Abstract 6806: Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden
Authors: McLaren Alistair S. , Fetit Rana , White Mark , Mills Megan L. , Lannagan Tamsin , Falconer John , Cortes-Lavaud Xabier , Gilroy Kathryn , Ridgway Rachel , Nixon Colin , Naiker Varushka , Njunge Renee , Clarke Cassie J. , Whyte Declan , Kirschner Kristina , Jackstadt Rene , Norman Jim C. , Carlin Leo M. , Campbell Andrew D. , Sansom Owen J. , Steele Colin W. 
Published: March 2024

Introduction: Neutrophils are a highly heterogenous, plastic cell population, known to play a vital role in the tumor microenvironment and progression of CMS4 colorectal cancer (CRC) metastases. We have performed a comprehensive examination of different neutrophil transcriptional states, through utilization of single-cell RNA sequencing of publicly available and independently generated data across health and malignancy in human and murine disease, and validated findings using in vivo and in vitro models of CMS4 subtype CRC.

Methods: Publicly available and independently generated breast, lung and CRC datasets from human disease mouse models were integrated to establish and validate reproducibility of neutrophil-gene signatures, and pseudo-time analysis was used to identify genes driving neutrophil development along a lineage from health to tumor using Seurat on R. Findings were validated in healthy and KrasG12D/+, Trp53fl/fl, Rosa26N1CD/+ (KPN) tumor-bearing wild type or Mrp8CreCxcr2fl/fl genetically engineered mouse models with neutrophil-specific loss of Cxcr2.

Results: We have identified distinct neutrophil subtypes in health and primary tumors, preserved in murine and human cancer and across multiple tumor types. In CRC metastases, discrete neutrophil transcriptomic subtypes are identified, with phenotypes from health to those identified in metastases driven by the IL-1β/CXCL8/CXCR2 signalling axis. Assessment of global signalling of single-cell transcripts has identified CD4+ T cells and macrophages as dominant regulators of the metastatic niche. Neutrophils sorted from the metastatic niche strongly suppress T cell proliferation, identifying them as immunosuppressive. Loss of neutrophil-specific Cxcr2 attenuates suppression of T cell proliferation in co-culture with neutrophils from the metastatic niche of tumor-bearing mice. T cell proliferation is unaffected in co-culture with healthy wild type or Cxcr2fl/fl neutrophils. We currently are running orthotopic intrasplenic models to assess the importance of loss of neutrophil-specific Cxcr2 in generation of metastases in vivo in CMS4 CRC.

Conclusions: We speculate that ablation of metastasis specific neutrophil subsets in CMS4 CRC in combination with other therapies, may alter the immunosuppressive nature of the immune microenvironment and allow for reduction in tumor burden in metastatic disease.

Citation Format: Alistair S. McLaren, Rana Fetit, Mark White, Megan L. Mills, Tamsin Lannagan, John Falconer, Xabier Cortes-Lavaud, Kathryn Gilroy, Rachel Ridgway, Colin Nixon, Varushka Naiker, Renee Njunge, Cassie J. Clarke, Declan Whyte, Kristina Kirschner, Rene Jackstadt, Jim C. Norman, Leo M. Carlin, Andrew D. Campbell, Owen J. Sansom, Colin W. Steele. Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6806.