Mmp10 is required for <i>Kras</i>-induced expansion and transformation of BASCs <i>in vitro.</i>

<p>BASCs isolated from Ntg, <i>Mmp10^−/−</i>, <i>LSL-Kras</i>, and <i>LSL-Kras/Mmp10^−/−</i> mice were treated with AdCre and plated in three-dimensional Matrigel culture as described in <i>Experimental Procedures</i>. <b>A</b>) Flow cytometry of isolated BASCs for SPC and CCSP <b>B</b>) QPCR for Mmp10 in BASCs from Ntg, <i>LSL-Kras,</i> and <i>LSL-Kras/Mmp10^−/−</i> mice. Fold of Ntg +/<i>−</i>SEM. n = 3, *p<0.000008. <b>C</b>) Morphology of BASC colonies from Ntg, <i>Mmp10^−/−</i>,<i>LSL-Kras</i>, and <i>LSL-Kras/Mmp10^−/−</i> mice. <b>D</b>) Analysis of BASC colony size. %Ntg +/−SEM. n = 85 Ntg, 56 <i>Mmp10^−/−</i>,30 (<i>LSL-Kras</i>) and 80 (<i>LSL-Kras/Mmp10^−/−</i>). *p<0.00001 Ntg vs, <i>LSL-Kras;</i> **p<0.00001 <i>LSL-Kras</i> vs. <i>LSL-Kras/Mmp10^−/−</i>.</p

Mmp10 expression correlates with cancer stem cell genotypes and metastasis in human lung tumors.

<p><b>A</b>) Gene expression data from primary human lung adenocarcinomas were divided into two groups of 30 samples consisting of lowest (Low) and highest (High) Mmp10 expressing lung tumors. n = 30; * p = 2.8×10⁻³³. <b>B</b>) Mmp10 in normal lung versus lung tumors with (<b><i>Met.</i></b>) and without (<b><i>No Met.</i></b>) bone metastases. n = 3, normals, n = 9, No Mets, n = 7, Met.; NS =  not significant, *p = 0.008; **p = 0.04. <b>C</b>) Mmp10 mRNA expression in human tumors. Data are expressed as fold-change from matched normal.</p

MMP10 is Overexpressed in many Human Cancer Types.

<p>MMP10 is Overexpressed in many Human Cancer Types.</p

Mmp10 plays a promotive role in urethane-induced lung tumorigenesis.

<p><i>Mmp10^−/−</i> mice and Ntg littermates were injected with urethane and analyzed as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#s4" target="_blank">Materials and Methods</a></i>. <b>A</b>) H & E and immunohistochemical staining for Mmp10 in urethane-induced lung tumors. Higher magnification image of Mmp10 immunostaining is shown in the inset. Quantitative analysis of tumor number <b>B</b>), tumor size <b>C</b>) and tumor burden <b>D</b>) in urethane-treated Ntg (n = 7) and <i>Mmp10^−/−</i> (n = 12) mice. Mean +/−SEM; p<. 0.012 tumor number; p<0.019 tumor burden; p = 0.034 tumor size). <b>E</b>) Urethane-induced tumors from Ntg and Mmp10<i>⁻</i>^/<i>−</i> mice were graded as hyperplasia, adenoma or adenocarcinoma using published criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone.0026439-KellySpratt1" target="_blank">[26]</a>. Results are presented as the percentage of total tumors of each grade. Statistical analysis using Mann-Whitney U test revealed no statistically significant difference in tumor grade between urethane-induced tumors in Ntg and Mmp10<i>⁻</i>^/<i>−</i> mice (p = 0.39).</p

Cancer Stem Cell Signatures correlate with high Mmp10 expression in lung cancer.

<p>Cancer Stem Cell Signatures correlate with high Mmp10 expression in lung cancer.</p

The 10 most highly correlated signatures associated with high Mmp10 in lung cancer.

<p>Gene sets marked in bold text contain Mmp10 as part of the gene signature.</p

Validation of the association between high Mmp10 in lung tumors and cancer stem cell signatures.

<p>Gene sets marked in bold text were also significantly enriched in the high Mmp10 lung tumor gene set analysis outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone-0026439-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone-0026439-t001" target="_blank">Table 1</a>.</p

Association of metastatic lung cancer genes with cancer stem cell signatures.

<p>Gene sets marked in bold text are cancer stem cell signatures also identified as highly correlated with lung tumors expressing high Mmp10.</p

Mmp10 is necessary for <i>Kras^LA2</i>-induced lung tumorigenesis <i>in vivo.</i>

<p><b>A</b>) Immunohistochemical staining of <i>Kras^LA2</i> lung tumor for mouse MMP10. Higher magnification image of Mmp10 immunostaining is shown in the inset. Quantitative analysis of <b>B</b>) tumor number, <b>C</b>) tumor size and <b>D)</b> tumor burden in <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice. Columns, mean; bars, SEM, n = 13, (*) denotes p = 0.04. E) Tumors from <i>Kras^LA2</i> and <i>Kras^LA2/Mmp10^−/−</i> mice were categorized as advanced adenomatous hyperplasia (AAH), or grade 1,2 or 3 adenomas using the published scoring criteria described by Jackson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026439#pone.0026439-Jackson1" target="_blank">[28]</a>. Results are presented as the percentage of total tumors of each grade. Statistical analysis using Mann-Whitney U test revealed a significant decrease in higher grade tumors in <i>Kras^LA2/Mmp10^−/−</i> mice; *p<0.002.</p