Macrophage migration inhibitory factor stimulates angiogenic factor expression and correlates with differentiation and lymph node status in patients with esophageal squamous cell carcinoma

Abstract

Objective: The objectives of this study were: 1) to examine the expression of macrophage migration inhibitory factor (MIF) in esophageal squamous cell carcinoma (ESCC); 2) to see if a relationship exists between MIF expression, clinicopathologic features, and long-term prognosis; and 3) to ascertain the possible biologic function of MIF in angiogenesis.

Summary background data: MIF has been linked to fundamental processes such as those controlling cell proliferation, cell survival, angiogenesis, and tumor progression. Its role in ESCC, and the correlation of MIF expression and tumor pathologic features in patients, has not been elucidated.

Methods: The expression of MIF in tumor and nontumor tissues was examined by immunohistochemical staining. Concentrations of MIF, vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in patients' sera and in the supernatant of tumor cells culture were examined by ELISA. Correlations with clinicopathologic factors were made.

Results: In 72 patients with ESCC, intracellular MIF was overexpressed in esophagectomy specimens. The expression of MIF correlated with both tumor differentiation and lymph node status. The median survival in the low-MIF expression group (<50% positively stained cancer cells on immunohistochemistry) and high expression group (>/=50% positively stained cancer cells) was 28.3 months and 15.8 months, respectively (P = 0.03). The 3-year survival rates for the 2 groups were 37.7% and 12.1%, respectively. MIF expression was related to microvessel density; increased MIF serum levels also correlated with higher serum levels of VEGF. In addition, in vitro MIF stimulation of esophageal cancer cell lines induced a dose-dependent increase in VEGF and IL-8 secretion.

Conclusions: These results demonstrate, for the first time, that human esophageal carcinomas express and secrete large amounts of MIF. Through its effects on VEGF and IL-8, MIF may serve as an autocrine factor in angiogenesis and thus play an important role in the pathogenesis of ESCC.

FIGURE 1. Immunohistochemical staining of ESCC. The expression of MIF, VEGF, and CD34 is stained brown. The purple color is counterstain, which indicates cell nuclei. A, MIF in well-differentiated tumor (original magnification ×100). B, MIF in nontumor tissue (original magnification ×100). C, MIF in poorly differentiated tumor (original magnification ×200). D, VEGF in tumor tissue (original magnification ×200). E, MVD in tumor tissue (original magnification ×100).

FIGURE 2. Box plots showing higher tumor MVD in tumors with high MIF expression than in tumors with low MIF expression (P = 0.0001).

FIGURE 3. Kaplan-Meier survival curves of patients in low-MIF expression group versus high-MIF expression group. Low-MIF expression group: <50% positively stained cancer cells; high-MIF expression group: >50% positively stained cancer cells, P = 0.03.

FIGURE 4. Serum concentrations of MIF in patients with ESCC and healthy controls.

FIGURE 5. MIF expression in esophageal tumor cell lines and normal epithelial cells detected by flow cytometry. Cell surface MIF expression (left panel) and intracellular expression (right panel) were evaluated by inmmunofluorescence staining with anti-MIF mAb. MIF was predominantly identified in the cytoplasm of HKESC-1 and HKESC-2 cell lines.

FIGURE 6. Secretion of MIF by esophageal tumor cell lines and normal epithelial cells. Cells were cultured in 24-well plates at 5 × 10⁴ cells for 24 hours. The culture supernatants were collected, and MIF was quantified by ELISA. Culture medium alone was used as control. Values represent the mean of 3 experiments conducted in triplicate and are expressed as mean ± SD (**P < 0.001).

FIGURE 7. MIF induces the secretion of VEGF and IL-8 by esophageal tumor cell lines and normal epithelial cells. Cells were cultured in 24-well plates at 5 × 10⁴ cells. The cells were treated with recombinant human MIF at concentrations as indicated for 24 hours. The culture supernatants were collected and IL-8 and VEGF were measured by ELISA. Values represent the mean of 3 experiments conducted in triplicate and are expressed as mean ± SD.

FIGURE 8. Effect of anti-MIF neutralizing mAb on production of IL-8 (A) and VEGF (B) by ESCC cell lines HKESC-1 and HKESC-2. Cells were cultured in 24-well plates at 5 × 10⁴ cells. The cells were treated with anti-MIF neutralizing mAb (100 μg/mL) or isotype control mAb (100 μg/mL) for 24 hours. IL-8 and VEGF in the supernatants were measured by ELISA (mean ± SD, n = 3). *P < 0.05.