P-01.17 Characterization of neutrophil subpopulations in pancreatic cancer through a high-dimensional surface marker screen

<h3>Background</h3> Pancreatic ductal adenocarcinoma (PDAC) remains one of the most difficult cancers to treat, with few effective therapies and a poor prognosis. One major reason for treatment failure is the immunosuppressive tumor microenvironment (TME), where neutrophil granulocytes can take on both pro- and anti-tumoral roles. However, the mechanisms that drive their transformation into immunosuppressive neutrophils are not well understood. A significant challenge is the lack of reliable surface markers to differentiate between pro- and anti-tumoral neutrophil subpopulations, which hinders the development of targeted therapies. Additionally, little is known about the factors that drive the reprogramming of neutrophils within the TME, making it difficult to counteract their immunosuppressive effects. <h3>Materials and Methods</h3> To explore neutrophil heterogeneity, we carried out a large-scale screen using the LegendScreen® assay combined with Infinity Flow (Becht et al., 2020), examining over 260 surface markers on myeloid immune cells from both an orthotopic murine PDAC model and peripheral blood samples from PDAC patients. Flow cytometry analysis was performed, and machine learning algorithms predicted co-expression patterns of surface markers, enabling high-dimensional clustering of neutrophil subpopulations. To investigate the role of tumor-secreted factors on neutrophil reprogramming, isolated neutrophils from both healthy and tumor-bearing mice were exposed to PDAC cell-derived supernatants, and changes in neutrophil marker profiles were assessed by flow cytometry. <h3>Results</h3> LegendScreen® analysis revealed distinct neutrophil subpopulations in both murine models and human PDAC samples. Tumor-derived neutrophils display a different phenotype than peripheral neutrophils based on marker expression, however, tumor bearing individuals already show neutrophils with different marker expression in the peripheral blood. We identified subpopulations expressing markers associated with immunosuppressive properties such as CD14, PD-L1, and Siglec-F in mice, and Lox-1 in humans. A neutrophil subpopulation with a comparable phenotype can be induced by stimulating neutrophils from healthy mice with PDAC cell conditioned medium. Additionally, this subpopulation shows significantly enhanced survival in vitro compared to neutrophils which were not stimulated with PDAC secreted factors. <h3>Conclusions</h3> We identified tumor-specific neutrophil subpopulations and showed that these subsets can be induced by tumor-secreted factors. Future research will focus on functionally characterizing these subpopulations and identifying the specific reprogramming factors within the tumor secretome. By gaining a deeper understanding of these mechanisms, we aim to develop new therapeutic strategies that target immunosuppressive neutrophil subpopulation while sparing the antitumoral counterparts. N. Abdul Razak: None. C. Marx: None. L. Delius: None. V. Kolarik: None. A. Sursanto: None. C. Rambuscheck: None. P. Metzger: None. N. Röhrle: None. D. Böhmer: None. C. Hörth: None. G. Beyer: None. J. Mayerle: None. S. Endres: None. M. Schnurr: None. L.M. König: None.