Using immunotherapy to enhance the response of a C3H mammary carcinoma to proton radiation

Abstract

Background

The benefit of combining immunotherapy with photon irradiation has been shown pre-clinically and clinically. This current pre-clinical study was designed to investigate the anti-tumour action of combining immunotherapy with protons.

Materials and Methods

Male CDF1 mice, with a C3H mammary carcinoma inoculated on the right rear foot, were locally irradiated with single radiation doses when tumours reached 200mm³. Radiation was delivered with an 83–107MeV pencil scanning proton beam in the centre of a 3 cm spread out Bragg peak. Following irradiation (day 0), mice were injected intraperitoneal with anti-CTLA-4, anti-PD-1, or anti-PD-L1 (10 mg/kg) twice weekly for two weeks. Endpoints were tumour growth time (TGT3; time to reach 3 times treatment volume) or local tumour control (percent of mice showing tumour control at 90 days). A Student’s T-test (tumour growth) or Chi-squared test (tumour control) were used for statistical analysis; significance levels of p < 0.05.

Results

Untreated tumours had a mean (± 1 S.E.) TGT3 of 4.6 days (± 0.4). None of the checkpoint inhibitors changed this TGT3. A linear increase in TGT3 was seen with increasing radiation doses (5–20 Gy), reaching 17.2 days (± 0.7) with 20 Gy. Anti-CTLA-4 had no effect on radiation doses up to 15 Gy, but significantly enhanced 20 Gy; the TGT3 being 23.0 days (± 1.3). Higher radiation doses (35–60 Gy) were investigated using a tumour control assay. Logit analysis of the dose response curve, resulted in a TCD50 value (radiation dose causing 50% tumour control; with 95% confidence intervals) of 48 Gy (44–53) for radiation only. This significantly decreased to 43 Gy (38–49) when mice were treated with anti-CTLA-4. Neither anti-PD-1 nor anti-PD-L1 significantly affected tumour control.

Conclusion

Checkpoint inhibitors enhanced the response of this C3H mammary carcinoma to proton irradiation. However, this enhancement depended on the checkpoint inhibitor and radiation dose.

Keywords:

• C3H mammary carcinoma
• proton radiation
• checkpoint inhibitors
• tumour growth delay
• local tumour control

Acknowledgements

The authors thank Ms. Dorthe Grand, Ms. Maria Lynnerup Bech, and Ms. Marianne Kristiansen for excellent technical help with the experiments and animal care.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data presented in this study are available on request from the corresponding author. The data are not publicly available because the results from these experiments and all other animal experiments at our institute are stored in a single data depository, therefore, access is limited to relevant qualified personnel only.

Additional information

Funding

This work was supported by the Danish National Board of Health’s research pool to strengthen research in immunotherapy of cancer under grant number J.nr. 4-1612-236/10; project 0300 and Kræftens Bekæmpelse (grant number: R302-A17231).