IL-6 receptor expression but not IL-6 expression correlates with poor prognosis in patients with ovarian cancer.

<p>(A) Immunohistochemical staining of tissue microarrays with malignant ovarian tissue sections. Representative areas of four different ovarian cancers stained using an anti-human IL-6R antibody and scored as “Low” or “High”. Placentae complicated with chorioamnionitis were used as a positive control. Arrows indicate membranous staining in trophoblasts. A negative control is nonimmune sera. (B) IL-6 receptor expression correlates with poor prognosis in patients with ovarian cancer. Kaplan-Meier curves of progression free survival (<i>left</i>) and overall survival (<i>right</i>) of ovarian cancer patients treated at Gifu University Hospital (n = 94). (C) Representative areas of four different ovarian cancers stained using an anti-human IL-6 antibody and scored as “Low” or “High”. Placentae complicated with chorioamnionitis were used as a positive control. Arrows indicate cytoplasmic staining in villous mesenchymal cells. A negative control is nonimmune sera. (D) IL-6 expression did not affect the prognosis in patients with ovarian cancer. Kaplan-Meier curves of progression free survival (<i>left</i>) and overall survival (<i>right</i>) of patients. Original magnification, ×200. Scale bar in each panel represents 50 μm.</p

Multivariate analysis for progression free survival of the patients.

<p>HR, hazard ratio;</p><p>95% CI, 95% confidence interval.</p><p>Multivariate analysis for progression free survival of the patients.</p

Expression of IL-6 and IL-6R in ovarian cancer cell lines.

<p>Real-time RT-PCR of IL-6 (A) and IL-6R (B). Total RNA was collected from seven different ovarian cancer cell lines using TRIzol and subjected to real-time RT-PCR. The 2^-ΔΔCT method was used to calculate the relative abundance with respect to GAPDH expression. Relative fold differences with respect to primary ovarian surface epithelium (OSE) are presented. (C) Western Blot. Cell lysates from seven ovarian cancer cells were resolved by SDS-PAGE and immunoblotted with an antibody against IL-6 and IL-6R. β-Actin was used as a loading control. (D) RT-PCR. RNA was collected and the expressions of full-length IL-6R (IL-6R) and soluble IL-6R (sIL-6R) expression in ovarian cancer cell lines were examined. PCR conditions were as described in Material and Methods. (E) ELISA assay of sIL-6R. Seven ovarian cancer cells (1 x 10⁵ each) were plated onto 24-well plates and cultured with 1 ml of serum-free medium for 72 h. Conditioned media were collected and the concentration of human sIL-6R was measured by ELISA. Experiments were repeated three times. n.d.; not detected. (F) Western Blot. Exogenous treatment of IL-6 activates IL-6/IL-6R signaling in ovarian cancer cell lines. SKOV3ip1 cells were stimulated with IL-6 (100 ng/mL) for 30 minutes with or without pretreatment using ranti-IL-6R antibody (1–100 μg/ml) non-immune IgG as control. Cell lysates were collected and equal amount of cell lysates (30 μg) was resolved by 10% SDS-PAGE and immunoblotted with anti–phosphorylated STAT3 (p-STAT3) antibody and anti–phosphorylated p44/42 MAPK (p-ERK1/2) antibody. The membranes were stripped and rehybridized with antibodies detecting the total forms of the protein. Blots are representative of three experiments.</p

Immunohistochemical analyses of IL-6 in high-grade serous ovarian cancer tissues.

<p>Serial sections of stage III high-grade serous ovarian cancer tissues were immunostained with anti-IL-6 antibody (A, C) and anti-CD-68 antibody (B, D). IL-6 was strongly expressed in stroma, while cancer cells little expressed IL-6. (A, B) Sections from a 56 year-old female with stage IIIC high-grade serous ovarian cancer. (C, D) sections from a 63 year-old female with stage IIIC high-grade serous ovarian cancer. CD68 staining identified macrophages. Arrows indicate macrophages. Arrowheads indicate ovarian cancer cells. Original magnification, x100 (upper panels), and x400 (bottom panels). Black bar; 200 μm, red bar; 50 μm.</p

Proposed paracrine mechanism.

<p>CD11b⁺CD14⁺ cells, which are predominantly macrophages, are one of the major sources of IL-6 production in an ovarian tumor microenvironment and promoted ovarian cancer invasion, proliferation and angiogenesis.</p

Exogenous treatment of IL-6 promotes ovarian cancer cell proliferation, invasion and VEGF production.

