An in vivo Investigation of Spatially Fractionated Radiation in Combination with Anti-PD-1 Blockade Immunotherapy

Abstract

Purpose: GRID therapy (Spatially Fractionated RT) has the potential to amplify systemic anti-tumor immune effect. The optimal GRID design, radiation dosage and combination with immunotherapies are not well understood. In this work, we characterized two novel, high-resolution GRIDs of smaller width and spacing had been was previously employed at Duke University. By combining these GRIDs with anti-PD-1 immune checkpoint blockade, we investigated the efficacy of this combination therapy in a preclinical mouse model. This work has two main aims. First, to observe any anti-tumor response from GRID therapy that arises from sparing T cell lymphocytes in the valleys adjacent to high peak doses of radiation facilitating tumor antigen presentation. Second, to investigate the robustness and replicability of a previously published influential work (Markovsky et al., 2019) which demonstrated that hemi-irradiation can produce similar levels of tumor control as conventional radiation therapy [1]. This is one of the first studies we are aware of that combines mini-GRID treatment with immunotherapies. Methods: Prior to in vivo studies, two novel, high-resolution in-house mini-GRIDs were characterized using the Small Animal Radiation Research Platform (SARRP). To perform this characterization, the SARRP (225 kV,13 mAs) irradiated EBT3 film. Using the Epson 11000XL scanner, EBT3 films were scanned prior to and post irradiation. Median filters were applied to avoid artificially suppressing peak and valley dose distributions. A calibration curve was generated using irradiations of a known dosage to determine what dose was delivered to the high and low-dose regions of the GRIDs. From this, peak-to-valley dose ratios as well as output factors were be calculated. Then, two pilot studies were performed using SARRP to deliver RT to C57BL/6J mice with subcutaneous LLC1 (Lewis Lung Carcinoma) flank tumors. The first study tested the therapeutic efficacy of single dose radiation while the second investigated fractionated radiation utilizing the newer, high-resolution GRIDs. In the first study, mice were randomized to four groups: 15 Gy to an open 20 mm x 20 mm field (n=5), 15 Gy to a GRID with 1mm width and spacing (n=5), and 24 Gy to a GRID with 1mm width and spacing (n=5). For the second study, mice were randomized to four groups: an open 20 mm x 20 mm field (n=6), the same field irradiating only half the tumor (n=6) (following Markovsky et al., 2019), a GRID with 1 mm width and spacing (n=6) and a GRID with 254 µm width and spacing (n=7). For both in vivo studies, all mice in this study were treated with 200 μg of anti-PD-1 antibody prior to 15 Gy of RT (single AP field) on days 0, 3, and 6. Anti-PD-1 was then administered weekly until mice reached humane endpoint (>15 mm in any dimension or ulceration). Tumor growth was measured thrice weekly using digital calipers. Results: • Film Characterizations: The peak to valley dose ratios for the 254 µm and 152 µm GRIDS were 19.8 ± 0.7 and 9.37 ± 0.33 respectively. The output factors for these GRIDs were 0.62 ± 0.09 and 0.59 ± 0.03. • First in vivo Study: Tumor quadrupling times (days, ± SD) were: 8.94 ± 1.17 (open field, 15 Gy), 7.75 ± 0.91 (1mm GRID, 15 Gy) and 7.98 ± 1.08 (1mm GRID, 24 Gy). Mean survival times (days, ± SD) were: 16.00 ± 0.00 (open field, 15 Gy), 12.8 ± 1.09 (1mm GRID, 15 Gy and 24 Gy). None of these differences were statistically significant. The width of the valleys for the 254 µm GRID is 544 ± 33.94 µm and for the 152 µm GRID is 548 µm ± 31.57. Assuming a clinically that 100 cells with a diameter of 5µm represent a clinically relevant sample for irradiation, this is a sufficient area for irradiation. • Second in vivo Study: Tumor quadrupling times (days, ± SD) were: 12.8 ± 2.6 (open field), 8.4 ± 2.8 (hemi-irradiation), 9.7 ± 2.4 (1mm GRID), and 6.4 ± 4.4 (0.25 mm GRID). Mean survival times (days, ± SD) were: 14.2 ± 2.1 (open field), 12.2 ± 1.0 (hemi-irradiation), 11.3 ± 1.6 (1mm GRID), and 10.4 ± 2.2 (254 µm GRID). Compared to the open field, time to tumor quadrupling was lower in all groups, significantly so in the hemi-irradiated and 0.25 mm GRID groups (p<0.05). Both the hemi-irradiated and GRID groups showed significantly shorter mean survival times compared to conventional open-field treatment (p<0.05 for 1 mm GRID, p<0.01 for hemi-irradiation and 0.25 mm GRID). Conclusion: Two novel mini-GRIDs were successfully characterized using the SARRP for preclinical work, and sufficiently kept valley doses below 1.5 Gy for infiltrative T cell function [2] with peak doses greater than 15 Gy, thereby enabling tumor antigen presentation. However, neither single dose nor fractionated GRID therapy with anti-PD-1 improved tumor growth delay or survival in a preclinical LLC flank model. In contrast to published data with this model, hemi-irradiation worsened tumor control compared to conventional treatment. Our work, therefore, does support the conclusion drawn in the Markovsky paper that hem-irradiation provides comparable tumor control using hemi-irradiation to conventional treatment [1]. The development of new technologies such as FLASH radiotherapy may present new opportunities for future studies utilizing GRID therapy.