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Induction of gastric cancer by successive oncogenic activation in the corpus

ABSTRACT: Background & Aims: Metaplasia and dysplasia in the corpus are reportedly derived from dedifferentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen Ctranscript expressing cells (PGC-transcript expressing cells) represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model. Methods: We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate PgcCreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments, histological and immunofluorescence staining. We further established Pgc-CreERT2;Kras G12D/+ mice and investigated whether PGCtranscript expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC-transcript expressing cells with activated Kras, inactivated Apc and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D , Apcflox , Trp53 flox mice. Results: Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;Kras G12D/+ mice, PGC-transcript expressing cells with Kras G12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven Kras G12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+ ;Apc flox/flox mice presented intramucosal dysplasia/carcinoma, while Pgc-CreERT2;KrasG12D/+ ;Apcflox/flox ;Trp53 flox/flox mice presented invasive and metastatic gastric carcinoma. Conclusions: The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.

ORGANISM(S): Mus musculus

PROVIDER: GSE166904 | GEO | 2021/02/17

REPOSITORIES: GEO

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Project description:We treated the following 3 groups of 8-16 week old mice with tamoxifen: 1) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+, Trp53 flox/flox mice (n=6); 2) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+,Trp53 R270H/flox mice (n=6); 3) Wild-type control mice (n=3). The CDX2P-CreERT2 transgene expresses a tamoxifen (TAM)-regulated Cre protein (CreERT2) under control of human CDX2 regulatory sequences, allowing for TAM-inducible targeting of flox alleles in adult mouse terminal ileum, cecum, and colon epithelium. Treating the mice having CDX2P-CreERT2 transgene with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the floxed alleles for Apc, Kras, and Trp53, resulting in deletion mutations in Apc and Trp53, and an activating, oncogenic mutation in Kras (G12D mutation). The Trp53 R270H allele carries a constitutive R270H mutation, which is the mouse equivalent of human TP53 R273H mutation. Laser capture microdissection was used to obtain adenocarcinoma tissue samples from the first two groups of mice and normal tissue from control mice. RNA was purified and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST plate arrays, which hold 41345 probe-sets, but we largely analyzed just those 24562 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the 3 groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file for those 24562 probe-sets, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups. It also shows data and analysis from a separate experiment of RNA purified from organoids cultured from samples from 3 groups of mice similar to those here, as well as an extra group of mice with CDX2P-CreERT2 Apc flox/flox genotype. It also joins a statistical summary of differences between 9 human tumors with TP53 missense mutations at codon 273 and 36 tumors with TP53 null mutations assayed with RNA-seq by the TCGA project. A separate supplementary file of the TCGA data is also provided. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

Project description:For organoid preparation, we first treated the following 4 groups of 8-12 week old mice with tamoxifen: 1) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+, Trp53 flox/flox mice (n=2); 2) CDX2P-CreERT2, Apc flox/+, Kras LSL-G12D/+,Trp53 R270H/flox mice (n=3); 3) CDX2P-CreERT2, Apc flox/flox mice (n=3); 4) Wild-type control mice (n=4). The CDX2P-CreERT2 transgene expresses a tamoxifen (TAM)-regulated Cre protein (CreERT2) under control of human CDX2 regulatory sequences, allowing for TAM-inducible targeting of flox alleles in adult mouse terminal ileum, cecum, and colon epithelium. Treating the mice having CDX2P-CreERT2 transgene with tamoxifen permits the Cre recombinase to enter the cell nucleus and recombine the floxed alleles for Apc, Kras, and Trp53, resulting in deletion mutations in Apc and Trp53, and an activating, oncogenic mutation in Kras (G12D mutation). The Trp53 R270H allele carries a constitutive R270H mutation, which is the mouse equivalent of human TP53 R273H mutation. Colon tumors were induced by TAM treatment in all the mice from the first three groups and organoids were derived from the tumors of each mouse. We also derived organoids from the normal colon epithelium in the 4th group of mice as controls. All organoids were generated and propagated using a slightly modified TMDU protocol as described in PMID:20872391. Organoids were cultured for 4 days and then harvested. RNA was purified from the organoids, and targets for Affymetrix arrays were synthesized from the mRNAs. We used Affymetrix Mouse Gene 2.1 ST plate arrays, which hold 41345 probe-sets, but we largely analyzed just those 24562 probe-sets that were mapped to Entrez gene IDs. Raw data was processed with the Robust Multi-array Average algorithm (RMA). Data is log2-transformed transcript abundance estimates. We fit a one-way ANOVA model to the 4 groups of samples. We supply a supplementary excel workbook that holds the same data as the data matrix file for those 24562 probe-sets, but also holds the probe-set annotation at the time we analyzed the data, and some simple statistical calculations, which selects subsets of the probe-sets as differentially expressed between pairs of groups. It also shows data and analysis from a separate experiment of RNA purified directly from 3 groups of mice with genotypes like those of the organoid data except that no group of mice with CDX2P-CreERT2 Apc flox/flox genotype were used. It also joins a statistical summary of differences between 9 human tumors with TP53 missense mutations at codon 273 and 36 tumors with TP53 null mutations assayed with RNA-seq by the TCGA project. A separate supplementary file of the TCGA data is also provided. Consumers should consider obtaining more up-to-date probe-set annotation for the array platform.

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Induction of gastric cancer by successive oncogenic activation in the corpus

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