Association Between Spatial Distribution of Leukocyte Subsets and Clinical Presentation of Head and Neck Squamous Cell Carcinoma

Background: Interactions between tumor cells and cells in the microenvironment contribute to tumor development and metastasis. The spatial arrangement of individual cells in relation to each other influences the likelihood of whether and how these cells interact with each other.

Methods: This study investigated the effect of spatial distribution on the function of leukocyte subsets in the microenvironment of human head and neck squamous cell carcinoma (HNSCC) using multiplex immunohistochemistry (IHC). Leukocyte subsets were further classified based on analysis of two previously published HNSCC single-cell RNA datasets and flow cytometry (FC).

Results: IHC revealed distinct distribution patterns of leukocytes differentiated by CD68 and CD163. While CD68hiCD163 and CD68CD163 cells accumulated near tumor sites, CD68CD163 cells were more evenly distributed in the tumor stroma. PD-L1 and PD-1 cells accumulated predominantly around tumor sites. High cell density of PD-L1 CD68CD163 cells or PD-1 T cells near the tumor site correlated with improved survival. FC and single cell RNA revealed high variability within the CD68/CD163 subsets. CD68CD163 and CD68CD163 cells were predominantly macrophages (MΦ), whereas CD68CD163 cells appeared to be predominantly dendritic cells (DCs). Differentiation based on CD64, CD80, CD163, and CD206 revealed that TAM in HNSCC occupy a broad spectrum within the classical M1/M2 polarization. Notably, the MΦ subsets expressed predominantly CD206 and little CD80. The opposite was observed in the DC subsets.

Conclusion: The distribution patterns and their distinct interactions via the PD-L1/PD-1 pathway suggest divergent roles of CD68/CD163 subsets in the HNSCC microenvironment. PD-L1/PD-1 interactions appear to occur primarily between specific cell types close to the tumor site. Whether PD-L1/PD-1 interactions have a positive or negative impact on patient survival appears to depend on both the spatial localization and the entity of the interacting cells. Co-expression of other markers, particularly CD80 and CD206, supports the hypothesis that CD68/CD163 IHC subsets have distinct functions. These results highlight the association between spatial leukocyte distribution patterns and the clinical presentation of HNSCC.