Background: A PDX bladder cancer model, BL0293-F563, grows large subcutaneous tumors, spontaneously metastasizes to the liver and bone, and sheds high numbers of circulating tumor cells (CTCs). This PDX model provides a unique opportunity to explore the relationships between primary tumors, CTCs and metastatic cell subpopulations. Methods: BL0293-F563 tumors (available from the NCI Patient-Derived Models Repository [] and originally developed by Jackson Laboratories) were implanted into NSG mice and and primary tumors, metastatic nodules in the liver, and blood were collected at maximal allowable tumor burden. Tumor tissue was dissociated using Miltenyi Tumor Dissociation Kit with OctoDissociator, and Human CTCs were enriched from whole mouse blood through negative selection with anti-mouse CD45 and anti-mouse MHC-1 magnetic beads. Single cell sequencing was done using 10X Genomics 3’ gene expression assay v3.1. Sequencing libraries were prepared using 10X Genomics Chromium and 3’ gene expression kit v3.1. Data processing and analysis was done using 10X Genomics’ Cell Ranger pipeline, Seurat, and consensus non-negative matrix factorization. Results: Using Seurat FindNeighbors, cells in the aggregated dataset were classified into 17 distinct clusters. All clusters were comprised of cells from multiple sites (primary tumor, CTCs, metastases), but three clusters were enriched in CTCs and one cluster was composed of mostly primary tumor cells. All clusters exhibited an epithelial-like gene expression signature score, suggesting that CTC shedding was occurring without prominent epithelial-mesenchymal transition. Consistent with expected differences in oxygenation states, CTC-enriched clusters exhibited a lower hypoxia gene expression score than primary tumor and metastasis-enriched clusters. CTC-enriched clusters also showed higher expression of oxidative phosphorylation genes, suggesting metabolic differences between CTCs and cells from primary tumors and metastases. Based on Human Primary Cell Atlas phenotype prediction, several clusters were associated with stem cell like phenotypes. Additionally, two of three CTC-enriched clusters had elevated expression of mitosis-associated genes, suggesting that at least some populations of CTCs are not quiescent but actively cycling. Conclusions: Utilizing single cell gene expression profiling, we have linked the gene expression profile of CTCs to specific cell subpopulations in primary tumors and metastases. We show that CTC-enriched cell clusters appear to maintain an epithelial phenotype. Subpopulations of CTC cells exhibited enrichment of stemness-associated transcripts and features of active cell cycling.

Citation Format: Tomas Vilimas, Brandie Fullmer, Alyssa Chapman, Li Chen, Ting-Chia Chang, Rini Pauly, Biswajit Das, Chris Karlovich, Yvonne A. Evrard, Howard Stotler, Michelle M. Gottholm‐Ahalt, Tara Grinnage-Pulley, Melinda G. Hollingshead, James H. Doroshow, P. Mickey Williams. Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P097.