Immunosuppression and Metabolic Rewiring in Tumor-infiltrating Neutrophils

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PROJECT SUMMARY/ABSTRACT Metastatic breast cancer (BC) remains largely resistant to immune checkpoint blockade (ICB) therapy. Tumor-infiltrating neutrophils (TINs) with immunosuppressive activity represent a major component in the tumor microenvironment (TME) to drive immunotherapy resistance. Therapeutic debilitation of immunosuppressive TINs is a promising approach to elicit synergistic efficacy when combined with immunotherapy. However, therapeutic targeting of TINs faces challenges in selectivity and safety, highlighted by the lack of TIN-specific targets. Recently, we used single-cell RNA sequencing to compare TINs and circulating neutrophils in BC models and identified aconitate decarboxylase 1 (Acod1) to be uniquely upregulated in TINs. Acod1 catalyzes the production of itaconate, a metabolite with anti-inflammatory activity in macrophages. But Acod1 function in neutrophils is poorly defined. Our preliminary results in BC mouse models suggest that TINs rely on Acod1 to sustain survival in the TME, and Acod1 loss leads to reduced TIN infiltration and metastasis. There are still significant knowledge gaps about the function, mechanism and therapeutic potential of Acod1 in TINs. Our central hypothesis is that Acod1 upregulated in TINs that infiltrate metastatic BC is essential for the TINs to persist in the TME and exert the immunosuppressive function, thus Acod1 ablation debilitates TINs, favors anti-tumor immunity, and sensitizes metastatic BC to immunotherapy. We propose to accomplish three Specific Aims: (Aim 1) Validate the pro-metastasis function of Acod1 in TINs in syngeneic and spontaneous murine mammary tumor models. (Aim 2) Identify the upstream and downstream molecular mechanisms underlying the upregulation and function of Acod1 in TINs. (Aim 3) Improve metastatic BC response to immunotherapy by Acod1 ablation and validate ACOD1 expression in clinical samples. To achieve our research goals, we have developed both whole-body and neutrophil-specific Acod1 knockout mice as hosts for BC syngeneic models, both mouse and human in vitro cell models of TINs, single- cell technologies for gene profiling and immune cell phenotyping, injection techniques that generate mammary tumors and metastases to lung and bone in the mice, and multiplex immunofluorescence staining techniques suitable for validation studies using clinical samples. We have assembled a strong research team with complementary expertise, which further ensures that the studies proposed are highly feasible to accomplish. Upon completion of the project, we expect to uncover the previous unknown function and mechanism of Acod1 in immunosuppressive TINs that are enriched in BC metastases, provide novel links between metabolic rewiring and immunoregulatory function of TINs, and generate the key preclinical evidence for targeting Acod1 to improve immunotherapy. In the long term, we envision that the bench-to-bedside translation of our findings through development of Acod1 inhibitors may accelerate the therapeutic application of combining agents that reprogram immunometabolism and immunotherapeutics to the curative treatment of BC and other cancers.