the-organoid-reconstitution-assay-ora-for-functional-analysis

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Erasmus University Medical Center

Among NSAIDs Celecoxib is one of the most efficient in triggering in vitro cancer cell death, and from this perspective has been subject of numerous studies. However, it is still controversial whether this in vitro-observed effect can also occur in vivo and contribute to the antitumor action of the drug. Moreover, besides common agreement on the involvement of COX-independent pathways, the mechanisms underlying Celecoxib toxicity are still unclear. In an attempt to shed light on these mechanisms, I found that cell death only occurs at insoluble concentrations of the drug, and follows irreversible binding and damage of the plasmamembrane by precipitates. This evidence strongly suggests that Celecoxib is devoid of true molecular toxicity. Moreover, since plasma levels reached during therapy are far below the threshold of toxic precipitation, direct cytotoxicity by Celecoxib is unlikely to occur on tumor cells in vivo. Thus the antitumor effect might be only due to COX inhibition, which requires significantly lower levels of the drug. Nonetheless, direct cytotoxicity might not be confined to an in vitro artifact, but contribute to the upper gastrointestinal side effects of Celecoxib. Overall, these findings represent an important basis for further studies on Celecoxib, where true molecular actions of the drug should be discriminated from the precipitate-dependent ones, and the relationship between in vitro and in vivo effects considered at the light of the precipitate-dependent model. Moreover, remarkably, this article indicates a model of critical analysis that can be extended to other poorly soluble drugs.

Cancer cell killing by Celecoxib: reality or just in vitro precipitation-related artifact?

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5(+) crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5(+) CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5(+) CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5(+) CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5(+) CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5(+) CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Andrea Sacchetti

Department of Pathology,
Erasmus MC Cancer Institute,
Department of Pathology, Erasmus MC Cancer Institute

Erasmus University Medical Center

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

Andrea Sacchetti

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,Erasmus MC Cancer Institute,Department of Pathology, Erasmus MC Cancer Institute

Erasmus University Medical Center

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

Department of Pathology,
Erasmus MC Cancer Institute,
Department of Pathology, Erasmus MC Cancer Institute