Abstract Backgrounds and aims Many studies have reported the existence of tumor stem cells (TSCs) in solid tumors including colorectal cancer, and targeting TSCs is believed to be a promising cancer therapy. However, previously reported TSC markers are often expressed in normal stem cells (NSCs), and using those markers for cancer therapy would severely affect the normal tissues. Therefore, it is necessary to identify the specific markers for TSCs. To identify a TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was a putative marker of intestinal NSCs, but it has been debated whether Dclk1 marks NSCs or differentiated cells (e.g., Tuft cells). There have been no reports which elucidate the stemness of Dclk1-positive cells in the intestine and in tumors by lineage tracing. The aim of this study is to examine the role of Dclk1-positive cells in both the normal intestines and intestinal tumors. Methods We generated the knock-in mice with the creERT2-IRES-EGFP cassette under the Dclk1 promoter (Dclk1-creERT2 mice). These mice were crossed with Rosa26-LacZ mice, and the stemness of Dclk1-positive cells was examined by lineage tracing analyses in the intestines under normal homeostasis and experimental injury conditions (chemically-induced colitis and radiation-induced enteritis). Next, the stemness of Dclk1-positive tumor cells was also evaluated by lineage tracing in the polyps of ApcMin/+ mice. Character of Dclk1-positve tumor cells was investigated by immunohistochemistory and FACS analyses. Results In the normal intestines of Dclk1-creERT2; Rosa26-LacZ mice, scattered LacZ-labeled Dclk1-positive cells were observed 1 day after tamoxifen injection. However, these cells did not produce progeny, and disappeared within a few weeks. Stemness of Dclk1-positive cells was also strictly limited even under experimental injury conditions. On the contrary, lineage tracing in the polyps of ApcMin/+; Dclk1-creERT2; Rosa26-LacZ mice showed that Dclk1-positive tumor cells continuously produce tumor progeny and maintained the polyps over the prolonged period. Immunohistochemistory showed that a population of Dclk1-positive cells at the tumor base expressed proliferation markers and Lgr5. FACS analyses showed that the Dclk1- and Lgr5- double positive fraction showed the strongest expression of CD44, CD133, and Bcl-2 compared to other cell fractions. Conclusions Our lineage tracing experiments suggested that Dclk1 can be used as a marker which distinguishes TSCs from NSCs in the intestine. These results support the development of novel therapeutic strategies in colorectal cancer through the targeting Dclk1-positive TSCs. Citation Format: Yuki Nakanishi, Hiroshi Seno, Taro Ueo, Tsutomu Chiba. Intestinal tumors are continuously renewed by Dclk1-positive tumor stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5012. doi:10.1158/1538-7445.AM2013-5012