Blocking IL-1β reverses the immunosuppression in mouse breast cancer and synergizes with anti-PD-1 for tumor abrogation

Abstract

Interleukin-1β (IL-1β) is abundant in the tumor microenvironment, where this cytokine can promote tumor growth, but also antitumor activities. We studied IL-1β during early tumor progression using a model of orthotopically introduced 4T1 breast cancer cells. Whereas there is tumor progression and spontaneous metastasis in wild-type (WT) mice, in IL-1β-deficient mice, tumors begin to grow but subsequently regress. This change is due to recruitment and differentiation of inflammatory monocytes in the tumor microenvironment. In WT mice, macrophages heavily infiltrate tumors, but in IL-1β-deficient mice, low levels of the chemokine CCL2 hamper recruitment of monocytes and, together with low levels of colony-stimulating factor-1 (CSF-1), inhibit their differentiation into macrophages. The low levels of macrophages in IL-1β-deficient mice result in a relatively high percentage of CD11b+ dendritic cells (DCs) in the tumors. In WT mice, IL-10 secretion from macrophages is dominant and induces immunosuppression and tumor progression; in contrast, in IL-1β-deficient mice, IL-12 secretion by CD11b+ DCs prevails and supports antitumor immunity. The antitumor immunity in IL-1β-deficient mice includes activated CD8+ lymphocytes expressing IFN-γ, TNF-α, and granzyme B; these cells infiltrate tumors and induce regression. WT mice with 4T1 tumors were treated with either anti-IL-1β or anti-PD-1 Abs, each of which resulted in partial growth inhibition. However, treating mice first with anti-IL-1β Abs followed by anti-PD-1 Abs completely abrogated tumor progression. These data define microenvironmental IL-1β as a master cytokine in tumor progression. In addition to reducing tumor progression, blocking IL-1β facilitates checkpoint inhibition.

Keywords: IL-1β; antitumor immunity; anti–PD-1; breast cancer; immunotherapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

IL-1β regulates mammary tumor growth and invasiveness. Tumor growth in BALB/c and IL-1β–deficient mice (IL-1β KO). A total of 2 × 105 4T1 cells were injected orthotopically into the mammary fat pad of either BALB/c (■) or IL-1β KO (□) mice as described (Materials and Methods). Tumor volume (A) and mouse survival (B) were monitored every 3–4 d (n = 6–12). (C) Lungs from BALB/c and IL-1β KO mice imaged ex vivo by an IVIS system 35 d after injection with 4T1-Luc cells. (D) Influence of IL-1β neutralization on 4T1 tumor growth. Tumor-bearing BALB/c mice were treated with either isotypic Abs (■) or anti–IL-1β Abs (○) twice a week from day 0 (downward arrows), and tumor volume was measured every 3–4 d as described (Materials and Methods; n = 3–10). (E) Primary tumor volume in BALB/c and IL-1β KO mice on day 21 after injection of 4T1 cells or IL-1β–overexpressing cells (4T1–IL-1β). (F) PyMT cells (2 × 105) were injected orthotopically into the mammary fat pads of either C57BL/6 (■) or C57BL/6-IL-1β KO (□) mice. Tumor volumes were monitored every 3–4 d. Graphs show mean ± SEM (n = 3–8). *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Fig. 2.

IL-1β affects the nature of myeloid cells in the mammary tumors. On day 12 after 4T1 cell injection, BALB/c and IL-1β KO mice were killed and single-cell suspensions from primary tumors were prepared as described (Materials and Methods). (A) Absolute number of macrophages (Left) and CD11b+ DCs (Right) was calculated from 100,000 CD11b+Ly6G−SSClow cells in tumors. (B) Percentage of macrophages (Left; CD11b+Ly6G−SSClowCD64+Ly6C−MHCII+/−) and CD11b+ DCs (Right; CD11b+Ly6G−SSClowCD64+Ly6C−MHCII+/−) in extracted tumors. Graphs show the percentage from CD11b+Ly6G−SSClow cells. (C) Percentage of macrophages (Left) and CD11b+ DCs (Right) in extracted 16-d old PyMT tumors. Graphs show the percentage from CD11b+Ly6G−SSClow cells. (D) BALB/c mouse treatment with either isotypic or anti–IL-1β Abs. On day 12, tumor single-cell suspensions were stained for macrophages (Left) or CD11b+ DCs (Right). Graphs show percentage from CD11b+Ly6G−SSClow cells. All graphs show mean ± SEM (n = 3–8). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Fig. 3.

IL-1β promotes accumulation of tumor-infiltrating CCR2+ cells through MCP-1. On day 12 after 4T1 cell injection, BALB/c and IL-1β KO mice were killed and single-cell suspensions from the primary tumors were prepared as described (Materials and Methods). (A) Percentage of CCR2+ cells from CD11b+ cells. (B) CCL2 protein concentration in 12-d 4T1 tumor extracts, measured by ELISA. Graphs in A and B show mean ± SEM (n = 4–8). **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) Correlation between IL1B expression and different CCR2 ligands (CCL2, CCL13, and CCL7) in human breast cancer samples from the TCGA dataset (n = 1,215).

