Abstract 5457: Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization
Authors: Gross Amy C. , Reinecke James B. , Saraf Amanda , Hinckley John , Cam Maren , Cannon Matthew V. , Vatelle Sophia , Roberts Ryan D. 
Published: March 2024

Abstract
Introduction: The development of lung metastasis drives the poor outcomes experienced by children and young adults with osteosarcoma. The mechanisms that facilitate metastatic colonization remain poorly understood. We previously determined that IL6 and CXCL8 are two osteosarcoma derived cytokines that are required for metastasis, though roles played by these cytokines have not been identified.
Methods: To explore the interactions that drive cytokine expression, the role these factors play in tumor cell trafficking, and the mechanisms that facilitate metastatic niche development, we utilized transwell and co-culture systems together with a panel of metastatic osteosarcoma mouse models that allowed tumor cell manipulation via lentiviruses, CRISPR-based gene editing, and pre-clinical therapeutic evaluation approaches.
Results: While metastatic osteosarcoma cells produced small amounts of IL6 and CXCL8 at baseline, lung epithelial cells markedly upregulated IL6/CXCL8 production. Ligand-receptor analysis utilizing single-cell transcriptomic data identified IL1 as an epithelial derived ligand that drives tumor IL6/CXCL8 production. Interestingly, IL6/CXCL8 production was limited to a small subset of hypo-proliferative osteosarcoma cells that have metastasis-initiating capacity. Utilizing CRISPR-mediated ablation of IL1R1 signaling within osteosarcoma cells and pharmacological inhibition with recombinant IL1R antagonist (anakinra), we elucidated that formation of pulmonary lesions in multiple mouse models of metastatic osteosarcoma required IL1 signaling. Recent work from our lab has demonstrated that osteosarcoma metastases are defined by marked phenotypic heterogeneity, though the mechanisms by which metastatic heterogeneity occurred were unclear. We hypothesized that hypo-proliferative metastasis-initiating cells may recruit circulating cells with higher proliferative capacity to establish phenotypic heterogeneity. Transwell migration assays showed that tumor cells induced the chemotactic recruitment of other tumor cells in an IL6/CXCL8-dependent manner. To test this hypothesis in vivo, we injected mice with GFP-tagged osteosarcoma cells, then injected the same mice 14 days later with RFP-labeled tumor cells from the same culture. Lungs harvested 14 days later exhibited RFP-dominant lesions surrounding a small core of GFP-labeled cells.
Conclusions: Collectively, these data suggest that cytokines expressed by a subgroup of hypo-proliferative tumor cells upon interaction with lung epithelium drive the trafficking of highly proliferative circulating tumor cells into developing metastatic lesions and that disrupting these signaling networks can prevent metastatic colonization.
Citation Format: Amy C. Gross, James B. Reinecke, Amanda Saraf, John Hinckley, Maren Cam, Matthew V. Cannon, Sophia Vatelle, Ryan D. Roberts. Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5457.