Osteosarcoma Exosome Priming of Primary Human Lung Fibroblasts Induces an Immune Modulatory and Protumorigenic Phenotype

ABSTRACT: Osteosarcoma (OS) is the most common malignant bone cancer. Thirty to forty percent of all patients with OS 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 tumor microenvironment that promote immune suppression, drug resistance, and tumor cell survival. Prior work shows tumor cells can co-opt fibroblasts to a protumorigenic phenotype via exosome-mediated intercellular communication. Currently, the mechanisms by which OS exosomes modulate resident lung fibroblast (LF) function have not been evaluated. To investigate this, we isolated exosomes from a panel of six OS cell lines. We assessed the uptake and response of human donor–derived primary LFs (n = 4) to OS exosome treatment in vitro via flow cytometry, confocal fluorescent microscopy, proliferation assays, phosphokinase array, multiplex cytokine analysis, and RNA sequencing. We observed that LFs efficiently take up OS exosomes, which are associated with the induction of MAPK pathway activation, fibroblast proliferation, and significantly enhanced secretion of IL-6, CXCL8, and CCL2 compared with untreated LFs. RNA sequencing 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 LF–OS coculture model, exosome-educated LFs conferred significantly increased OS cell survival and proliferation compared with untreated fibroblasts. Together, these data suggest that the OS-derived exosomes can induce a hallmark cancer-associated fibroblast–like inflammatory phenotype in LFs, providing valuable insights into mechanisms that may promote recurrent OS lung metastasis. SIGNIFICANCE: These findings provide a critical first step in characterizing the capacity of OS-derived exosomes to reprogram primary LFs toward a tumor-promoting inflammatory phenotype in vitro, offering novel molecular targets for the modulation of fibroblasts in the lung microenvironment as potential therapeutic strategies to prevent OS metastasis.