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Utrecht University
“Cancer Genomics and Developmental Biology” master’s programme
Thesis: Cancer Stem Cells
Giannis Ampatziadis-Michailidis, 3311864
Supervisor: Dr. R. Vries
Clevers Laboratory, Hubrecht Institute
Acknowledgments
I am truly grateful for the help and insightful comments of Dr. R. Vries and his inspiring
supervision. Moreover I have to thank in depth Prof. H. Clevers for approving my application
and for being the second reviewer of this thesis.
Lastly I have to pay my respects to Sofia and Nitsa for always being supportive and
encouraging and to Sofoklis because without his financial support I would not be able to
follow my dreams.
Utrecht, January 2010
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Index:
Chapter
1. Introduction
2. Stem cells
3. The niche
4. The identity of Cancer Stem Cell
5. Self-renewal and differentiation
6. Identification assays
6.1 Side population
6.2 Markers
6.3.1 In vitro demonstration of
tumorigenicity – sphere formation
6.3.2 In vivo demonstration of tumorigenicity
- xenotransplantation
7. Frequency of CSCs
8. Stochastic model versus CSCs model
9. CSCs and therapy resistance
10. Specific tumor types
10.1 CSCs and leukemia
10.2 CSCs and solid tumors
10.2.1 CSCs and breast cancer
10.2.2 CSCs and brain cancer
10.2.3 CSCs and colorectal cancer
10.2.4 CSCs in other solid tumors
11. CSCs and signal transduction
12. Cancer treatment – is there hope?
13. Future directions
14. References
Pages
4-5
5-6
6-7
7-8
9-10
10
10-11
11-13
13
13-15
15-16
16-17
18
19
19-21
22
22-24
24-26
26-29
29
29
30
30-31
32-36
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1. Introduction
Cancer is a disease that is caused by uncontrolled cell growth. It starts with the generation of
one or more tumor cells (oncogenesis) which have the potential to divide infinitively followed
by the formation of a mass of abnormally proliferating cells. Next some of the cells from the
tumor acquire extra mutations which allow some of the tumor cells to spread in other parts
of the body of the patient and establish secondary tumors (metastasis)1,2. Exactly this feature
of cancer, being able to spread to distant places, is characteristic of the disease’s
aggressiveness and is lethal for the patients. The current view on the oncogenesis field
suggests that for the first tumor cell to arise 6-10 events have to take place. These can be
grouped into two categories, mutations on tumor suppressor genes, or mutation on protooncogenes. The role of tumor suppressors is to regulate cell cycle and mutations causing loss
of function of these genes can lead to uncontrolled cell growth. On the other hand gain of
function mutations on proto-oncogenes that under normal conditions promote cell growth can
lead to their excessive over activation and then as oncogenes will drive uncontrolled cell
proliferation. In the case of tumor suppressors both alleles need to be mutated and hence
deactivated in order for the gene’s function to be lost but for a proto-oncogene to become
oncogene one mutated - overactivate allele is sufficient. Importantly the first of the series of
mutations needs to confer survival benefit on a normal cell and in that why allow for more
mutations to accumulate3,4.
Several ways to treat cancer have been discovered during the last century but in most cases
they do not lead to completely cure of the patients. Inspite of the increase of cancer incidents
during the last century, mainly because of
higher life expectancy rates and life style
parameters, such as smoking, improvement of screening methods and public awareness
together with discovery of new drugs and targeted therapy have actually increased the cure
rate since the last 50 years5. Different methods to treat cancer exist as chemotherapy,
radiotherapy, immunotherapy and surgery2. Surgery has the best success rates although is
most of times combined with one or more of the other options. Chemotherapy has also high
success rates but has major side-effects for the patiens6,7. The main reason that cancer is
incurable is that even when surgically removed and targeted by some kind of therapy the
tumor reappear most of the times with an even more aggressive phenotype. In general tumors
tend to be resistant to all possible combination of treatments. Some tumors are not being
4
affected by the application of treatment and some others even if they respond initially, they
finally develop acquired resistance. It is obvious that scientists are missing an important piece
of the puzzle and efforts are being made to discover the Achille’s heel of the cancer. Towards
this direction the last decade a theory has been brought to light, suggesting that in each tumor
only certain cells have tumorigenic potential and they are the ones that sustain the tumor mass
and cause metastasis. This theory, called the Cancer Stem Cells (CSCs) theory is not news for
the scientific community8-14. If the CSCs theory holds true then it will revolutionize the
oncology field and change radically the way of cancer treatment. Hopefully time will bring
more insightful publications and scientists will critically review new insights remaining
critical in order to prevent rushing into wrong conclusions.
2. Stem Cells
Before deciphering the cancer stem cell field it is important to be familiar with the stem cell
(SCs) area. According to the Collins english dictionary a stem cell is “a type of cell that can
produce other cells which are able to develop into any kind of cell in the body”. A stem cell
can be described as a cell that has self renewal properties and gives rise to more differentiated
populations, called the progenitor cells12,15-22. The progenitor or transit amplifying cells will
give rise to the differentiated cells and they still exhibit self renewal properties but in a less
extent, meaning that they will eventually stop proliferating after a certain number of divisions.
It is believed that SCs divide extremely rarely in order to preserve the integrity of DNA by
minimizing accumulation of mutations by cell division. The burden of further extending the
pool of differentiated cell is curried by the progenitor cells.
Furthermore the stem cells can be divided into two broad categories, embryonic and adult
SCs. The embryonic SCs are present in early embryos and originate from the zygote. The
second category adult SCs, which can be alternatively named tissue or somatic SCs
characterize SCs from fully developed tissues and organs. These cells are committed to
producing a more restricted repertoire of cells16-24.
Embryonic SCs are pluripotent and adult SCs are multipotent. These terms are used to
determine the differentiation potential of the cell. Topipotent cells are at the top of the
hierarchy giving rise to the 3 germ layers, the placenta and the primordial germ cells. Next in
the hierarchy are the pluripotent cells that can give rise to all 3 germ layers and further down
are multipotent cells which have the ability to produce various cell types but only within the
5
context of one germ layer. In mammals, topipotency show cells from the zygote until the 8
cells morula stage and pluripotency cells from the inner mass of the blastocyst, whereas
multipotent are the SCs that will give rise to a specific germ layer like the endoderm16-21.
Multipotent cells are needed in the adult organism in order to maintain the number of
differentiated cells in constant levels, by replacing the cells that die under normal or stress
conditions15,23,25. An example of multipotent cells are the hematopoietic SCs which constantly
renew the population of differentiated blood cells.
The main feature of SCs, self renewal is sustained by the asymmetric divisions of these cells.
