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Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis.

Yin SY, Jian FY, Chen YH, Chien SC, Hsieh MC, Hsiao PW, Lee WH, Kuo YH, Yang NS - Nat Commun (2016)

Bottom Line: Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression.Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel.Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression. Here we investigate the role of IL-25, an endogenous anticancer factor secreted from TAFs, in suppression of mouse 4T1 mammary tumour metastasis. We show that a synthetic dihydrobenzofuran lignan (Q2-3), the dimerization product of plant caffeic acid methyl ester, suppresses 4T1 metastasis by increasing fibroblastic IL-25 activity. The secretion of IL-25 from treated human or mouse fibroblasts is enhanced in vitro, and this activity confers a strong suppressive effect on growth activity of test carcinoma cells. Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel. Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

No MeSH data available.


Related in: MedlinePlus

Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system.(a) Effect of Q2-3 on population change of FSP-1+ER-TR7+ cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25+ fibroblasts in gated FSP-1+ER-TR7+ cells were compared with corresponding IL-25+ fibroblast population in PBS group. Data are reported as mean±s.d. (n=3). *P<0.05; ***P<0.001; NS, not significant (two-tailed t-test). FSP-1, fibroblast specific protein-1; ER-TR7, Erasmus University Rotterdam-thymic reticulum-7.IF staining; lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (b) IF staining, lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (c) Construction of 3D cell co-culture system for mammary tumour cells and tumour-associated fibroblasts. Western blot analyses of the expression of (d) mouse IL-25 in 3T3 fibroblasts and (e) human IL-25 in WI38 fibroblasts, in response to Q2-3 treatments in 3D culture. Some fibroblasts in the upper layer were co-cultured with mammary tumour cells as indicated. The expression level of IL-25 in 3T3 and WI38 fibroblasts were quantified and normalized using ImageJ software. Fold changes of IL-25 expression level in test samples were normalized with the value in fibroblasts of the 0 h group and were indicated by the number labelled in blue. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
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f3: Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system.(a) Effect of Q2-3 on population change of FSP-1+ER-TR7+ cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25+ fibroblasts in gated FSP-1+ER-TR7+ cells were compared with corresponding IL-25+ fibroblast population in PBS group. Data are reported as mean±s.d. (n=3). *P<0.05; ***P<0.001; NS, not significant (two-tailed t-test). FSP-1, fibroblast specific protein-1; ER-TR7, Erasmus University Rotterdam-thymic reticulum-7.IF staining; lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (b) IF staining, lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (c) Construction of 3D cell co-culture system for mammary tumour cells and tumour-associated fibroblasts. Western blot analyses of the expression of (d) mouse IL-25 in 3T3 fibroblasts and (e) human IL-25 in WI38 fibroblasts, in response to Q2-3 treatments in 3D culture. Some fibroblasts in the upper layer were co-cultured with mammary tumour cells as indicated. The expression level of IL-25 in 3T3 and WI38 fibroblasts were quantified and normalized using ImageJ software. Fold changes of IL-25 expression level in test samples were normalized with the value in fibroblasts of the 0 h group and were indicated by the number labelled in blue. β-Actin served as an internal control. Similar results were obtained from three independent experiments.

Mentions: In previous in vitro studies, Q2-3 was shown to confer high toxicity to various types of mammary tumour cells1415. However, in our current in vivo experiments, Q2-3 administration only slightly suppressed the growth of in situ caricinoma (Supplementary Fig. 1). By taking these results together with the potent anti-metastatic effect of Q2-3 (Fig. 2), we hypothesize that in vivo administration of Q2-3 may also confer a regulatory effect on the target metastatic tissues. To characterize the physiological significance of the modulatory activity of Q2-3, we analysed the expression of several secreted cytokines in vivo in lung tissue of test mice. The change in the expression level of IL-25 upon Q2-3 treatment was particularly striking. By comparison, we found that Q2-3 administration (100 μg kg−1) conferred a readily detectable stimulatory effect on IL-25 activity in lung fibroblasts, which were found to mainly surround the main pulmonary artery and vein tissue (Fig. 3b). In lung tissues of control and docetaxel-treated mice, little or no IL-25 expression was detected in lung fibroblasts. This result suggests that Q2-3-induced IL-25 expression is specifically expressed in the fibroblasts of the lung tissue microenvironment, which is not a pharmacological target of conventionally used anticancer drugs. To quantify the change in cell population of IL-25-expressing lung fibroblasts in response to Q2-3 treatment, populations of FSP-1+ER-TR7+ cells in test mouse lung tissues were quantified and compared for their IL-25 expression level at 3 weeks post tumour resection. Compared with the PBS (vehicle control)-treated mice, the cell population of IL-25+ fibroblasts (FSP-1+ER-TR7+IL-25+ cells) in Q2-3-treated mice was found to be drastically increased from 16.7 to 79.5% (Fig. 3a). In addition, the total FSP-1+ER-TR7+ fibroblast population in Q2-3-treated mice was also detected to be increased from 5.2 to 7.3%, in a dose-dependent manner (Fig. 3a). In contrast, treatment with docetaxel, a drug tested in parallel as a reference, did not increase the quantity and the IL-25 expression level of FSP-1+ER-TR7+ cells in lungs of test mice. This result is consistent with the data shown in Fig. 3a, and further quantifies and supports the finding that Q2-3 stimulates pulmonary fibroblasts in vivo.


Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis.

