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PANCREATIC CANCER STEM CELLS –

NEW INSIGHTS & PERSPECTIVES

Jorge Dorado, Enza Lonardo, Irene Miranda-Lorenzo, Christopher Heeschen

Stem Cells & Cancer Group, Clinical Research Programme, Spanish National Cancer Research

Centre (CNIO), Madrid, Spain

Correspondence: Dr. Christopher Heeschen, MD, PhD

Clinical Research Programme, Stem Cells & Cancer Group

Spanish National Cancer Research Centre (CNIO)

Madrid, Spain, E-mail: christopher.heeschen@cnio.es

Short title: Pancreatic cancer stem cells

Abbreviations: CSC: cancer stem cells; GEM: gemcitabine;

Keywords: cancer stem cells, tumor-initiating cells, pancreatic cancer

Total word count 5,781

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ABSTRACT

Since the identification of self-renewing cells in the hematopoietic system several decades ago,

stem cells have changed the way we study biology and medicine. Solid tumors contain a distinct

subpopulation of cells, which bear stem cell characteristics and are responsible for exclusively

driving tumorigenesis. This has led to the development of the stem cell concept for cancer, which

purports that a subpopulation of self-renewing tumor cells, also termed cancer stem cells is

responsible for tumorigenesis and metastasis. This contrasts with the stochastic model of tumor

development, which advances that all tumor cells are capable of tumor initiation. Different

subpopulations of cancer stem cells have also been identified in pancreatic ductal

adenocarcinoma based on the use of combinations of surface markers that allowed their isolation,

propagation, and further characterization. Importantly, cancer stem cells are not only capable of

self-renewal and differentiation, but also may confer virulence via immune evasion and multidrug

resistance, and potentially via vasculogenic mimicry and transition to migratory and

metastasizing derivatives. Therapeutic targeting of this subset of cells and the pathways defining

their virulence holds great promise for the development of more effective strategies for

amelioration and eradication of this most lethal form of cancer.

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INTRODUCTION

Pancreatic adenocarcinoma is the fourth most frequent cause of cancer-related deaths. Despite its

incidence is moderated when compared to the whole of the solid cancers, it accounts for one of

the highest mortality rates (1). Responsible for this are its late diagnosis due to lack of early

symptoms, common impossibility to resect the tumor, extensive metastasis and high resistance to

treatment. Despite intensive research activity in the field of pancreatic cancer and significant

advancements have been achieved in the understanding of the mechanisms underlying pancreatic

cancer, these advancements over the past decades have had modest clinical relevance. Little

progress has been done since the introduction in the late 90’s of the chemotherapeutic agent

gemcitabine, a nucleoside-analogue, which improved clinical response in terms of pain reduction

and loss of weight (2). Nowadays gemcitabine remains the first-line chemotherapeutic agent in

pancreatic cancer. However, the prognosis of patients diagnosed with pancreatic cancer remains

extremely poor: 5-year survival rate of 1-4% and median survival period of 4-6 months. The

incorporation of the only other approved agent, erlotinib, to the therapeutic cocktail has not

resulted in major improvement in survival (3). Very recent data suggest the existence of different

subtypes of pancreatic ductal adenocarcinoma with erlotinib being more effective in classical

subtype cell lines (4), although this still needs to be validated in clinical samples.

A new horizon has emerged in the therapeutic strategies against pancreatic cancer with the advent

of increasing evidence supporting the existence of cancer stem cells (CSCs) in both

hematopoietic and solid cancers, including pancreatic cancer. CSCs represent a subpopulation of

cells distinguishable from the bulk of the tumor based on their exclusive ability to drive

tumorigenesis and are blamed for their crucial role in relapse of the disease (5). Therefore, the

elucidation of the mechanisms underlying pancreatic tumorigenesis and specially pancreatic

CSCs is of crucial relevance for the development of clinically more efficient therapies.

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CANCER STEM CELL CONCEPT

Over the past years, different models were developed trying to explain the origin and progression

of carcinogenesis. Classical models of carcinogenesis imply that any mutant tumor cell of any

organ can be expanded during tumorigenesis (6) being the entire tumor a functionally

homogeneous cell mass. Emerging data suggest that the majority of tumors, including pancreatic

ductal adenocarcinomas, display significant functional heterogeneity in their respective cell

populations, where a small subpopulation of tumor cells that acquired the same somatic mutations

but are epigenetically distinct, are responsible for tumor initiation, growth and metastasis (5).

