We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response in HER2+ breast cancer. Here, we evaluated the role of the TNF/MUC4 axis in preclinical models of TNBC and the clinical impact of MUC4 in TNBC patients.
 Soluble and transmembrane TNF were blocked with etanercept (E), and the dominant negative protein INB03 (DN) was used to block soluble TNF. BT-549 and MDA-MB-231 TNBC cell lines treated with E or DN exhibited a decrease in MUC4 expression. Conditioned media of MDA-MB-231 and BT-549 cells treated with E or DN impaired the invasion of both cell lines (p<0.01). We performed an in vivo assay on female BALB/c mice using s.c. LMM3 tumor, and we demonstrated that DN in combination with PD-1 blocking antibody prevented the appearance of lung metastasis (p<0.05 vs. IgG, DN, and anti-PD-1 groups). 
 To explore how DN influences cell seeding, LMM3 cells pre-treated with DN or IgG were injected into the tail vein of mice. Animals injected with IgG-treated cells were treated with IgG, and the ones injected with DN-treated cells were treated with DN or DN+anti-PD-1. Mice treated with DN+anti-PD-1 showed less lung metastasis vs. IgG and DN treated groups (p<0.05). In a survival assay using female BALB/c bearing 4T1 tumors, we treated animals with the standard-of-care for TNBC patients, nab-paclitaxel+anti-PD-1 antibody, alone or with DN. We observed that the combined treatment significantly increased survival (p<0.05).
 In a cohort of 47 TNBC patients (stage I-III) we proved that MUC4 expression was inversely correlated with TILs (p=0.0003) and tumor PD-L1 expression (p=0.0003). MUC4 proved to be an independent predictor of poor overall survival (p=0.003) and was associated with a higher metastasis risk (p=0.04). We propose TNF as a novel target for the treatment of TNBC patients and MUC4 as a predictive biomarker to guide a combined treatment of TNF blocking agents with immunotherapy