Single-Cell Profiling Reveals the Impact of Genetic Alterations on the Differentiation of Inflammation-Induced Murine Colon Tumors

Author:

Ghobashi Ahmed H.123ORCID,Lanzloth Rosie12,Ladaika Christopher A.123ORCID,Masood Ashiq34,O’Hagan Heather M.235ORCID

Affiliation:

1. Genome, Cell, and Developmental Biology Graduate Program, Department of Biology, Indiana University Bloomington, Bloomington, IN 47405, USA

2. Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405, USA

3. Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indianapolis, IN 46202, USA

4. Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA

5. Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Abstract

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (MinApcΔ716/+) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAFV600E mutation (BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+, BLM) or knocking out Msh2 (Msh2LoxP/LoxPVil1-creMinApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.

Funder

Indiana University School of Medicine

IU Simon Comprehensive Cancer Center (IUSCCC) Tumor Microenvironment & Metastasis Program and the IUSCCC P30 Support

National Cancer Institute (NCI) SPORE Project, Epigenetic Therapies—New Approaches

Doane and Eunice Dahl Wright Fellowship

NCI

Publisher

MDPI AG

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