This model is based on a hierarchical organization of the tumor, where a stem cell can give rise to

the heterogeneous tumor cell mass, while maintaining the ability to self-renew (Figure 1).

Specifically, the latest consensus definition of a CSC represents a cell within a tumor that has

exclusively self-renewal capacities, can give rise the entire cancer cell lineages within a tumor,

and are exclusively tumorigenic in vivo (7, 8). First descriptive evidence for the cancer stem cell

hypothesis was generated in leukemia and myeloma (9, 10), but further proof has now been

provided in leukemia (11) as well as several solid cancers including breast cancer (7),

glioblastoma (12), colorectal (13), liver (14), and pancreatic cancer (8, 15).

Conceptually, CSCs are characterize by additional traits including the ability for

asymmetric/symmetric cell division, enabling CSCs to maintain and expand themselves (16), a

distinct surface markers expression profile, that is actually being linked with poor prognosis.

Chemo- or radiotherapy against pancreatic cancer are rather ineffective and are mostly following

by tumor relapse and spreading. Intriguingly, it has been shown that CSCs are highly resistant to

standard therapy (8, 17). Given these findings, many investigators have hypothesized that CSCs

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arise from somatic stem or progenitor cells with genetic, since normal stem cells survive longer in

tissues, they are more likely to accumulate mutations over the time.

Indeed, this has now been experimentally proven for several tumor entities (18). Accumulation of

sequential mutations has been defined as a main common mechanism giving rise to

tumorigenesis, especially in tissues such as the intestine (19). Alternatively, CSCs may arise via

dedifferentiation of lineage committed cells that (re-)acquire stem cell characteristics through

acquisition of genetic alterations (20). Tracking the “cell of origin” in tissues with a less clearly

defined hierarchy such as the pancreas is challenging and will also require a better understanding

of the processes involving tissue maintenance and regeneration. Indeed, the process of generating

CSC in different tissues may also vary on an individual basis considering the context of tumor

development including inflammation and exposure to carcinogens.

MARKERS FOR PROSPECTIVELY ITENTIFYING PANCREATIC CANCER STEM CELLS

First evidence for the existence of CSCs in pancreatic cancer was provided by Li et al, 2007 (15).

The authors identified a highly tumorigenic CD44+CD24

+EpCAM

+ subpopulation using a

xenograft model of immunocompromised mice for primary human pancreatic adenocarcinoma.

As few as 102 CD44

+CD24

+EpCAM

+ cells were able to generate a tumor in 50% of the animals

indicating strong enrichment for the tumorigenic fraction. In contrast, cells negative for all three

markers (CD44–CD24

–EpCAM

–) were not capable of initiating pancreatic cancer until 10

4 or

more cells were implanted. CD44+CD24

+EpCAM

+ cells displayed

typical stem cell features such

as self-renewal, activation of developmental

signaling pathways (e.g. Shh), generation of

progenies, and recapitulation of the phenotype of the parental tumor from which they were

derived. Unfortunately, the authors did not investigate the tumorigenicity of the remaining cell

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populations other than those that were negative for all three markers. Since EpCAM identifies

cells in the tumor that are committed to the epithelial lineage, their EpCAM negative counterparts

may be contaminated by non-epithelial inflammatory, stromal and vascular cells (15).

In a second study, Hermann et al. showed that expression of CD133 in freshly isolated primary

human pancreatic cancers discriminates for cells with the capacity for self-renewal, sphere

formation, and, most importantly, in vivo tumorigenicity (8). Importantly, CD133 positive cells

maintained their tumor-initiating capability during serial passaging in vivo. Interestingly, the

authors further showed partial, but not complete overlap of the CD133+

population with the

CD44+CD24

+EpCAM

+ subpopulation. Further studies will be required in order to determine

whether these markers (CD44+CD24

+EpCAM

+ and CD133

+) define two distinct pancreatic CSC

populations, or whether the use of a combination of these markers confers a higher enrichment in

pancreatic tumor initiating cells (Table 1).

