Background & AimsPatterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes.MethodsWe 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.ResultsThe 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.ConclusionsUsing 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.

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent “Warburg effect” blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.