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.

This article is featured in Highlights of This Issue, p. 243

Ovarian cancer is the deadliest gynecologic malignancy with 295,000 new cases and 185,000 deaths worldwide in 2018 (1). The majority of women present with advanced-stage disease, and due to high recurrence rates (70%–80%), prognosis remains poor with 5-year overall survival at approximately 46.5% (2). Standard treatment for ovarian cancer consists of surgical debulking followed by platinum–taxane chemotherapy (3). In recent years, there has been an influx of novel therapies including biologics and targeted therapies for the treatment of advanced ovarian cancer.

Immunotherapies, including anti-programmed cell death-1 (αPD-1) immune checkpoint inhibitors, are a promising therapeutic strategy for advanced-stage ovarian cancer (4). However, many tumors are either resistant or relapse following immune checkpoint inhibition, with clinical trials demonstrating at most a 15% response rate (5). T-cell infiltration into the tumor microenvironment is associated with improved immunotherapy efficacy, and is correlated with antitumor activity and prolonged survival in ovarian cancer (6, 7). Therefore, there is a critical need for treatment modalities that increase T-cell infiltration and reduce immunosuppressive features of the tumor microenvironment to improve immunotherapy responses in ovarian cancer.

Radiotherapy is an attractive strategy to optimize the efficacy of immune checkpoint blockade due to its ability to induce a cell death that is immunogenic and convert the tumor into an in situ vaccine (8). Preclinical and clinical studies show that radiotherapy can synergize with immune checkpoint blockade in multiple tumor types and can sensitize resistant tumors to immune checkpoint inhibition (8). Importantly, radiotherapy in combination with checkpoint blockade results in improved local control of the irradiated tumor, enhanced systemic tumor control, and durable responses due to the induction of immunologic memory (8). Thus, radiotherapy is a promising strategy to enhance the effects of immune checkpoint blockade in multiple tumor types. Although ovarian cancer is known to be radiosensitive, the use of abdominopelvic radiotherapy in metastatic peritoneal disease is limited by gastrointestinal and hematopoietic toxicities (9–14).

Over the past decade, major technological advances in radiation oncology have enabled the use of ultrahigh-dose rate irradiation (FLASH) to deliver dose rates of >40 Gy/s. Preclinical studies of FLASH irradiation in multiple tissue sites including lung, brain, and skin have shown reduced normal tissue toxicity while maintaining tumor control mice, minipigs, and cats (15–23). Clinically, FLASH irradiation has been used in a human study of cutaneous T-cell lymphoma (24). Although preclinical and clinical studies have demonstrated immunostimulatory effects with conventional radiotherapy, the immunomodulatory properties of FLASH irradiation remain unknown (25, 26).

We previously reported a preclinical FLASH irradiation platform using a clinical linear accelerator (LINAC) for electron FLASH irradiation of small animals (27). In addition, we demonstrated that abdominal FLASH irradiation reduces gastrointestinal morbidity and mortality while maintaining tumor control in a preclinical ovarian cancer model (15). In this study, we investigate the immunomodulatory role of FLASH radiotherapy in combination with αPD-1 immune checkpoint blockade in sensitive and resistant preclinical models of ovarian cancer. We demonstrate efficacy and tolerability of FLASH and αPD-1 combination therapy in preclinical models of advanced ovarian cancer.

Mice

All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Stanford University in accordance with institutional and NIH guidelines. Six- to -eight week-old female C57BL/6 mice were obtained from The Jackson Laboratory. Standard animal care and housing was provided by the Stanford University School of Medicine and is under the care and supervision of the Department of Comparative Medicine's Veterinary Service Center. Mice were maintained on the irradiated Envigo Teklad diet containing 18% protein and 6% fat.

All mice in this study were age matched, sex matched, and irradiated at the same time of day. All mice were irradiated 10 days following ID8 or UPK10 tumor cell intraperitoneal (i.p.) injection. During irradiation, mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) injected into the peritoneum. Control mice were treated with the same dose of ketamine/xylazine but were not exposed to radiation. Immediately after irradiation, mice were placed on a warming blanket until they woke up from the anesthesia. Mice were then placed back into their standard housing environment and were monitored daily for body weight, appearance, respiratory rate, general behavior, and provoked behavior using the Mouse Intervention Scoring System (MISS 3; ref. 28).

Mouse irradiation

As previously described, we developed a custom mouse stereotactic positioning frame made of polylactic acid (PLA) plastic using 3-D printing (15). Reproducible positioning within the frame was achieved by registering the front teeth on a nylon filament at a fixed location at the cranial end of the frame with extension of the hindlimbs and tail through designated slots at the caudal end of the frame. Positioning reproducibility to within 1 mm was confirmed by micro CT imaging in a representative cohort of mice. An abdominal irradiation shield was made by 3-D printing a PLA plastic shell with a central opening of 4 cm (lateral) by 4 cm (craniocaudal). Internally, a 3-cm-thick layer of aluminum oxide powder in tandem with a 1-cm-thick layer of tungsten spheres (2-mm diameter) was placed, a combination designed to minimize leakage dose from bremsstrahlung radiation produced in the shield materials. The stereotactic frame was registered to the shield such that the opening extended from the tenth rib at the cranial border to 4 cm caudally, encompassing all of the small intestine. EBT3 Gafchromic film (Ashland Advanced Materials) dosimetry confirmed that leakage dose to the shielded portions of the body when irradiating with 16 MeV electrons was <3.5% of the central dose at 10 mm from the field edge and further.