In an asymmetric division one SC produces a copy of itself (thus complying with the self
renewal) and another more committed cell following the differentiation pathway. SCs can also
undergo symmetric division and produce either two SCs or two differentiated cells when there
is a need to expand the SCs pool or the progenitors pool respectively16-20,23,26-28.
Figure 1: Stem cells self-renewal and differentiation properties, adapted from29
3. The niche
Niche is a term used to characterize an ill-defined population of cells which are not SCs but
provide the actual SCs with an essential homing microenvironment. It is proposed that the
niche works as a nest for the SCs, physically interacting with them and providing growth
factors required for their survival8,10,11,15,25,28,30. Interactions include paracrine signaling
pathways, suppression of the differentiation programme and pathways that inhibit mitogenic
stimuli15. Since CSCs exhibit properties of normal SCs scientists believe that the requirement
of niche applies to them as well. Moreover the niche has been implicated in the seed and soil
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theory which claims that the tumor cells that metastasize are able to nest and establish a
distant metastasis only when they meet the appropriate microenvironment – niche11,13,30.
Lately the niche has been under investigation because it has been proposed that targeting the
niche cells, could have an effect on the viability of CSCs. So the hypothesis is that the
destruction of the niche could indirectly lead to cancer regression by making the CSCs
incompetent13.
Finally if the niche hypothesis is true then some of the available in vitro data might have to be
re-examined to include the interactions between the tumor cells and their microenvironment10.
Figure 2: Possible carcinogenesis pathways involving the niche (a, b, c) or the progenitor cells
(d). Adapted from 25
4. The identity of Cancer Stem Cell
According to the Cancer Stem Cell theory, a cancer stem cell (CSC) is the cell that gives rises
to a tumor. Based on the findings that CSCs share common characteristics with normal SCS
the original hypothesis was that CSCs arise from normal SCs that acquire genetic mutations.
Nonetheless according to the literature a CSC does not necessarily come from a stem cell, but
in fact it can originate from different cell populations as diverse as normal stem cells,
progenitor cells or even differentiated cells8,9,12-15,21,31-33. The initial hypothesis that CSCs
originate from normal SCs was based on the fact that SCs have a longer life expectancy and
7
hence they have more chances to acquire mutations that will transform them into CSCs.
Additionally normal SCs by themselves exhibit the self renewal and differentiation properties
that characterize CSCs. For a progenitor cell the process is more complicated because it needs
to turn the differentiation clock backwards and acquire mutations that will enable it to show
the previously mentioned features. On the other hand SCs numbers are comparably low and
statistically it is more probably one of the non-SCs to gain mutations that will transform them
in to CSCs13.
Figure 3: Cancer stem cells can originate from normal stem cells, progenitor cells or even
from differentiated cells.
It is commonly appreciated that for a sell to be marked as CSC it needs to meet two criteria:
be able to self-renew and differentiate to the whole spectrum of cancer cells found in the
tumor from where they were isolated11-13,23,24,28,34. In other words only one CSC removed
from tumor biopsy should in theory have the potential to regrow an identical tumor in a
suitable environment.
An alternative name for the cells called CSCs, broadly used in the literature is Tumor
Initiating Cells (TICs). It has been suggested that this might be a more accurate term for the
cells in question because it does not lead to the assumption that these cells originate
exclusively from normal stem cells12,13,22,27,33. On the other hand using the term TIC can be
also misleading because one can believe that the reference is for the initial “unique” cell that
acquired the first mutation and lead to the formation of the tumor8.
In this review the term CSCs will be used to describe cells isolated from the tumor, primary or
metastatic, that have the ability to self-renew, differentiate and are experimentally identified
by exclusively recapitulating the tumor of origin with all its heterogeneity, mainly in
NOD/SCID mice8,9,13,31.
8
5. Self-renewal and differentiation
CSCs are special because they exhibit two special properties, self-renewal and differentiation
potential, properties that they share with normal stem cells23. Self-renewal can be described as
the ability to unlimited proliferation, in contrast with the conventional proliferation, where a
committed cell has a limited numbers of proliferation cycles after whom it stops dividing and
undergoes apoptosis. There are various mechanisms that dictate this behavior in the cell, with
the most dominant being the shrinkage of telomeres with each division. CSCs and stem cells
have developed mechanisms to circumvent this, by maintaining active telomerase for
example, allowing them to undergo unlimited rounds of divisions11,12,23,27.
If a stem cell (cancer or not) every time it divides produces two identical clones (symmetrical
division) then this will result in the expansion of the stem cell pool. For this reason stem cells
mostly undergo asymmetrical divisions which result in one daughter cell being identical clone
of the parental cell and a second daughter cell that has lost some of the properties of the
parental cell and deviates forming a new more differentiated cell subpopulation termed
progenitor cells. By following the asymmetrical division model constant numbers of stem
cells are maintained through time8,11,22,28.
Differentiation is very important in developmental biology because it makes feasible to get a
complex multicellular organism by only one cell, the zygote. The differentiation programme
for each cell is very complex and delicate with a plethora of molecules working on creating a
diversity of cells, each one with one particular task and astoundingly different from each other
although one common parental can be trashed back for all of them. The general principle is
that after zygote formation a pool of stem cells is established that will give rise to a
subpopulation of progenitor cells which next will produce a rich variety of fully differentiated
cells that eventually will form the diversity of tissues and organs.35
As discussed previously CSCs can originate from stem cells, progenitor cells or differentiated
cells
8,9,13,31
.
A stem cell can acquire mutations and become CSC since it already
demonstrates the two essential properties, self-renewal and differentiation potential and needs
only to be deregulated. In addition stem cells live longer than normal cells and thus they have
statistically more chances to accumulate mutations. By being present for longer periods they
are exposed to various oncogenic factors and for longer periods. Progenitor cells are
evolutionary close to stem cells and they need some additional events which will allow them
9
to change their phenotype back to stem cells. Differentiated cells need to go through multiple
events in order to become CSCs, they need to dedifferentiate, regain self-renewal and
differentiation potential and accumulate mutations which will make them tumorigenic. This is
why initially it was thought that CSCs derive exclusively from stem cells but today it is
established that progenitors and differentiated cells can be a source of origin as well. 8,9,13,31.
6. Identification assays
Right in the core of the CSCs theory lays the issue of the identification of such cells. To be
able to raise the case of CSCs scientists need to prove that such a subpopulation actually
exists. Furthermore to prove that the isolated population corresponds to the CSCs it is
necessary to demonstrate that these cells can self-renew and differentiate, resulting in the
formation of a tumor resembling the one that these cells were taken from.
For the identification of CSCs three methods are available to date36,37.
