Modeling, Mechanistic Understanding, and Therapeutic Targeting of Immunosuppressive Neutrophils

Myeloid-derived suppressor cells (MDSCs) represent a significant barrier to the success of cancer immunotherapy due to their potent immunosuppressive functions within the tumor microenvironment (TME). In this dissertation, I investigated two complementary strategies for targeting polymorphonuclear MDSCs (PMN-MDSCs): direct modulation of MDSCs through specific markers, and targeting tumor-intrinsic molecular mechanisms that drive their accumulation. First, I developed human isogenic neutrophil and PMN-MDSC models using the HL60 cell line, which enabled in vitro exploration of PMN-MDSC markers and functions. Proteomic profiling of these cell models identified a 41-protein signature that distinguishes PMN-MDSCs from neutrophils, while RNA-sequencing revealed VCAN (Versican) as a novel marker with significant upregulation in PMN-MDSCs. Clinical analysis demonstrated that VCAN expression in human cancers correlates with poor prognosis and enhanced tumor-associated neutrophil infiltration, positioning VCAN as a potential therapeutic target. In parallel, I established a murine isogenic PMN-MDSC model using ER-Hoxb8-transduced bone marrow precursors and employed CRISPR/Cas9 gene editing to investigate the function of Irg1 in PMN-MDSCs. Knockout of Irg1 provided insights into its role in regulating the immunosuppressive properties of PMN-MDSCs. Finally, I investigated tumor-intrinsic mechanisms that promote PMN-MDSC accumulation. I discovered that protein-arginine deiminase (PADI) activity, particularly PADI2 and PADI4, is upregulated in certain tumors, driving histone citrullination and cytokine expression that recruit immunosuppressive neutrophils to the TME. Inhibition of PADI activity not only reduced neutrophil accumulation in murine tumor models but also enhanced the efficacy of immune checkpoint blockade (ICB) therapy. This work establishes key markers and mechanisms underlying PMN-MDSC recruitment and function, providing potential therapeutic targets to enhance the effectiveness of cancer immunotherapy.