Data-analysis strategies for image-based cell profiling

Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences.Peer reviewe

E-Cadherin-Deficient Cells Are Sensitive to the Multikinase Inhibitor Dasatinib

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for the cancer syndrome hereditary diffuse gastric cancer (HDGC). CDH1-deficient gastric cancers exhibit high AKT serine/threonine kinase 3 (AKT3) expression, but specific drugs against this AKT isoform are not available. We therefore used two publicly available datasets to identify AKT3-associated genes which could be used to indirectly target AKT3. Reactome analysis identified an enrichment of extracellular matrix remodelling genes in AKT3-high gastric cancers. Of the 51 genes that were significantly correlated with AKT3 (but not AKT1), discoidin domain receptor tyrosine kinase 2 (DDR2) showed the strongest positive association. Treatment of isogenic human cells and mouse gastric and mammary organoids with dasatinib, a small molecule inhibitor of multiple kinases including SRC, BCR-ABL and DDR2, preferentially slowed the growth and induced apoptosis of E-cadherin-deficient cells. Dasatinib treatment also preferentially slowed the growth of gastric and mammary organoids harbouring both Cdh1 and Tp53 mutations. In organoid models, dasatinib treatment was associated with decreased phosphorylation of total AKT, with a stronger effect seen in Cdh1-deficient organoids. Treatment with combinations of dasatinib and an inhibitor of AKT, MK2206, enhanced the effect of dasatinib in breast MCF10A cells. In conclusion, targeting the DDR2-SRC-AKT3 axis with dasatinib represents a promising approach for the chemoprevention and chemotherapy of gastric and breast cancers lacking E-cadherin

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNF? and co-treatments

10.1038/srep43541Scientific Reports74354

E-Cadherin-Deficient Epithelial Cells Are Sensitive to HDAC Inhibitors

Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1−/− breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1+/− murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers

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