A. isolation of a side population (SP) based on the efflux of the Hoechst dye.
B. use of cell surface proteins – antigens, markers.
These methods are being currently used for the identification of CSCs resulting in different
degrees of enrichment and each one of them has certain advantages and disadvantages.
However the use of expression markers is the most popular. After the identification of cells by
either of the previously mentioned techniques these cells will be further analyzed by in vitro
sphere culture or in vivo xenotransplantation in mice model in order to demonstrate their true
CSCs properties.
6.1 Side population
Side population (SP) is a term used to describe the specific subpopulation of cells that upon
administration of the Hoechst 33342 dye, shows no accumulation of the dye, in contrast with
the rest of the cell population. The Hoechst 33342 dye binds to A-T regions in the DNA and
when excited by ultraviolet light emits a blue light. This is very useful because using flow
cytometry (FACS, Fluorescence Activated Cell Scanning38,39) analysis is easy to isolate this
SP. The interesting characteristic of this specific subpopulation is that SP from a variety of
10
tumors exhibit CSCs properties8,21,23. The conclusion is that when the SP assay is applied in
tumors it can enrich for CSCs although the enrichment rate may vary36,37,40.
The explanation as to why the SP cells are not accumulating the Hoechst dye needs further
validation but at present two possible scenarios try to interpret the findings based on
molecular functions. Firstly the SP population has been suggested to be in a quiescence state,
a property of SCs in general and because of that low or inefficient dye is entering the cells in
comparison with the rapid proliferating counterparts. An alternative hypothesis is that SP
although it originally takes up the dye, has the potential to pump it out due to the presence of
overactive pumps like the ABC drug transporters (ATP binding cassette family). None of the
hypotheses has been firmly proven although there is evidence to support both of them and
perhaps they are both valid.
As for the disadvantages selecting the SP assay as a way to enrich for CSCs, despite the
enrichment the identified cells are not homogeneous and thus only a minority within the SP is
true CSCs. Moreover in some tissues the SP assay fails completely to identify CSCs. Of
course as for all methods, the precise protocol used is very important and differences in the
dye concentration, staining time and temperature may cause misleading results and explain
inconsistencies between different experiments. However the major argument against the use
of the SP assay is that the Hoechst dye itself has been reported to be toxic and thus might
interfere with the outcome of the experiments, perhaps by selecting for resistant cells36,37.
6.2 Markers
Taking advantage of the similarities of SCs and CSCs unique phenotypes the following
method identifies cells based on the expression of special proteins on their cell surface. Some
markers are linked to the tumorigenic activity of the CSCs, in a way that they drive tumor
growth themselves, but others do not show to be functionally involved in the oncogenic
procedure. Most if not all CSCs markers have been identified as SCs markers in the past but
since these two categories share similar phenotypes the SCs markers have been extended in
the CSCs field as well36,37. Moreover for each tumor type there are specific markers which are
known to identify CSCs and a list of them is available in table 1, Nonetheless some markers
like CD133 and CD44 can be used for several tissues.9-11,13. The method to separate the
content of a biopsy into different subpopulations is to dissolve the whole mass and then sort
the cells by Fluorescence activated cell scanning (FACS38,39) analysis.
11
CD133 (Prominin 1) is broadly used in a variety of tumor types for identification of CSCs
although the function of the protein remains elusive. CD44, a cell surface glycoprotein is also
used for CSCs identification in several tumor types. The molecular function of this protein is
known and correlates with an oncogenic phenotype. CD44 activates many receptor tyrosine
kinases (RTKs) like EGFR and HER2 upon binding of hyaluroan (HA), leading to activation
of proliferation (MAPK) and survival (PI3K/AKT) pathways. The two markers CD133 and
CD44 will be reviewed later in the context of specific tumor types. More markers do exist and
ongoing efforts are aiming at making the selection criteria even stricter so that the markers
used could lead to enrichment up to 100% if possible.
The main disadvantage of the use of markers for identification of CSCs is that the number of
cells isolated each time is significantly low41 . This makes the identification of any CSC
impossible when it comes to tumor biopsies relatively small in size. Moreover for solid
tumors to be able to be screened for marker expression the tumor mass needs to be dissociated
so that all cells will become accessible. The treatment itself is chemically severe -proteinasesand can interfere with the expression phenotype41. Finally the main argument against the use
of this method is that the available proteins are not specific enough and more markers with
stringer expression are needed10.
Cancer type
Blood malignancies
Cell surface proteins
CD34+ CD38-
Breast
CD44+ CD24-/low Lin- EpCam+ CD90+
Brain
CD133+
Colon
CD133+ CD44+ EpCam+ CD166+ CD117+
Pancreatic
CD44+ EpCam+ CD24+ CD133+ CD14+
Prostate
CD44+ α2β1+ CD133+ CD24-
Multiple myeloma
CD138- CD34+ CD20+
12
Melanoma
CD20+ CD44+ CD133+ ABCB5+
Lung
CD133+ CD90+
Head and Neck
CD44+ Lin-
Liver
CD90+ CD133+ CD44+
Table 1: Based on data gathered from the following reviews: 14,26,36,40,42-49.
6.3.1 In vitro demonstration of tumorigenicity - sphere formation
When cultured in vitro in serum free media rich in EGF and FGF, the SCs tend to form a
characteristic structure called the sphere (from the greek word σφαίρα which means globe).
When viewed under the microscope these spheres are easily detectable and can be a helpful
tool to identify SCs or CSCs in a further extent8,14,23,34,36,37,50. This assay was first
demonstrated for cells from the central nervous system (CNS) and the structure was called
neurosphere, but since then it has been shown that SCs from other tissues follow this pattern
by forming mammospheres (breast tissue) and colonspheres (colon).
The main concern for the use of sphere culture for enrichment of CSCs is that the sphere itself
shows heterogeneity, meaning that there are issues of contamination by non CSCs. Again
protocol matters like culture medium, passages that he cells have undergone, sphere size may
differ between experiments and interfere with the outcome. Additionally the growth factors
present in the media have been charged with de-differentiation propertieas and may cause
artifacts generating CSCs by differentiated cells rather than selecting the CSCs present in the
biopsy taken13.
6.3.2 In vivo demonstration of tumorigenicity - xenotransplantation
No matter which technique is for enrichment of CSCs the next step is to prove that the
isolated subpopulation demonstrates in fact CSCs properties: self renewal, tumorigenic and
differentiation potential. The best way up to date to show that a cell is in fact a CSC is to
transplant it to immunocompromised mice models8,9,13,14,31,36,37,50. If truly a CSC should be
able to proliferate and give rise to a tumor that recapitulates the histological phenotype of the
13
parental one40. The main mice models currently used for this purpose are the SCID (severe
combined immunodeficiency) mouse and the NOD/SCID (nonobese diabetic SCID) mouse.
