Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer

Abstract

Background: Stereotactic body radiotherapy (SBRT) has been increasingly used as adjuvant therapy in pancreatic ductal adenocarcinoma (PDAC), and induces immunogenic cell death, which leads to the release of tumor antigen and damage-associated molecular patterns. However, this induction often fails to generate sufficient response to overcome pre-existing tumor microenvironment (TME) immunosuppression. Toll-like receptor (TLR) 7/8 ligands, such as R848, can amplify the effect of tumor vaccines, with recent evidence showing its antitumor effect in pancreatic cancer by modulating the immunosuppressive TME. Therefore, we hypothesized that the combination of R848 and SBRT would improve local and systemic antitumor immune responses by potentiating the antitumor effects of SBRT and reversing the immunosuppressive nature of the PDAC TME.

Methods: Using murine models of orthotopic PDAC, we assessed the combination of intravenous TLR7/8 agonist R848 and local SBRT on tumor growth and immune response in primary pancreatic tumors. Additionally, we employed a hepatic metastatic model to investigate if the combination of SBRT targeting only the primary pancreatic tumor and systemic R848 is effective in controlling established liver metastases.

Results: We demonstrated that intravenous administration of the TLR7/8 agonist R848, in combination with local SBRT, leads to superior tumor control compared with either treatment alone. The combination of R848 and SBRT results in significant immune activation of the pancreatic TME, including increased tumor antigen-specific CD8⁺ T cells, decreased regulatory T cells, and enhanced antigen-presenting cells maturation, as well as increased interferon gamma, granzyme B, and CCL5 along with decreased levels of interleukin 4 (IL-4), IL-6, and IL-10. Importantly, the combination of SBRT and systemic R848 also resulted in similar immunostimulatory changes in liver metastases, leading to improved metastatic control. CD8⁺ T cell depletion studies highlighted the necessity of these effector cells at both the local and hepatic metastatic sites. T cell receptor (TCR) clonotype analysis indicated that systemic R848 not only diversified the TCR repertoire but also conditioned the metastatic foci to facilitate entry of CD8⁺ T cells generated by SBRT therapy.

Conclusions: These findings suggest that systemic administration of TLR7/8 agonists in combination with SBRT may be a promising avenue for metastatic PDAC treatment.

Keywords: Combined Modality Therapy; Gastrointestinal Neoplasms; Immunotherapy; Radiotherapy; Tumor Microenvironment.

Figure 1

R848 significantly enhances the antitumor efficacy of SBRT in the murine orthotopic model of pancreatic cancer. (A) Schematic of the experimental design. C57BL/6J mice were injected with KCKO-Luc or KP2.1-Luc cells in the tail of pancreas and treated with SBRT and/or R848. Tumor growth was analyzed by IVIS twice a week. Representative IVIS images from day 19 after KCKO-Luc (B) or KP2.1-Luc (E) implantation. Tumor growth curve based on IVIS imaging, with (C) and (F) being KCKO-Luc and KP2.1-Luc, respectively. Data represent at least two independent experiments (n=5–7 mice/group). *p<0.05, SBRT+R848 compared with SBRT or R848 alone. Kaplan-Meier survival curves of mice bearing KCKO-Luc (D) or KP2.1-Luc (G) tumors. Data combined from two individual experiments. *p<0.05; **p<0.01; ****p<0.0001. SBRT+R848 compared with no treatment group or monotherapy groups by log-rank (Mantel-Cox) test. IVIS, in vivo imaging system; i.v., intravenous; KCKO-Luc, luciferase-expressing KCKO; KP2.1-Luc, luciferase-expressing KP2 cells; p/sec/cm²/sr, photons/second/cm²/steradian; SBRT, stereotactic body radiation therapy.

Figure 2

Treatment of SBRT, but not R848, induced tumor immunogenic cell death in the orthotopic model of murine pancreatic cancer. Mice bearing KP2.1-Luc orthotopic pancreatic tumors were treated with SBRT and/or R848, and ICD was determined by immunohistochemistry (IHC) staining of cleaved caspase 3 (A), HMGB1 (B), and calreticulin (C). Results are expressed as mean±SEM from five mice/group and analyzed by one-way analysis of variance (ANOVA) with Dunnett posttest. *p<0.05, compared with no treatment group or monotherapy groups. ICD, immunogenic cell death; KP2.1-Luc, luciferase-expressing KP2 cells; SBRT, stereotactic body radiation therapy.

