Background and Aims

Little is known about the mechanism of the biologically proton versus photon irradiation in healthy tissue. Differences in DNA double strand breaks could initiate a distinct secondary response of normal tissue cells, such as the initiation of senescence. To investigate these potential differences between proton and photon irradiation, we used our murine salivary gland organoid model that closely resembles the in vivo response after irradiation.

Methods

First, we established the induction of senescence and senescence-related genes upon both proton and photon irradiation using SA-b-Gal, p16 and p21 as markers.

Results

Interestingly, no differences were observed in the initiation of senescence. However, bulk RNA sequencing showed clear distinctive transcriptome profiles at different time points and radiation doses, especially the inflammatory response. Significant differences were observed in genes related to cytoplasmatic dsDNA and dsRNA recognition, immunity activation, Interferon 1 (IFN1) response and tissue development. Next to this, proton irradiation enhanced cGAS-related signalling, the number of micronuclei containing both cGAS and dsDNA and both cytoplasmic dsRNA and genes related to cytosolic dsRNA sensing. All this coincided with a higher expression of Interferon stimulated genes.

Conclusions

Our study suggests that the DNA damage induced by proton irradiation might lead to more dsDNA leakage into the cytoplasm and consequently more cGAS activation. This might lead to a more pronounced activation of Interferon stimulated genes and dsRNA release that act as positive feedback for the activation of immune responses and inflammation. This enhanced cellular immune response could lead to dissimilar normal tissue response to proton versus photon irradiation.

Background and Aims

Little is known about the mechanism of the biologically proton versus photon irradiation in healthy tissue. Differences in DNA double strand breaks could initiate a distinct secondary response of normal tissue cells, such as the initiation of senescence. To investigate these potential differences between proton and photon irradiation, we used our murine salivary gland organoid model that closely resembles the in vivo response after irradiation.

Methods

First, we established the induction of senescence and senescence-related genes upon both proton and photon irradiation using SA-b-Gal, p16 and p21 as markers.

Results

Interestingly, no differences were observed in the initiation of senescence. However, bulk RNA sequencing showed clear distinctive transcriptome profiles at different time points and radiation doses, especially the inflammatory response. Significant differences were observed in genes related to cytoplasmatic dsDNA and dsRNA recognition, immunity activation, Interferon 1 (IFN1) response and tissue development. Next to this, proton irradiation enhanced cGAS-related signalling, the number of micronuclei containing both cGAS and dsDNA and both cytoplasmic dsRNA and genes related to cytosolic dsRNA sensing. All this coincided with a higher expression of Interferon stimulated genes.

Conclusions

Our study suggests that the DNA damage induced by proton irradiation might lead to more dsDNA leakage into the cytoplasm and consequently more cGAS activation. This might lead to a more pronounced activation of Interferon stimulated genes and dsRNA release that act as positive feedback for the activation of immune responses and inflammation. This enhanced cellular immune response could lead to dissimilar normal tissue response to proton versus photon irradiation.

Background and Aims

We present the current status and outcomes of the joint radiobiological experiment performed at eight European proton therapy centers or research institutes. The study aims to spot the potential differences in the in vitro proton RBE values measured by different groups sharing a similar setup and identify its causes.

Methods

A phantom and a protocol for sample preparation and post-processing are shared among the participants to ensure minimal differences in the biological part of the experimental procedure. In this phantom, V79 cells grow on the polyester slides that can be inserted at different depths, which enables their simultaneous irradiation at multiple positions within the radiation field. The setup is irradiated with proton beams with two SOBP configurations (6 cm, 6 Gy, and 4 cm, 8 Gy), followed by the reference photon irradiation (LINAC or x-ray), and the biological effect is evaluated using a colony-forming assay.

Results

The study is still ongoing, and the spread of data for measured cell survival is yet to be evaluated. However, some non-obvious differences in the experimental procedures and setups are already revealed, e.g. post-processing timing or varying dose distributions in the beam plateau/fall-off regions.

Conclusions

As an outcome of the experiment, we plan to summarize the details of the experimental procedure for biological experiments with proton beams, differing between the centers across Europe. Accounting for these details would help to harmonize future studies in the field.

This work was supported by EU Horizon2020 grant 730983 (INSPIRE).