Single-cell multi-omics analysis reveals heterogeneity and plasticity of neutrophil states in response to immunotherapies
BackgroundTumor-associated neutrophils (TANs) are emerging as functionally heterogeneous and plastic cells in the tumor microenvironment. In immunologically cold tumors, elevated neutrophil abundance correlates with poor prognosis and resistance to immune checkpoint inhibition (ICI). Whether distinct anti-tumoral neutrophil states can be induced by different immunotherapies and how they relate to treatment efficacy remains unclear. MethodsUsing the syngeneic MOC2-huEGFR (M2h) mouse model of head and neck squamous cell cancer (HNSCC), we treated tumor-bearing mice with agonistic anti-CD40 monoclonal antibody (mAb) (aCD40), TNF, Cetuximab, or a combination of all three, designated Neutrophil Activating Therapy (NAT). In addition to evaluating anti-tumor efficacy, we performed single-cell multiomics RNA and protein sequencing, followed by bioinformatics analyses and flow cytometry validation. NAT-induced anti-tumor efficacy and related neutrophil states were also assessed in another cold tumor model, 9464D-GD2 neuroblastoma. Murine treatment-induced neutrophil gene signatures were then evaluated using clinical, proteomic, and transcriptomic data from HNSCC patients. ResultsFive transcriptionally distinct neutrophil states (N0-N4), including precursor state CD49d+ N4, were identified using the M2h model. N0 neutrophils (immunosuppressive/quiescent) dominated untreated tumors, but not in successful treatments. ISG+ N1 neutrophils and CCR3+ N3 neutrophils expanded by aCD40, TNF, and NAT treatment with anti-tumoral gene signatures and found more interacting with CD8+ T cells from bioinformatics analysis. N2 neutrophils reflected a recently established hypoxia-adapted state found in all treatments. ICAM1 (CD54) emerged as a marker of treatment-induced neutrophil activation, discriminating N1, N2, and N3 neutrophils from N0 neutrophils, validated by flow cytometry. In the 9464D-GD2 neuroblastoma model, NAT treatment also reduced the N0 dominance seen in untreated tumors in the HNSCC model but failed to induce anti-tumoral neutrophil states. In 23 HNSCC patients who received ICI therapy, ICAM1 protein expression in neutrophils trended toward association with responder status (TMA-level p=0.029), and ICAM1 neutrophil gene expression also trended toward association with improved overall survival in TCGA data (HR=0.75, p=0.059). ConclusionsDistinct immunotherapy-induced neutrophil states are defined by transcriptional profiles enriched in different functional pathways, associated with both anti-tumor and pro-tumor signatures. ICAM1 identifies activated neutrophils and potentially serves as a biomarker of ICI response in HNSCC, warranting further clinical validation. WHAT IS ALREADY KNOWN ON THIS TOPICNeutrophil heterogeneity has received increasing attention, with studies identifying antitumoral neutrophil populations, either at baseline or induced by treatment. Several effective treatment regimens involve an anti-CD40 agonist (aCD40) antibody, among them Neutrophil Activating Therapy (NAT), which combines aCD40, TNF, and a tumor antigen binding antibody designed to reprogram neutrophils. NAT could thus be particularly effective in cold, myeloid-rich tumors that are largely unresponsive to conventional immunotherapies such as checkpoint blockade, enacting these anti-tumoral effects through similar and different mechanisms; however, this has not been tested. WHAT THIS STUDY ADDSThis study adds a single-cell multi-omics framework for defining treatment-induced neutrophil heterogeneity in MOC2-huEGFR and 9464D-GD2 tumors, two immunologically cold models. It highlights ICAM1/CD54 and interferon-stimulated genes as markers of a dominant antitumor neutrophil state, while showing that neutrophil state composition variy across tumor models. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYThese results support the efficacy of a myeloid-modulating therapy built around aCD40 and TNF in a cold murine head and neck cancer model, and to a lesser extent in a cold murine neuroblastoma model. ICAM1/CD54 expression in neutrophils was also identified as a promising marker of antitumor activity and treatment response. More broadly, this work suggests that incorporating aCD40 and/or TNF into existing treatment regimens could improve outcomes, while ICAM1/CD54-high neutrophils may serve as a useful therapeutic readout.