Silent Waters Run Deep. Quiescent stem cells in homeostasis and cancer

__Abstract__ The Introduction summarizes the current literature on quiescence in adult stem cell niches and the various methods for the isolation of quiescent stem cells, outlines the complexity of the intestinal stem cell niche, and formulates the hypothesis that quiescent stem cells are involved in inflammation-associated tumours. Moreover, quiescent cancer stem cells and their clinical relevance are reviewed. In Chapter 1 and 2 we describe the generation of tightly regulated doxycyclineinducible models for studying mouse intestinal and esophageal biology respectively. The mouse model developed and characterized in Chapter 1 was employed in Chapter 3 to isolate and characterize a quiescent label-retaining cell population present within the intestinal epithelium. Chapters 4 to 6 describe the role of quiescent cells during tissue injury and their presence in full-blown tumors. In Chapter 4, we describe the induction of colonic inflammation in tumor-prone Apc1638N/+/villin-KRASG12V animals. These mice develop colon cancer characterized by the presence of Paneth-like cells. In Chapter 5, a rare villin-expressing, quiescent population of tumor cells is shown to be present in stomach tumors of the intestinal type from Apc1638N/+ animals. Moreover, we characterized quiescent, label-retaining tumor cells in small intestinal adenocarcinomas of Apc1638N/+/villin-KRASG12V animals, as outlined in Chapter 6. Finally, the Discussion summarizes the main finding of this thesis and gives directions for future research

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

Background: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. Results: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we ubsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 710 days. Conclusions: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. oreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 710 days

Cancer Stemness in Apc- vs. Apc/KRAS-Driven Intestinal Tumorigenesis

Constitutive activation of the Wnt pathway leads to adenoma formation, an obligatory step towards intestinal cancer. In view of the established role of Wnt in regulating stemness, we attempted the isolation of cancer stem cells (CSCs) from Apc- and Apc/KRAS-mutant intestinal tumours. Whereas CSCs are present in Apc/KRAS tumours, they appear to be very rare (®-catenin intracellular stabilization