Identification of a spatially resolved neighborhoods in HNSCC that limit response to immunotherapy

Immunotherapy (IO) has transformed treatment of Head and Neck Squamous Cell Carcinoma (HNSCC), yet many patients do not benefit. While scRNAseq has advanced our understanding of tumor, stromal, and immune cell states, the spatial organization of the tumor microenvironment is poorly defined. We hypothesize that a distinct cellular neighborhood at the tumor margin contributes to immune exclusion and IO resistance. To investigate, we performed spatial transcriptomics (ST) on a tumor microarray (TMA) of 20 treatment-naïve HNSCC patients using Brüker’s CosMx platform. We integrated an HNSCC scRNAseq atlas with the ST data to enable robust cell identification. Neighborhoods were identified by performing clustering on a cell adjacency matrix. We identified 6 recurrent spatially organized neighborhoods. Neighborhood 5 (N5) represented the tumor core and contained more exhausted CD8+ T cells versus other neighborhoods. Neighborhood 3 (N3) at the tumor-stromal interface displayed immunosuppressive signatures (e.g., TGFβ signaling) and extracellular matrix remodeling, suggesting a role in immune exclusion. Cox regression analysis showed that a high enrichment of a N3 signature correlated with worse outcomes following IO. Exhausted CD8+ T cells were enriched in N5, suggesting that adjacent N3 may restrict access to tumor antigens and limit response to IO. To explore neighborhood function, we developed 3D spheroids with HNSCC tumor cells, fibroblasts, endothelial cells, and peripheral blood immune cells to model immune infiltration in vitro. This spheroid platform enables mechanistic exploration of immune exclusion and allows for identification of strategies to enhance IO response. In short, ST revealed novel immune exclusionary features that may be targeted to improve antitumor immunity. 

Learning Objectives:

1. Discuss the strengths and weaknesses of scRNA-seq vs spatial transciptomics 

2. Summarize the knowledge that can be gained from spatial transcriptomics data

3. Explore a novel cellular neighborhood which may limit anti-cancer immune response in HNSCC