IFI35 limits antitumor immunity in triple-negative breast cancer via CCL2 secretion

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis due to the lack of therapeutic targets. Although immunotherapy brings survival benefits to patients diagnosed with TNBC, it remains limited and treatment resistance is widespread. Here we demonstrate that IFI35 is highly expressed in tumor tissues and can be induced by Interferon-γ in a time-dependent and concentration-dependent manner in breast cancer cells. In xenograft models, we reveal that IFI35 dramatically increases myeloid-derived suppressor cells infiltration in tumors, along with depletion and anergy of CD8⁺T cells. IFI35 ablation leads to prolonged survival of the mice. Mechanistically, RNA-sequencing reveals that IFI35 promotes CCL2 secretion, resulting in the remodeling of TNBC immune microenvironment. Ablation of IFI35 promotes the infiltration of effector CD8⁺T cells, and thereby sensitizes TNBC to anti-PD-1 immunotherapy. Our data suggest that IFI35 limits antitumor immunity and may be expected to become a new immunotherapy target in TNBC.

Fig. 1. IFN-γ induces IFI35 expression in tumor microenvironment.

A Representative immunostainings for IFI35 in TNBC patients’ sample (n = 45). B Histological quantification of IFI35 expression in tumor cells, fibroblasts, immune cells within (A) (one-way ANOVA, Tukeys’s multiple comparison test, mean with SEM is plotted). C Correlation of IFI35 expression in tumor and immune cells (Pearson, n = 45 pairs, two-tailed). D, E RT-qPCR and Immunoblot analysis of BT-549 or MDA-MB-231 with indicated IFN-γ for IFI35 and β-actin as loading control (one-way ANOVA, Tukeys’s multiple comparison test, mean with SEM is plotted).

Fig. 2. IFI35 promotes TNBC progression via MDSCs recruitment.

A Immunoblot analysis of IFI35 protein level in 4T1 or EMT6 mouse TNBC cells transduced with sgRNA targeting Ifi35 and control. B Tumor growth curves of balb/c mice inoculated mammary fat pad with WT or fi35^ko 4T1 and EMT6 tumor cells (two-way ANOVA test, n = 8). C Tumor weight 30 days after tumor inoculation (n = 8, one-way ANOVA test with Turkey’s multiple comparisons, Mean with SD is plotted). D Kaplan–Meier curves for the survival of balb/c mice with Ifi35^ko or WT 4T1 and EMT6 tumor cells (n = 10 per group, log-rank test). The frequencies of MDSCs and CD8⁺T cells population in balb/c mice inoculated with Ifi35^ko or WT 4T1 (E) and EMT6 (F) tumor cells (n = 6-7, one-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted).

Fig. 3. CD8⁺T cells exert anti-tumor effector functions.

A The frequencies of IFNγ⁺CD8⁺, GzmB⁺CD8⁺ and Ki-67⁺CD8⁺ T cells population in balb/c mice inoculated with Ifi35^ko or WT 4T1 and EMT6 tumor cells (n = 6-7, one-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted). B Tumor growth curves of nude mice inoculated mammary fat pad with Ifi35^ko or WT 4T1 or EMT6 tumor cells (n = 6-8 per group, two-way ANOVA test, mean with SD is plotted). C Ifi35^ko or WT 4T1 and EMT6 tumor growth curves of Balb/c mice treated with anti-mouse CD8 antibody or isotype control (n = 5 per group, two-way ANOVA test, mean with SD is plotted).

Fig. 4. IFI35 orchestrates tumor immune microenvironment by CCL2.

A Heatmap showing log2 fold change in Il1α, Ccl2, Cxcl5, Cxcl10 expression in Ifi35^ko or WT 4T1 cell lines (n = 3 per group). B RT-qPCR and ELISA analysis for CCL2 from Ifi35^ko or WT 4T1 cell lines (n = 3) (one-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted). C, D. RT-qPCR analysis, ELISA analysis, and tumor growth curves of Ifi35^ko, Ifi35^ko +Ccl2, and WT 4T1 (C) and EMT6 (D) cell lines (n = 3) (one-way or two-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted). The frequencies of MDSCs and CD8⁺T cells population in balb/c mice inoculated with Ifi35^ko, Ifi35^ko +Ccl2, and WT 4T1 (E) or EMT6 (F) cell lines (n = 6) (one-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted).

Fig. 5. IFI35 ablation combined with PD-1 blockade synergistically inhibits TNBC progression.

A Schematic of tumor inoculation and antibody injection in mice. B Tumor growth curves of indicated 4 groups (WT + Isotype, WT + αPD-1, Ifi35^ko + Isotype, Ifi35^ko + αPD-1) (n = 6 per group, two-way ANOVA test, mean with SD is plotted). C Tumor weight 30 days after tumor inoculation in indicated 4 groups (WT+Isotype, WT + αPD-1, Ifi35^ko + Isotype, Ifi35^ko + αPD-1) (n = 6 per group, one-way ANOVA test with Turkey’s multiple comparisons, Mean with SD is plotted). D Kaplan–Meier curves for the survival of indicated 4 groups of balb/c mice (WT + Isotype, WT + αPD-1, Ifi35^ko + Isotype, Ifi35^ko + αPD-1) (n = 8 per group, log-rank test). E The frequencies of IFNγ⁺CD8⁺ and GzmB⁺CD8⁺ T cells population in indicated 4 groups of balb/c mice (WT + Isotype, WT + αPD-1, Ifi35^ko + Isotype, Ifi35^ko + αPD-1) (n = 6 per group, one-way ANOVA test with Turkey’s multiple comparisons, mean with SD is plotted). F Model illustrating how IFI35 orchestrates tumor microenvironment to promote TNBC progression.