Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency

Developers

Kerstin Keysselt, Karen Rother, Joerg Galle, Gabriela Aust, etc.

Description of the technology

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer, including its development in process of ageing. This technology was created for study of formation of Lynch syndrome/hereditary nonpolyposis colorectal cancer. It originates in heterozygous germline mutations in mismatch repair (MMR) genes. Among them, mutations of MutS homolog 2 (MSH2) and MutL homolog 1 (MLH1) are the most common.

In order to develop this technology the culture cell model of intestinal epithelium was used. Intestinal epithelium is maintained by intestinal stem cells (ISCs). The identification of signaling pathways responsible for their self-renewal enabled ISC cultures from isolated intestinal crypts to form organoids. In this context, the term «organoid» is used for designation of the structures, which are formed in culture in vitro from intestinal stem cells, and resemble the structures of normal intestine or intestinal tumors. Kinetics and cell-type composition of these organoids reflect the in vivo situation, making them an advantageous model to study ISC transformation following MMR gene inactivation over time.

The time scale, sequence and extension of molecular and phenotypic changes in the epithelium preceding the onset of intestinal tumors were examined. To analyze and understand the profile of intestinal stem cell (ISC) transformations, MSI and growth characteristics of organoids were quantified in Msh2-deficient and control mice from birth until tumor formation. Then these characteristics were related to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency.

In summary, as highlighted by organoid model, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation. The accumulation of the mutations, particularly in Msh2−/− tissue or organoids, can also reflect the ageing process, so the used model is applicable for study of formation of the colorectal cancer in process of ageing.

Practical application

The technology, using intestinal organoids of mice with MMR deficiency and those of control mice, enables the study and understanding the accumulation of genetic imbalances during ageing, long-term culture and growth factor withdrawal of ISCs.

This study allows to develop early interventions that suppress essential steps in pre-neoplastic transformation in the intestine and created novel approaches to anti-cancer therapy.

Laboratories

• Department of Surgery, Research Laboratories, University of Leipzig, Leipzig (Germany)
• Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, Leipzig (Germany)
• Interdisciplinary Center for Clinical Research Leipzig (IZKF), Core-Unit DNA Technologies, University of Leipzig, Leipzig (Germany)
• Institute for Medical Statistics, Informatics and Epidemiology, University of Leipzig, Leipzig (Germany)

Links

Publications

• Keysselt, K. et al. «Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency." Oncogene, 2016 Dec 12. doi:10.1038/onc.2016.429. (Advance online publication).
• Hamidouche, Z. et al. «Bistable Epigenetic States Explain Age-Dependent Decline in Mesenchymal Stem Cell Heterogeneity." Stem Cells. 2016 Oct 13. doi: 10.1002/stem.2514
• Thalheim, T. et al. «Stem cell competition in the gut: insights from multi-scale computational modelling." 13.121 J R Soc Interface. (2016): 20160218.