Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1ß/CXCR2 Axis in Generation of Metastasis-specific Neutrophils.

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand-receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1ß/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1ß/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states. SIGNIFICANCE: We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1ß/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.

Single cell sequencing of neutrophils demonstrates phenotypic heterogeneity and functional plasticity in health, disease, and cancer.

A vital constituent of innate immunity, neutrophils had previously been considered functionally rigid with a fixed, defined role in host pathogen response, in part due to their fleeting lifespan. However, that consensus opinion has changed with evidence of functional neutrophil plasticity in a range of diseases including cancer. Typically difficult to sequence due to their low level of transcriptomic activity, advances in single cell RNA sequencing has allowed for closer examination of the neutrophil transcriptome in humans and mouse models and their interaction with other immune system constituents, both in health and disease, allowing for description of neutrophil phenotypes beyond previous descriptions reliant upon microscopic appearance, surface marker expression, and function. Transcriptomic analysis shows that neutrophils develop and mature along a fixed trajectory, but their transcriptome varies based on maturity, the insult that has provoked release from the bone marrow, and the tissue to which they are recruited. Thus neutrophil heterogeneity increases with maturity, with immature neutrophils being more transcriptomically rigid. Here, we review work done in neutrophil single cell RNA sequencing in mice and humans in health and a range of disease states including coronavirus disease 2019 (COVID-19) infection, and solid cancers to provide a template for understanding neutrophil biology in context.