(2009) Pancreatic cancer stem cells: insights and perspectives. profiling analysis showed that CSLCs (CD44+/CD133+/EpCAM+) exhibit differential expression of more than 1,600 mRNAs, including (2,C4). A large number of studies have shown clear evidence in support of the presence of CSLCs and their clinical implications because the rare subpopulations of CSLCs have been recognized from most tumors, such as prostate, lung, breast, pancreas, brain, gastric, and colorectal tumors. These CSLCs are involved in cell growth, migration/invasion, and apoptosis resistance, attributing to treatment resistance and metastasis, leading to poor clinical end result (2,C4). However, the pathogenesis of CSLCs during tumorigenesis and tumor progression has not been well documented. Although significant improvements have been made in the fight against cancers, pancreatic malignancy (PC) remains one of the most aggressive and lethal malignant diseases in the world, and remains the 4th leading cause of cancer-related death in the United States (5). For example, it was estimated that 45,220 people would be newly diagnosed with PC, and 38,460 patients would die in 2013 (5). Due to the lack of specific signs and symptoms and the lack of early detection techniques for PC, the majority of patients are diagnosed at an advanced LY450108 stage (80% of newly diagnosed cases). The conventional treatments, including surgical resections and chemo-radiotherapy are not effective, which is usually in part due to therapeutic resistance and greater potential for locally advanced and metastatic disease. The majority of patients will pass away within an average of 5C6 months after diagnosis. The overall 5-12 months disease-free survival rate is usually 1C4%. It has been reported that very small subpopulations of CSCs (CSLCs), positive for CD133, can be recognized from PC tissues (6). These CSLCs exhibit more aggressive phenotypes, such as increased tumorigenic and metastatic potentials and studies using a mouse xenograft tumor model showed that CSLSs derived from MiaPaCa-2 cells display a 100-fold higher potential for tumor formation and also faster tumor growth, which was consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental cells. The inhibition of FoxQ1 by its siRNA attenuated tumor formation and growth, consistent with the down-regulation of CSC markers/mediators in xenograft tumor derived from CSLCs of MiaPaCa-2 cells. Our observation suggests that pathways that are activated in CSLCs could be targeted as novel therapies for PC. MATERIALS AND METHODS Cell Lines and Culture Conditions CD44+/CD133+/EpCAM+ (triple-marker-positive cells) were isolated as the CSLCs from human pancreatic malignancy cell collection MiaPaCa-2 and L3.6pl cells by the fluorescence-activated cell sorting (FACS) technique and LY450108 cultured in the serum-free sphere formation medium (1:1 DMEM/F-12K medium plus B27 and N2 supplements, Invitrogen) to maintain its undifferentiated status. Moreover, triple-marker-negative (CD44?/CD133?/EpCAM?) cells were isolated from MiaPaCa-2 and L3.6pl cells by the FACS technique and were also cultured in 5% fetal bovine serum (FBS)-DMEM at 37 C in standard culture conditions, as described previously (8, 9). CD44+, CD133+, and EpCAM+ are known as stem cell surface proteins, which have been considered as the pancreatic CSLC (CSC) markers (3, 6, 10). Sphere Formation Assay The sphere formation assay was conducted to assess the CSLC self-renewal capacity, as explained previously (8, 9). Briefly, 1,000 single suspended cells were seeded around the ultralow attachment wells of Costar 6-well plates (Corning Inc.) in 2 ml of sphere formation medium. After 7 days of incubation, the sphere cells termed pancreatospheres were harvested by centrifugation (300 for 5 min). The number of pancreatospheres was counted under a converted microscope. Cell Growth Assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to assess the cell survival or growth. Briefly, 5,000 cells/well were seeded in a 96-well plate and incubated Mouse monoclonal antibody to Annexin VI. Annexin VI belongs to a family of calcium-dependent membrane and phospholipid bindingproteins. Several members of the annexin family have been implicated in membrane-relatedevents along exocytotic and endocytotic pathways. The annexin VI gene is approximately 60 kbplong and contains 26 exons. It encodes a protein of about 68 kDa that consists of eight 68-aminoacid repeats separated by linking sequences of variable lengths. It is highly similar to humanannexins I and II sequences, each of which contain four such repeats. Annexin VI has beenimplicated in mediating the endosome aggregation and vesicle fusion in secreting epitheliaduring exocytosis. Alternatively spliced transcript variants have been described in 5% FBS-DMEM medium overnight. After changing the medium, LY450108 the cells were continued for the incubation. After 3 days of incubation, the cells were harvested for the standard MTT assay, as explained previously (8, 11). Colony Formation Assay The colony formation assay was conducted to assess clonogenic potential of the cells, as explained previously (8, 11). Briefly, 1,000 single viable cells were seeded in 10 ml of 5% FBS-DMEM in 10-cm Petri dishes. The cells were then incubated at 37 C in a tissue culture incubator for 14 days. LY450108 Colonies were stained with 2% crystal violet, washed with water, and counted. Wound Healing Assay The wound healing assay was conducted to assess the migration capacity of the LY450108 cells under different experimental conditions, as.