Modern life increasingly forces individuals to live “out of sync” with their internal biological clocks. Shift work, frequent travel, and irregular sleep schedules disrupt circadian rhythms that regulate sleep, hormone secretion, metabolism, immune function, and tissue homeostasis. Epidemiological studies link circadian rhythm disruption (CRD) to increased breast cancer risk, yet the biological mechanisms driving this association remain poorly understood. Here, we investigated how chronic CRD alters the mammary microenvironment and promotes aggressive breast cancer progression.

Using a genetically engineered mouse model of aggressive mammary tumorigenesis, animals were maintained under standard light–dark (LD12:12) or chronic CRD conditions. Tumor growth, mammary gland morphology, and lung metastasis were assessed. Tumor immune landscapes were profiled using single-cell RNA sequencing, flow cytometry, multiplex immunostaining, and quantitative PCR. Untargeted metabolomics and 16S rRNA sequencing were performed, followed by integrative microbiome–metabolome modeling (MIMOSA2). Therapeutic targeting of LILRB4 and arginase-1 (ARG1) was evaluated pharmacologically. Human breast cancer datasets were analyzed to assess clinical relevance.

Chronic CRD induced abnormal mammary gland morphology, increased tumor burden, and significantly enhanced lung metastasis. CRD profoundly remodeled the tumor immune microenvironment, suppressing cytotoxic immune responses and expanding immunosuppressive myeloid populations. We identified marked upregulation of the immune-inhibitory receptor LILRB4 under CRD. Targeted blockade of LILRB4 restored antitumor immunity and reduced metastatic spread. Mechanistically, LILRB4 activated a previously unrecognized non-canonical WNT signaling pathway that drove aggressive tumorigenesis under CRD. Metabolomic profiling revealed CRD-associated accumulation of immunosuppressive metabolites converging on the arginine–polyamine axis and inducing ARG1 expression. ARG1 inhibition restored CD8⁺ T-cell infiltration and reduced metastasis. CRD also altered the intratumoral microbiome, enriching polyamine-producing taxa that correlated with immunometabolic suppression. Human tumor analyses confirmed associations with poor clinical outcomes.

These findings establish a direct mechanistic link between circadian disruption and aggressive breast cancer progression. CRD promotes coordinated immune, metabolic, and microbial reprogramming that facilitates immune evasion and metastasis. Targeting LILRB4 and immunometabolic pathways represents a promising therapeutic strategy for circadian disruption–associated breast cancer and underscores the importance of circadian health in cancer prevention.

Thanks to Zymo Research and Creative Proteomics for analyzing the microbiome and metabolomics data.