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Mesenchymal-transitioned cancer cells instigate the invasion of epithelial cancer cells through secretion of WNT3 and WNT5B.

Kato S, Hayakawa Y, Sakurai H, Saiki I, Yokoyama S - Cancer Sci. (2014)

Bottom Line: In this study, we show that the mesenchymal-transitioned cancer cells instigate the invasive ability and metastatic potential of the neighboring epithelial cancer cells in vitro and in vivo.We identify WNT3 and WNT5B as critical factors secreted from Transforming growth factor-induced mesenchymal cancer cells for instigating the epithelial cancer cell invasion along with the induction of secondary EMT phenotype.These results shed light on the significance of cancer heterogeneity and the interaction between epithelial and mesenchymal-transitioned cancer cells within the tumor microenvironment in promoting metastatic disease through the WNT-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

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Epithelial cancer cells acquire metastatic potential upon co-culture with mesenchymal-transitioned cancer cells. (a) A549 (left) or Panc-1 (right) cells were subjected to Matrigel invasion assay after 24-h co-culture of epithelial cancer cells (E-cells) with mesenchymal-transitioned cancer cells (M-cells) at the indicated cell numbers. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of four independent experiments. (b) Labeled E-cells (E-A549/Luc2; left panel or E-Panc/EGFP; right panel) were subjected to Matrigel invasion assay after 24-h-co-culture with M-cells. Invasive abilities were determined by measuring luciferase activity (A549) or counting invaded EGFP+ cells (Panc-1), respectively. Invasion ratio was calculated by the division of invaded E-cells by total E-cells applied. Data represented as the mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 versus E-cells alone group by Dunnett's test. (c) Epithelial A549 cells overexpressing Luc2 gene (E-A549/Luc2) were co-cultured with either epithelial A549 (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 5).
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fig01: Epithelial cancer cells acquire metastatic potential upon co-culture with mesenchymal-transitioned cancer cells. (a) A549 (left) or Panc-1 (right) cells were subjected to Matrigel invasion assay after 24-h co-culture of epithelial cancer cells (E-cells) with mesenchymal-transitioned cancer cells (M-cells) at the indicated cell numbers. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of four independent experiments. (b) Labeled E-cells (E-A549/Luc2; left panel or E-Panc/EGFP; right panel) were subjected to Matrigel invasion assay after 24-h-co-culture with M-cells. Invasive abilities were determined by measuring luciferase activity (A549) or counting invaded EGFP+ cells (Panc-1), respectively. Invasion ratio was calculated by the division of invaded E-cells by total E-cells applied. Data represented as the mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 versus E-cells alone group by Dunnett's test. (c) Epithelial A549 cells overexpressing Luc2 gene (E-A549/Luc2) were co-cultured with either epithelial A549 (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 5).

Mentions: To directly evaluate the role of heterogeneity of cancer cells in their invasive potential, we performed Matrigel invasion assays in human epithelial lung adenocarcinoma A549 and pancreatic ductal adenocarcinoma Panc-1 cell co-culture at various ratios of epithelial cancer cells (E-cells) and TGF-β-induced mesenchymal-transitioned cancer cells (M-cells), because TGF-β is known to trigger EMT in both A549 and Panc-1, which is the most potent EMT-inducing cytokine (Fig. S1).25,26 After 24 h co-culture of E-cells and M-cells, the invasive potential of the E-cell and M-cell mixture was more enhanced in both A549 and Panc-1 cell lines, in an M-cell dose-dependent manner, than that in M-cells alone (Fig.1a). To determine the cell population, either E-cells or M-cells, responsible for the enhanced invasive potential after the co-culture, we employed luminescence-labeled E-A549 (E-A549/Luc2) or fluorescent-labeled E-Panc-1 (E-Panc/EGFP) to distinguish E-cells from M-cells within the co-culture. By measuring the invasiveness of labeled E-cells in either A549 or Panc-1, we have found that the invasive potential of E-cells co-cultured with M-cells was higher than that of E-cells alone in both A549 and Panc-1 (Fig.1b) and such induction of invasiveness was observed in an M-cell dose-dependent manner (Fig.1b). We have also found that the invasion potential of E-cells was not altered without the co-culture with M-cells (Fig. S2). Of note, the invasive potential of M-Panc cells was not affected by the co-culture with E-Panc cells as compared with M-Panc cells alone (data not shown). These results clearly indicate that the co-culture of E-cells with M-cells selectively enhances the invasiveness of E-cells in both A549 and Panc-1 cell lines.


Mesenchymal-transitioned cancer cells instigate the invasion of epithelial cancer cells through secretion of WNT3 and WNT5B.

