Using Spatial Epigenomics and Transcriptomics to Characterize the Tumor Microenvironment of Nonmelanoma Skin Cancer

Introduction: Non-melanoma skin cancer (NMSC) has a higher incidence and worse outcomes in immunosuppressed (IS) patients. The objective of this study is to evaluate epigenetic alterations and changes in gene expression associated with spatial distribution of cells within the tumor microenvironment (TME) in immunocompetent (IC) and IS patients with NMSC.

Methods: Tumor samples and samples of normal skin from patients with NMSC undergoing Mohs micrographic surgery or biopsy at the University of Texas MD Anderson Cancer Center were collected prospectively. The Deterministic Barcoding in Tissue sequencing (DBiT-seq) platform was used to perform spatial ATAC (assay for transposase-accessible chromatin) analysis of samples from 8 IC and 8 IS patients. The 10x Genomics Xenium in situ RNA hybridization platform was used to perform spatial transcriptomic analysis of 26 IC and 65 IS samples from 48 patients using a custom panel of 350 target genes.

Results: Spatial ATAC identified populations of M2 macrophages within the stroma and CD68+ tumor-associated macrophages (TAMs) in the TME of both IC and IS patients. Gene accessibility scores for CD68+ TAMs were significantly higher in IC compared to IS patients. Neighborhood analysis demonstrated significant interactions between clusters of tumor cells and clusters of fibroblasts and M2 macrophages in IC patients that were not observed in IS patients. Transcription factor motif analysis further identified specific epigenetic regulatory programs for the different spatial clusters. Preliminary results from the Xenium spatial transcriptomic analysis have identified tumor cells, fibroblasts, TAMs, and stromal macrophages in the TME of IC and IS patients, validating the spatial ATAC results.

Conclusions: Differences in epigenetic modifications and spatial proximity of TAMs in the TME between IC and IS patients suggest that differences in innate immunity are key drivers of immunosuppression in patients with NMSC. These data support the use for agents such as TLR (toll-like receptor) agonists to augment the innate immune system to improve efficacy of treatment of IS patients.