Co-treatment with palmitic and oleic acids reprograms tumour-associated macrophages to enhance anti-cancer activity against breast cancer cells

Recent research has highlighted the overexpression of fatty acids (FAs) like palmitic acid (PA) and oleic acid (OA) in breast cancer (BC) patients, particularly those with obesity. Secreted by cancer-associated adipocytes (CAAs) within the tumour microenvironment (TME), these FAs disrupt metabolic and immune pathways by modulating tumour-associated macrophages (TAMs) function, creating a feedback loop that drives tumour progression. While PA induces pro-inflammatory responses, OA promotes anti-inflammatory signalling and M2 polarisation, contributing to an immunosuppressive TME. However, the combined effects of PA and OA on macrophage polarisation remain underexplored. This study aimed to evaluate the therapeutic potential of reprogramming tumour-associated macrophages (TAMs) toward an anti-tumour M1 phenotype through co-treatment with PA and OA, with the goal of enhancing phagocytic activity, inducing apoptosis, and suppressing metastasis in 4T1 BC cells. To investigate, M0 macrophages (resting RAW 264.7 macrophages) and M2-like TAMs (differentiated using 4T1-conditioned medium) were treated with PA-BSA and OA-BSA, either individually or in combination. M1 (iNOS and CD86) and M2 (ARG1 and CD206) macrophage-associated markers were assessed using immunofluorescence staining, real-time quantitative PCR (qPCR) and flow cytometry. Levels of nitric oxide (NO), reactive oxygen species (ROS) and cytokines were also measured. The study further explored the anti-cancer activity of the reprogrammed macrophages, including phagocytosis, apoptosis induction, and inhibition of migration and invasion in 4T1 cells, using fluorescence microscopy and flow cytometry. LPS/IFN-γ-induced M1 macrophages served as a positive control for comparative analysis. Cotreatment with PA-BSA and OA-BSA robustly induced M1 polarisation, evidenced by increased iNOS and CD86 expression, alongside reduced ARG1 and CD206 levels. This shift was accompanied by elevated production of proinflammatory cytokines (TNF-α and IL-1β), NO and ROS, including hypoxia inducible alpha-1 (HIF-1α) activation. Reprogrammed macrophages demonstrated significantly enhanced phagocytic activity and induced substantial apoptosis in 4T1 cells compared to respective controls, as shown by AO/PI staining and Annexin V/FITC assays. Co-culture with these reprogrammed macrophages also significantly upregulated NO and TNF-α expression, supporting their role in apoptosis induction. Furthermore, CM from these macrophages also inhibited 4T1 cell migration and invasion, with a marked reduction in wound healing capacity. These findings highlight the potential of PA and OA co-treatment to reprogram TAMs toward an M1 phenotype, enhancing their anti-tumour capabilities through increased phagocytosis, apoptosis induction, and suppression of metastasis. This strategy represents a promising immunotherapeutic approach for BC, offering a novel way to target the TME and overcome tumour progression.

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