Maximizing tumor immunity with fractionated radiation

Abstract

Purpose: Technologic advances have led to increased clinical use of higher-sized fractions of radiation dose and higher total doses. How these modify the pathways involved in tumor cell death, normal tissue response, and signaling to the immune system has been inadequately explored. Here we ask how radiation dose and fraction size affect antitumor immunity, the suppression thereof, and how this might relate to tumor control.

Methods and materials: Mice bearing B16-OVA murine melanoma were treated with up to 15 Gy radiation given in various-size fractions, and tumor growth followed. The tumor-specific immune response in the spleen was assessed by interferon-γ enzyme-linked immunospot (ELISPOT) assay with ovalbumin (OVA) as the surrogate tumor antigen and the contribution of regulatory T cells (Tregs) determined by the proportion of CD4(+)CD25(hi)Foxp3(+) T cells.

Results: After single doses, tumor control increased with the size of radiation dose, as did the number of tumor-reactive T cells. This was offset at the highest dose by an increase in Treg representation. Fractionated treatment with medium-size radiation doses of 7.5 Gy/fraction gave the best tumor control and tumor immunity while maintaining low Treg numbers.

Conclusions: Radiation can be an immune adjuvant, but the response varies with the size of dose per fraction. The ultimate challenge is to optimally integrate cancer immunotherapy into radiation therapy.

Figure 1

Radiation dose-dependently increases tumor control, tumor-specific IFNγ⁺ splenocytes and alters splenic Tregs. Mice were implanted with 0.8×10⁶ B16-OVA cells s.c. in the leg and treated 10 days later with various doses of radiation. 7 days after treatment tumor size and splenic responses were measured. A) Tumor size as mean mm diameter (2 dimension) of n=4-16 ± s.e.m. B) Splenocytes were mixed ex vivo with EG7.OVA cells and the number of IFNγ-producing cells determined by ELISPOT. Data are mean number of spots per 10⁵ splenocytes of n=3-16 ± s.e.m. C) Splenocytes were stained for CD4, CD25 and Foxp3 and enumerated by flow cytometry. Data are mean CD4+, CD25+ and Foxp3+ Tregs as fraction of CD4+ Splenocytes of n=1-11 ± s.e.m. * p<0.05 compared to 0Gy.

Figure 2

Fractionated radiation affects tumor control, tumor-specific IFNγ⁺ splenocytes and alters splenic Tregs. Mice bearing B16-OVA tumors of 4mm in size were given fractionated radiation in 6h intervals of a total dose of 15Gy and left for 7 days to recover. A) Tumor size as mean diameter of n=12 ± s.e.m. B) Mean number of IFNγ- ELISpots per 10⁵ splenocytes of n=3-6 ± s.e.m. in response to EG7.OVA ex vivo re-stimulation. C) Mean percent of splenic CD4⁺CD25⁺Foxp3⁺ Tregs as analyzed by FACS of n=8-12 ± s.e.m. * p<0.05 compared to 0Gy.