846 – Interleukin-27-Producing Immune Cells in the Gastric Mucosa Protect the Stomach from Inflammation-Induced Atrophy and Metaplasia

Intestinal type gastric cancer develops from pre-cancerous lesions by a multistep process of pre-cancerous cell progression and evolution.Cancer stem cells (CSCs) have self-renewal capacity and can give rise to heterogeneous lineages in tumors.While many previous investigations have identified CSC markers in human gastric cancer, it remains unknown whether the CSCs are already present in pre-cancerous stages and if those CSCs can give rise to gastric cancer cells.In this study, we examined whether putative CSC populations exist in pre-cancerous stages and show stemness potential.We have recently established pre-neoplastic organoids (Meta4) from Mist1-Kras mice, which develop pre-neoplastic glands within 4 months following induction of constitutively-active Kras expression in chief cells.We treated the Meta4 organoids with Selumetinib, a MEK inhibitor, to target downstream mediators of Kras signaling pathway and evaluated altered cellular behaviors, structural changes and survival.The Meta4 organoids treated with Selumetinib for 3 days died or did not show an increase in size, while DMSO-treated organoids grew continuously.We next performed inDrop single cell-RNA sequencing (scRNA-seq) in Meta4 cells treated with either DMSO or Selumetinib to define cellular heterogeneity and subpopulations of Meta4.The scRNA-seq data analysis identified four distinct cell subpopulations in both samples.Interestingly, two subpopulations were associated with stem cell phenotypes and expressed several cancer stem cell markers, such as CD44, CD133 and CD166.We isolated two distinct subpopulations of Meta4 cells using the CSC markers, CD44+CD133+CD166+ (triple positive) and CD44-CD133+CD166+ (double positive), by flow cytometry analysis and performed clonal analysis to define the stemness of the isolated cells.The clonal analysis demonstrated that double-positive cells produced spheres more efficiently than triple-positive cells.We also co-stained for the CSC markers with Ki67, a proliferation marker, in pre-neoplastic stomach tissues of Mist1-Kras mice.We identified the two sub-populations at the base of glands in the mucosa.Many double-positive cells rather than triple-positive cells were copositive for Ki67.We additionally examined the expression of these CSC markers in human normal, metaplastic and gastric cancer tissues and observed the presence of the CSCs from the metaplastic stage.Collectively, these results indicate the functional heterogeneity of Meta4 cells and the presence of putative CSC populations in Meta4.Therefore, our study suggests that the putative CSC populations, present in the pre-cancerous stages, may be responsible for maintaining pre-cancerous heterogeneity and the cell state transition during pre-cancerous progression towards gastric cancer.