Targeted Killing of CSCs by CDF investigating the greater potential of tumor growth by pancreato- spheres, we injected only 5,000 cells in mice as a proof-of-concept study. The tumor weight was remarkably increased as the days progressed (Figure 8A). The level of miR-21 was increased between tumors implanted with one million of parental cells compared to pancreatospheres (Figure 8B). The animal was euthanized after 30 days because of tumor burden, and gross tumors are shown in Figure 8C indicating larger tumors as well as loco-regional lymph node metastasis whereas tumors derived from parental cells did not show any metastasis over a period of 30 days. The tumor-derived cells showed significant inhibition of pancrea- tospheres when treated with CDF (Figure 8D). Overall, these results suggest that CSCs (pancreatospheres) can be grown in mice and CDF could be useful for the killing of these drug resistant cells (Figure. 8). Discussion In this study, we have demonstrated that a synthetic analogue of curcumin, CDF, is significantly more effective compared to curcumin in the killing of gemcitabine-resistant pancreatic cancer (PC) cells that consists high proportion of cells with cancer stem cells (CSCs) or cancer stem-like cells characteristics. The inhibition of cell growth could in part be due to better cellular uptake, retention and reduced metabolic inactivation of CDF by PC cells, which is consistent with our published findings on cellular and animal pharmacokinetics data [10,11]. Our previous reports indicate that CDF inhibits NF-kB and COX-2 activity in PC cells in vitro [12]. Here we confirm these observations in vivo using a mouse xenograft model. Thus, the killing of gemcitabine-resistant PC cells by CDF is associated with inactivation of NF-kB and COX-2 signaling pathway which is very important because these Figure 8. Tumor growth pattern of pancreatospheres derived from MIAPaCa-2 cells. (A). 5,000 pancreatospheres were inoculated in mice using 1:1 matrigel, progressive tumor growth over a period of 30 days. Moderate increase in the expression of miR-21 as measured by real-time RT- PCR was observed in tumors derived from pancreatospheres compared to tumors derived from parental cells by injecting one million cells and tumor was assessed over the same period of time (B). Photographs showing tumor growth, arrow points to tumor and asterisk (*) refers to loco-regional lymph node metastasis whereas we did not find any metastasis when one million parental cells were injected (C). Tumor cells harvested from the tumors derived from pancreatospheres were treated with CDF showed significant inhibition in the formation of pancreatospheres (D). doi:10.1371/journal.pone.0017850.g008 PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17850