Content uploaded by Jan Skoda
Author content
All content in this area was uploaded by Jan Skoda on Apr 01, 2016
Content may be subject to copyright.
MASARYK UNIVERSITY
Faculty of Science
Department of Experimental Biology
Identification of cancer stem cells
in cell lines derived from selected
types of solid tumors
Ph.D. dissertation
Jan Škoda
Supervisor
prof. RNDr. Renata Veselská, Ph.D., M.Sc. Brno 2016
Bibliographic entry ◄ ii
Bibliographic entry
Author
RNDr. Jan Škoda
Faculty of Science, Masaryk University
Department of Experimental Biology
Title of dissertation:
Identification of cancer stem cells
in cell lines derived from selected types of solid tumors
Degree programme: Biology
Field of study: Molecular and Cellular Biology
Supervisor: prof. RNDr. Renata Veselská, Ph.D., M.Sc.
Academic year: 2015/2016
Number of pages: 197
Keywords:
cancer stem cells, pediatric sarcomas, osteosarcoma, Ewing’s
sarcoma, rhabdomyosarcoma, pancreatic ductal adenocarcinoma,
marker, Sox2, CD133, ABCG2, nestin
Bibliographic entry in Czech ◄ iii
Bibliografický záznam
Autor:
RNDr. Jan Škoda
Přírodovědecká fakulta, Masarykova univerzita
Ústav experimentální biologie
Název práce:
Identifikace nádorových kmenových buněk v buněčných
liniích derivovaných z vybraných typů solidních nádorů
Studijní program: Biologie
Studijní obor: Molekulární a buněčná biologie
Školitel: prof. RNDr. Renata Veselská, Ph.D., M.Sc.
Akademický rok: 2015/2016
Počet stran: 197
Klíčová slova:
nádorové kmenové buňky, sarkomy dětského věku,
osteosarkom, Ewingův sarkom, rabdomyosarkom,
duktální adenokarcinom pankreatu, marker, Sox2,
CD133, ABCG2, nestin
Abstract ◄ iv
Abstract
According to the cancer stem cell (CSC) model, CSCs are responsible for driving the tumor
growth and are considered a cause of tumor relapse and disease progression. Therefore,
therapeutic strategies targeting these cells might increase effectiveness of anticancer therapy
and could provide a reduction of its toxicity. Identification of a phenotype that specifically
defines CSCs in an individual tumor type represents the essential step toward such treatment
options.
This dissertation summarizes the results of our studies that sought to identify CSCs in
the most frequent pediatric sarcomas and in pancreatic ductal adenocarcinoma (PDAC). For
this purpose, we used CSC markers that were previously described in these or related tumor
types. Expression of CD133, ABCG2 and nestin was analyzed in pediatric sarcomas; CD24,
CD44, EpCAM, CD133 and nestin were examined in PDAC. In all experiments, we used cell
lines derived directly from primary tumors of patients with the respective tumor types. This
gave us a unique opportunity to compare results obtained in vitro with the expression of CSC
markers in the respective tumor tissues, and correlate these results with patient clinical
outcome.
We demonstrated that expression of CD133, ABCG2 or nestin does not have potential
to distinguish CSCs in pediatric sarcomas, despite these proteins were previously proposed as
CSC markers. In contrast, in vivo tumorigenicity assays strongly suggested that only the cells
displaying high levels of Sox2 are key mediators of sarcoma tumorigenesis. Thus, the Sox2
pathway could be considered as a target for new anticancer drugs in pediatric sarcomas.
Although not useful as CSC markers, CD133 and nestin were identified as significant
negative prognostic factors in Ewing’s sarcoma and rhabdomyosarcoma, respectively. In
addition, atypical subcellular localization of certain putative CSC markers, especially CD133,
was described in both sarcomas and PDAC, providing novel insights into possible functions
of these molecules in cell signaling. Finally, we were the first to demonstrate that
CD24+/CD44+/EpCAM+/CD133+ cells are present in human PDAC cell lines derived from
primary tumors. Although these cells were common under in vitro conditions, we showed that
their increased proportion corresponded to a pro-tumorigenic gene expression profile.
To conclude, our results suggest that previously described CSC markers are more
informative with regard to the clinical outcome or tumor progression than to the CSC
phenotype. Defining more selective markers, such as Sox2, is needed for further
investigations of CSCs.
Abstract in Czech ◄ v
Abstrakt
Podle modelu nádorových kmenových buněk (CSCs) jsou tyto buňky zdrojem
nádorového růstu a jsou proto považovány za příčinu relapsu a progrese nádorových
onemocnění. Terapeutické strategie, které by byly zaměřeny na CSCs, by mohly v budoucnu
zvýšit efektivitu protinádorové léčby a zároveň snížit její toxicitu. Prvním a nezbytným
krokem pro dosažení takových léčebných metod je však identifikace fenotypu, který
v jednotlivých typech nádorů specificky odliší CSCs.
Tato dizertační práce shrnuje výsledky vlastních studií, jejichž cílem bylo identifikovat
CSCs u duktálního adenokarcinomu pankreatu (PDAC) a nejčastějších pediatrických
sarkomů. Pro dané účely byly využity markery, které byly dříve popsány jako specifické pro
CSCs u těchto nebo podobných typů nádorů. U pediatrických sarkomů byla analyzována
exprese CD133, ABCG2 a nestinu. U PDAC pak exprese CD24, CD44, EpCAM, CD133
a nestinu. V rámci všech experimentů byly využity buněčné linie derivované z primárních
nádorů odebraných pacientům s příslušným typem nádoru, což umožnilo porovnat výsledky
získané in vitro s expresí markerů CSCs v příslušné nádorové tkáni a korelovat tyto výsledky
s klinickým průběhem onemocnění daného pacienta.
Ačkoliv exprese CD133, ABCG2 nebo nestinu byla dříve považována za specifický
marker CSCs, naše výsledky prokázaly, že tyto proteiny nemají u pediatrických sarkomů
potenciál rozlišit CSCs. Naopak, testy tumorigenicity in vivo naznačily, že pouze buňky, které
vykazují zvýšené hladiny Sox2, jsou zodpovědné za tumorigenezi sarkomů. Signální dráhu
Sox2 lze proto u pediatrických sarkomů zvažovat jako vhodný cíl nových protinádorových
léčiv. Přestože CD133 ani nestin nepředstavovaly vhodné markery CSCs, zvýšená exprese
CD133 byla identifikována jako významný negativní prognostický faktor u Ewingova
sarkomu a nestin byl obdobně asociován s horším přežitím u rabdomyosarkomu. Jak u
pediatrických sarkomů, tak u PDAC byla navíc objevena atypická intracelulární lokalizace
některých z navrhovaných markerů CSCs, zejména CD133, což nově poukazuje na možné a
dosud neprozkoumané funkce těchto molekul v buněčné signalizaci. V neposlední řadě pak
analýza markerů CSCs u PDAC vůbec poprvé prokázala v lidských buněčných liniích
derivovaných z primárních nádorů pankreatu přítomnost buněk s fenotypem
CD24+/CD44+/EpCAM+/CD133+. Ačkoliv se obecně tyto buňky vyskytovaly v podmínkách
in vitro ve velkém počtu, jejich zvýšené zastoupení v rámci buněčné linie korespondovalo
s pro-tumorigenním expresním profilem.
Abstract in Czech ◄ vi
Z výsledků studií souhrnně prezentovaných v této práci tak vyplývá, že význam dříve
navrhovaných markerů CSCs spočívá spíše v jejich možném využití pro účely stanovení
prognózy onemocnění než pro identifikaci CSCs samotných. Nalezení selektivnějších
markerů, jako např. Sox2, je proto nezbytné pro další výzkum CSCs.
Acknowledgements ◄ vii
Acknowledgements
First and foremost, I would like to thank my supervisor prof. RNDr. Renata Veselská, Ph.D.,
M.Sc. for giving me the opportunity to work on this intriguing topic of cancer stem cell
research. I am deeply grateful for all her contributions of time, ideas, and funding, which
helped me through this challenging Ph.D. pursuit. I also appreciate that she gave me the
chance to participate on several research projects; on many of them from the early beginning
of the grant proposal writing, which is an invaluable experience. Most of all, I would like to
thank her for the trust she gave me during my Ph.D. years, and especially for being a great
scientific mentor, who taught me to narrow and focus my efforts.
I thank to all my colleagues from the Laboratory of Tumor Biology (Masaryk
University, Brno, Czech Republic), who created very friendly, motivating and cooperative
atmosphere. My special thanks belong to RNDr. Jakub Neradil, Ph.D., and Mgr. Alena
Nunukova who helped me by insightful discussions and motivated me many times. I really
enjoyed being a part of this team.
I would also express my acknowledgements to prof. MUDr. Jaroslav Štěrba, Ph.D.,
MUDr. Peter Múdry, Ph.D., and other colleagues from the Department of Pediatric Oncology
(University Hospital Brno, Czech Republic) for providing us with tumor tissue samples and
relevant clinical data. Without this excellent and friendly collaboration it would be impossible
to perform our research. Thank you all.
Similarly, I wish to express my deep gratitude to prof. MUDr. Markéta Hermanová,
Ph.D., and MUDr. Iva Zambo, Ph.D. from the 1st Institute of Pathologic Anatomy (St. Anne’s
University Hospital, Brno, Czech Republic) for their expertise, which always extended the
value of our results. I really appreciate your enthusiastic efforts to fulfill our needs.
Last but not least, I would like to warmly thank to my parents, sister and friends for
supporting me on my studies. Especially, I would like to thank my wife for her understanding
and love during the past demanding years, and to my daughters, who light up my life. Thank
you!
This research was supported by the project No. NT13443-4 from the Internal Grant Agency of
the Czech Ministry of Healthcare, by the project Translational Medicine (No. LQ1605) from
the National Program of Sustainability II, and by the European Regional Development Fund
– projects CEB (No. CZ.1.0/2.3.00/20.0183), and RECAMO (No. CZ.1.05/2.1.00/03.0101).
Declaration and copyright ◄ viii
Declaration
Hereby I declare that I worked on this Ph.D. dissertation on my own under the supervision of
prof. RNDr. Renata Veselská, Ph.D., M.Sc., and I used only primary and secondary literature,
which is properly cited and listed in the References.
Brno 29. 2. 2016
………………………………
Jan Škoda
© Jan Škoda, Masaryk University, 2016
Content ◄ ix
Content
List of abbreviations ................................................................................................................. x
1 Introduction ...................................................................................................................... 1
2 Background ....................................................................................................................... 2
2.1 Cancer stem cells ......................................................................................................... 2
2.1.1 Cancer stem cell model ......................................................................................... 2
2.1.2 Cancer stem cell markers ...................................................................................... 5
2.1.3 Detection of cancer stem cells ............................................................................ 12
2.1.4 Implications for clinical practice ........................................................................ 16
2.2 Pediatric sarcomas ..................................................................................................... 18
2.2.1 Osteosarcoma ...................................................................................................... 18
2.2.2 Ewing’s sarcoma ................................................................................................. 22
2.2.3 Rhabdomyosarcoma ........................................................................................... 25
2.3 Pancreatic ductal adenocarcinoma ............................................................................. 28
3 Objectives ........................................................................................................................ 34
4 Results and discussion .................................................................................................... 35
5 Conclusion ....................................................................................................................... 41
6 References ........................................................................................................................ 42
7 Publications related to the thesis ................................................................................... 57
7.1 Manuscript I ............................................................................................................... 60
7.2 Manuscript II .............................................................................................................. 77
7.3 Manuscript III ............................................................................................................ 83
7.4 Manuscript IV ............................................................................................................ 90
7.5 Manuscript V ............................................................................................................. 99
7.6 Manuscript VI .......................................................................................................... 114
7.7 Manuscript VI I ......................................................................................................... 147
List of abbreviations ◄ x
List of abbreviations
ABC
ATP binding cassette
ALDH
aldehyde dehydrogenase
BAA
BODIPY amino acetate
BAAA
BODIPY aminoacetaldehyde
BrdU
5-bromo-2’-deoxyuridine
CD
cluster of differentiation
CFU
colony forming unit
CSCs
cancer stem cells
CSFE
carboxyfluorescein diacetate succinimidyl ester
DEAB
diethylaminobenzaldehyde
ΔTK
truncated version of the herpes simplex virus thymidine kinase
EMT
epithelial-mesenchymal transition
EpCAM
epithelial cell adhesion molecule
ESA
epithelial-specific antigen
ESCs
embryonic stem cells
ETS
E26 transformation-specific
FACS
fluorescence-activated cell sorting
FGFR3
fibroblast growth factor receptor 3
FoxO1
forkhead box protein O1
GFP
green fluorescent protein
HGF
hepatocyte growth factor
HMG
high mobility group
iPSCs
induced pluripotent stem cells
MSCs
mesenchymal stem cells
NOD/SCID
non-obese diabetic/severe combined immunodeficiency
NOG
NOD/ShiJic-scid/IL2Rγnull immunodeficient strain of mice
NSG
NOD/ShiLtSz-scid/IL2Rγnull immunodeficient strain of mice
PanIN
pancreatic intraepithelial neoplasia
PDAC
pancreatic ductal adenocarcinoma
SP
side population
TICs
tumor-initiating cells
Introduction ◄ 1
1 Introduction
The purpose of this Ph.D. thesis was to identify cancer stem cells (CSCs) in selected cell
lines, derived predominantly from pediatric solid tumors. As a part of a long-term
collaboration, these tumor cell lines have been routinely derived in our Laboratory of Tumor
Biology from tumor samples of patients treated at the Department of Pediatric Oncology,
University Hospital Brno (Czech Republic). To date, we keep more than 350 pediatric tumor
cell lines, which give us a great opportunity to investigate biological characteristics of tumors
affecting this particular age group of patients.
The focus of this Ph.D. thesis originates from initial observations published by our
group concerning expression of nestin and CD133 in glioblastoma and osteosarcoma cell lines
(Veselska et al., 2006; Veselska et al., 2008; Loja et al., 2009). At that time, both nestin and
CD133 were discussed as markers of stem-like phenotype of cancer cells in various
malignancies. However, little was known about the role of these proteins in pediatric
sarcomas; even less evidence was available regarding CSCs in these tumor types frequent in
children. Therefore, we sought to determine whether cells with stem-like phenotype can be
identified in pediatric sarcomas. Studies that aimed at this goal were performed in
collaboration with colleagues from the 1st Institute of Pathologic Anatomy, St. Anne’s
University Hospital (Brno, Czech Republic).
Expression of nestin in pancreatic ductal adenocarcinoma (PDAC) has been studied by
this group (Lenz et al., 2011). During the progress of their study, we successfully derived 24
PDAC cell lines. As CSCs have been proposed to play key role in PDAC tumor initiation and
maintenance (Li et al, 2007; Hermann et al., 2007), this tumor type represented other subject
of this Ph.D. thesis.
The first part of this dissertation briefly introduces the model of CSC-driven
tumorigenesis; overview of the methods used to study CSCs and implications of this model
for cancer treatment will follow. Next, summary of the current knowledge about the cancer
stem cells in the most common types of pediatric sarcomas – i.e. rhabdomyosarcoma,
osteosarcoma and Ewing’s sarcoma – and in PDAC is given. The main part of this
dissertation constitutes of five original and two review articles aimed at identification and
characterization of cells with CSC phenotype in pediatric sarcomas and PDAC: the results of
these studies are presented and discussed; all these articles are also attached in full texts.
Background – Cancer stem cells ◄ 2
2 Background
2.1 Cancer stem cells
Cancer stem cells (CSCs), also termed tumor-initiating cells (TICs), are defined as a
subpopulation of cancer cells with self-renewing capacity that possess high tumorigenic
potential and can undergo multilineage differentiation to give rise to all types of cells present
within the malignancies (Clarke et al., 2006). According to the CSC model, CSCs drive tumor
growth and these cells are considered to be a cause of tumor relapse and disease progression,
perhaps through their resistance to therapy and metastatic potential. Therefore, CSCs are
currently extensively studied in various cancers.
Although the concept of CSC hypothesis is very old (for further details concerning the
history of CSC hypothesis see Cogle, 2011; Wicha et al., 2006), the field of CSC research
truly started about 20 years ago when Dick and colleagues published their pioneer studies
demonstrating that only minor subpopulation of acute myeloid leukemia cells with
CD34+/CD38− phenotype presents the potential to initiate leukemia in the immunodeficient
mice (Lapidot et al., 1994; Bonnet and Dick, 1997). Since then, large body of evidence has
been published that supports the essential role of CSCs in initiation and progression of several
hematological malignancies and of a wide range of solid tumors (Beck and Blanpain, 2013).
2.1.1 Cancer stem cell model
Cellular heterogeneity is a common feature of a spectrum of human malignancies from solid
tumors to hematological malignancies and this intratumoral heterogeneity represents one of
the greatest challenges in cancer therapeutics (Kleppe and Levine, 2014; Zellmer and Zhang,
2014; O’Connor et al., 2014; Brooks et al., 2015). As this thesis focus on CSCs in solid
tumors, tumor-oriented terminology is used in the following paragraphs. However, the
principles of cancer development described further are applicable to both solid tumors and
hematological malignancies.
In general, two models are usually proposed to explain tumor growth and heterogeneity
(Fig. 1). According to the stochastic model (Fig. 1a), all tumor cells are equipotent and
proliferate to fuel tumor growth while some of the tumor cells differentiate (Beck and
Blanpain, 2013). Conversely, CSC model proposes that only certain cells contribute to long-
term tumor growth (Fig. 1b) and these CSCs generate, analogously to normal stem cells, more
Background – Cancer stem cells ◄ 3
restricted progenitor cells with limited replicative/self-renewal capacity. However, it is
believed that these progenitors may transiently extensively proliferate and thus constitute the
mass of the tumor (Kreso and Dick, 2014).
Fig. 1 – Tumor growth models. (a) In the stochastic model of tumor growth, all tumor cells are
equipotent and stochastically self-renew or differentiate, leading to intratumoral heterogeneity. (b) In
the CSC model, only a subset of cells with the capacity of long-term self-renewal is responsible for
sustained tumor growth. These CSCs give rise to more committed progenitors with limited proliferative
potential that eventually terminally differentiate. (c, d) Clonal evolution resulting from new somatic
mutations may further increase intratumoral cellular heterogeneity in both stochastic and CSC model.
As suggested by Driessens et al. (2012) clonal evolution in the CSC model may also originate from
intrinsic nature of CSCs themselves, as every CSC within a tumor is equally likely to clonally expand
due to neutral completion between these cells. (Illustrations created based on Beck and Blanpain,
2013).
It is widely accepted, that tumor cell subpopulations (clones) evolve through
accumulation of genetic and epigenetic mutations, some of which may increase the fitness and
survival of the individual clone, and thus promote its expansion in the respective tumor
microenvironment (Greaves and Maley, 2012). Importantly, clonal evolution may occur in
Background – Cancer stem cells ◄ 4
both stochastic model (Fig. 1c) and CSC model (Fig. 1d) of tumorigenesis. However,
considerable controversy remains regarding the hierarchical organization proposed by the
original CSC model (Fig. 1b,d). Several studies have shown that differentiated or neoplastic
non-stem cells are able to re-enter CSC state under certain circumstances. Such cell plasticity
has been shown for breast carcinoma (Chaffer et al., 2011; Iliopoulos et al., 2011; Chaffer et
al., 2013), colorectal carcinoma (Vermeulen et al., 2010; Schwitalla et al., 2013), and gliomas
(Charles et al., 2010). These findings led to the proposal of a fluid CSC model where the cell
hierarchy is more transient than previously suggested (Fig 2; O’Connor et al., 2014).
Fig. 2 – Fluid CSC model. In this extended CSC model of tumor growth, both progenitor cells and
differentiated cells are able to re-acquire self-renewal potential (indicated by red arrows), thus
becoming CSCs. That means that although the cells are hierarchically organized with the respect to
their stem-like characteristics in a certain time point, this hierarchy is more transient and may change
during tumor progression. (Illustration created based on O’Connor et al., 2014 and Beck and Blanpain,
2013).
However, recent lineage-tracing studies (all reviewed in Rycaj and Tang, 2015)
provided clear evidence of CSCs across three different types of solid tumors: papilloma and
squamous cell carcinoma (Driessens et al., 2012), glioblastoma (Chen et al., 2012), and
intestinal adenoma (Schepers et al., 2012). Parada and co-workers showed that
nestin-expressing glioblastoma cells are the source of the new glioblastoma growth after
transient tumor growth arrest induced by temozolomide treatment (Chen et al., 2012; this
study is described in detail in Chapter 2.1.2). Clevers and colleagues crossed R26R-Confetti
mice into Lgr5 knock-in tamoxifen-inducible Cre-expressing mouse strain to obtain Lgr5+
cells randomly labeled by one of the four fluorescent colors encoded in the R26R-Confetti
allele (Schepers et al., 2012). By this approach, the researchers demonstrated the role of
single Lgr5+ intestinal stem cells in the initiation and maintenance of intestinal adenoma.
Background – Cancer stem cells ◄ 5
Finally, the group of Cedric Blanpain demonstrated cellular hierarchy in a mouse model of a
benign skin papilloma showing that only about 20% of papilloma cells present long-term
self-renewal capacity (Driessens et al., 2012). Remarkably, neutral competition was found
between these CSCs, indicating that clonal expansion is continuous process that may not be
driven by rare mutations but may originate in CSCs themselves. Moreover, Driessens et al.
showed that progression of papilloma to squamous cell carcinoma was associated with an
expansion of the CSCs and reduced production of non-stem cells. These results suggest that
hierarchical organization of the tumor cells may diminish during malignant progression.
