Metastases following subcutaneous xenograft.

a<p>mice were injected with 10×10⁶ cells.</p>b<p>the presence of macrometastases was evaluated by histological analysis.</p>*<p><i>P</i> = 0.02. Mice injected with MCF7 clones/population expressing Mini5B were grouped and mice injected with clones/cell population not expressing Mini5B were grouped.</p

Impact of Mini5B expression on the dissemination of MCF7 cell xenografts in immunodeficient mice.

<p>(<b>A</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of tumors from mice injected with the Ires-Luc clone, the Mini5B-Luc CYS shRNA clone, the Mini5B-Luc clone and the Mini5B-Luc empty shRNA clone and stained with anti-MUC5B antibody. Mini5B was expressed and secreted by tumoral cells in tumors of Mini5B-Luc group and Mini5B-Luc empty shRNA. (<b>B</b>) Serial sections of livers of mice injected with the Ires–Luc clone and stained with HE and anti-PCNA antibody, and serial sections of lungs of mice injected with the Mini5B–Luc clone and stained with HE and anti-PCNA antibody. Metastases in the liver and the lung were visualized. Metastatic cells were PCNA positive. (<b>C</b>) Histological analysis of a macrometastasis in lung of a mouse injected with the Mini5B–Luc clone using HE staining and E-cadherin immunostaining (serial sections). Mt; metastasis. Nuclei were counterstained with Hoechst 33258. Scale bar 50 µm.</p

Histological analysis of mice sacrificed during the experimentation.

<p>(<b>A</b>) HE staining was performed on paraffin-embedded sections of the liver, lungs, and lymph nodes of mice injected with Mini5B–Luc clones. Metastasis is showed in the magnified image. (<b>B</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of liver from mice injected with Mini5B–Luc clones and stained with anti-PCNA antibody. Metastatic cells were PCNA positive. (<b>C</b>) Double immunofluorescence analysis using the anti-human E-cadherin and anti-MUC5B antibodies were performed on paraffin-embedded sections of thoracic lymph nodes from mice injected with Mini5B–Luc clones. Metastatic cells expressed both the epithelial marker E-cadherin and secreted MUC5B protein. Nuclei were counterstained with Hoechst 33258 or propidium iodide. Mt, metastasis. Scale bar 50 µm.</p

Clinical scores of xenografted SCID mice.

<p>The empty box and the box with the diagonal lines are for the Ires–Luc clone and the Mini5B KD groups, respectively. The black box and the box with vertical lines are for the Mini5B–Ires empty shRNA and the Mini5B–Ires–Luc groups, respectively.</p

Generation of stable clones expressing or not expressing the mini-mucin Mini5B.

<p>(<b>A</b>) Schematic representation of the control Ires–Luc and Mini5B–Luc vectors. The insert encoding the Mini5B was made by a signal sequence, 11 TR of 29 aa, one R-end domain of 111 aa, and two CYS domains of 110 aa. (<b>B</b>) Using the luciferase activity assay, four stable clones expressing the Ires–Luc transgene (clones B2, C5, C7, and C10) and two stable clones expressing the Mini5B–Luc transgene (clones A3 and D6) were obtained. (<b>C</b>) <i>MUC5B</i> mRNA expression was studied by qRT–PCR (TaqMan) in MCF7 parental cell line (white box), in MCF7 Ires-Luc clone (grey box) and in Mini5B-Luc clone (black box). Expression levels were normalized to mRNA levels of 18S and shown as x-fold relative to the normalized expression of <i>MUC5B</i> gene in MCF7 parental cells. Relative amounts of target genes were calculated using the ΔΔCt method. Values are means ± standard deviation from four to six independent samples. (<b>D</b>) MUC5B immunofluorescence analysis of MCF7 parental cells, Ires–Luc and Mini5B–Luc clones. Mini5B was secreted at the cell surface of Mini5B–Luc clones. Nuclei were counterstained with propidium iodide. Scale bar 50 µm. (<b>E</b>) Expression of the Mini5B was evaluated by RT–PCR. A PCR product of 159 bp was detected only in the Mini5B–Luc clone. (<b>F</b>) Western blot analysis of lysates from Ires–Luc clones, Mini5B–Luc clones, and Mini5B–Luc CYS shRNA subpopulation. (<b>G</b>) Inhibition of Mini5B production detected by Western blotting was quantified. Mini5B production of the clone Mini5B–Luc was set at 100 and the production of Ires–Luc was set at 0.</p

Impact of Mini5B expression on the growth of MCF7 cell xenografts in immunodeficient mice.

<p>(<b>A</b>) Mini5B–Luc, Ires–Luc, Mini5B–Luc empty shRNA, or Mini5B–Luc CYS shRNA xenografts. Tumor growth was monitored once a week for 118 days. *<i>P<</i>0.02, ** <i>P</i><0.005. (<b>B</b>) Examples of tumor growth and metastasis on days 62 and 118 in anesthetized mice using the Xenogen apparatus.</p