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. 2014 Jan 21;110(2):469-78.
doi: 10.1038/bjc.2013.748. Epub 2013 Dec 17.

Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction

T Nagasaki  1 , M Hara  1 , H Nakanishi  2 , H Takahashi  1 , M Sato  1 , H Takeyama  1
Affiliations

Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction

T Nagasaki et al. Br J Cancer. .

Abstract

Background: Interleukin-6 (IL-6) has an important role in cancer progression, and high levels of plasma IL-6 are correlated with a poor prognosis in a variety of cancers. It has also been reported that tumour stromal fibroblasts are necessary for steps in cancer progression, such as angiogenesis. There have been few reports of a correlation between fibroblast actions and IL-6 levels. In this study, we examined the correlation between cancer stromal fibroblasts and IL-6 and the utility of IL-6 as a therapeutic target in human colon cancer.

Methods: The expression levels of IL-6 and VEGF of fibroblasts and cancer cell lines were evaluated using real-time PCR and ELISA. The anti-angiogenic effect of inhibiting IL-6 signalling was measured in an angiogenesis model and animal experiment.

Results: We demonstrate that stromal fibroblasts isolated from colon cancer produced significant amounts of IL-6 and that colon cancer cells enhanced IL-6 production by stromal fibroblasts. Moreover, IL-6 enhanced VEGF production by fibroblasts, thereby inducing angiogenesis. In vivo, anti-IL6 receptor antibody targeting stromal tissue showed greater anti-tumour activity than did anti-IL6 receptor antibody targeting xenografted cancer cells.

Conclusion: Cancer stromal fibroblasts were an important source of IL-6 in colon cancer. IL-6 produced by activated fibroblasts induced tumour angiogenesis by stimulating adjacent stromal fibroblasts. The relationship between IL-6 and stromal fibroblasts offers new approaches to cancer therapy.

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Figures

Figure 1
Figure 1
Localisation of IL-6 and α-SMA in the stroma of colon cancer tissues and normal colonic mucosa as revealed by double immunofluorescent staining of fresh frozen tissues. (A and B) In the tumour tissue, both IL-6 and α-SMA were largely colocalised in the cancer stromal compartment, and no significant expression by the tumour cells was observed. (C) Merged image demonstrates coexpression of both proteins mainly in the stromal fibroblast-like cells. (D–F) In normal colonic mucosa, IL-6 and α-SMA expression was weaker than tumour tissue, but the still weakly observed expression colocalised in the pericryptal and stromal fibroblast-like cells. (G–I) In the tumour tissue, no significant CD45 expression was observed in the tumour stoma. Bars=100 μm. S=stroma, C=crypt, T=tumour tissue.
Figure 2
Figure 2
Isolated fibroblasts. (A) All cultivated cells were positive for expression of CD90 and vimentin, whereas all were negative for cytokeratin. IL-6 expression by cancer cells (HT29, COLM5), fibroblasts isolated from normal colon (NFs) and those from cancer (CAFs). (B) Immunofluorescent staining of IL-6 and α-SMA. Whereas CAFs stained for both IL-6 and α-SMA, NFs did not stain for either. (C) IL6 mRNA levels measured with RT–PCR (relative to NFs as a control). IL6 mRNA expression in CAFs was 11.8-fold higher relative to that in NFs. Compared with fibroblasts, IL6 mRNA expression in cancer cells was <1/100 as great (COLM5: 0.01; HT29: not detected. **P<0.01). (D) ELISA assay of IL-6 protein secretion of cancer cell lines (HT-29 and COLM5) and various fibroblasts. Whereas IL-6 secretion from cancer cell lines was barely detected, that from fibroblasts was significantly higher. Especially, IL-6 secretion from CAFs was 3.5-fold greater than NFs.
Figure 3
Figure 3
IL-6 secretion in response to various stimuli. IL6 mRNA expression levels were calculated relative to fibroblasts without any stimunlation. (A–C) Co-culture of fibroblasts with cancer cells or stimulation by LPS increased the expression of IL6 mRNA from fibroblasts significantly more than control. However, cancer cell lines were not induced by conditions that induced fibroblasts to express IL-6 (**P<0.01). (D–F) IL-6 protein levels in the supernatants measured with ELISA. As observed with mRNA, IL-6 protein levels secreted from fibroblasts cocultured with cancer cells were much higher than DFs or NFs alone. Cancer cell lines barely secreted IL-6 protein. (G and H) IL-1β and TNF-α significantly increased mRNA expression and IL-6 production of DFs and NF.
Figure 4
Figure 4
The expression and secretion of VEGF induced by IL-6. Expression levels of VEGF mRNA 24 h after treatment and VEGF protein levels 48 h after treatment with IL-6 and MRA were measured using RT–PCR (relative to control) and ELISA, respectively. VEGF mRNA levels in (A) CAFs and (B) NFs were increased by stimulation with IL-6 and decreased by treatment with MRA. IL-6 (1 ng ml−1) induced VEGF mRNA expression in both CAFs and NFs. MRA inhibited IL-6-induced VEGF expression. (D and E) The increases in VEGF secretion mediated by IL-6 stimularion were more remarkable in NFs than CAFs. IL-6 induced VEGF secretion by both dermal and colon fibroblasts. MRA inhibited VEGF secretion of fibroblasts. (C and F) IL-6 scarcely induced colon cancer cell lines to express VEGF (**P<0.01).
Figure 5
Figure 5
Angiogenic response to IL-6 stimulation. Stimulation of angiogenesis by IL-6 was estimated using an HUVEC plus colon fibroblast model (see Materials and Methods). Both (A) tubular area and (B) the number of joints were significantly increased in the presence of IL-6 (P<0.05). These increases were inhibited by the addition of anti-IL-6 receptor antibody, MRA, to the control level. The increases were not significantly diffferent between 1 and 20 ng ml−1 IL-6. (C) Representative images of immunohistochemistry 9 days after treatment with IL-6 and MRA.
Figure 6
Figure 6
Anti-tumour effect of anti-IL-6 receptor antibody on xenografted colon cancer cell line. (A) Xenografted tumour volume was measured after various treatments (control, MRA: anti-human IL-6 receptor antibody, MR16-1: anti-mouse IL-6 receptor antibody, and combination). Harvested tumours after 5 weeks of treatment were examined. (B) Harvested tumours weights were significantly less in the groups with MR16-1 (MR16-1 and the combination group) (P<0.05). (C) VEGF mRNA expression and (D and E) micro-vessel density in harvested tumours were significantly less in the MR16-1 group and combination group. However, those in the MRA group were not significantly different from those of the control group (*P<0.05, **P<0.05).
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