Fig. 4.

IL-1β promotes tumor-infiltrating inflammatory monocyte (Mono.) differentiation to macrophages. On day 7 after 4T1 cell injection, BALB/c and IL-1β KO mice were injected with 50 μL of liquid Matrigel mixed with 200,000 blood mononuclear cells extracted from naive BALB/c or IL-1β KO mice, respectively. Mice were killed 24 h, 48 h, and 72 h after Matrigel plug transplantation, and single-cell suspensions from Matrigel plugs were prepared as described (Materials and Methods). (A) Representative contour plot of CD11b+Ly6G−SSClowLy6C+/−/CD64+ cells in BALB/c and IL-1β KO mice. (B) Percentage of mature macrophages (CD11b+Ly6G−SSClowLy6C−CD64+MHCII+/−) in Matrigel plugs from CD11b+ cells (Right) and percentage of CD11b+ DCs (CD11b+Ly6G−SSClowLy6C−CD64−MHCII+) in Matrigel plugs from CD11b+ cells (Left). Graphs show mean ± SEM (n = 3–4). *P < 0.05. ns, not significant. (C) Expression of the CSF1 gene in 12-d 4T1 tumors from BALB/c and IL-1β KO mice was assessed using qPCR. Gene expression was normalized based on the expression of ACTB. The graph shows mean ± SEM (n = 3). *P < 0.05. (D) Correlation between IL1B expression and CSF1, FL3LG, and CSF2 in human breast cancer samples from the TCGA dataset (n = 1,215).

Fig. 5.

Tumor regression in IL-1β KO mice is CD8+ T cell-dependent. BALB/c and IL-1β KO mice were injected with 2 × 105 4T1 cells. The 4T1-bearing BALB/c and IL-1β KO mice were killed on day 12 after injection of 4T1 cells. (A) Percentage of CD8+ T cells from CD3+ cells in primary tumor single-cell suspensions (n = 3). (B) Histological section from primary tumor stained with anti-CD8 Abs (green) and DAPI (blue). (Magnification: B, 60×.) (C) Depletion of CD8+ cells was achieved by multiple i.p. injections of anti-CD8 Abs. Primary tumor volumes of BALB/c mice (■), BALB/c mice treated with anti-CD8 Abs (□), IL-1β KO mice (●), and IL-1β KO mice treated with anti-CD8 Abs (○) are shown. Tumor volume was measured every 3–4 d as described (Materials and Methods; n = 4–5). (D) Representative contour plot of intracellular production of IFN-γ and TNF-α by tumor-derived CD8+ T cells. (E) Expression of GZMB gene in 12-d 4T1 tumors from BALB/c and IL-1β KO mice was assessed using qPCR. Gene expression was normalized based on the expression of CD8A (n = 3). Graphs show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0007.

Fig. 6.

Macrophage-derived IL-10 leads to tumor promotion through CD8+ T cell suppression. On day 12 after 4T1 cell injection, BALB/c and IL-1β KO mice were killed and single-cell suspensions from the primary tumors were prepared as described (Materials and Methods). (A) Representative histograms of different cell populations within the primary tumor stained intracellularly with anti–IL-10 Abs, anti–IL-12p40 Abs (white), or isotype control Abs (gray). Imm., immature; Eosinoph., eosinophil; Mono., monocyte; Neutro., neutrophil. Mean fluorescence intensity of anti–IL-10 Abs in MHClow macrophages (B) and anti–IL-12p40 Abs in CD11b+ DCs (C) (n = 4). Tumor-bearing mice were treated i.p. with anti–IL-10 or control IgG Abs (Materials and Methods). After 14 d, mice were killed. (D) Primary tumor weight (n = 4). (E) Expression of CD8A and GZMB genes in primary tumors was assessed using qPCR. Gene expression was normalized based on the expression of ACTB (n = 4). (F) Expression of IL12A and IL12B genes in primary tumors. Gene expression was normalized based on the expression of ITGAM (n = 4). (G) Expression of IL10 and GZMB genes in PyMT tumors. Gene expression was normalized based on the expression of ACTB. Graphs show mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 7.

Combination of anti–IL-1β with anti–PD-1 treatment improves the outcome of 4T1 mammary carcinoma in mice. BALB/c mice were injected with 2 × 105 4T1 cells. On days 4 and 7 after tumor cell injection, some of the mice were injected i.p. with anti–IL-1β or isotype control Abs. On day 10, some mice received a single injection of anti–PD-1 or isotype control Abs. (A) Tumor volume of control (■) mice, mice treated with only anti–IL-1β Abs (□), mice treated with only anti–PD-1 Abs (△), and mice treated with both anti–IL-1β and anti–PD-1 Abs (○). On day 30, mice were killed. (B) Weight of primary tumors. (C) Tumors were enzymatically digested, and cells were analyzed by FACS. The graph shows the percentage of CD8+ T cells from tumor-derived CD45+ leukocytes. All graphs show mean ± SEM (n = 4–6). *P < 0.05; ***P < 0.001. ns, not significant.