One of the earliest metastatic events in human ovarian cancer, tumor spread to the omentum, may be influenced by expression of interleukin 6 (IL6) and its cognate receptor (IL6Rα). Previous reports have shown that IL6 and IL6Rα expression is elevated in the serum and ascites of patients with ovarian cancer and that this can influence in vitro processes such as cell survival, proliferation and migration. In this study, overexpression of IL6Rα, and to a lesser extent IL6, enhanced tumor growth on the omentum. Moreover, adherence to plastic and to peritoneal extracellular matrix components was enhanced in tumor cells overexpressing IL6 or IL6Rα. Host production of IL6 and IL6Rα was also sufficient to influence tumor adherence to the omentum. Expression of LY75/CD205/DEC205, a collagen-binding mannose family receptor, was directly influenced by IL6Rα expression. Blocking LY75 with antibody reduced the adherence of tumor cells overexpressing IL6Rα to matrices in vitro and to the omentum. The association between IL6Rα expression and LY75 expression has not been previously reported, and the promotion of cellular adherence is a novel role for LY75. These studies indicate that overexpression of LY75 may be an additional mechanism by which IL6 signaling influences the progression of ovarian cancer, and suggests that blocking LY75 could be a valuable clinical strategy for reducing the early metastasis of ovarian cancer.
Supplemental Fig. 1Altered expression of IL6 and IL6Rα in Skov-3 cells. Q-PCR confirms altered mRNA expression in 2 variant Skov-3 cell lines (A and B) with overexpression (A) or knockdown shRNA (B) constructs for human IL6 (left panels) or IL6Rα (right panels). Data are expressed as fold change compared with cells expressing empty (control) vector (CV) or a scrambled sequence (pScramble). (C) Increased protein expression of IL6Rα was detected by ELISA of conditioned media collected from Skov-3 (1000 cells/well; 7 days). *p < 0.05. (D) Representative in vivo fluorescent images of omenta 48 h after injection of ES-2 cells. Arrows indicate the omentum (TIFF 6451 kb)
Supplemental Fig. 2IL6 and IL6Rα expression increase proliferative activity but not cell migration. (A) Expression of IL6 or IL6Rα was associated with increased proliferation/survival as assessed by MTT assay and (B) increased anchorage-independent growth on soft agar. (C) There were no differences in anoikis rates after 24 hours incubation on polyHEMA-coated plates among control cells and cells with altered expression of IL6 or IL6Rα. (D) The migration of cells through tissue culture inserts with 8 μm pores was not different for cells expressing control vector or IL6 or IL6Rα overexpression vectors. *p < 0.05 (TIFF 10627 kb)
Supplemental Fig. 3Addition of exogenous IL6 and IL6Rα increases adherence of tumor cells. Exogenous soluble IL6 (ng/mL) and IL6Rα (μg/mL) were added at the concentrations indicated to ES-2 cells expressing empty vector (CV) or IL6 or IL6Rα overexpression vectors, and adherence to plastic was assessed after 30 minutes (TIFF 2044 kb)