Figure 3

SBRT combined with R848 enhances antitumor immune response in the orthotopic model of murine pancreatic cancer. Mice bearing KCKO-Luc orthotopic pancreatic tumors were treated with SBRT on day 7, day 8, day 9 and day 10, and/or R848 on day 6 and day 11. Mice were sacrificed on day 12, tumor-infiltrating immune cells were determined by flow cytometry, and tumor cytokines and chemokines were determined by multiplex Luminex assay. (A) Tumor-infiltrating CD8⁺ T cells, CD8⁺IFNγ⁺, CD8⁺GzmB⁺, CD8⁺CTLA4⁺, and CD8⁺programmed cell death protein 1 (PD1⁾⁺ cells were analyzed. (B) Tumor-infiltrating CD4⁺ T cells, Tregs, and natural killer (NK) cells were analyzed. (C)TAMs and their expression of CD80 and CD86 were analyzed. (D) MDSCs, including Mo-MDSC and G-MDSC populations, were shown. (E) Tumor-infiltrating DCs, CD80⁺DCs, and CD86⁺DCs were analyzed. Results are expressed as mean±SEM from five mice/group and analyzed by one-way analysis of variance (ANOVA) with Dunnett posttest. Significance is indicated by *p<0.05 and **p<0.01, compared with no treatment group or monotherapy. (F) Fold changes of cytokines and chemokines in the tumor following treatment of SBRT/R848. Levels of cytokines and chemokines of tumors from mice of no treatment group were set as 1, and data were fold changes as compared with no treatment group. DCs, dendritic cells; IFNγ, interferon gamma; G-MDSC, granulocytic myeloid derived suppressor cell; GzmB, granzyme B; KCKO-Luc, luciferase-expressing KCKO; MDSCs, myeloid derived suppressor cells; Mo-MDSC, monocytic myeloid derived suppressor cell; SBRT, stereotactic body radiation therapy; TAMs, tumor-associated macrophages; Tregs, regulatory T cells.

Figure 4

CD8⁺ T cells are vital for the antitumor effect of SBRT/R848 in the orthotopic model of murine pancreatic cancer. (A) Schematic of experimental design. KCKO-OVA pancreatic tumor bearing mice were treated with SBRT and/or R848 and mice were sacrificed on day 12, antigen presentation was detected by B3Z T-cell activation, and antigen-specific CD8⁺ T cells were determined by flow cytometry. (B) Enhanced antigen presentation following SBRT/R848 treatment. Antigen presentation was detected by B3Z T-cell hybridoma activation assay after coculture with cells from draining or non-draining lymph nodes or spleen. Results are expressed as mean±SEM from five mice/group and analyzed by one-way analysis of variance (ANOVA) with Dunnett posttest. *p<0.05 and **p<0.01, compared with no treatment group or monotherapy group. Representative of flow cytometry plots (C) and quantitative analysis (D) for H2K^b/SIINFEKL⁺Dextramer⁺CD8⁺ T cells. (D) Data are represented as mean±SE (n=5 for each group). *p<0.05 and **p<0.01, compared with no treatment group or monotherapy. Representative of IVIS image on day 19 (E) and tumor growth curve (F) based on IVIS after treated with SBRT+R848 with/without CD8⁺ T cell depletion. Data are represented as mean±SE (n=5 for each group). *p<0.05; SBRT+R848+CD8 depletion group compared with SBRT+R848+IgG group. IVIS, in vivo imaging system; KCKO-OVA, OVA-expressing KCKO; SBRT, stereotactic body radiation therapy.

