* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005 indicated statistical significance. Selleck Ro 61-8048 Data are presented as mean ± standard deviation. Each experiment was repeated at least three times. Multiple group comparison experiments were validated by ANOVA. Results Single cell cloning Four CX-5461 solubility dmso clones were isolated from the pancreatic cell line, MiaPaCa-2 and successfully established as cell lines.

The invasion status of the clones was tested using the Boyden chamber assay with inserts coated with matrigel. Two sub-populations, Clone #3 and Clone #8, showed a significant increase (Clone #3, 2.5-fold increase, p = 0.001) and decrease (Clone #8, 12-fold decrease, p = 0.00001), ANOVA (p < 0.001), (Fig 1A(i-ii) and 1B) in invasion through matrigel, compared to the parental MiaPaCa-2 cells. These two

clonal populations also displayed distinct morphological differences (Fig 1A(iii-iv)). The invasive cell line, Clone #3 displayed an elongated spindled shaped AZ 628 morphology, similar to mesenchymal cells. Clone #8, low invasion, was similar to epithelial cells in tight clustered colonies. Figure 1 A. Morphology of the highly invasive (i) Clone #3 with elongated and spindle-like phenotype and low-invasive (ii) Clone #8 with epithelial tight colonies. Cell invasion assay representing (iii) Clone #3 and (iv) Clone #8 invading through ECM coated Boyden chamber, stained with crystal violet. Magnification 200×. Scale bar, 200 μm. B. Total number of invading cells. Results shown are a minimum of three repeats ± standard deviation (n = 3). Invasion and adhesion to ECM proteins Invasion of MiaPaCa-2 and sub-populations, Clone #3 and Clone #8, through a range of ECM proteins was examined (Fig 2A). The Carnitine palmitoyltransferase II invasion

of MiaPaCa-2 and Clone #3 is comparable through laminin and fibronectin whereas Clone #8 showed a significant decrease in invasion, 6.3 and 4.0-fold (p = 0.002, p = 0.008) through laminin and fibronectin, respectively, ANOVA (all p < 0.001). Low invasion was observed for Clone #3 through collagens type I and IV; Clone #8 showed significantly decreased invasion through the collagens (1.6 and 1.6-fold (p = 0.03, p = 0.02)), ANOVA (p = 0.007, p = 0.001). Interestingly, the lowest level of invasion displayed by the cell lines was through the collagens, type IV and I, which is in agreement with previous studies indicating MiaPaCa-2 does not express collagen-binding integrins [23]. The highest level of invasion was observed through fibronectin. Clone #3 also displayed significantly increased motility (p = 0.00005) whereas the motility of Clone #8 was similar to that of MiaPaCa-2, ANOVA (p < 0.001) (Fig 2A). Figure 2 A. Invasion assay of MiaPaCa-2, Clone #3 and Clone #8 through ECM proteins. Motility assay refers to invasion assay without the presence of ECM. Results are displayed as the total mean number of cells invading at 200× magnification (n = 3). B.