Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.


Statistics
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Data analysis
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability We performed at least 3 independent experiments and capture multiple images on multiple tissues or wells. We specified the number of cells and independent experiments in the figure legends and/or methods. Fifteen to twenty wells of Meta4 organoids per condition were used for FACS analysis. A total of 5,000 cells per group were used for clonality analysis using CRA microwell system. About 50 organoids were used for in vitro assays. A total of 50 to 100 organoids were considered from three representative images taken from three wells of each organoid line for quantitative analyses.
We only exclude analyses if there is an obvious reason for poor data, such as dead or sick-looking organoids prior to drug treatment.
All attempts at replication are successful. We established 3 metaplastic and 3 dysplastic organoids lines from independent mouse stomach tissues. We performed at least 3 independent experiments and capture multiple images on multiple tissues or wells.
Both male and female mice were randomly used to establish organoid lines.
The mouse alleles which were used for this study are C57BL/6 wild-type or Kras-induced mice to establish metaplastic or dysplastic organoid lines. Thus, investigators were not blinded to mouse genotypes during experiments. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation