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Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

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Selection of DU145 human prostate cancer cells with increased metastatic potential.(A) Schematic of the experimental approach. DU145 prostate cells were injected orthotopically into the prostate. Lymph nodes were removed and cultured, and selected tumor cells subjected to repeated rounds of orthotopic injection. Illustrations by Kristin Johnson (Vascular Biology Program, Boston Children's Hospital). (B) Photography of gross specimens (tumors and sentinel lymph nodes). DU145 parental cells and DU145-LN sublines (DU145-LN4 shown) were reinjected into the prostate and the prostate and lymph nodes were removed after 5 wks. Scale bar = 1 cm. (C) Representative H&E staining of lymph nodes from mice bearing orthotopic parental DU145 tumors (left panel, P) and DU145-LN4 tumors (center and right panels, LN4). Metastatic nodule indicated by arrowhead in center panel, magnification shown in right panel.
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f1: Selection of DU145 human prostate cancer cells with increased metastatic potential.(A) Schematic of the experimental approach. DU145 prostate cells were injected orthotopically into the prostate. Lymph nodes were removed and cultured, and selected tumor cells subjected to repeated rounds of orthotopic injection. Illustrations by Kristin Johnson (Vascular Biology Program, Boston Children's Hospital). (B) Photography of gross specimens (tumors and sentinel lymph nodes). DU145 parental cells and DU145-LN sublines (DU145-LN4 shown) were reinjected into the prostate and the prostate and lymph nodes were removed after 5 wks. Scale bar = 1 cm. (C) Representative H&E staining of lymph nodes from mice bearing orthotopic parental DU145 tumors (left panel, P) and DU145-LN4 tumors (center and right panels, LN4). Metastatic nodule indicated by arrowhead in center panel, magnification shown in right panel.

Mentions: To select for prostate cancer cells with increased metastatic potential we used an in vivo cycling strategy2327 to establish a series of sublines from the DU145 human prostate cancer cell line28. To establish this model, 2 × 106 DU145 cells were injected orthotopically into the prostate of nude mice. Tumor growth was monitored by abdominal palpation. Once the tumor was 0.5–1 cm in diameter (5–12 weeks), the mice were euthanized and necropsied in a sterile environment. The sentinel paraaortic lymph nodes were excised, minced and the cells were placed into culture (schematic in Figure 1A), as described in Methods. Primary lymph node cultures contained tumor cells and fibroblasts, but after several passages only tumor cells remained and were named DU145-LN1. Repeated rounds of lymph node excision and tumor cell reinjection were performed to establish the DU145-LN2, DU145-LN3 and DU145-LN4 cell lines. RT-PCR was used to confirm that the cell cultures were not contaminated with cells of mouse origin (e.g. fibroblasts) which might affect tumor growth (Supplementary Figure 1A).


Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

Selection of DU145 human prostate cancer cells with increased metastatic potential.(A) Schematic of the experimental approach. DU145 prostate cells were injected orthotopically into the prostate. Lymph nodes were removed and cultured, and selected tumor cells subjected to repeated rounds of orthotopic injection. Illustrations by Kristin Johnson (Vascular Biology Program, Boston Children's Hospital). (B) Photography of gross specimens (tumors and sentinel lymph nodes). DU145 parental cells and DU145-LN sublines (DU145-LN4 shown) were reinjected into the prostate and the prostate and lymph nodes were removed after 5 wks. Scale bar = 1 cm. (C) Representative H&E staining of lymph nodes from mice bearing orthotopic parental DU145 tumors (left panel, P) and DU145-LN4 tumors (center and right panels, LN4). Metastatic nodule indicated by arrowhead in center panel, magnification shown in right panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818652&req=5

f1: Selection of DU145 human prostate cancer cells with increased metastatic potential.(A) Schematic of the experimental approach. DU145 prostate cells were injected orthotopically into the prostate. Lymph nodes were removed and cultured, and selected tumor cells subjected to repeated rounds of orthotopic injection. Illustrations by Kristin Johnson (Vascular Biology Program, Boston Children's Hospital). (B) Photography of gross specimens (tumors and sentinel lymph nodes). DU145 parental cells and DU145-LN sublines (DU145-LN4 shown) were reinjected into the prostate and the prostate and lymph nodes were removed after 5 wks. Scale bar = 1 cm. (C) Representative H&E staining of lymph nodes from mice bearing orthotopic parental DU145 tumors (left panel, P) and DU145-LN4 tumors (center and right panels, LN4). Metastatic nodule indicated by arrowhead in center panel, magnification shown in right panel.
Mentions: To select for prostate cancer cells with increased metastatic potential we used an in vivo cycling strategy2327 to establish a series of sublines from the DU145 human prostate cancer cell line28. To establish this model, 2 × 106 DU145 cells were injected orthotopically into the prostate of nude mice. Tumor growth was monitored by abdominal palpation. Once the tumor was 0.5–1 cm in diameter (5–12 weeks), the mice were euthanized and necropsied in a sterile environment. The sentinel paraaortic lymph nodes were excised, minced and the cells were placed into culture (schematic in Figure 1A), as described in Methods. Primary lymph node cultures contained tumor cells and fibroblasts, but after several passages only tumor cells remained and were named DU145-LN1. Repeated rounds of lymph node excision and tumor cell reinjection were performed to establish the DU145-LN2, DU145-LN3 and DU145-LN4 cell lines. RT-PCR was used to confirm that the cell cultures were not contaminated with cells of mouse origin (e.g. fibroblasts) which might affect tumor growth (Supplementary Figure 1A).

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

Show MeSH
Related in: MedlinePlus