Investigating Proton and Electron Flash in novel 3D cultures

Background: There is world-wide interest in ultra-high dose rate radiotherapy (FLASH) (typically > 40 Gy/s) as it appears to offer increased normal tissue sparing, compared to conventional radiotherapy while still suppressing tumour growth.

CRUK has funded a RadNet FLASH infrastructure which draws on the strengths of the RadNet partners and provides a national resource for FLASH research.

This proposal will use this national FLASH Infrastructure to access novel 3D cultures developed in Cambridge; electron FLASH (in Oxford) and proton FLASH (in Manchester).

This will enable gaps in the mechanistic understanding of FLASH, where there is currently a lack of high-quality data to be directly addressed.

Aims: to investigate the influence of the FLASH physical delivery parameters and oxygenation conditions on DNA repair and cellular response mechanisms in tumour and normal tissue over timescales from seconds to days.

Methods: The novel primary 3D culture system developed in Cambridge takes advantage of the regeneration properties of epithelia and allows the establishment of trypsin-free, long-term, self-maintaining and expandable primary 3D cultures of mouse and human epithelial tissues.

This cutting-edge 3D system shows several key advantages compared to previous methods used in many other laboratories, such as its passage-free feature, which allows for maintaining fully confluent like-tissue primary cells in culture, under homeostatic conditions and its long-term culture (for up to 2-years).

It is widely assumed that the mechanisms for FLASH are agnostic of the radiation modality, but there are few studies that directly compare this.

This study will directly compare electron FLASH with proton FLASH in the same novel tissue culture, over timescales ranging from seconds to days.

A panel of experimental techniques will be used to investigate the mechanisms of DNA repair and cell behaviour (e.g. proliferation, differentiation and cell competition) using immunohistochemistry, confocal imaging and the Sartorius Incucyte Live cell analysis Instrument.

The 3 objectives of this proposal which build on mathematical modelling studies at Manchester and Oxford investigate: 1) Radiochemical response (seconds to minutes) this allows similarities and differences in the beam delivery parameters for FLASH to be elucidated. 2) Extending the timescale from minutes to hours allows the DNA repair response to be characterised to enable the optimal timing and fractionation for FLASH delivery. 3) Further extending the timescale to days enables the normal tissue and tumour cellular response to be studied to bridge the mechanistic knowledge-gap between in vivo literature results and the results of this study.