This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT- CA-24-029. Cancer is clearly a disease of aging. This is especially true in the case of skin cancer, which is the most common form of human cancer, accounting for more than all other cancers combined in the USA. The major objective of this U01 Supplement application is a mechanistic understanding of the interactions between age-related changes in the dermal microenvironment and infiltrating immune cells in the initiation of keratinocyte cancer. This application is based on the findings from our humanized mouse model of tumor initiation in our accelerated dermal aging mouse model. This model expresses the matricellular protein CCN1 in dermal fibroblasts and oncogenic HRas in epidermal keratinocytes. CCN1 expression in dermal fibroblasts (the source of elevated CCN1 in aged human skin) drives strikingly accelerated dermal aging, characterized by fragmentation and disorganization of the collagenous dermal extracellular matrix, and an aged fibroblast phenotype typified by reduced ECM production and increased expression of pro-inflammatory mediators. The age-related dermal microenvironment enhances the tumorigenic potential of oncogenic HRas and results in squamous cell cancer-like tumorigenesis. Importantly, at the early stage of tumor initiation, we find substantial infiltration of activated immune cells into the dermis in close approximation to the dysplastic precancerous epidermis. Therefore, we hypothesize that the interactions between the aged-dermal microenvironment and infiltrating immune cells critically enable tumor initiation. We propose to test this hypothesis by determining the global gene expression profiles in individual dermal fibroblasts and infiltrating immune cells in situ in the context of their localization within the precancerous dermis. This aim will be accomplished by using the newly developed most advanced spatial gene expression technology, Visium HD. Visium HD has a spatial resolution of 2µm, which can reveal the comprehensive gene expression of individual cells and their locations within the dermis. Thus, we will investigate the impact of the interactions between immune cells, fibroblasts, and the dermal microenvironment during tumor initiation. For these studies Dr. Evan Keller will join our research team. Dr. Keller is a Professor of Urology and Pathology and the Director of the University of Michigan Medical School Single Cell Spatial Analysis Program and Co-director of The Rogel Cancer Center Single Cell and Spatial Analysis Shared Resource. He will provide oversight of the Visium HD spatial studies, data acquisition, quality control, data interpretation and management of personnel. We are confident that the proposed study will provide novel insights into the cellular and molecular mechanisms by which the interactions between the aged dermal microenvironment and infiltrating immune cells act in concert to initiate...