More recently, additional markers have also been associated with pancreatic CSCs like ALdehyde

DeHydrogenase 1 (ALDH-1), that has been associated with the tumorigenic cells in pancreatic

cancer (21-23), although more recent data suggest an abundant expression of ALDH-1 in normal

pancreas tissue (24), which may limit the utility of ALDH-1 as a marker for CSCs. Indeed,

ALDH1 can be best used for tumors whose corresponding normal tissues express ALDH1 in

relatively restricted or limited levels such as breast, lung, ovarian, or colorectal. Moreover, side

populations (SP) cells, which are identified by their ability to exclude the dye Hoechst 33342

through the over expression of the ABCG2 transporters, proved to be enriched for cancer-

initiating cells in several cancers (25). Consistently, Kabashima and colleagues reported for

established pancreatic a CSCs that the contained SP cells are enriched for cells with cancer stem

cell features and bear strong potential of the epithelial to mesenchymal transition (EMT),

invasion, and metastasis (26). Since the homeobox gene MSX2 appears to represent an important

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inducer of EMT in pancreatic cancer and MSX2 turned out to correlate with chemoresistance,

subsequent analysis of the MSX2-target gene identified ABCG2, a defining feature of SP cells, as

the responsible gene (27). Together these findings do not only suggest that SP cells are strongly

enriched in CSC, but also validate an intriguing regulatory mechanism of the cancer stem cell-

related phenotype, which could represent novel therapeutic target.

Table 1: CSC markers in pancreatic cancer

Pancreatic ductal adenocarcinoma Markers Reference

EpCAM+ CD44

+ CD24

+ (15)

CD133+ (8)

Tumor-initiating population

ALDH-1+

Side population / ABCG2

(21-23)

(26, 27)

Migrating cancer stem cells CD133+ CXCR4

+ (8)

Taken together, none of the above listed markers appear to be capable of selectively identifying a

pure population of CSCs rather than enrich for a population containing CSC. Single cell

experiments have mostly failed to produce tumor formation and largely depend on the use of the

immunological competence of the secondary recipient (28, 29). Importantly, exposure of cancer

cells to therapeutic interventions prior to their isolation may lead to changes in the expression of

these makers, which could affect their utility to identify tumor-initiating cells. A broader

consensus on the use of the most appropriate markers is certainly desirable and would increase

the probabilities for producing consistent data in pancreatic cancer and other tumor entities.

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CANCER STEM CELLS ARE HETEROGENOUS POPULATIONS OF CELLS

CSCs are unlikely to represent a homogenous population rather than evolve over time into

different clones, some of them providing them with advantages features including proliferation,

anti-apoptosis, DNA damage response, resistance to hypoxia, invasion, and metastasis (Figure 2).

Based on the CSC concept, only CSC should be equipped with metastatic potential as the bear

exclusive tumorigenic capacity, Indeed, while many cancer cells appear to circulate in the

peripheral blood, only a subset of these cells are eventually directed through lymphatic or blood

vessels toward specific secondary sites to form metastases and establish secondary lesions.

Whether all CSC are equipped with this potential or whether this is restricted to a subpopulation

had not been investigated until recently.

Herman et al. identified two distinct subsets of CD133+ CSC based on the additional expression

of the chemokine receptor CXCR4 (8). CXCR4 is a chemokine receptor responding to

chemotactic gradients of its specific ligand stromal cell derived factor 1 (SDF-1) and was

originally identified in the context of homing of leukocyte and hematopoietic progenitors. Out of

the heterogeneous population of CD133+ cells, two different subpopulations were identified by

means of expression of CXCR4. While CD133+ CXCR4

– were responsible for the initiation and

growth promotion of the primary tumor, the other subset of cells characterize by co-expression of

CXCR4 resulting in CD133+CXCR4

+ cells had exclusive metastatic potential. Depletion of the

CSC population for CD133+CXCR4

+ cells did not affect the tumor initiating potential of the cells

but completely abrogated their metastatic capacity. Consistently, significantly higher numbers of

CD133+CXCR4

+ migrating CSCs were observed in patients with lymph node metastasis (pN1+),

demonstrating the clinical relevance and prognostic potential for the detection of migrating CSCs.

