Interleukin-6 from Ovarian Mesenchymal Stem Cells Promotes Proliferation, Sphere and Colony Formation and Tumorigenesis of an Ovarian Cancer Cell Line SKOV3

Abstract

The origin of the majority of epithelial ovarian cancers (EOC) is regarded as extraovarian, with the ovary being the secondary site. The aim of this study was to explore the possible role of ovarian mesenchymal stem cells (OvMSCs) and secreted IL-6 in the development of EOC. OvMSCs were derived from normal ovarian stroma. Cell surface markers and differentiation capability were determined. The effects of IL-6 and conditioned medium of OvMSCs on the malignant phenotype of SKOV3 ovarian cancer cells were tested, and the status of STAT3 and ERK phosphorylation was investigated. OvMSCs had similar surface marker profiles as bone marrow mesenchymal stem cells, i.e., CD44 (+), CD90 (+) and CD45 (-), and was readily inducible to osteogenic, adipogenic and chondrogenic differentiation. OvMSCs secreted an extremely high level (>2500 pg/ml) of IL-6. Treatment of SKOV3 cells with conditioned media from OvMSCs increased cell proliferation, tumor sphere formation and anchorage independent growth, and resulted in activation of STAT3 but not ERK. Coinjection of OvMSCs with SKOV3 cell enhanced tumorigenesis in NOD-SCID mice. All of these behaviors were blocked by IL-6 receptor blocking antibody administered in vitro or in vivo. The OvMSCs alone injected into mice had no tumor growth after 3 months. By secreting high levels of IL-6, OvMSCs enhance the proliferation, sphere and colony formation and tumorigenesis of SKOV3 cells.

Keywords: IL-6, proliferation; SKOV3.; STAT3; tumorigenesis.

Figure 1

Cell surface marker profile and differentiation of ovarian mesenchymal stem cells (OvMSCs). (A) In flow cytometry, OvMSCs were positive for CD44, CD73, CD90, CD105 and HLA-ABC, and negative for CD34, CD45 and HLA-DR. (B) OvMSCs were induced for differentiation with appropriate culture medium, as indicated. Induction of adipogenesis (A), osteogenesis (O), and chondrogenesis (C) were demonstrated by staining with Oil Red, Alizarin Red and Alcian blue, respectively, and by the expressions of mRNA of PPARγ, alkaline phosphotase (ALPL), aggrecan (ACAN) and type 2 collagen (COL2A1) (C), respectively, **p<0.01, ***p<0.001.

Figure 2

Enzyme-linked immuno-sorbent assay (ELISA) analysis of interleukin-6 (IL-6) concentration in conditioned medium (CM) of various cell lines, *p<0.05, ***p<0.001. RL95-2: endometrial cancer, HCT116: colon cancer, FF: follicular fluid, SKOV3: ovarian cancer, FE25RAS, FE25: immortalized and cancerous fallopian tube epithelial cells, FTSC: fallopian tube mesenchymal stem cells, OvMSC: ovarian mesenchymal stem cell.

Figure 3

In vivo tumor growth of cancer cells with or without OvMSCs (n=6). SKOV3 cells alone or with OvMSCs were subcutaneously injected to NOD-SCID mice. After 35 days, the mice were sacrificed and the tumors collected. Tumor weight was measured. (A) A significantly larger tumor (**p<0.01) was noted on the co-injected side compared to the SKOV3 only side. IL-6R antibody intraperitoneal injection could inhibit tumor growth after 6 weeks (**p<0.01). Representative tumor tissues were obtained at day 35 after inoculation into NOD-SCID mice in the upper panel. (B) Hematoxylin & Eosin staining of representative section of xenografts with or without OvMSCs. Scale bar: 100 μm.

Figure 4

Transformation phenotype of ovarian cancer cells (SKOV3) treated with OvMSCs conditioned medium (CM) and/or anti-IL-6 antibody. (A) Proliferation of ovarian cancer cells after treatment with OvMSC CM, IL-6 and/or anti-IL-6 neutralizing antibody. (B) Comparison of cell proliferation on day 3. (C) Sphere formation assays of ovarian cancer cells with the same treatment as above. (D) Anchorage independent growth (AIG) of ovarian cancer cells with the same treatment as in (A). *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Activation of STAT3 in response to IL-6 and conditioned medium (CM) of OvMSCs. Expression of activated and total STAT3 (A) or ERK (C) pre- and post-IL-6, IL-6 receptor antibody (IL-6R Ab) and CM treatment in SKOV3 cell lines. SKOV3 cell lysates were treated with 3ng IL-6, 1500ng/ml anti-IL-6R Ab and OvMSC CM for 24 h and Western blot analysis was performed. Experiments were performed three times and blots are representative of one independent experiment. (B) Graphs are represented as the sum of the mean density of p-STAT3/STAT3 normalized against actin from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.