2018 Fiscal Year Annual Research Report
Developing mouse models of inflammation-driven invasive gastric cancer to reveal novel therapeutic targets
| Project/Area Number |
17H01399
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| Research Institution | Kanazawa University |
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Principal Investigator |
NICHOLAS BARKER 金沢大学, その他部局等, リサーチ・プロフェッサー (30787651)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | gastric cancer / mouse model / inflammation / Lgr5 / organoid |
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| Outline of Annual Research Achievements |
Our mechanistic understanding of gastric cancer remains limited, hampering the development of more effective therapeutics. Using proprietary new mouse models and ex vivo culture assays, we will i) evaluate the contribution of Lgr5-expressing corpus stem cells to inflammation-driven cancer initiation ii) generate the first inflammation driven mouse models of metastatic gastric cancer to functionally evaluate Lgr5-expressing tumor cells as cancer stem cells iii) evaluate the contribution of TCGA derived driver mutations such as RhoA and RNF43 to in vivo gastric cancer formation. These research avenues will deliver invaluable mechanistic insight into gastric cancer progression and will reveal novel opportunities for therapeutic intervention.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
1. We activated Lgr5-driven lineage tracing in adult Gan/Lgr5-2A-CreERT2/RosatdTomato mice with established chronic gastric inflammation via IP injection of tamoxifen. The tdTomato expression in the corpus epithelium was subsequently analyzed via confocal imaging/IHC to determine the in vivo output of the Lgr5+ corpus cells within the inflamed stomach epithelium. In parallel, we isolated the Lgr5-EGFP+ corpus cells from inflamed stomachs via FacSorting and evaluated their ability by generating epithelial organoids ex vivo. Organoid forming capacity of Lgr5+ cells from healthy and inflamed stomach epithelia was directly compared using in vitro assay.
2. We tried to generate the first inflammation-driven mouse model of invasive, metastatic gastric cancer to facilitate the functional evaluation of Lgr5-expressing tumor cells as cancer stem cells. Although, we succeeded to established a new invasive, metastatic gastric cancer mouse model, we couldn’t incorporate the inflammation setting in that model by March 2018. Finally, we succeeded to establish the inflammation-driven mouse model by July 2018. Using this mouse model, the functional evaluation of the cancer stem cell identity of the tumor-resident Lgr5-expressing cells was achieved by administering diphtheria toxin to mice with established gastric cancer/metastases. This achieve the selective ablation of the tumor-resident Lgr5+ cells in vivo. Any resulting effect on tumor growth will be subsequently evaluated via histological/marker analysis of the corpus regions from treated mice over months.
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| Strategy for Future Research Activity |
1. We will evaluate any inflammation-driven changes to the Lgr5+ corpus stem cells by isolating the EGFP+ cells from inflamed corpus tissues from adult Gan/Lgr5-DTR-EGFP mice via FacSorting and performing RNA-sequencing expression profiling.
2. We will evaluate the cancer stem cell identity of tumor-resident Lgr5+ cells via ex vivo culture/transplantation. Lgr5-EGFPhi and Lgr5-EGFPneg populations will then be isolated by FacSorting for GFP expression and evaluated their ability to generate epithelial cancer organoids ex vivo and in vivo.
3. Lgr5-EGFPhi and neg populations will be isolated from established gastric tumors organoids and RNA-sequencing will be performed. The resulting tumor cell transcriptomes will be compared with one another and with existing healthy Lgr5+ corpus stem cell transcriptomes to reveal novel mechanistic insight into the cancer stem cells. Surface expressed genes and druggable enzymes selectively expressed on the cancer stem cells will be validated via IHC/in-situ hybridization on mouse/ Human gastric tumors/metastases.
4. To evaluate the contribution of candidate TCGA derived driver mutations such as RhoA and RNF43 to in vivo gastric cancer formation, successful activation of RhoA mutations will be confirmed by IHC for active RhoA. Successful loss of RNF43 function will be confirmed by IHC/in-situ hybridization for RNF43. We will isolate tumor tissue from the various models for generating epithelial organoids ex vivo to determine any phenotypic changes to their growth characteristics as compared to non-cancer organoids.
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