Poster #29 - Jiazhen Rong

Mitochondrial clone tracing within spatially intact human tissues.

Sydney A. Bracht 1†, Jiazhen Rong 2†, Rodrigo A. Gier 1,10, Maureen DeMarshall 4, Hailey Golden 5, Diya Dhakal 5,Jayne C. McDevitt 7,8, Feiyan Mo 7,8, Emma E. Furth 3,9, Alexandra Strauss Starling 4, Amanda B. Muir 6, Gary W. Falk 4, Bryson W. Katona 4, Ben Z. Stanger 7,8,9, Nancy R. Zhang 6*, Sydney M. Shaffer 1,3,8* 1 Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA 2 Graduate Group in Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4 Division of Gastroenterology and Hepatology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 5 Gastrointestinal Epithelium Modeling Program, Division of Gastroenterology, Hepatology and Nutrition, Children's Hospital of Philadelphia, Philadelphia, PA, USA 6 Department of Statistics, The Wharton School, University of Pennsylvania, Philadelphia, PA, USA 7 Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 8 Abramson Cancer Center and Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA USA 9 Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA USA 10 Present address: Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California, San Francisco, CA, USA. *Co-Correspondence: nrzhang@gmail.com, sydshaffer@gmail.com † Authors contributed equally to this work, listed alphabetically.

Understanding tissue development and intra-tissue evolution requires the ability to trace clones in intact tissues coupled with high-plex molecular profiling preserving spatial context. However, current lineage tracing tools are incompatible with spatial omics. Here, we present SUMMIT (Spatially Unveiling Mitochondrial Mutations In Tissues), a spatially-resolved lineage tracing technology that integrates gene expression profiling with mitochondrial mutation-based clone identification. Unlike synthetic lineage recording methods, SUMMIT relies only on endogenous mutations and thus can be applied to human tissues. To address the compositional mixing of cell types within spatial spots, SUMMIT includes a rigorous statistical framework to confidently assign variants to specific cell subpopulations and achieves high power for spatially localized clones by pooling information across neighboring spots. We validated SUMMIT using a controlled model in which we mixed two cancer cell lines in a mouse tumor, then demonstrated it on multiple human tissues including Barrett's esophagus, gastric cardia, small bowel, and colorectal cancer. Across these samples, we distinguished between global mutations and mutations marking locally restricted clones. The coupled transcriptomic data allowed us to characterize the cell type composition within each clone and delineate their spatial configuration. This integrated approach provides a framework to understand spatially-defined clonal evolution in preserved human tissue.