Evidence for Early Stage Anti-Tumor Immunity Elicited by Spatially Fractionated Radiotherapy-Immunotherapy Combinations

Abstract

The combination of radiotherapy and immunotherapy may generate synergistic anti-tumor host immune responses and promote abscopal effects. Spatial fractionation of a radiation dose has been found to promote unique physiological responses of tumors, which might promote synergy with immunotherapy. To determine whether spatial fractionation may augment immune activity, whole-tumor or spatial fractionation grid radiation treatment (GRID) alone or in combination with antibodies against immune checkpoints PD1 and CTLA-4 were tested in an immunocompetent mouse model using a triple negative breast tumor (4T1). Tumor growth delay, immunohistochemistry and flow cytometry were used to characterize the effects of each treatment type. Whole-beam radiation with immune checkpoint inhibition significantly restrained tumor growth in the irradiated tumor, but not abscopal tumors, compared to either of these treatments alone. In mice that received spatially fractionated irradiation, evidence of abscopal immune responses were observed in contralateral tumors with markedly enhanced infiltration of both antigen-presenting cells and activated T cells, which were preceded by increased systemic IFNγ production and led to eventual tumor growth delay. These studies suggest that systemic immune activation may be triggered by employing GRID to a primary tumor lesion, promoting anti-tumor immune responses outside the treatment field. Interestingly, PD-L1 was found to be upregulated in abscopal tumors from GRID-treated mice. Combined radio-immunotherapy therapy is becoming a validated and novel approach in the treatment of cancer. With the potential increased benefit of GRID to augment both local and metastatic disease responses, further exploration of GRID treatment as a part of current standards of care is warranted.

FIG. 1.

Irradiation setup. Panel A: Preirradiated film in position for whole-tumor irradiation. Panel B: Mouse in the treatment position. Panel C: GRID collimator. Panel D: GRID irradiated film. Panel E: Schematic representation of the experiment design. Shown here are mice with primary and abscopal tumors implanted on the same day. Additional analysis of mice with abscopal tumors implanted on the day of irradiation are not shown here.

FIG. 2.

Tumor growth curves when abscopal (nonirradiated) tumors were inoculated on the day of primary (1°) tumor irradiation. Each group consisted of 5–8 4T1-bearing mice and the error bars represent ±1 SEM. Panel A: Comparable growth rates in abscopal tumors after whole-beam irradiation to the primary (1°) tumors. Panel B: Decreased growth rate of abscopal tumors after GRID to the primary (1°) tumors. Panel C: Immune cell composition with selected IHC images in primary (1°) and abscopal tumors from WTRT-treated mice and GRID-treated mice. Panel D: Post-treatment changes in CD8:CD4 ratio in primary (1°) vs. abscopal tumors.

FIG. 3.

Comparison of tumor growth for (panels A–D) WTRT, WTRT + ICI, GRID and GRID + ICI, respectively, in primary (1°) (irradiated) or abscopal (nonirradiated) lesions. Each group consisted of 5–8 4T1-bearing mice and error bars represent ±1 SEM.

FIG. 4.

Panels A and B: CD8⁺ and CD4⁺ T-cell activation, respectively, in a nonirradiated tumor at day 6 and day 12. Each group consisted of 5–8 4T1-bearing mice and error bars represent ± 1 SEM. Panel C: Collective T-cell activation changes in nonirradiated tumor from day 6 to day 12.

FIG. 5.

Panels A and B: MHCII⁺ cells in abscopal tumors measured at day 6 and day 12, respectively. Each group consisted of 5–8 4T1-bearing mice and error bars represent ±1 SEM. Panel C: Collective MHCII⁺ changes from day 6 to day 12. Panel D: Dendritic cells (MHCII⁺/CD11c⁺) in abscopal tumors at day 12.

FIG. 6.

Panel A: IFNγ concentration in blood. Each group consisted of serum samples from 5–8 4T1-bearing mice and error bars represent ±1 SEM. Panel B: Collective change in IFNγ concentration from each treatment group at day 6 compared to baseline. Note that untreated mice were measured only at baseline and at day 12

FIG. 7.

PD-L1% measured by flow cytometry in the nonirradiated tumor cell population at day 6 and day 12. Each group consisted of 5–8 4T1-bearing mice and error bars represent ±1 SEM.