Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer

Abstract

Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise; however, current clinical trials are limited by modest response rates. Radiotherapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. Ultrahigh-dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, however, the immunomodulatory properties of FLASH irradiation remain unknown. Here, we demonstrate that single high-dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH-treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8⁺ T cells. Compared with conventional irradiation, FLASH irradiation increased intratumoral T-cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8⁺ T-cell infiltration and enhance the efficacy of αPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer.

Figure 1.. Abdominopelvic FLASH irradiation promotes intestinal regeneration and has similar tumor control compared to CONV irradiation in the ID8 ovarian cancer mouse model.

(A) CT images (coronal and sagittal slices) showing reproducible mouse positioning within the stereotactic frame. The yellow shaded rectangle (4 cm × 4 cm) highlights the irradiated region in the abdomen; the 4 cm length with the cranial border at the 10^th rib encompasses the entire small intestine and pelvic region. The distance from the abdominal wall to the ventral surface of the spine, representing the most posterior extent of the abdominal cavity, is 1.3 cm (yellow arrow). (B) Depth dose, craniocaudal and lateral profiles at the entrance surface for FLASH and CONV setups using EBT3 Gafchromic films between layers of polystyrene. The doses are uniformly distributed (within <10% heterogeneity) within the abdominopelvic region. (C) Quantification of the average number of regenerating crypts per jejunal circumference 96 hours after 14 Gy abdominopelvic irradiation, demonstrating over double the number of regenerating crypts after FLASH vs. CONV irradiation (n=10 mice in CONV; n=7 in FLASH; 3 circumferences per mouse were analyzed). **p<0.01, CONV vs. FLASH compared by unpaired 2-tailed Student’s t-test. Scale bar shows 200 μm (top) and 100 μm (bottom). (D) Representative images showing metastatic tumor burden in mice that were intraperitoneally injected with ID8 ovarian tumor cells (left) and the quantification of total tumor weight and ascites volume in sham (Control), 14 Gy CONV, or 14 Gy FLASH irradiated mice demonstrating similar tumor control efficacy with both FLASH and CONV irradiation (n=8 mice per group). *p<0.05, **p<0.01 compared by one-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent standard deviation of the mean.

Figure 2.. Abdominopelvic CONV and FLASH irradiation decrease intratumoral immunosuppressive regulatory T cells and increase cytolytic CD8⁺ T cells in ID8 ovarian tumors.

(A) Experimental design where C57BL/6 mice were injected with 5×10⁶ ID8 cancer cells. Ten days post-injection, mice were irradiated with either 14 Gy CONV or FLASH radiotherapy. Mice were analyzed at early (96 hours post irradiation) and late (17 days post irradiation). (B) Flow cytometry analysis for lymphocytes (CD45⁺), T cells (TCRβ⁺), CD4⁺, CD8⁺, and Treg (CD4⁺ FoxP3⁺) infiltration, Ki-67⁺ proliferation, and CD107a⁺ staining in the tumor bearing omentum at 96 hours and 17 days post irradiation (n= 5 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. Error bars represent standard deviation of the mean. At least 2 independent experiments showed similar results.

Figure 3.. Abdominopelvic CONV and FLASH irradiation enhance the efficacy of αPD-1 therapy in the ID8 ovarian cancer tumor model.

(A) Experimental design where C57BL/6 mice were injected with 5×10⁶ ID8 cancer cells. On days 7, 10, and 13 post-injection, mice were treated with αPD-1 or IgG control. On day 10 post-injection, mice were treated with 14 Gy CONV or 14 Gy FLASH irradiation. On day 27, mice were analyzed. (B) Macroscopic images of the tumor-bearing mice in each treatment arm. (C) Intraperitoneal tumor weight and volume of ascites fluid (n= 5 mice per group). *p<0.05, **p<0.01, ***p<0.001 compared by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. Error bars represent standard deviation of the mean.

Figure 4.. Abdominopelvic CONV and FLASH irradiation in combination with αPD-1 therapy enhance intratumoral CD8⁺ infiltration and decrease the Treg to T effector ratio in the ID8 ovarian tumor microenvironment.

Flow cytometry analysis for lymphocytes (CD45⁺), T cells (TCRβ⁺), CD4⁺, CD8⁺, and Treg (CD4⁺ FoxP3⁺) infiltration, Ki-67⁺ proliferation, and CD107a⁺ staining in the tumor bearing omentum at 96 hours and 17 days post irradiation (n= 5 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. Error bars represent standard deviation of the mean. At least 2 independent experiments showed similar results.

Figure 5.. Abdominopelvic CONV and FLASH irradiation in combination with αPD-1 therapy enhance intratumoral CD8⁺ infiltration and decrease the Treg to T effector ratio in the UPK10 ovarian tumor microenvironment.

(A) Experimental design where C57BL/6 mice were injected with 5 million UPK10 cancer cells. On days 7, 10, and 13 post-injection, mice were treated with αPD-1 or IgG control. On day 10 post-injection, mice received 14 Gy CONV or 14 Gy FLASH irradiation. On day 22, mice were analyzed. (B) Macroscopic images of the tumor-bearing mice. (C) Intraperitoneal tumor nodule weight. (D) Flow cytometry analysis for lymphocytes (CD45⁺), T cells (TCRβ⁺), CD4⁺, CD8⁺, and Treg (CD4⁺ FoxP3⁺) infiltration, Ki-67⁺ proliferation, and CD107a⁺ staining in the tumor bearing omentum at 96 hours and 17 days post irradiation (n= 5 mice per group) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. Error bars represent standard deviation of the mean. At least 2 independent experiments showed similar results.

Figure 6.. Abdominopelvic FLASH irradiation combination with αPD-1 therapy does not increase toxicity compared to FLASH irradiation alone in the ID8 ovarian cancer model.

(A) Body weight of mice following treatment with IgG, 14 Gy abdominal FLASH irradiation, αPD-1, or the FLASH and αPD-1 combination treatment. (B) Stool pellet counts (24 hr) from mice in treatment groups at 96 hours or 17 days post irradiation. (C) Complete blood cell (CBC) analysis of blood collected at day 27 of the experiment after tumor injection.