IL-6 and IL-10 are associated with good prognosis in early stage invasive breast cancer patients

Abstract

Macrophage-associated cytokines play an important role in cancer metastasis; however, the functions of interleukins (IL) 6 and 10 in breast cancer (BC) progression and metastasis are not clear. In this study the roles of IL-6/IL-10 in regulating vascular invasion and their prognostic significance in BC are investigated. MDA-MB-231 and MCF-7 migration (± IL-6 or IL-10) was assessed by scratch wound assay. Cancer cell adhesion to IL-6/IL-10 stimulated blood and lymphatic endothelial cells (EC) was investigated. Expression of IL-6 /IL-10 was assessed using immunohistochemistry in an annotated cohort of early stage BC (n = 1380) and associations with clinicopathological variables and clinical outcome evaluated. IL-6 did not alter BC cell migration however a dose-dependent inhibition in MDA-MB-231 migration with IL-10 treatment was observed (P = 0.03). BC cells were more adhesive to blood vs lymphatic EC, however, IL-6/IL-10 had no effect on adhesion patterns. High expression of IL-6/IL-10 was associated with clinicopathological criteria (e.g. hormone receptor status, all P < 0.05), improved disease-free survival (DFS; P < 0.05) and improved BC-specific survival (BCSS; only IL-6, P = 0.017). However, neither IL-6 nor IL-10 expression were independent prognostic factors from multivariate analysis. In BC subgroups, IL-6 and IL-10 were good prognosticators in terms of DFS in non-basal, non-triple-negative (non-TN), ER-positive, PgR-positive (only IL-10), and Her-2-negative (only IL-6) BC (all P < 0.05). IL-6 was associated with improved BCSS in non-basal, ER-positive and non-TN BC (all P < 0.05).

Keywords: Breast cancer; IL-10; IL-6; Macrophage-associated cytokines; Metastasis.

Fig. 1

Effect of recombinant human IL-6 or IL-10 stimulation on breast cancer cell migration rate. IL-6 treatment caused a slight decrease in MDA-MB-231 (a) and MCF-7 (b) migration after 24 h treatment; however, the reduction in migration did not reach statistical significance. IL-10 (15 ng/mL) was associated with a significant reduction in MDA-MB-231 migration (P = 0.03) (c), but has no significant effect on MCF-7 migration (d). Data are presented as mean ± SD of three independent experiments each carried out in triplicate, and P values evaluated by paired sample t test (asterisk represent a significant P value)

Fig. 2

Effect of IL-6 and IL-10 on endothelial adhesion patterns of MDA-MB-231 and MCF-7. IL-6 or IL-10 stimulation of blood (hMEC-1) and lymphatic (hTERT-LEC) endothelial cells did not significantly alter adhesion patterns compared to the unstimulated controls. a MDA-MB-231 and b MCF-7 adhesion to IL-6 stimulated endothelial cells, c MDA-MB-231 and d MCF-7 adhesion to IL-10 stimulated endothelia. Data represent the mean of adhered cells ± SD of three independent experiments, each carried out in duplicate (n = 6)

Fig. 3

IL-6 and IL-10 expression in breast cancer and stromal cells. Representative images of tumour staining with IL-6 (a–c) and IL-10 (d–f). Staining pattern: a, d weak, b, e moderate, and c, f strong. g, h, j, k Representative images of specificity tests of IL-6 and IL-10 antibodies, respectively: BC staining with IL-6 antibody (g) or IL-6 antibody blocked overnight with 1 µg of rIL-6 (h); BC staining with IL-10 antibody alone (j) or IL-10 antibody blocked overnight with 2 µg of rIL-10 (k). Examples of stromal expression of IL-6 and IL-10 are shown in i, l, respectively (black arrows). Photomicrographs: a–f; × 100 and g–l × 200 magnification; inset boxes at × 200 magnification; scale bars 100 µm

Fig. 4

Kaplan–Meier analysis of association between IL-6 and IL-10 expression with breast cancer prognosis. High IL-6 expression is significantly associated with improved disease-free survival (P = 0.007, a) and improved breast cancer-specific survival (P = 0.017, b). High IL-10 expression is significantly associated with improved disease-free survival (P = 0.027, c) but not breast cancer-specific survival (P = 0.150, d). Significance was determined using the log-rank test. Black represents high expression and grey represents low expression of the cytokine