Abstract B015: RNA-seq analysis to uncover drivers of cancer cells forming tunneling nanotubes for intercellular communication

Abstract Tunneling nanotubes (TNTs) are dynamic membranous protrusions that are critical for intercellular communication in cancer, yet their transient nature and lack of definitive molecular markers pose challenges for systematic study. To address this issue gap in knowledge, we used bulk and single-cell transcriptomic sequencing approaches to identify differentially expressed genes in ovarian cancer cells enriched for TNTs to identify molecular pathways and genes potentially implicated in TNT formation, maintenance and function. We used edgeR and Seurat to perform differential expression analysis on ovarian cancer cells grown in conditions favoring upregulation of TNT formation and further performed single-cell sequencing of the same cell type isolated via spatiotemporal genomic analysis (SaGA) and fluorescence-activated cell sorting (FACS). We found 771, 32 and 279 genes upregulated in bulk sequenced TNT forming SKOV3, COV362 and ES-2 ovarian carcinoma cell lines, respectively. Gene families that repeatedly came up across all three of the cell lines included carbonic anhydrases, matrix metalloproteinases, SERPIN (Serine Protease Inhibitor-30), and SNORA (Small Nucleolar RNAs). We narrowed down candidate genes of interest to 23 differentially expressed genes (DEGs), many of which are involved in cellular stress response, organization of the extracellular matrix or intracellular cytoskeleton, cell adhesion, and other pathways. Further analyzing the single cell sequenced ES-2 cells, we found the differential expression of KISS1 gene, a metastasis suppressor implicated in cytoskeletal organization, significantly upregulated in TNT-enriched cells, highlighting its potential role in TNT formation. Other genes, such as EPHB1, MMP3, and WNT7B, were also upregulated in TNT-enriched cells, suggesting their involvement in regulating cell adhesion, migration, and differentiation. Conversely, we observed downregulation of BGN, a gene associated with promoting metastatic potential and NFkB signaling in cancer cells, as well as SYNGR1, a gene traditionally linked to synaptic transmission but also involved in vesicle exocytosis, which may influence TNT formation. Citation Format: Rojina Sapkota. RNA-seq analysis to uncover drivers of cancer cells forming tunneling nanotubes for intercellular communication [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B015.