The number of dots per cell was obtained by counting all dots ROI having more than 50% of their surface included in each segmented cell perimeter defined previously. of Notch1+ tumour cells highly correlates with ISCs, suggestive of their origin from normal crypt cells. Surprisingly, Notch1+ expression labels a subset of CSCs that shows reduced levels of Lgr5, a reported CSCs marker. The existence of distinct stem cell populations within intestinal tumours highlights the necessity of better understanding their hierarchy and behaviour, to identify the correct cellular targets for therapy. Introduction Intestinal crypts have been reported to harbour two distinct types of stem cells: homeostatic stem cells, marked by the G-protein coupled receptor Lgr51, that continuously generate new progenitors to ensure efficient renewal of the intestinal mucosa, and presumably quiescent stem cells, thought to provide a reserve source of stem cells that can be activated upon injury2,3. We have shown that the Notch1 receptor is expressed in both homeostatic and reserve stem cells populations studies provide evidence for the existence of different types of CSCs in intestinal tumours, which might have different origins and/or exhibit differential response to treatment. Results Notch1-CreERT2 labels undifferentiated and proliferative tumour cells To track Notch1+ intestinal adenoma cells tumour suppressor gene and spontaneously develop intestinal adenomas, initially detectable at around six months of age, due to loss of heterozygosity (LOH) at the locus. In the compound N1-Cre/R26mTmG/Apc mice, the membrane-associated red fluorescent protein (mT) is expressed in all cells, while membrane-associated GFP (mG) marks Cre-targeted cells. To identify the cells expressing the Notch1 receptor within tumours, N1-Cre/R26mTmG/Apc tumour-bearing mice received a single dose of tamoxifen and were analysed 24?h later (Fig.?1b). Quantification by flow cytometry of the proportion of Notch1+ cells within tumour epithelial cells (selected with the markers EpCAM+/Lin-, see gate strategies in Supplementary Fig.?1), indicated, in agreement with our immunofluorescence results, that Notch1-expressing epithelial cells represent a rare tumour cell population comprising 1,2%??0,3% of tumour cells (Fig.?1c). It should be noted that, as the N1-Cre line also labels other types of stromal cells, we exclusively focused our analysis on epithelial cells, expressing the epithelial marker EpCAM (Epithelial cell adhesion molecule15) (Fig.?1d). Since mutant intestinal tumours present differentiated tumour cells, we evaluated if Notch1 is expressed in such cells by immunostaining for differentiation markers for secretory cells, such as Agglutinin (Ulex Europaeus Agglutinin, labelling both Paneth and Goblet SOX18 cells), Lysozyme116 (a specific marker of Paneth 4-Aminohippuric Acid cells) and Mucin217 (expressed in Goblet cells) (Fig.?1d). None of these markers was expressed in GFP+ cells, consistently with the lack of Notch1 expression in secretory cells in the normal intestinal epithelium9. We also assessed the expression of secretory and enterocyte (alkaline phosphatase intestinal, Alpi18) markers by qRT-PCR on sorted tumour cells and confirmed that GFP+ cells show strongly reduced levels of expression for all of these markers (Fig.?1e), indicating that the N1-Cre mouse line labels undifferentiated tumour cells. Open in a separate window Figure 1 Notch1-CreERT2 labels undifferentiated and proliferative tumour cells. (a) Schematic representation of the triple transgenic mouse model used in this study. Notch1CreERT2 knock-in mice (referred to as N1-Cre) were crossed to Rosa26mTmG reporter mice and to Apc+/1638N mice (termed Apc). (b) Representative section of an intestinal tumour from N1-Cre/R26mTmG/Apc mice, 24?h post tamoxifen injection. The inset shows a higher magnification of a Notch1-expressing tumour cell (marked by GFP 4-Aminohippuric Acid in green). DNA is labelled by DAPI in blue. Scale bars represent 200?m and 15?m in the magnification panel. (c) FACS analysis (see Supplementary Fig.?1 for gate strategy details) of tumour cells dissociated from N1-Cre/R26mTmG/Apc mice 24?h post induction. Lin+ cells were excluded and single epithelial tumour cells were gated as epithelial cells (Epcam+/Lin?), allowing the quantification of Notch1+ tumour cells. Note that GFP+ cells also display Tomato fluorescence 24?h after induction (GFP+/Tom+), as the Tomato protein is still present at this time point, even if recombination has occurred. (d) Immunofluorescence analysis of N1-Cre/R26mTmG/Apc tumour sections using anti-EpCAM, Agglutinin (UEA), 4-Aminohippuric Acid anti-lysozyme (Lyz1) and anti-Mucin2, all in red. Notch1-expressing tumour cells are labelled in green (GFP+) and DNA is marked by DAPI in blue. Magnifications insets are shown in the right panels. Arrows indicate Notch1-expressing tumour cells and asterisks show secretory tumour cells. (e) qRT-PCR showing the relative RNA expression of.