International Journal of Radiation Biology, 2014; Early Online: 1–8 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.920968

Report on the International Workshop ‘Cancer stem cells: The mechanisms of radioresistance and biomarker discovery’

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

Anna Dubrovska OncoRay – National Center for Radiation Research in Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, German Cancer Consortium (DKTK) Dresden, and German Cancer Research Center (DKFZ) Heidelberg, Germany

(Kwon et al. 2007, Murat et al. 2008, Pallini et al. 2008, de Jong et al. 2010, Wang et al. 2009, Sung et al. 2010, Kim et al. 2011, Metellus et al. 2011, Avoranta et al. 2012, Kawamoto et al. 2012, Koukourakis et al. 2012, Smit et al. 2013, Sprenger et al. 2013). Despite some limitations of these studies such as a low number of enrolled patients, using volume-related parameters as an endpoint, and lack of functional characterization of the marker positive cells for their tumorigenic properties, these studies have paved an avenue for future development of CSC-related predictive biomarkers and their clinical translation. In addition to the impact of CSC density on tumor radiocurability, recent preclinical studies suggest a number of different intrinsic and extrinsic mechanisms that confer tumor radioresistance and which were also described for CSC populations, including quiescence, increased DNA repair capability, activation of the cell survival pathways and scavenging of the reactive oxygen species (ROS) that can induce DNA damage (Pajonk et al. 2010, Peitzsch et al. 2013, Rycaj and Tang 2014). A broad variety of the microenvironmental CSC niches as well as genetic and epigenetic changes of CSC during tumor development make molecular mechanisms of radioresistance dynamic in nature (Tang 2012, Peitzsch et al. 2013, Kreso and Dick 2014, Rycaj and Tang 2014). Identification of the predictive biomarkers for individualized radiotherapy and characterization of the molecular mechanisms by which tumor initiating cells survive treatment may ultimately lead to more efficient cancer therapy. The aim of the Workshop ‘Cancer stem cells: The mechanisms of radioresistance and biomarker discovery’, which was held on 23–24 September 2013 at OncoRay – National Center for Radiation Research in Oncology in Dresden, Germany, was to bring together the most recent viewpoints and insights about: (i) the molecular characterization and

Abstract The aim of the Workshop “Cancer stem cells: The mechanisms of radioresistance and biomarker discovery”, which was held on 23–24 September 2013 at OncoRay – National Center for Radiation Research in Oncology in Dresden, Germany, was to bring together the most recent viewpoints and insights about: (i) the molecular characterization and regulation of CSC, (ii) the mechanisms of CSC radioresistance, and (iii) the discovery of new CSC targeting therapeutics and biomarkers. In this report some research aspects presented in these three topics are highlighted. Keywords: Radioresistance, Cancer Stem Cells, Circulating Tumor Cells, Metastasis, Biomarkers, Hypoxia, Integrins

Introduction In many cases radiotherapy can completely destroy the tumor. Nevertheless, cancer patients can still relapse after radiation therapy. The radiotherapy failure might be related to cancer stem cells (CSC) or a cancer progenitor population which was not completely sterilized during the treatment and caused tumor re-growth (Baumann et al. 2008, Bütof et al. 2013, Peitzsch et al. 2013). The fraction of CSC is highly variable even in the tumors of the same entity. During the last decade, different techniques have been developed for the identification of CSC in cell cultures and tumor tissues, including immunological staining, analysis of CSC-specific protein activity and label-free techniques (Brunner et al. 2012, Harkness et al. 2012, Peitzsch et al. 2013, Uckermann et al. 2014). Recent findings suggest that estimation of the number of CSC in pre-therapeutic tumor biopsies might be predictive of tumor radiocurability in cervical squamous cell carcinoma (CSCC), head and neck squamous cell carcinoma (HNSCC), rectal carcinoma and glioma patients

Correspondence: Dr Anna Dubrovska, OncoRay – National Center for Radiation Research in Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden and Helmholtz-Zentrum Dresden-Rossendorf, Fetscherstrasse 74, 01307 Dresden. E-mail: Anna.Dubrovska@ OncoRay.de (Received 23 April 2014; revised 28 April 2014; accepted 30 April 2014)

1

2

A. Dubrovska

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

regulation of CSC, (ii) the mechanisms of CSC radioresistance, and (iii) the discovery of new CSC targeting therapeutics and biomarkers, and to work on advancing the field. The limitations of the current models to study cancer stem cell properties including their putative radioresistance were also discussed. In the next sections some research aspects presented in these three topics are highlighted. The talks of Dean G. Tang, Ingeborg Tinhofer, Michael H. Muders, Ira-Ida Skvortsova, Serhiy Souchelnytskyi, Nils Cordes and Andreas Androutsellis-Theotokis, and poster presentations of Lovisa Lundholm, Ina Kurth, Franziska Trautmann and Ortrud Uckermann are present in this issue as original papers.