The shield and positioning frame were loaded into a polystyrene cradle registered to specified locations relative to the LINAC treatment head for FLASH and CONV irradiation. For both FLASH and CONV setups, we placed EBT3 Gafchromic films between layers of polystyrene to measure transverse and depth dose profiles to characterize dose homogeneity throughout the treatment volume. In addition, entrance dose for every individual mouse irradiation was recorded by EBT3 Gafchromic films (1 × 2 inch) placed inside the positioning frame. For both FLASH and CONV irradiation, the dose was prescribed at the entrance surface of the mouse.

CONV irradiation setup

We used a Varian Trilogy radiotherapy system (Varian Medical Systems) to perform both CONV and FLASH irradiation. For CONV irradiation, the gantry was rotated to 180° (beam direction from floor to ceiling), and the collimator was rotated to 0°. The cradle with the shield and mouse jig was placed on top of a 15 cm electron applicator such that the distance from the electron scattering foil to the shield was 77.6 cm. Under service mode, irradiation was delivered using a clinical 16 MeV electron beam in the 400 MU/minute dose rate mode (pulse repetition rate 72 Hz). Calibration by film dosimetry determined that the entrance dose after the shield was 1.91 cGy/MU, with a resulting average dose rate of 0.126 Gy/s.

FLASH irradiation setup

We configured the Varian Trilogy radiotherapy system to perform FLASH irradiation as previously described (27). The gantry was rotated to 180°, the treatment head cover was removed, and the jaws were fully opened (40 × 40 cm). The cradle with radiation shield and mouse stereotactic frame was loaded and registered to fixed points on the face of the gantry, such that the distance from the electron scattering foil to the shield was 14.6 cm. Beam parameters were configured on a dedicated electron beam control board. We used an electron beam energy of approximately 16 MeV with the 16 MeV scattering foil (confirmed by depth dose measurements) and adjusted the radiofrequency power and gun current settings to produce a dose per pulse of 2.0 Gy at the entrance surface of the mouse. We controlled pulse delivery using a programmable controller board (STEMlab 125-14, Red Pitaya) and relay circuit to count the number of delivered pulses detected by the internal monitor chamber and impose beam hold and release through the respiratory gating system of the LINAC. We used an external ion chamber positioned after the mouse and 10 cm of solid water (where the dose rate did not saturate the chamber), calibrated to film measurements of entrance dose, to provide immediate dose readout per mouse. The pulse repetition rate was set to 90 Hz for an average dose rate of 210 Gy/s at 2 Gy/pulse at the entrance surface of the mouse.

Tissue processing and histologic analysis

Animals were euthanized via CO2 asphyxiation and secondary cardiac exsanguination. Soft tissues were harvested and immersion-fixed in 10% neutral buffered formalin for 24 hours, followed by PBS for 24 hours, and then stored in 70% ethanol. Three transverse sections of the small intestine from the jejunum (mid-segment) were collected. Formalin-fixed tissues were processed, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin.

Regenerating crypt counts

A total of three transverse sections of jejunum were analyzed per mouse for the number of regenerating crypts by the crypt microcolony assay (29). Transverse sections were analyzed if they met the following criteria: (i) a complete jejunal circumference was present and (2) the mucosa was oriented perpendicular to the long axis of the intestine. Crypts were considered regenerating if they comprised >10 basophilic crypt epithelial cells.

Ovarian cancer tumor models

The murine ID8 ovarian cancer cell line was obtained from Dr. Katherine F. Roby (University of Kansas Medical Center, Kansas City, KS; ref. 30). The murine UPK10 ovarian cancer cell line was obtained from Dr. Jose Conejo-Garcia (The Wistar Institute, Philadelphia, PA; ref. 31). Cell lines were authenticated from the original source and used within early passage numbers. In addition, cells were tested upon receipt for viability, cell morphology, and the presence of mycoplasma and viruses (Charles River Laboratories). ID8 cells were passaged in DMEM supplemented with 10% FCS and Pen/Strep. UPK10 cells were passaged in RPMI supplemented with 10% FCS, Pen/Step, and 50 μmol/L beta-mercaptoethanol. ID8 and UPK10 cells (5 × 106 cells in 200 μL PBS) were i.p. injected using a 27-gauge needle on day 0.

Injections

For immunotherapy treatments, 200 μg (ID8 model) or 20 μg (UPK10 model) of αPD-1 (clone 29F.1A12, Bio X Cell) or rat IgG2α isotype control antibody (clone 2A3, Bio X Cell) was suspended in 200-μL PBS and i.p. injected on days 7, 10, and 13 after tumor cell injection.

Tumor response and intestinal function

Tumor response was determined by counting tumor nodule numbers, measuring tumor weight, and measuring ascites volume (ID8 model) on necropsy on day 22 (UPK10 model) or 27 (ID8 model) following tumor cell injection. The number of formed stool pellets made over 24 hours at specified time points in the study was collected to determine intestinal function in live animals.

