The Contribution of Interleukin-34 in Inflammation and Osteosarcoma

Interleukin-34 is a cytokine sharing functional similarities with the macrophage-colony stimulating factor (M-CSF). It is responsible for the proliferation/differentiation/survival of myeloid cells. Upregulation of IL-34 contributes to disease progression and growth of osteosarcoma. IL-34 is therefore a novel prognostic biomarker, and a target for therapeutic intervention of osteosarcoma. This study aimed to investigate the role of IL-34 and its signalling/communication with the tumour stroma via extracellular vesicles, and in development and function using a zebrafish model. To determine the therapeutic effect of IL-34 targeting in osteosarcoma murine pre-clinical models where used. METHODS: Extracellular vesicles were isolated from osteosarcoma cells and mesenchymal stem cells. Proliferation, differentiation of lineage abilities and protein contents were assessed. In the second part of the study, anti-IL-34 monoclonal blocking antibodies were used in xenograft and allograft murine models, at 4mg/kg and administered (i.p) three times weekly. The effects on tumour growth and histological markers were investigated. We also evaluated the potential therapeutic benefit of combining the IL-34 blocking agent, in combination with doxorubicin as a combination treatment. Finally, using CRISPR/Cas9 system a loss of function model of IL34 was developed. Expression of the cytokine was determined by RT-qPCR and in-situ hybridization, while tail-fin injury assays were used characterize the expression of IL-34 in response to inflammation. RESULTS: Exosomes from osteosarcoma cells induced the commitment of mesenchymal stem cells towards adipogenesis whilst exosomes isolated from mesenchymal stem cells induced the proliferation of osteosarcoma cells indicating a tumour supportive role. In murine models, administration of blocking IL-34 antibody, inhibited tumour progression in both syngeneic and xenogeneic models. The treatment had no effect on bone associated remodelling after analysis by microCT. IHC analysis showed an increase in F4/80 infiltrates, and a tendency towards lower vascularisation as marked by CD31. CRISPR/Cas9 created a stable loss of function zebrafish mutant line deficient of IL-34. Investigation of IL-34 expression revealed low expression of the cytokine in larval development, that becomes upregulated in response to inflammation. A decrease in the number of macrophages in IL-34 morphants was also seen. CONCLUSION: This study represents the first characterization of IL-34 function and expression in zebrafish by a loss of function model. In osteosarcoma, the inhibition of IL-34 specific blocking antibodies demonstrates that the therapeutic benefit to abrogate IL-34.