Spatially fractionated radiotherapy has shown potential to improve therapeutic outcomes possibly with an immunogenic mechanistic component. Here we report on in vivo mouse studies investigating mini-GRID pencil-beam radiotherapy combined with anti-PD-1 immune checkpoint blockade. Methods: mini-GRID therapy was delivered at 225kV using the XStrahl Small Animal Radiation Research Platform with two custom lead mini-GRIDs, each consisting of an array of equally spaced holes: 1 mm diameter with 1mm center-to-center spacing and 254 µm diameter with 508 µm spacing. Mini-GRID dosimetry was characterized using EBT3 film to determine peak-to-valley dose ratios and output. Two studies were performed with C57BL/6J mice bearing subcutaneous LLC1 lower extremity tumors. In the first, mice (n=5/group) were treated in 3 groups with a single fraction: 15 Gy open field, 15 Gy 1 mm mini-GRID, or 24 Gy 1 mm mini-GRID. In the second, mice (n=6-7/group) were treated with fractionated mini-GRID radiotherapy in 5 groups: 15 Gy open field x 3 fractions, 15 Gy hemi-irradiation x 3 fractions, 15 Gy 1 mm mini-GRID x 3 fractions, or 15 Gy 254 µm mini-GRID x 3 fractions. All mice were treated with 200 μg anti-PD-1 antibody on days 0, 3, and 6, then weekly until humane endpoint (tumor >15 mm in any dimension or ulceration). Results: Peak to valley ratios were 24.5 ± 0.6 and 19.8 ± 0.7 for the 1 mm and 254 µm mini-GRIDs, respectively. Tumor growth and mean survival times in both studies were significantly reduced for all non-open field arms (p < 0.05; Log Rank for survival; 2-way ANOVA for tumor growth). Conclusions: Two novel mini-GRIDs were characterized and tested in combination with anti-PD-1 therapy. In conclusion, in this study, when anti-PD-1 was combined with either mini-GRID therapy (single or fractionated) or hemi-irradiation no improvement was observed in tumor growth delay or survival when compared to anti-PD-1 combined with conventional open field radiotherapy.