Both lacking T and B lymphocytes but the latter is additionally deficient in producing natural
killer cells (NKs) and antigen presenting cells (APCs). This means that SCID mice show
deficiency in the adaptive immune system and NOD/SCID in the adaptive and innate immune
system8,23,27. The most useful feature of both mice models is that they are unable to reject
xenografts and thus candidate CSCs can be transplanted in orthotopic sites - the same area
where the parental tumor was located-. Usually in experiments with mice models after an
initial successful tumor formation, cells from the mice’s tumor this time are being isolated
based on the same criteria that were used for the initial selection. Those cells are transplanted
in other mice - syngenic transplanation- and then the new cells in other mice (with the same
genotype although) and undergo what is called serial transplantation, rounds of several
transplantations, usually 2-313,23. This is to ensure that the cells isolated from the parental
tumor can self renew, differentiate and reconstitute the original tumor’s heterogeneity not
only upon xenotransplantation but also when transplanted from one mouse to another40. Self
renewal properties can alternatively be tested in vitro with non adherent sphere culture or
culture in soft agar. However culture in 3D matrix gel has the potential to cause epigenetic
changes and to promote tumorigenesis50,51. Taking everything into account mice models
although more laborious, timing consuming and expensive still give insightful information by
resembling the condition of a tumor growing inside patients. .
Figure 4: Cells are isolated from solid tumors and the sorted cells can be next tested in vivo
for demonstration of CSCs properties. Here only the cells depicted in green are true CSCs and
14
can reproduce the heterogeneity of the original tumor upon xenotransplantation. Adapted
from44
Up to date scientists have managed to enrich for CSCs so that as few as 100 selected cells can
induce tumor formation in mice models when more than 105 non-selected cells are needed to
have the same outcome. These results can be interpreted in two ways. Either the isolation
methods are not sensitive enough, reaching only up to 0.1% or there is a niche dependency
issue. The latter hypothesis is based on the assumption that CSCs as SCs are dependent on
their microenvironment, so upon xenotransplantation even if the selected subpopulation are
indeed CSCs only 1% can adopt to the new mouse microenvironment and actually establish a
tumor(xenotransplantation issue)13,31. Moreover the debate against the use of mice models in
the CSCs area lies in the fact that although these mice resemble the conditions in a human
organism still are not optimal and other factors may interfere in the survival of a specific pool
of cells For example there is incompatibility in respect of the growth factors and chemokines
and their receptors between the human and mouse10,13,28,50. All considered, the NOD/SCID
mouse model is very useful in the CSCs field making possible to test for self renewal,
tumorigenic and differentiation potential resulting in formation of a tumor histologically
similar to the parental. Despite all the arguments against their use, mice models are the model
which is closest to human patients.
Overall until reliable methods for isolating cells from a tumor, without interfering with the
cells, and a accurate, suitable assay for self-renewal and differentiation are found all data
gathered in the field of CSCs need to looked at with great care. Needless to say working with
biopsies means that scientists are allowed to “see” the tumor only during only one particular
stage, most of the times when it has already grown and has made its presence known. This
also adds more uncertainty to the experiments done because it introduces more variability.
7. Frequency of CSCs
Based on the theory of CSCs, those cells are rarely found in a tumor. This is of course not
definite and numbers greatly depend on the tumor type and vary from patient to patient. But in
principle CSCs represent a minor subpopulation of the tumor. This assumption derives from
the CSCs theory itself. Contradicting the stochastic model where each cell has the potential to
lead to carcinogenesis, the CSCs suggests that the cells capable to cause tumor formation can
15
only be found in the CSCs pool. Therefore the CSCs need to be a subpopulation of the whole
cell number of a tumor. Furthermore based on the majority of available data, the CSCs
detected until now suggest that the CSCs represent a minor subpopulation. Hence CSCs are
characterized as rare based on the or their low frequency. As the tumor mass expands and
tumor cells continue proliferating it makes sense that the stage and aggressiveness of a tumor
will correlate with the percentage of CSCs in the tumor.
The number of CSCs identified in the first attempts was indeed low but some groups lately
have discovered that the percentage of CSCs may vary significantly and may rich levels that
do not comply with the “rare” description9,28. The key behind this inconsistency is mainly the
felicitous selection of the markers for the selection of the CSCs. It has to be mentioned that
the finding that in some cases the percentage of CSCs is very high is one of the main
arguments against the existence of CSCs10.
8. Stochastic model vs CSCs model
Before the CSCs theory was introduced the main theory for tumor formation was the
stochastic model. According to the stochastic model each cell in the organism has the
potential to acquire mutations, gain of function for oncogenes and loss of function for tumorsuppressors, that will lead to tumor formation1,2,11,13,15,23,40. Moreover within the tumor
accumulative mutations happening in a random fashion lead to clonal expansion of a
subpopulation of cells that is the most capable of surviving under the given conditions in each
case. Based on this assumption one can not predict which cell is going to start the oncogenic
process, because the model is characterized by randomness.
According to the CSCs theory there is a specific subpopulation of cells that is inducing tumor
formation and sustains the tumor’s growth. The main principle of this theory is that there is a
hierarchical organization within the tumor which could also explain the functional
heterogeneity observed. Based on the CSCs theory each tumor consists of a subpopulation of
cells that are the mother cells of all tumor cells. All other cells form the bulk of the tumor and
do not have the potential to sustain the tumor mass. In conclusion not all cells are important
for studying cancer induction but only the few with CSCs properties.
16
Figure 5: The two models explaining tumor heterogeneity and tumorigenic potential. Adapted
from 52
Finally both theories the clonal expansion and the CSCs may be applicable and may even
work together promoting carcinogenesis by not being necessarily mutually exclusive13. In
other words oncogenesis could be started only by the CSCs subpopulation but then stochastic
events will select for the CSC that will establish the tumor or even the CSC that will be
implicated in metastasis and tumor resistance, figure 5 (d)
Figure 6: Cellular hierarchy (a) and oncogenesis explained by the stochastic (b) and CSCs
model (c). CSCs model incorporating the clonal evolution theory (d). Adapted from50
17
9. CSCs and therapy resistance
The CSCs theory has drawn the attention of scientists because it indicates which
subpopulation of the tumor is the one that will be resistant to therapy and will further expand
and differentiate to repopulate the tumor mass, resembling the phenotype of the original
tumor. If the CSCs model holds true, then resistance to therapy could be circumvent by
specifically targeting the CSCs pool11,13-15,21-23,40. It is assumed that chemotherapy and
radiotherapy fail to kill CSCs48 and only cause shrinkage of the tumor because they target the
bulk cells11,12,27,34. CSCs are resistant to chemotherapy because they are proliferating less
frequently than the normal cells and so chemotherapeutics whose mechanism of function
depends on interferences with the cells cycle, efficiently kill dividing cells but fail to kill
CSCs11,12,21,23. Moreover CSCs are resistant to radiotherapy because their DNA damage
response is more efficient compared to differentiated cells. It is like conventional therapy can
only destroy the differentiated cancer cells when the CSCs being resistant by nature can give
birth to the tumor again and again. This raises serious questions concerning the routine
treatment that patients get and also the reliability of the assays used in laboratories and
clinical trials to measure tumor regression11-13,27. There is a possibility that the majority of the
drugs used in the clinic and radiotherapy are unsuitable for cancer treatment and in no way
they aim at full recovery of the patient10,12. They only save time but reducing the tumor size
(which is the standard criteria of a successful treatment12) but the “source of all evil” the
CSCs are still present after the treatment and they strike back as soon as the treatment has
stopped or even during the administration. In some cases the new tumor is even more
aggressive.
