Characterising neutrophil subtypes in cancer using scRNA sequencing demonstrates the importance of IL-1β/CXCR2 axis in generation of metastasis specific neutrophils.

Neutrophils are a highly heterogenous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy, has not been performed. We utilised 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 (CRC) 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 signalling pathways between neutrophils and other immune cell populations in primary CRC and metastatic CRC were investigated. We define two main neutrophil subtypes in primary tumours: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumour-specific subtype. This signature is conserved in murine and human cancer, across different tumour types. In CRC metastases, neutrophils are more heterogenous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL-1β/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-tumouroid co-cultures 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 IL-1β/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 optimise immunotherapeutic interventions into early and late interventions, targeting different neutrophil states.