Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium.

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Menin Maintains Cholesterol Content in Colorectal Cancer via Repression of LXR-Mediated Transcription.

BACKGROUND AND AIMS: Menin is a nuclear scaffold protein that regulates gene transcription in an oftentimes tissue-specific manner. Our previous work showed that menin is over-expressed in colorectal cancer (CRC); however, the full spectrum of menin function in colonic neoplasia remains unclear. Herein, we aimed to uncover novel menin-regulated pathways important for colorectal carcinogenesis. METHODS: RNA-Seq analysis identified that menin regulates LXR-target gene expressions in CRC cell lines. Isolated colonic epithelium from Men1f/f;Vil1-Cre and Men1f/f mice was used to validate the results in vivo. Cholesterol content was quantified via an enzymatic assay. RESULTS: RNA-Seq analysis in the HT-29 CRC cell line identified that menin inhibition upregulated LXR-target genes, specifically ABCG1 and ABCA1, with protein products that promote cellular cholesterol efflux. Similar results were noted across other CRC cell lines and with different methods of menin inhibition. Consistent with ABCG1 and ABCA1 upregulation, and similarly to LXR agonists, menin inhibition reduced the total cellular cholesterol in both HT-29 and HCT-15 cells. To confirm the effects of menin inhibition in vivo, we assessed Men1f/f;Vil1-Cre mice lacking menin expression in the colonic epithelium. Men1f/f;Vil1-Cre mice were found to have no distinct baseline phenotype compared to control Men1f/f mice. However, similarly to CRC cell lines, Men1f/f;Vil1-Cre mice showed an upregulation of Abcg1 and a reduction in total cellular cholesterol. Promoting cholesterol efflux, either via menin inhibition or LXR activation, was found to synergistically suppress CRC cell growth under cholesterol-depleted conditions and when administered concomitantly with small molecule EGFR inhibitors. CONCLUSIONS: Menin represses the transcription of LXR-target genes, including ABCA1 and ABCG1 in the colonic epithelium and CRC. Menin inhibition conversely upregulates LXR-target genes and reduces total cellular cholesterol, demonstrating that menin inhibition may be an important mechanism for targeting cholesterol-dependent pathways in colorectal carcinogenesis.