708)) subjected to single doses of 10 or 15 Gy by 50% (partial) or 100% (full) TV irradiation. The experimental data included effects from anti-CD8+antibodies and immunosuppressive drugs. Using a new optimization method, promising fits were obtained where the lowest and highest root-mean-squared error values were observed for anti-CD8+treatment and unirradiated control data, respectively, for both cell types. Additionally, predictive capabilities of the model were tested by using the estimated model parameters to predict scenarios for higher doses and different TV irradiation fractions. Here, mean relative deviations in the range of 19%-34% from experimental data were found. However, more validation data is needed to conclude on the model's predictive capabilities. In conclusion, the model was found useful in evaluating the impact from partial and full TV irradiation on the immune response and subsequent tumor growth. The model shows potential to support and guide spatially fractionated radiotherapy in future pre-clinical and clinical studies.

Low dose-rate irradiation with [3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model.

BACKGROUND: Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model. METHODS: Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [3H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography. RESULTS: Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [3H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue. CONCLUSION: Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.

Low-Dose-Rate Irradiation for 1 Hour Induces Protection Against Lethal Radiation Doses but Does Not Affect Life Span of DBA/2 Mice.

Prior findings showed that serum from DBA/2 mice that had been given whole-body irradiation for 1 hour at a low dose rate (LDR) of 30 cGy/h induced protection against radiation in reporter cells by a mechanism depending on transforming growth factor ß3 and inducible nitric oxide synthase activity. In the present study, the effect of the 1 hour of LDR irradiation on the response of the preirradiated mice to a subsequent lethal dose and on the life span is examined. These DBA/2 mice were prime irradiated for 1 hour at 30 cGy/h. Two experiments with 9 and 9.5 Gy challenge doses given 6 weeks after priming showed increased survival in primed mice compared to unprimed mice followed up to 225 and 81 days after challenge irradiation, respectively. There was no overall significant difference in life span between primed and unprimed mice when no challenge irradiation was given. The males seemed to have a slight increase in lifespan after priming while the opposite was seen for the females.