While these cells could also be detected in the portal vein of the mice orthotopically transplanted

with CD133+CXCR4

+ cells, it remains to be demonstrated whether these cells can also be

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detected in the circulation of patients with advanced pancreatic cancer. They may serve as

independent predictors for the clinical course of the patients and may be used as novel markers

for monitoring of CSC-targeted treatment.

Interestingly, CSC can acquire an invasive phenotype as a consequence of epigenetic changes

suggesting that the evaluation of metastatic CSC may not only require genetic alterations but may

also be achieved by epigenetic changes responding to microenvironmetal clues (Figure 2).

Indeed, Epithelial-Mesenchymal-Transition (EMT) as a reversible dedifferentiation process has

been demonstrated to equip cancer (stem) cells with strong migratory capacity, while maintaining

their stemness features, allowing the production of progenies during metastasis. This is associated

with loss of E-cadherin and de novo expression of vimentin. In this context, Wellner et al.

recently showed for pancreatic cancer that the EMT-activator ZEB1 represents an important

promoter of metastasis by suppressing E-cadherin. Most interestingly, the stem cell phenotype

was maintained in the context of EMT by suppression of miR-200 family members that usually

target stem cell factors such as Sox2 and Klf4 (30).

It remains to be determined, which cell populations in the heterogeneous populations of the tumor

are actually capable of undergoing EMT in vivo. In fact, if also the progenies of the CSC may be

able to undergo EMT and, in this context, obtain the capacity to self-renew, the CSC population

would represent a moving target making their elimination even more challenging. Future studies

will have to elucidate whether transit-amplifying progenitors as the immediate progenies of CSC

or even differentiated epithelial cells can undergo EMT.

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CANCER STEM CELL-TARGETED THERAPY

Despite huge efforts for the identification of more effective therapies, pancreatic cancer continues

to be one of the deadliest cancer-related diseases in the world (31). The development of advanced

therapeutic strategies still represents a prerequisite to eventually achieve better outcome as

sufficiently early detection of pancreatic cancer is unlikely to occur in the near future. Recent

studies base on mathematical modeling for the evaluation of metastatic pancreatic cancer suggest

that metastatic processes in pancreatic cancer take place many years prior to current diagnosis

(32). Thus, an improvement of the currently available imaging methods used for the diagnosis of

pancreatic cancer is of crucial need for improving the prognosis of these patients.

Nevertheless, treatment of pancreatic cancer with the standard chemotherapy agent gemcitabine is

still not capable of eliminating the CSC subpopulation rather than resulted in a remaining G0/1

population of CSC. These cells may represent the source for rapid disease relapse.

Comprehensive cell tracking experiments will be needed to determine whether this is a remaining

quiescent CSC population that does not respond to chemotherapy and/or whether chemotherapy

results in cell cycle arrest. Most importantly, (a subpopulation of) CSC did not undergo apoptosis

and, as soon as gemcitabine was withdrawn, these cells immediately started to expand again

resulting in tumor relapse (8). The mechanistic basis of resistance to chemotherapy might not

only be linked to a quiescent subpopulation but also to their enhanced anti-apoptotic mechanisms

(33), distinct DNA repair mechanism (34), or the presence of membrane transporters that rapidly

eliminated administered drugs from these cells (35).

There are mainly two non-exclusive scenarios for depleting CSC populations. First, developing

therapeutic agents that selectively kill CSCs by targeting their self-renewal machinery without

affecting normal stem cells. Secondly, identifying cues to force CSCs into differentiation, since

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this process may be reversible due to enhanced plasticity of cancer cells, these treatment

modalities most certainly need to be accompanied by cytotoxic or other targeted therapies.

Stem cells and CSC share features as they both strongly express ABC membrane transporters, in

particular ABCG2 and MDR1, that can exclude cytotoxic substances from the cell (35) and have

an extraordinarily high capacity to repair DNA damage. CSC also display a reduced

immunogenicity and are characterized by an increased expression of anti-apoptotic genes such as

Bcl-XL that can allow the incorporation of normal nucleosides, but not toxic analogues (36).

Most importantly, however, CSC seem to contain a quiescent subpopulation, that will evade the

effects of most cytotoxic drugs simply because they are not proliferating. As a consequence, it

seems that stem cells survive conventional cancer chemotherapy and radiation (37). Of note, CSC

resistance to radiation in glioblastoma and breast cancer could be counteracted by a preferential

activation of DNA damage response mechanisms, which seem to be restricted to CSC (38, 39).

Importantly, recent studies suggested that CSCs also express high levels of telomerase despite the

quiescence of at least a subpopulation of these cells (40-43). Therefore telomerase, which is

essential for tumor progression, appears to be a critical marker in many cancers and most likely

for all cancer cells despite the inherent cellular heterogeneity of solid tumors. Because of this,

telomerase inhibition has emerged as an almost universal tumor target. It has been reported that

the combination of standard chemotherapy with telomerase inhibitors is more effective for solid

tumors such as prostate cancer (44). Several therapeutic approaches for telomerase inhibition are

now being developed and tested in solid tumors including pancreatic cancer, although most of the

candidate molecules are still in preclinical development (42). Most importantly, whether this new

treatment modality will also be capable of eliminating CSCs still remains to be determined.

Successful targeted CSC elimination may require the inhibition of multiple stemness pathways as

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a consequence of their redundancy and/or non-exclusiveness. One of the most promising

approaches to target stem cells is certainly the inhibition of stem cell-associated pathways (e.g.

sonic hedgehog, mTOR, notch, BMI, BMP). The Sonic Hedgehog (Shh) pathway has been shown

to be critical for the embryonic development of the pancreas (45), but has also been deeply

implied in the maintenance and progression of pancreatic cancer (46, 47). More recently, this

pathway has been considered as a crucial element for the maintenance of CSCs. Inhibition of Shh

signaling improved survival in a mouse model of pancreatic cancer (48) and therapeutic blockade

of this pathway in a xenograft model induced tumor regression and decreased the CSC content

(22). Moreover, Feldmann and colleagues (23) also showed that Hedgehog signaling blockade

abrogates pancreatic cancer metastases, a process which has been linked to the evasion of

migrating CSCs from the primary tumor.

Although several genetically engineered mouse models have been established to investigate a

causative role of Shh signaling in pancreatic tumorigenesis, none of them distinguished paracrine

versus autocrine canonical versus ligand-independent Shh signaling in pancreatic cancer. Yauch

et al. recently demonstrated a paracrine requirement for the Shh pathway in xenograft models of

pancreatic cancer, where Shh ligand is produced by tumor cells and the pathway is activated by

the adjacent stroma (49). To address whether a paracrine Shh signal is present in autochthonous

mouse pancreatic tumors, and to test if epithelial cancer cells is competent to transduce the Shh

signal, Tian el al. used an oncogenic form of smoothened to activate the pathway cell

autonomously (50). These data indicated that Shh signaling is restricted to tumor stroma,

contradicting previous reports that suggest a key role for ligand-driven epithelial Shh signaling in

tumor cell growth. However, these studies are still consistent with the observation that a

subpopulation of CSCs, which do not express markers of epithelial differentiation, rely on Shh

signaling (15, 23). Therefore, more work is needed to comprehensively define the precise

mechanism by which Hh pathway activation in stromal cells may also generate a

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microenvironment providing a CSC niche in pancreatic cancer.

Interestingly, Mueller et al. have recently shown that neither Shh inhibition alone nor as a

supplement to chemotherapy were capable of effectively diminishing the CSC pool (51). They

were also able to show that CD133 positive cells in pancreatic cancers show particularly high

activity for mTOR signaling suggesting that this pathway may also play are dominant role in the

CSC population. The mammalian Target Of Rapamycin (mTOR) is a serine/threonine kinase,

which belongs to the phosphatidylinositol 3-kinase (PI3K) superfamily, and is the target of a

widely branched signaling pathway that activates mTOR among other downstream effectors (52).

For pancreatic CSCs, the authors were indeed able to demonstrate that single-agent therapy with

rapamycin alone resulted in a significant decrease in CD133 positive CSCs.

However, inhibition of the mTOR pathway by rapamycin, as also observed for Shh pathway

inhibition, was not sufficient to eliminate CSCs completely. Only the combined inhibition of Shh

and mTOR together with chemotherapy resulted in the desired complete targeting of the CSC.

This triple (CRG) therapy resulted in a virtually complete depletion of the pancreatic CSC pool.

