Abstract 2593: Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution

Abstract Primary cutaneous melanomas develop in stages from melanoma precursor cells that may progress to melanoma in situ (MIS) and eventually to invasive disease. Molecular changes, such as tumor cell state switches from melanocytic to undifferentiated and neural-crest-like phenotypes, have been associated with the development of melanoma metastases and therapeutic resistance. However, it is unknown whether cell states or gene expression changes enriched in advanced tumors exist at the premalignant stage and if they can elicit tumor-infiltrating lymphocytes (TILs). To address this gap, we analyzed a cohort of clinical samples from 43 patients with primary cutaneous melanoma using whole-slide cyclic multiplex immunofluorescence imaging (CyCIF), morphology-guided micro-regional RNA sequencing, and high-resolution 3D imaging. Each specimen contained multiple histologically-distinct regions representing a progression axis from normal to precursor to melanoma in situ to invasion. Subsequently, we examined the molecular profiles of both atypical melanocytes and tumor cells, and the gene expression and tumor microenvironmental (TME) changes that occurred during these progression stages. We identified major intra- and inter-specimen heterogeneity in the tumor intrinsic and extrinsic features across progression stages. Multiple tumor samples contained regions where publicly available gene signatures linked to both melanocytic and undifferentiated cell phenotypes were enriched. CyCIF analysis confirmed that various tumor cell phenotypes can coexist in close proximity to each other in early-stage untreated melanomas, and that the majority of undifferentiated tumor cells were located within regions of dermal invasion. Moreover, the cellular neighborhoods around undifferentiated tumor cells had a higher proportion of CD8+ T cells and cells of myeloid lineage compared to the neighborhoods around melanocytic tumor cell phenotypes. When assessing the localization and abundance of various tumor cell phenotypes, the levels of spatial and regional heterogeneity increased proportionally along normal to premalignant to malignant progression. Furthermore, the regional entropy of tumor cell phenotypes decreased along tumor depth in the majority (63%) of the samples, and even the melanoma in situ regions showed varying levels of spatial heterogeneity. Our in-depth multimodal molecular analyses highlight the presence of spatial heterogeneity among tumor cell phenotypes as well as the organization of early-stage primary melanomas, and are suggestive of tumor cell adaptation to the local environment. Citation Format: Tuulia Vallius, Edward Novikov, Ajit Nirmal Johnson, Shi Yingxiao, Roxanne Pelletier, Shishir Pant, Zoltan Maliga, Alyce Chen, Clarence Yapp, Sabrina Chan, Christine Lian, George F. Murphy, Sandro Santagata, Peter Sorger. Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2593.