Abstract PO-012: Metastatic bottleneck in osteosarcoma selects for certain clonal populations, while metastatic lesions exhibit diverse phenotypes

This study focuses on identifying if the metastatic niche in osteosarcoma (OS) enriches for cells that have a survival advantage either from pre-defined genetic programs or adaptive responses to environmental cues. OS patients who develop metastases experience a dismal survival rate that is lower than twenty percent, a statistic that remains unchanged despite four decades of efforts to improve these outcomes. While therapies that treat metastasis could dramatically improve outcomes, we understand too little about the mechanisms of lung colonization to propose any rational intervention. To simultaneously evaluate changes in clonal diversity and phenotype during the metastatic bottleneck, cell by cell, we combined prospective lineage tracing with single cell transcriptomics in several murine models of metastatic OS. Single cell suspensions of metastatic tumor tissues were processed for single-cell RNA sequencing (scRNA-Seq). For lineage tracing studies, OS cells were virally-transduced to contain a high diversity, heritable and scRNA-Seq compatible lineage tag before inoculating mice. Single-cell libraries were processed bioinformatically to overlay phenotype and lineage information for each cell. Our findings support the following conclusions: First, that the metastatic bottleneck selects for certain clones with increased colonizing capacity, but this selection does not narrow the overall phenotypic diversity of those cells–clones of many different phenotypes can be found within the metastases. Second, the metastatic lung enriches for certain features that are conserved across different cell line and PDX models of metastatic OS. Gene set enrichment analysis of genes enriched within the surviving clones identified novel activated signaling pathways and upstream regulators that appear critical for metastatic progression. This signature includes activated IL6, TGF-β, PDGF signaling, response to a previously unidentified upstream regulator, IL-1β, and the acquisition of enhanced glycolytic activity. Whether each lesion becomes colonized by a phenotypically-distinct clone giving rise to phenotypically-diverse lesions or whether cooperation between phenotypically-distinct clones is required to effect colonization remains a point of active study. In summary, metastatic colonization of the lung by OS involves the selection of phenotypically-diverse tumor cell clones that share characteristics that engender fitness within the lung microenvironment. Citation Format: Sanjana Rajan, Maren Cam, Amy Gross, James Reinecke, Cenny Taslim, Ryan Roberts. Metastatic bottleneck in osteosarcoma selects for certain clonal populations, while metastatic lesions exhibit diverse phenotypes [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-012.