Migration and Invasion of Cancer Cells following MSC phenotype co-culture.

<p>Transwell migration and matrigel invasion assays were performed with 3 µM Falcon fluoroblok transwell inserts as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Zwezdaryk1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt3" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Tomchuck1" target="_blank">[45]</a>. MSCs were added at a 10∶1 ratio of SKOV3 to MSC. These were co-cultured on traditional 2D dishes 72 hr prior to placing the dissociated cells within the transwell inserts. Representative micrographs of <b>A.</b> transwell migrating and <b>B.</b> matrigel invading cells were visualized and obtained on an inverted fluorescence microscope (A. 100X and B. 200X, Olympus, MetaMorph analysis software). Data are representative of duplicates in at least three independent experiments<b>. C.</b> Representative bar graph of quantitative real-time PCR (qPCR) assays carried out as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045590#pone.0045590-Coffelt4" target="_blank">[39]</a>. Gene expression of <i>mmps</i> among the MSC samples is expressed by the normalized cumulative threshold method (ΔΔC(t)). *<i>P</i><0.05 versus the normalized values for MSC. Statistically significant differences were not measured among the other samples. Samples were run in triplicate for at least four different MSC donors. <b>D.</b> Representative micrograph following gelatin zymography of the condition medium from MSC-SKOV3 co-cultures (1∶10) or SKOV3 and MSC samples cultured alone as indicated for 72 hr. Bands are of pro-MMP2 (72 kDa) and active MMP2* (62 kDa). The numbers below micrograph are the fold changes relative to SKOV3 alone sample obtained following densitometric analysis (ImageJ). Data are representative of at least three independent experiments.