Final Report Summary - ICSC LGR5 (Identification and Genetic Profile Characterization of Lgr5 positive Colon Cancer Stem Cells)
Project context and objectives
It is firmly established that inappropriate activation of the Wnt signalling pathway via mutations in core pathway components such as APC is one of the initiating events in colon cancer. The cancer stem cell hypothesis postulates that a small reservoir of self-sustaining cells is exclusively able to self-renew and generates the cell population that constitutes the bulk of the tumour. Importantly, cancer stem cells (CSC) not only fuel tumour growth but also tumour metastases. In 2002, Lgr5 was identified as a gene expressed in colon cancer. By using mouse genetic engineering we deleted APC exclusively in the Lgr5 intestinal stem cells. We observed transformation of the epithelia within days. Transformed stem cells remain located at the crypt bottoms, while fuelling a growing microadenoma. We also observed that cell hierarchy between stem cells, progenitors and differentiated cells was still maintained in early stem-cell-derived adenomas, lending support to the 'cancer stem cell' concept. These results were published in the journal Nature on 29 January 2009 (Barker, Nature 2009).
Lgr5 expression is not only restricted to a subset of cells in the small intestine but we have also found Lgr5 expression in other adult tissues, suggesting that it may be a more general marker of adult stem cells. Therefore, we next set out to investigate whether Lgr5 could indeed be a bona fide marker for adult stem cells and cancer stem cell populations in other tissues. The stomach shares a number of features with the intestine, including a common endodermal origin and a constantly renewing epithelium. Using mouse genetic manipulation and in vivo lineage tracing we found that indeed the intestinal stem cell marker Lgr5 also marks an active adult stem cell population at the base of the stomach glands. We isolated single Lgr5 cells from normal stomach epithelia and analysed their genetic profile using microarray gene expression analysis. We found robust canonical Wnt signalling activity on the Lgr5 stem cell population whereas several lineage-specific genes (enteroendocrine-pit cell) were absent in the stem cells but rapidly up-regulated in their daughter cells.
Since it had been previously described that spontaneous inactivation of APC also promotes the formation of gastric adenomas, we next sought to determine the tumorigenic potential of the Lgr5 stomach cells. We specifically deleted APC in the Lgr5 stem cells and observed a clear expansion of the stem cell compartment and formation of adenomas in the stomach epithelia. Similarly, as in the small intestine, the Lgr5-derived adenomas fulfilled the criteria of the CSC hypothesis, since they were mainly formed by stem cells as well as differentiated cells. These results were published in Cell Stem Cell 2010.
Since no long-term culture system had been established that allow for the gastric physiology in vitro to be studied, we next wondered whether these Lgr5 stomach cells could be maintained in culture while forming the basic structure of the gastric gland units. When isolated Lgr5 stomach cells were cultured under the right growth media and growth factors, they formed gastric organoid structures (mini-stomachs) that developed gland-like units and could be maintained for months in culture. This gastric culture system not only self-renews in vitro, but also contains the differentiated adult cells present in the normal adult stomach epithelia. This was the first time that an adult stomach culture that self-renews and differentiates in vitro could be maintained in culture for many months without losing its properties. These results were published in Cell Stem Cell 2010.
Project impact
This work will have an important impact on the development of new therapies for gastric pathologies, such as gastric epithelial infection, inflammation and stomach cancer. It also represents a major step forward for the development of new strategies for regenerative medicine.
Contact details
E-mail m.huch@hubrecht.eu for more information.
It is firmly established that inappropriate activation of the Wnt signalling pathway via mutations in core pathway components such as APC is one of the initiating events in colon cancer. The cancer stem cell hypothesis postulates that a small reservoir of self-sustaining cells is exclusively able to self-renew and generates the cell population that constitutes the bulk of the tumour. Importantly, cancer stem cells (CSC) not only fuel tumour growth but also tumour metastases. In 2002, Lgr5 was identified as a gene expressed in colon cancer. By using mouse genetic engineering we deleted APC exclusively in the Lgr5 intestinal stem cells. We observed transformation of the epithelia within days. Transformed stem cells remain located at the crypt bottoms, while fuelling a growing microadenoma. We also observed that cell hierarchy between stem cells, progenitors and differentiated cells was still maintained in early stem-cell-derived adenomas, lending support to the 'cancer stem cell' concept. These results were published in the journal Nature on 29 January 2009 (Barker, Nature 2009).
Lgr5 expression is not only restricted to a subset of cells in the small intestine but we have also found Lgr5 expression in other adult tissues, suggesting that it may be a more general marker of adult stem cells. Therefore, we next set out to investigate whether Lgr5 could indeed be a bona fide marker for adult stem cells and cancer stem cell populations in other tissues. The stomach shares a number of features with the intestine, including a common endodermal origin and a constantly renewing epithelium. Using mouse genetic manipulation and in vivo lineage tracing we found that indeed the intestinal stem cell marker Lgr5 also marks an active adult stem cell population at the base of the stomach glands. We isolated single Lgr5 cells from normal stomach epithelia and analysed their genetic profile using microarray gene expression analysis. We found robust canonical Wnt signalling activity on the Lgr5 stem cell population whereas several lineage-specific genes (enteroendocrine-pit cell) were absent in the stem cells but rapidly up-regulated in their daughter cells.
Since it had been previously described that spontaneous inactivation of APC also promotes the formation of gastric adenomas, we next sought to determine the tumorigenic potential of the Lgr5 stomach cells. We specifically deleted APC in the Lgr5 stem cells and observed a clear expansion of the stem cell compartment and formation of adenomas in the stomach epithelia. Similarly, as in the small intestine, the Lgr5-derived adenomas fulfilled the criteria of the CSC hypothesis, since they were mainly formed by stem cells as well as differentiated cells. These results were published in Cell Stem Cell 2010.
Since no long-term culture system had been established that allow for the gastric physiology in vitro to be studied, we next wondered whether these Lgr5 stomach cells could be maintained in culture while forming the basic structure of the gastric gland units. When isolated Lgr5 stomach cells were cultured under the right growth media and growth factors, they formed gastric organoid structures (mini-stomachs) that developed gland-like units and could be maintained for months in culture. This gastric culture system not only self-renews in vitro, but also contains the differentiated adult cells present in the normal adult stomach epithelia. This was the first time that an adult stomach culture that self-renews and differentiates in vitro could be maintained in culture for many months without losing its properties. These results were published in Cell Stem Cell 2010.
Project impact
This work will have an important impact on the development of new therapies for gastric pathologies, such as gastric epithelial infection, inflammation and stomach cancer. It also represents a major step forward for the development of new strategies for regenerative medicine.
Contact details
E-mail m.huch@hubrecht.eu for more information.