Flow cytometry

For flow cytometric analysis of tumor-bearing omentum and/or mesenteric, lymph nodes were harvested on day 22 or 27 after tumor inoculation. Gating and analysis were performed using FlowJo software (TreeStar). Single-cell suspensions were prepared in PBS with 2% BSA, and red blood cells were lysed using ACK Lysis Buffer (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA). Live/dead cell discrimination was performed using Zombie NIR Fixable Viability Kit (BioLegend). Cells were treated with Fc block (Miltenyi Biotec). Cell surface staining was done for 20 minutes. Immune cells were identified as CD45+ (eBioscience, 64-0451-82). B cells were identified as B220+ (eBioscience, 17-0452-81), macrophages as CD11b+ (eBioscience, 56-0112-82), F4/80+ (BioLegend, 123132), M-MDSCs as CD11b+Ly6C+ (BD Biosciences, 560593), PMN-MDSCs as CD11b+Ly6CloLy6G+ (BD Biosciences, 746448), M2 macrophage marker CD206 (BioLegend, 141727), and M1 macrophage marker iNOS (eBioscience, CXNFT). T cells were identified as TCRβ+ (eBioscience, 45-5961-82) and divided as CD8+ (eBioscience, 25-0081-82) or CD4+ (eBioscience, 11-0041-82). T cells were stained intracellularly after using a fixation/permeabilization kit (eBioscience) for FoxP3 (eBioscience, 50-5773-80) and Ki-67 (BD Biosciences, 561227) and CD107a (BioLegend, 121631). Samples were run on an LSR Fortessa X-20 (BD) and analyzed using FlowJo software (TreeStar).

Complete blood count analysis

At the time of sacrifice, blood was collected via cardiac puncture and processed using a Hemavet 950FS (Drew Scientific). Complete blood count (CBC) analysis was performed on the IgG, FLASH, αPD-1, and aPD-1 + FLASH-treated mice harboring ID8 or UPK10 tumors. Data for the hematocrit, hemoglobin, white blood cells, and platelets are shown.

Statistical analysis

Statistical significance was conducted using GraphPad Prism. Results were reported as mean ± SD. Analysis of two groups was performed using a Student t test. Analyses of three groups or more were performed using an ANOVA, and pair-wise comparisons were performed in a post hoc analysis with a Tukey or Sidak adjustment for multiple comparisons. All statistical tests were two-sided with an alpha level of 0.05.

Conventional and FLASH irradiation reduce regulatory T cells and increase T cells with cytolytic potential in the ovarian cancer tumor microenvironment

FLASH irradiation has emerged as a new treatment modality to reduce radiation-induced intestinal injury. Moreover, FLASH irradiation maintains similar tumor control to conventional irradiation in multiple tumor models (15–23). However, the immunomodulatory effects of FLASH irradiation remain unknown. To compare the immunomodulatory effects of total abdominopelvic conventional (CONV) and FLASH irradiation in preclinical models of ovarian cancer, we used a clinical linear accelerator modified to generate a 16 MeV electron beam to deliver both uniform treatment across the mouse and a homogenous depth dose (within <10% heterogeneity) throughout the mouse abdominal cavity (1.3 cm maximum depth) in both FLASH (210 Gy/s) and conventional mode (0.126 Gy/s; Fig. 1A and B; Supplementary Fig. S1). We developed a mouse stereotactic positioning frame with an irradiation field that extends 4 cm in the cranial/caudal direction starting at the 10th rib allowing for total abdominopelvic treatment (Fig. 1A). In agreement with our previous study, a sublethal dose of 14-Gy FLASH irradiation enhanced intestinal regeneration and maintained tumor control in the ID8 model of ovarian cancer metastasis (Fig. 1D and E; ref. 15). The ID8 ovarian cancer model is syngeneic in C57BL/6 mice, metastasizes throughout the peritoneal cavity, and forms tumor nodules along the small and large intestine (30). Ten days after ID8 cancer cell injection, the mice were randomized into sham-irradiated control, 14-Gy CONV, or 14-Gy FLASH-irradiation treatment groups. At 96 hours after irradiation, ID8 tumor–bearing mice treated with FLASH irradiation showed increased numbers of regenerating crypts in the jejunum compared with mice treated with CONV irradiation (Fig. 1D). Analysis of tumor burden at day 27 after irradiation revealed a decrease in the total tumor weight and ascites volume in CONV- and FLASH-irradiated mice compared with the sham-irradiated control mice (Fig. 1E). No significant differences in tumor weight or ascites volume were observed when comparing CONV- and FLASH-irradiated mice, indicating that FLASH and CONV irradiations have similar efficacy in the treatment of ovarian cancer peritoneal tumors (Fig. 1E).

Figure 1.

Abdominopelvic FLASH irradiation promotes intestinal regeneration and has similar tumor control compared with 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 10th 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 versus CONV irradiation (n = 10 mice in CONV; n = 7 in FLASH; three circumferences per mouse were analyzed). **, P < 0.01, CONV versus FLASH compared by unpaired two-tailed Student t test. Scale bar, 200 μm (top) and 100 μm (bottom). D, Representative images showing metastatic tumor burden in mice that were i.p. 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 multiple comparisons test. Error bars, SD of the mean.

