The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time

Abstract

Background: Tumor-associated macrophages (TAMs) play an important role in growth, progression and metastasis of tumors. In non-small cell lung cancer (NSCLC), TAMs' anti-tumor or pro-tumor role is not determined. Macrophages are polarized into M1 (with anti-tumor function) and M2 (with pro-tumor function) forms. This study was conducted to determine whether the M1 and M2 macrophage densities in NSCLC are associated with patient's survival time.

Methods: Fifty patients with an average of 1-year survival (short survival group) and 50 patients with an average of 5-year survival (long survival group) were included in this retrospective study. Paraffin-embedded NSCLC specimens and their clinicopathological data including up to 8-year follow-up information were used. Immunohistochemical double-staining of CD68/HLA-DR (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded fashion. The M1 and M2 macrophage densities in the tumor islets, stroma, or islets and stroma were determined using computer-aided microscopy. Correlation of the macrophage densities and patient's survival time was analyzed using the Statistical Package for the Social Sciences.

Results: Approximately 70% of TAMs were M2 macrophages and the remaining 30% were M1 macrophages in NSCLC. The M2 macrophage densities (approximately 78 to 113 per mm2) in the tumor islets, stroma, or islets and stroma were not significantly different between the long survival and short survival groups. The M1 macrophage densities in the tumor islets (approximately 70/mm2) and stroma (approximately 34/mm2) of the long survival group were significantly higher than the M1 macrophage densities in the tumor islets (approximately 7/mm2) and stroma (13/mm2) of the short survival group (P < 0.001 and P < 0.05, respectively). The M2 macrophage densities were not associated with patient's survival time. The M1 macrophage densities in the tumor islets, stroma, or islets and stroma were positively associated with patient's survival time in a univariate analysis (P < 0.01 or 0.001). In a multivariate Cox proportional hazards analysis, the M1 macrophage density in the tumor islets was an independent predictor of patient's survival time.

Conclusions: The M1 macrophage density in the tumor islets is an independent predictor of survival time in NSCLC patients.

Figure 1

Immunohistochemical detection of macrophages in NSCLC tumor islets. a) The M1 macrophage double stained with the anti-CD68 (red) and anti-HLA-DR (black-purple) antibodies (arrow). b) The M2 macrophages double stained with the anti-CD68 (red) and anti-CD163 (black-purple) antibodies (arrow). c) The M1 macrophage marker HLA-DR stained black-purple (arrowhead) and the M2 macrophage marker CD163 stained red (arrow). Original magnification, × 1000 for a & b, and × 400 for c.

Figure 2

Scatter plots of the macrophage density versus patient's survival time. r_s represents the Spearman's rank correlation coefficient.

Figure 3

Kaplan-Meier survival curves. One hundred patients (N = 100) are divided into two groups with the macrophage densities above or below the median value. P values are obtained in comparisons of the two groups through a univariate analysis using the log-rank test. [M1] and [M2] represent the M1 macrophage density and M2 macrophage density, respectively.