IL-1β blockade prevents cardiotoxicity and improves the efficacy of immune checkpoint blockers and chemotherapy against pancreatic cancer in mice with obesity

Abstract

Background: Immune checkpoint blockers (ICBs) have revolutionized cancer therapy, yet they remain largely ineffective in treating pancreatic ductal adenocarcinoma (PDAC). Moreover, ICBs can cause severe immune-related adverse events (irAEs), including fatal cardiac toxicity. Finally, obesity is a risk factor in PDAC that may differentially modulate ICB efficacy in a malignancy-dependent manner.

Methods: We investigated the mechanisms underlying irAEs induced by dual ICB therapy and sought to identify strategies to mitigate them while improving ICB efficacy in the obese setting. To this end, we used a clinically relevant mouse model that integrated key features of human PDAC: (1) high-fat diet-induced obesity, (2) an orthotopic PDAC, and (3) a therapeutic regimen combining chemotherapy (FOLFIRINOX) with ICBs (α-programmed cell death protein-1 + α-cytotoxic T-lymphocyte associated protein-4 antibodies).

Results: Obese mice developed cardiac irAEs and had elevated serum interleukin (IL)-1β levels after chemoimmunotherapy. IL-1β blockade not only prevented myocarditis and reduced cardiac fibrosis but also enhanced the antitumor efficacy of the combination of chemotherapy plus dual ICB therapy and significantly improved the overall survival of PDAC-bearing obese mice.

Conclusions: Our findings provide the rationale and compelling data to test a Food and Drug Administration-approved anti-IL-1β antibody in combination with chemotherapy and dual ICB therapy in patients with pancreatic cancer with obesity.

Keywords: Cardiotoxicity; Immune Checkpoint Inhibitor; Immune related adverse event - irAE.

Figure 1. Development of a clinically relevant model to study cardiac irAE. (A) Schematic diagram showing experiment plan. Chow or high-fat diet-fed mice were orthotopically implanted with pancreatic ductal adenocarcinoma cell line (KPC001s) expressing Guassia luciferase (Gluc). Mice were randomized and treated with one dose of FFX and three subsequent doses of either “IgG” or “ICB” (α-programmed cell death protein-1 and α-cytotoxic T-lymphocyte associated protein-4). (B) Tumor progression monitored via circulating Gluc levels is plotted over days post tumor implantation for chow diet (n=8–10 per treatment group) and high-fat diet-fed mice receiving IgG or ICB cocktail (n=6 per treatment group). (C) Endpoint tumor weights measured on the dissection day (day 29 post-implantation) are plotted (n=7–15 per treatment group). (D) irAEs measured by body-weight change are plotted over days post-tumor implantation for chow diet (n=8–9 per treatment group) and high-fat diet-fed mice (n=5 per treatment group), receiving IgG or ICB cocktail. Note that this is a separate experiment, where mice were allowed to survive longer to monitor systemic and cardiac toxicity. (E–F) Representative echocardiograms measured on day 35 post-tumor implantation demonstrating the thickness of the left ventricular posterior wall (LVPW) (E) and its quantification in different treatment/diet groups (F) (n=6–8 per treatment group). (G) Ejection fraction percentage calculated from echocardiograms in different treatment/diet groups (n=6–8 per treatment group) (H) Heart weight to body weight ratio of mice treated with combination therapy, measured on the dissection day (day 29 post-implantation) (n=6–13 per treatment group). *p<0.05, **p<0.01 and *** p<0.001; error bars with SEM, multiple groups. ICB, immune checkpoint blockade; irAE, immune-related adverse event.

