Spatial profiling of melanocytic tumors and their microenvironment Understanding tissue structure is fundamental for biological sciences and for distinguishing benign versus malignant neoplasms, but histopathological assessment alone is inaccurate for the diagnosis of certain tumors, including a subset of melanocytic neoplasms (melanocytic nevi and melanomas), resulting in diagnostic errors and worsened patient outcomes. Therefore, novel biomarkers for the diagnosis of melanoma are needed. To identify such markers, it is imperative to better understand the interaction between melanocytes and neighboring keratinocytes, immune cells, and other components of the complex tumor microenvironment in nevi and melanoma. Nevi and early primary melanoma display intratumoral heterogeneity, often coupled with low cellularity and purity. Therefore, the tumor-microenvironment interactions could be missed by bulk approaches or single-cell sequencing of advanced/metastatic tumors only, which has been the focus of most prior studies. The goal of this research is to build high-resolution spatial maps of gene expression of the tumor and its microenvironment in morphologically preserved nevi and melanomas to identify novel diagnostic biomarkers. The hypothesis is that melanocytic tumors and their microenvironments contain subpopulations of cells with characteristic gene expression patterns that differ between nevi and melanoma. We hypothesize that some of these differentially expressed genes are spatially confined and cell-type specific and could be used as potential diagnostic markers. Our prior data demonstrated differences in CDK2 gene expression between melanocyte- rich regions of nevi and melanoma. In Aim 1, we will assess spatial expression of CDK2 by immunohistochemistry in a tumor panel of over 200 nevi versus melanoma, comparing it to proliferative markers, as well as established melanoma biomarkers, including PRAME. In Aim 2, to identify novel biomarkers, we will establish high-resolution spatial maps of gene expression of tumor and microenvironment subpopulations in nevi versus melanoma by performing a spatial whole transcriptome analysis and a high-plex single-cell imaging. Top differentially expressed genes in these subpopulations will be validated via immunohistochemistry in the tumor panel described above. This study improves current theoretical concepts by investigating tumor and microenvironment populations in nevi and early primary melanoma – the common, yet previously understudied tumor types. Furthermore, this study utilizes improved state-of-the-art approaches, including high-plex single-cell spatial gene expression profiling. This research will improve the understanding of tumor-microenvironment subpopulations in their spatially correct context, relevant for tumor biology and biomarker development, ultimately leading to improved diagnostic accuracy of melanoma and improved patient outcomes.