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posted on 2024-02-29, 14:21 authored by Rana Fetit, Alistair S. McLaren, Mark White, Megan L. Mills, John Falconer, Xabier Cortes-Lavaud, Kathryn Gilroy, Tamsin R.M. Lannagan, Rachel A. Ridgway, Colin Nixon, Varushka Naiker, Renee Njunge, Cassie J. Clarke, Declan Whyte, Kristina Kirschner, Rene Jackstadt, Jim Norman, Leo M. Carlin, Andrew D. Campbell, Owen J. Sansom, Colin W. Steele Characterization of neutrophils in metastasis. A, UMAP of neutrophils in CRCLM. B, Coexpression of H_enriched and T_enriched signatures. One cluster is not enriched for either signature (blue arrow). C and D, Unsupervised pseudotime analysis and estimated smoothers for TXNIP expression over the different numbered pseudotime lineages. E, Coexpression of TXNIP and CXCR2. F and G, Expression and estimated smoothers for CXCL8 over pseudotime. H, Coexpression of CXCL8 and IL1β. I, IHC staining of TXNIP in a patient CRCLM sample at 4x (left) and 10x (right). Scale bars = 50 µm. J and K, IHC staining of ITGAM (Neutrophils) and CD3 (T cells) in a patient CRCLM sample at 4x, scale bars = 50 µm. Dashed squares indicate regions where immune cells cluster. L, Differentially expressed genes in metastasis-specific neutrophil cluster. M, H_enriched, T_enriched, and M_enriched gene signatures in mouse bulk-RNA-seq neutrophil dataset. L, Healthy liver tissue, PT: Primary tumor, LMET: Liver metastasis. N, Averaged expression of the individual genes of the three signatures. O, UMAP plot of integrated human neutrophils from PT and metastatic (M) datasets of different cancers. P, Coexpression of CXCR2 with IL1β (top) and TXNIP (bottom). Q, Differential gene expression between neutrophils in malignancy compared with PT. R and S, GO and KEGG analysis of M_CRC neutrophils.
Funding
UK Research and Innovation (UKRI)
Cancer Research UK (CRUK)
Blood Cancer UK
UKRI | Medical Research Council (MRC)
CRUK | Beatson Institute for Cancer Research (The Beatson Institute)
History
ARTICLE ABSTRACT
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 IL1β/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 IL1β/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.
We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1β/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.
