Cytokines are mediators of inflammation that could lead to fibrosis. The aim was to monitor cytokine levels in saliva and serum after locally fractionated radiotherapy of the head and neck in mice and investigate associations with salivary gland fibrosis and hyposalivation. C57BL/6 mice were randomized to sham or X-ray irradiation of 66 Gy in 10 fractions over 5 days. Blood and saliva were collected on days −7, 5, 35, 80, and 105 following cytokine analysis. The harvested submandibular salivary gland was assessed for the presence of fibrosis. Decision tree regression analysis was used to investigate whether cytokine levels could predict late endpoints in terms of hyposalivation or fibrosis. Significant formation of fibrosis in gland tissue and reduced saliva production was found after irradiation. The pro-inflammatory cytokines IL-1α, TNF, TIMP1, G-CSF, KC, and MIP-1α showed increased levels in saliva in irradiated mice and a strong correlation with late endpoints. The decision tree analysis largely separated controls from irradiated animals, with IL-1α being the strongest predictor. Pro-inflammatory cytokines in saliva, but not in serum, were associated with late endpoints. This indicates that cytokine expression in saliva is a good biomarker for local salivary gland damage with IL-1α as the strongest single predictor.

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-β) family are key players in the fibrotic response. While TGF-β1 is known to be pro-fibrotic, TGF-β3 has mainly been considered anti-fibrotic. Moreover, TGF-β3 has been shown to act protective against acute toxicities after radio- and chemotherapy. In the present study, we investigated the effect of TGF-β3 treatment during fractionated H&N RT in a mouse model. 30 C57BL/6J mice were assigned to three treatment groups. The RT + TGF-β3 group received local fractionated H&N RT with 66 Gy over five days, combined with TGF-β3-injections at 24-hour intervals. Animals in the RT reference group received identical RT without TGF-β3 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. A subset of mice in the RT + TGF-β3 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-β3 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-β3 treatment did not impact saliva production. When repeatedly administered during fractionated RT at the current dose, TGF-β3 treatment increased acute H&N radiation toxicities and increased mortality. Furthermore, TGF-β3 treatment may increase the severity of radiation-induced salivary gland fibrosis.

The purpose of this study was to investigate acute normal tissue responses in the head and neck region following proton- or X-irradiation of a murine model. Female C57BL/6J mice were irradiated with protons (25 or 60 MeV) or X-rays (100 kV). The radiation field covered the oral cavity and the major salivary glands. For protons, two different treatment plans were used, either with the Bragg Peak in the middle of the mouse (BP) or outside the mouse (transmission mode; TM). Delivered physical doses were 41, 45, and 65 Gy given in 6, 7, and 10 fractions for BP, TM, and X-rays, respectively. Alanine dosimetry was used to assess delivered doses. Oral mucositis and dermatitis were scored using CTC v.2.0-based tables. Saliva was collected at baseline, right after end of irradiation, and at day 35. The measured dose distribution for protons (TM) and X-rays was very similar. Oral mucositis appeared earlier, had a higher score and was found in a higher percentage of mice after proton irradiation compared to X-irradiation. Dermatitis, on the other hand, had a similar appearance after protons and X-rays. Compared to controls, saliva production was lower right after termination of proton- and X-irradiation. The BP group demonstrated saliva recovery compared to the TM and X-ray group at day 35. With lower delivered doses, proton irradiation resulted in similar skin reactions and increased oral mucositis compared to X-irradiation. This indicates that the relative biological effectiveness of protons for acute tissue responses in the mouse head and neck is greater than the clinical standard of 1.1. Thus, there is a need for further investigations of the biological effect of protons in normal tissues.

Extracellular vesicles (EVs) are membrane-bound particles released from cells, and their cargo can alter the function of recipient cells. EVs from X-irradiated cells have been shown to play a likely role in non-targeted effects. However, EVs derived from proton irradiated cells have not yet been studied. We aimed to investigate the proteome of EVs and their cell of origin after proton or X-irradiation. The EVs were derived from a human oral squamous cell carcinoma (OSCC) cell line exposed to 0, 4, or 8 Gy from either protons or X-rays. The EVs and irradiated OSCC cells underwent liquid chromatography–mass spectrometry for protein identification. Interestingly, we found different protein profiles both in the EVs and in the OSCC cells after proton irradiation compared to X-irradiation. In the EVs, we found that protons cause a downregulation of proteins involved in cell growth and DNA damage response compared to X-rays. In the OSCC cells, proton and X-irradiation induced dissimilar cell death pathways and distinct DNA damage repair systems. These results are of potential importance for understanding how non-targeted effects in normal tissue can be limited and for future implementation of proton therapy in the clinic.

Background Radiotherapy of head and neck cancer may cause detrimental late side effects such as fibrosis and hyposalivation. Our aim was to investigate late radiation-induced cellular and molecular changes of the salivary glands after fractionated irradiation to the head and neck in a murine model. Methods 12-week-old female C57BL/6J mice were irradiated with X-rays to a total dose of 66 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity and major salivary glands. Salivary gland function was assessed by collecting saliva at baseline and at various time points after irradiation. The submandibular (SMG), sublingual (SLG), and parotid glands (PG) were dissected at day 105. Using different staining techniques, morphological, cellular, and molecular changes were investigated in the salivary glands. Results Saliva production was significantly reduced in irradiated compared to control mice at day 35, 80, and 105. We observed a significant decrease in total gland area and a significant increase in fibrotic area in irradiated compared to control SMG at day 105. Atrophy of acinar cells was observed in all irradiated SMG and SLG. Increased amount of chronic inflammatory cells, increased cell proliferation and altered expression of apoptotic markers were found in atrophic areas of irradiated glands. Conclusion Acinar and duct cells in irradiated salivary glands show increased cell proliferation and altered expression of apoptotic markers, proposing an attempt to overcome or withstand tissue damage caused by irradiation. This suggests a potential for regeneration of salivary glands after radiation therapy.

Background and purpose: Radiotherapy of head and neck cancer may cause detrimental late side effects such as fibrosis and hyposalivation. We investigated mouse salivary glands after fractionated irradiation, with the aim to elucidate cellular plasticity and potential regeneration. Methods: 12-week-old female C57BL/6JRj mice were irradiated with X-rays to a total dose of 66 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity and major salivary glands. The submandibular (SMG), sublingual (SLG), and parotid glands (PG) were dissected at day 100 after finishing irradiation. Using different histological staining techniques, morphological, cellular, and molecular changes were investigated in irradiated and control SMG, SLG, and PG. Results: Atrophy of acinar cells was observed in irradiated SMG and SLG, but not in PG. Surprisingly, the acinar atrophy was confined to one region of each irradiated gland. These atrophic regions showed distinct cellular and molecular compositions compared to non-atrophic regions and control glands. Increased expression of the ductal cell markers keratin 5 and 19 (K5 and K19), along with increased percentages of proliferating myofibroblasts, fibroblasts, leukocytes, and acinar cells (Nkcc1+) were observed in the atrophic regions compared to controls. In addition, some of the K19+ and K5+ duct-like cells also co-expressed Nkcc1. Interpretation: Through a detailed histological assessment of the cellular and molecular changes in the major salivary glands of irradiated mice, we observed signs of cellular plasticity where ductal cells adopt an acinar cell phenotype upon irradiation. This suggests a regenerative potential of salivary glands after irradiation.