The “Direct Connection” section at “The MolBio Hut” includes blog posts discussing primary research articles in the field, but these posts are written by the authors themselves. This allows them to discuss the background, results and implications of their work with a wider audience and in a more relaxed format. We hope that this direct link between the authors and the scientific community (hence its name), promotes discussion and interaction with scientists in other fields.
The intestinal epithelium has the highest self-renewal rate in our body (and in mammals in general). It has been estimated that the entire intestinal epithelial lining is renewed every 5-7 days (which essentially means that around 5 grams of cells are discarded every day). Tissue regeneration in the intestine is ultimately sustained by actively proliferating intestinal stem cells (ISCs) that reside at the base of mucosal invaginations called crypts of Lieberkühn (see Figure 1).These cells give rise to all intestinal cell lineages.
Colorectal cancer (CRC) is the second cause of death by cancer. It has been known for over 20 years that mutations that activate the Wnt signaling pathway, essential for intestinal homeostasis, can give rise to benign lesions called adenomas. Adenomas are not dangerous per se, but are the substrate for further mutations, a process that can lead to the development of CRC and eventually metastasis, which is the main cause of death in cancer patients.
The current therapeutic strategy for most CRC patients consists of surgical extraction of the tumor followed by preventive chemotherapy. Clinicians have been aware, however, for a very long time, that several patients relapse (cancer recurs in 30%–50% of all cases) and eventually develop tumors, usually in the form of metastasis.
The fact that there is disease relapse, means that there are cells that are able to survive for extended periods of time, even after chemotherapy, and resume growth and lead to the development of new (metastatic) tumors, which resemble the primary tumor.
Given the fact that these “relapse-causing cells” are long lived and able to regenerate whole tumors, we sought out to find whether there was a relationship between cancer recurrence and intestinal stem cells. In this “Direct Connection”, I will describe our work entitled “The Intestinal Stem Cell Signature Identifies Colorectal Cancer Stem Cells and Predicts Disease Relapse”, published earlier this year on Cell Stem Cell1.
The first thing we did was analyze the transcriptome of the various populations of intestinal cells in order to study the differences between the ISCs and their progeny.
By using fluorescence-activated cell sorting (FACS), we were able to separate the different populations of cells based on the surface expression of EphB2, a tyrosine-kinase receptor whose expression is highest in the cells at the base of the crypt (in ISCs) and gradually decreases towards the villi (where differentiated cells are, see Figure 1). We then globally analyzed gene expression on these populations by using microarrays and determined genes whose expression was specifically restricted to ISCs, proliferating cells or differentiated cells.
Using these “gene signatures”, we analyzed several cohorts of colorectal cancers and basically found two main things:
- Late stage cancer has an expression profile more similar to ISCs than early stage cancer.
Tumors are divided in stages which reflect how advanced they are (based on size and invasiveness). This classification is called the American Joint Cancer Committee [AJCC] staging system. We found that tumors that are in the latter stages (most invasive and dangerous) display high expression of an ISC-like program, while early stage tumors do so to a much lower extent.
2. Tumors that relapse have a higher expression of ISC-specific genes than tumors that don’t.
Further, tumors that display expression profiles resembling the ones from proliferating cells have a lower incidence of relapse (which makes sense, since chemotherapy is highly effective against these cells).
Basically, the data appeared to indicate that most aggressive CRCs expressed high levels of ISC-specific genes and that relapsing tumors had an overall ISC-like phenotype.
What we didn’t know, however, was whether the ISC gene expression program was characteristic of all cells in the tumor or restricted to specific subpopulations. As Lgr5, a G protein-coupled receptor whose function has just been reported2, is to date the best ISC marker, we used it to identify the localization of tumor cells with an ISC-like phenotype. We also investigated the expression of intestinal differentiation markers in CRC samples by analyzing the expression of Krt20, a widely described marker of intestinal cell differentiation. Notably, we found that both Lgr5+ and Krt20+ cells were present in tumors, but their expression patters were mutually exclusive (i.e tumor cells expressed either Lgr5 or Krt20).
Further, as we found these cell types in very close proximity to one another, we considered unlikely that they were derived from independent tumor subpopulations. With this in mind, we postulated that advanced CRCs are organized in a hierarchical fashion, reminiscent of the normal intestinal epithelium, in which we can find cells with two main mutually exclusive phenotypes: ISC-like or differentiated cell-like phenotypes.
To functionally validate the relevance of these different phenotypes, we once again turned to EphB2. We investigated whether EphB2 expression could distinguish between ISC-like and differentiated-like cells in CRCs, just as it does in the normal intestinal mucosa. Indeed, we saw that tumor EphB2-expressing cells were also enriched in the expression of ISC genes, while the majority of cells expressing markers of differentiation (i.e Krt20+ cells) were EphB2 negative.
To further test our model, we sought to determine the tumor forming capacity of ISC-like and differentiated-like tumor cells. We purified epithelial tumor cells expressing high, medium, or low surface EphB2 levels and injected them into immune-deficient mice. Notably, we found that EphB2 positive populations retained the capacity to generate tumors with high efficiency (i.e. are enriched in tumor initiating cells), whereas EphB2 negative populations displayed reduced or null tumorigenic capacity. In case you are wondering, the EphB2med cells showed an intermediate behavior. Further, the EphB2 derived tumors recapitulated the organization of the tumor of origin. We concluded that “ISC-like tumor cells hold high tumor-initiating potential as well as display long-term self-renewal and differentiation capacity”.
Overall, we described the transcriptional landscape of normal intestinal populations and showed that the risk of developing recurrent CRC is proportional to the expression of ISC-specific genes. Furthermore, we showed that colorectal tumors are organized in a hierarchical structure, similar to that present in normal crypts, and that the ISC-like cells within these tumors have tumor-initiating and self-renewal capacity.
With all this evidence, we postulate that CRC follows a “cancer stem cell” model of growth (See “Cancer Stem Cells: the root of all evil?“).
As with any research project, there are several open questions of which I’ll only highlight three:
Is the enrichment in late stage cancer of the ISC signature due to increased expression of these genes by a small subset of cells? Or is there an expansion of the ISC-like population?
Which ISC genes are actually relevant for tumor behavior? Which are only markers?
Will the targeting of cancer stem cells actually improve the treatment of cancer? (I think this is the most relevant question in the field of cancer stem cells)
Finally, I´d like to thank my PhD supervisor Eduard Batlle and the post-doc who led this project, Anna Merlos-Suárez, for the opportunity to work on this project and for all the support and help during my PhD.
I hope that before my PhD is over I’ll get to write another article for the “Direct connection” section explaining the project I’m currently working on.
-Francisco M. Barriga
1Merlos-Suárez A, Barriga FM, Jung P, Iglesias M, Céspedes MV, Rossell D, Sevillano M, Hernando-Momblona X, da Silva-Diz V, Muñoz P, Clevers H, Sancho E, Mangues R, & Batlle E (2011). The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell stem cell, 8 (5), 511-24 PMID: 21419747
2de Lau, W., Barker, N., Low, T.Y., Koo, B.-K., Li, V.S.W., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M., et al. (2011). Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature advance online publication. PMID: 21727895