Abstract 2511: Metastatic lung colonization by osteosarcoma requires cooperation between tumor subpopulations with distinct phenotypes

Four decades of intense effort have not improved outcomes for patients with osteosarcoma. Even though 35-40% of young people so treated still die from the disease, usually from complications related to lung metastasis, our standard of care remains unchanged since 1982. While effective metastasis-targeting interventions could transform clinical care, our incomplete understanding of the mechanisms driving tumor spread have hampered efforts to develop rational, targeted therapeutic interventions. Working to address these gaps, our lab previously showed that concurrent inhibition of IL6 and CXCL8 could prevent metastases in up to 85% of osteosarcoma-bearing mice, though the mechanisms remain unclear. Seeking to understand the mechanism, we generated single-cell RNA-seq datasets from the lungs of osteosarcoma bearing mice collected at multiple time points during lung colonization. We employed niche labeling technology to isolate both tumor and tumor-associated cells and generated single cell transcriptomic datasets from the isolated cells. We used this data to perform inter-cellular signaling analyses and to characterize changes occurring in the composition and phenotype of cells occupying the metastatic niche over developmental time. Immunofluorescent staining of both murine and human metastatic lesions confirmed key findings from the scRNA-seq data. We found that a small subpopulation of osteosarcoma cells triggers an IL1-IL6 paracrine loop upon interaction with lung epithelial cells, driving IL6/CXCL8 production within the early metastatic niche. This subset of tumor cells is hypo-proliferative, hyper-secretory, p21+, and survives the stresses of dissemination to the lung. We call these metastasis initiating cells "anchor" cells and their rapidly proliferative counterparts "growth cells." Very early metastases show near-complete enrichment for anchor-phenotype osteosarcoma cells, while growth cells become dominant as lesions expand. Anchor derived signals can recruit circulating growth cells into a developing niche. However, it remains unclear whether this or state changes in anchor cell progeny are the primary mechanism for the appearance of growth cells within a lesion. Intriguingly, anchor cells appear resistant to chemotherapy, providing a potential mechanism for relapse after treatment. Taken together, the data suggest that the establishment of lung metastases arises through cooperation among tumor cells with distinct phenotypes. Anchor cells survive th