Stem cells as the root of pancreatic ductal adenocarcinoma

    Stem Cells as the Root of Pancreatic Ductal Adenocarcinoma Anamaria Balic, Jorge Dorado, Mercedes Alonso-G´omez, Christopher Heeschen PII: DOI: Reference:

S0014-4827(11)00449-6 doi: 10.1016/j.yexcr.2011.11.007 YEXCR 8880

To appear in:

Experimental Cell Research

Received date: Revised date: Accepted date:

23 September 2011 5 November 2011 8 November 2011

Please cite this article as: Anamaria Balic, Jorge Dorado, Mercedes Alonso-G´ omez, Christopher Heeschen, Stem Cells as the Root of Pancreatic Ductal Adenocarcinoma, Experimental Cell Research (2011), doi: 10.1016/j.yexcr.2011.11.007

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ACCEPTED MANUSCRIPT Stem Cells as the Root of Pancreatic Ductal Adenocarcinoma

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Anamaria Balic, Jorge Dorado, Mercedes Alonso-Gómez, Christopher Heeschen*

Centre (CNIO), Madrid, Spain

Correspondence should be addressed to:

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*

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Clinical Research Programme

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Dr. Christopher Heeschen

Stem Cells & Cancer Group

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

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Spanish National Cancer Research Centre (CNIO) C/ Melchor Fernandez Almagro 3, 28029 Madrid, Spain Email: [email protected]

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ATP binding cassette

ALDH1

Aldehyde dehydrogenase 1a1

Arx

Aristalless-related homeobox

EMT

Epithelial-to-mesenchymal transition

CAC

Centroacinar cells

CD

Cluster of differentiation

CDKN2A

Cyclin-dependent kinase inhibitor 2A

CSC

Cancer stem cells

CTC

Circulating tumor cells

CXCR4

CXC chemokine receptor 4

EpCAM

Epithelial cell adhesion molecule

Hes1

Hairy enhancer of split 1

IL-4

Interleukin 4

IPMN

Intraductal mucinous neoplasm

Klf4

Krueppel-like factor 4

MDR1

Multi-drug resistance 1

mTOR

mammalian target of rapamycin

NF-B PanIN Pax4 PDAC Pdx1

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Ngn3

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ABC

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LIST OF ABBREVIATIONS

Neurogenin 3

Nuclear factor kappa light chain enhancer of activated B cells Pancreatic intraepithelial neoplasia Paired box gene 4 Pancreatic ductal adenocarcinoma Pancreatic and duodenal homeobox 1

PTEN

Phosphatase and tensin homolog

Ptf1

Pancreas-specific transcription factor 1

RBP-J

Recombination signal-binding protein 1 for J-kappa

SDF-1

Stromal-derived factor 1

Shh

Sonic hedgehog

Sox2

Sex determining region Y-box 2

SP

Side population

TP53

Tumor protein 53 gene

ZEB1

Zinc finger E-box-binding homeobox 1

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ACCEPTED MANUSCRIPT ABSTRACT Emerging evidence suggests that stem cells play a crucial role not only in the generation

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and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct

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subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed

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cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that

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pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC

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rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver

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as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative

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enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.

Keywords  Pancreatic cancer  Cancer stem cells  Tumor-initiating cells  Inflammation  Targeted therapy

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ACCEPTED MANUSCRIPT 1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is the deadliest solid cancer and currently the

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fourth most frequent cause of cancer-related deaths. PDAC is characterized by late diagnosis due to lack of early symptoms, extensive metastasis, and high resistance to chemotherapy and

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radiation. Despite expanding research activities in the field of pancreatic tumor and vascular

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biology, there has been little substantial therapeutic progress regarding clinical endpoints over the past decades. Indeed, in contrast to the general trend of decreasing incidences for most cancers,

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the incidence and death rates for PDAC continue to increase ("Cancer Facts & Figures 2011",

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American Cancer Society, www.cancer.org). Since 1998, incidence rates of PDAC have been increasing by 0.8% per year in men and by 1.0% per year in women. This year, an estimated 44,030 people in the United States will be diagnosed with PDAC and 37,660 will die from this

reported.

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disease. Worldwide, around 200,000 new cases of PDAC per annum having similar outcomes are

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In the 1990s, the introduction of the anti-metabolite, gemcitabine, has improved clinical response in terms of pain reduction and weight loss (Figure 1) [1]. However, with a 5-year survival rate of 1-4% and a median survival period of 4-6 months, the prognosis of patients with PDAC remains extremely poor [2-7]. For patients with metastatic disease but good performance status, a new combination therapy has recently proven quite effective. Compared with gemcitabine, combination therapy FOLFIRINOX (oxaliplatin, irinotecan, fluorouracil, and leucovorin) was associated with a survival advantage (11.1 months in the FOLFIRINOX group as opposed to 6.8 months in the gemcitabine group) but had increased toxicity [8] (Figure 1). As targeted therapy, erlotinib has been tested in PDAC. However, its addition to gemcitabine as the current standard therapy has not resulted in a major improvement in survival [9]. Interestingly, more recent cell line-based data suggest the existence of different subtypes of PDAC, with erlotinib being more effective in classical subtype cell lines, although this still needs to be validated in clinical samples [10]. 4

ACCEPTED MANUSCRIPT In conclusion, currently used therapies only slightly improve survival (mostly in small subsets of patients) and rarely result in long-term progression-free survival. Therefore, to

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substantially increase therapeutic response, the comprehensive elucidation of the mechanisms governing pancreas biology during development and tissue maintenance during health and

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disease, e.g. chronic inflammation, is mandatory. This will eventually allow us to understand the

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deregulation of critical pathways in the cell-of-origin of PDAC, which could lead to new preventive strategies. Additionally, comprehensive investigations into the hierarchical nature of

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PDAC, including therapy-resistant CSC and their protective niche as the putative root of the

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disease, should result in higher response rates or even cure from this deadly disease.

