Abstract 3768: Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma

Abstract

Abstract A critical challenge for the effective treatment of pediatric solid tumors is tumor recurrence. In pediatric rhabdomyosarcoma (RMS), 70% of patients with metastatic disease will experience relapse despite treatment with chemotherapy, radiation and/or surgery. Those patients with relapse have particularly dismal outcomes with less than 20% survival over 5 years. These poor outcomes underscore the need for a better understanding of RMS, particularly the biologic processes responsible for recurrence and progression. Currently, a major obstacle towards improving RMS treatment has been that we, as a field, do not know which cells within RMS tumors evade treatment toxicity, and how these cells are selected for in the presence of chemotherapy. We have previously developed a panel of orthotopic patient-derived xenografts (O-PDXs) that encompass the clinical and molecular diversity of RMS. In this study, we have leveraged single-cell RNA-sequencing (scRNA-seq) of our O-PDX system to characterize the intratumoral heterogeneity within this panel. Analysis of single-cell transcriptomic data demonstrate that fusion-negative RMS O-PDXs bear two distinct subpopulations: (1) a major subpopulation encompassing approximately 99% of the tumor cells, and (2) a minor population that are non-cycling and express genes from a variety of mesenchymal lineages. This two-population intratumoral structure is stable within O-PDXs during passaging of xenografts. To extend these findings, we have developed a workflow using lentiviral transduction of O-PDX cells with a high complexity barcode library. This library integrates a unique 18-mer oligonucleotide barcode into each transduced cell within the 3’-untranslated region of blue fluorescent protein. Thus, all cells within an O-PDX with the same barcode are progeny of the same ancestral cell. We have validated longitudinal tracking of barcode diversity using genomic DNA and within the libraries generated during scRNA-seq. Mice bearing barcoded O-PDXs have undergone chemotherapy dosed to pharmacokinetically mimic pediatric therapy regimens; O-PDXs regress during treatment and ultimately recur following completion of therapy, akin to the clinical experience with RMS. Recurrent O-PDXs bear divergent barcodes, indicating that chemotherapy selects for tumor cells independent of the originating lineage of those cells. Further studies to evaluate the changes that occur in recurrent O-PDXs cells are underway. Citation Format: Anand G. Patel, Elizabeth Stewart, Michael Clay, Xiang Chen, Michael Dyer. Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3768.