Abstract 973: Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model
Authors: Vilimas Tomas , Fullmer Brandie , Chapman Alyssa , Pauly Rini , Chang Ting-Chia , Chen Li , Das Biswajit , Karlovich Chris , Evrard Yvonne , Hollingshead Melinda , Stotler Howard , Ahalt-Gottholm Michelle , Grinnage-Pulley Tara , Williams Mickey , Doroshow James H. 
Published: June 2022

Abstract
Background: A PDX bladder cancer model, BL0293-F563, 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 metastases.
Methods: BL0293-F563 tumors (available from the NCI Patient-Derived Models Repository [https://pdmr.cancer.gov/] and originally developed by Jackson Laboratories) were implanted into NSG mice, 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 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. Data processing and analysis was done using 10X Genomics’ Cell Ranger pipeline, Seurat, and cNMF.
Results: single cell RNAseq data from primary tumors, CTCs, and metastases from 9 mice were aggregated into a single dataset, and cells were classified into 17 clusters using Seurat FindNeighbors. All clusters contained 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 epithelial-like gene expression signature scores, suggesting that CTC shedding was occurring without prominent epithelial-mesenchymal transition. CTC-enriched clusters showed elevated expression of RHO pathway genes, implicating ameboid-like migration in CTC shedding in this PDX model. 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 other sites. Additionally, two of three CTC-enriched clusters had elevated expression of mitosis-associated genes, indicating that at least some subpopulations of CTCs are actively cycling. A metastasis suppressor gene KISS1 was expressed in a subset of primary tumor cells but undetectable in CTCs, suggesting that KISS1 expression loss occurs before CTC shedding.
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 exhibit enrichment of motility-associated transcripts and features of active cell cycling. Our results implicate a known metastasis suppressor gene KISS1 in CTC shedding and metastatic dissemination in this PDX model.
Citation Format: Tomas Vilimas, Brandie Fullmer, Alyssa Chapman, Rini Pauly, Ting-Chia Chang, Li Chen, Biswajit Das, Chris Karlovich, Yvonne Evrard, Melinda Hollingshead, Howard Stotler, Michelle Ahalt-Gottholm, Tara Grinnage-Pulley, Mickey Williams, James H. Doroshow. Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model [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 973.