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Tumour Stem Cell Marker Identified

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“Dclk1 distinguishes between tumor and normal stem cells in the intestine”

The study of tumour stem cells (TSCs) has been hindered by the lack of selective markers which can differentiate between normal stem cells (NSCs) and TSCs. In the intestine, many markers of NSCs and TSCs are shared (Delarba et alO’Brien et alZhu et al and Todaro et al) and so identifying TSC-specific markers is an important challenge. In the following study, researchers from the laboratory of Tsutomu Chiba at Kyoto University Graduate School of Medicine, Japan have studied the doublecortin-like kinase 1 (Dclk1) gene, previously identified in mouse and human intestine (May et al 2008 and May et al 2009) and associated with mouse intestinal tumors and human colorectal carcinomas (May et al 2008 and Gerbe et al), and have found that Dclk1 does not mark NSCs in the intestine but instead marks TSCs, with specific ablation of Dclk1-positive TSCs leading to the regression of intestinal polyps (Nakanishi et al).

Knockin mice were generated by integrating a CreERT2-IRES-EGFP cassette into Dclk1 (creating Dclk1CreERT2/+ mice), and Dclk1-positive cells detected in the intestine were distinct from other epithelial cell populations. Tamoxifen-induced Cre-mediated lineage tracing (Sauer et al and Soriano) in the small intestine and colon of Dclk1CreERT2/+; Rosa26R mice found scattered LacZ-labelled blue cells mainly in the lower crypt, which migrated toward the villus tip on a daily basis, accompanied by a decrease in their numbers.   Additionally, these cells did not incorporate bromodeoxyuridine (BrdU) altogether suggesting that Dclk1 is not a stem cell marker in the normal intestine. Also, Dclk1-positive cells rarely produced progeny during mucosal regeneration after mucosal injury, and their potential stemness was strictly limited. Moving onto analysis of Dclk1 in TSCs in a mouse tumour model, lineage-tracing analyses of Dclk1CreERT2/+; Rosa26R; ApcMin/+ mice found LacZ-labelled blue cells in intestinal polyps. At day 1 of tamoxifen injection, 3.1% of tumor cells were labelled with LacZ, while at 5-7 days labelling had spread to occupy the entire polyp.

Dclk1-positive tumor cells expressed proliferation markers, particularly at the polyp base, and some were also positive for Lgr5, which suggested that these double-positive cells were candidate TSCs in ApcMin/+ mice.  Analysis of another tumour model system (Lgr5CreERT2/+; Ctnnb1lox(ex3)/+ mice) found double-positive cells at the base of intestinal polyps, but this was not observed in Dclk1CreERT2/+; Ctnnb1lox(ex3)/+ mice, which did not develop intestinal polyps, suggesting that Dclk1-positive cells in the normal intestine do not transform into TSCs directly and that Dclk1 expression arises secondarily in Lgr5-positive TSCs. Analysis of differing cell populations (Dclk1−Lgr5−, Dclk1+Lgr5−, Dclk1−Lgr5+ and Dclk1+Lgr5+) from Lgr5CreERT2/+ and Ctnnb1lox(ex3)/+ mice polyps found the proportion of Dclk1+Lgr5+ tumor cells in the total Dclk1-positive cell population to be 5.3%, similar to immunohistochemical data. Of the 4 cell populations sorted, TSC markers (CD44 and CD133) and anti-apoptotic markers (Bcl2) were highest in the double-positive population which also contained occasional Ki67-positive proliferative cells.

Finally, the targeting of these cells as a potential anti-tumour therapy was investigated in Dclk1CreERT2/+; Rosa26R; ApcMin/+; Rosa26iDTR/+ mice, in which Cre activation induces the diphtheria toxin receptor in Dclk1-positive cells and subsequent administration of diphtheria toxin results in the death of these cells (Buch et al and Saito et al). In normal tissues, no damage was observed upon tamoxifen and diphtheria toxin treatment, however severely injured or collapsed polyps were observed;  apoptotic Dclk1-positive tumor cells were seen  at 12 h after diphtheria toxin injection, while at later time points, Dclk1-positive tumor cells were absent, and the proportion of total apoptotic cells returned to the basal level, suggest that diphtheria toxin successfully eliminated only Dclk1-positive cells (TSCs) without affecting other tumor cells.

These data suggest that Dclk1 represents an important marker of tumour stem cells in the mouse intestine when analysed alongside another potential marker, Lgr5. Tumour regression after specific ablation of Dclk1-positive TSCs suggests that targeting this marker alone may be sufficient to halt tumorigenesis and, importantly, the anti-tumour therapy used did not seem to affect normal stem cells.  

References

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  • Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. USA 104, 10158–10163 (2007).
  • Gerbe, F. et al. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. J. Cell Biol. 192, 767–780 (2011).
  • May, R. et al. Doublecortin and CaM kinase-like-1 and leucine-rich-repeat–containing G-protein–coupled receptor mark quiescent and cycling intestinal stem cells, respectively. Stem Cells 27, 2571–2579 (2009).
  • May, R. et al. Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice. Stem Cells 26, 630–637 (2008).
  • O’Brien, C.A. et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106–110 (2007).
  • Saito, M. et al. Diphtheria toxin receptor–mediated conditional and targeted cell ablation in transgenic mice. Nat. Biotechnol. 19, 746–750 (2001).
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  • Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).
  • Todaro, M. et al. Colon cancer stem cells: promise of targeted therapy. Gastroenterology 138, 2151–2162 (2010).
  • Zhu, L. et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 457, 603–607 (2009).

Study originally appeared in Nature Genetics.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.