Cancer Stem Cells: the root of all evil?

A recent series of papers [1-3] have prompted me to write about the cancer stem cell (CSC) hypothesis, since during the last years there has been a lot of controversy regarding the existence, function and clinical implications of cancer stem cells. This confusion stems (no pun intended) from the lack of clarity in the field nowadays, arising mainly from misconceptions regarding the origin and function of these cells. On top of all of this is the confusion derived from media-hype and some not-so knowledgeable scientific journalists.

1.Background

The classical view of tumor biology is that transformed cells, which carry mutations that confer them selective advantages under certain conditions, divide continuously forming a homogeneous tumor. This concept entailed the idea that every clone of the original tumor proliferates indefinitely as an autonomous entity within the whole malignant cell population, and if removed from the tumor and placed somewhere else it could generate a new tumor.

The cancer stem cell hypothesis challenges the aforementioned idea by stating that there is a distinct subset of cells within the whole population capable of initiating and sustaining the growth of the tumor. The descendants of these tumor stem cells are the so-called “tumor bulk”, which are only short lived cells. This then means that it is only these ‘cancer stem cells’ that could lead to the formation of new tumors.

2. Experimental evidence from different models of cancer

2.1 Leukemia

The evidence for this hypothesis came from the group led by John Dick [4], which showed that acute myeloid leukemia (AML) cells differ in their tumor initiating abilities. The experimental approach was straightforward: sorting the cells of the whole tumor through FACS, and then injecting different subpopulations into immunocompromised mice. They later followed which subpopulation was able to reconstitute the original tumor and saw that a small fraction of them had this ability.
The subpopulations arise from using surface markers to separate them. I will come back to this point later to point out the technical problems and advantages associated to this approach).
This pioneering work showed that tumors possess a hierarchical organization, and suggested the existence of cancer stem cells. It is noteworthy that at this point the authors only referred to tumor initiating cells (the term cancer stem cell was coined later).

2.2 Solid tumors

Early work on the hematopoietic system (mainly through the clear characterization of surface markers) allowed tracing of populations with high accuracy. In the case of solid tumors much less is known about the differentiation process, and even less is known about surface markers that characterize each stage. Furthermore, FACS analysis is much more complicated due to the disaggregation step that must be performed (which can alter the surface markers).

Despite the lack of clear ways to trace populations, characterization of CSCs has been done for several types of solid tumors including breast cancer, pancreatic cancer, brain tumors and colon cancer [5-8].
Usually the approach used in most of these studies is based on the usage of known CSC markers from some type of tumor, to separate populations in another one. This rationale has proven useful in some cases, however it cannot be generalized:

-CD44 was known to be a marker of leukemic stem cells and it has been recently shown to be also a marker for breast cancer stem cells.
-CD133 was known to be a marker of glioblastoma stem cells, yet in colon cancer it has been shown to be a marker of a broader population, thus biasing the results.
In general, more knowledge about the dynamics of tissue hierarchy in both normal and pathological conditions will give rise to better tools for understanding potential populations of cancer stem cells in solid tumors.

3. Unresolved issues

3.1 Origin

Several authors claim that the term “cancer stem cell” is inaccurate since not only stem cells can give rise to cancer. This statement is incorrect. The cancer stem cell denomination is given only on a functional basis, which means that these tumor initiating cells have stem-cell properties (self-renewal and the ability to generate all lineages within the tumor), not that they exclusively arise from stem cells.

A recent work by the Clevers group [9] shows that only intestinal stem cells bearing the Lgr5 surface marker are capable of initiating adenomas, the earliest stage of colorectal cancer. The most interesting observation was that differentiated Lgr5- cells were unable to initiate tumors. Whether Lgr5+ cells within the tumor are maintained up until late-stage cancer remains unknown, but this study shows that for this model of tumor the substrate of cancer initiation is indeed an adult stem cell.

On the other hand, work done in leukemia has suggested that progenitor cells (which give rise to only a subset of cells within the tissue) are also capable of tumor initiation, particularly in chronic myeloid leukemia, where although the hematopoietic stem cell carries a mutation in Wnt signaling, only the myeloid progenitors effectively give rise to tumors.

3.2 Experimental Caveats

So far the assessment of stemness remains a highly complex issue that lacks a straightforward answer (i.e. how do you quantify stemness? I welcome any ideas).
CSC quantification is done by the capability of these cells to initiate a tumor in immunocompromised mice, although this may reflect only the ability of certain cells to adapt to a foreign environment.

