Lgr5‐expressing stem cells are not the cells of origin of pyloric neuroendocrine carcinomas in mice

Abstract

In intestinal and pyloric epithelia, leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-expressing cells represent long-lived adult stem cells that give rise to all epithelial cell types, including endocrine cells. Ablation of the Apc gene in Lgr5-expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5-positive stem cells, we crossed Lgr5-EGFP-IRES-creER(T2) mice, which express EGFP and Cre recombinase driven by the Lgr5 promoter, with CEA424-SV40-TAg mice, which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19 day-old mice, single SV40 T antigen (TAg)-positive cells were identified preferentially at the the bases of pyloric glands, close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5-positive cells in gastrointestinal neoplasia, we could not detect Lgr5-EGFP-positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg, since β-catenin-mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424-SV40-TAg tumours, we performed tracing experiments using Lgr5-EGFP-IRES-creERT2:CEA424-SV40-TAg:ROSA26-tdRFP mice. Following tamoxifen induction, it was possible to efficiently trace the progeny of Lgr5-expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP-positive tumour cells were detected, even when RFP gene activation occurred in 7 day-old mice well before the appearance of TAg-positive tumour cells. Hence, we conclude that Lgr5-expressing stem cells probably do not constitute the cells of origin in CEA424-SV40-TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5-positive stem cells. Thus, additional long-lived LGR5-negative stem cells or progenitor cells with a low turnover rate might exist in the pyloric region, which could give rise to tumours.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer

“…After having collected evidence that both drug and target are present in the tumor, the preclinical efficacy of fasudil was monitored in living animals by PET/CT and post mortem by histopathological examination. Consistent with the known high metabolic and proliferative activity of the gastric tumor [27] , [28] , [45] , [ 18 F]-FDG uptake was clearly visible in an anatomical area corresponding to the localization of the pylorus, below the heart and above the two kidney signals. The 3D tumor volume (SUV (ccm)) was significantly reduced in therapy vs. control mice.…”

“…We resorted to a transgenic mouse model of GC [27] , where the viral oncogene SV40 large T-antigen is expressed as a transgene under the human CEA promoter specifically in the lower stomach (pylorus) of the mice, and which has been successfully subjected to pharmacological intervention by us before [32] , [43] , [44] . This tumor is characterized by early onset (within 4 weeks of age), a high proliferation index and up-regulation of stem cell (Wnt/Notch) [45] and neuroendocrine differentiation signatures [28] culminating in a small cell carcinoma of the lower part of the mouse stomach, the pylorus. At late stages, microinvasion and prolaps of luminal tumor nests toward the upper duodenum can be observed.…”

Models of Gastroenteropancreatic Neuroendocrine Neoplasms: Current Status and Future Directions

“…By means of inducible lineage tracing of LGR5-positive adult stem cells in the CEA424-SV40-Tag model, the origin of the neuroendocrine carcinomas was more directly addressed. Results clearly separated SV40-Tag and LGR5 expression, respectively, consistent with long-lived pre-enteroendocrine precursor cells or reserve stem cells as putative tumor originating cells [185].…”

Gastric Stem Cells: Physiological and Pathological Perspectives

“…As stated before, some stem cells contribute to the production of enteroendocrine cells in the gastric epithelium (Table 1), but whether these stem cells can directly give rise to neuroendocrine tumors is not well determined. For example, results from animal models indicated that Lgr5 + cells may not be the origin cells for pyloric neuroendocrine carcinomas, though this subtype stem cells give rise to endocrine cells in this region (Vetter et al, 2016). Besides, evidence for the interaction between enteroendocrine cells and stem cells is limited.…”