Kato S, Hayakawa Y, Sakurai H, Saiki I, Yokoyama S - Cancer Sci. (2014)

Epithelial cancer cells acquire metastatic potential upon co-culture with mesenchymal-transitioned cancer cells. (a) A549 (left) or Panc-1 (right) cells were subjected to Matrigel invasion assay after 24-h co-culture of epithelial cancer cells (E-cells) with mesenchymal-transitioned cancer cells (M-cells) at the indicated cell numbers. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of four independent experiments. (b) Labeled E-cells (E-A549/Luc2; left panel or E-Panc/EGFP; right panel) were subjected to Matrigel invasion assay after 24-h-co-culture with M-cells. Invasive abilities were determined by measuring luciferase activity (A549) or counting invaded EGFP+ cells (Panc-1), respectively. Invasion ratio was calculated by the division of invaded E-cells by total E-cells applied. Data represented as the mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 versus E-cells alone group by Dunnett's test. (c) Epithelial A549 cells overexpressing Luc2 gene (E-A549/Luc2) were co-cultured with either epithelial A549 (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Epithelial cancer cells acquire metastatic potential upon co-culture with mesenchymal-transitioned cancer cells. (a) A549 (left) or Panc-1 (right) cells were subjected to Matrigel invasion assay after 24-h co-culture of epithelial cancer cells (E-cells) with mesenchymal-transitioned cancer cells (M-cells) at the indicated cell numbers. Total invaded cells were counted after H&E staining. Data represented as the mean ± SD of four independent experiments. (b) Labeled E-cells (E-A549/Luc2; left panel or E-Panc/EGFP; right panel) were subjected to Matrigel invasion assay after 24-h-co-culture with M-cells. Invasive abilities were determined by measuring luciferase activity (A549) or counting invaded EGFP+ cells (Panc-1), respectively. Invasion ratio was calculated by the division of invaded E-cells by total E-cells applied. Data represented as the mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 versus E-cells alone group by Dunnett's test. (c) Epithelial A549 cells overexpressing Luc2 gene (E-A549/Luc2) were co-cultured with either epithelial A549 (E-A549) cells or mesenchymal-transitioned A549 (M-A549) cells and i.v. inoculated into mice. Mice were killed 24 h after the tumor inoculation and lungs were subjected to bioluminescent imaging to determine total flux (photon/s) for lung metastasis quantification. The representative ex vivo images are shown. Data represented as the mean ± SEM (n = 5).
Mentions: To directly evaluate the role of heterogeneity of cancer cells in their invasive potential, we performed Matrigel invasion assays in human epithelial lung adenocarcinoma A549 and pancreatic ductal adenocarcinoma Panc-1 cell co-culture at various ratios of epithelial cancer cells (E-cells) and TGF-β-induced mesenchymal-transitioned cancer cells (M-cells), because TGF-β is known to trigger EMT in both A549 and Panc-1, which is the most potent EMT-inducing cytokine (Fig. S1).25,26 After 24 h co-culture of E-cells and M-cells, the invasive potential of the E-cell and M-cell mixture was more enhanced in both A549 and Panc-1 cell lines, in an M-cell dose-dependent manner, than that in M-cells alone (Fig.1a). To determine the cell population, either E-cells or M-cells, responsible for the enhanced invasive potential after the co-culture, we employed luminescence-labeled E-A549 (E-A549/Luc2) or fluorescent-labeled E-Panc-1 (E-Panc/EGFP) to distinguish E-cells from M-cells within the co-culture. By measuring the invasiveness of labeled E-cells in either A549 or Panc-1, we have found that the invasive potential of E-cells co-cultured with M-cells was higher than that of E-cells alone in both A549 and Panc-1 (Fig.1b) and such induction of invasiveness was observed in an M-cell dose-dependent manner (Fig.1b). We have also found that the invasion potential of E-cells was not altered without the co-culture with M-cells (Fig. S2). Of note, the invasive potential of M-Panc cells was not affected by the co-culture with E-Panc cells as compared with M-Panc cells alone (data not shown). These results clearly indicate that the co-culture of E-cells with M-cells selectively enhances the invasiveness of E-cells in both A549 and Panc-1 cell lines.

Bottom Line: In this study, we show that the mesenchymal-transitioned cancer cells instigate the invasive ability and metastatic potential of the neighboring epithelial cancer cells in vitro and in vivo.We identify WNT3 and WNT5B as critical factors secreted from Transforming growth factor-induced mesenchymal cancer cells for instigating the epithelial cancer cell invasion along with the induction of secondary EMT phenotype.These results shed light on the significance of cancer heterogeneity and the interaction between epithelial and mesenchymal-transitioned cancer cells within the tumor microenvironment in promoting metastatic disease through the WNT-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Show MeSH
Related in: MedlinePlus