2. Pancreatic Stem Cells in Tissue Maintenance and Disease

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The pancreas is a gland of both exocrine and endocrine nature and is formed by a complex branching network of ducts that end in globular structures (acini) where the production

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and secretion of digestive fluids occurs. The exocrine compartment of the pancreas produces precursors of multiple digestive enzymes, which are released via the main pancreatic duct into the duodenum. The endocrine compartment is formed by islets of cells, responsible for the secretion of hormones implicated in the regulation of carbohydrate metabolism. Islets group α- and β-cells produce glucagon and insulin respectively in response to varying blood glucose levels. Extensive efforts have been made to identify pancreatic stem cells, which could be involved in the maintenance and/or regeneration of the pancreas in response to injury (e.g. chronic pancreatitis) and loss of -cell mass, respectively. The characterization of such an elusive stem cell population could lead to the development of therapeutic strategies for the replacement of -cells in patients with type I diabetes. Despite lacking a clear definition of postnatal pancreas stem cells for the different cell types within the pancreas, comprehensive knowledge has been accumulated regarding the characteristics of pancreatic stem cells during embryonic

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ACCEPTED MANUSCRIPT development. Thus, all pancreatic cells, both from exocrine and endocrine lineages, are believed to originate from an initial cell progenitor expressing the transcription factor pancreatic and

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duodenal homeobox 1 (Pdx1) (Figure 2). The expression of this factor together with silencing of signaling mediated by Sonic hedgehog (Shh) in the surrounding mesenchymal tissue initiates

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embryonic pancreas development. The implication of Shh in this process is supported by several

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observations, including a lack of Pdx1 expression in embryos with constitutively active hedgehog signaling [11]. In addition, Pdx1 null mutant mice form pancreas during embryonic development.

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Although aberrant, their pancreas contains insulin and glucagon expressing cells. Thus, Pdx1 can

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be considered a critical transcription factor in pancreatic commitment, although there might be more actors implicated, since absence of this factor does not result in complete impairment of pancreas formation.

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Another transcription factor was recently shown to play important role in pancreas development in humans. Malfunctioning mutations of pancreas-specific transcription factor 1

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(Ptf1) in humans result in impaired pancreas development [12], while studies in mice showed that forced expression of Ptf1 induces pancreas development at ectopic locations [13]. Ptf1 is activated in a subset of pancreatic stem cells expressing Pdx1, shortly after these cells acquired Pdx1 expression, but despite the apparent temporal sequence, the expression of Pdx1 and Ptf1 occurs in an independent manner [14]. Ptf1 expression has been implicated in the commitment of precursor cells towards an exocrine phenotype because Ptf1 null mutant mice show impaired pancreas development but are still capable of developing endocrine cells [14]. In addition, commitment towards an exocrine fate seems to be potentiated through signaling of the surrounding mesenchyme on Pdx1 positive cells. Mesenchymal cells would enhance Notch signaling in progenitor cells via its downstream target hairy enhancer of split 1 (Hes1) and inhibit the expression of the pro-endocrine differentiation factor Neurogenin 3 (Ngn3) [15]. Thus, null mutant mice for both Notch ligand delta-like and Notch target RBP-J transcription factor are

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ACCEPTED MANUSCRIPT enriched in cells of the endocrine lineage, and Hes1 null mice display severe hypoplasia of the pancreas as a result of lack of exocrine progenitor cells [15].

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Determination of endocrine fate is induced by expression of the transcription factor Ngn3. In fact, Ngn3-positive cells represent the origin of all the heterogeneity of pancreatic

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endocrine cells [16]. Both - and -cells can be derived from Ngn3-positive cells, although they

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are generated in a different ratio. In early pancreatic development during mouse embryogenesis, the vast majority of cells derived from Ngn3-positive cells are glucagon-secreting -cells,

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supporting a notion that Pdx1-Ngn3 forced expression primarily leads to the development of

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glucagon cells [17]. The -cells down-regulate Pdx1 expression and progress towards a nonepithelial phenotype through a process that strongly resembles Epithelial-to-Mesenchymal

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Transition (EMT). On the other hand, -cells retain Pdx1 expression and remain in low numbers

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as compared to glucagon secreting cells, until later in development when branching morphogenesis and acinar cell differentiation occur and require an amplification of the pool of -

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cells [16, 18].

Commitment towards - or -cell fate seems to depend on the mutually exclusive action of the transcription factors, Aristalless-related homeobox (Arx) and paired box gene 4 (Pax4). Expression of Arx may induce the formation of -cells, since deletion of this gene results in impaired generation of this cell type [19], whereas Pax4 appears to be responsible for -cell formation [20]. The existence of different sequential progenitor cells raises the question of whether these cells can also be reverted to a less differentiated phenotype in order to give rise to a broader number of cell types (plasticity). However, accumulating evidence suggests that -cells are differentiated cells with very limited expansion capability. In fact, most -cells seem to originate from a pool of already existing -cell precursors rather than from expansion of ancient -cells [21]. Notch is not capable of compelling mature endocrine cells to revert towards a progenitor-like state [22]. In contrast, Ngn3-positive cells demonstrate greater plasticity, since

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ACCEPTED MANUSCRIPT they can be reverted to a ductal progenitor phenotype [23]. Therefore, while the pancreas lacks a clear hierarchical organization and a final definition of a putative pancreatic stem cell is still

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missing, it has been shown that a number of cellular compartments bear the potential to regenerate the different subsets of the pancreas and are putative targets for the cell-of-origin for

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PDAC.

3. Cell-of-Origin for Pancreatic Ductal Adenocarcinoma

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The cell from which PDAC arises still remains elusive. A possible scenario for tumor

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initiation in solid organs is the malignant transformation of stem cells resident in the normal tissue. Somatic stem cells are intrinsically endowed with the capacity of self-renewal and would therefore only need to accumulate sequential mutations to undergo malignant transformation and

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give rise to a tumor. Indeed, this hypothesis has just recently been validated for intestinal cancer [24]. However, putative pancreatic stem cells, although proposed for mice several years ago [25],

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still cannot be genetically tracked due to their rather vague description as mentioned above. This has hampered the field in providing definitive proof for this hypothesis. Until this stem cell model for the development of PDAC is authenticated or otherwise by the accumulation of more evidence, other models will need to be considered as the putative mechanism. To clarify these aspects of tumor initiation and progression, a diverse set of genetically engineered mouse models has been developed, most of them based on the expression of mutated Kras as the initiating mutation (Figure 3). Almost every pancreatic premalignant lesion or adenocarcinoma in humans is associated with activating mutations in Kras oncogene, suggesting Kras activation as one of the crucial and most likely initiating genetic events leading to tumorigenic transformation. Independently of the cell-of-origin, expression of an activated mutant Kras allele (KrasG12V) in the mouse pancreas recapitulates formation of premalignant pancreatic intraepithelial neoplasia (PanIN), some of which progress towards PDAC. However, it is remarkable that, while all the pancreatic cells in this model express activated Kras, only a minor 8

ACCEPTED MANUSCRIPT subset of these cells eventually progress to neoplastic lesions after a considerable delay. This has been attributed to a process of Kras-mediated senescence, by which cells with a mutated Kras are

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maintained in a senescent state that is overcome only when p21 is suppressed by means of mutated p53, thus allowing progression of the tumor-initiating process [26-28]. When this allele

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is conditionally activated in Pdx1-positive cells by use of a Cre system, mutated Kras is expressed

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in virtually all pancreatic cells starting on embryonic day 8.5. These mice develop PDAC in a process that mirrors tumor progression in humans. Additional mutations such as loss of TP53,

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INK4A, CDKN2D, and SMAD4 are also found in a smaller percentage of PDAC and accelerate

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progression to PDAC in mouse models [26, 27].

Considering the fact that PDAC has a ductal morphology and that its gene expression pattern is similar to that of ductal cells, it is tempting to speculate that a ductal cell represents the

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target for tumorigenic transformation. Unfortunately, the current lack of ductal promoters for in vivo mouse models renders this hypothesis difficult to prove, and the evidence obtained so far is

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not yet conclusive. Specifically, expression of KrasG12V under the control of the ductal promoter cytokeratin-19 in a transgenic mouse model did not produce an apparent malignant phenotype [29]. However, despite the ductal histology of PDAC, the lesions, which can be appreciated at the earliest stages of tumorigenesis, are actually embedded in islets mainly formed by clusters of and -cells [30]. This observation raises the possibility that transdifferentiation of -cells represents a putative root for PDAC, a hypothesis which is further supported by the observation that chemical depletion of -cells impairs tumor initiation. Indeed, transdifferentiation of cells in the pancreas has already been suggested by the expression of markers of foregut differentiation in some premalignant pancreatic lesions [31]. While tracing experiments suggested that -cells may not contribute to the generation of cells with acinar or ductal phenotype during tumorigenesis [32], a new mouse model of Kras activation in -cells has provided new insights. While exclusive Kras activation in -cells on its own was not sufficient for transformation of these cells,

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ACCEPTED MANUSCRIPT concomitant induction of pancreatitis eventually lead to the development of exocrine neoplasia [30].

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During the early stages of pancreatic tumorigenesis, transdifferentiation of acinar cells towards a ductal phenotype (ductal metaplasia) is also frequently detected; implicating those

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acinar cells could also represent the cell-of-origin for PDAC [32]. Mouse studies using the acinar-

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specific promoter of the Elastase gene have revealed that conditional activation of Kras exclusively in acinar cells results in tumors of mixed acinar and ductal morphology [33].

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Moreover, Notch signaling cooperates with Kras activation during tumor initiation and

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progression [34]. Another observation pointing to acinar cells as the pancreatic cell type where tumorigenesis may initiate has been the recent discovery of a Bmi1-positive population within the acinar subset of cells, which is capable of maintaining pancreatic cell homeostasis [35]. Finally,

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centroacinar cells, which are located at the junction of ductal and acinar compartments, have emerged as another putative cell type for the origin of PDAC. The fact that Notch signaling and

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its target gene, Hes1, remain active in these cells during adulthood [35, 36], together with the observation that Notch signaling maintains an undifferentiated state during pancreas development [23], has lead to the hypothesis that these centroacinar cells are putative targets for tumor initiating events. Consistent with this hypothesis, it has been observed that different Notch signaling mediators, including Hes1, are overexpressed in PDAC. Furthermore, specific deletion of phosphatase and tensin homolog (PTEN) gene in pancreatic tissue leads to expansion of centroacinar cells and eventual progression to carcinoma, a fact that may suggest that these cells could constitute the origin of tumorigenic processes within this organ in mice [37]. However, in this model, mice develop tumors morphologically distinct from PDAC and more related to human intraductal mucinous neoplasm (IPMN), an infrequent premalignant lesion in ductal cells that can derivate to PDAC.

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ACCEPTED MANUSCRIPT 4. Role of Chronic Pancreatitis Pancreatitis is an inflammatory response of the pancreas towards autoimmune antibodies

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and the liberation of pro-enzymatic content of the exocrine pancreas following external injury or damage caused by xenobiotics such as caerulein [38]. There is growing evidence that this

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inflammatory state facilitates pancreatic tumorigenesis, indicating that physiological context can

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exert a strong influence on the susceptibility of a cell towards transforming events., Chronic pancreatitis has been consistently identified as a prominent risk factor for PDAC in humans, a

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link which has been well documented in several epidemiological studies [39-41]. Accumulating

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experimental evidence from mouse models also supports this relationship. An inflammatory stimulus was necessary for the induction of tumorigenesis through activation of Kras in both acinar and -cells of adult mice, which otherwise are refractory to this oncogenic input [30],[42].

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Interestingly, this process was not mediated by the pro-inflammatory transcription factor, NF-B, although such a mechanism has been demonstrated to be operative for other similar processes

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such as colitis-associated colon carcinogenesis [43]. Pancreatitis has not only been shown to have synergistic effects in promoting malignant transformation, but has also been implicated in mobilization of tissue progenitor cells and induction of their proliferative capacity. Specifically, partial duct ligation of the pancreas results in activation of -cell progenitors together with their expansion [44]. In a similar manner, pancreatic injury leading to pancreatitis has been demonstrated to affect the endocrine status of insulin-secreting cells, enabling them to behave as starting points for exocrine neoplasias [30]. Since, under normal conditions, pancreatic stem cells are likely to constitute only a minor population, which is difficult to detect, experimental induction of chronic pancreatitis may increase the number of these cells, thereby facilitating their detection, characterization and further investigation. Therefore, future studies should address the possibility of pancreatic stem cells being expanded or generated de novo by dedifferentiation of cells that were committed to

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ACCEPTED MANUSCRIPT different lineages as a response to pancreatitis. Such cells are rendered more susceptible to transforming events and their subsequent conversion into CSC as the proposed origin of PDAC as

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discussed in the following section.

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5. Pancreatic Cancer Stem Cells

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Irrespective of the still-ongoing debate about the cell-of-origin in PDAC, increasing evidence suggests that, eventually, cells with stemness features, also termed CSC, exclusively

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drive pancreatic tumorigenesis in humans. The CSC hypothesis is the subject of great interest

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within the field of PDAC as well as other malignancies, since it also provides a rationale for the phenomenon of high resistance to chemotherapy leading to relapse of disease after treatment. In this context, a thorough understanding of the biological characteristics of the subpopulation of

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CSC will be crucial for their identification and their tracking during treatment, representing a novel measure of treatment response. Additionally, increased understanding of the biology of the

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CSC could lead to the development of new treatments specifically directed against these cells as the putative root of PDAC.

Currently, pancreatic CSC are mainly identified by flow cytometry using cell surface markers that are poorly defined and non-exclusively expressed on CSC. Up until now, pancreatic CSC have been identified and characterized using the surface markers, CD44, CD24, EpCAM, CD133, CXCR4, and c-Met (Figure 4) [45-47]. More recently, other identification methods such as the side population assay or the ALdehyde DeHydrogenase-1a1 (ALDH1) activity assay have emerged [48]. CSC can also be functionally enriched by their capability to form spheres in vitro [46]. It is important to note that the process of isolating putative CSC populations from resected tumors, whether for studying their in vitro behavior or in order to obtain single cells for further analysis, is potentially prone to artifacts. Tumor digestion consists of mechanical and chemical disruption that can be harsh on the cells, impairing their viability. Therefore, the cells of 12

ACCEPTED MANUSCRIPT interest may be lost and/or damaged during the isolation process. Moreover, modifications in cell behavior and marker expression are to be expected, due to changes in the CSC environment.

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Once isolated CSC may loss their properties because they are not interacting with the stromal environment or circulating stromal or endothelial cells. Furthermore, the low incidence of CSC

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requires sensitive techniques for their identification and isolation. Thus, development of

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comprehensive and corresponding in vitro and in vivo working models that recapitulate the whole heterogeneity of the resected tumor and mimic its complex network of relationships with the

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surrounding environment is of crucial importance for studying human CSCs. These models

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should correlate to the responding in vivo situation of the patient in order to develop and test efficient therapies targeting CSC populations. In this context, a great effort has been made on the development of primary tissue xenograft models and corresponding in vitro primary cell cultures

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as a platform for expansion of fresh tumor samples. These xenografts were proven highly relevant for several cancers as they accurately recapitulate the features of the patient tumor, including

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retaining the genetic features of the tumor, faithfully maintaining the heterogeneity of tumor cell composition, and its microenvironment including the stroma [49, 50]. Based on the outstanding clinical relevance of the original tumor composition, tissue xenografts have become important working models for the CSC field including pancreatic CSCs [51-53]. In addition, in vivo imaging constitutes an important tool in the future working systems to study CSCs. Direct visualization of CSCs using reporter constructs provides a novel opportunity for a better understanding of tumor initiation and progression in their in vivo environment. This constitutes a crucial starting point for the evaluation of the treatment response to novel directed therapies. Therefore, this model system is providing important information on tumor biology and on the role on CSCs in the tumorigenic process, with minimal artifacts and alterations in comparison with the primary tissue [54]. The characteristics of the CSC population could determine the response to treatment or outcomes from cancer. To support this principle a CSC biomarker is required and has to be 13

ACCEPTED MANUSCRIPT reproducible and measurable in patient samples. Ideal markers would be those that, while the cells remain viable, could be studied in a longitudinal fashion in order to correlate the presence of

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CSC and disease outcome. The identification of CSC markers fulfilling these criteria would indeed represent a mayor breakthrough that could allow the development of a personalized

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therapeutic approach to the different types of CSC that are resistant to chemo- and radiotherapy

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treatments.

The first evidence for a CSC population in PDAC was provided by Li and colleagues

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[45]. The authors identified a highly tumorigenic CD44+CD24+EpCAM+ subpopulation using a xenograft model of immunocompromised mice for primary human PDAC. In contrast to their

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CD44–CD24–EpCAM– counterparts, these CD44+CD24+EpCAM+ cells were able to form tumors at low numbers and displayed typical stem cell features such as self-renewal, activation of

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developmental signaling pathways (e.g. hedgehog), generation of differentiated progeny, and the ability to recapitulate the phenotype of the parental tumor from which they were derived [45].

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Interestingly, the finding that tumorigenicity in PDAC is confined to CD44 +CD24+ cells is in stark contrast to the original findings in breast cancer, where only CD44+CD24–/low cells were tumorigenic [55]. However, these different findings have now been extended to other tumor entities such as ovarian cancer [56]. In a second study, Hermann et al. showed that CD133 also reproducibly discriminates for cells with capacity for self-renewal, sphere formation, and, most importantly, in vivo tumorigenicity in secondary and tertiary recipients [46]. Although CD133+ cells show some overlap with the CD44+CD24+EpCAM+ subpopulation, these data indicate that the putative CSC isolated by different research groups are not identical. Further studies will be required in order to determine whether these markers (CD44+CD24+EpCAM+ and CD133+) define two distinct pancreatic CSC populations, or whether the use of a combination of these makers markers confers a higher enrichment in PDAC initiating cells (Figure 4). Very recently, a follow-up study

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ACCEPTED MANUSCRIPT revealed that CD44+ cells derived from PDAC xenografts can be further enriched for CSC properties using the Hepatocyte Growth Factor Receptor c-Met[47].

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More recently, additional markers have also been associated with pancreatic CSCs. Specifically, ALDH1 has been shown by several groups to label tumorigenic cells in PDAC [48,

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57, 58]. However, although ALDH1 may indeed enrich for a tumorigenic population within the

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tumor tissue, ALDH1 has also been found to be abundantly expressed in normal pancreas tissue [59]. Therefore, ALDH1 can be best used for tumors whose corresponding normal tissues express

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ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

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Since the currently available cell surface markers are merely enriching for CSC populations, and therefore their use remains controversial, functional assays such as sphere-formation capacity in vitro and tumorigenicity in vivo are becoming even more important for the identification and

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subsequent characterization of CSC and may also serve as a platform to find better CSC markers. It is important to note that CSC do not represent a homogenous clonal population of cells

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with equal capabilities but have undergone genetic evolution during the many years of tumor development and subsequent progression. While earlier studies in pancreatic cancer already pointed towards distinct populations of CSC with distinct features including the capability to metastasize [46], genetic evolution has now also been shown to occur in distant metastasis of pancreatic cancer, with genomic instability being the cause for different subclones of metastasisinitiating cells, although its relation to CSC subpopulations has not been determined yet [60, 61]. However, the issue of clonal heterogeneity of CSC has just recently been comprehensively addressed in acute lymphoblastic leukemia by studying DNA copy number alteration [62]. These studies demonstrate that different subclones of CSCs are present in individual patients suggesting that there is not a single CSC subset with a static phenotype, but rather that clonal evolution within CSCs is a common event. Genetic instability can cause rise of different CSC subclones originating from a common progenitor (precursor) that display different properties including their proliferative properties as well as invasive features. Due to selection pressure, one or several 15

ACCEPTED MANUSCRIPT subclones may play a dominant role in the tumorigenic and/or metastasis process. The evolving concept CSC heterogeneity also indicates that therapeutic approaches need to be designed to

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target and eradicate all the CSC subclones in order to be clinically efficient as spared subclones will lead to relapse of the disease. This multiclonality may at least in part rationalize eventual

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relapse of the diseas even though the initiating oncogenic event has been clearly defined and

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targeted. This may be exemplified by the high recurrence rate in patients with chronic myeloid

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6. Metastatic Pancreatic Cancer Stem Cells

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leukemia treated with imatinib [63].

Essential properties of CSC are, self-renewal, evidenced as in vitro sphere formation or in vivo tumorigenicity, as well as differentiation capacity to generate the heterogeneous cancer cell

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population within a tumor, a process that is also recapitulated in metastatic spread. Metastasis is the major cause of death in PDAC patients and currently there is no effective treatment available

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for patients with advanced PDAC. Importantly, not all cells within a tumor (or even within the CSC population) possess the same metastatic potential, and only a small subset of cells is directed through lymphatic or blood vessels toward specific secondary sites to form metastases. In order to be able to establish secondary lesions, the migrating cells would require features similar to the cells initiating the primary tumor. For this reason, CSC were proposed to represent the only cell population capable of spreading and giving rise to metastases. Indeed, Hermann et al. for the first time identified two distinct subsets of CD133+ CSC based on their ability to form metastasis. Only one of them being capable of driving metastasis was characterized by the additional expression of the chemokine receptor CXCR4 [46]. CXCR4 is a chemokine receptor that specifically binds to stromal cell derived factor 1 (SDF-1) expressed in a gradient that enables chemotaxis. SDF-1 was originally found to be responsible for leukocyte and hematopoietic progenitor cell homing. Both molecules are also obligatory players in the maintenance of pancreatic duct survival, proliferation and migration during pancreatic organogenesis and 16

ACCEPTED MANUSCRIPT regeneration [64]. Emerging evidence suggests that CXCR4 plays a pivotal role in the metastatic process of different tumor entities towards a gradient of SDF-1, which is highly expressed in

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secondary sites usually associated with metastasis such as lymph nodes, lung, liver and bone marrow [65, 66].

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Hermann et al. identified a “stationary” population expressing CD133, but not CXCR4,

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which is responsible for the initiation and maintenance of the primary tumor, and a “migrating” and highly metastatic population characterized by co-expression of CD133 and CXCR4 [46].

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Only CD133+CXCR4+ cells had metastatic potential, while depletion of the CSC population for

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CD133+CXCR4+ cells completely abrogated the usually strong metastatic phenotype of the implanted tumors. Consequently, pharmacological inhibition of the CXCR4 receptor by AMD3100 also prevented the metastatic activity of transplanted CSCs. These data provide

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convincing evidence for a crucial role of the CXCL12 (SDF-1alpha)/CXCR4 axis in metastasis. Since most cancers initially spread to local lymph nodes long before solid organ colonization, the

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lymphatic system and lymph node metastases also need to be investigated for the presence and contribution of migrating CSCs. Indeed, Hermann et al. also found significantly higher numbers of CD133+CXCR4+ migrating CSC in the primary tumor of patients with lymph node metastasis (pN1+), demonstrating a close clinical correlation between migrating CSC and advanced disease [46]. More recently, two different studies have linked PDAC metastasis to two other cytokine receptors. Nakata et al. suggested that CCR7 (also known as BLR2 or CD197), is associated with lymph node metastasis in PDAC and, based on multivariate survival analysis, could serve as an independent prognostic factor [67]. Li et al. demonstrated that surface expression of c-Met further enriches for CSCs, and may also represent a therapeutic target as blocking of c-Met in combination with gemcitabine resulted in decreased growth rates of PDAC xenografts and, most importantly, abrogation of metastatic spread following intracardiac injection of pretreated cells [47].

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ACCEPTED MANUSCRIPT CSC may acquire a migrating phenotype through EMT in primary tumors, since the mesenchymal phenotype is usually associated with strong migration capacity while maintaining

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stemness, thus allowing the production of progenies during metastasis. Recently, Wellner et al. showed in pancreatic and colon cancer that the EMT-activator ZEB1 represents an important

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promoter of metastasis by suppressing E-cadherin. Furthermore, the stem cell phenotype was

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maintained by suppression of miR-200 family members that usually target stem cell factors such as Sox2 and Klf4 [68]. Together, these results suggest that the metastatic process is not random,

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but rather regulated by specific mechanisms related to the expression of adhesion molecules,

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chemokine receptors and their respective ligands. Whether this is a reversible process in PDAC remains to be determined. Indeed, Hermann et al. did not find any evidence for the generation of CD133+CXCR4+ from CD133+CXCR4- cells in the utilized model systems [46]. Indeed, the

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plasticity of CSC versus non-CSC is a matter of intensive debate [69]. Some investigators have reported the ability of non-CSC to generate CSC, although most of these studies were performed

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using rather artificial models of stable cell lines that were subsequently transformed by overexpression of a single oncogene [70]. While this raises the possibility that during the proceeding many years of culture already resulted in the accumulation of many unknown genetic alterations, these studies also neglect the putative contentment of diverse cell populations including transient amplifying progenitors in the non-CSC fraction of the transformed cell line (Figure 4). Therefore, for more definitive conclusions on this important aspect, experiments need to be performed at the single cell level using primary cells.

7. Identification of circulating cancer (stem) cells Despite encouraging experimental findings on the existence of metastatic CSCs, the clinical routine for evaluating cancer patients includes only the assessment of clinical manifestations of cancer, radiologic evaluations and measurement serum tumor markers. However, these methods do not provide early enough timely information of metastatic spread 18

ACCEPTED MANUSCRIPT before they become detectable by current imaging technologies or cause clinical symptoms. More recently, however, the detection of circulating tumor cells (CTC) has been added to the clinical

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armamentarium. While the first description of the presence of CTC in the peripheral blood of cancer patients was documented already in 1869, the technology for the reproducible detection of

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CTC was not ready for clinical application until recently. As new devices using different

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approaches for the detection of CTC are arising at increasing pace, the identification and detection of CTCs could eventually become a real-time parameter for diagnosis and prognosis of

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cancer patients. Importantly, however, current technology does not differentiate between

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circulating CSC and non-CSC. Indeed, it has not been proven yet whether CTC or any contained subpopulation is actually capable of tumor initiation in distant sites. CTC in most patients are extremely rare events. The expected concentration is one cell

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per 105-107 mononuclear cells [71] and therefore their detection and isolation is technically challenging. Although inherently biased towards a specific subset of cancer cells, most methods

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used for the separation of CTC involve an enrichment step followed by a detection step [71]. CTC enrichment is performed based on morphological and immunological characteristics of the cells of interest. Morphology-based isolation techniques are based on the CTC physical characteristics size or density. Isolation could be performed by filtration considering that epithelial cells are regularly slightly larger in diameter as compared to leukocytes. This technique is not very specific taking into account that at least a subset of circulating cancer cells could actually be smaller than leukocytes based on their undifferentiated state. Another technique for separation of CTC is based on density gradient separation commonly used for isolation of polymorphonuclear monocytes (PBMCs) and granulocytes from erythrocytes and plasma. The drawback of this approach is the possible loss of CTC due to the migration or presence of these cells in different layers due to their distinct characteristics. On the other hand, enrichment can also be achieved based on immunological characteristics. These methods involve the detection of (surface) markers by antibody linked to 19

ACCEPTED MANUSCRIPT magnetic beads or fluorescent dyes. These markers may be broadly expressed by most tumor cells of epithelial origin (e.g. Epithelial Cell Adhesion Molecule or cytokeratins) or are restricted to a

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-neu, MUC1/MUC2, mammaglobulin, and CarcinoEmbryonic Antigen). Once labeled, CTC can be separated from the rest of the cells using

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immunomagnetic separation or fluorescent activated cell sorting. One of the main problems of

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these techniques based on a positive selection represents the possibility of missing cells that do not express markers of epithelial commitment such as CSC and/or cells that have undergone

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EMT.

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CTC including circulating CSC could serve as an early marker for the assessment of therapeutic response in overt cancers and could hopefully lead to refinement of clinical management of cancer patients. Indeed, several CTC detection platforms have now been

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validated in various clinical settings and strongly suggest that CTC detection bears a strong potential of assisting malignancy diagnosis, estimating prognosis, monitoring disease recurrence,

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and response of the anticancer therapy [72]. Accurate and unbiased detection of CTC may not only allow the assessment of early stages of metastasis and predict clinical outcome but may also represent a novel platform for improving outcome in cancer patients. Using refined microfluidic technologies for the isolation of viable CTC based on their physical properties [73] and/or circulating CSC, investigators may eventually obtain low-risk access to a patients’ tumor material. CTC could be characterized as single cells as well as expanded in vitro or, preferably, in vivo using established xenograft models. Their direct comparison with CSC isolated from primary tumor material obtained during surgery may proof as an invaluable tool for advancing our understanding of the metastatic process.

8. Therapeutic implications of pancreatic cancer stem cells The identification and subsequent isolation and characterization of CSC from PDAC suggested this population to be highly responsible for the failure of conventional therapy in 20

ACCEPTED MANUSCRIPT pancreatic tumors. CSC display resistance to classic cytotoxic agents due to high expression of efflux pumps (ABC membrane transporters that can exclude toxic substances from the cell) [74],

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strong ability to repair DNA damage, reduced immunogenicity, and anti-apoptotic properties [75, 76]. In addition, this population, or part of it, remains in a quiescent state that makes them

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resistant to most cytotoxic drugs due to lack of proliferation. Thus, treatment of the pancreatic

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cancer cells with the cytotoxic drug gemcitabine did not affect the CSC population and, as soon as the drug is withdrawn, these cells repopulate the cancer (stem) cell pool leading to tumor

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relapse [76]. For these reasons it is of utmost importance to discover novel treatment modalities

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that are capable of either depleting CSC and/or induction of their differentiation into nontumorigenic progenies that should also be more susceptible to cytotoxic agents. This approach would suggest that most likely multi-modal treatment regimens will be necessary for irreversible

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eliminating CSC as the root of PDAC (Figure 5). PDAC demonstrates altered signaling pathways that are normally active during embryonic

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development, such as hedgehog, Notch, mTOR, Wnt, Bmp2, etc. During the embryonic development of the pancreas the hedgehog pathway plays an important role, but in adult pancreas this pathway is shut down [77, 78]. This signaling cascade involves three Hedgehog molecules: Sonic, Indian and Desert that bind to the Patched receptor molecule and un-inhibit Smoothened, a transmembrane protein that subsequently activates the Gli transcription factor and its downstream targets [79]. Abnormal activation of the hedgehog pathway, mainly Sonic hedgehog (Shh), has been found in a high number of PDAC. Several studies using the Smoothened inhibitors have now demonstrated inhibition of proliferation and induction of apoptosis of pancreatic cancer cells translating into significant size reduction of tumor xenographs in vivo as well as abrogation of metastatic potential [80-82]. It is important to note, however, that up-regulation of the hedgehog pathway has also been linked to the mutation of the upstream regulator Kras resulting in induction of PDAC growth (autocrine effect) [83]. Finally, Shh is highly expressed in the CSC subpopulations and inhibition of its pathway negatively affects the CSC subpopulation, 21

ACCEPTED MANUSCRIPT demonstrated as a decrease in ALDH1+ cells [80-82]. These data suggest an important role for the hedgehog pathway in the maintenance of CSC. It is worth noting, however, that the tumor

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regression observed in these studies could also at least in part be rationalized by inhibiting Shh signaling in the surrounding stromal tissue (paracrine effect). It has been shown that the Shh

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pathway is able to promote motility of cancer fibroblasts, enhance tumor angiogenesis and

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lymphangiogenesis, and induce expression of hypoxia inducible factor-1 alpha (HIF1-a), as well as vascular endothelial growth factor (VEGF) [81, 84]. These studies illustrate that hedgehog

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signaling is important for tumor induction, maintenance, and progression as it impacts both

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highly tumorigenic CSC population and cells constituting the tumor microenvironment. Regardless of how promising the target hedgehog pathway seemed to be in the outlined studies, Mueller et al. have shown in primary human CSC that inhibition of the hedgehog

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pathway alone, or as a supplement to chemotherapy (gemcitabine), is not capable of completely eliminating CSC [52]. This is most likely due to the cross-talk and synergistic effects between

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hedgehog and other signaling pathways. Indeed, the authors showed that CD133+ CSC display high activity of mTOR signaling. The mammalian Target of Rapamycin (mTOR) belongs to the phosphatidyl-inositol 3-kinase (PI3K) family of enzymes that regulate growth and proliferation of PDAC cells and contributes to their drug resistance [85, 86]. Mueller et al. have shown that the mTOR inhibitor rapamycin alone already significantly decreased the number of CD133 + CSC in vivo using patient-derived primary PDAC, but as noted for hedgehog inhibition, rapamycin was also insufficient to completely eliminate this population. Intriguingly, the authors then showed that, the combined inhibition of the hedgehog pathway and the mTOR pathway together with gemcitabine administration resulted in complete abrogation of CSC in patient-derived PDAC offering a promising novel treatment approach for PDAC. Other molecules have recently also gained attention in the CSC field. Curcumin, a naturally occurring compound that can be isolated from turmeric, has been shown to inhibit cell proliferation and induce apoptosis of PDAC cells through inactivation of Notch signaling [87]. 22

ACCEPTED MANUSCRIPT Bao et al. have recently shown that curcumin and its synthetic analogue CDF are able to decrease the expression of CSC surface markers CD44 and EpCAM, which coincided with reduced sphere-

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forming ability of the treated PDAC cells [88]. The treatment effect of the synthetic compound was much stronger as that of the natural compound curcumin and effects were even more

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pronounced in combination with gemcitabine. This could be accounted to the ability of curcumin

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and derivatives to inhibit multidrug resistance-associated protein 5 (MRP5), an ABC transporter strongly expressed in PDAC cells [45]. Moreover, studies on various tumors have demonstrated

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the ability of curcumin and derivatives to inhibit various signaling pathways including MAPK

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and PI3K/Akt pathways, which are involved in tumor initiation, growth and progression [87, 89, 90].

CSC share many features with healthy adult stem cell. For example, telomerase is

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responsible for the longevity of stem cells (in healthy tissue and cancer) as it allows for unlimited cell divisions without shortening of the telomeres. The enzyme is present at low levels in healthy

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adult stem cells, but while normal tissue stem cells show progressive telomere shortening with increased age, it is in CSC where stable telomere length is maintained [91-93]. Recent studies showed that treatment of pancreatic and breast cancer cell lines with the specific telomerase inhibitor imetelstat results in depletion of CSC [94]. However, despite these promising results, the effect of imetelstat appeared to be predominantly mediated through a yet unidentified telomereshortening independent pathway. Thus, further studies are necessary to uncover the mechanisms through which telomerase inhibitors work. The downside of targeting signaling pathways involved in maintenance and support of CSC could be that it may also affect healthy adult stem cells present in and important for maintenance of numerous tissues such as bone marrow, intestine, and skin. Thus, identifying pathways exclusively active and functionally relevant in CSC may create saver ways of exclusively targeting CSC. Alternatively, specific delivery systems such as targeted nanocarriers

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ACCEPTED MANUSCRIPT for delivery of such treatments to cancer tissue may be mandatory for stem cell-targeting therapies that are also relevant for normal stem cell function.

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As the majority of patients with PDAC succumb from metastatic disease and a distinct subpopulation of CSC residing in the invasive front of the tumor has been identified, the specific

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targeting of CXCR4+ CD133+ CSC may inhibit disease progression and metastasis [44]. Indeed,

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earlier studies already implicated the CXCR4 receptor and its specific ligand CXCL12 play in cancer biology, by driving metastatic properties of cancer cells, as well as modulating tumor

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vascularization and cancer cell survival [95]. Moreover, other molecules involved in cancer

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development and/or maintenance, including HIF-1a, VEGF, NF-kB and various oncoproteins induce expression of CXCR4 [96]. In PDAC, the cytokine CXCL12 is expressed at high levels in the surrounding stroma but only at very low levels in the cancer cells itself suggesting a paracrine

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effect of CXCL12 on cancer cells [97-99]. In 2010, Singh et al. reported that CXCL12/CXCR4 interaction activates Akt and ERK signaling pathways and thereby directly affecting cell survival

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[100]. In addition these authors show that CXCR4/CXCL12 signaling plays an important role in establishing the pool of resistant cells in response to gemcitabine treatment. The CXCR4 antagonist AMD3100 can block CXCR4/CXCL12 signaling. Intriguingly, AMD3100 is also capable of blocking metastasic spread of the above-mentioned migrating CXCR4+ CD133+ CSC suggesting that, this could be a novel treatment option for targeting the cell compartment that actually drives metastasis in PDAC. Although AMD3100 remains in clinical use for short-term hematopoietic stem cell mobilization, its chronic administration has been associated with significant cardiotoxicity [101]. Recent studies have also shown that AMD3100 may paradoxically bind and activate chemokine receptor CXCR7 [102]. To date, various novel CXCR4 inhibitors have been tested, including small peptide antagonist T140 and its analogues, and natural compounds such as AKBA (Acetyl-11-keto-b-boswelic acid) and celastrol [103-105] and therefore should be tested in this context.

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ACCEPTED MANUSCRIPT 9. Summary and Perspectives According to the cancer progression model postulated by Fearon and Vogelstein back in

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1990, at least 4-5 genetic events are required for the progression from normal epithelium to carcinoma [106]. It is important to note that, while Jones et al. identified an average of 63 genetic

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alterations in advanced PDAC, the majority of these genetic alterations are in fact bystander

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mutations [107]. While 4-5 initial genetic events may indeed be sufficient to transform the cellof-origin, these cells will sequentially accumulate more alterations due their progressive genomic

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instability leading different subclones defined by partially differently emerging genetic profiles

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[60, 61]. Due to the long life span, stem cells could represent a target for the accumulation of these initiating genetic events, although this has not been demonstrated for the pancreas. However, irrespective of their actual cell-of-origin, sufficient evidence has now been

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accumulated demonstrating the existence of CSC in several epithelial tumors including PDAC. Importantly, CSC seem to harbor mechanisms protecting them from standard therapy. To further

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foster our understanding of CSC biology including their interactions with the microenvironment and a putative niche, it will be required to study them in vivo using either whole tissue human xenografts or genetically engineered mouse models. The development of novel probes such as targeted nanoparticles and corresponding intravital imaging modalities could be of paramount importance for in vivo tracking of CSC. These studies will also pave the way to better elucidate the underlying regulatory mechanisms of CSC and develop platforms for targeted theragnostics [108]. As evidence is now accumulating for novel therapeutic targets that are capable of eliminating CSC, newly emerging treatment regimens that include this knowledge arising from the CSC concept may eventually lead to a better outcome for patients suffering from this currently deadly disease.

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Figure 1: Chemotherapy for treatment of metastatic pancreatic ductal adenocarcinoma.

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Gemcitabine significantly improves median survival in patients with advanced pancreatic cancer from 4.4 to 5.6 months (left panel). Ten years later, so far the only other approved targeted

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treatment modality using the EGF receptor inhibitor Erlotinib enhanced survival by no more than

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10 days (right panel). In 2011, FOLFRINOX was tested against gemcitabine, which increased median survival from 6.8 to 11.1 months but was only tolerated by patients with good

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Figure 2: Development of the pancreas. Different transcription factors are responsible for the determination of cell fate during pancreas development. Cells retaining Pdx1 expression and

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initiate the expression of Ptf1 and Notch signaling progress towards an exocrine lineage. In contrast, the expression of Ngn3 determines an endocrine fate associated with differential

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expression of Arx and Pax4, which will then further differentiate these committed cells into αcells and β-cell, respectively.

Figure 3: Experimental models targeting different pancreatic cell types. To study the development of pancreatic intraepithelial neoplasias (PanIN) or pancreatic ductal adenocarcinoma (PDA) a large variety of mouse models have been developed providing experimental evidence for a diverse set of putative sources of pancreatic cancer in mice.

Figure 4: Distinct populations of cancer stem cells in pancreatic ductal adenocarcinoma. In addition to the tumor resident EpCAM+CD44+CD24+, CD44+c-Met+, CD133+, and/or ALDH1+ CSC, a subpopulation of migrating CSC, identified by CD133+CXCR4+ expression, can be detected in the invasive front in the pancreas as well as in the circulating blood. Typically, metastatic lesions in pancreatic cancer are found in organs with strong expression of Stromal33

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Figure 5. Model of advanced treatment strategies for pancreatic ductal adenocarcinoma. PDAC is heterogeneous tissue containing a pool of CSC that represents the source for highly

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proliferative populations of more differentiated cancer cells that comprise the bulk of the tumor,

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albeit lacking tumor-initiating potential. Current therapies mainly target highly proliferative cancer cells, and have little or no effect on the CSC population, resulting in relapse of the tumor (column 2). Novel treatment strategies include drugs that are capable of eliminating CSC by

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inhibiting/abolishing their self-renewal (column 3) or by drugs that induce differentiation of CSC

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(column 4) into a more therapy susceptible progeny. Combination of both (column 5).

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