Effect of osteosarcoma-derived exosomes on human primary lung fibroblasts

Osteosarcoma (OS) is the most common malignant bone cancer. 30-40% of all OS patients develop tumor recurrence, almost exclusively in the form of lung metastasis, which is associated with a dismal 20% 5-year survival rate. Cancer-associated fibroblasts are a critical cell type within the lung TME, which promote immune suppression, drug resistance, and tumor cell survival. Prior work shows tumor cells can co-opt fibroblasts to a pro-tumorigenic phenotype via exosome mediated intercellular communication. Currently, the mechanisms by which OS exosomes modulate resident lung fibroblast function has not been evaluated. To investigate this, we isolated exosomes from a panel of 6 OS cell lines. We assessed the uptake and response of human donor-derived primary lung fibroblasts (LFs; n=4) to OS exosome treatment in vitro via flow cytometry, confocal fluorescent microscopy, proliferation assays, phospho-kinase array, multiplex cytokine analysis and RNA-sequencing. We observed that LFs efficiently take up OS exosomes, which is associated with induction of MAPK pathway activation, fibroblast proliferation and significantly enhanced secretion of IL-6, CXCL8 and CCL2 compared to untreated LFs. RNA-seq of exosome treated LFs confirmed these responses and revealed significant enrichment of pathways related to cytokine secretion, proliferation, immune cell chemotaxis, migration, proinflammatory and profibrotic mediators. Finally, in an exosome-educated lung fibroblast-OS co-culture model, exosome educated LFs conferred significantly increased OS cell survival and proliferation as compared to untreated fibroblasts. Together, these data suggest OS-derived exosomes can induce a hallmark, CAF-like inflammatory phenotype in lung fibroblasts providing valuable insights into mechanisms that may promote recurrent OS lung metastasis.