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974 Single cell analysis of neutrophil heterogeneity reveals anti-tumoral phenotypes and its implications for immune-checkpoint blockade therapy in head and neck cancer
  1. Anqi Gao1,
  2. Wei Wang2,
  3. Athena Golfinos2,
  4. Paul Lambert2 and
  5. Huy Q Dinh2
  1. 1University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
  2. 2University of Wisconsin-Madison, Madison, WI, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Only ~20% of recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) patients respond to immune-checkpoint blockade (ICB) therapy. A major roadblock is the immunosuppressive nature of the tumor microenvironment. Increased infiltration of neutrophils in blood and tumors of HNSCC patients is associated with worse prognosis and ICB therapy resistance. However, depleting neutrophils has not improved ICB in mice bearing MOC2 cells, which are phenotypically analogous to immunologically ‘cold’ HNSCC tumors. We hypothesize that this is due to the heterogeneity of tumor-infiltrated neutrophils with both pro- and anti-tumoral phenotypes that prevent efficient promoting ICB-responsive TME from happening. This study aims to characterize HNSCC neutrophil heterogeneity to promote its anti-tumoral phenotypes aiding ICB therapy response.

Methods We used single-cell RNA-sequencing (scRNA-seq) and combined protein/RNA single-cell profiling (CITE-Seq) to evaluate neutrophil heterogeneity in MOC2 tumors compared to an immunological ‘hot’ tumor and ICB-responsive tumors established by knocking out stress keratin K17 from MOC2 cells (Wang et al., 2022). We then employed downstream integrated bioinformatics analyses with cell-cell interaction inferences to identify neutrophil signaling pathways associated with ICB response. Additionally, we applied the recently published neutrophil activating therapy (Linde et al., 2023) by injecting MOC2-huEGFR, a syngeneic mouse HNSCC cancer line expressing humanized EGFR, into the flanks of WT C57BL/6 mice with recombinant TNF-alpha, anti-CD40 agonist, and anti-EGFR (Cetuximab). Various in vitro assays, including T-cell activation, cytotoxicity, and neutrophil killing (ROS-mediated or T-cell mediated), will be used to characterize anti-tumoral neutrophils in murine and human patient samples.

Results In our scRNA-seq analysis, we identified four neutrophil subsets in MOC2 tumors. Among these subsets, one was two times more abundant in K17KOMOC2 tumors, which also exhibited interferon-stimulated and TNF gene expression, similar to anti-tumoral neutrophil phenotypes observed in other types of cancer from the work published this year (Gungabeesoon et al., 2023; Hirschhorn et al., 2023). Additionally, we identified neutrophil cell-cell signaling specific to K17KOMOC2 tumors. We also found that NAT altered neutrophil subsets and phenotypes while also slowing down MOC2 tumor growth.

Conclusions We showed that distinct pro and anti-tumoral neutrophil subsets exist in the HNSCC murine models, and our preliminary data indicated that modulating neutrophils toward anti-tumoral phenotypes could promote an ICB-responsive TME. Ongoing and future work include identifying the mechanism by which anti-tumoral neutrophils are induced and if these multifaceted phenotypes could be recapitulated with patient samples.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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