Expression of macrophage migration inhibitory factor relates to survival in high-grade osteosarcoma

Abstract

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is implicated in many aspects of tumor progression, including cell proliferation, invasion, and angiogenesis. We asked if MIF expression predicts survival and if it is associated with angiogenesis and cell invasion in osteosarcoma. We performed immunohistochemistry for MIF expression in prechemotherapy biopsy specimens of 58 patients with osteosarcoma. To investigate the role of MIF in angiogenesis, microvessel density was measured and compared with MIF expression. We also treated osteosarcoma cell lines (U2-OS and MG63) with MIF and measured vascular endothelial growth factor, a potent proangiogenic factor, by enzyme-linked immunosorbent assay. To study the role of MIF in cell invasion, Boyden chamber assay was performed after knockdown of MIF by short interfering RNA. MIF independently predicted overall survival and metastasis-free survival. MIF expression correlated with microvessel density and induced a dose-dependent increase in vascular endothelial growth factor. Knockdown of MIF by short interfering RNA resulted in decreased cell invasion. These results suggest MIF could serve as a prognostic marker and a potential therapeutic target for osteosarcoma.

Level of evidence: Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.

Fig. 1A–C

Representative sections show expression of migration inhibitory factor (MIF) by immunohistochemistry. (A) Positive control (hepatocellular carcinoma) shows strong cytoplasmic expression of MIF (Stain, diaminobenzidine and Meyer’s hematoxylin; original magnification, ×400). (B) A representative case of high MIF expression is shown (Stain, diaminobenzidine and Meyer’s hematoxylin; original magnification, ×200). (C) A representative case of low MIF expression is shown (Stain, diaminobenzidine and Meyer’s hematoxylin; original magnification, ×200).

Fig. 2A–B

(A) A Kaplan-Meier overall survival curve is illustrated according to the level of migration inhibitory factor (MIF) expression. Patients with high MIF-expressing tumors had worse (p < 0.0001) overall survival than those with low MIF-expressing tumors. (B) A Kaplan-Meier metastasis-free survival curve is illustrated according to the level of MIF expression. There was a trend (p = 0.018) toward increased metastasis in patients with high MIF expression.

Fig. 3A–B

(A) A representative immunohistochemical staining of osteosarcoma specimen with anti-CD34 antibodies for microvessel density (MVD) quantification is shown (Stain, diaminobenzidine and Meyer’s hematoxylin; original magnification, ×100). (B) Specimens with high migration inhibitory factor (MIF) expression had higher (p = 0.016) MVD than those with low MIF expression.

Fig. 4A–B

Migration inhibitory factor (MIF) induced the secretion of vascular endothelial growth factor (VEGF) by U2-OS (A) and MG63 (B) cells in a dose-dependent manner. *p < 0.05 when compared with 0 ng/mL of MIF.

Fig. 5A–B

(A) Migration inhibitory factor (MIF) short interfering RNA (siRNA) suppressed MIF expression in a dose-dependent manner. Representative result of three experiments is shown. (B) Knockdown by MIF siRNA decreased the migration of U2-OS (p = 0.03) and MG63 cells (p = 0.04).