Figure 1.

Abdominopelvic FLASH irradiation promotes intestinal regeneration and has similar tumor control compared with 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 10th 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 versus CONV irradiation (n = 10 mice in CONV; n = 7 in FLASH; three circumferences per mouse were analyzed). **, P < 0.01, CONV versus FLASH compared by unpaired two-tailed Student t test. Scale bar, 200 μm (top) and 100 μm (bottom). D, Representative images showing metastatic tumor burden in mice that were i.p. 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 multiple comparisons test. Error bars, SD of the mean.

Close modal

Next, we compared the effects of 14-Gy CONV and FLASH irradiation in modulating the ovarian tumor immune microenvironment at both early (96 hours after irradiation) and late (17 days after irradiation) timepoints. ID8 tumor–bearing mice were treated with sham irradiation, 14-Gy conventional (CONV) irradiation, or 14-Gy FLASH irradiation at 10 days after cancer cell injection to allow for tumor seeding throughout the peritoneal cavity. At 96 hours and 17 days after irradiation, immune cell populations were analyzed in the tumor-bearing omentum and the draining mesenteric lymph nodes (Fig. 2A; Supplementary Fig. S2). At early timepoints, CONV and FLASH irradiation reduced the frequency of CD45+ leukocytes, T and B cells in the tumor microenvironment compared with the sham-irradiated control (Fig. 2B; Supplementary Fig. S3A). Notably, CONV and FLASH irradiation showed a shift in the Treg-to-T-effector ratio with a decrease in regulatory T cells and an increase in CD8+ T-cell proliferation compared with the sham-irradiated control (Fig. 2B). When comparing CONV and FLASH irradiation, there was an increase in CD4+ cells in FLASH-treated tumors compared with CONV-treated tumors (Fig. 2B). At later timepoints, CONV and FLASH irradiation showed an enhancement in CD107a+ CD8+ T cells with cytolytic potential compared with the sham-irradiated control (Fig. 2B). There were no differences found in the frequency of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), monocytic myeloid-derived suppressor cells (M-MDSCs), or the M2/M1 macrophage ratio within the tumor microenvironment with comparing sham-irradiated with CONV- or FLASH-irradiated mice (Supplementary Fig. S3A). Within the mesenteric lymph node, CONV and FLASH irradiation increased CD8+ T-cell proliferation consistent with a priming T-cell response in the local draining lymph node (Supplementary Fig. S3B). Overall, these data demonstrate that abdominopelvic FLASH irradiation maintains the ability to decrease regulatory T cells and increase cytolytic CD8+ T cells in the tumor microenvironment, suggesting an enhanced antitumor immune response with both treatment modalities in the ovarian cancer microenvironment.

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 after injection, mice were irradiated with either 14-Gy CONV or FLASH radiotherapy. Mice were analyzed at early (96 hours after irradiation) and late (17 days after 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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two independent experiments showed similar results.

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 after injection, mice were irradiated with either 14-Gy CONV or FLASH radiotherapy. Mice were analyzed at early (96 hours after irradiation) and late (17 days after 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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two independent experiments showed similar results.

Close modal

CONV and FLASH irradiation enhance the efficacy of αPD-1 therapy in the ID8 ovarian cancer model

Given the immunomodulatory properties of abdominopelvic CONV and FLASH irradiation, we hypothesized that both treatment modalities may enhance the efficacy of αPD-1 immune checkpoint blockade in preclinical models of ovarian cancer. We first used the ID8 syngeneic model of ovarian cancer, which is resistant to αPD-1 therapy even though the tumors express PD-L1 (Supplementary Fig. S4A; ref. 32). Mice were i.p. injected with ID8 ovarian cancer cells and randomized into six cohorts: isotype control antibody (IgG), IgG + 14-Gy CONV, IgG + 14-Gy FLASH, αPD-1, αPD-1 + 14-Gy CONV, or αPD-1 + 14-Gy FLASH using the treatment scheme shown in Fig. 3A. On day 27 after injection, mice were euthanized to analyze tumor burden and immune infiltration. CONV and FLASH irradiation reduced tumor weight and ascites volume in both the IgG and αPD-1 treatment arms (Fig. 3B and C). However, the FLASH + αPD-1 combination therapy had superior efficacy compared with FLASH + IgG with a significant reduction in tumor weight and ascites volume (Fig. 3C). These data indicate that CONV and FLASH irradiation enhance the efficacy of αPD-1 in a resistant ovarian cancer model.

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 after injection, mice were treated with αPD-1 or IgG control. On day 10 after 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 multiple comparisons post hoc test. Error bars, SD of the mean.

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 after injection, mice were treated with αPD-1 or IgG control. On day 10 after 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 multiple comparisons post hoc test. Error bars, SD of the mean.

