# Combinational Radiotherapies Improve Brain Cancer Treatment at High Dose Rates In Vitro

## TL;DR

Combining high-dose radiation with drugs improves brain cancer treatment by increasing DNA damage and killing cancer cells more effectively.

## Contribution

Demonstrates synergistic effects of ultra-high dose rate radiation with radiosensitisers and chemotherapy for brain cancer.

## Key findings

- Combinational treatment with IUdR, MTX, and UHDR X-rays increased 9L cell killing 5.4 times compared to conventional X-rays.
- γH2AX imaging showed increased DNA damage and dying cells after UHDR radiation.
- UHDR synchrotron X-rays showed enhanced cancer-killing effects even in resistant 9L cells.

## Abstract

Brain cancer is notoriously resistant to conventional treatments. New and improved methods have long been needed to improve outcomes. This has included the use of radiosensitisers combined with chemotherapy drugs to enhance conventional radiotherapies (RTs) and novel techniques. This includes ultra-high dose rate (UHDR) RT, which has been known to spare normal tissue whilst retaining tumour control. Using a clonogenic cell survival assay and γH2AX confocal imaging, we show a significant increase in cancer killing and DNA damage in 9L gliosarcoma brain cancer cells using UHDR X-rays (produced by synchrotron light sources). These dose rate effects demonstrate significant additive effects with high-Z iododeoxyuridine radiosensitisers combined with methotrexate drugs. Notable increases in DNA damage and possible cell death were observed following combinational treatment with these drugs and UHDR synchrotron X-ray fields. While UHDR effects are normally associated with tissue sparing, our results show an increased cancer-killing effect even with highly resistant 9L cells. Particularly, we show significant synergy when drugs are used to enhance the UHDR fields. This suggests a potential and interesting new option for using high dose rates and combinational RTs to improve brain cancer treatment.

Background/objectives: Brain cancer remains difficult to treat, with survival statistics stagnant for decades. The resistance of glioblastoma brain tumours can greatly challenge the effectiveness of conventional cancer radiotherapy. However, high dose rate radiotherapy has unique effects that allow for normal tissue sparing whilst maintaining tumour control. The addition of targeted radiosensitisers, such as the chemotherapeutic drug methotrexate (MTX) or the high-Z halogenated pyrimidine drug iododeoxyuridine (IUdR), can improve radiotherapy outcomes. Combining these radiosensitiser agents with ultra-high dose rate (UHDR) synchrotron X-rays can bear synergistic effects to enhance the efficacy of these multi-modal UHDR therapies, providing a means to overcome the radioresistance of brain cancer. Methods: Here, we use controlled in vitro assays following treatment, including a clonogenic assay to determine long-term cell survival and γH2AX immunofluorescent confocal microscopy to quantify double-strand DNA breaks (DSBs). Results: We find significant enhancement for highly synergistic combinations of IUdR+MTX with synchrotron X-rays. Cell survival results demonstrate 5.4 times increased 9L gliosarcoma cell killing when these agents are combined with UHDR synchrotron X-rays compared with conventional X-rays alone at the same 5 Gy dose. The underlying mechanisms are unveiled using γH2AX imaging and reveal significant increases in DSBs and dying cells following exposure to UHDR radiation. Conclusions: Our results demonstrate that highly synergistic combination treatments using UHDR synchrotron radiation can yield significantly improved brain cancer killing compared with conventional radiotherapy. We anticipate that these additive, multi-modal combination therapies will provide options for more targeted and effective use of radiotherapies for the future treatment of brain cancer.