SPP1^hi Macrophages Interact with CXCR4^hi CD62L^lo Aged Neutrophils via SPP1-CD44 Axis to Shape the Immunosuppressive Phenotype of the Tumor Microenvironment in HNSCC

Introduction

The immunosuppressive phenotype of the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) significantly undermines the efficacy of immune checkpoint inhibitors, such as anti-PD1/L1 therapy. Notably, there is substantial evidence highlighting the contribution of myeloid cells, particularly tumorassociated macrophages (TAMs) and tumor-associated neutrophils (TANs), to the immunosuppressive landscape of the TME. These cells facilitate immune evasion and tumor progression by modulating anti-tumor immune responses and promoting an immune-suppressive niche.

Methods

Flow cytometry was used to detect CXCR4^hi CD62L^lo aged TAN, SPP1^hi TAMs, and T cell exhaustion markers (PD-1, TIM-3, LAG-3) in HNSCC tumor samples (laryngeal and hypopharyngeal cancer).scRNA-seq was performed on tumor tissues from laryngeal and hypopharyngeal cancers.Multiplex immunofluorescence staining was performed for CD44 on CD66b⁺ TANs and SPP1 on CD68⁺ TAMs

Results

Flow cytometry revealed upregulated PD-L1 expression on CXCR4hi CD62L^lo aged TANs and SPP1^hi TAMs, which correlated with the degree of T cell exhaustion.scRNA-seq revealed that CXCR4^hi CD62L^lo aged TANs and SPP1^hi TAMs exhibit significant immunosuppressive phenotypes. Cell-cell communication analysis indicates their interaction through the CD44-SPP1 axis.Multiplex immunofluorescence confirmed the spatial colocalization of CD44 on CD66b⁺ TANs and SPP1 on CD68⁺ TAMs, both of which were associated with poor patient prognosis.

Conclusion

In our investigation, we identified CXCR4^hi CD62L^lo aged TAN with a prolonged-survival phenotype interacting with SPP1^hi TAMs via the CD44-SPP1 axis, collaboratively shaping the immunosuppressive TME in HNSCC.

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