81P Exosomal HMGB1 induce PD-L1+-tumor associated macrophages via glycometabolic reprogramming to promote lung-tropic metastasis of triple-negative breast cancer

The aim of this study was to investigate whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and promote a pre-metastatic immunosuppressive niche. Exosomes from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Mass spectrometry was used to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Significance of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed. Functions of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche. HMGB1 was significantly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling suggested a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages incubated with EO771/exo exhibited an enhanced glycolysis and lactate production. Cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy demonstrated that EO771/exo were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were detected in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche.