Inflammasome Deletion Promotes Anti-tumor NK Cell Function in an IL-1/IL-18 Independent Way in Murine Invasive Breast Cancer

Abstract

Inflammasomes are molecular complexes that trigger an inflammatory response upon detection of pathogens or danger signals. Recent studies suggest that they are also involved in cancer progression. However, their roles during tumorigenesis remain poorly understood and controversial. Here, we investigated whether inflammasome activation supports mammary tumor growth. Using mouse models of invasive breast cancer, our results demonstrate that the absence of a functional inflammasome impairs tumor growth. Importantly, tumors implanted into inflammasome-deficient mice recruited significantly less neutrophils and more natural killer (NK) cells, and these latter cells displayed a more active phenotype. Interestingly, NK cell depletion abolished the anti-tumoral effect observed in inflammasome-deficient mice, although inflammasome-regulated cytokine neutralization had no effect. Thus, our work identifies a novel role for the inflammasome in supporting mammary tumor growth by attenuating NK cell recruitment and activity. These results suggest that inflammasome inhibition could be a putative target for treating invasive breast cancers.

Keywords: ASC; Caspase-1; NK cells; breast cancer; inflammasome; inflammation.

Figure 1

Caspase-1 deficiency delays tumor appearance in MMTV-Neu^V664E BALB/c mouse model. Kaplan–Meier curves depicting tumor growth latency of MMTV-Neu^V664E mice, defined as time from birth until appearance of the first palpable tumor. **P < 0.01 (Comparison of survival curves: Gehan–Breslow–Wilcoxon test).

Figure 2

Loss of caspase-1 and ASC expression reduces 4T1 tumor growth in BALB/c mice. (A) WT, Caspase-1 KO, and Asc KO mice (n = 5) were injected orthotopically with 4T1 mammary tumor cells. Tumor growth was measured over 30 days. (B) Individual growth curves depicted in A. (C) WT, Caspase-1 KO, and Nlrp3 KO mice (n = 8) were injected orthotopically with 4T1 mammary tumor cells. Tumor growth was measured over 30 days. (D) Individual growth curves depicted in C. Data represent mean ± SD ***P < 0.001 (Two-way ANOVA analysis; n.s, non-significant).

Figure 3

The presence of the inflammasome in the hematopoietic compartment promotes mammary tumor growth. Irradiated mice of indicated genotypes were grafted with bone marrow stem cells from WT or Caspase-1 KO mice and were injected with 4T1 mammary tumor cells 28 days later. (A) Tumor growth was measured over 30 days. (B) Individual growth curves depicted in A. Data represent mean ± SD ***P < 0.001 (Two-way ANOVA analysis).

Figure 4

Caspase-1 or ASC deficiency improves NK cell recruitment 14 days post-injection. (A) Flow cytometry analysis of myeloid cell populations in 4T1 tumors at day 14 post-injection from WT (n = 6), Caspase-1 KO (n = 6), and Asc KO (n = 6) mice. The displayed dotplots were obtained by gating live and CD45+ cells. (B) Quantification and analysis of myeloid cell populations in the different mouse genotypes. (C) Flow cytometry analysis of lymphoid cell populations in 4T1 tumors from the same mice as in A. The displayed dotplots were gated from live and CD45+ cells. (D) Quantification and analysis of lymphoid cell populations in the different mouse genotypes. Data represent mean ± SD *P < 0.05; **P < 0.01 (One-way ANOVA test followed by Bonferroni's Multiple Comparison Test).

Figure 5

IL-1β, IL-18 or IL-1R inhibition does not affect 4T1 tumor growth in vivo. (A) WT (n = 5) were injected with IgG control, anti-IL1β antibody, IL-18BP, or both anti-IL1β and IL-18BP the day before tumor inoculation and then every 3 days. Treated mice were orthotopically injected with 4T1 mammary tumor cells. Tumor growth was measured over 28 days. (B) WT, caspase-1 KO were injected with PBS or Anakinra (IL-1 receptor inhibitor) the day before tumor inoculation and then every 2 days. Treated mice were orthotopically injected with 4T1 mammary tumor cells. Tumor growth was measured over 14 days. Data represent mean ± SD *P < 0.05 (Two-way ANOVA analysis; n.s, non-significant).

Figure 6

NK cells control tumor growth and are more active in caspase-1 KO mice. (A) Tumor growth of 4T1 cells was measured in NK-depleted (anti-asialo GM1) or PBS-treated WT and caspase-1 KO mice (n = 5). Each value represents mean ± SD **P < 0.01; ***P < 0.001 (Two-way ANOVA analysis). (B) Individual growth curves depicted in (A). (C–E) Flow cytometry analysis of NK cell size in tumors and spleens (C) and activation markers in 4T1 tumors (D) and in spleen (E) from WT (n = 5) and Caspase-1 KO (n = 5) mice at day 7 post-injection. (F) Cell suspensions from digested tumors of the indicated mouse genotype were cultured in the presence of cytokines (IL-12/IL-18), antibodies (NKp46, Ly49D, NKG2D) or tumor cells (YAC-1, 4T1) and NK cell IFN-γ production (F) or IFN-γpositive cells (G) were measured by flow cytometry. Data from (C–G) represent mean ± SD *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Two-way ANOVA test followed by Bonferroni's Post-test; ns, non-significant).