Dynamic cell populations are responsible for sarcoma propagating potential.

Self-renewal can maintain tumor viability and play a role in recurrence and metastasis, yet little is known about the control of self-renewal in sarcomas. We previously found that cells that exclude Hoechst dye (side population or SP cells) are enriched for tumor propagating potential in serial transplantations in a variety of sarcoma types, and thus might be enriched for renewal capacity. Here we used a mouse in which soft tissue sarcomas were driven by Trp53 deletion and Kras overexpression to trace individual clones of cells. Expression of a confetti allele or expression of barcoding in the Rosa26 locus was used to trace clones. Tumors were observed in situ in the mice, and in serial translations. Intravital microscopy in transplantations of tumors from mice expressing the confetti allele was also undertaken. While there were multiple fluorescent colors in the primary tumors, over time a dominant clone was apparent. This usually arises from a small population of cells in the early tumor. This finding was verified with serial intravital microscopy, as well as tracing the clones using barcoding. SP cells were sorted from the tumors and found to be significantly enriched for tumor propagating potential as demonstrated using serial transplantations. Over multiple transplantations, the fluorescent color expressed by cells that sorted to the SP, and were enhanced for tumor propagating potential, changed. This suggests that the tumor propagating population is dynamic, rather than being limited to a specific clone. Barcoding in additional experiments showed the same result. This was further verified by genetically expressing different-colored fluorescent markers in SP and non-SP cells. While at a given time the SP expressed one fluorescent color, over serial transplantations, they expressed different colors. Taken together, this shows that the small subpopulation of cells that are enhanced for tumor propagating potential are not derived from the same clone, but a dynamic population that changes over time. Similar to findings in human sarcoma SP cells, RNA sequencing data showed that the murine SP cells were enhanced for Wnt and Notch signaling, even in different clones over time from the same initial population. Blocking these pathways suppresses the tumor propagating potential. Thus, epigenetic changes that activate Wnt and Notch in dynamic cell populations derived from different clones in the initial tumor are likely responsible for enhancing tumor propagating potential and for cells to achieve an SP cell phenotype. Citation Format: Jackie Tang, Kirsch David, Benjamin Alman. Dynamic cell populations are responsible for sarcoma propagating potential [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA13.