Figure 7: Cancer stem cells involved in carcinogenesis, tumorigenesis and resistance to
current therapies. Adapted from 43
18
10. Specific tumor types
As mentioned the CSCs field has still a lot of unclear aspects. Each tumor type exhibits
specific characteristics and this contributes to the variability of the data. Historically the
malignancy that first provided evidence for the CSCs theory was leukemia
8,13,31,53
.The last
couple of years more information has been brought to light from the examination of solid
tumors like breast54,brain55 and colorectal56,57 cancer. Many of the issues of the CSCs model,
like the origin of the CSCs, the most appropriate markers and assays used to identify them
should more appropriately be addressed in specific tumor types. This is the reason certain
malignancies will be discussed here based on their location.
10.1 CSCs and leukemia
Why do blood malignancies play such a pivotal role in CSCs field? Cells with tumor initiating
properties were historically first presented in blood tumors11-13,40,58. The hematopoietic system
is a perfect candidate to investigate the CSCs hypothesis because the heterogeneity and
hierarchy among the normal blood cells has long been established23,27,59. The various cell
subpopulations across the differentiation lineage are represented in figure 8 where the
hierarchical differentiation is depicted35.
Figure 8: Hematopoietic stem cells differentiate and lead to the formation of the variety of
blood cells. Adapted from25
19
Hematopoietic stem cells (HSCs) are on the top of the differentiation pyramid, producing
myeloid and lymphoid progenitor cells. Those are more restricted in their developmental
potential and will go through cycles of differentiation generating at the end the whole
spectrum of blood cells. In more details the myeloid precursors will establish the lines of
gralulocytes, erythrocyes, monocytes and megacaryocytes whereas the lymphoid precursors
will differentiate leading to the formation of all lymphocyte populations. As differentiation
continues the cells become more morphologically distinct, acquiring special properties
distinctive of the their cell identity35.
The question that remains to be answered is if the same hierarchy can be observed in
leukemias. Efforts to identify one single leukemic cell that can propagate and transmit the
systemic disease upon transplantation in mice are tracing back to the past. Taking advantage
of the development of tools for identifying HSCs59, several groups demonstrated that only a
minority of the malignant blood cells can induce tumor formation in vivo60, in NOD/SCID
mice models or form colonies in vitro61. These experiments might be subjected to the survival
of particular cells due to the nature of the assay used. This is why the fact that hierarchy exists
in blood malignancies was first addressed when diverse blood diseases like acute myeloid
leukemia (AML), chronic myeloid leukemia (CML), essential thrombocythemia and
polycythemia vera, were found to be characterized as clonal diseases58,62,63. These
publications contributed to the popularity of the CSCs theory, but the pioneering work in the
CSCs field performed by J. Dick and his colleagues53,64.
The main issue in the CSCs field was to identify the specific subpopulation that bears the
tumorigenic properties. The approach of J. Dick’s group was to use FACS analysis to sort the
various subtypes of cell populations from AML patient’s peripheral blood,. Several antibodies
were used with known targets, anti-CD15 (granulocytes), anti-CD14 (monocytes), anti-CD34
(primitive progenitors and stem cells), anti-CD33 and anti-CD38 (myeloid cells). So after the
subpopulations of blood cells were sorted out they transplanted them into NOD/SCID mice.
The fascinating finding was that only one subpopulation that had the capacity to engraft in the
mouse model was characterized as CD34+CD38- . These cells were able to recapitulate the
phenotypic heterogeneity of the original tumor, and showed self-renewal and differentiation
properties. This subpopulation was named leukemic stem cells (LSCs) and such cells were
found in low frequency in the original pool of cells, only 1 in 250.00053. Altogether this work
was the first experimental proof of the CSCs model because in a human malignancy the
cancer population was found to follow a hierarchic organization (as is the case with normal
20
hematopoiesis and HSC). Furthermore a study on the CD34+CD38− population showed that
the number of these cells could be of important prognostic significance for diagnosis in AML
patients. The hypothesis of the authors is that as larger the CSCs number the higher the
percentage of cells resistant to chemotherapy which predicts poor survival65.
A very important issue is the origin of the CSCs in hematological malignancies. Originally it
was believed that the LSCs derive exclusively from normal HSCs because they shared
phenotypic characteristics, both being CD34+CD38-. Later it was observed that LSCs
phenotype actually resembles more the progenitor cells rather than HSCs66. On the other hand
it has been suggested that is reasonable for HSCs and LSCs to have distinct phenotypic
differences due to the oncogenic events that lead to the formation of the latter and
subsequently change the repertoire of the cell’s markers67.
Studying the CSCs in hematological cases, various articles have been published,
experimentally approaching the subject mainly by transducing mouse models with leukemia
related genes23. However the main conclusion is that progenitor cells are able to become LSCs
but, as expected they first have to go through some additional rounds of mutagenesis in order
to gain the self-renewal properties. Studies on AML tend to show a HSC origin of the LSC23
but different types of blood malignancies should be examined separately as they may have
constitutional differences and the most probable scenario is that both normal SCs and
progenitor cells are involved in the origin of the LSCs
Following disease progression, this time with experiments focused on human CML (mainly
cells from patients with chronic phase versus blast crisis) revealed that the LSC might be
changing during the time course of the disease68. It was observed that in chronic phase CML
the cells with oncogenic potential were similar to normal blood SCs whereas in blast crisis
these cells they resembled the blood progenitor cells. These findings led to the assumption
that the two phases of CML are driven by different cell subpopulations. The chronic phase
seems to be coordinated by cells originating from the normal SCs compartment and the latter
phase of blast crisis by cells originating from more differentiated cells like the progenitor
cells69
21
10.2 Solid tumors
Blood malignancies were the first to be examined for the CSCs theory. The next step was to
further validate the theory in solid tumors, something that has been more complicated8,42,50,70.
