Abstract A065: Spatial and single cell transcriptomics uncovers metabolic reprogramming and NETosis of neutrophils in human pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma is the most hypovascular solid tumor, imposing harsh constraints on tumor metabolism. Cancer cells adapt to reduced access to vascular-derived nutrients and O2 by cooperating with abundant stromal cells. Specifically, critical nutrients including alanine, proline, glycosaminoglycans, branched chain ketoacids, lysophosphatidic acid, and unsaturated fatty acids can be provided by fibroblasts in vitro and in vivo. However, the extent of metabolic support provided by other cell types in the PDAC TME is relatively unexplored. We hypothesized that cell types that are cooperating metabolically with cancer cells will be spatially positioned near cancer cells in these tumors. We performed Xenium spatial transcriptomics on 8 treatment-naive PDAC resections and generated a spatial atlas of nearly 700, 000 cells across a range of expected cell types. We noted striking neutrophil aggregates, nucleating in the glands of classical-like malignant cells and elaborating extraglandularly. Cellular neighborhood analysis demonstrates neutrophil aggregation is strongly correlated with malignant cell proliferation and low endothelial content, suggesting neutrophils may be an important source of pro-tumoral, non-vascular nutrients in the PDAC TME. Reanalysis of published scRNAseq data revealed 3 neutrophil metabolic states in human PDAC tumors. Metabolic cluster 7 neutrophils were found only in PDAC patient samples but not healthy controls and were enriched for Type I interferon response as well as pentose phosphate pathway and iron uptake. Metabolic cluster 2 neutrophils were strikingly depleted from PDAC tumor samples compared to blood, suggesting decreased infiltration or increased cell death. Cluster 2 neutrophils were enriched for several catabolic pathways including glycogenolysis, autophagy, and proteasome, and selectively express the NETosis gene PADI4. Multiplexed immunofluorescence demonstrated that neutrophil aggregates in PDAC are citH3+/MPO+ large neutrophil extracellular traps (NETs). We reasoned pancreatic cancer cells could proliferate under tumor-like metabolic stress by repurposing amino acids liberated by autophagy, glucose liberated by glycogenolysis, or simply consuming bulk neutrophil dead cell debris via macropinocytosis. Using tumor interstitial fluid media (TIFM) and 0. 5% O2 to mimic tumor-like metabolic stress, we cocultured pancreatic cancer cells with human primary neutrophils and HL60-derived neutrophil-like cells. However, under tumor-like metabolic conditions, coculture with human primary neutrophils failed to substantially increase cancer cell proliferation and NETotic neutrophils were cytotoxic. However, we noticed enhanced neutrophil viability in tumor-like metabolic conditions, suggesting metabolic regulation of neutrophil cell death. Together, these data shed light on the metabolic phenotypes and interactions with cancer cells of neutrophils in the PDAC tumor microenvironment. Citation Format: Carson D Poltorack, Sydney M Shaffer, M Celeste Simon. Spatial and single cell transcriptomics uncovers metabolic reprogramming and NETosis of neutrophils in human pancreatic cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84 (17 Suppl₂): Abstract nr A065.