Mouse Models of Human Gastric Cancer Subtypes With Stomach-Specific CreERT2-Mediated Pathway Alterations.

BACKGROUND & AIMS: Patterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes. METHODS: We searched databases to identify genes with unique expression in the stomach epithelium, resulting in the identification of Anxa10. We generated mice with tamoxifen-inducible Cre recombinase (CreERT2) in the Anxa10 gene locus. We created 3 mouse models with alterations in pathways that characterize the chromosomal instability (CIN) and the genomically stable (GS) subtypes of human gastric cancer: Anxa10-CreERT2;KrasG12D/+;Tp53R172H/+;Smad4fl/f (CIN mice), Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Smad4fl/fl (GS-TGBF mice), and Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Apcfl/fl (GS-Wnt mice). We analyzed tumors that developed in these mice by histology for cell types and metastatic potential. We derived organoids from the tumors and tested their response to chemotherapeutic agents and the epithelial growth factor receptor signaling pathway inhibitor trametinib. RESULTS: The gastric tumors from the CIN mice had an invasive phenotype and formed liver and lung metastases. The tumor cells had a glandular morphology, similar to human intestinal-type gastric cancer. The gastric tumors from the GS-TGFB mice were poorly differentiated with diffuse morphology and signet ring cells, resembling human diffuse-type gastric cancer. Cells from these tumors were invasive, and mice developed peritoneal carcinomatosis and lung metastases. GS-Wnt mice developed adenomatous tooth-like gastric cancer. Organoids derived from tumors of GS-TGBF and GS-Wnt mice were more resistant to docetaxel, whereas organoids from the CIN tumors were more resistant to trametinib. CONCLUSIONS: Using a stomach-specific CreERT2 system, we created mice that develop tumors with morphologic similarities to subtypes of human gastric cancer. These tumors have different patterns of local growth, metastasis, and response to therapeutic agents. They can be used to study different subtypes of human gastric cancer.

Human gastric cancer modelling using organoids.

OBJECTIVE: Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. DESIGN: Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. RESULTS: Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. CONCLUSION: We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing.

Gastrointestinal organoids: How they gut it out.

The gastrointestinal tract is characterized by a self-renewing epithelium fueled by adult stem cells residing at the bottom of the intestinal crypt and gastric glands. Their activity and proliferation is strongly dependent on complex signaling pathways involving other crypt/gland cells as well as surrounding stromal cells. In recent years organoids are becoming increasingly popular as a new and powerful tool to study developmental or other biological processes. Organoids retain morphological and molecular patterns of the tissue they are derived from, are self-organizing, relatively simple to handle and accessible to genetic engineering. This review focuses on the developmental processes and signaling molecules involved in epithelial homeostasis and how a profound knowledge of these mechanisms allowed the establishment of a three dimensional organoid culture derived from adult gastrointestinal stem cells.

Sensitization of Patient-Derived Colorectal Cancer Organoids to Photon and Proton Radiation by Targeting DNA Damage Response Mechanisms.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient's tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells.

The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.

Co-application of canavanine and irradiation uncouples anticancer potential of arginine deprivation from citrulline availability.

The moderate anticancer effect of arginine deprivation in clinical trials has been linked to an induced argininosuccinate synthetase (ASS1) expression in initially ASS1-negative tumors, and ASS1-positive cancers are anticipated as non-responders. Our previous studies indicated that arginine deprivation and low doses of the natural arginine analog canavanine can enhance radioresponse. However, the efficacy of the proposed combination in the presence of extracellular citrulline, the substrate for arginine synthesis by ASS1, remains to be elucidated, in particular for malignant cells with positive and/or inducible ASS1 as in colorectal cancer (CRC). Here, the physiological citrulline concentration of 0.05 mM was insufficient to overcome cell cycle arrest and radiosensitization triggered by arginine deficiency. Hyperphysiological citrulline (0.4 mM) did not entirely compensate for the absence of arginine and significantly decelerated cell cycling. Similar levels of canavanine-induced apoptosis were detected in the absence of arginine regardless of citrulline supplementation both in 2-D and advanced 3-D assays, while normal colon epithelial cells in organoid/colonosphere culture were unaffected. Notably, canavanine tremendously enhanced radiosensitivity of arginine-starved 3-D CRC spheroids even in the presence of hyperphysiological citrulline. We conclude that the novel combinatorial targeting strategy of metabolic-chemo-radiotherapy has great potential for the treatment of malignancies with inducible ASS1 expression.