(P 120) THE FEASIBILITY OF CD47-BASED IMMUNOTHERAPY FOR OSTEOSARCOMA

Objective: Macrophages account for the majority of immune cells infiltrated in sarcoma tissues. CD47 is expressed on many cancer cells and acts as a macrophage immune checkpoint by interacting with SIRPα of the macrophage surface. In this study, we evaluated the feasibility of CD47 as a potential candidate for immunotherapy of osteosarcoma.

Methods: CD47 immunohistochemical staining was performed in 24 biopsy samples obtained from patients younger than 20 years, and the relationships between CD47 expression and clinicopathologic characteristics were evaluated. For KHOS/NP and MG-63 osteosarcoma cells, CD47 protein expression was analyzed using western blotting. From fresh peripheral blood obtained from healthy adult volunteers, monocytes were isolated using human CD14 magnetic microbeads, and cultured with macrophage colony-stimulating factor to differentiate into macrophages. KHOS/NP and MG-63 osteosarcoma cells were labeled with CFSE dyes and pre-treated with CD47 antibody (B6H12) or isotype control and then co-cultured with differentiated macrophages. The efficacy of the CD47 antibody on the phagocytic activity of macrophages was evaluated using flow cytometry.

Results: Of the 24 biopsy samples of osteosarcoma, CD47 protein was expressed in 5 samples. Positive staining for CD47 protein was related to the presence of metastasis at diagnosis (P=0.04). Patients with tumors stained positive for CD47 protein tended to be older than those with tumors stained negative (16.56.2 years vs. 15.73.0 years, P=0.07). However, sex, tumor size, and histologic response to preoperative chemotherapy were not associated with CD47 protein expression. Western blot showed that CD47 protein is expressed in both KHOS/NP and MG-63 osteosarcoma cells, with a slightly higher expression level in KHOS/NP cells. CD47 antibody pre-treatment did not influence the proliferation and apoptosis of two osteosarcoma cells. Peripheral blood-derived CD14-positive monocytes were effectively differentiated into macrophages. The phagocytic activity of differentiated macrophages on KHOS/NP osteosarcoma cells was significantly higher in cells pretreated with CD47 antibody, compared to the control. In contrast, the phagocytic activity on MG-63 osteosarcoma cells tended to increase, but there was no significant difference.

Conclusion: Our study results suggest the possibility of CD47 as a new immunotherapeutic target of osteosarcoma. To increase the anti-osteosarcoma efficacy of the CD47 antibody, further studies are necessary to couple the macrophage phagocytosis and effector T cell function.