Close modal

CONV and FLASH irradiation combined with αPD-1 decreases the Treg-to-T-effector ratio and increases intratumoral CD8+ T-cell infiltration in ovarian cancer peritoneal metastases

We next investigated the effects of each treatment on the ovarian tumor immune microenvironment. In the ID8 model, the combination of CONV or FLASH irradiation with αPD-1 were the only treatment groups that decreased the Treg-to-T-effector ratio that was associated with an increase in tumor-infiltrating CD8+ T cells (Fig. 4). In addition, CONV or FLASH irradiation in combination with αPD-1 reduced the immunosuppressive PMN-MDSC and M2 to M1 macrophage ratios in the tumor microenvironment (Supplementary Fig. S4B). These findings demonstrate that similar to CONV irradiation, FLASH irradiation in combination with αPD-1 is an effective strategy to reduce tumor progression and simultaneously enhance intratumoral CD8+ T-cell infiltration and reduce immunosuppressive monocyte populations in the αPD-1–resistant ID8 model of ovarian cancer.

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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two independent experiments showed similar results.

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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two independent experiments showed similar results.

Close modal

Next, we sought to determine if CONV or FLASH irradiation would enhance immune cell responses to αPD-1 treatment in the UPK10, αPD-1–sensitive model of ovarian cancer (33). UPK10 cells were i.p. injected into C57BL/6 mice, and mice were randomized into treatment groups as described previously (Fig. 5A). Mice were sacrificed at 22 days after injection for analysis. In this αPD-1–sensitive model of ovarian cancer, mice treated with αPD-1, 14-Gy CONV, 14-Gy FLASH, 14-Gy CONV + αPD-1, and 14-Gy FLASH + αPD-1 showed a similar decrease in tumor weight compared with mice treated with the IgG control (Fig. 5B and C). Despite similar tumor control, the combination CONV + αPD-1 and FLASH + αPD-1 treatments were the only treatment groups that increased intratumoral CD8+ T-cell infiltration compared with the IgG control (Fig. 5D; Supplementary Fig. S5). These findings demonstrate that similar to CONV irradiation, FLASH irradiation in combination with PD-1 blockade therapy improves intratumoral CD8+ T-cell infiltration in αPD-1–sensitive ovarian cancer models.

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 after injection, mice were treated with αPD-1 or IgG control. On day 10 after 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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two 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 after injection, mice were treated with αPD-1 or IgG control. On day 10 after 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 after 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 multiple comparisons post hoc test. Error bars, SD of the mean. At least two independent experiments showed similar results.

Close modal

FLASH irradiation and αPD-1 combination therapy does not increase toxicity compared with FLASH irradiation alone

Collectively, our data demonstrate that FLASH irradiation reduces radiation-induced intestinal injury, while maintaining similar tumor control and immunomodulation compared with CONV irradiation. These data raise the intriguing possibility that FLASH irradiation may be an effective strategy to enhance immune checkpoint blockade responses in ovarian cancer. However, both radiotherapy and checkpoint blockade therapy are known to cause gastrointestinal toxicity (26, 34). Therefore, we determined the safety and tolerability of the FLASH irradiation and αPD-1 combination therapy. As we have previously observed, 14-Gy abdominal FLASH irradiation in the FLASH + IgG and the FLASH + αPD-1 treatment groups reduced body weight over 7 days after irradiation that returned to baseline levels by 10 days after irradiation (Fig. 6A; ref. 15). Reduced body weight in the FLASH + IgG and FLASH + αPD-1–treated mice was associated with a decrease in formed stool pellets at 96 hours after irradiation that recovered to control levels by day 17 (Fig. 6B; Supplementary Fig. S6). Importantly, there were no significant differences in body weight or stool production between the FLASH + IgG and FLASH + αPD-1–treated mice (Fig. 6A and B). Similar results were observed in the UPK10 model (Supplementary Fig. S7). CBC analysis at 14 days after treatment revealed comparable hemoglobin, hematocrit, white blood cell counts, and platelets among all treatment groups indicating that abdominal FLASH and αPD-1 treatments are not associated with hematologic toxicity (Fig. 6C). These findings demonstrate that combination of FLASH irradiation with αPD-1 inhibition does not increase gastrointestinal or hematologic toxicities in mice compared with FLASH treatment alone.

Figure 6.

Abdominopelvic FLASH irradiation combination with αPD-1 therapy does not increase toxicity compared with 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 hours) from mice in treatment groups at 96 hours or 17 days after irradiation. C, CBC analysis of blood collected at day 27 of the experiment after tumor injection.

Figure 6.

Abdominopelvic FLASH irradiation combination with αPD-1 therapy does not increase toxicity compared with 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 hours) from mice in treatment groups at 96 hours or 17 days after irradiation. C, CBC analysis of blood collected at day 27 of the experiment after tumor injection.

Close modal

As immunotherapy becomes an increasingly utilized tool in our armamentarium against cancer, efforts to understand its interaction with traditional therapies are underway. In ovarian cancer, checkpoint inhibition is a promising therapeutic strategy. However, many tumors are either resistant or relapse following immune checkpoint monotherapy. Thus, optimization of immunotherapies through the identification of safe and effective immunomodulatory combination therapies is needed. Here, we demonstrate that combining abdominal FLASH radiotherapy with PD-1 blockade enhanced tumor control associated with increased immunomodulation, while demonstrating safety and tolerability.

