Abstract A013: Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells

Abstract

Abstract Characterization of colon cancer stem cells in vivo is extremely challenging. Our mouse model provides the opportunity to do so, as it displays oncogenic dedifferentiation. In this mouse model, Wnt and TGF signaling are deregulated by simultaneously mutating their transcriptional effectors, b-catenin and Smad4 respectively. This mouse model referred to as b-catenin gain-of-function and Smad4 loss-of-function (b-cateninGOF-Smad4LOF) conditional mutant displays oncogenic dedifferentiation in the intestinal epithelium. Within a week of inducing the mutation, villi cells (the mature epithelial compartment) express proliferative and stem cell markers; eventually developing adenomas (reported previously). Two striking observations, however, remain unexplained: firstly, fate reversal to stemness is seen only in a minor fraction of the epithelial cells despite the mutation being pan-epithelial; secondly, when the mutation is induced in a subset of stem cells, selective pressure is seen against the retention of mutant stem cells in the crypt s(progenitor compartment), and adenomas occur preferentially near the luminal epithelium. Our goal is to a) determine the basis for the selective pressure against mutant stem cells, and b) the differences between the normal and the b-cateninGOF-Smad4LOF mutant intestinal stem cells. To determine the gene expression profile unique to the dedifferentiating cells, we performed scRNA-seq on the dedifferentiating cells. To sort these cells–which express CD44 or proliferative markers–we integrated a reporter gene that expresses red fluorescent protein (RFP) into the b-cateninGOF-Smad4LOF mice. FACS for CD44 and RFP was performed seven days after the mutation to isolate the dedifferentiating cells from the mutant villi and the proliferating and/or CD44 positive cells in the mutant crypts. scRNA-seq confirmed enrichment of stem cell markers in the dedifferentiating cells. Gene set enrichment analyses revealed significant enrichment for the molecular signatures associated with Myc targets and active OXPHOS and Mtorc1 signaling in the dedifferentiating cells. The increase in OXPHOS is contrary to the expected metabolism in tumors. However, bulk RNA-seq for differential gene expression in the mutant villi epithelium and molecular analyses show enrichment of hypoxia gene targets and transient increase in hypoxia. These contradictory results are consistent with the spatial and temporal heterogeneity of hypoxia and metabolism observed in tumors and support the notion that intestinal stem cells rely on OXPHOS using the metabolites derived from the glycolytic metabolism of the neighboring cells. To determine the differences between normal intestinal stem cells and mutant stem cells, we cultured epithelium in 3D-matrigel to derive organoids from wild-type and mutant epithelium. The mutant organoids show higher expression of the CD44 splice variant implicated in cancer. Since stem cells are attributed to the organoid formation, our data indicate that stem cells from the mutant cells are oncogenic stem cells. Citation Format: Ansu O. Perekatt, Kylee Wrath, Zahra Hashemi, Dahlia Matouba, Christina Li. Single-cell RNA sequencing of dedifferentiating cells from a mouse model that display oncogenic plasticity: Implications in understanding colon cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A013.