Relationships between spatial distribution in the tumour microenvironment and functions of macrophage subtypes in squamous cell carcinomas of the head and neck

Tumour development and metastasis are significantly influenced by interactions with cells in the microenvironment. The spatial arrangement of the individual cells in relation to each other influences the probability of whether and in what way these cells interact with each other. We investigated the spatial relationships of different macrophage (MΦ) subsets, T cells and tumour cells in human head and neck squamous cell carcinoma (HNSCC) using multiplex immunohistochemistry (IHC). To further characterise the MΦ we used flow cytometry (FC) and reanalysed two published HNSCC single cell RNA datasets. We found different spatial distributions of MΦ subpopulations based on CD68 and CD163 expression. CD68hi and CD68hiCD163hi MΦ accumulated in HNSCCs mainly in tumour nests and at the tumour-stroma boundary, whereas CD163hi MΦ were relatively evenly distributed in the tumour stroma. Among the MΦ CD68hi and CD68hiCD163hi, a high proportion expressed PD-L1 and PD-1 as well as CD206. Colocalisation measurements suggest that PD-L1/PD-1 interactions are multidirectional between MΦ, T cells and tumour cells immediately at the tumour-stroma boundary. Single-cell RNA sequencing data also supported high variability within MΦ subsets in HNSCCs. These results highlight the importance of including spatial cell-cell relationships when considering cell dynamics in the microenvironment of HNSCCs. In particular, more sophisticated investigation of PD-L1/PD-1 interactions could improve the prognostic sensitivity of immune checkpoint therapy response.