In fact, the Morrison group showed that depending on the murine model utilized, the frequency of alleged CSCs within melanomas derived from patients varies [10]. This suggests that the microenvironment is the limiting step and partially disproves that CSCs are a rare population within certain tumor types. This was also seen by the Strasser group utilizing murine leukemias in immunocompetent mice [11].

Although this may suggest that CSCs are not as rare as previously reported, this does not contradict in any sense the CSC hypothesis, since the idea of a rare population came from the experimental models used and may vary considerably between tumors.
In order to properly assess which subpopulation (if any) contains CSCs, the tumor initiation experiments must be done in immunocompetent animals that share the same genetic background than that of the host (the immune system plays a major role in tumor appearance and progression, so this should be integrated into the experimental framework), and serial transplantations must be done to show that this ability is retained in time by the same population of cells (serial transplantation assays are a robust method to follow the tumor initiating capacity of a population of cells in extended periods of time). Furthermore, it has to be shown that these putative CSCs are the only ones able to generate all other lineages within the tumor.
(Of course these concepts would only apply to murine tumors, but is the proof of principle from where the field can resolve most of the current issues).

3.3 Therapeutic Relevance

Whether the direct targeting of CSCs by new treatments will effectively give rise to better therapies still remains to be proven, and so it is essential to realize that, at present, this would only be an adjuvant therapy to conventional methods. The reason for this is quite clear: although the CSCs putatively maintain the growth of the tumor, it is still the bulk of the tumor which is responsible for the disease (assuming that CSCs are a minor population of course). Nevertheless, some groups have shown that targeting the CSCs could be sufficient to stop the growth of the tumor. Work done in the Frank lab showed that by targeting the melanoma stem cell marker ABCB5, they could effectively treat the progression of this disease[12]. The same was shown for CD44 in acute myeloid leukemia by the Dick lab[13].

Moreover, in a very recent paper, the group of Michael Clarke[14] showed that the CSC population in breast cancer is more resistant to radiation, and that this is due to reduced reactive oxygen species within these cells. This could partly explain why current therapies are inefficient, as treatment-resistant CSCs would lead to relapse.
Taken together, it is evident that further studies on the nature and function of CSCs for the development of new approaches against cancer, are needed.

4. Concluding Remarks

I hope that with this brief summary of the CSC hypothesis, some light has been shed in the understanding of this concept and how it may influence both basic and clinical oncology in the future.
My impression is that further work is required to understand the dynamics of tumor initiation and growth through the CSC hypothesis, but the overall evidence seems to point towards the validation of this model. Hopefully the elucidation of how CSCs regulate tumor properties will lead to better therapeutic approaches and a deeper knowledge of the basic mechanisms that control cancer origin and development.

Francisco Barriga.


5. References

1. Jordan, C. (2009). Cancer Stem Cells: Controversial or Just Misunderstood? Cell Stem Cell, 4 (3), 203-205 DOI: 10.1016/j.stem.2009.02.003
2. Rosen, J., & Jordan, C. (2009). The Increasing Complexity of the Cancer Stem Cell Paradigm Science, 324 (5935), 1670-1673 DOI: 10.1126/science.1171837
3. Visvader, J., & Lindeman, G. (2008). Cancer stem cells in solid tumours: accumulating evidence and unresolved questions Nature Reviews Cancer, 8 (10), 755-768 DOI: 10.1038/nrc2499
4. Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
5. Al-Hajj, M., et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 2003. 100(7): p. 3983-8.
6. Dalerba, P., et al., Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A, 2007. 104(24): p. 10158-63.
7. Li, C., et al., Identification of pancreatic cancer stem cells. Cancer Res, 2007. 67(3): p. 1030-7.
8. Singh, S.K., et al., Identification of human brain tumour initiating cells. Nature, 2004. 432(7015): p. 396-401.
9. Barker, N., et al., Crypt stem cells as the cells-of-origin of intestinal cancer. Nature, 2009. 457(7229): p. 608-11.
10. Quintana, E., et al., Efficient tumour formation by single human melanoma cells. Nature, 2008. 456(7222): p. 593-8.
11. Kelly, P.N., et al., Tumor growth need not be driven by rare cancer stem cells. Science, 2007. 317(5836): p. 337.
12. Schatton, T., et al., Identification of cells initiating human melanomas. Nature, 2008. 451(7176): p. 345-9.
13. Jin, L., et al., Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med, 2006. 12(10): p. 1167-74.
14. Diehn, M., et al., Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature, 2009. 458(7239): p. 780-3.