FLASH irradiation is emerging as a new treatment paradigm that has the potential to enhance the therapeutic index of radiotherapy through its ability to reduce normal tissue toxicity. FLASH irradiation has been shown to reduce radiation-induced injury in multiple tissues (skin, lung, brain, and intestine) and in multiple species (mice, minipig, and human; refs. 15–23). In the intestine, preclinical studies have demonstrated that FLASH irradiation reduces intestinal injury and promotes regeneration in both naïve and tumor-bearing mice (15–18). Here, we provide further data to support that FLASH irradiation promotes intestinal regeneration after radiation injury in ovarian tumor–bearing mice.

Although preclinical and clinical studies have demonstrated the immunostimulatory effects of conventional radiotherapy, the immunomodulatory properties of FLASH remain largely unknown. Kim and colleagues recently reported increased intratumoral CD8+ T cells at 6 hours after irradiation in a Lewis lung carcinoma model (35). Our study reveals that at 96 hours after irradiation, FLASH irradiation increases intratumoral T cells, most notably CD4+ T cells, compared with CONV irradiation in a preclinical model of ovarian cancer. Both FLASH and CONV irradiation increased CD4+ and CD8+ T-cell proliferation and increased CD8+ T cells with cytolytic potential in the ovarian cancer tumor microenvironment and mesenteric lymph node. In addition, FLASH and CONV irradiation decreased the frequency of immunosuppressive regulatory T cells and reduced the regulatory to effector T-cell ratio both in the tumor and the mesenteric lymph node. Overall, our findings indicate that although FLASH irradiation reduces radiation-induced intestinal injury, it maintains the ability to increase T-cell infiltration and reduce immunosuppressive cells in the tumor microenvironment.

Conventional radiotherapy combined with checkpoint blockade has demonstrated improved tumor control in preclinical and clinical studies. However, the efficacy and safety of abdominal FLASH irradiation combined with immunotherapy are not known. We show that similar to conventional irradiation, combining FLASH and αPD-1 inhibition enhanced tumor control in an αPD-1–resistant model of ovarian cancer. Although the precise mechanism of therapeutic efficacy is unknown, the enhanced intratumoral effector to regulatory T-cell ratio following FLASH irradiation and αPD-1 therapy likely contributes to improved tumor control. Studies have shown the ovarian cancer microenvironment correlates with patient outcomes. Increased levels of CD3+ tumor-infiltrating lymphocytes and decreased levels of regulatory T cells in ovarian cancer correspond with improved prognosis and survival (36, 37).

Our findings have important clinical implications for the treatment of ovarian cancer and other abdominal/pelvic tumors. The overall low success of immunotherapy highlights the need to find alternate therapies that can modulate and improve immune treatment therapies. We have shown that combining FLASH irradiation with PD-1 blockade therapy is an effective and safe strategy in preclinical models of ovarian cancer. Future studies are needed to further elucidate mechanisms and long-term effects of FLASH exposure combined with immunotherapy use.

J.T. Eggold reports grants from the Department of Defense during the conduct of the study and grants from the National Science Foundation outside the submitted work. O. Dorigo reports personal fees from Merck, PACT, and GlaxoSmithKline; personal fees and other support from IMV and Genentech; and other support from AstraZeneca, Millenium, Pharmamar, and Bioeclipse outside the submitted work. B.W. Loo reports grants from the NIH during the conduct of the study; grants from Varian Medical Systems; and other support from TibaRay outside the submitted work. E.B. Rankin reports grants from the Department of Defense (CDMRP), National Science Foundation, National Cancer Institute, Carol and Doug Kimmelman Fund, and My Blue Dots; and nonfinancial support from Varian during the conduct of the study. No disclosures were reported by the other authors.