Figure 2. ICB increases IL-1β-dependent cardiac immune infiltrates. (A) Schematic diagram showing mice fed with a high-fat diet and implanted with KPC001S tumors were randomized and treated with combination therapy (FFX+IgG/ICB). At the end of therapy (day 20 post-implantation), serum was profiled for cytokines, and the heart was analyzed for immune infiltrates. (B) Serum cytokine levels in high-fat-diet-fed mice receiving FFX and ICB as compared with FFX and IgG. Combination therapy of FFX+ICB in high-fat diet-fed mice resulted in increased levels of circulating IL-1β in serum (n=16 per treatment group). (C–G) Flow cytometry analysis demonstrated increased Gr1⁺ cells (C), F4/80⁺ cells (D), IFN-γ⁺CD4⁺ cells (F), and IL-17⁺CD4⁺ cells (G) in the myocardium of mice. (H) Schematic diagram showing high-fat diet-fed mice bearing KPC001S tumors were randomized and started treatment with α-IL-1β or α-IL-1R or isotype control (IgG) before combination therapy. α-IL-1β or α-IL-1R was administered intraperitoneally every other day until day 20 post-implantation when flow cytometry was performed. (I–N) IL-1β inhibition resulted in decreased Gr1⁺ cells (I), F4/80⁺ cells (J), TCRβ⁺ T cells (K), CD4⁺IFN-γ⁺ T cells (L), CD4⁺IL-17⁺ T cells (M), and CD8⁺ T cells (N) in the myocardium of mice. (n=5–17 mice per treatment group for (C–N); *p<0.05, **p<0.01, and ***p<0.001; error bars with SEM, two groups were compared with Student’s t-test, multiple group comparison done with one-way analysis of variance with Dunn’s multiple testing correction). CTLA-4, cytotoxic T-lymphocyte associated protein-4; ICB, immune checkpoint blockade; IFN, interferon; IL, interleukin; PD-1, programmed cell death protein-1; TCR, T cell receptor.

Figure 3. IL-1β blockade reduces ICB-induced cardiac immune-related adverse events. (A) Schematic diagram showing high-fat diet-fed mice bearing KPC001S tumors randomized and treated every other day with isotype control (IgG), α-IL-1β or α-IL-1R, with the first dose starting before combination therapy (FFX+ICB). (B) Representative echocardiograms measured on day 35 post-implantation demonstrating thickness of the left ventricular posterior wall (LVPW). (C) Quantification of LVPW, showing rescue of posterior wall thickness post IL-1β signaling inhibition (n=6 per treatment group). (D) Ejection fraction percentage calculated from echocardiograms in different treatment groups (n=6 per treatment group). (E) Body weight change plotted over time in mice treated with isotype control/ α-IL-1β/ α-IL-1R plus FFX and ICB. Note the body weight loss induced by the combination therapy gets rescued by IL-1β inhibition (n=6 per treatment group). (F) Heart weight to body weight ratio measured on day 20 post-implantation (n=6–14 per treatment group). (G) Representative cardiac myocardium sections stained with Picrosirius red. The dark red areas show collagen. (H) Quantification of the collagen-positive area in heart histological sections (dissected on day 20 post-implantation) stained with picrosirius red (n=6–7 per treatment group). Note the reduced collagen content (cardiac fibrosis) post IL-1β signaling blockade. (*p<0.05, multiple group comparison done with one-way analysis of variance with Dunn’s multiple testing correction). HFD, high-fat diet; ICB, immune checkpoint blockade; IL, interleukin.

Figure 4. Preventative IL-1β blockade improves ICB efficacy and enhances survival. (A) Schematic diagram showing high-fat diet-fed mice bearing KPC001S tumors randomized and treated every other day with isotype control (IgG), α-IL-1β or α-IL-1R, with the first dose starting before combination therapy (FFX+ICB). (B) Gluc levels showing tumor growth over time in different treatment groups. Note the reduced tumor growth as a result of IL-1β signaling inhibition (n=6–12 per treatment group). (C) Kaplan-Meier survival analysis shows improved survival following treatment with α-IL-1β or α-IL-1R compared with isotype control (IgG) (n=9–13 per treatment group). (D–E) Flow cytometry analyses of pancreatic ductal adenocarcinoma tumor and tumor microenvironment, performed at day 20 post-tumor implantation. Gr1⁺ cells (D) and CD8⁺IFN-γ⁺ cells (E) (n=6 per treatment group). Note the decrease in Gr1⁺ cells and increase in effector CD8⁺ T cells in the tumor of mice receiving α-IL-1β or α-IL-1R. (*p<0.05, multiple group comparison done with one-way analysis of variance with Dunn’s multiple testing correction, survival data analyzed by Mantel-Cox log-rank test). HDF, high-fat diet; ICB, immune checkpoint blockade; IFN, interferon; IL, interleukin.