Abstract A120: Single-Cell RNA Sequencing Combined with Lineage Tracing Identify Clonal Populations Across Melanoma Organ-Specific Metastasis

Abstract Melanoma, a highly metastatic skin cancer, exhibits variations in prognosis and response to therapy based on the site of metastasis. Despite the success of immunotherapy and targeted therapies in melanoma, over half of metastatic melanoma patients will experience disease progression due to therapy resistance. The heterogeneity and plasticity of melanoma cells contribute to metastatic dissemination and therapy resistance. We aim to determine whether distinct clones and/or their transcriptional cell states can predict the formation of tumours in various organs. Lineage tracing and clonal heterogeneity were evaluated using the state-of-the-art technology, SPLINTR (Single-cell Profiling and LINeage Tracing), enabling us to match a cells evolution with changes in transcriptional states. To identify melanoma clones across different metastatic sites, specifically subcutaneous, lungs and brain, C57BL/6 mice were injected subcutaneously, intravenously or intracranially with the same pool of cells of barcoded luciferase expressing YUMMER1.7PV1 murine melanoma cells. Transduction conditions ensured that one DNA barcode integrated into cell genomes at one barcode per cell, serving as a lineage tag. Bioluminescence imaging was performed once a week to monitor the tumour growth of mice injected intravenously and intracranially. Subcutaneous tumours were measured by calliper. Mice were euthanized before ethical endpoints. Tumors were harvested and single cell analysis as well as DNA sequencing was performed to identify barcodes expressed by the tumor cells across different tumor-sites. Barcode analysis was conducted both before cell implantation and at tumor harvest. Clonality exhibited significant variability between sites. Single-cell RNA sequencing will unveil states associated with dominant clones present at different metastatic sites. This information will provide a new understanding of melanoma transcriptional phenotype behaviours and biological characteristics at different metastatic sites and in response to therapy. Citation Format: Veronica Aedo-Lopez. Single-Cell RNA Sequencing Combined with Lineage Tracing Identify Clonal Populations Across Melanoma Organ-Specific Metastasis [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A120.