Figure 5

Systemic R848 and local SBRT treatment exert synergistic antitumor effects on liver metastasis of pancreatic cancer. (A) and (B) Naïve mice or mice that cured of KP2.1-Luc tumors after SBRT/R848 treatment were given KP2.1-Luc via splenic injection. Representative IVIS imaging of hepatic tumor metastasis on day 19 post-tumor injection and tumor growth curve based on IVIS were shown. (B) Results are expressed as mean±SEM from five mice/group. (C) Schematic of orthotopic pancreatic cancer and hepatic metastasis establishment, and treatment with SBRT and R848. Representative IVIS imaging of hepatic tumor metastasis on day 14 following KCKO-Luc (D) or KP2.1-Luc (F) tumor injection. The growth of liver metastases of KCKO-Luc (E, left panel) or KP2.1-Luc (G, left panel) was analyzed by IVIS imaging. Tumor burden was observed at random either at the pancreas or liver, and in some case, both sites exhibited tumor growth. Data are expressed as mean±SEM from 5 mice/group, representing 2 individual experiments. Kaplan-Meier survival curves of mice bearing KCKO and KCKO-Luc (E, right panel) or KP2 and KP2.1-Luc (G, right panel) tumors are combined from 2 individual experiments. *p<0.05, **p<0.01, and ***p<0.001, compared with no treatment group or monotherapy by one-way ANOVA with Dunnett posttest for growth curve or by log-rank (Mantel-Cox) test for survival curve. IVIS, in vivo imaging system; KCKO-Luc, luciferase-expressing KCKO; KP2.1-Luc, luciferase-expressing KP2 cells; p/sec/cm²/sr, photons/second/cm²/steradian; SBRT, stereotactic body radiation therapy.

Figure 6

CD8⁺ T cells are essential for the antitumor efficacy of SBRT and R848 in liver metastasis. (A) and (B) Mice bearing orthotopic pancreatic tumors and hepatic metastases were treated with SBRT and R848, with or without CD8⁺ T cell depletion. Shown in (A) is representative IVIS imaging of hepatic tumor metastases on day 14 following KCKO-Luc injection. The growth of liver metastases was analyzed by IVIS imaging as in (B). Data are expressed as mean±SEM from 4 mice/group to 5 mice/group. *p<0.05; SBRT+R848+CD8 depletion group compared with SBRT+R848+IgG group. (C) Schematic of experimental design. KCKO-OVA pancreatic and hepatic tumor bearing mice were treated with SBRT and/or R848. Mice were sacrificed on day 12 and antigen-specific CD8⁺ T cells from liver metastasis were determined by flow cytometry. (D) and (E) Representative of flow cytometry plots (D) and quantitative analysis (E) for H2K^b/SIINFEKL⁺Dextramer⁺CD8⁺ T cells after treatment with or without SBRT and/or R848. Data are expressed as mean±SEM from 5 mice/group. *p<0.05 and **p<0.01, compared with no treatment group. IVIS, in vivo imaging system; KCKO-Luc, luciferase-expressing KCKO; KP2.1-Luc, luciferase-expressing KP2 cells; KCKO-OVA, OVA-expressing KCKO; p/sec/cm²/sr, photons/second/cm²/steradian; SBRT, stereotactic body radiation therapy.

Figure 7

Combination of SBRT and R848 increases overlapping TCR clonotypes between the primary pancreatic tumor and liver metastasis. Mice bearing KCKO tumors in pancreas and liver were treated with SBRT only at primary tumor site (pancreas), R848 systemically, or combination of SBRT/R848 as illustrated in (A). On day 12 after tumor inoculation, mice were sacrificed and TCR sequence of tumor infiltrating CD8⁺ T cells sorted from pooled tumor samples (5 mice/group) were analyzed. Summary proportion of clonotypes with specific indices were analyzed in pancreatic tumor (B) and liver metastases (D) showing SBRT or SBRT/R848 increased frequency of top 5 TCRβ clone types in pancreatic tumor. The diversity (chao 1) of TCRβ clonotypes in pancreatic tumor (C) and liver metastases (E) from different treatment groups was calculated from randomly downsampling of each sample to the least reads (liver mets—SBRT+R848 group), showing R848 or SBRT/R848 increased diversity of TCRβ clonotypes in pancreatic tumor but not in liver metastasis. (F) TCRβ repertoire overlap (Morisita’s overlap index) was analyzed showing that combination of SBRT and R848 resulted in highest TCRβ overlap between pancreatic tumor and liver metastasis. (G) Clonotype tracking of 10 top TCRβ clonotypes in pancreatic tumor infiltrating CD8⁺ T cells was analyzed in liver metastasis showing highest similarity of respective clonotype frequency between pancreatic tumor and liver metastasis following SBRT/R848 treatment. i.v., intravenous; KCKO-Luc, luciferase-expressing KCKO; KP2.1-Luc, luciferase-expressing KP2 cells; KCKO-OVA, OVA-expressing KCKO; SBRT, stereotactic body radiation therapy; TCR, T cell receptor; PC, pancreatic cancer.