Abstract Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer related deaths in the Unites States. Several therapies are currently being used to treat the disease but recurrence remains a challenge. It is believed that cancer stem cells (CSCs) may be resistant to currently used radiation and chemotherapeutic treatments, and may thus be the likely cause of relapse. It is postulated that CSCs may arise from normal adult stem cells and, therefore, preventing aberrant proliferation of stem/progenitor cells and/or targeting CSCs may improve treatment outcomes. However, to-date, there are no assays for examining response of ‘stem cells’ in vitro. Therefore one of our goals was to isolate stem cells from colorectal cancers, and establish stem/progenitor cell bioassays to examine inhibitory efficacy of chemo preventive agents and stem cell specific siRNA. In recent years, several putative stem/progenitor cell markers have been identified, including DCAMKL-1, CD44 and LGR5. Curcumin is being used in clinical trials for treating CRCs. For our studies we chose to isolate CSCs using the above indicated stem cell markers, each of which expresses an extracellular domain. We successfully isolated stem cell populations enriched in the indicated markers, and established tumorospheres from a colon cancer cell line (HCT-116); these growths are selective for stem cells, and can therefore be used in bioassays for examining inhibitory efficacy of drugs on stem cells in vitro. We examined the inhibitory efficacy of curcumin±DCAMKL-1 siRNA against growth of HCT-116 tumorospheres. Spheres were fixed and stained for DCAMKL-1, CD44, and LGR5, and examined by fluorescence confocal microscopy. The effect on growth signaling pathways was quantified by Western Blot analysis. Our results reveal that both DCAMKL-1 siRNA and curcumin exert inhibitory effects on growth of HCT-116 tumorospheres, and reduce relative expression levels of NFκBp65 and β-catenin in tumorospheres. Importantly, DCAMKL-1 siRNA potentiated inhibitory potency of Curcumin. In conclusion, we have successfully established in vitro methods for examining proliferative/inhibitory efficacy of various molecules on the biology and response of cancer stem cells, which can be expected to impact cancer treatment strategies. This work is supported by NIH grants CA97959 and CA114264 to PS and NASA grants NNX09AM08G and NNJ04HD83G to RU. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2011-4363