The Establishment and Characterisation of Gastric Organoids as a Model for Hereditary Diffuse Gastric Cancer

Cancer is an exceptionally complex disease which requires an appropriately sophisticated model to facilitate its research. Organoids are 3D structures generated from stem cells, which recapitulate a target organ. These spherical cell cultures have the delicate organisation of the in vivo tissue, albeit on a smaller, simpler scale. Organoids are emerging in the field as invaluable tools for studying normal organogenesis and disease. Additionally, they can be used as an intermediate step between 2D cell lines and animal models in drug screens. This project describes the establishment and characterisation of mouse-derived gastric organoids as a disease model and medium-throughput drug screening tool for E-cadherin negative (CDH1-/-) hereditary diffuse gastric cancer (HDGC). Their primary use will be to identify drugs that can be used as chemopreventative treatments for HDGC. Organoids were cultured using an air-liquid interface (ALI) method from the gastric stem cells of conditional Cdh1 knockout mice. Cdh1 deletion was induced by co-culturing with endoxifen. Immunofluorescence and confocal microscopy have been used in the characterisation of the organoids and to validate the efficacy of Cdh1 knockout. For drug screening, organoids were first exposed to the drug 48h post-seeding, and effects were recorded at 24 h intervals for 96 h. Our gastric organoids harbour functional populations of epithelial cells and proliferating stem cells. They are spherical and cystic in shape, containing an inner lumen surrounded by epithelium. Cdh1-negative cells accumulate in the lumen of the organoids, possibly due to their impaired cell-cell adhesion ability relative to the Cdh1-positive cells. ARQ-092 and MK2206, both pan-AKT inhibitors, have been shown to reduce growth and induce death in the organoids in a synthetic lethal manner, validating their use as a drug screening tool for the development of a treatment for HDGC

The Establishment and Characterisation of Gastric Organoids as a Model for Hereditary Diffuse Gastric Cancer

Cancer is an exceptionally complex disease which requires an appropriately sophisticated model to facilitate its research. Organoids are 3D structures generated from stem cells, which recapitulate a target organ. These spherical cell cultures have the delicate organisation of the in vivo tissue, albeit on a smaller, simpler scale. Organoids are emerging in the field as invaluable tools for studying normal organogenesis and disease. Additionally, they can be used as an intermediate step between 2D cell lines and animal models in drug screens. This project describes the establishment and characterisation of mouse-derived gastric organoids as a disease model and medium-throughput drug screening tool for E-cadherin negative (CDH1-/-) hereditary diffuse gastric cancer (HDGC). Their primary use will be to identify drugs that can be used as chemopreventative treatments for HDGC. Organoids were cultured using an air-liquid interface (ALI) method from the gastric stem cells of conditional Cdh1 knockout mice. Cdh1 deletion was induced by co-culturing with endoxifen. Immunofluorescence and confocal microscopy have been used in the characterisation of the organoids and to validate the efficacy of Cdh1 knockout. For drug screening, organoids were first exposed to the drug 48h post-seeding, and effects were recorded at 24 h intervals for 96 h. Our gastric organoids harbour functional populations of epithelial cells and proliferating stem cells. They are spherical and cystic in shape, containing an inner lumen surrounded by epithelium. Cdh1-negative cells accumulate in the lumen of the organoids, possibly due to their impaired cell-cell adhesion ability relative to the Cdh1-positive cells. ARQ-092 and MK2206, both pan-AKT inhibitors, have been shown to reduce growth and induce death in the organoids in a synthetic lethal manner, validating their use as a drug screening tool for the development of a treatment for HDGC

Loss of E-Cadherin Leads to Druggable Vulnerabilities in Sphingolipid Metabolism and Vesicle Trafficking

Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC

Hereditary diffuse gastric cancer: updated clinical practice guidelines

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.No Stomach for Cancer DeGregorio Foundation DD & DF Heads Charitable Trust University of Otago New Zealand Health Research Council Programme 17/610 Portuguese Foundation for Science and Technology POCI-01-0145-FEDER-3016