2.1.2 Cancer stem cell markers
Identification and/or isolation of cells with CSC phenotype represent the essential step in CSC
studies. For this purposes, various molecules have been evaluated to mark selectively CSC
population. In the initial studies, different cell surface proteins were used to distinguish CSCs
from other cancer cells. In mid-1990’s, the research group of John Dick sorted acute myeloid
leukemia cells for CD34+/CD38− phenotype and showed that this cell fraction exhibited
significantly higher capacity to initiate leukemia after injection into immunodeficient mice
(Lapidot et al., 1994; Bonnet and Dick, 1997). These studies gave the theoretical and
methodological bases for the subsequent efforts to identify CSCs in other malignancies.
Tumorigenicity assay in mice, as used by Dick’s group, still stays the most definitive
test of CSC phenotype (the functional assays used for detection of CSCs are reviewed in
chapter 2.1.3). As immunophenotyping using cluster of differentiation (CD) antigens is
routinely used in diagnosis of leukemia, the use of these cell surface molecules for isolation of
specific leukemia cell fractions was obvious. However, the same approach was later applied
also in solid tumors, which resulted in the first evidence that CD44+/CD24− phenotype
identifies CSCs in breast cancer (Al-Hajj et al., 2003) and that CD133 molecule marks CSCs
in human brain tumors (Singh et al., 2003) . Since then, a wide range of cell surface proteins
has been proposed as CSC markers in many types of tumors. Nevertheless, the validity of
these markers is partly controversial as there is no evidence that any combination of CSC
markers isolates any CSC population to a high degree of purity (Meacham and Morrison,
2013; Lathia, 2013). Currently, common pluripotency markers such as Oct4, Sox2, and
Nanog are extensively studied in relation to CSCs (Liu et al., 2013a). Activation targets of
these transcription factors were found to be more frequently overexpressed in poorly
differentiated tumors than in well-differentiated tumors (Ben-Porath et al., 2008). These
initial data revealed a link between genes associated with embryonic stem cell (ESC) identity
Background – Cancer stem cells ◄ 6
and tumor pathogenesis and supported the possibility that these genes contribute to stem
cell-like phenotype exhibited by many tumors.
Following paragraphs provide a brief overview of the most important putative CSC
markers in solid tumors with respect to those studied in sarcomas and PDAC. A
comprehensive list of CSC markers reported in sarcomas and PDAC is indicated in Table 1.
Tab. 1 – Putative CSC markers in pediatric sarcomas and PDAC
CSC marker Tumor type Reference
ABCG2 Osteosarcoma
Martins-Neves et al. (2016); Tirino et al. (2008);
Di Fiore et al. (2009); Saini et al. (2012)
PDAC Olempska et al. (2007); Matsuda et al. (2014)
ALDH
Osteosarcoma
Honoki et al. (2010); Wang et al. (2011);
Martins-Neves et al. (2015)
Ewing’s sarcoma Awad et al. (2010)
Rhabdomyosarcoma Nakahata et al. (2015)
PDAC Rasheed et al. (2010); Kim et al. (2011a); Kim et al.
(2013); Song et al. (2015); Singh et al. (2016)
c-Met PDAC
Li et al. (2011); Hage et al. (2013);
Delitto et al. (2014)
CD24 PDAC
Li et al. (2007); Rasheed et al. (2010);
Hage et al. (2013)
CD44 PDAC
Li et al. (2007); Rasheed et al. (2010);
Li et al. (2011); Hage et al. (2013);
Herreros-Villanueva et al. (2013)
CD49f Osteosarcoma Ying et al. (2013); Penfornis et al. (2014)
CD57 Ewing’s sarcoma Wahl et al. (2010); Leuchte et al. (2014)
CD117 (c-kit) Osteosarcoma Adhikari et al. (2010)
CD133
(prominin-1)
Osteosarcoma
Tirino et al. (2008); Veselska et al. (2008);
Tirino et al. (2011); Yang et al. (2011);
He et al. (2012); Zambo et al. (2012)
Ewing’s sarcoma
Suva et al. (2009); Riggi et al. (2010);
Jiang et al. (2010); Leuchte et al. (2014)
Rhabdomyosarcoma
Walter et al. (2011); Pressey et al. (2013);
Hirotsu et al. (2009)
PDAC
Hermann et al. (2007); Immervoll et al. (2008);
(Immervoll et al, 2011); Kure et al. (2012); Hou et al.
(2014); Chen et al. (2014); Kim et al. (2012); Li et al.
(2015); Song et al. (2015)
Background – Cancer stem cells ◄ 7
Tab. 1 – Continued
CSC marker Tumor type Reference
CXCR4 Osteosarcoma Adhikari et al. (2010); He et al. (2012)
PDAC
Hermann et al. (2007); Thomas et al. (2008);
Shen et al. (2013); Krieg et al. (2015)
EpCAM PDAC
Li et al. (2007); Hage et al. (2013);
Herreros-Villanueva et al. (2013)
FGFR3 Rhabdomyosarcoma Hirotsu et al. (2009)
FoxO1 negative PDAC Song et al (2015)
Lgr5 Ewing’s sarcoma Scannell et al. (2013)
Nanog
Osteosarcoma
Tirino et al. (2011); Wang et al. (2011);
He et al. (2012)
Ewing’s sarcoma Suva et al. (2009); Riggi et al. (2010)
Rhabdomyosarcoma Walter et al. (2011)
Nestin Osteosarcoma Veselska et al. (2008); Tirino et al. (2011);
Zambo et al. (2012)
PDAC Hagio et al. (2013); Matsuda et al. (2014)
Oct4
Osteosarcoma
Tirino et al. (2011); Wang et al. (2011);
He et al. (2012)
Ewing’s sarcoma Suva et al. (2009);
Riggi et al. (2010)
Rhabdomyosarcoma Walter et al. (2011)
Sox2
Osteosarcoma
Tirino et al. (2011); Wang et al. (2011) ;
Basu-Roy et al. (2012); Yang et al. (2014a);
Martins-Neves et al. (2015); Basu-Roy et al. (2015)
Ewing’s sarcoma Riggi et al. (2010)
Rhabdomyosarcoma Walter et al. (2011); Nakahata et al. (2015)
PDAC Hage et al. (2013); Herreros-Villanueva et al. (2013);
Singh et al. (2015)
If not indicated otherwise, enhanced expression or activity of respective molecule served as CSC
marker. Note that some contradictory references are listed.
CD133 (also known as prominin-1) is a pentaspan transmembrane glycoprotein belonging to
the prominin family. At least seven CD133 isoforms resulting from alternative splicing of
PROM1 gene transcript have been described (Grosse-Gehling et al., 2013; Corbeil et al.,
2013). These molecules may further differ in their glycosylation status, thus the molecular
weight of CD133 ranges from 92 to 120 kDa. CD133 is widely used to identify various stem
Background – Cancer stem cells ◄ 8
cells and has been reported as a marker of CSCs in a wide range of human malignancies
(Grosse-Gehling et al., 2013). However, the validity of such reports remains questionable,
because there is also plenty of evidence arguing against CD133 as a specific marker of CSCs
even in those previously reported tumor types, such as gliomas or colorectal carcinoma (for a
comprehensive review, see Table 3 in Grosse-Gehling et al., 2013). The very similar
discrepancies can be found concerning the prognostic significance of CD133 expression in
tumor tissue. Although CD133 is preferentially localized in highly curved plasma membrane
protrusions such as microvilli and primary cilia (Corbeil et al., 2010), the physiological
function of this protein remains largely unknown. It was demonstrated that CD133 molecule
can be phosphorylated on cytoplasmic tyrosine sites (Boivin et al., 2009). Another studies
suggested an involvement of CD133 in the Wnt or the PI3K/Akt signaling pathways (Mak et
al., 2012; Takenobu et al., 2011; Wei et al., 2013; Shimozato et al., 2015). Recently, CD133
has been directly linked to p53 in a study showing that p53 can bind to CD133 promoter
which leads to downregulation of CD133 expression (Park et al., 2015). Finally, atypical
subcellular localization of CD133 that suggests its previously unknown biological functions
have been observed in tumor cells; our results concerning this phenomenon that were obtained
as a part of this thesis are discussed in the Chapter 4.
CD24 is a small cell surface protein (consisting of 27 amino acids only) with highly variable
glycosylation depending on the cell or tissue type (Fang et al., 2010). CD24 functions as a
ligand in cell-cell and cell-matrix interactions, however its ligand specificity seems to be
dependent on distinct glycosylation status (Fang et al., 2010; Jaggupilli and Elkord, 2012).
Correlation of CD24 expression with tumor progression and worse prognosis has been
reported for a variety of human carcinomas including breast, colorectal, gastric, lung, ovarian,
pancreatic, and prostate carcinomas (Jaggupilli and Elkord, 2012). Correspondingly, CD24
has been proposed as a CSC marker in these carcinomas; however, the role of CD24
expression status as marker of CSCs is ambiguous (Fillmore and Kuperwasser, 2007; Keysar
and Jimeno, 2010; Jaggupilli and Elkord, 2012). For example, although several studies
suggested CD44+/CD24−/low phenotype as CSC marker in breast carcinoma (Al-Hajj et al.,
2003; Sheridan et al., 2006; Yan et al., 2013), it has been demonstrated that CD24 expression
in breast cancer cells is dynamically regulated and interconversion between CD44+/CD24+
and CD44+/CD24− phenotypes may occur (Meyer et al., 2009). Finally, upregulated
expression of CD24 has been shown to associate with worse prognosis in breast carcinoma
patients (Kristiansen et al., 2003; Kim et al., 2011b; Kwon et al., 2015).
Background – Cancer stem cells ◄ 9
CD44 is a multifunctional transmembrane glycoprotein expressed as a wide variety of
isoforms in many cells (Zöller, 2011; Yan et al., 2015). CD44, which is implicated in cell–cell
and cell–matrix interactions, is an important receptor for hyaluronan and a co-receptor for
array of growth factors and cytokines. The multiple cellular functions of CD44 rely on its
association with partner proteins that regulate cell migration and adhesion, growth, survival,
and differentiation (Misra et al., 2011). Expression of CD44 has been identified as a typical
CSC surface marker in several malignancies of hematopoietic and epithelial origin, including
PDAC, breast carcinoma, colorectal or lung carcinoma (Yan et al., 2015). CD44 is involved in
homing of hematopoietic stem cells and leukemia–initiating cells into the niche (Zöller,
2011). In solid tumors, CD44 induces migration, epithelial-mesenchymal transition (EMT)
and extravasation, thus it promotes metastasizing of tumor cells (Zöller, 2011; Yan et al.,
2015).
Epithelial cell adhesion molecule (EpCAM), previously termed as epithelial-specific
antigen (ESA), is a transmembrane glycoprotein of 314 amino acids, consisting of a large
N-terminal extracellular domain, a single-spanning transmembrane domain and a short
C-terminal cytoplasmic domain (Strnad et al., 1989). EpCAM was originally described as
colorectal carcinoma-specific antigen (Herlyn et al., 1979). Since then, EpCAM was detected
in a wide range of normal human epithelial tissues and human carcinomas (Schnell et al.,
2013). Although EpCAM is able to promote cell–cell interactions, these are relatively weak
and increased expression of EpCAM is often associated with malignant transformation that is
promoted by weakening cadherin-mediated cell–cell adhesions (Schnell et al., 2013).
Overexpression of EpCAM has been associated with worse overall survival in various
carcinomas, and has also been linked to metastases and CSCs (Patriarca et al., 2012).
EpCAM has been shown as an effective marker of circulating tumor cells and is currently
widely used for their detection and isolation (Raimondi et al., 2015), although EpCAM
sensitivity in these assays have been questioned recently (de Wit et al., 2015; Schneck et al.,
2015).
ATP binding cassette (ABC) transporters are found in the plasma membrane of numerous
cell types giving them protection against xenobiotics (Wilkens et al., 2015). ABC transporters,
especially ABCC1 and ABCG2, are often overexpressed in transformed cells and represent
the major cause of multidrug resistance phenotype of various cancers. Interestingly, high
expression of ABC transporters has been identified in side populations of hematopoietic stem
Background – Cancer stem cells ◄ 10
cells and other tissue-specific stem cells (Bunting, 2002; Dean, 2009; Ding et al., 2010; the
method of side population analysis is described in Chapter 2.1.4). Recent study demonstrated
high levels of different ABC proteins also in ESCs and their derivatives, with ABCG2 being
the most prevalent in the undifferentiated ESCs (Erdei et al., 2014). These results show that
ABC transporters are commonly expressed in stem cells suggesting an important role of these
proteins in stem-cell physiology. Not surprisingly, elevated expression of ABC transporters,
especially ABCG2, has been linked to CSC phenotype in a wide range of tumors (Ding et al.,
2010; Tirino et al., 2008; Di Fiore et al., 2009; Saini et al., 2012; Leccia et al., 2014; Cioffi et
al., 2015).
Aldehyde dehydrogenase (ALDH) actually represents the whole family of enzymes
comprising of at least 19 isoforms (reviewed in Marchitti et al., 2008). ALDHs are
responsible for oxidizing aldehydes to carboxylic acids, thus prevent cells from cytotoxic
effects caused by many aldehydes. ALDHs are also involved in retinoid metabolism and were
demonstrated to be essential in embryonic development. ALDH activity, as measured by
ALDH assay (described in Chapter 2.1.4), is considered to be a universal marker of normal
stem cells as well as CSCs (Marcato et al., 2011; Ma and Allan, 2011). This enhanced ALDH
activity may be partially the cause of inherent chemoresistance of CSCs. It has been
demonstrated that overexpression of ALDH1A1 and ALDH3A1 in cell lines and normal
hematopoietic progenitor cells results in increased resistance to cyclophosphamide; however,
the role of ALDH in resistance to another chemotherapeutic agents remains controversial
(Januchowski et al., 2013). Nevertheless, overexpression of specific ALDH isoforms,
especially ALDH1A1 and ALDH1A3, has been associated with worse prognosis in breast
carcinoma, lung carcinoma, pancreatic adenocarcinoma, and carcinomas of ovaries, prostate,
bladder and rectum (Marcato et al., 2011; Liu et al., 2013b; Deng et al., 2014).
Nestin, a class VI intermediate filament protein is widely described as an important marker of
CSCs, especially in tumors of neurogenic origin (Krupkova et al., 2010; Neradil and
Veselska, 2015). Intermediate filaments represent an important cytoskeletal component
responsible for mechanical integrity of the cell, thus providing the scaffold for other
components of the cytoskeleton and for some organelles (Dey et al., 2014). Nestin is
primarily regarded as a marker of neural stem/progenitor cells, although its expression has
been shown also in various embryonic tissues and at tissue/organ-specific sites in adults
(Wiese et al., 2004). The re-expression or upregulation of nestin was observed during tissue
Background – Cancer stem cells ◄ 11
reparation processes and in many types of human malignancies. Besides a spectrum of
neurogenic tumors, nestin expression has been shown to identify CSCs in a variety of tumors
of mesenchymal and epithelial origin (comprehensively reviewed in Neradil and Veselska,
2015). As mentioned above, the group of Luis Parada recently reported nestin expression as a
marker of relatively quiescent glioblastoma cells that were demonstrated to be the source of
new tumor growth after temozolomide treatment (Chen et al., 2012). For the purpose of their
study, the authors used glioma-prone mice model harboring the additional Nes-ΔTK-GFP
transgene comprising of truncated version of the herpes simplex virus (HSV) thymidine
kinase (ΔTK) and green fluorescent protein (GFP) under the control of nestin promoter. Thus,
GFP expression selectively labeled nestin expression in the mouse cells and expression of
ΔTK sensitized the GFP+ cells to ganciclovir. GFP+ cells were found temozolomide-resistant
and relatively quiescent compared with their GFP− counterparts. More importantly, lineage
tracing of the GFP+ cells revealed that these cells were responsible for the re-initiation of the
tumor growth after the administration of temozolomide to tumor-bearing mice.
Sox2 is a member of SOX family of transcription factors, which are characterized by the
presence of DNA-binding high-mobility-group (HMG) domain with amino acid similarity of
50% or higher to the HMG domain of the mammalian testis-determining factor, Sry (Sarkar
and Hochedlinger, 2013). Sox2 has been shown to be crucial for stem cell maintenance and
cell lineage fate determination, and it is often referred to as a core ESC pluripotency factor
(together with Oct4 and Nanog) (Yeo et al., 2013). The essential role Sox2 in pluripotency has
been clearly demonstrated by its use for the reprogramming of differentiated adult cells to
induced pluripotent stem cells (iPSCs) (reviewed in Liu et al., 2014a). In 2006, the research
group of Shinya Yamanaka used four transcription factors – Oct4, Sox2, Klf4, and c-Myc – to
reprogram adult mouse fibroblasts into iPSCs (Takahashi and Yamanaka, 2006). A year later,
the same group of researchers used these factors for derivation of iPSCs from adult human
fibroblasts (Takahashi et al., 2007). An independent study, published on the exactly same
day, showed that iPSCs can be also generated from human somatic cells using Oct4, Sox2,
Lin28, and Nanog transcription factors (Yu et al., 2007). Therefore, expression of Sox2 has
been recently extensively studied in various malignancies (reviewed in Weina and Utikal,
2014). These studies, including ours, that suggest Sox2 as a common CSC marker are
discussed in the Chapter 4.
Background – Cancer stem cells ◄ 12
2.1.3 Detection of cancer stem cells
Although CSC markers have been proven useful for the enrichment of CSC populations, their
utility is still limited by variability in expression and regulation by environmental factors. As
a result, definitive identification of CSCs requires functional assays demonstrating their
essential features – the ability to self-renew and the tumorigenic potential. Based on the
methodical approaches used, these functional assays can be classified into two main
categories: in vivo assays and in vitro assays (Skoda et al., 2014 – Manuscript III).
In vivo tumorigenicity assay. This method represents a gold standard test of CSC phenotype,
because it enables to tests (directly in an animal model) the ability of cancer cells to self-
renew and to form a tumor that resembles cellular heterogeneity of the primary tumor (Clarke
et al., 2006). In general, cells tested by this assay are implanted into immunodeficient mice
and the possible tumor growth is examined. After the first appearance of the tumor or after
defined time period, mice are sacrificed and the tumorigenicity is evaluated as a ratio of
number of mice that developed a tumor to number of all injected mice. Tumor size, time to
the first appearance of the tumor, and the number of cells implanted are other criteria usually
used for the comparison of different cell fractions tested in these experiments. The capacity of
cells to self-renew need to be further confirmed by serial xenotransplantations, i.e., by
repeated isolation of CSCs from the xenograft tumors and their implantation into secondary
and tertiary recipient mice (Fig. 3).
Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were used
in the initial CSC studies that demonstrated the presence of very limited fraction of CSCs
(Quintana et al., 2008). However, the subsequent studies showed that tumorigenicity read out
may be significantly increased with the use of severely immunodeficient mice strains bearing
the mutation in gene coding the γ-chain of the interleukin-2 receptor: NOD/SCID/IL2Rγnull,
NOD/ShiLtSz-scid/IL2Rγnull (NSG), and NOD/ShiJic-scid/IL2Rγnull (NOG) (McDermott et
al., 2010). Through the use of NSG mice, the assessed frequency of melanoma CSCs
increased from previously published 0.0001% (Schatton et al., 2008) to 27% and 30%, while
the tumors were formed even after injection of the single cells (Quintana et al., 2008;
Quintana et al., 2010). Similarly, much higher tumorigenicity of leukemia cells was reported
when tested in NSG mice (Agliano et al., 2008). Although the change in the frequency of
CSCs has not been observed in some types of solid tumors, such as PDAC, usage of NSG
mice generally shortened the time to the first occurrence of the tumor.
Background – Cancer stem cells ◄ 13
Fig. 3 – Schematic overview of in vivo tumorigenicity assay. The CSC phenotype is definitively
confirmed by serial xenotransplantation of sorted cell fractions. Only cells that initiate tumors in mice fit
the definition of CSCs.
In vitro functional assays. The main advantage of all in vitro assays for testing of the CSC
phenotype is the fact that the results are obtained in shorter time than those of in vivo assays.
However, it is always necessary to confirm the results of in vitro approaches using the in vivo
tumorigenicity assay because the in vitro methods are always simplified and they cannot
mimic all aspects of the tumorigenesis in one assay. The most frequently used in vitro
functional assays include (i) sphere formation assay, (ii) colony-forming unit assay, also
Background – Cancer stem cells ◄ 14
termed as clonogenic assay, (iii) side population analysis, (iv) aldehyde dehydrogenase
activity assay, and (v) label retention assay.
Sphere formation assay test the ability of single cells to form spherical cell colonies,
and quantifies the activity and self-renewal capacity of the examined cells (Shaw et al., 2012).
This assay is typically performed in non-adherent conditions to evaluate the anchorage-
independent growth of the tested cells, and in serum-free medium supplemented with defined
growth factors to minimize the influence of external signals on cell signaling. To confirm that
putative CSCs maintain their self-renewal ability, spheres need to be passaged serially:
spheres are enzymatically dissociated to single-cell suspension (optionally sorted for specific
phenotype) and are cultured again under the above-defined conditions (Reynolds et al., 1992;
Dontu et al., 2005). This approach also enables to determine the clonogenicity of the
examined cells as assessed according to the number of spheres in the second and further
generations.
Spheres have been derived from variety of tumors and are commonly named (indicated
in square brackets) according to the tissue of origin or type of the tumor: neurogenic tumors
[neurospheres] (Singh et al., 2003; Hemmati et al., 2003), breast carcinoma [mammospheres]
(Ponti et al., 2005), rhabdomyosarcoma [rhabdospheres] (Walter et al., 2011), osteosarcoma
[osteospheres] (Tirino et al., 2008; Di Fiore et al., 2009), colorectal carcinoma
[colonospheres] (Kanwar et al., 2010), prostate carcinoma [prostaspheres] (Lee et al., 2011),
or hepatocellular carcinoma [hepatospheres] (Cao et al., 2011).
During the initial experiments, cells were cultured in liquid media, which permitted the
cell aggregation. This compromised the results of such sphere formation assays because
spheres could arise from a cluster of cells more than from individual cell. Usage of semi-solid
medium, which restricts the cell motion and aggregation, enabled researchers to overcome
this problem (Tirino et al., 2008; Di Fiore et al., 2009). Soft agar colony-forming assay may
be used as an alternative to the sphere formation assay (Tirino et al., 2008; Lee et al., 2011).
In this method, single cells are plated in soft (low concentration) agar onto the solid agar
bottom layer. Formation of 3D cell colonies arising from individual cells in soft agar provides
an evidence of clonogenicity and anchorage-independent growth capacity of the assayed cells.
Similarly to sphere assay, colony forming unit (CFU) assay also termed as clonogenic
assay is used to evaluate the clonogenic capacity of the tested cells. However, in CFU assay,
single-cell suspension of cells is seeded on conventional cell culture plates, thus cultured
under adherent conditions in serum-containing medium. Alternatively, culture plates may be
pre-coated with Matrigel™ (Cao et al., 2011). Usually, colonies comprising more than 30–70
Background – Cancer stem cells ◄ 15
cells are counted after defined time and the frequency of clonogenic cells in the tested cell
fraction is determined (Cao et al., 2011; Yang et al., 2011; Ye et al., 2012; Fedr et al., 2013).
Recently, Fedr et al. (2013) published an elegant method of automatic cell cloning assay.
Using fluorescence-activated cell sorting (FACS), cells can be sorted according to the
expression of putative CSC markers and automatically plated onto 96-well or 384-well
microplates by an automatic cell deposition unit. This rapid approach simplifies the CFU
assay protocol and enables hi-throughput screening of putative CSC marker or anti-cancer
drugs, especially in combination with automated imaging and quantification systems (Wylie
and Bowen, 2007; Single et al., 2015).
Side population (SP) is defined as a population of cells that have the ability to efflux
fluorescent DNA-binding dye Hoechst 33342 (Goodell et al., 1996); alternatively, rhodamine
123 has been used for SP detection (Bunting et al., 2002). Unbound Hoechst 33342 has the
maximum emission at 510 nm, whereas after binding to the minor groove of DNA its
emission maximum shifts to 460 nm (Petersen et al., 2004). Importantly, at higher
concentrations, the dye molecules can no longer bind to DNA without overlapping which
results in decrease of blue fluorescence and increase of red fluorescence. As SP assays use
typically four to five times more dye molecules than there are DNA minor-groove binding
sites, cells that cannot effectively efflux Hoechst dye therefore exhibit significant amount of
fluorescence in both blue and red spectrum (Petriz, 2013). Conversely, cells that efflux
Hoechst 33342 shows overall dim fluorescence signal shifted to blue spectrum. This cell
fraction represents the SP and is generally characterized by upregulated expression of ABCG2
which has been demonstrated to be the major Hoechst 33342 efflux pump (Scharenberg et al.,
2002). Thus, control experiments with selective inhibitors of ABCG2, mainly verapamil
(Mayol et al., 2009) or fumitremorgin C (Hiraga et al., 2011), should be used to verify the
specificity of gated SP during cytometric analysis and sorting.
Aldehyde dehydrogenase activity assay measures the activity of ALDH, which is
considered to be a stem-cell specific marker, as mentioned above (Ma and Allan, 2011;
Marcato et al., 2011). This assay uses a specific fluorescent substrate BAAA (BODIPY
aminoacetaldehyde) that can diffuse into the cells, where ALDH converts this compound to
BAA (BODIPY amino acetate) which leads to emission of fluorescence (Storms et al., 1999).
The fluorescence intensity of individual cells is quantified by flow cytometry, and can be used
for isolation of cells with ALDH high (ALDH+/bright/high) and ALDH low (ALDH−/low) activity
by FACS. Similarly to SP detection, negative controls with ALDH specific inhibitor
diethylaminobenzaldehyde (DEAB) are needed to determine background fluorescence in the
Background – Cancer stem cells ◄ 16
analyzed sample. Currently, ALDEFLUOR™ is the only commercially available kit for
analysis of ALDH activity.
Finally, label-retention assays provide a powerful tool to evaluate the cycling
properties within a given population (Hsu and Fuchs, 2012). Label retention assays are
sometimes referred to as pulse-chase experiments, because these assays contain two essential
parts: a pulse period and a chase period. Generally, in the first step of the label retention
assay, the cells are labeled (in vitro or in vivo) by specific reagents (the pulse). The labeling
reagents are then taken away for a prolonged period (the chase) before the cells or tissues are
examined. Fast-cycling cells are constantly dividing and thus dilute the label through each
round of division. Conversely, slow-cycling cells divide infrequently during the chase period.
Therefore, after the chase, the label intensity corresponds inversely to a proliferation rate of
the respective cell. Generally, three main approaches are used for cell labeling in label-
retention assays: (i) labeling DNA with 5-bromo-2’-deoxyuridine (BrdU) or radiolabeled
nucleoside analogues (Fillmore and Kuperwasser, 2008; Duque and Rakic, 2011); (ii)
labelling cells with vital fluorescent dyes such as DiI and PKH26 membrane stains (Willan
and Farnie, 2011), or carboxyfluorescein diacetate succinimidyl ester (CSFE) that diffuses
through the cell membrane and covalently binds to intracellular molecules (Wang et al.,
2005); and (iii) using transgenes expressing GFP-tagged histone, expression of which can be
suppressed by doxycycline (Tumbar et al., 2004). The main reason why this method has been
used for detection of CSCs originates from the fact, that many stem cells are slow-cycling.
However, label retention does not necessarily imply stemness, therefore the results of this
assay need to be interpreted with caution (Hsu and Fuchs, 2012).
2.1.4 Implications for clinical practice
As proposed by CSC model, intratumoral heterogeneity is generated by CSCs which give rise
to the different populations of cells (clones). These cells are more differentiated, may
extensively proliferate for a certain time period, and they are thought to represent the tumor
bulk; however, this may not be true in all cases, as suggested by Driessens et al. (2012).
Current conventional anti-cancer therapies target the tumor bulk, but have limited or no effect
on the CSCs. If CSCs are the primary drivers of tumorigenesis and metastasizing, then
effective anti-cancer therapies must target these cells (Fig 4).
In clinical practice, patients often relapse and their prognosis is poor despite the initial
remission of the tumor; examples are given for pediatric sarcomas and PDAC in the following
chapters. CSCs are considered to be a cause of the observed tumor recurrence because their
Background – Cancer stem cells ◄ 17
resistance to therapy and metastatic potential (Cojoc et al., 2015). Several mechanisms of this
resistance were suggested: (i) efflux of chemotherapeutic mediated by upregulation of ABC
transporters; (ii) high ALDH activity that allow CSCs to metabolize different
chemotherapeutic; (iii) enhanced response to DNA damage and prevention of this damage by
an efficient scavenging of reactive oxygen species; (iv) autophagy that enables CSCs to
overcome microenvironmental insults like hypoxia, starvation or treatment; (v)
microenvironmental stimuli provided by the specific CSC niche (comprehensively reviewed
in Cojoc et al., 2015).
Targeting CSCs itself or exploiting these mechanisms of CSC resistance might improve
cancer treatments (Chen et al., 2013). Nevertheless, to achieve these improvements, a key
step is to identify and characterize the targets of such treatments – CSCs – in the respective
tumor types.
Fig. 4 – Implications of CSCs in cancer treatment. Conventional anti-cancer therapies that kill
primarily non-tumorigenic cells (blue, gray) can shrink the tumor, but will not eradicate the tumor
because these therapies do not target CSCs, which will eventually regenerate the tumor or initiate
metastases. CSC-targeted therapies represent potential treatment improvements because they will kill
or differentiate CSCs, thus targeting tumorigenesis, tumor growth and tumor metastasizing. However,
it is evident that treatment combining both CSC-targeted and conventional therapy will be necessary
to completely eradicate the tumor (Illustration created based on Pardal et al., 2003).
Background – Pediatric sarcomas ◄ 18
2.2 Pediatric sarcomas
Sarcomas represent a very heterogeneous group of tumors that arise from primitive
mesenchymal cells (Doyle, 2014). Generally, sarcomas are classified into two main groups:
bone and soft tissue sarcomas. These tumors are relatively rare and represent only 1% of all
malignancies. However, substantially higher incidence is observed in children.
Pediatric sarcomas comprise about 13% of all pediatric malignancies and they are the
third most common type of childhood cancers. Rhabdomyosarcoma, osteosarcoma and
Ewing’s sarcoma have the highest incidence among pediatric sarcomas. These neoplasms
often display highly aggressive behavior with tendency to form metastases (Anderson et al,
2012). Intensive multimodal therapy, typically surgery with systemic chemotherapy, results in
5-year complete remission of about 70% of patients with localized disease (Perkins et al.,
2014). However, the prognosis of patients diagnosed with advanced disease remains poor.
Significant proportion of children with sarcoma suffers recurrence and dissemination of
disease despite aggressive treatment with subsequent rates of survival no higher than 20%
(Dela Cruz, 2013). Sarcoma treatment failures and disease recurrences led the researchers to
hypothesize that CSC model may account for this group of tumors and CSCs have been
intensively studied in the past decade.
2.2.1 Osteosarcoma
Osteosarcoma is malignant mesenchymal tumor of bone that is histologically characterized by
the presence of osteoid-producing neoplastic osteoblasts. Osteosarcoma is the most common
primary bone tumor in children and young adults, with an incidence of 4.8 per million per
year, peaking in the adolescent age range (Weiss et al., 2014). Primary osteosarcomas
typically occur in the metaphyses of long bones (Fig. 5a). These tumors are generally locally
aggressive and tend to produce early systemic metastases (Luetke et al., 2014). Therapeutic
protocols incorporating multi-agent cytotoxic chemotherapy and surgery has resulted in cure
rates of 60–70% in the localized disease setting (Dela Cruz, 2013). However, at the time of
osteosarcoma diagnosis, about 20% of patients present with macroscopic evidence of
metastatic disease, with the lung being the most common site of metastases, followed by bone
and rarely lymph nodes (Luetke et al., 2014). Moreover, about 80–90% of patients are
assumed to have micrometastatic disease that is undetectable using current diagnostic
modalities. A total of 30–40% of patients with localized osteosarcoma will develop a local or
Background – Pediatric sarcomas ◄ 19
distant recurrence. Despite the use of intensive chemotherapy, surgery, and/or radiation
therapy, prognosis of these patients with metastatic or recurrent osteosarcoma remains poor
and it seems that plateau in the survival and cure rates has been reached in these patients with
presently available treatment.
Fig. 5 – Most common primary tumor sites in osteosarcoma, Ewing’s sarcoma and rhabdo-
myosarcoma. (a) Osteosarcoma (blue) preferentially develops in distal femur, proximal tibia or
proximal humerus (Luetke et al., 2014). The most common sites for Ewing’s sarcoma (red) are the
pelvis, femur, and tibia (Hong et al., 2010). (b) The most common sites of rhabdomyosarcoma are
presented including relative incidence according to Breitfeld and Meyer (2005). (Image from Skeletal
muscles, 2016, http://www.cliparthut.com/human-internal-organs-with-skseleton-clipart-GbWTTa.html)
The evidence that CSCs may be present in osteosarcoma was first reported by Gibbs et al.
(2005) who found that osteosarcoma cell lines contain small population of self-renewing cells
with stem-like properties. In their study, Gibbs et al. showed that about 1 in 100–1000
osteosarcoma cells were capable of growth in vitro under anchorage-independent and growth-
constraining conditions to form spherical colonies, termed sarcospheres. Cells within these
sarcospheres expressed increased levels of stem cell markers (Oct4 and Nanog) and single
cells repeatedly generated spheres during serial recloning.
Background – Pediatric sarcomas ◄ 20
Later, Tsuchida et al. (2008) showed that treatment of osteosarcoma HOS cell line with
cisplatin led to an expansion of highly tumorigenic side-population (SP) cells. Exposure of
HOS cells to cisplatin resulted in the increase of colony-forming and migratory abilities of
these cells in vitro. Moreover, when SP cells were isolated, only cisplatin-treated SP cells
were able to form tumors in mice. This suggests that SP in cisplatin-treated cells was enriched
for cells with CSC properties but this population did not defined CSCs absolutely. Similarly
to these results, another group reported a selection of stem-like osteosarcoma cell line 3AB-
OS by long-term treatment of MG-63 cells with of 3-aminobenzamide (Di Fiore et al., 2009).
3AB-OS cells were characterized by increased capacity to form spheres, stronger self-renewal
ability, upregulated expression of genes required for maintaining stem cell state, and
prominent positivity for CD133.
Expression of CD133 in osteosarcomas was firstly evaluated by Tirino et al. (2008).
Previously, CD133 has been proposed to be a marker of CSCs in several human malignancies,
and therefore this research group tested whether expression of this molecule might be used to
identify and isolate a subpopulation of cells with CSC characteristics also in osteosarcoma.
Examination of osteosarcoma cell lines identified a subpopulation of CD133+ cells that
showed self-renewal characteristics, formed spherical colonies during cultivation in serum-
free medium, and expressed the stem cell-associated POU5F1 gene coding Oct3/4. In the
same year, another research group demonstrated that cells co-expressing CD133 and nestin
are present in osteosarcoma cell lines (Veselska et al., 2008). The identification of
CD133+/nestin+ cells suggested the possible occurrence of a cell population with a stem-like
phenotype. A subsequent study by Zambo et al. (2012) supported this hypothesis – high levels
of nestin tended to indicate a worse clinical outcome in osteosarcoma patients. However, the
aforementioned studies did not verify the CSC phenotype of CD133+ and nestin+ populations
through the in vivo tumorigenicity assays. Surprisingly, when the tumorigenicity of
osteosarcoma cells was tested, two cell lines that were shown to express CD133 did not form
tumors after injection into NOD/SCID mice (Tirino et al., 2011). Another study did not find
any difference in expression of CD133 between SP cells that were enriched in tumorigenic
cells and non-SP cells (Yang et al., 2011). Nevertheless, the expression of CD133 has been
shown to correlate with lung metastasis and poor prognosis in osteosarcoma patients (He et
al., 2012). Although CD133 seems to be of importance in osteosarcoma progression, its role
in osteosarcoma CSCs remains controversial.
Other cell surface markers have been identified to indicate tumorigenic phenotype in
osteosarcoma cell lines. Adhikari et al. (2010) reported that double positivity for CD117
Background – Pediatric sarcomas ◄ 21
(c-kit) and Stro-1 (a marker of osteogenic progenitors in bone marrow) marked CSCs in
mouse and human osteosarcoma cell lines. The CD117+/Stro-1+ cells efficiently formed
serially transplantable tumors, whereas CD117−/Stro-1− cells initiated tumors only rarely.
Moreover, CD117+/Stro-1+ cells had higher metastatic potential in NOD/SCID mice and
exhibited drug-resistant phenotype when exposed to doxorubicin. These results suggested
CD117 and Stro-1 to be potential therapeutic targets in osteosarcoma. However, no further
study has been published to support the utility of CD117 and Stro-1 as markers of CSCs in
osteosarcoma. Recently, two independent groups have reported that CD49f may serve in
osteosarcoma as another marker that can distinguish CSCs from the cells with limited
tumorigenic capacity (Ying et al., 2013; Penfornis et al., 2014). Nevertheless, these two
studies brought contradictory results. Whereas Ying et al. (2013) initially identified
CD49f−/CD133+ cells that possessed strong tumorigenic activity, the other study suggested
that high levels of CD49f correlate with stemness (Penfornis et al., 2014). Thus, the
significance of CD49f in identifying CSCs in osteosarcoma remains to be seen.
ABC transporters are considered to cause the resistance of CSCs to chemotherapy and
are therefore studied as prospective CSC markers (Wilkens et al., 2015). The results
concerning expression of ABC transporters in osteosarcoma seem to be partly controversial
(reviewed in Veselska et al., 2012 – Manuscript II). Nevertheless, recent study demonstrated
that exposure of osteosarcoma cells to chemotherapeutic agents (doxorubicin, cisplatin and
methotrexate) induce their stem-like phenotype and result in upregulation of ABC transporters
and ALDH through activation of Wnt/β-catenin signaling (Martins-Neves et al., 2016).
Examinations of ALDH activity showed the presence of subpopulation of cells with
high ALDH activity (ALDH+) in several osteosarcoma cell lines (Honoki et al., 2010; Wang
et al., 2011; Martins-Neves et al., 2015). Wang et al. (2011) found that ALDH+ cells
possessed increased tumorigenic capacity compared with their ALDH− counterparts. ALDH+
cells also showed increased cell growth rate, clone formation ability, and expression of stem
cell marker genes (Oct4, Nanog and Sox2) in vitro. However, these results were obtained only
when ALDH+ cells were isolated directly from osteosarcoma xenograft tumors but not from
the parental cell line. Furthermore, previous study showed that both ALDH+ and ALDH− cells
were able to reconstitute the same pattern of ALDH activity as the parental cell population
(Honoki et al., 2010). These observations questioned the validity of ALDH activity as a
specific osteosarcoma CSC marker and showed the limits of specificity and/or sensitivity of
ALDH activity assay for isolation of the pure population of CSCs. Nevertheless, further
Background – Pediatric sarcomas ◄ 22
studies reported that ALDH activity was associated with metastatic potential in murine and
human osteosarcomas (Mu et al., 2013; Greco et al., 2014).
Recently, Martins-Neves et al. (2015) provided evidence that ALDH+ cells overexpress
Sox2 in osteosarcoma. During the last 5 years, Sox2 has been shown to associate with clinical
outcome and/or mediate the maintenance of CSC subpopulation in various types of cancer
including osteosarcoma (Weina and Utikal, 2014). Basu-Roy et al. (2012) demonstrated that
Sox2 is required for self-renewal of CSCs in human and murine osteosarcoma cell lines.
Depletion of Sox2 by shRNA decreased the ability of cells to form colonies and osteospheres,
and reduced their migration and invasiveness. When compared with parental cells, Sox2
knockdown in murine osteosarcoma cells had drastically reduced ability to form tumors in
vivo. Additionally, Sox2 overexpression enhanced osteosphere formation by murine primary
osteoblasts. The involvement of Sox2 in osteosarcoma tumorigenesis was indirectly
illustrated in the study of miR-126, which acts as a tumor suppressor in osteosarcoma cells via
the targeting of Sox2 (Yang et al., 2014a). Recent study demonstrated that Sox2 interferes
with the tumor-suppressive Hippo pathway to maintain CSCs in osteosarcoma (Basu-Roy et
al., 2015). Thus, blocking of Sox2 function may be considered as a novel therapeutic strategy
for these tumors.
2.2.2 Ewing’s sarcoma
Ewing’s sarcoma family tumors are the second most common malignant tumors of bone and
soft tissue in children and young adults (Balamuth and Womer, 2010). This group of
malignancies comprises a spectrum of aggressive tumors, including Ewing’s sarcoma or
peripheral primitive neuroectodermal tumor, that are diagnostically characterized by the
presence of specific fusion oncoproteins. These oncoproteins typically result from
chromosomal translocation t(11;22) fusing EWS and FLI1 genes (85% of Ewing’s sarcomas);
otherwise, EWS protein is usually joined to another member of the ETS (E26 transformation-
specific) family of transcription factors. Although the exact functions of these fusion
oncoproteins are still a matter of research, expression of EWS-Fli-1 has been demonstrated to
be essential for the Ewing’s sarcoma oncogenesis (Prieur et al., 2004; Owen et al., 2008;
Kinsey et al., 2009). Tirode et al. (2007) demonstrated EWS-Fli-1 to block terminal
mesenchymal differentiation of mesenchymal stem cells (MSCs) and suggested that these
cells may represent the origin of Ewing’s sarcoma cells. Thus, MSCs have been recently
utilized as a model to investigate and manipulate oncogenesis in Ewing’s sarcoma (reviewed
in Monument et al., 2013). The most frequent primary sites of Ewing’s sarcomas include
Background – Pediatric sarcomas ◄ 23
pelvis and femur; metastatic disease often affects lungs (Fig. 5a; Hong et al., 2010). Although
the 5-year overall survival of children with localized disease reaches 80%, about a quarter of
patients have detectable metastases at diagnosis and their survival remain low at 40% (Karski
et al., 2016).
For the first time, the presence of CSCs in Ewing’s sarcoma was reported using
isolation CD133+ cells from primary tumors (Suva et al., 2009). CD133+ cells displayed the
capacity to initiate and maintain tumor growth through serial xenotransplantations, and were
able to re-establish the parental tumor phenotype and cell hierarchy (CD133+ and CD133−
cells) at each in vivo passage. The CD133+ cells also expressed significantly higher levels of
OCT4 and NANOG, but not SOX2 and EWS-FLI1, than their CD133− counterparts. Consistent
with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell
population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic
lineages. In the subsequent study, the same research group expressed fusion gene EWS-FLI1
in pediatric MSCs (MSCsEWS-FLI-1) and demonstrated that EWS-Fli-1 induced expression of
CD133 in 6–10% of these cells (Riggi et al., 2010). Sorted CD133+ MSCsEWS-FLI-1 displayed
higher expression levels of OCT4 and NANOG, but did not differ in EWS-FLI1 expression
level compared with CD133− fraction. Additionally, unsorted MSCsEWS-FLI-1 were able to form
spheres and more importantly these cells expressed significantly reduced expression of
miR-145 than wild-type pediatric MSCs. Downregulation of miR-145 has been observed in
many types of cancers, suggesting that it may serve as a tumor suppressor (reviewed in Cui et
al., 2014). Indeed, miR-145 expression was found low in self-renewing human ESCs but
highly upregulated during differentiation, repressing expression of SOX2, OCT4 and KLF4
(Xu et al., 2009). Inhibition of miR-145 in dermal skin fibroblasts led to upregulation of
pluripotency-associated genes including SOX2, KLF4, OCT4 and MYCC, and increased
efficiency of reprogramming these fibroblasts to induced pluripotent stem cells (Barta et al.,
2015). Given the role of miR-145 in silencing pluripotency, loss of function of this miRNA
may also potentiate tumorigenesis by promoting a program of aberrant self-renewal in
preneoplastic cells (Fig. 6). It is not surprising that several studies have demonstrated anti-
proliferative and differentiating effects of miR-145 onto CSCs in various cancers (Yin et al.,
2011; Wu et al., 2011; Polytarchou et al., 2012; Jia et al., 2012; Xu et al., 2013; Panza et al.,
2014; Hu et al., 2014). In the aforementioned study of pediatric MSCs, repression of miR-145
upon EWS-FLI1 expression resulted in upregulation of SOX2 (Riggi et al., 2010). More
importantly, overexpression of miR-145 or depletion of SOX2 in Ewing’s sarcoma cell lines
led to reduced tumorigenicity of the cells in vivo. Consistent with this study, knockdown of
Background – Pediatric sarcomas ◄ 24
EWS-FLI1 in Ewing’s sarcoma cell lines dramatically increased the levels of miR-145, and
forced miR-145 expression halted growth of the cells (Ban et al., 2011). In the light of these
findings, “EWS-Fli-1/miR-145/Sox2” axis may represent the key regulatory pathway in
Ewing’s sarcoma tumorigenesis reprogramming preneoplastic cells towards the CSC
phenotype.
Fig. 6 – Regulation of self-renewal and pluripotency is mediated by miR-145. miR-145 targets
multiple ESC factors including SOX2, OCT4, and KLF4. Enhanced expression of miR-145 inhibits
self-renewal and pluripotency, and contributes to ESC differentiation. Conversely, many tumors
downregulate miR-145, suggesting that this microRNA might function more generally to maintain
differentiated non-proliferative states. (Adapted and modified from Chivukula and Mendell, 2009)
In contrast to the first study reporting CD133 as marker of CSCs in Ewing’s sarcoma
(Suva et al., 2009), Jiang et al. (2010) did not find any differences in the tumorigenicity or
chemoresistance between CD133+ and CD133− cell fractions in three of four Ewing’s sarcoma
cell lines tested. Additionally, they found only 4 of the 48 Ewing’s sarcomas examined that
exhibited high expression of CD133. Among these four cases, two patients rapidly succumbed
to aggressive drug-resistant disease and two were long-term survivors. Thus, the significance
of CD133 as CSC marker in Ewing’s sarcoma remains elusive. An alternative surface marker
proposed to reflect enhanced tumorigenicity in Ewing’s sarcoma is CD57 (Wahl et al., 2010),
which was originally described as the neural crest stem cell marker. CD57high cells were more
tumorigenic, formed spheres at higher frequency and had enhanced migratory potential than
CD57low cells. Interestingly, only partial overlap was observed among CD57high and CD133+
populations of cells, suggesting that CD57 identify different population of Ewing’s sarcoma
cells with CSC phenotype. Recently, Leuchte et al. (2014) argued against a role of CD133 and
CD57 as markers of CSCs in Ewing’s sarcoma. In their study, expression of CD133 or CD57
Background – Pediatric sarcomas ◄ 25
did not vary significantly between the cells cultivated as spheres or monolayers. Additionally,
sphere-derived cells did not exhibit higher in vivo tumorigenicity than monolayer cells.
Although sphere culture conditions have been shown to enrich CSCs in numerous cancers,
findings of Leuchte et al. (2014) questioned the validity of this approach in Ewing’s sarcoma.
LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) has been recently
suggested as another CSC marker in Ewing’s sarcoma (Scannell et al., 2013). Lgr5 is a
receptor for the R-spondin family of ligands and its activation potentiates Wnt/β-catenin
signaling, contributing to stem cell proliferation and self-renewal in various tissues
(Leushacke and Barker, 2012). In Ewing’s sarcoma, expression of LGR5 was identified in
both tumor tissues and cell lines, and elevated levels of LGR5 were associated with clinically
aggressive tumors (Scannell et al., 2013). Increased expression of LGR5 also corresponded
with CD133 positivity and high ALDH activity in Ewing’s sarcoma cell lines.
Similarly to osteosarcoma, high ALDH activity was reported to identify stem-like
chemotherapy-resistant population in Ewing’s sarcoma (Awad et al., 2010). ALDHhigh cells
were enriched for clonogenic and sphere forming activity. More importantly, these cells were
highly tumorigenic in vivo. As few as 160 of ALDHhigh cells were sufficient to initiate tumors
in NOG mice whereas the same number of CD133+ cells did not result in tumor formation,
suggesting superior enrichment of ALDHhigh population for CSCs compared with CD133+
cells. Interestingly, although ALDHhigh cells were resistant to cytotoxic chemotherapy, these
cells demonstrated sensitivity to small molecule inhibitor of EWS-Fli-1 (YK-4-279). Direct
cytotoxicity and loss of clonogenic activity after treatment with YK-4-279 indicated the
dependence of the ALDHhigh cells on EWS-FLI1 oncogene expression. These findings further
support the crucial role of the aforementioned “EWS-Fli-1/miR-145/Sox2” axis for
maintenance of CSC phenotype in Ewing’s sarcoma.
2.2.3 Rhabdomyosarcoma
Rhabdomyosarcoma is the most common malignant mesenchymal tumor encountered in
children with the peak of incidence in patients younger than 5 years (Yang et al., 2014b).
Because rhabdomyosarcoma is derived from immature striated skeletal muscle, this disease
can occur at any site in the body (Fig. 5b).
Recently revised classification of rhabdomyosarcoma distinguishes four subtypes: (i)
alveolar rhabdomyosarcoma; (ii) embryonal rhabdomyosarcoma; (iii) pleomorphic
rhabdomyosarcoma; and (iv) sclerosing/spindle cell rhabdomyosarcoma (Fletcher et al.,
2013). Embryonal subtype represents about 70% of all childhood rhabdomyosarcomas,
Background – Pediatric sarcomas ◄ 26
mainly affecting infants and children under 10 years of age, and is usually associated with a
favorable prognosis (Punyko et al., 2005; Yang et al., 2014b). Embryonal
rhabdomyosarcomas predominantly localize to the head and neck, the genitourinary tract and
the retroperitoneum. In contrast, alveolar rhabdomyosarcoma is a high-grade malignancy with
5-year survival of <50% and occurs mostly in adolescents and young adults, usually arising in
the extremities and trunk. This subtype of rhabdomyosarcoma typically harbors one of two
characteristic chromosomal translocations t(2;13)(q35;q14) or t(1;13)(p36;q14) that juxtapose
PAX3 or PAX7 and FOXO1A genes, resulting in Pax3-FKHR and Pax7-FKHR fusion proteins
(Marshall and Grosveld, 2012).
Similarly to Ewing’s sarcomas, MSCs were suggested as the cells of origin of alveolar
rhabdomyosarcomas. Ren et al. (2008) showed that Pax3-FKHR and Pax7-FKHR induced
skeletal myogenesis in murine MSCs, although additional secondary genetic event was
needed for their transformation towards alveolar rhabdomyosarcoma and tumor formation in
vivo. No characteristic cytogenetic abnormality has been associated with tumorigenesis of
embryonal rhabdomyosarcoma. Nevertheless, this rhabdomyosarcoma subtype may probably
develop from a whole range of muscle cells, including muscle satellite cells and downstream
myogenic progenitors such as maturing myoblasts (Rubin et al., 2011; Soleimani and
Rudnicki, 2011).
Embryonal rhabdomyosarcoma cell lines cultured as spherical colonies (rhabdospheres)
were reported to be enriched for cells that possessed stem cell properties including elevated
expression of stem cell markers POU5F1, NANOG, MYCC, SOX2, and PAX3 (Walter et al.,
2011). Rhabdosphere cells were highly tumorigenic compared with adherent cells and showed
upregulated CD133 expression both on RNA and protein levels. CD133+ sorted cells formed
tumors at lower cell densities than CD133− and unsorted cells, and were more resistant to
treatment with the chemotherapy drugs cisplatin and chlorambucil. Furthermore, high
expression of CD133 in tumor tissue samples correlated with poor survival of embryonal
rhabdomyosarcoma patients. Later, Pressey et al. (2013) suggested CD133 as a marker of
CSCs also in alveolar rhabdomyosarcoma. The authors showed that both alveolar and
embryonal rhabdomyosarcoma-derived CD133+ cells have enhanced colony-forming ability
and resistance to chemotherapy, and are characterized by a myogenically primitive phenotype.
In contrast, no difference in tumorigenicity of CD133+ and CD133− cells was found in a
previous study of three embryonal rhabdomyosarcoma cell lines (Hirotsu et al., 2009). The
investigators tested a panel of potential CSC markers and found that only cell fractions
positive for fibroblast growth factor receptor 3 (FGFR3) were enriched for CSCs.
Background – Pediatric sarcomas ◄ 27
Recently, ALDH1 has been found to mark population of embryonal rhabdomyosarcoma
cells showing higher capacity for self-renewal and tumor formation (Nakahata et al., 2015).
ALDHhigh cells were more chemoresistant and expressed higher levels of SOX2 than their
ALDHlow counterparts. Thus, ALDH1 is a potential marker of CSCs at least in embryonal
subtype of rhabdomyosarcoma.
Background – Pancreatic ductal adenocarcinoma ◄ 28
2.3 Pancreatic ductal adenocarcinoma
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Although
its incidence is relatively low, PDAC represents the fourth and eight leading cause of cancer-
related deaths in Western countries (Siegel et al., 2015) and worldwide (Jemal et al., 2011),
respectively. PDAC is rarely diagnosed in persons younger than 40 years of age, and the
median age at diagnosis is 71 years (Ryan et al., 2014). Approximately 60–70% of pancreatic
cancers are located in the head of the pancreas, and 20–25% are located in the body and tail of
the pancreas (Fig. 7).
Fig. 6 – Anatomy of human pancreas. (Image from OpenStax, Exocrine and Endocrine Pancreas,
2013, http://cnx.org/content/col11496/1.6/. Licenced under CC BY 3.0.)
Background – Pancreatic ductal adenocarcinoma ◄ 29
Despite recent advances in the diagnosis and treatment of pancreatic cancer, its
incidence almost equals its mortality rate, and the 5-year survival rate does not generally
reach 5% (Balic et al., 2012). More than 90% of PDAC patients die from the disease – about
70% of these patients die from extensive metastatic disease; the other 30% have limited
metastatic disease (Ryan et al., 2014).
The first evidence for the existence of CSCs in PDAC was reported by two groups in
2007 (Li et al., 2007; Hermann et al., 2007). First, Li et al. (2007) demonstrated that the
combination of cell surface markers CD44, CD24, and epithelial cell adhesion molecule
(EpCAM; epithelial-specific antigen, ESA) identified a highly tumorigenic subpopulation of
PDAC cells with stem cell properties. As few as 100 CD44+/CD24+/EpCAM+ cells were
sufficient to form tumors in NOD/SCID mice, whereas CD44−/CD24−/EpCAM− cells did not
developed tumors until 104 of these cells were engrafted.
Later, Hermann et al. (2007) reported pancreatic CSCs that were defined by the
expression of CD133. Distinct subpopulation of CD133+ cells that co-expressed CXCR4 was
further identified in the invasive front of the PDAC tumors. These CD133+CXCR4+ cells were
shown to have migratory capacity in vitro and were demonstrated to be essential for
metastatic phenotype of the PDAC in vivo. Although CD133+CXCR4− formed tumors at the
same rate, only mice injected with CD133+CXCR4+ cells developed metastases. In
accordance with these results, another study showed that CXCR4 is expressed in pancreatic
intraepithelial neoplasias (PanIN) and its expression is increased during PanIN progression
towards invasive carcinoma (Thomas et al., 2008; Fig. 8). The possible prognostic
significance of CXCR4 in PDAC was further confirmed by a meta-analysis study showing
correlation between CXCR4 expression and poor prognosis (Krieg et al., 2015). More
importantly, strong association of CXCR4 expression and metastatic disease was found in this
study. Consistent with these findings, previous experimental data demonstrated increased
proliferation and invasiveness of pancreatic cancer cells after induction of CXCR4 by its
ligand CXCL12 (Shen et al., 2013).
Although CD133 was initially suggested as a CSC marker in PDAC (Hermann et al.
2007), further published studies argued against the usefulness of this protein alone to
specifically identify pancreatic CSCs. Immervoll et al. (2008) showed that CD133 is
expressed not only in pancreatic cancer cells but also in normal pancreas. Moreover, no
correlation of CD133 and patient survival was found in subsequent studies (Immervoll et al.,
2008; Kure et al., 2012). Co-expression of CD44 and CD133 was then proposed as more
specific phenotype of CSCs (Immervoll et al, 2011), and was shown to predict worse survival
Background – Pancreatic ductal adenocarcinoma ◄ 30
in PDAC patients (Hou et al., 2014; Chen et al., 2014). However, significance of CD133
expression in PDAC tumorigenesis has been recently supported by two independent studies
reporting CD133 as efficient negative prognostic factor (Kim et al., 2012; Li et al., 2015).
Fig. 8 – Progression of normal pancreatic ductal epithelial cells to PanIN lesions to PDAC. (a)
Schematic illustration of normal exocrine pancreas progressing into increasingly dysplastic PanIN and
PDAC (Image adapted and modified from Logsdon et al., 2010). (b) Hematoxylin-eosin-stained tissue
sections (Images adapted and modified from Hezel et al., 2006)
Expression of ALDH isoenzymes and their enhanced activity represent another putative
marker of CSCs that has been evaluated in PDAC. ALDH1-positive cells were detected in
primary tumor tissues, and their presence was associated with shorter survival (Rasheed et al.,
2010). Importantly, ALDH1 positivity was found in metastatic lesions of primary PDAC
tumors that were ALDH1 negative. Further experiments demonstrated that sorted ALDHhigh
cells were considerably more clonogenic in vitro and tumorigenic in vivo than ALDHlow cells.
Interestingly, only minor overlap of ALDHhigh and CD44+/CD24+ cell populations was found
in PDAC cell lines – about 2% ALDHhigh cells co-expressed CD44 and CD24 (Rasheed et al.,
Background – Pancreatic ductal adenocarcinoma ◄ 31
2010). However, these ALDHhigh/CD44+/CD24+ cells showed increased tumorigenic potential
compared to ALDHhigh or CD44+/CD24+ cells only. Contrary to these results, another study
reported much higher rates of tumor formation after injection of ALDHhigh cells into
NOD/SCID mice (Kim et al., 2011a). In some cases, as few as 100 ALDHhigh cells were able
to initiate tumor growth in 100% of mice, suggesting that sorting for ALDHhigh cells alone is
sufficient to enrich for CSCs. ALDHhigh cells possessed substantially increased tumorigenic
potential than CD133+ cells, and no significant difference in tumorigenicity of
ALDHhigh/CD133+ and ALDHhigh/CD133− cells was shown in this study. Additionally, the
authors also questioned the role of ALDHhigh/CD44+/CD24+ in PDAC initiation. Given their
low incidence, as only about 0.5% of ALDHhigh cells co-expressed CD44 and CD24,
ALDHhigh/CD44+/CD24+ cells unlikely accounted for the 100% incidence of tumor formation
observed after implantation of 100 ALDHhigh cells. Nevertheless, tumorigenicity of
ALDHhigh/CD44+/CD24+ cells was not tested in this study. Thus it still needs to be determined
whether ALDHhigh/CD44+/CD24+ cells may represent more primitive cells that give rise to
phenotypically distinct but still (to a certain extent) tumorigenic pancreatic cancer cells.
Recently, ALDH1B1 expression was shown to correlate with invasiveness of PDAC
tumors and proliferation of PDAC-derived cells (Singh et al., 2016). These observations are in
agreement with previously detected high positivity of ALDH1 in PDAC metastases (Rasheed
et al., 2010). Kim et al. (2013) observed that tumors of PDAC patients who had undergone
preoperative gemcitabine-based chemoradiation contained increased proportion of ALDHhigh
cells compared to tumors of patients treated only surgically. Using CFPAC-1 cell line,
gemcitabine treatment was further demonstrated to enrich for ALDHbright cells (strongly
positive subpopulation of ALDHhigh cells) also in vitro. These cells were sensitive to
disulfiram, an ALDH inhibitor. In vivo experiments in mice showed that administration of
disulfiram in combination with low-dose gemcitabine significantly suppressed tumor growth
and reduced ALDH positivity of xenografted CFPAC-1 cells. Thus targeting ALDHhigh
therapy-resistant CSCs with specific inhibitors, such as disulfiram, may provide better
therapeutic response and reduced toxicity of chemotherapy in PDAC patients.
Recently, CD133+ and ALDHhigh cells were demonstrated to express high levels of
forkhead box protein O1 (FoxO1), whereas both negative fractions expressed nearly null
FoxO1 (Song et al., 2015). FoxO1-negative cells formed tumor spheres and maintained the
capacity to initiate tumor growth during serial xenotransplantations in mice, whereas
depletion of these cells completely inhibited the growth of PDAC cells. Although limited to
one study, these experiments suggested FoxO1-negative phenotype as a potential marker of
Background – Pancreatic ductal adenocarcinoma ◄ 32
pancreatic CSCs. Similarly, c-Methigh pancreatic cell population was shown to be enriched
with CSCs (Li et al., 2011). c-Met is a receptor tyrosine kinase activated by hepatocyte
growth factor (HGF). HGF/c-Met pathway signaling induces complex of biological responses,
collectively termed as invasive growth, which are essential during embryonic development as
well as for wound healing or liver regeneration in adulthood (Gentile et al., 2008). Previous
studies reported that c-Met is overexpressed in PDAC and its expression has been associated
with invasion, metastasis, and chemoresistance (reviewed in Delitto et al., 2014). Li et al.
(2011) showed that high levels of c-Met corresponded with increased sphere formation
capacity and tumorigenicity of PDAC cells. Small numbers of c-Methigh cells were able to
initiate tumor growth in mice, and even higher tumorigenic potential was detected when
c-Methigh/CD44+ cells were tested. Inhibition of c-Met using cabozantinib significantly
inhibited tumor sphere formation in vitro and reduced tumor growth in vivo. Moreover,
tumors in mice treated with cabozantinib exhibited lower proportion of CD44+/CD24+/
EpCAM+ cells and these mice did not developed any metastases. Correspondingly,
cabozantinib was found to downregulate CSC markers Sox2, c-Met and CD133 (Hage et al.,
2013). Inhibition of c-Met induced apoptosis in gemcitabine-resistant PDAC cell lines and
increased efficacy of gemcitabine treatment. Together, these findings suggest c-Met as
another pancreatic CSC marker and indicate the possible therapeutic potential of c-Met-
targeted therapy.
Nestin expression has also been examined in PDAC (reviewed in Neradil and Veselska,
2015). Knockdown of nestin in PDAC cells reduced their migratory potential and
invasiveness in vitro, and resulted in decreased incidence of liver metastases when tested in
mice (Matsuda et al., 2011). In agreement with these results, high levels of nestin together
with ALDH1A1 and ABCG2 were detected in metastatic cells that were obtained after
injection of human PDAC cells into the spleen of NOG mice (Matsuda et al., 2014). These
metastatic cells exhibited increased motility and invasion, and formed a greater number of
spheres than their parental counterparts. It was further demonstrated in vivo that cells isolated
from PDAC metastases have enhanced metastatic potential and that silencing of nestin
expression by shRNA attenuates metastatic potential of these cells. Nestin was also suggested
to be involved in EMT and finally, its expression was proposed as pancreatic CSC marker
(Hagio et al., 2013; Matsuda et al., 2014). However, these conclusions are in sharp contrast
with studies published by other research groups, which have not found any significant
association of the nestin expression with survival or other clinicopathological parameters of
PDAC patients (Yang et al., 2008; Kawamoto et al., 2009; Lenz et al., 2011; Kim et al., 2012)
Background – Pancreatic ductal adenocarcinoma ◄ 33
Recently, it has been shown that human ESC markers Sox2, Oct4, and Nanog are
expressed in PDAC and that these transcription factors may associate with drug resistance,
metastasis and overall worse prognosis (reviewed in Herreros-Villanueva et al., 2014).
Regarding pancreatic CSCs, particularly Sox2 expression seems to be crucial for stem-like
features of PDAC cells (Herreros-Villanueva et al., 2013; Singh et al., 2015). Overexpression
of Sox2 in vitro induced cell dedifferentiation and promoted EMT reprogramming, which is
necessary for PDAC progression. Moreover, CD44+/EpCAM+ cell fractions isolated from two
different tumor samples were enriched for Sox2-positive cells (Herreros-Villanueva et al.,
2013). Thus, Sox2 may serve as potential CSC marker in PDAC.
Objectives ◄ 34
3 Objectives
CSCs are widely accepted to be involved in tumor initiation, progression, drug resistance and
recurrence. Therefore, the first step in developing more effective anti-cancer therapeutic
strategies is to target CSCs. Several molecules that should specifically identify CSCs have
been described repeatedly in numerous tumor types. However, the validity of some of these
CSC markers remains unclear.
Although a lot of papers concerning CSCs in hematological malignancies, neurogenic
tumors and the most frequent carcinomas have been published, relatively few studies focused
on the identification of CSCs in sarcomas and PDAC. Clinical experience emphasizes the
relevance of such studies as pediatric sarcomas represent the third most common cancer
among children, and PDAC is one of the most lethal malignancies at all.
Thus, this thesis focuses on identification and characterization of CSCs in pediatric
sarcomas and PDAC. Based on our preliminary data concerning expression of some CSC
markers, especially nestin in osteosarcoma (published in Veselska et al., 2008, Zambo et al.,
2012) and PDAC (published in Lenz et al. 2011), the specific objectives of this thesis were
defined as follow:
1) Analysis of expression of putative CSC markers in cell lines derived from pediatric
sarcomas and PDAC.
2) Comparison of expression levels detected in cell lines with expression of CSC markers in
respective primary tumor tissues.
3) Detailed study and characterization of cells or cell lines expressing CSC markers.
4) Validation of possible CSC phenotype identified in vitro using in vivo tumorigenicity
assay in mice.
5) Correlation of obtained results with patient clinical outcomes to identify potential
prognostic values of selected markers.
Results and discussion ◄ 35
4 Results and discussion
Seven peer-reviewed papers concerning identification of CSCs – five original articles and two
review articles – consitute the main part of this dissertation. The results obtained in these
studies are summarized and discussed in this chapter. List of the articles related to the thesis
including author’s contribution is provided in Chapter 7, followed by full texts of the
respective manuscripts (further referred to as Manuscript I–VII).
Sox2 identifies cells with CSC phenotype in sarcoma cell lines
Despite the progress in treatment of patients with localized tumors, the survival of patients
diagnosed with advanced disease remains low at about 30% (Perkins et al., 2014). Previously,
our research group demonstrated in osteosarcoma cell lines the presence of cells that co-
expressed CD133 and nestin, which were suggested as markers of CSCs (Veselska et al.,
2008). Therefore, we decided to examine the expression of these molecules in another subtype
of sarcoma, rhabdomyosarcoma, which represents the most frequent soft-tissue sarcoma
affecting children and adolescents.
In our initial study, we identified CD133 and nestin expression in all 10 primary tumor
tissue samples and in all 5 patient-derived rhabdomyosarcoma cell lines examined (Sana et
al., 2011 – Manuscript I). This study was the first report showing expression of CD133 in
rhabdomyosarcoma. Furthermore, we showed that all cell lines contained small population of
CD133+/nestin+ cells, and expressed Oct4 (POU5F1) and nucleostemin (GNL3) that are
considered to be markers of the hESCs. Preliminary in vivo tumorigenicity assay further
demonstrated that NSTS-11 cells were able to form tumors in the immunodeficient mice and
suggested a possible presence of cells with a CSC phenotype in rhabdomyosarcoma. In the
same year, another research group supported these findings by reporting CD133+ cells with
tumorigenic potential and enhanced chemoresistance that were enriched in embryonal
rhabdomyosarcoma cell lines cultured as spheres (Walter et al., 2011). Later, CD133 was
described as the CSC marker also in alveolar rhabdomyosarcoma (Pressey et al., 2013). In
contrast, Hirotsu et al. (2009) noted no difference between tumorigenicity of CD133+ and
CD133− rhabdomyosarcoma cells; however, no data and detailed information about these
experiments were shown in their study.
Based on these results, we performed a comprehensive study of putative CSC markers
on a panel of Ewing’s sarcoma, osteosarcoma and rhabdomyosarcoma (Skoda et al., 2016 –
Results and discussion ◄ 36
Manuscript V). Three cell lines derived from three primary tumor samples were analyzed for
each of these tumor types. This experimental design provided an important opportunity to
compare the pattern of CD133, ABCG2 and nestin expression in nine tumor samples paired
with nine cell lines. We showed that the frequency of ABCG2+ and CD133+ cells was
predominantly increased in the respective cell lines but that the high levels of nestin
expression were reduced in both osteosarcoma and rhabdomyosarcoma under in vitro
conditions. Although we demonstrated that expression of these markers changed under in
vitro conditions, suggesting the selection advantage of certain cells or population of cells, in
vivo tumorigenicity assay did not show any association of tumorigenic capacity of sarcoma
cells and expression level of any of three CSC markers examined. Further analysis including
common stem cell markers – Sox2, Oct4, Nanog, and ALDH1 – revealed an evident
association of the transcription factor Sox2 with tumorigenic potential of sarcoma cells.
Interestingly, we found that regardless of the expression of ABCG2, CD133, nestin, Oct4,
Nanog and ALDH1 only cell lines displaying increased Sox2 expression were tumorigenic.
Although surprising, these results are in agreement with recently published studies showing
that Sox2 is necessary for tumorigenicity and stemness of osteosarcoma (Basu-Roy et al.,
2012; Basu-Roy et al., 2015) and Ewing’s sarcoma cells (Riggi et al., 2010). Increased Sox2
levels were also detected in sarcospheres derived from osteosarcoma (Honoki et al., 2010) and
rhabdomyosarcoma cell lines (Walter et al., 2011), even though the correlation of Sox2
expression with tumorigenic potential was not analyzed in these studies.
As mentioned before, Sox2 is a transcription factor that has a crucial role in stem cell
maintenance, controls cell lineage fate decision, and it is necessary factor to reprogram
differentiated cells back towards pluripotency (Sarkar and Hochedlinger, 2013). Sox2 has
been demonstrated to be involved in development of all three germ layers, including
mesoderm from which sarcomas are derived. For example, deletion of Sox2 in cultured
osteoblast cell lines leads to a senescence-like phenotype, while its overexpression prevents
differentiation (Mansukhani et al., 2005). Accumulating evidence suggests that dysregulation
of Sox2 is associated with numerous cancers and Sox2 has been even proposed as oncogene
in some cases (reviewed in Weina and Utikal, 2014). Moreover, besides aforementioned
studies concerning osteosarcoma and Ewing’s sarcoma, recently published data show that
Sox2 expression is required for self-renewal and tumorigenicity of CSCs in other tumor types,
including glioblastoma (Gangemi et al., 2009; Ikushima et al., 2011; Berezovsky et al., 2014),
melanoma (Santini et al., 2014), ovarian carcinoma (Bareiss et al., 2013), cervical carcinoma
(Liu et al., 2014b), prostatic carcinoma (Rybak et al., 2013), lung carcinoma (Nakatsugawa et
Results and discussion ◄ 37
al., 2011; Chou et al., 2013), and squamous-cell carcinoma of the skin (Siegle et al., 2014).
High levels of Sox2 expression were also found in nestin+ glioblastoma cells that were proven
to be CSCs by lineage-tracing (Chen et al., 2012). Finally, two lineage-tracing studies have
recently demonstrated Sox2 as a functional CSC marker in medulloblastoma (Vanner et al.,
2014) and squamous cell carcinoma (Boumahdi et al., 2014), and showed that targeting Sox2
abrogated tumor initiation and growth.
All of these findings support our conclusion that cells displaying elevated expression of
Sox2 are key mediators of sarcoma tumorigenesis (Skoda et al., 2016 – Manuscript V). In
addition, our study is the first to show that Sox2 identifies cells with CSC phenotype not only
in osteosarcoma and Ewing’s sarcoma, but also in rhabdomyosarcoma. In contrast with
previously published papers, we demonstrated on a panel of nine patient-derived sarcoma cell
lines that expression of putative CSC markers – CD133, ABCG2, and nestin – is not
associated with tumorigenicity and therefore does not reflect CSC phenotype in sarcomas.
Nevertheless, we found that some of these proteins may have prognostic potential in some
types of sarcomas, as discussed below.
CD133 and nestin correlate with shorter survival in certain pediatric sarcomas
CD133, nestin, and ABCG2 have been described as putative CSC markers in sarcomas based
on the results of studies using sarcoma cell lines. We sought to determine the expression
levels of these molecules in primary tumor tissues and correlate them with clinical outcome.
In a study of 56 pediatric sarcomas, we identified that high expression levels of CD133
correlate with worse survival in rhabdomysarcoma patients (Zambo et al., under review –
Manuscript VI). These results confirmed the previous report of another group, who found
association of CD133 expression with poor survival in embryonal rhabdomyosarcoma (Walter
et al., 2011). Similarly, CD133 has been identified as an independent negative prognostic
factor in osteosarcoma patients (He et al., 2012). Although not significant, we found the same
trend in our cohort. In contrast, we showed that expression of CD133 does not have any
prognostic value in Ewing’s sarcoma; the same finding was reported also by another research
group (Jiang et al., 2010).
Nestin has been detected in a wide range of cancers and its increased levels have been
identified as negative prognostic factor in breast carcinoma, ovarian carcinoma, ependymoma,
melanoma, and multiple myeloma (Neradil and Veselska, 2015). In contrast, nestin was
recently reported as a positive prognostic factor for rhabdomyosarcomas (Glumac et al.,
2015). Nevertheless, we observed markedly high nestin expression in all 24
Results and discussion ◄ 38
rhabdomyosarcoma samples, as well as in normal skeletal muscle, suggesting nestin as a
potential diagnostic marker of myogenic differentiation in rhabdomyosarcoma (Zambo et al.,
under review – Manuscript VI). Correspondingly, abundance of cells expressing nestin has
been reported in tissue samples of pediatric rhabdomyosarcoma (Kobayashi et al., 1998), and
nestin has been detected in myoblasts during skeletal muscle regeneration (Vaittinen et al.,
2001). In Ewing’s sarcoma, we identified nestin expression in 14 of 22 cases and were the
first to show that high expression of nestin is significantly correlated with shorter survival in
these patients.
Until now, ABCG2 expression and its prognostic significance in sarcoma patients have
not been reported. We observed expression of ABCG2 in more than 50% of
rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma cases (Zambo et al., under review –
Manuscript VI). However, ABCG2 was not found to be significant prognostic factor in our
cohort, although trends toward shorter overall survival have been revealed in Ewing’s
sarcoma.
Our study represents the first complex analysis of CD133, nestin, and ABCG2 together
in three different types of pediatric sarcomas (Zambo et al., under review – Manuscript VI).
We demonstrated that these putative CSC markers are expressed in primary tumor tissues of
osteosarcoma, rhabdomyosarcoma and Ewing’s sarcoma. Furthermore, we showed their
possible prognostic values in these tumors.
CD133 is present in nuclei of sarcoma cells
During our first experiments with NSTS-11 cell line derived from rhabdomyosarcoma, we
noted not only membranous but also cytoplasmic localization of CD133 (Sana et al., 2011 –
Manuscript I). Human CD133 is traditionally described as a five transmembrane single-chain
glycoprotein that preferentially localizes in highly curved plasma membrane protrusions such
as microvilli and primary cilia (Grosse-Gehling et al., 2013). However, very little is known
about the physiological function of CD133 in the plasma membrane and even less about the
possible roles of CD133 located within the cancer cells. Several studies, including ours, have
shown in various cancers that although not detected at the cell surface, CD133 expression can
be identified within the cell (Sana et al., 2011 – Manuscript I; Skoda et al., under review –
Manuscript VII; Tirino et al., 2008; Campos et al., 2011; Bauer et al., 2011; Barrantes-Freer
et al., 2015).
In the course of our study of putative CSC markers in pediatric sarcomas, we observed a
surprising phenomenon: a stable subset of cells in each of five rhabdomyosarcoma cell lines
Results and discussion ◄ 39
examined exhibited an exclusive nuclear localization of CD133 (Nunukova et al., 2015 –
Manuscript IV). We confirmed these observations using three different anti-CD133 antibodies
and three independent methods: (i) indirect immunofluorescence and confocal microscopy
followed by software cross-section analysis; (ii) immunogold labeling and transmission
electron microscopy; and (iii) immunoblotting of the nuclear and cytoplasmic fractions. Later,
we identified this atypical nuclear localization of CD133 also in other two subtypes of
sarcoma – osteosarcoma and Ewing’s sarcoma (Skoda et al., 2016 – Manuscript V). CD133
was observed in nuclei of some tumor samples and in all cell lines examined. Especially
Ewing’s sarcoma ESFT-04 cell line, in which absolute nuclear positivity for CD133 was
found, appears as promising model to further study the role of CD133 in the cell nucleus of
tumor cells.
The studies of intracellular CD133 should be of great interest as these may elucidate its
biological functions that have not been well understood so far. Unexpectedly, Boivin et al.
(2009) demonstrated that CD133 is phosphorylated on cytoplasmic tyrosine sites (tyrosine-
828 and tyrosine-858) by Src and Fyn tyrosine kinases. Recent studies showed that
internalized CD133 could be involved in cell signaling, specifically in the canonical Wnt
pathway (Mak et al., 2012) or the PI3K/Akt pathway (Takenobu et al., 2011; Wei et al., 2013;
Shimozato et al., 2015). Nevertheless, although the exact role of CD133 localized within the
cell still remains to be elucidated, our results clearly showed that neither cytoplasmic nor
nuclear localization of CD133 is associated with the tumorigenic potential of sarcoma cells
(Skoda et al., 2016 – Manuscript V).
Pro–tumorigenic expression profile in PDAC corresponds to proportion of
CD24+/CD44+/EpCAM+/CD133+ cells
Pancreatic CSCs were described nearly ten years ago as CD44+/ CD24+/EpCAM+ cells (Li et
al., 2007) or CD133+ cells (Herman et al., 2007). However, no study has evaluated the co-
expression of all 4 of these markers in PDAC in detail. Similar to other combinations of CSC
markers, this approach might more accurately define the phenotype of CSCs. Therefore, we
performed detailed analysis of the expression of putative CSC markers (CD24, CD44,
EpCAM, CD133 and nestin) in 3 pairs of matched primary PDAC tissue samples and derived
cell lines (Skoda et al., under review – Manuscript VII).
We found that all of the examined markers are expressed in all PDAC cell lines. IHC
confirmed the expression patterns of the CSC markers in the corresponding tumor tissues,
although expression of CD24 and CD44 increased considerably in cultures. This finding is in
Results and discussion ◄ 40
accordance with other studies that reported high percentages of CD44+ cells in pancreatic cell
lines compared with PDAC tumor tissues (Kure et al., 2012; Bunger et al, 2012). Similarly to
our sarcoma studies, we observed an alternative localization of CSC markers that were
examined in our experiments on PDAC cells. Whereas these molecules are described as
plasma membrane proteins, we detected substantial amount of them to be localized within the
cell. Flow cytometric analysis of fixed cells showed that CD24, CD44, CD133, or EpCAM, as
evaluated separately, are expressed in more than 80% of cells irrespective of the cell line.
These values were much higher than those that have been previously reported. However,
dramatic decrease in the proportion of CD24+, CD133+ and EpCAM+ cells was obtained using
live cells in flow cytometric analysis, suggesting that only limited proportion of cells
expressed these markers on the cell surface. Real-time PCR results supported this suggestion
showing that all markers were expressed among the cell lines at the mRNA level. Our study
provides further evidence that the subcellular localization of CSC markers can vary
considerably. These alterations may lead to the completely different effects on cell signaling,
proliferation, invasiveness, and metastatic potential, and may result in different patient
outcomes (discussed in Skoda et al., under review – Manuscript VII).
Most notably, we provided the first evidence that cells with CD24+/CD44+/
EpCAM+/CD133+ phenotype are present in PDAC-derived cell lines. Considerably large
proportions of these cells (range, 43 to 72%) were detected in all 3 cell lines, to some extent
opposing the hypothesis that CD24+/CD44+/EpCAM+/CD133+ phenotype could specifically
identify pancreatic CSCs. Nevertheless, gene expression profiling revealed that a higher
proportion of these cells in the PDAC cell line corresponded with a pro-tumorigenic
expression profile. These results indicate that CD24+/CD44+/EpCAM+/CD133+ cells may be
involved in PDAC development. Further functional studies are needed to determine whether
these cells eventually possess CSC characteristics.
Conclusion ◄ 41
5 Conclusion
Our results suggest that previously described CSC markers are more informative with regard
to clinical outcome or tumor progression than to the CSC phenotype. At least in pediatric
sarcomas and PDAC, more selective marker is needed for further investigations of CSCs.
Currently, essential role of Sox2 in maintenance of CSC phenotype and tumorigenesis
have been extensively studied in numerous types of tumors. Those studies included also
osteosarcoma, Ewing’s sarcoma and PDAC. However, we are the first to demonstrate that
Sox2 is associated with tumorigenicity of rhabdomyosarcoma cells. Moreover, we showed in
pediatric sarcomas that high levels of Sox2 expression may better distinguish tumorigenic
cells than originally reported CSC markers.
Thus, it seems possible that pluripotency factors, such as Sox2, may represent the key
common regulators reprogramming premalignant cells into CSCs. Usage of such essential
factors as CSC markers may provide more consistent results than previously reported by
studies which utilized cell surface markers. Although methodologically difficult, sorting of
tumor cells based on the Sox2 expression may yield high purity fraction of CSCs (Larsson et
al., 2012). Finally, studies of CSCs defined by high expression of Sox2 (or by another
pluripotency marker) may provide new insights into tumor initiation and maintenance, and
this knowledge could potentially be transformed into development of novel CSC-targeted
therapies.
References ◄ 42
6 References
Adhikari, A.S., Agarwal, N., Wood, B.M., Porretta, C., Ruiz, B., Pochampally, R.R., and Iwakuma, T.
(2010). CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis
and drug resistance. Cancer Res 70, 4602-4612.
Agliano, A., Martin-Padura, I., Mancuso, P., Marighetti, P., Rabascio, C., Pruneri, G., Shultz, L.D.,
and Bertolini, F. (2008). Human acute leukemia cells injected in NOD/LtSz-scid/IL-2Rgamma
null mice generate a faster and more efficient disease compared to other NOD/scid-related strains.
Int J Cancer 123, 2222-2227.
Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., and Clarke, M.F. (2003). Prospective
identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983-3988.
Anderson, J.L., Denny, C.T., Tap, W.D., and Federman, N. (2012). Pediatric sarcomas: translating
molecular pathogenesis of disease to novel therapeutic possibilities. Pediatr Res 72, 112-121.
Awad, O., Yustein, J.T., Shah, P., Gul, N., Katuri, V., O'neill, A., Kong, Y., Brown, M.L., Toretsky,
J.A., and Loeb, D.M. (2010). High ALDH activity identifies chemotherapy-resistant Ewing's
sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition. PLoS One 5, e13943.
Balamuth, N.J., and Womer, R.B. (2010). Ewing's sarcoma. Lancet Oncol 11, 184-192.
Balic, A., Dorado, J., Alonso-Gomez, M., and Heeschen, C. (2012). Stem cells as the root of
pancreatic ductal adenocarcinoma. Exp Cell Res 318, 691-704.
Ban, J., Jug, G., Mestdagh, P., Schwentner, R., Kauer, M., Aryee, D.N., Schaefer, K.L., Nakatani, F.,
Scotlandi, K., Reiter, M., Strunk, D., Speleman, F., Vandesompele, J., and Kovar, H. (2011). Hsa-
mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in
Ewing's sarcoma. Oncogene 30, 2173-2180.
Bareiss, P.M., Paczulla, A., Wang, H., Schairer, R., Wiehr, S., Kohlhofer, U., Rothfuss, O.C., Fischer,
A., Perner, S., Staebler, A., Wallwiener, D., Fend, F., Fehm, T., Pichler, B., Kanz, L., Quintanilla-
Martinez, L., Schulze-Osthoff, K., Essmann, F., and Lengerke, C. (2013). SOX2 expression
associates with stem cell state in human ovarian carcinoma. Cancer Res 73, 5544-5555.
Barrantes-Freer, A., Renovanz, M., Eich, M., Braukmann, A., Sprang, B., Spirin, P., Pardo, L.A.,
Giese, A., and Kim, E.L. (2015). CD133 Expression Is Not Synonymous to Immunoreactivity for
AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells. PLoS One 10,
e0130519.
Barta, T., Peskova, L., Collin, J., Montaner, D., Neganova, I., Armstrong, L., and Lako, M. (2016).
Brief Report: Inhibition of miR-145 Enhances Reprogramming of Human Dermal Fibroblasts to
Induced Pluripotent Stem Cells. Stem Cells 34, 246-251.
Basu-Roy, U., Bayin, N.S., Rattanakorn, K., Han, E., Placantonakis, D.G., Mansukhani, A., and
Basilico, C. (2015). Sox2 antagonizes the Hippo pathway to maintain stemness in cancer cells. Nat
Commun 6, 6411.
Basu-Roy, U., Seo, E., Ramanathapuram, L., Rapp, T.B., Perry, J.A., Orkin, S.H., Mansukhani, A.,
and Basilico, C. (2012). Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas.
Oncogene 31, 2270-2282.
Bauer, N., Wilsch-Bräuninger, M., Karbanová, J., Fonseca, A.V., Strauss, D., Freund, D., Thiele, C.,
Huttner, W.B., Bornhäuser, M., and Corbeil, D. (2011). Haematopoietic stem cell differentiation
promotes the release of prominin-1/CD133-containing membrane vesicles—a role of the
endocytic–exocytic pathway. EMBO Mol Med 3, 398-409.
Beck, B., and Blanpain, C. (2013). Unravelling cancer stem cell potential. Nat Rev Cancer 13, 727-
738.
References ◄ 43
Ben-Porath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W., Regev, A., and Weinberg, R.A.
(2008). An embryonic stem cell-like gene expression signature in poorly differentiated aggressive
human tumors. Nat Genet 40, 499-507.
Berezovsky, A.D., Poisson, L.M., Cherba, D., Webb, C.P., Transou, A.D., Lemke, N.W., Hong, X.,
Hasselbach, L.A., Irtenkauf, S.M., Mikkelsen, T., and Decarvalho, A.C. (2014). Sox2 promotes
malignancy in glioblastoma by regulating plasticity and astrocytic differentiation. Neoplasia 16,
193-206, 206.e119-125.
Boivin, D., Labbe, D., Fontaine, N., Lamy, S., Beaulieu, E., Gingras, D., and Beliveau, R. (2009). The
stem cell marker CD133 (prominin-1) is phosphorylated on cytoplasmic tyrosine-828 and tyrosine-
852 by Src and Fyn tyrosine kinases. Biochemistry 48, 3998-4007.
Bonnet, D., and Dick, J.E. (1997). Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med 3, 730-737.
Boumahdi, S., Driessens, G., Lapouge, G., Rorive, S., Nassar, D., Le Mercier, M., Delatte, B.,
Caauwe, A., Lenglez, S., Nkusi, E., Brohee, S., Salmon, I., Dubois, C., Del Marmol, V., Fuks, F.,
Beck, B., and Blanpain, C. (2014). SOX2 controls tumour initiation and cancer stem-cell functions
in squamous-cell carcinoma. Nature 511, 246-250.
Breitfeld, P.P., and Meyer, W.H. (2005). Rhabdomyosarcoma: new windows of opportunity.
Oncologist 10, 518-527.
Brooks, M.D., Burness, M.L., and Wicha, M.S. (2015). Therapeutic Implications of Cellular
Heterogeneity and Plasticity in Breast Cancer. Cell Stem Cell 17, 260-271.
Bunger, S., Barow, M., Thorns, C., Freitag-Wolf, S., Danner, S., Tiede, S., Pries, R., Gorg, S., Bruch,
H.P., Roblick, U.J., Kruse, C., and Habermann, J.K. (2012). Pancreatic carcinoma cell lines reflect
frequency and variability of cancer stem cell markers in clinical tissue. Eur Surg Res 49, 88-98.
Bunting, K.D. (2002). ABC transporters as phenotypic markers and functional regulators of stem cells.
Stem Cells 20, 11-20.
Campos, B., Zeng, L., Daotrong, P.H., Eckstein, V., Unterberg, A., Mairbaurl, H., and Herold-Mende,
C. (2011). Expression and regulation of AC133 and CD133 in glioblastoma. Glia 59, 1974-1986.
Cao, L., Zhou, Y., Zhai, B., Liao, J., Xu, W., Zhang, R., Li, J., Zhang, Y., Chen, L., Qian, H., Wu, M.,
and Yin, Z. (2011). Sphere-forming cell subpopulations with cancer stem cell properties in human
hepatoma cell lines. BMC Gastroenterol 11, 71.
Chaffer, C.L., Brueckmann, I., Scheel, C., Kaestli, A.J., Wiggins, P.A., Rodrigues, L.O., Brooks, M.,
Reinhardt, F., Su, Y., Polyak, K., Arendt, L.M., Kuperwasser, C., Bierie, B., and Weinberg, R.A.
(2011). Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc
Natl Acad Sci U S A 108, 7950-7955.
Chaffer, C.L., Marjanovic, N.D., Lee, T., Bell, G., Kleer, C.G., Reinhardt, F., D'alessio, A.C., Young,
R.A., and Weinberg, R.A. (2013). Poised chromatin at the ZEB1 promoter enables breast cancer
cell plasticity and enhances tumorigenicity. Cell 154, 61-74.
Charles, N., Ozawa, T., Squatrito, M., Bleau, A.M., Brennan, C.W., Hambardzumyan, D., and
Holland, E.C. (2010). Perivascular nitric oxide activates notch signaling and promotes stem-like
character in PDGF-induced glioma cells. Cell Stem Cell 6, 141-152.
Chen, J., Li, Y., Yu, T.S., Mckay, R.M., Burns, D.K., Kernie, S.G., and Parada, L.F. (2012). A
restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522-
526.
Chen, K., Huang, Y.H., and Chen, J.L. (2013). Understanding and targeting cancer stem cells:
therapeutic implications and challenges. Acta Pharmacol Sin 34, 732-740.
Chen, K., Li, Z., Jiang, P., Zhang, X., Zhang, Y., Jiang, Y., He, Y., and Li, X. (2014). Co-expression
of CD133, CD44v6 and human tissue factor is associated with metastasis and poor prognosis in
pancreatic carcinoma. Oncol Rep 32, 755-763.
References ◄ 44
Chivukula, R.R., and Mendell, J.T. (2009). Abate and switch: miR-145 in stem cell differentiation.
Cell 137, 606-608.
Chou, Y.T., Lee, C.C., Hsiao, S.H., Lin, S.E., Lin, S.C., Chung, C.H., Kao, Y.R., Wang, Y.H., Chen,
C.T., Wei, Y.H., and Wu, C.W. (2013). The emerging role of SOX2 in cell proliferation and
survival and its crosstalk with oncogenic signaling in lung cancer. Stem Cells 31, 2607-2619.
Cioffi, M., D'alterio, C., Camerlingo, R., Tirino, V., Consales, C., Riccio, A., Ierano, C., Cecere, S.C.,
Losito, N.S., Greggi, S., Pignata, S., Pirozzi, G., and Scala, S. (2015). Identification of a distinct
population of CD133(+)CXCR4(+) cancer stem cells in ovarian cancer. Sci Rep 5, 10357.
Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., Visvader, J.,
Weissman, I.L., and Wahl, G.M. (2006). Cancer stem cells–perspectives on current status and
future directions: AACR Workshop on cancer stem cells. Cancer Res 66, 9339-9344.
Cogle, C.R. (2011) Cancer stem cells: historical perspectives and lessons from leukemia. In A.L. Allan
(Ed.), Cancer stem cells in solid tumors (pp. 3–11). New York, NY: Springer New York.
Cojoc, M., Mabert, K., Muders, M.H., and Dubrovska, A. (2015). A role for cancer stem cells in
therapy resistance: cellular and molecular mechanisms. Semin Cancer Biol 31, 16-27.
Corbeil, D., Karbanova, J., Fargeas, C.A., and Jaszai, J. (2013). Prominin-1 (CD133): Molecular and
Cellular Features Across Species. Adv Exp Med Biol 777, 3-24.
Corbeil, D., Marzesco, A.M., Wilsch-Brauninger, M., and Huttner, W.B. (2010). The intriguing links
between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical
plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell
differentiation. FEBS Lett 584, 1659-1664.
Cui, S.Y., Wang, R., and Chen, L.B. (2014). MicroRNA-145: a potent tumour suppressor that
regulates multiple cellular pathways. J Cell Mol Med 18, 1913-1926.
De Wit, S., Van Dalum, G., Lenferink, A.T., Tibbe, A.G., Hiltermann, T.J., Groen, H.J., Van Rijn,
C.J., and Terstappen, L.W. (2015). The detection of EpCAM(+) and EpCAM(-) circulating tumor
cells. Sci Rep 5, 12270.
Dean, M. (2009). ABC transporters, drug resistance, and cancer stem cells. J Mammary Gland Biol
Neoplasia 14, 3-9.
Dela Cruz, F.S. (2013). Cancer stem cells in pediatric sarcomas. Front Oncol 3, 168.
Delitto, D., Vertes-George, E., Hughes, S.J., Behrns, K.E., and Trevino, J.G. (2014). c-Met signaling
in the development of tumorigenesis and chemoresistance: potential applications in pancreatic
cancer. World J Gastroenterol 20, 8458-8470.
Deng, Y., Zhou, J., Fang, L., Cai, Y., Ke, J., Xie, X., Huang, Y., Huang, M., and Wang, J. (2014).
ALDH1 is an independent prognostic factor for patients with stages II-III rectal cancer after
receiving radiochemotherapy. Br J Cancer 110, 430-434.
Dey, P., Togra, J., and Mitra, S. (2014). Intermediate filament: structure, function, and applications in
cytology. Diagn Cytopathol 42, 628-635.
Di Fiore, R., Santulli, A., Ferrante, R.D., Giuliano, M., De Blasio, A., Messina, C., Pirozzi, G., Tirino,
V., Tesoriere, G., and Vento, R. (2009). Identification and expansion of human osteosarcoma-
cancer-stem cells by long-term 3-aminobenzamide treatment. J Cell Physiol 219, 301-313.
Ding, X.W., Wu, J.H., and Jiang, C.P. (2010). ABCG2: a potential marker of stem cells and novel
target in stem cell and cancer therapy. Life Sci 86, 631-637.
Dontu, G., and Wicha, M.S. (2005). Survival of mammary stem cells in suspension culture:
implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia 10, 75-86.
Doyle, L.A. (2014). Sarcoma classification: an update based on the 2013 World Health Organization
Classification of Tumors of Soft Tissue and Bone. Cancer 120, 1763-1774.
References ◄ 45
Driessens, G., Beck, B., Caauwe, A., Simons, B.D., and Blanpain, C. (2012). Defining the mode of
tumour growth by clonal analysis. Nature 488, 527-530.
Duque, A., and Rakic, P. (2011). Different effects of bromodeoxyuridine and [3H]thymidine
incorporation into DNA on cell proliferation, position, and fate. J Neurosci 31, 15205-15217.
Erdei, Z., Lorincz, R., Szebenyi, K., Pentek, A., Varga, N., Liko, I., Varady, G., Szakacs, G., Orban,
T.I., Sarkadi, B., and Apati, A. (2014). Expression pattern of the human ABC transporters in
pluripotent embryonic stem cells and in their derivatives. Cytometry B Clin Cytom 86, 299-310.
Fang, X., Zheng, P., Tang, J., and Liu, Y. (2010). CD24: from A to Z. Cell Mol Immunol 7, 100-103.
Fedr, R., Pernicova, Z., Slabakova, E., Strakova, N., Bouchal, J., Grepl, M., Kozubik, A., and Soucek,
K. (2013). Automatic cell cloning assay for determining the clonogenic capacity of cancer and
cancer stem-like cells. Cytometry A 83, 472-482.
Fillmore, C., and Kuperwasser, C. (2007). Human breast cancer stem cell markers CD44 and CD24:
enriching for cells with functional properties in mice or in man? Breast Cancer Res 9, 303.
Fillmore, C.M., and Kuperwasser, C. (2008). Human breast cancer cell lines contain stem-like cells
that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast
Cancer Res 10, R25.
Fletcher, C.D., Hogendoorn, P., Mertens, F., Bridge, J. (2013). WHO Classification of Tumours of Soft
Tissue and Bone. Lyon: IARC Press.
Gangemi, R.M., Griffero, F., Marubbi, D., Perera, M., Capra, M.C., Malatesta, P., Ravetti, G.L., Zona,
G.L., Daga, A., and Corte, G. (2009). SOX2 silencing in glioblastoma tumor-initiating cells causes
stop of proliferation and loss of tumorigenicity. Stem Cells 27, 40-48.
Gentile, A., Trusolino, L., and Comoglio, P.M. (2008). The Met tyrosine kinase receptor in
development and cancer. Cancer Metastasis Rev 27, 85-94.
Gibbs, C.P., Kukekov, V.G., Reith, J.D., Tchigrinova, O., Suslov, O.N., Scott, E.W., Ghivizzani, S.C.,
Ignatova, T.N., and Steindler, D.A. (2005). Stem-like cells in bone sarcomas: implications for
tumorigenesis. Neoplasia 7, 967-976.
Glumac, S., Pejic, S., Kovacevic, R., Dundjerovic, D., Davidovic, R., Ristic, D., and Sopta, J. (2015).
Immunohistochemical expression of nestin in rhabdomyosarcoma: implications for
clinicopathology and patient outcome. Genet Mol Res 14, 14649-14659.
Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. (1996). Isolation and
functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med
183, 1797-1806.
Greaves, M., and Maley, C.C. (2012). Clonal evolution in cancer. Nature 481, 306-313.
Greco, N., Schott, T., Mu, X., Rothenberg, A., Voigt, C., Mcgough, R.L., 3rd, Goodman, M., Huard,
J., and Weiss, K.R. (2014). ALDH Activity Correlates with Metastatic Potential in Primary
Sarcomas of Bone. J Cancer Ther 5, 331-338.
Grosse-Gehling, P., Fargeas, C.A., Dittfeld, C., Garbe, Y., Alison, M.R., Corbeil, D., and Kunz-
Schughart, L.A. (2013). CD133 as a biomarker for putative cancer stem cells in solid tumours:
limitations, problems and challenges. J Pathol 229, 355-378.
Hage, C., Rausch, V., Giese, N., Giese, T., Schonsiegel, F., Labsch, S., Nwaeburu, C., Mattern, J.,
Gladkich, J., and Herr, I. (2013). The novel c-Met inhibitor cabozantinib overcomes gemcitabine
resistance and stem cell signaling in pancreatic cancer. Cell Death Dis 4, e627.
Hagio, M., Matsuda, Y., Suzuki, T., and Ishiwata, T. (2013). Nestin regulates epithelial-mesenchymal
transition marker expression in pancreatic ductal adenocarcinoma cell lines. Mol Clin Oncol 1, 83-
87.
References ◄ 46
He, A., Qi, W., Huang, Y., Feng, T., Chen, J., Sun, Y., Shen, Z., and Yao, Y. (2012). CD133
expression predicts lung metastasis and poor prognosis in osteosarcoma patients: A clinical and
experimental study. Exp Ther Med 4, 435-441.
Hemmati, H.D., Nakano, I., Lazareff, J.A., Masterman-Smith, M., Geschwind, D.H., Bronner-Fraser,
M., and Kornblum, H.I. (2003). Cancerous stem cells can arise from pediatric brain tumors. Proc
Natl Acad Sci U S A 100, 15178-15183.
Herlyn, M., Steplewski, Z., Herlyn, D., and Koprowski, H. (1979). Colorectal carcinoma-specific
antigen: detection by means of monoclonal antibodies. Proc Natl Acad Sci U S A 76, 1438-1442.
Hermann, P.C., Huber, S.L., Herrler, T., Aicher, A., Ellwart, J.W., Guba, M., Bruns, C.J., and
Heeschen, C. (2007). Distinct populations of cancer stem cells determine tumor growth and
metastatic activity in human pancreatic cancer. Cell Stem Cell 1, 313-323.
Herreros-Villanueva, M., Zhang, J.S., Koenig, A., Abel, E.V., Smyrk, T.C., Bamlet, W.R., De
Narvajas, A.A., Gomez, T.S., Simeone, D.M., Bujanda, L., and Billadeau, D.D. (2013). SOX2
promotes dedifferentiation and imparts stem cell-like features to pancreatic cancer cells.
Oncogenesis 2, e61.
Herreros-Villanueva, M., Bujanda, L., Billadeau, D.D., and Zhang, J.S. (2014). Embryonic stem cell
factors and pancreatic cancer. World J Gastroenterol 20, 2247-2254.
Hezel, A.F., Kimmelman, A.C., Stanger, B.Z., Bardeesy, N., and Depinho, R.A. (2006). Genetics and
biology of pancreatic ductal adenocarcinoma. Genes Dev 20, 1218-1249.
Hiraga, T., Ito, S., and Nakamura, H. (2011). Side population in MDA-MB-231 human breast cancer
cells exhibits cancer stem cell-like properties without higher bone-metastatic potential. Oncol Rep
25, 289-296.
Hirotsu, M., Setoguchi, T., Matsunoshita, Y., Sasaki, H., Nagao, H., Gao, H., Sugimura, K., and
Komiya, S. (2009). Tumour formation by single fibroblast growth factor receptor 3-positive
rhabdomyosarcoma-initiating cells. Br J Cancer 101, 2030-2037.
Hong, W.K., Bast, R.C. Jr, Hait, W.N., Kufe, D.W., Pollock, R.E., Weichselbaum, R.C., Holland, J.F.
(Eds.). (2010). Holland-Frei Cancer Medicine. Shelton: PMPH-USA.
Honoki, K., Fujii, H., Kubo, A., Kido, A., Mori, T., Tanaka, Y., and Tsujiuchi, T. (2010). Possible
involvement of stem-like populations with elevated ALDH1 in sarcomas for chemotherapeutic
drug resistance. Oncol Rep 24, 501-505.
Hou, Y.C., Chao, Y.J., Tung, H.L., Wang, H.C., and Shan, Y.S. (2014). Coexpression of CD44-
positive/CD133-positive cancer stem cells and CD204-positive tumor-associated macrophages is a
predictor of survival in pancreatic ductal adenocarcinoma. Cancer 120, 2766-2777.
Hsu, Y.C., and Fuchs, E. (2012). A family business: stem cell progeny join the niche to regulate
homeostasis. Nat Rev Mol Cell Biol 13, 103-114.
Hu, J., Qiu, M., Jiang, F., Zhang, S., Yang, X., Wang, J., Xu, L., and Yin, R. (2014). MiR-145
regulates cancer stem-like properties and epithelial-to-mesenchymal transition in lung
adenocarcinoma-initiating cells. Tumour Biol 35, 8953-8961.
Ikushima, H., Todo, T., Ino, Y., Takahashi, M., Saito, N., Miyazawa, K., and Miyazono, K. (2011).
Glioma-initiating cells retain their tumorigenicity through integration of the Sox axis and Oct4
protein. J Biol Chem 286, 41434-41441.
Iliopoulos, D., Hirsch, H.A., Wang, G., and Struhl, K. (2011). Inducible formation of breast cancer
stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion. Proc Natl
Acad Sci U S A 108, 1397-1402.
Immervoll, H., Hoem, D., Sakariassen, P.O., Steffensen, O.J., and Molven, A. (2008). Expression of
the "stem cell marker" CD133 in pancreas and pancreatic ductal adenocarcinomas. BMC Cancer 8,
48.
References ◄ 47
Immervoll, H., Hoem, D., Steffensen, O.J., Miletic, H., and Molven, A. (2011). Visualization of CD44
and CD133 in normal pancreas and pancreatic ductal adenocarcinomas: non-overlapping
membrane expression in cell populations positive for both markers. J Histochem Cytochem 59,
441-455.
Jaggupilli, A., and Elkord, E. (2012). Significance of CD44 and CD24 as cancer stem cell markers: an
enduring ambiguity. Clin Dev Immunol 2012, 708036.
Januchowski, R., Wojtowicz, K., and Zabel, M. (2013). The role of aldehyde dehydrogenase (ALDH)
in cancer drug resistance. Biomed Pharmacother 67, 669-680.
Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., and Forman, D. (2011). Global cancer
statistics. CA Cancer J Clin 61, 69-90.
Jia, Y., Liu, H., Zhuang, Q., Xu, S., Yang, Z., Li, J., Lou, J., and Zhang, W. (2012). Tumorigenicity of
cancer stem-like cells derived from hepatocarcinoma is regulated by microRNA-145. Oncol Rep
27, 1865-1872.
Jiang, X., Gwye, Y., Russell, D., Cao, C., Douglas, D., Hung, L., Kovar, H., Triche, T.J., and Lawlor,
E.R. (2010). CD133 expression in chemo-resistant Ewing sarcoma cells. BMC Cancer 10, 116.
Kanwar, S.S., Yu, Y., Nautiyal, J., Patel, B.B., and Majumdar, A.P. (2010). The Wnt/beta-catenin
pathway regulates growth and maintenance of colonospheres. Mol Cancer 9, 212.
Karski, E.E., Mcilvaine, E., Segal, M.R., Krailo, M., Grier, H.E., Granowetter, L., Womer, R.B.,
Meyers, P.A., Felgenhauer, J., Marina, N., and Dubois, S.G. (2016). Identification of Discrete
Prognostic Groups in Ewing Sarcoma. Pediatr Blood Cancer 63, 47-53.
Kawamoto, M., Ishiwata, T., Cho, K., Uchida, E., Korc, M., Naito, Z., and Tajiri, T. (2009). Nestin
expression correlates with nerve and retroperitoneal tissue invasion in pancreatic cancer. Hum
Pathol 40, 189-198.
Keysar, S.B., and Jimeno, A. (2010). More than markers: biological significance of cancer stem cell-
defining molecules. Mol Cancer Ther 9, 2450-2457.
Kim, M.P., Fleming, J.B., Wang, H., Abbruzzese, J.L., Choi, W., Kopetz, S., Mcconkey, D.J., Evans,
D.B., and Gallick, G.E. (2011a). ALDH activity selectively defines an enhanced tumor-initiating
cell population relative to CD133 expression in human pancreatic adenocarcinoma. PLoS One 6,
e20636.
Kim, H.J., Kim, M.J., Ahn, S.H., Son, B.H., Kim, S.B., Ahn, J.H., Noh, W.C., and Gong, G. (2011b).
Different prognostic significance of CD24 and CD44 expression in breast cancer according to
hormone receptor status. Breast 20, 78-85.
Kim, H.S., Yoo, S.Y., Kim, K.T., Park, J.T., Kim, H.J., and Kim, J.C. (2012). Expression of the stem
cell markers CD133 and nestin in pancreatic ductal adenocarcinoma and clinical relevance. Int J
Clin Exp Pathol 5, 754-761.
Kim, S.K., Kim, H., Lee, D.H., Kim, T.S., Kim, T., Chung, C., Koh, G.Y., and Lim, D.S. (2013).
Reversing the intractable nature of pancreatic cancer by selectively targeting ALDH-high, therapy-
resistant cancer cells. PLoS One 8, e78130.
Kinsey, M., Smith, R., Iyer, A.K., Mccabe, E.R., and Lessnick, S.L. (2009). EWS/FLI and its
downstream target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's
sarcoma. Cancer Res 69, 9047-9055.
Kleppe, M., and Levine, R.L. (2014). Tumor heterogeneity confounds and illuminates: assessing the
implications. Nat Med 20, 342-344.
Kobayashi, M., Sjoberg, G., Soderhall, S., Lendahl, U., Sandstedt, B., and Sejersen, T. (1998).
Pediatric rhabdomyosarcomas express the intermediate filament nestin. Pediatr Res 43, 386-392.
Kreso, A., and Dick, J.E. (2014). Evolution of the cancer stem cell model. Cell Stem Cell 14, 275-291.
References ◄ 48
Krieg, A., Riemer, J.C., Telan, L.A., Gabbert, H.E., and Knoefel, W.T. (2015). CXCR4--A Prognostic
and Clinicopathological Biomarker for Pancreatic Ductal Adenocarcinoma: A Meta-Analysis.
PLoS One 10, e0130192.
Kristiansen, G., Winzer, K.J., Mayordomo, E., Bellach, J., Schluns, K., Denkert, C., Dahl, E., Pilarsky,
C., Altevogt, P., Guski, H., and Dietel, M. (2003). CD24 expression is a new prognostic marker in
breast cancer. Clin Cancer Res 9, 4906-4913.
Krupkova, O., Jr., Loja, T., Zambo, I., and Veselska, R. (2010). Nestin expression in human tumors
and tumor cell lines. Neoplasma 57, 291-298.
Kure, S., Matsuda, Y., Hagio, M., Ueda, J., Naito, Z., and Ishiwata, T. (2012). Expression of cancer
stem cell markers in pancreatic intraepithelial neoplasias and pancreatic ductal adenocarcinomas.
Int J Oncol 41, 1314-1324.
Kwon, M.J., Han, J., Seo, J.H., Song, K., Jeong, H.M., Choi, J.S., Kim, Y.J., Lee, S.H., Choi, Y.L.,
and Shin, Y.K. (2015). CD24 Overexpression Is Associated with Poor Prognosis in Luminal A and
Triple-Negative Breast Cancer. PLoS One 10, e0139112.
Lapidot, T., Sirard, C., Vormoor, J., Murdoch, B., Hoang, T., Caceres-Cortes, J., Minden, M.,
Paterson, B., Caligiuri, M.A., and Dick, J.E. (1994). A cell initiating human acute myeloid
leukaemia after transplantation into SCID mice. Nature 367, 645-648.
Larsson, H.M., Lee, S.T., Roccio, M., Velluto, D., Lutolf, M.P., Frey, P., and Hubbell, J.A. (2012).
Sorting live stem cells based on Sox2 mRNA expression. PLoS One 7, e49874.
Lathia, J.D. (2013). Cancer stem cells: moving past the controversy. CNS Oncol 2, 465-467.
Leccia, F., Del Vecchio, L., Mariotti, E., Di Noto, R., Morel, A.P., Puisieux, A., Salvatore, F., and
Ansieau, S. (2014). ABCG2, a novel antigen to sort luminal progenitors of BRCA1- breast cancer
cells. Mol Cancer 13, 213.
Lee, E.K., Cho, H., and Kim, C.W. (2011). Proteomic analysis of cancer stem cells in human prostate
cancer cells. Biochem Biophys Res Commun 412, 279-285.
Lenz, J., Karasek, P., Jarkovsky, J., Muckova, K., Dite, P., Kala, Z., Veselska, R., and Hermanova, M.
(2011). Clinicopathological correlations of nestin expression in surgically resectable pancreatic
cancer including an analysis of perineural invasion. J Gastrointestin Liver Dis 20, 389-396.
Leuchte, K., Altvater, B., Hoffschlag, S., Potratz, J., Meltzer, J., Clemens, D., Luecke, A., Hardes, J.,
Dirksen, U., Juergens, H., Kailayangiri, S., and Rossig, C. (2014). Anchorage-independent growth
of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like
phenotype and function. Oncol Rep 32, 845-852.
Leushacke, M., and Barker, N. (2012). Lgr5 and Lgr6 as markers to study adult stem cell roles in self-
renewal and cancer. Oncogene 31, 3009-3022.
Li, C., Heidt, D.G., Dalerba, P., Burant, C.F., Zhang, L., Adsay, V., Wicha, M., Clarke, M.F., and
Simeone, D.M. (2007). Identification of pancreatic cancer stem cells. Cancer Res 67, 1030-1037.
Li, C., Wu, J.J., Hynes, M., Dosch, J., Sarkar, B., Welling, T.H., Pasca Di Magliano, M., and Simeone,
D.M. (2011). c-Met is a marker of pancreatic cancer stem cells and therapeutic target.
Gastroenterology 141, 2218-2227.e2215.
Li, X., Zhao, H., Gu, J., and Zheng, L. (2015). Prognostic value of cancer stem cell marker CD133
expression in pancreatic ductal adenocarcinoma (PDAC): a systematic review and meta-analysis.
Int J Clin Exp Pathol 8, 12084-12092.
Liu, A., Yu, X., and Liu, S. (2013a). Pluripotency transcription factors and cancer stem cells: small
genes make a big difference. Chin J Cancer 32, 483-487.
Liu, S., Liu, C., Min, X., Ji, Y., Wang, N., Liu, D., Cai, J., and Li, K. (2013b). Prognostic value of
cancer stem cell marker aldehyde dehydrogenase in ovarian cancer: a meta-analysis. PLoS One 8,
e81050.
References ◄ 49
Liu, K., Song, Y., Yu, H., and Zhao, T. (2014a). Understanding the roadmaps to induced pluripotency.
Cell Death Dis 5, e1232.
Liu, X.F., Yang, W.T., Xu, R., Liu, J.T., and Zheng, P.S. (2014b). Cervical cancer cells with positive
Sox2 expression exhibit the properties of cancer stem cells. PLoS One 9, e87092.
Logsdon, C.D., Ji, B., and Hwang, R.F. (2010). Molecular Relationships Between Chronic Pancreatitis
and Cancer. In Pancreatic Cancer (pp. 285-315). New York, NY: Springer New York.
Loja, T., Chlapek, P., Kuglik, P., Pesakova, M., Oltova, A., Cejpek, P., and Veselska, R. (2009).
Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic
and nuclear localization of nestin. Oncol Rep 21, 119-127.
Luetke, A., Meyers, P.A., Lewis, I., and Juergens, H. (2014). Osteosarcoma treatment - where do we
stand? A state of the art review. Cancer Treat Rev 40, 523-532.
Ma, I., and Allan, A.L. (2011). The role of human aldehyde dehydrogenase in normal and cancer stem
cells. Stem Cell Rev 7, 292-306.
Mak, A.B., Nixon, A.M., Kittanakom, S., Stewart, J.M., Chen, G.I., Curak, J., Gingras, A.C.,
Mazitschek, R., Neel, B.G., Stagljar, I., and Moffat, J. (2012). Regulation of CD133 by HDAC6
promotes beta-catenin signaling to suppress cancer cell differentiation. Cell Rep 2, 951-963.
Mansukhani, A., Ambrosetti, D., Holmes, G., Cornivelli, L., and Basilico, C. (2005). Sox2 induction
by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation. J
Cell Biol 168, 1065-1076.
Marcato, P., Dean, C.A., Giacomantonio, C.A., and Lee, P.W. (2011). Aldehyde dehydrogenase: its
role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle 10, 1378-1384.
Marchitti, S.A., Brocker, C., Stagos, D., and Vasiliou, V. (2008). Non-P450 aldehyde oxidizing
enzymes: the aldehyde dehydrogenase superfamily. Expert Opin Drug Metab Toxicol 4, 697-720.
Marshall, A.D., and Grosveld, G.C. (2012). Alveolar rhabdomyosarcoma - The molecular drivers of
PAX3/7-FOXO1-induced tumorigenesis. Skelet Muscle 2, 25.
Martins-Neves, S.R., Corver, W.E., Paiva-Oliveira, D.I., Van Den Akker, B.E., Briaire-De-Bruijn,
I.H., Bovee, J.V., Gomes, C.M., and Cleton-Jansen, A.M. (2015). Osteosarcoma Stem Cells Have
Active Wnt/beta-catenin and Overexpress SOX2 and KLF4. J Cell Physiol.
Martins-Neves, S.R., Paiva-Oliveira, D.I., Wijers-Koster, P.M., Abrunhosa, A.J., Fontes-Ribeiro, C.,
Bovee, J.V., Cleton-Jansen, A.M., and Gomes, C.M. (2016). Chemotherapy induces stemness in
osteosarcoma cells through activation of Wnt/beta-catenin signaling. Cancer Lett 370, 286-295.
Matsuda, Y., Naito, Z., Kawahara, K., Nakazawa, N., Korc, M., and Ishiwata, T. (2011). Nestin is a
novel target for suppressing pancreatic cancer cell migration, invasion and metastasis. Cancer Biol
Ther 11, 512-523.
Matsuda, Y., Yoshimura, H., Ueda, J., Naito, Z., Korc, M., and Ishiwata, T. (2014). Nestin delineates
pancreatic cancer stem cells in metastatic foci of NOD/Shi-scid IL2Rgamma(null) (NOG) mice.
Am J Pathol 184, 674-685.
Mayol, J.F., Loeuillet, C., Herodin, F., and Wion, D. (2009). Characterisation of normal and cancer
stem cells: one experimental paradigm for two kinds of stem cells. Bioessays 31, 993-1001.
Mcdermott, S.P., Eppert, K., Lechman, E.R., Doedens, M., and Dick, J.E. (2010). Comparison of
human cord blood engraftment between immunocompromised mouse strains. Blood 116, 193-200.
Meacham, C.E., and Morrison, S.J. (2013). Tumour heterogeneity and cancer cell plasticity. Nature
501, 328-337.
Meyer, M.J., Fleming, J.M., Ali, M.A., Pesesky, M.W., Ginsburg, E., and Vonderhaar, B.K. (2009).
Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell
lines. Breast Cancer Res 11, R82.
References ◄ 50
Misra, S., Heldin, P., Hascall, V.C., Karamanos, N.K., Skandalis, S.S., Markwald, R.R., and Ghatak,
S. (2011). Hyaluronan-CD44 interactions as potential targets for cancer therapy. Febs j 278, 1429-
1443.
Monument, M.J., Bernthal, N.M., and Randall, R.L. (2013). Salient features of mesenchymal stem
cells-implications for Ewing sarcoma modeling. Front Oncol 3, 24.
Mu, X., Isaac, C., Greco, N., Huard, J., and Weiss, K. (2013). Notch Signaling is Associated with
ALDH Activity and an Aggressive Metastatic Phenotype in Murine Osteosarcoma Cells. Front
Oncol 3, 143.
Nakahata, K., Uehara, S., Nishikawa, S., Kawatsu, M., Zenitani, M., Oue, T., and Okuyama, H.
(2015). Aldehyde Dehydrogenase 1 (ALDH1) Is a Potential Marker for Cancer Stem Cells in
Embryonal Rhabdomyosarcoma. PLoS One 10.
Nakatsugawa, M., Takahashi, A., Hirohashi, Y., Torigoe, T., Inoda, S., Murase, M., Asanuma, H.,
Tamura, Y., Morita, R., Michifuri, Y., Kondo, T., Hasegawa, T., Takahashi, H., and Sato, N.
(2011). SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments
the tumorigenicity. Lab Invest 91, 1796-1804.
Neradil, J., and Veselska, R. (2015). Nestin as a marker of cancer stem cells. Cancer Sci 106, 803-811.
O'connor, M.L., Xiang, D., Shigdar, S., Macdonald, J., Li, Y., Wang, T., Pu, C., Wang, Z., Qiao, L.,
and Duan, W. (2014). Cancer stem cells: A contentious hypothesis now moving forward. Cancer
Lett 344, 180-187.
Olempska, M., Eisenach, P.A., Ammerpohl, O., Ungefroren, H., Fandrich, F., and Kalthoff, H. (2007).
Detection of tumor stem cell markers in pancreatic carcinoma cell lines. Hepatobiliary Pancreat
Dis Int 6, 92-97.
Owen, L.A., Kowalewski, A.A., and Lessnick, S.L. (2008). EWS/FLI mediates transcriptional
repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma. PLoS One 3, e1965.
Panza, A., Votino, C., Gentile, A., Valvano, M.R., Colangelo, T., Pancione, M., Micale, L., Merla, G.,
Andriulli, A., Sabatino, L., Vinciguerra, M., Prattichizzo, C., Mazzoccoli, G., Colantuoni, V., and
Piepoli, A. (2014). Peroxisome proliferator-activated receptor gamma-mediated induction of
microRNA-145 opposes tumor phenotype in colorectal cancer. Biochim Biophys Acta 1843, 1225-
1236.
Pardal, R., Clarke, M.F., and Morrison, S.J. (2003). Applying the principles of stem-cell biology to
cancer. Nat Rev Cancer 3, 895-902.
Park, E.K., Lee, J.C., Park, J.W., Bang, S.Y., Yi, S.A., Kim, B.K., Park, J.H., Kwon, S.H., You, J.S.,
Nam, S.W., Cho, E.J., and Han, J.W. (2015). Transcriptional repression of cancer stem cell marker
CD133 by tumor suppressor p53. Cell Death Dis 6, e1964.
Patriarca, C., Macchi, R.M., Marschner, A.K., and Mellstedt, H. (2012). Epithelial cell adhesion
molecule expression (CD326) in cancer: a short review. Cancer Treat Rev 38, 68-75.
Penfornis, P., Cai, D.Z., Harris, M.R., Walker, R., Licini, D., Fernandes, J.D., Orr, G., Koganti, T.,
Hicks, C., Induru, S., Meyer, M.S., Khokha, R., Barr, J., and Pochampally, R.R. (2014). High
CD49f expression is associated with osteosarcoma tumor progression: a study using patient-
derived primary cell cultures. Cancer Med 3, 796-811.
Perkins, S.M., Shinohara, E.T., Dewees, T., and Frangoul, H. (2014). Outcome for children with
metastatic solid tumors over the last four decades. PLoS One 9, e100396.
Petersen, T.W., Ibrahim, S.F., Diercks, A.H., and Van Den Engh, G. (2004). Chromatic shifts in the
fluorescence emitted by murine thymocytes stained with Hoechst 33342. Cytometry A 60, 173-
181.
Petriz, J. (2013). Flow cytometry of the side population (SP). Curr Protoc Cytom Chapter 9, Unit9.23.
References ◄ 51
Polytarchou, C., Iliopoulos, D., and Struhl, K. (2012). An integrated transcriptional regulatory circuit
that reinforces the breast cancer stem cell state. Proc Natl Acad Sci U S A 109, 14470-14475.
Ponti, D., Costa, A., Zaffaroni, N., Pratesi, G., Petrangolini, G., Coradini, D., Pilotti, S., Pierotti, M.A.,
and Daidone, M.G. (2005). Isolation and in vitro propagation of tumorigenic breast cancer cells
with stem/progenitor cell properties. Cancer Res 65, 5506-5511.
Pressey, J.G., Haas, M.C., Pressey, C.S., Kelly, V.M., Parker, J.N., Gillespie, G.Y., and Friedman,
G.K. (2013). CD133 marks a myogenically primitive subpopulation in rhabdomyosarcoma cell
lines that are relatively chemoresistant but sensitive to mutant HSV. Pediatr Blood Cancer 60, 45-
52.
Prieur, A., Tirode, F., Cohen, P., and Delattre, O. (2004). EWS/FLI-1 silencing and gene profiling of
Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-
like growth factor binding protein 3. Mol Cell Biol 24, 7275-7283.
Punyko, J.A., Mertens, A.C., Baker, K.S., Ness, K.K., Robison, L.L., and Gurney, J.G. (2005). Long-
term survival probabilities for childhood rhabdomyosarcoma. A population-based evaluation.
Cancer 103, 1475-1483.
Quintana, E., Shackleton, M., Foster, H.R., Fullen, D.R., Sabel, M.S., Johnson, T.M., and Morrison,
S.J. (2010). Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is
reversible and not hierarchically organized. Cancer Cell 18, 510-523.
Quintana, E., Shackleton, M., Sabel, M.S., Fullen, D.R., Johnson, T.M., and Morrison, S.J. (2008).
Efficient tumour formation by single human melanoma cells. Nature 456, 593-598.
Raimondi, C., Nicolazzo, C., and Gradilone, A. (2015). Circulating tumor cells isolation: the "post-
EpCAM era". Chin J Cancer Res 27, 461-470.
Rasheed, Z.A., Yang, J., Wang, Q., Kowalski, J., Freed, I., Murter, C., Hong, S.M., Koorstra, J.B.,
Rajeshkumar, N.V., He, X., Goggins, M., Iacobuzio-Donahue, C., Berman, D.M., Laheru, D.,
Jimeno, A., Hidalgo, M., Maitra, A., and Matsui, W. (2010). Prognostic significance of
tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst
102, 340-351.
Ren, Y.X., Finckenstein, F.G., Abdueva, D.A., Shahbazian, V., Chung, B., Weinberg, K.I., Triche,
T.J., Shimada, H., and Anderson, M.J. (2008). Mouse mesenchymal stem cells expressing PAX-
FKHR form alveolar rhabdomyosarcomas by cooperating with secondary mutations. Cancer Res
68, 6587-6597.
Reynolds, B.A., and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the
adult mammalian central nervous system. Science 255, 1707-1710.
Riggi, N., Suva, M.L., De Vito, C., Provero, P., Stehle, J.C., Baumer, K., Cironi, L., Janiszewska, M.,
Petricevic, T., Suva, D., Tercier, S., Joseph, J.M., Guillou, L., and Stamenkovic, I. (2010). EWS-
FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell
reprogramming toward Ewing sarcoma cancer stem cells. Genes Dev 24, 916-932.
Rubin, B.P., Nishijo, K., Chen, H.I., Yi, X., Schuetze, D.P., Pal, R., Prajapati, S.I., Abraham, J.,
Arenkiel, B.R., Chen, Q.R., Davis, S., Mccleish, A.T., Capecchi, M.R., Michalek, J.E., Zarzabal,
L.A., Khan, J., Yu, Z., Parham, D.M., Barr, F.G., Meltzer, P.S., Chen, Y., and Keller, C. (2011).
Evidence for an unanticipated relationship between undifferentiated pleomorphic sarcoma and
embryonal rhabdomyosarcoma. Cancer Cell 19, 177-191.
Ryan, D.P., Hong, T.S., and Bardeesy, N. (2014). Pancreatic adenocarcinoma. N Engl J Med 371,
1039-1049.
Rybak, A.P., and Tang, D. (2013). SOX2 plays a critical role in EGFR-mediated self-renewal of
human prostate cancer stem-like cells. Cell Signal 25, 2734-2742.
Rycaj, K., and Tang, D.G. (2015). Cell-of-Origin of Cancer versus Cancer Stem Cells: Assays and
Interpretations. Cancer Res 75, 4003-4011.
References ◄ 52
Saini, V., Hose, C.D., Monks, A., Nagashima, K., Han, B., Newton, D.L., Millione, A., Shah, J.,
Hollingshead, M.G., Hite, K.M., Burkett, M.W., Delosh, R.M., Silvers, T.E., Scudiero, D.A., and
Shoemaker, R.H. (2012). Identification of CBX3 and ABCA5 as putative biomarkers for tumor
stem cells in osteosarcoma. PLoS One 7, e41401.
Santini, R., Pietrobono, S., Pandolfi, S., Montagnani, V., D'amico, M., Penachioni, J.Y., Vinci, M.C.,
Borgognoni, L., and Stecca, B. (2014). SOX2 regulates self-renewal and tumorigenicity of human
melanoma-initiating cells. Oncogene 33, 4697-4708.
Sarkar, A., and Hochedlinger, K. (2013). The sox family of transcription factors: versatile regulators
of stem and progenitor cell fate. Cell Stem Cell 12, 15-30.
Scannell, C.A., Pedersen, E.A., Mosher, J.T., Krook, M.A., Nicholls, L.A., Wilky, B.A., Loeb, D.M.,
and Lawlor, E.R. (2013). LGR5 is Expressed by Ewing Sarcoma and Potentiates Wnt/beta-Catenin
Signaling. Front Oncol 3, 81.
Scharenberg, C.W., Harkey, M.A., and Torok-Storb, B. (2002). The ABCG2 transporter is an efficient
Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic
progenitors. Blood 99, 507-512.
Schatton, T., Murphy, G.F., Frank, N.Y., Yamaura, K., Waaga-Gasser, A.M., Gasser, M., Zhan, Q.,
Jordan, S., Duncan, L.M., Weishaupt, C., Fuhlbrigge, R.C., Kupper, T.S., Sayegh, M.H., and
Frank, M.H. (2008). Identification of cells initiating human melanomas. Nature 451, 345-349.
Schepers, A.G., Snippert, H.J., Stange, D.E., Van Den Born, M., Van Es, J.H., Van De Wetering, M.,
and Clevers, H. (2012). Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal
adenomas. Science 337, 730-735.
Schneck, H., Gierke, B., Uppenkamp, F., Behrens, B., Niederacher, D., Stoecklein, N.H., Templin,
M.F., Pawlak, M., Fehm, T., and Neubauer, H. (2015). EpCAM-Independent Enrichment of
Circulating Tumor Cells in Metastatic Breast Cancer. PLoS One 10, e0144535.
Schnell, U., Cirulli, V., and Giepmans, B.N. (2013). EpCAM: structure and function in health and
disease. Biochim Biophys Acta 1828, 1989-2001.
Schwitalla, S., Fingerle, A.A., Cammareri, P., Nebelsiek, T., Goktuna, S.I., Ziegler, P.K., Canli, O.,
Heijmans, J., Huels, D.J., Moreaux, G., Rupec, R.A., Gerhard, M., Schmid, R., Barker, N.,
Clevers, H., Lang, R., Neumann, J., Kirchner, T., Taketo, M.M., Van Den Brink, G.R., Sansom,
O.J., Arkan, M.C., and Greten, F.R. (2013). Intestinal tumorigenesis initiated by dedifferentiation
and acquisition of stem-cell-like properties. Cell 152, 25-38.
Shaw, F.L., Harrison, H., Spence, K., Ablett, M.P., Simoes, B.M., Farnie, G., and Clarke, R.B. (2012).
A detailed mammosphere assay protocol for the quantification of breast stem cell activity. J
Mammary Gland Biol Neoplasia 17, 111-117.
Shen, B., Zheng, M.Q., Lu, J.W., Jiang, Q., Wang, T.H., and Huang, X.E. (2013). CXCL12-CXCR4
promotes proliferation and invasion of pancreatic cancer cells. Asian Pac J Cancer Prev 14, 5403-
5408.
Sheridan, C., Kishimoto, H., Fuchs, R.K., Mehrotra, S., Bhat-Nakshatri, P., Turner, C.H., Goulet, R.,
Jr., Badve, S., and Nakshatri, H. (2006). CD44+/CD24- breast cancer cells exhibit enhanced
invasive properties: an early step necessary for metastasis. Breast Cancer Res 8, R59.
Shimozato, O., Waraya, M., Nakashima, K., Souda, H., Takiguchi, N., Yamamoto, H., Takenobu, H.,
Uehara, H., Ikeda, E., Matsushita, S., Kubo, N., Nakagawara, A., Ozaki, T., and Kamijo, T.
(2015). Receptor-type protein tyrosine phosphatase kappa directly dephosphorylates CD133 and
regulates downstream AKT activation. Oncogene 34, 1949-1960.
Siegel, R.L., Miller, K.D., and Jemal, A. (2015). Cancer statistics, 2015. CA Cancer J Clin 65, 5-29.
Siegle, J.M., Basin, A., Sastre-Perona, A., Yonekubo, Y., Brown, J., Sennett, R., Rendl, M., Tsirigos,
A., Carucci, J.A., and Schober, M. (2014). SOX2 is a cancer-specific regulator of tumour initiating
potential in cutaneous squamous cell carcinoma. Nat Commun 5, 4511.
References ◄ 53
Singh, S., Arcaroli, J.J., Orlicky, D.J., Chen, Y., Messersmith, W.A., Bagby, S., Purkey, A.,
Quackenbush, K.S., Thompson, D.C., and Vasiliou, V. (2016). Aldehyde Dehydrogenase 1B1 as a
Modulator of Pancreatic Adenocarcinoma. Pancreas 45, 117-122.
Singh, S.K., Chen, N.M., Hessmann, E., Siveke, J., Lahmann, M., Singh, G., Voelker, N., Vogt, S.,
Esposito, I., Schmidt, A., Brendel, C., Stiewe, T., Gaedcke, J., Mernberger, M., Crawford, H.C.,
Bamlet, W.R., Zhang, J.S., Li, X.K., Smyrk, T.C., Billadeau, D.D., Hebrok, M., Neesse, A.,
Koenig, A., and Ellenrieder, V. (2015). Antithetical NFATc1-Sox2 and p53-miR200 signaling
networks govern pancreatic cancer cell plasticity. Embo j 34, 517-530.
Singh, S.K., Clarke, I.D., Terasaki, M., Bonn, V.E., Hawkins, C., Squire, J., and Dirks, P.B. (2003).
Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821-5828.
Single, A., Beetham, H., Telford, B.J., Guilford, P., and Chen, A. (2015). A Comparison of Real-Time
and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation. J Biomol
Screen 20, 1286-1293.
Skoda, J., Neradil, J., and Veselska, R. (2014). [Functional assays for detection of cancer stem cells].
Klin Onkol 27 Suppl 1, S42-47.
Soleimani, V.D., and Rudnicki, M.A. (2011). New insights into the origin and the genetic basis of
rhabdomyosarcomas. Cancer Cell 19, 157-159.
Song, W., Li, Q., Wang, L., and Huang, W. (2015). FoxO1-negative cells are cancer stem-like cells in
pancreatic ductal adenocarcinoma. Sci Rep 5, 10081.
Storms, R.W., Trujillo, A.P., Springer, J.B., Shah, L., Colvin, O.M., Ludeman, S.M., and Smith, C.
(1999). Isolation of primitive human hematopoietic progenitors on the basis of aldehyde
dehydrogenase activity. Proc Natl Acad Sci U S A 96, 9118-9123.
Strnad, J., Hamilton, A.E., Beavers, L.S., Gamboa, G.C., Apelgren, L.D., Taber, L.D., Sportsman,
J.R., Bumol, T.F., Sharp, J.D., and Gadski, R.A. (1989). Molecular cloning and characterization of
a human adenocarcinoma/epithelial cell surface antigen complementary DNA. Cancer Res 49,
314-317.
Suva, M.L., Riggi, N., Stehle, J.C., Baumer, K., Tercier, S., Joseph, J.M., Suva, D., Clement, V.,
Provero, P., Cironi, L., Osterheld, M.C., Guillou, L., and Stamenkovic, I. (2009). Identification of
cancer stem cells in Ewing's sarcoma. Cancer Res 69, 1776-1781.
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S.
(2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell
131, 861-872.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126, 663-676.
Takenobu, H., Shimozato, O., Nakamura, T., Ochiai, H., Yamaguchi, Y., Ohira, M., Nakagawara, A.,
and Kamijo, T. (2011). CD133 suppresses neuroblastoma cell differentiation via signal pathway
modification. Oncogene 30, 97-105.
Thomas, R.M., Kim, J., Revelo-Penafiel, M.P., Angel, R., Dawson, D.W., and Lowy, A.M. (2008).
The chemokine receptor CXCR4 is expressed in pancreatic intraepithelial neoplasia. Gut 57, 1555-
1560.
Tirino, V., Desiderio, V., D'aquino, R., De Francesco, F., Pirozzi, G., Graziano, A., Galderisi, U.,
Cavaliere, C., De Rosa, A., Papaccio, G., and Giordano, A. (2008). Detection and characterization
of CD133+ cancer stem cells in human solid tumours. PLoS One 3, e3469.
Tirino, V., Desiderio, V., Paino, F., De Rosa, A., Papaccio, F., Fazioli, F., Pirozzi, G., and Papaccio,
G. (2011). Human primary bone sarcomas contain CD133+ cancer stem cells displaying high
tumorigenicity in vivo. Faseb j 25, 2022-2030.
Tirode, F., Laud-Duval, K., Prieur, A., Delorme, B., Charbord, P., and Delattre, O. (2007).
Mesenchymal stem cell features of Ewing tumors. Cancer Cell 11, 421-429.
References ◄ 54
Tsuchida, R., Das, B., Yeger, H., Koren, G., Shibuya, M., Thorner, P.S., Baruchel, S., and Malkin, D.
(2008). Cisplatin treatment increases survival and expansion of a highly tumorigenic side-
population fraction by upregulating VEGF/Flt1 autocrine signaling. Oncogene 27, 3923-3934.
Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W.E., Rendl, M., and Fuchs, E. (2004).
Defining the epithelial stem cell niche in skin. Science 303, 359-363.
Vaittinen, S., Lukka, R., Sahlgren, C., Hurme, T., Rantanen, J., Lendahl, U., Eriksson, J.E., and
Kalimo, H. (2001). The expression of intermediate filament protein nestin as related to vimentin
and desmin in regenerating skeletal muscle. J Neuropathol Exp Neurol 60, 588-597.
Vanner, R.J., Remke, M., Gallo, M., Selvadurai, H.J., Coutinho, F., Lee, L., Kushida, M., Head, R.,
Morrissy, S., Zhu, X., Aviv, T., Voisin, V., Clarke, I.D., Li, Y., Mungall, A.J., Moore, R.A., Ma,
Y., Jones, S.J., Marra, M.A., Malkin, D., Northcott, P.A., Kool, M., Pfister, S.M., Bader, G.,
Hochedlinger, K., Korshunov, A., Taylor, M.D., and Dirks, P.B. (2014). Quiescent sox2(+) cells
drive hierarchical growth and relapse in sonic hedgehog subgroup medulloblastoma. Cancer Cell
26, 33-47.
Vermeulen, L., De Sousa, E.M.F., Van Der Heijden, M., Cameron, K., De Jong, J.H., Borovski, T.,
Tuynman, J.B., Todaro, M., Merz, C., Rodermond, H., Sprick, M.R., Kemper, K., Richel, D.J.,
Stassi, G., and Medema, J.P. (2010). Wnt activity defines colon cancer stem cells and is regulated
by the microenvironment. Nat Cell Biol 12, 468-476.
Veselska, R., Hermanova, M., Loja, T., Chlapek, P., Zambo, I., Vesely, K., Zitterbart, K., and Sterba,
J. (2008). Nestin expression in osteosarcomas and derivation of nestin/CD133 positive
osteosarcoma cell lines. BMC Cancer 8, 300.
Veselska, R., Kuglik, P., Cejpek, P., Svachova, H., Neradil, J., Loja, T., and Relichova, J. (2006).
Nestin expression in the cell lines derived from glioblastoma multiforme. BMC Cancer 6, 32.
Wahl, J., Bogatyreva, L., Boukamp, P., Rojewski, M., Van Valen, F., Fiedler, J., Hipp, N., Debatin,
K.M., and Beltinger, C. (2010). Ewing's sarcoma cells with CD57-associated increase of
tumorigenicity and with neural crest-like differentiation capacity. Int J Cancer 127, 1295-1307.
Walter, D., Satheesha, S., Albrecht, P., Bornhauser, B.C., D'alessandro, V., Oesch, S.M., Rehrauer, H.,
Leuschner, I., Koscielniak, E., Gengler, C., Moch, H., Bernasconi, M., Niggli, F.K., and Schafer,
B.W. (2011). CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched
in rhabdospheres. PLoS One 6, e19506.
Wang, L., Park, P., Zhang, H., La Marca, F., and Lin, C.Y. (2011). Prospective identification of
tumorigenic osteosarcoma cancer stem cells in OS99-1 cells based on high aldehyde
dehydrogenase activity. Int J Cancer 128, 294-303.
Wang, X.Q., Duan, X.M., Liu, L.H., Fang, Y.Q., and Tan, Y. (2005). Carboxyfluorescein diacetate
succinimidyl ester fluorescent dye for cell labeling. Acta Biochim Biophys Sin (Shanghai) 37, 379-
385.
Wei, Y., Jiang, Y., Zou, F., Liu, Y., Wang, S., Xu, N., Xu, W., Cui, C., Xing, Y., Cao, B., Liu, C., Wu,
G., Ao, H., Zhang, X., and Jiang, J. (2013). Activation of PI3K/Akt pathway by CD133-p85
interaction promotes tumorigenic capacity of glioma stem cells. Proc Natl Acad Sci U S A 110,
6829-6834.
Weina, K., and Utikal, J. (2014). SOX2 and cancer: current research and its implications in the clinic.
Clin Transl Med 3, 19.
Weiss, A., Gill, J., Goldberg, J., Lagmay, J., Spraker-Perlman, H., Venkatramani, R., and Reed, D.
(2014). Advances in therapy for pediatric sarcomas. Curr Oncol Rep 16, 395.
Wicha, M.S., Liu, S., and Dontu, G. (2006). Cancer stem cells: an old idea--a paradigm shift. Cancer
Res 66, 1883-1890; discussion 1895-1886.
References ◄ 55
Wiese, C., Rolletschek, A., Kania, G., Blyszczuk, P., Tarasov, K.V., Tarasova, Y., Wersto, R.P.,
Boheler, K.R., and Wobus, A.M. (2004). Nestin expression--a property of multi-lineage progenitor
cells? Cell Mol Life Sci 61, 2510-2522.
Wilkens, S. (2015). Structure and mechanism of ABC transporters. F1000Prime Rep 7, 14.
Willan, P.M., and Farnie, G. (2011). Application of stem cell assays for the characterization of cancer
stem cells. In A.L. Allan (Ed.), Cancer stem cells in solid tumors (pp. 259−282). New York, NY:
Springer New York.
Wu, Y., Liu, S., Xin, H., Jiang, J., Younglai, E., Sun, S., and Wang, H. (2011). Up-regulation of
microRNA-145 promotes differentiation by repressing OCT4 in human endometrial
adenocarcinoma cells. Cancer 117, 3989-3998.
Wylie, P.G., and Bowen, W.P. (2007). Determination of cell colony formation in a high-content
screening assay. Clin Lab Med 27, 193-199.
Xu, F., Wang, H., Zhang, X., Liu, T., and Liu, Z. (2013). Cell proliferation and invasion ability of
human choriocarcinoma cells lessened due to inhibition of Sox2 expression by microRNA-145.
Exp Ther Med 5, 77-84.
Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A., and Kosik, K.S. (2009). MicroRNA-145
regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells.
Cell 137, 647-658.
Yan, W., Chen, Y., Yao, Y., Zhang, H., and Wang, T. (2013). Increased invasion and tumorigenicity
capacity of CD44+/CD24- breast cancer MCF7 cells in vitro and in nude mice. Cancer Cell Int 13,
62.
Yan, Y., Zuo, X., and Wei, D. (2015). Concise Review: Emerging Role of CD44 in Cancer Stem
Cells: A Promising Biomarker and Therapeutic Target. Stem Cells Transl Med 4, 1033-1043.
Yang, C., Hou, C., Zhang, H., Wang, D., Ma, Y., Zhang, Y., Xu, X., Bi, Z., and Geng, S. (2014a).
miR-126 functions as a tumor suppressor in osteosarcoma by targeting Sox2. Int J Mol Sci 15,
423-437.
Yang, L., Takimoto, T., and Fujimoto, J. (2014b). Prognostic model for predicting overall survival in
children and adolescents with rhabdomyosarcoma. BMC Cancer 14, 654.
Yang, M., Yan, M., Zhang, R., Li, J., and Luo, Z. (2011). Side population cells isolated from human
osteosarcoma are enriched with tumor-initiating cells. Cancer Sci 102, 1774-1781.
Yang, X.H., Wu, Q.L., Yu, X.B., Xu, C.X., Ma, B.F., Zhang, X.M., Li, S.N., Lahn, B.T., and Xiang,
A.P. (2008). Nestin expression in different tumours and its relevance to malignant grade. J Clin
Pathol 61, 467-473.
Ye, J., Wu, D., Shen, J., Wu, P., Ni, C., Chen, J., Zhao, J., Zhang, T., Wang, X., and Huang, J. (2012).
Enrichment of colorectal cancer stem cells through epithelial-mesenchymal transition via CDH1
knockdown. Mol Med Rep 6, 507-512.
Yeo, J.C., and Ng, H.H. (2013). The transcriptional regulation of pluripotency. Cell Res 23, 20-32.
Yin, R., Zhang, S., Wu, Y., Fan, X., Jiang, F., Zhang, Z., Feng, D., Guo, X., and Xu, L. (2011).
microRNA-145 suppresses lung adenocarcinoma-initiating cell proliferation by targeting OCT4.
Oncol Rep 25, 1747-1754.
Ying, M., Liu, G., Shimada, H., Ding, W., May, W.A., He, Q., Adams, G.B., and Wu, L. (2013).
Human osteosarcoma CD49f(-)CD133(+) cells: impaired in osteogenic fate while gain of
tumorigenicity. Oncogene 32, 4252-4263.
Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J.,
Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, Ii, and Thomson, J.A. (2007). Induced
pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
References ◄ 56
Zambo, I., Hermanova, M., Adamkova Krakorova, D., Mudry, P., Zitterbart, K., Kyr, M., Vesely, K.,
Sterba, J., and Veselska, R. (2012). Nestin expression in high-grade osteosarcomas and its clinical
significance. Oncol Rep 27, 1592-1598.
Zellmer, V.R., and Zhang, S. (2014). Evolving concepts of tumor heterogeneity. Cell Biosci 4, 69.
Zöller, M. (2011). CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?
Nat Rev Cancer 11, 254-267.
Publications related to the thesis ◄ 57
7 Publications related to the thesis
This dissertation consists of seven peer-reviewed papers – five original articles and two
review articles. A list of attached manuscripts including author’s contribution is provided
below and is followed by full texts of these manuscripts.
Manuscript I (Original article) For full text see page 60
Sana, J., Zambo, I., Skoda, J., Neradil, J., Chlapek, P., Hermanova, M., Mudry, P., Vasikova,
A., Zitterbart, K., Hampl, A., Sterba, J. and Veselska, R. (2011). CD133 expression and
identification of CD133/nestin positive cells in rhabdomyosarcomas and rhabdomyosarcoma
cell lines. Analytical Cellular Pathology, 34(6), 303–18.
− JCR impact factor (2011): 0.917
Author’s contribution (10%): performed FISH analysis, assisted with in vivo experiments,
and participated in manuscript preparation.
Manuscript II (Review) For full text see page 77
Veselska, R., Skoda, J. and Neradil, J. (2012). Detection of cancer stem cell markers in
sarcomas. Klinicka Onkologie, 25(Suppl 2), 2S16–2S20.
Author’s contribution (20%): participated in manuscript preparation.
Manuscript III (Review) For full text see page 83
Skoda, J., Neradil, J. and Veselska, R. (2014). Functional assays for detection of cancer stem
cells. Klinicka Onkologie, 27(Suppl 1), S42–7.
Author’s contribution (80%): drafted the manuscript.
Publications related to the thesis ◄ 58
Manuscript IV (Original article) For full text see page 90
Nunukova, A., Neradil, J., Skoda, J., Jaros, J., Hampl, A., Sterba, J. and Veselska, R. (2015).
Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma
cell lines. International Journal of Molecular Medicine, 36(1), 65–72.
− JCR impact factor (2014): 2.088
Author’s contribution (20%): performed software cross-section analysis of confocal
microscopy data, participated in data analysis and manuscript preparation.
Manuscript V (Original article) For full text see page 99
Skoda, J., Nunukova, A., Loja, T., Zambo, I., Neradil, J., Mudry, P., Zitterbart, K.,
Hermanova, M., Hampl, A., Sterba, J. and Veselska, R. (2016). Cancer stem cell markers in
pediatric sarcomas: Sox2 is associated with tumorigenicity in immunodeficient mice. Tumor
Biology, doi: 10.1007/s13277-016-4837-0
− JCR impact factor (2014): 3.611
Author’s contribution (30%): participated in the design of the study, performed all
experiments with osteosarcoma cell lines, conducted real-time PCR experiments, performed
data collection and analysis, and drafted the manuscript.
Manuscript VI
Zambo, I., Hermanova, M., Zapletalova, D., Skoda, J., Mudry, P., Kyr, M., Zitterbart, K.,
Sterba, J. and Veselska, R. Cancer stem cell markers in pediatric sarcomas: expression of
nestin, CD133 and ABCG2 in relation to the clinical outcome. Cancer Biomarkers, under
review.
(Original article) For full text see page 114
Author’s contribution (10%): participated in data analysis, prepared all figures and critically
revised the manuscript.
Publications related to the thesis ◄ 59
Manuscript VII
Skoda, J., Hermanova, M., Loja, T., Nemec, P., Neradil, J., Karasek, P. and Veselska, R.
Co-expression of cancer stem cell markers corresponds to a pro-tumorigenic expression
profile in pancreatic adenocarcinoma. PLoS One, under review (revision submitted).
(Original article) For full text see page 147
Author’s contribution (50%): designed the study, performed all experiments with the cell
lines, performed data collection and analysis, and drafted the manuscript.