<p>(A) A matrigel invasion assay was done using a modified Boyden chamber system. 1 x 10⁵ of SKOV3ip1 (<i>left</i>) or RMUS-S (<i>right</i>) cells were placed on the top chamber in serum-free medium and allowed to invade for 72 h. Various concentrations of IL-6 (1–100 ng/ml) or 60 ng/ml of sIL-6R were applied in the bottom chamber as a chemoattractant. 10 μg/ml of anti-IL-6R antibody or non-immune IgG was co-treated. Non-invading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated. Relative numbers of invading cells with respect to the control (no IL-6 treatment) are shown. (B) <i>In vitro</i> cell proliferation assay. 1 x 10⁴ cells of SKOV3ip1 (<i>left</i>) or RMG1 (<i>right</i>) cells were plated in 24-well plates in 10% FBS/DMEM for 24 h and then incubated in serum-free medium in the presence or absence of various concentrations of IL-6 (1–100 ng/ml) with or without anti-IL-6R antibody or non-immune IgG as control for 72 h. Cell proliferation was evaluated by a modified MTS assay. Cell proliferation was expressed as the ratio of the number of viable cells. (C) ELISA assay of VEGF-A. 1 x 10⁵ SKOV3ip1 cells were plated onto 6-well plates and cultured with 2 ml of serum-free medium in the presence or absence of 100 ng/ml of IL-6 for 72 h. Anti-IL-6R antibody or control IgG was co-treated. Conditioned media were collected and the concentration of human VEGF-A was measured by ELISA. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, *; P < 0.05, **; P < 0.01.</p

CD11b⁺CD14⁺ cells from ovarian cancer ascites promote ovarian cancer cell invasion and proliferation <i>via</i> producing IL-6.

<p>(A) The protocol of isolation of CD11b^-, CD11b⁺CD14^- and CD11b⁺CD14⁺ cells using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotech). ELISA assay of IL-6 (B) and sIL-6R (C). 1 x 10⁵ of SKOV3ip1 cells and primary cells indicated in the figure were plated onto 6-well plates and cultured with 2 ml of serum-free medium for 72 h. Conditioned media were collected and the concentrations of human IL-6 (B) as well as sIL-6R (C) were measured by ELISA. Experiments were repeated three times and values are means (±SD) of triplicates. (D) Matrigel invasion assay. 1 x 10⁵ SKOV3ip1 cells were placed on the upper chamber with the same number of primary cells indicated in the figure seeded on the bottom chamber as a chemoattractant, and were allowed to invade for 72 h. The relative number of invading cells when no cells were plated on the bottom chamber was set as 1.0. (E) Anti-IL-6R antibody inhibited ovarian cancer cell invasion induced by CD11b⁺CD14⁺ cells. In this experiment, the co-culture experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4D</a> was repeated with the addition of the 10 μg/ml of anti-IL-6R antibody or non-immune IgG in the bottom chamber. Representative pictures of transwells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">4E</a> (<i>bottom</i>). (F) In vitro cell proliferation assay. 1 x 10⁴ SKOV3ip1 cells were plated in 24-well plates. Thereafter, polycarbonate filters with 1-μm pores were placed onto 24-well plates and the same number of primary cells indicated in the figure were seeded as a stimulant and cells were cultured for 72 h. Cell proliferation was expressed as the ratio of the number of viable cells. (G) Anti-IL-6R antibody inhibited ovarian cancer cell proliferation induced by CD11b⁺CD14⁺ cells. In this experiment, the co-culture experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4F</a> was repeated with the addition of the 10 μg/μl of anti-IL-6R antibody or non-immune IgG in the upper chamber. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, n.d.; not detected, *; P < 0.05, **; P < 0.01.</p

Characteristics of the patients whose ascites were collected and used for analyses.

<p>Characteristics of the patients whose ascites were collected and used for analyses.</p

Characteristics of the patients (n = 94).

<p>Characteristics of the patients (n = 94).</p

Characterization of primary cells from ovarian cancer ascites.

<p>(A) FACS analyses. Ovarian cancer ascites were collected aseptically, and cells were isolated by standard Ficoll-Paque density-gradient. Thereafter, cells were labelled with cell surface markers CD11b (PE, x-axis) and CD14 (APC, y-axis). Cells were divided into 3 groups, CD11b^-CD14^-, CD11b⁺CD14^- and CD11b⁺CD14⁺ cells. (B) The majority of CD11b⁺CD14⁺ cells are M2-polalized macrophages. These three populations of cells were further labeled with cell surface markers CD68 (Alexa Fluor-488, x-axis) and CD206 (eFluor-450, y-axis). 87.8% of CD11b⁺CD14⁺ cells (<i>right</i>) were CD68 and CD206 positive.</p