Implantation of cells that were pre-treated ex vivo consistently demonstrated that the in vivo

tumorigenic activity as the most important endpoint was completely abrogated. In a clinically

most relevant setting, the authors then investigated the effects of the triple therapy on established

pancreatic cancers utilizing patient-derived pancreatic cancer tissues (51). For the first time, the

authors were thus able to show that a multimodal therapy, involving the inhibition of two relevant

stem cell pathways and additional chemotherapy, represents a very promising approach, resulting

in virtually complete elimination of CSCs, significantly reduced tumorigenic and metastatic

activity, and long-term event-free survival. Future studies will now have to demonstrate the

transferability of these findings to the clinical scenario.

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The results from a recent study in glioblastoma indicate that inhibition of Notch by use of an

inhibitor of γ-secretase (GSI-18) is also capable of significantly reducing the CD133+ Notch

+ cell

population and consistently led to the eradication of cmedulloblastoma SP cells (53). Since

medulloblastoma cells treated with GSI-18 did no longer expand in vivo, it was concluded that

CSCs were effectively eradicated. These findings might be also applicable to pancreatic CSCs as

Notch 2 has also been implicated in pancreatic cancer progression (54), but further studies are

certainly warranted to determine the role of Notch in pancreatic CSCs.

CONCLUSIONS

The increasing evidence demonstrating a hierarchical organization for many cancers and the

identification of CSC, their prospective isolation, and characterization in pancreatic cancer has

now paved the way for the development of novel therapies targeting CSC as the putative root.

Still much work needs to be done in order to completely understand their origin in pancreatic

cancer as well as mechanisms regulating their stemness features in the tumorigenic process and

their increased resistance to chemo- and radiotherapy. But the knowledge gained during the last

years now provides an increasingly solid starting-point for the development of new research lines.

Eventually, specifically eliminating this subpopulation of cells should improve the clinical

outcome of patients suffering from the deadly pancreatic cancer. For this purpose, new clinical

endpoints need to be defined as tumor regression per se is unlikely to serve as a good predictor of

successful CSC targeting. While tracking of circulating CSC is still hampered by technological

challenges, treatment of patients in a neo-adjuvant setting with tumor biopsies prior to initiation

of treatment and a second tissue collection after treatment during surgical resection of the primary

tumor could serve as a suitable platform to track changes in CSC content.

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FIGURE LEGENDS

Figure 1. Cancer stem cell model. Like somatic stem cells, cancer stem cells are on top of a

hierarchical organization. They possess the ability to self-renew and therefore maintain the pool

of cancer stem cells within a tumor. Upon sequential differentiation they generate progenitor cells

representing the bulk of the tumor. Depending on the plasticity of the progenies, they may be

capable of contributing to the replenishment of the cancer stem cell pool. Epithelial-

Mesenchymal-Transition (EMT) may lead to the generation of a more mesenchymal stem cell

population bearing strong metastatic capacity.

Figure 2. Cancer stem cell heterogeneity. CSCs can undergo both symmetric and asymmetric

divisions. Symmetric divisions result in two daughter CSCs, contributing to the expansion of the

CSC pool. In an asymmetric division, a CSC generates another CSC plus a differentiated cell.

Moreover, CSCs evolve within the tumor due to genetic and epigenetic changes and influences

from their microenvironment that can result in the acquisition of no features of the CSC. A more

invasive phenotype could lead to the evasion of the CSC from the primary tumor, circulation and

eventually generation of metastatic lesion.

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Stem

cells

Progenitor

cells

Transit-

amplifying

cells

Differentiated

cells

Somatic

stem cells

Cancer

stem cells

Mesenchymal

phenotype

Increased

plasticity ?

Figure 1 – Dorado et al.

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CSC

CSC

CSC

CSC

Cancer stem cells

CSC

Mos

tly

Sym

met

ric

divi

sion

CSC

High percentage of CSCs

Undifferentiated state

Poor clinical outcome

Symm

etric &

asymm

etric

division

Low percentage of CSCs

More differentiated state

Better clinical outcome

CSC evolution

Genetic & epigenetic changes

Microenvironment

CSC’

CSC

Tumor evasion

CSC’

CSC’

CSC’

Metastasis

Circulating

CSC

Figure 2 – Dorado et al.

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