Yin SY, Jian FY, Chen YH, Chien SC, Hsieh MC, Hsiao PW, Lee WH, Kuo YH, Yang NS - Nat Commun (2016)

Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system.(a) Effect of Q2-3 on population change of FSP-1+ER-TR7+ cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25+ fibroblasts in gated FSP-1+ER-TR7+ cells were compared with corresponding IL-25+ fibroblast population in PBS group. Data are reported as mean±s.d. (n=3). *P<0.05; ***P<0.001; NS, not significant (two-tailed t-test). FSP-1, fibroblast specific protein-1; ER-TR7, Erasmus University Rotterdam-thymic reticulum-7.IF staining; lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (b) IF staining, lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (c) Construction of 3D cell co-culture system for mammary tumour cells and tumour-associated fibroblasts. Western blot analyses of the expression of (d) mouse IL-25 in 3T3 fibroblasts and (e) human IL-25 in WI38 fibroblasts, in response to Q2-3 treatments in 3D culture. Some fibroblasts in the upper layer were co-cultured with mammary tumour cells as indicated. The expression level of IL-25 in 3T3 and WI38 fibroblasts were quantified and normalized using ImageJ software. Fold changes of IL-25 expression level in test samples were normalized with the value in fibroblasts of the 0 h group and were indicated by the number labelled in blue. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
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f3: Q2-3 treatment significantly upregulates IL-25 expression in tumour-associated fibroblasts, as evaluated by 3D cell co-culture system.(a) Effect of Q2-3 on population change of FSP-1+ER-TR7+ cells and their IL-25 expression level in lungs of test mice, quantified using flow cytometry. The percentage of IL-25+ fibroblasts in gated FSP-1+ER-TR7+ cells were compared with corresponding IL-25+ fibroblast population in PBS group. Data are reported as mean±s.d. (n=3). *P<0.05; ***P<0.001; NS, not significant (two-tailed t-test). FSP-1, fibroblast specific protein-1; ER-TR7, Erasmus University Rotterdam-thymic reticulum-7.IF staining; lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (b) IF staining, lung tissues of control (0.1% DMSO in PBS), Q2-3-treated and docetaxel-treated mice were collected at 21 days post tumour resection and stained for the presence of IL-25-expressing cells (red and indicated with arrowheads), FSP-1 (green) and DAPI (blue). T, tumour; A, alveoli. (c) Construction of 3D cell co-culture system for mammary tumour cells and tumour-associated fibroblasts. Western blot analyses of the expression of (d) mouse IL-25 in 3T3 fibroblasts and (e) human IL-25 in WI38 fibroblasts, in response to Q2-3 treatments in 3D culture. Some fibroblasts in the upper layer were co-cultured with mammary tumour cells as indicated. The expression level of IL-25 in 3T3 and WI38 fibroblasts were quantified and normalized using ImageJ software. Fold changes of IL-25 expression level in test samples were normalized with the value in fibroblasts of the 0 h group and were indicated by the number labelled in blue. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
Mentions: In previous in vitro studies, Q2-3 was shown to confer high toxicity to various types of mammary tumour cells1415. However, in our current in vivo experiments, Q2-3 administration only slightly suppressed the growth of in situ caricinoma (Supplementary Fig. 1). By taking these results together with the potent anti-metastatic effect of Q2-3 (Fig. 2), we hypothesize that in vivo administration of Q2-3 may also confer a regulatory effect on the target metastatic tissues. To characterize the physiological significance of the modulatory activity of Q2-3, we analysed the expression of several secreted cytokines in vivo in lung tissue of test mice. The change in the expression level of IL-25 upon Q2-3 treatment was particularly striking. By comparison, we found that Q2-3 administration (100 μg kg−1) conferred a readily detectable stimulatory effect on IL-25 activity in lung fibroblasts, which were found to mainly surround the main pulmonary artery and vein tissue (Fig. 3b). In lung tissues of control and docetaxel-treated mice, little or no IL-25 expression was detected in lung fibroblasts. This result suggests that Q2-3-induced IL-25 expression is specifically expressed in the fibroblasts of the lung tissue microenvironment, which is not a pharmacological target of conventionally used anticancer drugs. To quantify the change in cell population of IL-25-expressing lung fibroblasts in response to Q2-3 treatment, populations of FSP-1+ER-TR7+ cells in test mouse lung tissues were quantified and compared for their IL-25 expression level at 3 weeks post tumour resection. Compared with the PBS (vehicle control)-treated mice, the cell population of IL-25+ fibroblasts (FSP-1+ER-TR7+IL-25+ cells) in Q2-3-treated mice was found to be drastically increased from 16.7 to 79.5% (Fig. 3a). In addition, the total FSP-1+ER-TR7+ fibroblast population in Q2-3-treated mice was also detected to be increased from 5.2 to 7.3%, in a dose-dependent manner (Fig. 3a). In contrast, treatment with docetaxel, a drug tested in parallel as a reference, did not increase the quantity and the IL-25 expression level of FSP-1+ER-TR7+ cells in lungs of test mice. This result is consistent with the data shown in Fig. 3a, and further quantifies and supports the finding that Q2-3 stimulates pulmonary fibroblasts in vivo.

Bottom Line: Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression.Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel.Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression. Here we investigate the role of IL-25, an endogenous anticancer factor secreted from TAFs, in suppression of mouse 4T1 mammary tumour metastasis. We show that a synthetic dihydrobenzofuran lignan (Q2-3), the dimerization product of plant caffeic acid methyl ester, suppresses 4T1 metastasis by increasing fibroblastic IL-25 activity. The secretion of IL-25 from treated human or mouse fibroblasts is enhanced in vitro, and this activity confers a strong suppressive effect on growth activity of test carcinoma cells. Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel. Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

No MeSH data available.


Related in: MedlinePlus