WT or IRF8 cKO NLDAMs were transfected with a full-length murine IRF8 expression plasmid driven by the CMV promoter in comparison to a control pcDNA3.1+ plasmid using the jetOPTIMUS and the Polyplus protocol. Briefly, the transfection mix consisted of 1 μg DNA and 1 μL jetOPTIMUS reagent in 500 μL jetOPTIMUS buffer. After confluent NLDAMs were incubated with the transfection mix for 1.5 h, the transfection mix was removed and NLDAMs were cultured in fresh complete RPMI with GM-CSF supplementation. Transfection efficiency and stability was evaluated with an EGFP expression plasmid. EGFP-transfected NLDAMs were observed with the ZOE Fluorescent Cell Imager (Bio-Rad) for proliferation and maintained EGFP expression for at least a month post-transfection. Control- or IRF8-transfected NLDAMs were utilized 4 days post-transfection for flow cytometry, indirect co-culture or in vivo adoptive transfer studies.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.