Experimentally the main obstacle is to get access to all cells in a tumor mass, since the mass
itself is rather heterogeneous23,27. Within the mass the cells are attached to one another and
some are well hidden in the core. The inevitable dissociation of the tumor mass in order to get
single cells may interfere with the experiments, as it can induce anoikis71. Moreover the
physicochemical procedures used to break the cell – cell interaction within a tumor may alter
the phenotype of the tumor cells41. Furthermore in solid tumors it has been suggested that the
CSCs are dependent on the presence of a special microenvironment called the niche, which
mainly consists of normal cells. So by removing them from their niche scientists select for
niche independent cells13,41,71. Finally it is common practice to use normal SCs marker’s to
identify CSCs50. Nonetheless the field of normal SCs in solid tumors is not as much
investigated as in the hematopoietic system and there is a lack of proper markers for SCs
which makes things even more complicated for the identification of CSCs8,13,41.
All in all the CSCs theory is more laborious to be verified in solid tumors in comparison with
blood malignancies. However efforts have been made and the available data so far are more
than promising. Since each tumor type demonstrates special features, the main tumor types in
which progress has been made in the CSCs field will be individually reviewed.
10.2.1 CSCs and breast cancer
Breast cancer was the first solid tumor that exhibited the presence of a CSC
population8,14,23,31,40,42,44,50,70,72-76. Cells taken from human tumors, pleural effusion samples,
were first FACS sorted according to their phenotype and then xenografted in NOD/SCID
mice54. From this experiment it was observed that only one subpopulation had the potential to
regenerate tumors in mice which were phenocopies of the original cancer, even after being
serially passaged several times. These cells were reported to be CD44+CD24- actually in the
original article CD44+CD24-/lowLineage-) and as few as 100 in number could induce tumor
formation when as much as 104 from the rest of the cancer cells failed to54. This was the first
in vivo proof that in solid tumors, a subpopulation of cells from the tumor exclusively posses
22
self renewal and tumorigenic potential while at the same time they are able to produce the
whole spectrum of cells present in the parent tumor.
Research is still ongoing in this field and scientists try to gain more insights into both the
normal breast SCs and CSCs and in some cases exciting data come into light.
Work performed on mouse models revealed that a subpopulation of cells exists from which
even only one cell is able to generate a functional mammary gland in vivo77.In this publication
these stem cells were called mammary stem cells (MaSCs) and were CD29hiCD24+Lin- and
single cells with this phenotype (marked with a LacZ transgene) could restructure a complete
mammary gland when transplanted in mice with surgically cleared mouse breast fat pads77.
Moreover these particular cells were both multipotent and self renewing since a single cells
could recapitulate the whole spectrum of heterogeneity of the breast tissue and it could also
divide indefinitely proved by serial transplantation clonal outgrowths77. In another article the
same cells were characterized as mammary repopulating units (MRUs) and were
CD24medCD49fhi 78. In addition the latter group demonstrated that these cells are SCs that can
differentiate into progenitor cells which can form adherent colonies in vitro.
Just recently more information about the issue of SCs and CSCs in breast has come under the
spotlight. When breast cancer patients where examined after treatment, the tumor cells that
remained exhibited a distinctive signature in their gene expression profile79. The
subpopulation of the resistant tumor cells showed the same signature independently from the
kind of treatment, chemotherapy or hormone (endocrine) treatment. This indicates that the
mechanism of resistance is a general feature of these cells and is not treatment specific.
Interestingly this signature is claimed to resemble the signatures of the CD44+CD24/low
Lineage- cells, identified as the CSCs54 and of self renewing cells, as proven by in-vitro
mammosphere assays80. Furthermore this specific signature is proposed to work as a
prognostic tool for metastasis77 since it has been correlated with a specific uncommon breast
cancer type, claudin low, where the cells undergo epithelial mesenchymal transition (EMT)81.
These findings are in agreement with previous efforts comparing normal and cancerous breast
tissue82.
A second publication in the same month brought more data about the phenotypic resemblance
of normal breast SCs, breast CSCs and embryonal carcinoma cells. All these subtypes showed
a down regulation of some micro-RNAs with the most important being miR-200c83 which
normally inhibits the BMI1 protein which itself is repressing apoptotic and differentiation
23
signals84. Elevating dose of this micro-RNA suppressed self-renewal and induced
differentiation in not only normal CSs but also in CSCs opening new ways for treatment.
Furthermore there were identified more than 30 micro-RNAs that were differentially
expressed between normal SCs and CSCs which also can lead to the delineation of molecules
that only interfere with CSCs and not normal SCs. These findings can lead to therapies
targeting exclusively the CSCs and leaving the SCs unaffected.
10.2.2 CSCs and brain cancer
Soon after the publication of evidence for CSCs in breast cancer54 more information was
brought for CSCs in solid tumors by work focused in brain tumors8,11,14,23,27,40,46-48,50,55,85-89.
Initially the CSCs were defined in vitro, with the help of the neurosphere assay, in fact from
various types of human brain tumors (medulloblastoma, ganglioglioma, pilocytic
astrocytoma, ependydoma). CSCs were exclusively found in the subpopulation expressing the
CD133 marker, a glycoprotein also known as prominin-155.
These subpopulation demonstrated proliferation, self renewal and differentiation properties
reconstituting the phenotype of the patient’s tumor in vitro when the CD133 - failed to do so.
With further experiments the same group tested the CD133+ cells in vivo using a xenograft
assay85. The CD133+ was the only subpopulation that could initiate tumor formation in
NOD/SCID mice models that was a phenocopy of the parental tumor, even after serial
transplantation. In fact, as few as 100 CD133 positive cells were enough, when as many as
105 did not lead to carcinogenesis. For these studies only one marker was used, CD13386,87. In
the future the need for more markers to validate the identity of the CSCs and to achieve even
better enrichment rates is imperative.
CD133 was not randomly selected for the prospective isolation of CSCs. It had been
established before as a marker for SCs or even progenitors cells in the hematopoietic90 and
central nervous system (CNS)87,91,92. Since it is characteristic for both stem and progenitor
cells the cell or origin of the CSCs in brain cancer remains elusive89. Moreover when the
induced tumors in the NOD/SCID mice were investigated CD133- were present, meaning that
CD133+ not only capable to self renew, proliferate and induce tumor formation but in addition
they can differentiate and give rise to more committed cells23. Furthermore the number of
CD133+ varies among the available publications, 5-30 %27, 19-22 %23, 0.2-10.4 %45. This
24
difference in number might be explained based on the various brain tumor types and
aggressiveness of each individual tumor85. Finally it has to be highlighted that the CD133
positive population most probably contains another subpopulation that are the true CSCs87.
Since the number of CD133+ cells needed to induce tumor formation in NOD/SCID mice is
unreasonably high, 100 cells, it has been proposed that the CSCs can be found in the CD133 +
population in a frequency of 0.1-1%.
In chronological order CD133 marker has been found to be associated with CSCs properties
in cells isolated from ependymoma in combination with other markers, in the context of
CD133+/Nestin+/RC2+/BLB2+ cells93. Arguing that the CSCs are found exclusively in the
CD133+ pool, CD133- cells isolated from glioblastomas were shown to be as much
tumorigenic as CD133+ using xenograft methods94. Further on CD133 has been correlated
with the patient’s outcome in glioma patients. In fact not only has the proportion of CD133
positive cells been shown to be an important prognostic factors for adverse progression free
survival and overall survival, independently from tumor grade, extent of resection, or the
patient’s age, but at the same time to work as an independent risk factor for tumor regrowth
and period to tumor progression for grade 2-3 tumors95. Additional evidence for correlation of
CD133 expression and poor prognosis for patients with gliomas has been published short after
the last publication96. In the same year work on glioblastomas revealed that the CD133- can be
tumorigenic as well, using mice models and specifically this subpopulation can produce upon
passaging CD133+ cells, an event that accompanied angiogenesis and marked aggressiveness.
In conclusion the authors claim that their results suggest that a positive CD133 cells is not
prerequisite for tumor induction but that its detection rather confirms tumor progression97.
Further evidence linking CSCs and angiogenesis have been presented with CD133+ forming
tumors which exhibit elevated levels of angiogenesis via an VEGF mediated mechanism98 and
most recently, for CSCs selected based on the expression of an alternative marker (serine)99.
Finally a recent review apart from questioning the special identity of CD133+ being the solely
CSCs cells in brain, based on publications that prove that cells lacking the marker show CSCs
characteristics, introduces a new controversy by gathering data that CD133 is also expressed
in normal cells, in contrast with the belief that marks only stem cells and progenitors45.
Additional work done on brain CSCs, isolated based on CD133 expression has revealed the
important clinical significance of these cells in terms of radioresistance100. In gliomas these
25
cells are enriched after radiation treatment and contribute to tumor radioresistance via a
mechanism that activate two responses. The DNA damage checkpoint and that of DNA repair.
After the initial experiments in brain cancer that showed that CD133+ in contrast to the
CD133- are the cells that harbor the ability to induce tumor formation in vitro55 and in vivo85
some publications presented evidence that suggest that the later category has also tumorigenic
potential, at least in some brain tumor types. At the same point data have been gathered that
prove the CD133+ protein as a CSCs marker. The most probably explanation is that in brain
tumors the phenotype of the CSCs is specific to each subtype and also that the methods used
for the identification of the CSCs need to be further optimized50,87.
The important message from all the above articles is that no publication claimed that the
CD133+ are not CSCs but the main argument against them is being the sole CSCs
subpopulation and that CD133- can also exhibit CSCs properties.
10.2.3 CSCs and colorectal cancer
Colorectal cancer was the third solid cancer, after breast and brain to be investigated for the
presence of CSCs14,48,76,101. Colorectal cancer has been examined in details and a model
describing the process of tumorigenesis is depicted in figure 9. Some publication have shed
light on the CSCs model but still the identification assays are under criticism because of the
use of the debatable marker CD133. However recent publications using more CSCs markers
seem to validate the CSCs model in the gut tumorigenesis.
Figure 9: The adenoma-carcinoma model describing colorectal carcinogenesis according to4.
Adapted from 76
26
The first evidence of the presence of human CSCs in colon was based on their isolation using
the CD133 marker56, which as mentioned before is used also in brain cancer. According to the
results of this group all CSCs in this publication referred to as colon cancer initiating cells
(CC-IC) were CD133+. As in previous experiments the criteria that CSCs had to meet were
self-renewal, differentiation potential and recapitulation of the original tumor’s heterogeneity.
In contrast the CD133- cells, although were redundant within the tumor mass did not have the
potential to induce tumor formation when xenotransplanted in NOD/SCID mice under the
renal capsule. Moreover the researchers calculated the frequency of the CC-IC cells which
was enriched more than 200-fold within the CD133+ subpopulation. This means that not every
cell in the CD133+ pool is a true CSC.
On the next article in the same issue of the Nature journal the theory of tumor organization in
colon in a hierarchic way was addressed57. The main finding of the second publication was
that CD133+ show CSCs properties whereas their counterparts CD133- did not, when
xenotransplanted in SCID mice (and not NOD/SCID this time) subcutaneously. Additional in
vitro work proved that the CD133+ cells could proliferate continuously for one year as
undifferentiated tumor spheres in serum-free medium, still being able to demonstrate tumor
formation upon transplantation. Furthermore CD133+cells were found in areas of the tumor
which demonstrated high cellular density and not frequently found in normal colon tissues.
Finally CD133 positive cells would lose their oncogenic ability once started to differentiate
but in contrast would exhibit a more aggressive phenotype after serial in vivo passages.
Moving forward, the third publication presented evidence about the CSCs, not using the
CD133 marker but this time the cells that showed CSCs properties were isolated based on the
EpCAMhighCD44+ phenotype102. In fact according to the authors using these two markers
instead of the CD133 is a more reliable method for the identification of CSCs based on the
facts that CD133- cells did show CD44 expression and moreover the CD44 marker could be
used for further enrichment within the CD133+ subpopulation. Furthermore by analyzing the
EpCAMhighCD44+ cells, which were also positive for CD49f and ALDH (regarded also as
CSCs marker14,103) activity, a new marker was identified in colon, the CD166 protein. In
summary the result of this work is that CD133+ cells can be enriched for CSCs by using
EpCAM and then the new subpopulation can be enriched a step further with the use of
CD166. The EpCAM and CD44 proteins have been also utilized in isolation of breast CSCs
27
and CD166, a mesenchymal SC marker has been associated with poor clinical outcome in
colon cancer104.
As in brain CSCs the use of CD133 as a marker for CSCs has been a field of controversy.
