Targeted Killing of CSCs by CDF CDF remarkably increased pancreatosphere disintegration of PC cells To examine the effect of treatments on the sphere forming ability of PC cells (pancreatosphere) and disintegration of pancreato- spheres, we conducted sphere disintegration assay for 10 days to generate the formation of pancreatospheres, followed by 5 days of drug treatment. The results show that there was a remarkable increase of sphere disintegration by curcumin and CDF treatment, not by gemcitabine treatment (Figure 4). However, the greatest effect on disintegration was observed in response to the CDF treatment (Figure 4), once again suggesting that CDF is much more superior in inhibiting the functions of cancer stem-like cells. CDF inhibited pancreatospheres formation in PC cells To examine the effect of drugs on CSC self-renewal capacity in PC cells, we conducted sphere formation assay for 1 week and four weeks (Figure 5A and B). The results indicated that CDF in combination with gemcitabine completely eliminated pancreatospheres formation after four weeks of treatment compared to gemcitabine and curcumin combination in PC cells even in gemcitabine-resistant PC cells, suggesting that CDF may cause the pancreatospheres more sensitive to gemcitabine than that of curcumin treatment, and could be useful for targeted killing of CSCs. Figure 5C shows the effect of different concentration of gemcitabine and CDF on 2nd passage of pancreato- spheres in pre-treated primary pancreatospheres of AsPC-1 cells. CDF treatment remarkably inhibited 2nd passage of pancreato- spheres in a dose-dependent manner. Furthermore, CDF-pre-treated cells exhibited a greater effect than non-CDF-pre-treated cells. CDF decreased CD44 and EpCAM expression in pancreatospheres of PC cells We examined the effect of drugs on CSC biomarkers, CD44 and EpCAM in pancreatospheres of AsPC-1 and AsPC-1-GTR cells by confocal microscopy (Figure 6). The results indicate that CDF decreased CD44 and EpCAM expression in pancreato- spheres, suggesting the inhibitory effect of CDF on pancreato- Figure 4. CDF remarkably increased disintegration of pancreatospheres in AsPC-1, AsPC1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR cells. P values were calculated by the paired t test. doi:10.1371/journal.pone.0017850.g004 PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17850