Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&T-seq

Abstract Extrachromosomal DNA circularization is a common event in cancer cells and frequently serves as a vehicle for cancer oncogene amplification. Random segregation of oncogene-containing extrachromosomal circular DNA promotes rapid intercellular heterogeneity, conferring tumors the ability to rapidly evolve and escape therapy. Smaller, copy-number neutral extrachromosomal circular DNAs are also abundantly identified in both healthy and malignant tissues, but their function in cancer is still unknown. Understanding how extrachromosomal circular DNAs contribute to intercellular heterogeneity in cancer cells remains crucial, however methods for an unbiased characterization of extrachromosomal circular DNAs in single cells are lacking. We introduce scEC&T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cells. We demonstrate the ability of our method to isolate and detect extrachromosomal circular DNAs genome-wide from all range of sizes in single cells. We observed that whereas large oncogene-containing circular DNAs are clonally present in most cancer cells, only a very small fraction of small circular DNAs are recurrently identified in single cells, indicating yet unknown prerequisites for maintenance and propagation. Our method was able to capture and recapitulate the structural complexity of oncogene-containing extrachromosomal circular DNAs in single cells, and the matching transcriptomic data allowed us to identify fusion transcripts resulting from the rearranged extrachromosomal structures. In addition, we observed that whereas the main structure of extrachromosomal circular DNAs is mostly stable in single cells, intercellular differences in extrachromosomal circular DNAs’ content can drive differences in oncogene transcription levels in single cells. We envision that by integrating extrachromosomal circular DNA and mRNA sequencing, our method will not only be useful to investigate the impact of intercellular heterogeneity in extrachromosomal circular DNA in tumor evolution, but also to interrogate its function in other biological and pathological processes. Citation Format: Rocio Chamorro Gonzalez, Thomas Conrad, Robin Xu, Madalina Giurgiu, Maja Cwikla, Katharina Kasack, Lotte Brückner, Eric van Leen, Elias Rodriguez-Fos, Konstantin Helmsauer, Heathcliff Dorado Garcia, Yi Bei, Karin Schmelz, Sascha Sauer, Angelika Eggert, Johannes H. Schulte, Roland F. Schwarz, Kerstin Haase, Richard P. Koche, Anton G. Henssen. Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&T-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1693.