After the previous publications more followed that supported the case that CD133+ are
enriched for CSCs105 and that the marker is of high prognostic significance106, but evidence
started to appear suggesting that CD133+ is the not the most appropriate marker for the
identification of CSCs107 with some publications suggesting that the use of the CD133 antigen
for this purpose should be avoided108. The last article revealed new data concerning the
CD133 marker that strongly argued against its use for identification of colorectal CSCs. In
more details the researchers found that the expression of CD133 in colon is not restricted to
stem cells but is largely spread in all differentiated epithelial cells and furthermore they
showed that CD133+ but also CD133- from samples isolated from metastases can induce
tumor formation. So, according to their data, selecting for CD133+ is not necessary leading to
enrichment for CSCs in colon cancer. Perhaps in the metastatic population some of the
CD133+ cells have differentiated into CD133- but they still are CSCs. That would mean that
different markers should be applied in biopsies taken by metastatic sites. Finally the model
that they propose is that CD133 antigen can be used for identification of mature ciliated ductal
and luminal epithelia cells and to distinguish cells from the CD133- stromal tumor
components. Moreover they believe that CD133 expression is reduced in the metastatic
compartments and both CD133 positive and negative cells isolated from metastatic sites can
induce tumor formation, both being candidates for CSCs in colon cancer. This last publication
raises some important issues about the selection of the CSCs markers and how strict the
criteria need to be. However the methods used differ from the ones used in the previous
publications. In particular the expression of CD133 in cells was measured using a mice model
which was designed to have a knock-in lacZ reporter whose expression was driven by
endogenous CD133 promoters (CD133lacZ/+). This is entirely different from using antibodies
against CD133. It is possible that mRNA and/or antibody affinity issues have caused
misleading results by missing out CD133+ cells in the previous experiments.
Alternative markers need to be discovered in colon cancer in order to be positive that the cells
identified in each experiment are true CSCs. Towards this direction work on colon stem cells
is necessary. In fact the knowledge in this field is constantly expanding especially with the
discovery of Lgr5, the leucine rich repeat containing G-protein coupled receptor 5. Lgr5 has
28
been found to be solely expressed in cycling columnar cells in the base of the colon crypt109.
These cells were demonstrated to be stem cells with lineage trace experiments using an
inducible Cre knock-in allele and as a reporter the Rosa26-lacZ. The Lgr5 positive stem cells
could generate the whole spectrum of epithelial cells (enterocytes, goblet and paneth cells)
within 2 months. Taking advantage of the identification of this new SCs marker, the same
group proved that the Lgr5 protein can be of great value for the identification of CSCs in
colon cancer110.
10.2.4 CSCs in other solid tumors
Apart from the before mentioned tumor types, leukemias, breast, brain and colorectal there
has been research on others trying to identify CSCs to sustain a universal model of their
existence. Such efforts include work on prostate14,111 and pancreatic cancer14,112,113, head neck
squamous cell carcinoma114, lung cancer115, melanoma14,116, liver14,117 and ovarian cancer118.
Evidence from all studies should be viewed as specific cases, especially because of the
different methods or markers used each time. However certain general conclusions can be
drawn indicating that the CSCs theory is valid.
11. CSCs and signal transduction
Several developmental pathways are implicated in the CSCs field which makes perfect sense
because of the self renewal properties of these cells. Cancer specimens show over activation
of many molecular pathways like the Notch, Wnt/β-catenin, mTOR, Bmi-1, Pten and
Hedgehog pathways whose role has long been established in developmental processes9,14,23.
In more details, the Notch pathway is found to be over activated (mainly by mutations) in
blood malignancies. The Wnt signaling is up regulated in many human cancers and
accumulation of β-catenin is depicted in melanoma, sarcoma, breast and brain cancer.
Especially mutation in the β-catenin and APC genes are commonly found in colon cancer.
Pten a tumor suppressor gene shows loss of function mutations in various malignancies
whereas over activation of Hedgehog is associated with skin and brain cancer whereas Bmi-1
is over expressed in LSCs23. Targeted inhibition of these pathways could lead to tumor
regression although their regulation should be monitored closely since they are important for
normal cells as well.
29
12. Cancer treatment - is there hope?
There have been theories about the resistance of the CSCs and SCs in general, to apoptotic
stimuli and how chemotherapy fails to kill them and chemo-resistance emerges as a result119.
This is why some scientists believe that is impossible to kill the CSCs without killing all other
cells too, since it would be necessary to take very drastic measurements in terms of the
toxicity of chemotherapeutics. A new publication just last year seems to give an alternative
way to selectively kill CSCs, in particular breast CSCs and brings more optimism for the
future120. The interesting point about this work is that the researchers developed a method to
create artificially CSCs in the laboratory. This is most convenient because CSCs are both rare
in numbers but also difficult to extract from patients. Practically the method they used
allowed them to grow immortal human breast cells and then induce mutations until the cells
would become CSCs. Actually it is an artificial condition but allows for experimentation
when samples of CSCs are not available. Furthermore, when the CSCs population was
established this way, a screening was performed for a variety of chemical compounds. In total
16000 chemicals, commercial and natural, were tested for epithelial CSCs specific toxicity on
the breast CSCs. In fact only 32 were selectively toxic and 4 out of them showed consistency.
One of them in particular, salinomycin proved to be 100 times more effective than paclitaxel,
a drug widely used in the clinic. Salinomycin was next tested in mice models and proved to
cause inhibition of the tumorigenesis, induction of the differentiation and correlated with loss
of expression of genes related to poor prognosis. The future goal is to test salinomycin in
clinical trials to determine if it has clinical significance and then to optimize the conditions for
treatment of human patients.
13. Future directions
The CSCs theory has drawn a lot of attention during the last decade and many research
laboratories have invested significant amounts of time and money in order to delineate the
model. The necessity of finding new treatments for cancer may have caused an early
excitement and high expectations from the public after the first evidence of the existence of
CSCs. Many people idealize CSCs as the root of all evil, the reason of tumor formation and
recurrence and believe that by specifically targeting them, the true cure of cancer can be
found. Humanity has gained a great tool to fight the disease of the century and may even win
30
the war against cancer, declared several years back. However many issues in CSCs research
need to be refined and optimized, especially the issue of the identification assays. Known
markers should be used wisely and only after their validation by several experiments.
Needless to say the search for more optimal markers for the CSCs is one of the main goals for
the future. However in the case that CSCs actually exist then new treatments should be
designed taking into consideration the CSC role in tumor resistance. Such treatments would
be mainly be targeted therapy against the CSC subpopulation. Apart from the direct targeting
treatment, alternative ways are differentiation of all CSCs which then could be targeted by
conventional therapies as differentiated cells and therapies that target the niche leaving the
CSCs unable to survive and proliferate.
Figure 10: strategies to eliminate the CSCs population by directly targeting them, forcing
them to differentiate or targeting their niche adapted from 71
The CSCs theory seems very fascinating and promising but scientists need to stay rational and
test the hypothesis before rushing into any conclusions. There is also the possibility that the
CSCs is not applicable for all tumor types and thus has to be investigated if it is a universal
model or not. Time will show how solid and universal the CSCs theory is and if in fact will
help in the elimination of treatment resistance.
31
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