J.T. Eggold: Conceptualization, resources, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing, co-first author. S. Chow: Conceptualization, resources, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing, co-first author. S. Melemenidis: Data curation, formal analysis. J. Wang: Resources, investigation, methodology. S. Natarajan: Data curation, investigation. P.E. Loo: Investigation. R. Manjappa: Resources, investigation, methodology. V. Viswanathan: Data curation, formal analysis. E.A. Kidd: Conceptualization. E. Engleman: Conceptualization, funding acquisition. O. Dorigo: Conceptualization, funding acquisition. B.W. Loo: Conceptualization, resources, funding acquisition. E.B. Rankin: Conceptualization, resources, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Department of Defense Ovarian Cancer Research Program under Award No. W81XWH-17-1-0042; the National Science Foundation Graduate Research Fellowship under DGE-1656518 (J.T. Eggold); 1R01CA23395801 (E. Engleman and B.W. Loo); Carol and Doug Kimmelman Scholarship (E.B. Rankin); and the My Blue Dots fund (E.B. Rankin). The authors would like to thank Miguel Jimenez, Daniel Pawlak, and James Clayton from Varian Medical Systems for their technical assistance on the FLASH irradiation system.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Ferlay
J
,
Colombet
M
,
Soerjomataram
I
,
Mathers
C
,
Parkin
DM
,
Pineros
M
, et al
Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods
.
Int J Cancer
2019
;
144
:
1941
53
.
2.
Matulonis
UA
,
Sood
AK
,
Fallowfield
L
,
Howitt
BE
,
Sehouli
J
,
Karlan
BY
. 
Ovarian cancer
.
Nat Rev Dis Primers
2016
;
2
:
16061
.
3.
Morgan
RJ
 Jr.
,
Alvarez
RD
,
Armstrong
DK
,
Boston
B
,
Burger
RA
,
Chen
LM
, et al
Epithelial ovarian cancer
.
J Natl Compr Canc Netw
2011
;
9
:
82
113
.
4.
Matulonis
UA
,
Shapira-Frommer
R
,
Santin
AD
,
Lisyanskaya
AS
,
Pignata
S
,
Vergote
I
, et al
Antitumor activity and safety of pembrolizumab in patients with advanced recurrent ovarian cancer: Results from the phase II KEYNOTE-100 study
.
Ann Oncol
2019
;
30
:
1080
7
.
5.
Hamanishi
J
,
Mandai
M
,
Ikeda
T
,
Minami
M
,
Kawaguchi
A
,
Murayama
T
, et al
Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer
.
J Clin Oncol
2015
;
33
:
4015
22
.
6.
Spranger
S
,
Gajewski
TF
. 
Impact of oncogenic pathways on evasion of antitumour immune responses
.
Nat Rev Cancer
2018
;
18
:
139
47
.
7.
Zhang
L
,
Conejo-Garcia
JR
,
Katsaros
D
,
Gimotty
PA
,
Massobrio
M
,
Regnani
G
, et al
Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer
.
N Engl J Med
2003
;
348
:
203
13
.
8.
Sharabi
AB
,
Lim
M
,
DeWeese
TL
,
Drake
CG
. 
Radiation and checkpoint blockade immunotherapy: Radiosensitisation and potential mechanisms of synergy
.
Lancet Oncol
2015
;
16
:
e498
509
.
9.
Klaassen
D
,
Shelley
W
,
Starreveld
A
,
Kirk
M
,
Boyes
D
,
Gerulath
A
, et al
Early stage ovarian cancer: a randomized clinical trial comparing whole abdominal radiotherapy, melphalan, and intraperitoneal chromic phosphate: A National Cancer Institute of Canada Clinical Trials Group report
.
J Clin Oncol
1988
;
6
:
1254
63
.
10.
Sell
A
,
Bertelsen
K
,
Andersen
JE
,
Stroyer
I
,
Panduro
J
. 
Randomized study of whole-abdomen irradiation versus pelvic irradiation plus cyclophosphamide in treatment of early ovarian cancer
.
Gynecol Oncol
1990
;
37
:
367
73
.
11.
Chiara
S
,
Conte
P
,
Franzone
P
,
Orsatti
M
,
Bruzzone
M
,
Rubagotti
A
, et al
High-risk early-stage ovarian cancer. Randomized clinical trial comparing cisplatin plus cyclophosphamide versus whole abdominal radiotherapy
.
Am J Clin Oncol
1994
;
17
:
72
6
.
12.
Dembo
AJ
. 
Abdominopelvic radiotherapy in ovarian cancer. A 10-year experience
.
Cancer
1985
;
55
:
2285
90
.
13.
Hepp
R
,
Baeza
MR
,
Olfos
P
,
Suarez
E
. 
Adjuvant whole abdominal radiotherapy in epithelial cancer of the ovary
.
Int J Radiat Oncol Biol Phys
2002
;
53
:
360
5
.
14.
Cmelak
AJ
,
Kapp
DS
. 
Long-term survival with whole abdominopelvic irradiation in platinum-refractory persistent or recurrent ovarian cancer
.
Gynecol Oncol
1997
;
65
:
453
60
.
15.
Levy
K
,
Natarajan
S
,
Wang
J
,
Chow
S
,
Eggold
JT
,
Loo
PE
, et al
Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice
.
Sci Rep
2020
;
10
:
21600
.
16.
Ruan
JL
,
Lee
C
,
Wouters
S
,
Tullis
ID
,
Verslegers
M
,
Mysara
M
, et al
