Single-cell RNA-seq analysis of early melanoma development and treatment resistance
Final Report Abstract
To identify and characterize early molecular events in melanoma development, we performed single-cell sequencing of melanoma and skin cells using the 10x Genomics platform. This enabled a transcriptomic and epigenetic analysis of over 200,000 cells. In our sample selection, we focused on early, treatment-naive primary tumors, which in some cases were nevus-associated, and complemented the study with the addition of benign melanocytic nevi. This combination allowed us to capture various stages of melanoma development and their malignant transition using advanced bioinformatic tools. The first part of our analysis addresses the cellular sub-classification of melanoma cells based on their differentiation status. These cellular melanoma subtypes were then characterized using RNA velocity, Latent time and self-organizing maps (SOM). Since treatment approaches underwent a strong shift towards targeted immunotherapy in recent years, we implemented an analysis of cell-cell communication. We utilized LIANA to find novel ligand-receptor interactions (LRI) between melanoma cells and T lymphocytes. The most promising candidates were subjected to further investigation by immunohistochemistry and in situ hybridization. This showed the colocalization of the candidates within the tumor microenvironment (TME) validating their potential to interact. Additionally, the binding ability of the LRIs was measured with biolayer interferometry. To assess the functionality of these predicted interactions, we established a co-culture system of gp100-expressing melanoma cell lines and TCR-transgenic Jurkat cells. Using siRNA, we downregulated validated melanoma targets and assessed the changes in Jurkat cell activity upon co-culture. Our data also predicted two potential ligands for the co-activation receptor CD2 on T lymphocytes. As part of a collaboration, the group of Annette Paschen (Essen) assessed the role of these ligands in tumor immunity using an autologous melanoma co-culture system. By adding ligand-blocking antibodies, the activity of the patient derived CD8+ T cells was reduced as well as their cytotoxicity towards cancer cells. The scATACseq data showed an increased accessibility of enhancers upstream of the CD2 gene in lymphocytes derived from melanoma compared to nevi. We were able to identify motifs in these accessible enhancer regions and hypothesize that one of the respective transcription factors impacts on the expression of CD2 in tumor-infiltrating lymphocytes (TILs) to enhance anti-tumor immunity.
Publications
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Single‐cell transcriptomics and epigenomics point to CD58‐CD2 interaction in controlling primary melanoma growth and immunity. Cancer Communications, 45(4), 465-470.
Stubenvoll, Antonia; Schmidt, Maria; Moeller, Johanna; Chango, Max Alexander Lingner; Schultz, Carolyn; Antoniadou, Olga; Loeffler‐Wirth, Henry; Bernhart, Stephan; Große, Florian; Thier, Beatrice; Paschen, Annette; Anderegg, Ulf; Simon, Jan C.; Ziemer, Mirjana; Schoeder, Clara T.; Binder, Hans & Kunz, Manfred
