Radiotherapy induces tumor cell killing by generating DNA double strand breaks (DSBs). The effectiveness of radiotherapy is significantly influenced by the repair of DSBs, which counteracts this lethal effect. Current investigations are focused on determining whether non-homologous end joining (NHEJ) or homologous recombination is the predominant repair pathway following proton and photon radiation. In this study, we examined the response of FaDu cells, a head and neck squamous cell carcinoma model, to spread-out Bragg peak (SOBP) proton and photon radiation combined with mild hyperthermia (42 °C for one hour) to induce homologous recombination deficiency or NHEJ inhibition by AZD7648. Hyperthermia resulted in stronger radiosensitization after proton radiation (SR = 1.53) compared to photon radiation (SR = 1.32). Conversely, NHEJ inhibition did not produce a significant differential effect between photon and proton radiation. This indicates a greater reliance on homologous recombination following proton radiation compared to photon radiation. We found that the number of DSBs formed after photon versus proton irradiation is comparable. Interestingly, the homologous recombination protein Rad51 accumulated more frequently at DSBs following proton irradiation than photon irradiation. These findings support the hypothesis that cells rely more on homologous recombination to repair proton-induced DNA damage compared to photon-induced DNA damage. As clinically applied hyperthermia enhances the therapeutic effect of photon irradiation by, among other factors, inducing homologous recombination deficiency, our results suggests that hyperthermia could be more effective in combination with proton irradiation than photon irradiation.

Understanding the cellular and molecular effect of proton radiation, particularly the increased DNA damage complexity at the distal end of the Bragg curve, is current topic of investigation. This work aims to study in vitro clonogenic survival and DNA damage foci kinetics of a head and neck squamous cell carcinoma cell line at various positions along a double passively scattered Bragg curve. Complementary in silico studies are conducted to gain insights into the link between cell survival variations, experimentally yielded foci and the number and complexity of double strand breaks (DSBs). Proton irradiations are performed at the HollandPTC R&D proton beamline, using a double passively scattered setup. A custom water phantom setup is employed to accurately position the samples within the Bragg curve. FaDu cells are irradiated at the proximal 36 % point of the Bragg peak, (P36), proximal 80 % point of the Bragg peak (P80) and distal 20 % point of the Bragg peak (D20), with dose-averaged mean lineal energies ( y D ¯ ) of 1.10 keV/μm, 1.80 keV/μm and 7.25 keV/μm, respectively. Clonogenic survival correlates strongly with y D ¯ , showing similar survival for P36 (D_37%=3.0 Gy) and P80 (D_37%=2.9 Gy), but decreased survival for D20 (D_37% = 1.6 Gy). D20 irradiated samples exhibit increased 53BP1 foci shortly after irradiation, slower resolution of the foci, and larger residual 53BP1 foci after 24 h, indicating unrepaired complex breaks. These experimental observations are supported by the in silico study which demonstrates that irradiation at D20 leads to a 1.7-fold increase in complex DSBs with respect to the total number of strand breaks compared to P36 and P80. This combined approach provides valuable insights into the cellular and molecular effect of proton radiation, emphasizing the increased DNA damage complexity at the distal end of the Bragg curve, and has the potential to enhance the efficacy of proton therapy.

Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delays, leading to worse outcomes and a decreased quality of life. Therefore, there is an urgent need for an approach to predicting normal mucosal responses in patients prior to treatment. We here describe an assay to detect irradiation responses in healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated and cultured for three days. Seven samples were irradiated with X-ray, and three additional samples were irradiated with both X-ray and protons. Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample using standardized automated image analysis. Furthermore, increased levels of inflammation-inducing factors—major drivers of mucositis development—could be detected after irradiation. This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.

High linear energy transfer (LET) radiation is more harmful than low LET radiation because it deposits energy in a concentrated manner, resulting in clustered DNA damage (CDD). Double strand breaks (DSBs) are among the most damaging types of DNA damage, and if not repaired, they may trigger cell death. DSBs can be repaired through three mechanisms: non-homologous end joining (NHEJ), homologous recombination (HR), and theta-mediated end joining (TMEJ). This study aimed to assess how these pathways contribute to repairing DSBs induced by low LET X-ray and proton radiation, and high LET alpha-particle radiation. We used mouse embryonic stem (mES) cells lacking key repair proteins to examine clonogenic survival and the formation and resolution of 53BP1 foci, a DNA damage marker, after exposure to X-ray, proton, and alpha-particle radiation. The results showed increased sensitivity to X-ray and proton radiation in NHEJ, HR, and TMEJ repair-deficient cell lines compared to wild-type cells, with similar trends for both radiation types. Notably, Rad54-deficient cells showed slower resolution of 53BP1 foci after proton exposure, indicating increased reliance on HR for repairing proton-induced DSBs. Clonogenic survival assays revealed a relative biological effectiveness (RBE) of 4.6-5.8 for alpha-particles compared to protons and X-rays, confirming that alpha-particles are more effective at causing cell death. Our findings suggest that TMEJ is important for repairing DSBs caused by alpha-particles. This study highlights differences in repairing low LET versus high LET DNA damage, offering new insights for radiation biology and therapeutic strategies.

Proton-induced cell death is primarily driven by the induction and repair of DNA double strand breaks. While DNA damage dynamics have been extensively studied, the early cellular responses to proton irradiation remain underexplored. To address this, we developed a novel live-cell microscopy platform that enables real-time visualization of cellular responses to DNA damage induced by proton therapy. We designed a modular set-up with the requirement that it can be assembled and disassembled within 30 minutes, allowing for efficient deployment in an R&D proton beam line. An inverted fluorescence microscope was mounted at a 90-degree angle relative to the horizontal proton beam, enabling accurate irradiation at various depths along the spread-out Bragg peak with precise dosimetry and control over dose rates. As a proof-of-concept, we investigated the formation of 53BP1 foci following proton irradiation and determined the foci dynamics over time. With this setup, we observed endogenous 53BP1 foci pre-irradiation, with radiation-induced foci appearing as early as 4 minutes post-irradiation. The maximum number of 53BP1 foci was observed 12 minutes after irradiation, and the foci could be tracked up to 30 minutes post-irradiation. Our platform enabled precise dosimetry and real-time monitoring of 53BP1-mClover-labeled FaDu cells during proton exposure. This robust setup holds significant potential for studying DNA damage repair dynamics at various positions along the Bragg peak and across different dose rates, including ultrahigh dose rates (FLASH).

Background: Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delay leading to worse outcomes and decreased quality of life. Therefore, there is an urgent need for an approach to predict normal mucosa response in patients prior to treatment. Methods: We here describe an assay to detect irradiation response in patient healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated, and cultured for three days. Seven samples were irradiated with X-ray and three additional samples were irradiated with both X-ray and protons. Results: Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample, using standardized automated image analysis. Furthermore, increased levels of inflammation inducing factors - major drivers in mucositis development - could be detected after irradiation. Conclusion: This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.