Irradiation at ultra-high (FLASH) dose rates reduces acute normal tissue toxicity in the mouse gastrointestinal system
.
Int J Radiat Oncol Biol Phys
2021
;
111
:
1250
61
.
17.
Diffenderfer
ES
,
Verginadis
II
,
Kim
MM
,
Shoniyozov
K
,
Velalopoulou
A
,
Goia
D
, et al
Design, implementation, and in vivo validation of a novel proton FLASH radiation therapy system
.
Int J Radiat Oncol Biol Phys
2020
;
106
:
440
8
.
18.
Kim
MM
,
Verginadis
II
,
Goia
D
,
Haertter
A
,
Shoniyozov
K
,
Zou
W
, et al
Comparison of FLASH proton entrance and the spread-out bragg peak dose regions in the sparing of mouse intestinal crypts and in a pancreatic tumor model
.
Cancers (Basel)
2021
;
13
:
4244
.
19.
Vozenin
MC
,
De Fornel
P
,
Petersson
K
,
Favaudon
V
,
Jaccard
M
,
Germond
JF
, et al
The advantage of FLASH radiotherapy confirmed in mini-pig and cat-cancer patients
.
Clin Cancer Res
2019
;
25
:
35
42
.
20.
Soto
LA
,
Casey
KM
,
Wang
J
,
Blaney
A
,
Manjappa
R
,
Breitkreutz
D
, et al
FLASH irradiation results in reduced severe skin toxicity compared to conventional-dose-rate irradiation
.
Radiat Res
2020
;
194
:
618
24
.
21.
Favaudon
V
,
Caplier
L
,
Monceau
V
,
Pouzoulet
F
,
Sayarath
M
,
Fouillade
C
, et al
Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice
.
Sci Transl Med
2014
;
6
:
245ra93
.
22.
Montay-Gruel
P
,
Petersson
K
,
Jaccard
M
,
Boivin
G
,
Germond
JF
,
Petit
B
, et al
Irradiation in a flash: Unique sparing of memory in mice after whole brain irradiation with dose rates above 100Gy/s
.
Radiother Oncol
2017
;
124
:
365
9
.
23.
Simmons
DA
,
Lartey
FM
,
Schuler
E
,
Rafat
M
,
King
G
,
Kim
A
, et al
Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation
.
Radiother Oncol
2019
;
139
:
4
10
.
24.
Bourhis
J
,
Sozzi
WJ
,
Jorge
PG
,
Gaide
O
,
Bailat
C
,
Duclos
F
, et al
Treatment of a first patient with FLASH-radiotherapy
.
Radiother Oncol
2019
;
139
:
18
22
.
25.
Twyman-Saint Victor
C
,
Rech
AJ
,
Maity
A
,
Rengan
R
,
Pauken
KE
,
Stelekati
E
, et al
Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer
.
Nature
2015
;
520
:
373
7
.
26.
Herrera
FG
,
Irving
M
,
Kandalaft
LE
,
Coukos
G
. 
Rational combinations of immunotherapy with radiotherapy in ovarian cancer
.
Lancet Oncol
2019
;
20
:
e417
e33
.
27.
Schuler
E
,
Trovati
S
,
King
G
,
Lartey
F
,
Rafat
M
,
Villegas
M
, et al
Experimental platform for ultra-high dose rate FLASH irradiation of small animals using a clinical linear accelerator
.
Int J Radiat Oncol Biol Phys
2017
;
97
:
195
203
.
28.
Koch
A
,
Gulani
J
,
King
G
,
Hieber
K
,
Chappell
M
,
Ossetrova
N
. 
Establishment of early endpoints in mouse total-body irradiation model
.
PLoS One
2016
;
11
:
e0161079
.
29.
Withers
HR
,
Elkind
MM
. 
Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation
.
Int J Radiat Biol Relat Stud Phys Chem Med
1970
;
17
:
261
7
.
30.
Roby
KF
,
Taylor
CC
,
Sweetwood
JP
,
Cheng
Y
,
Pace
JL
,
Tawfik
O
, et al
Development of a syngeneic mouse model for events related to ovarian cancer
.
Carcinogenesis
2000
;
21
:
585
91
.
31.
Rutkowski
MR
,
Stephen
TL
,
Svoronos
N
,
Allegrezza
MJ
,
Tesone
AJ
,
Perales-Puchalt
A
, et al
Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation
.
Cancer Cell
2015
;
27
:
27
40
.
32.
Duraiswamy
J
,
Kaluza
KM
,
Freeman
GJ
,
Coukos
G
. 
Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors
.
Cancer Res
2013
;
73
:
3591
603
.
33.
Miao
YR
,
Thakkar
KN
,
Qian
J
,
Kariolis
MS
,
Huang
W
,
Nandagopal
S
, et al
Neutralization of PD-L2 is essential for overcoming immune checkpoint blockade resistance in ovarian cancer
.
Clin Cancer Res
2021
;
27
:
4435
48
.
34.
de Malet
A
,
Antoni
G
,
Collins
M
,
Soularue
E
,
Marthey
L
,
Vaysse
T
, et al
Evolution and recurrence of gastrointestinal immune-related adverse events induced by immune checkpoint inhibitors
.
Eur J Cancer
2019
;
106
:
106
14
.
35.
Kim
YE
,
Gwak
SH
,
Hong
BJ
,
Oh
JM
,
Choi
HS
,
Kim
MS
, et al
Effects of ultra-high dose rate FLASH irradiation on the tumor microenvironment in lewis lung carcinoma: Role of myosin light chain
.
Int J Radiat Oncol Biol Phys
2021
;
109
:
1440
53
.
36.
Sato
E
,
Olson
SH
,
Ahn
J
,
Bundy
B
,
Nishikawa
H
,
Qian
F
, et al
Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer
.
Proc Natl Acad Sci U S A
2005
;
102
:
18538
43
.
37.
Curiel
TJ
,
Coukos
G
,
Zou
L
,
Alvarez
X
,
Cheng
P
,
Mottram
P
, et al
Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival
.
Nat Med
2004
;
10
:
942
9
.