Abstract 1702: SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools
Authors: Guo Qiuchen , Spasic Milos , Maynard Adam , Goreczny Gregory , Bizuayehu Amanuel , Olive Jessica , McAllister Sandra 
Published: June 2022

Abstract
Single cell “omics” and lineage tracing analyses are valuable tools for studying heterogeneity and clonal dynamics. However, current clonal analyses do not easily enable live cell retrieval. That is a particular issue when further study of cells that were targeted for elimination or otherwise not selected during an experiment could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and retrieval of live cells. Single cell-derived clonal populations are generated from complex cell population, each is infected with a unique, heritable molecular barcode, and stocks of each individual barcoded clone (BC) are retained. BCs can be combined to create a version of the original cell population or to generate custom BC pools. We developed a highly sensitive, accurate, rapid, and inexpensive qPCR-based method for identifying and quantifying BCs in vitro and in vivo.We applied SunCatcher to various breast cancer cell lines, including murine Met1 triple negative breast cancer line. A heterogeneous pool comprised of 31 Met1 BCs reliably reproduced the proliferation rate, tumor-forming capacity, and cancer progression as the original parental cell line. Individual BCs, however, displayed significant differences in proliferation, tumor formation, and breast cancer cell hallmarks: cytokeratin 8/14, Zeb1 (mesenchymal marker), EpCAM (epithelial marker) PD-L1 (immune checkpoint protein) and MHC-I (antigen presentation). Five BCs consistently dominated the primary tumors at the ethical end point of 21 days: BC2, BC8, BC25, BC53, BC67. Interestingly, not all 5 BCs were the fastest growing clones, suggesting in vitro proliferation does not correlate with clone expansion in vivo. Two clones (BC43, 18) were not detected in the primary tumors at ethical endpoint, yet when injected alone, had tumor-forming capacity after a long latency (~60 days). Therefore, SunCatcher enabled identification of clones that are consequential for disease progression that would otherwise not be identified during pre-clinical experimentation. Using SunCatcher, we successfully detected Met1 spontaneous metastases as early as 3 weeks in the lung, long bone, and mandible. Long bone had the highest incidence and metastatic burden and were dominated by BC53 and 8 in 3/5 mice. Lung had lower metastatic burden and BC53 was the dominant clone in 4/4 mice. BC8 was exclusive to bone, suggesting site-specific metastasis capacity for different tumor clones. SunCatcher is a reliable, inexpensive, and rapid clonal barcoding method that enables deeper understanding of heterogeneity and clonal dynamics. We identified and analyzed BCs that were responsible for cancer progression, and, for the first time, could analyze BCs that were negatively selected during cancer progression. We envision that SunCatcher will be a useful cancer research tool.
Citation Format: Qiuchen Guo, Milos Spasic, Adam Maynard, Gregory Goreczny, Amanuel Bizuayehu, Jessica Olive, Sandra McAllister. SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools [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 1702.