Topic 1. Molecular characterization and regulation of cancer stem cells The CSC hypothesis provides a strong clinical rationale for the identification of CSC specific antigens to develop new predictive biomarkers and therapeutic strategies (Baumann et al. 2008, Bütof et al. 2013, Peitzsch et al. 2013, Mäbert et al. 2014). However, despite a large number of CSC markers having been identified in the experimental studies, very few of them were validated in a clinical setting. This could be due to the lack of CSC markers with a high level of specificity, since almost all of them were also found in normal stem cells making development of CSC-specific predictive signatures a challenging task. On the other hand recent findings revealed that non-stem cancer cell populations can be reprogrammed into CSC by various microenvironmental insults such as hypoxia, inflammation and anti-cancer therapy, including irradiation. In addition, a high genetic and phenotypic plasticity of the CSC population suggests that it can be a dynamic and multi-faced target, which might be hard to eradicate by conventional therapy (Tang 2012, Peitzsch et al. 2013, Kreso and Dick 2014). Dean G. Tang (The University of Texas M.D. Anderson Cancer Center, USA) previously demonstrated that prostate cancer cells, which express a low level of the differentiation marker prostate-specific antigen (PSA) are quiescent, resistant to therapy, including androgen deprivation and standard chemotherapy and possess long-term tumor propagating potential (Qin et al. 2012). Studies of Tang and colleagues demonstrated plasticity of the pool of PSA-positive cells, which can be converted into the androgen independent PSA-negative tumor initiating population by overexpression of embryonic stem cell self-renewal gene Nanog or by persistent androgen deprivation (Badeaux et al. 2013, Chen et al. 2013). The prostate CSC population defined by a low PSA expression is heterogenous and contains many other tumor initiating cells whose phenotypes warrant further investigation. Furthermore, metastatic prostate cancer cells are enriched for CSC phenotype and functions suggesting the involvement of CSC in metastatic cancer spread (Li and Tang 2011). Ingeborg Tinhofer (Charité University Hospital, Berlin, Germany) discussed several studies aiming at molecular characterization of circulating tumor cells (CTC) with particular focus on epithelial-mesenchymal transition (EMT) and CSC features of CTC (Tinhofer et al. 2014). These find-

ings demonstrated a relationship between stemness and EMT, which on one hand plays a critical role in the maintenance and generation of CSC, and on the other hand leads to cancer cell dissemination and metastasis. In a xenograft mouse model, CTC from breast cancer patients were able to initiate metastasis (Baccelli et al. 2013). The number of CTC from the blood of patients with metastatic breast cancer was correlated with progression-free and overall survival and with a number of metastatic sites (Baccelli et al. 2013, Bidard et al. 2014). Analysis of CTC phenotype using a murine breast cancer xenograft model revealed a loss of the expression of epithelial cell adhesion molecule EpCAM and upregulation of the mesenchymal markers (Gorges et al. 2012). A few independent studies demonstrated a CSC phenotype of CTC in peripheral blood of the patients with breast, colon cancer, oral squamous cell carcinoma (OSCC) and hepatocellular carcinoma e.g., expression of cluster of differentiation 133 (CD133), CD44, ATP-binding cassette sub family G member 2 (ABCG2), Nanog, CD117 and a high aldehyde dehydrogenase (ALDH) activity (Armstrong et al. 2011, Kasimir-Bauer et al. 2012, Pilati et al. 2012, Sinha et al. 2013, Sun et al. 2013). Taken together, current data indicate that CSC population can contribute to tumor dissemination, and metastasis initiating cells can derive from CSC clones during local tumor progression (Klein 2009, Baccelli and Trumpp 2012). The ability of CTC to survive in the circulation for a long time and tumor initiating properties of certain subsets of CTC could potentially enable them to escape radiotherapy and eventually develop more aggressive tumors which are difficult to treat (Kurth et al. 2014). Lack of the reliable CSC markers limits their clinical translation and gives an impetus to employ new technologies and animal models to identify CSC specific antigens (Wurdak 2012). Heiko Wurdak (Leeds Institute of Molecular Medicine, Leeds, UK) described identification of the adapter protein TRRAP (transformation/transcription domain associated protein) as a new regulator of glioblastoma stem cells (GSC) using a kinome-wide RNA interference (RNAi) screen of primary glioblastoma cells (Wurdak et al. 2010). Genetic silencing of TRRAP decreases self-renewal and proliferation of GSC and improves survival of animals carrying xenograft brain tumors that makes TRRAP an attractive therapeutic target. However, one of the major challenges to the development of CSC-specific biomarkers, CSC-targeted therapy and personalized medicine might lie within tumor heterogeneity. Recent findings demonstrated that many types of tumors including glioblastoma, leukemia, breast and renal cancer contain multiple cell lineages arising from a single clonogenic cell (Hwang-Verslues et al. 2009, Gerlinger et al. 2012, Sottoriva et al. 2013, Kreso and Dick 2014). Using single cell profiling technologies may help to determine the optimal treatment strategies to eradicate all tumor-initiating cell populations. The major concerns regarding CSC analysis by using xenograft mouse models are related to the role of the xenogeneic microenvironment, normal stem cell compartment and immune system in tumor development (Cheng et al. 2010, Kreso and Dick 2014). Rapid development of genetic engineering provides a favorable ground for more accurate

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

Report on the Cancer Stem Cell Workshop CSC assays in autochthonous and syngeneic models. Haikun Liu (DKFZ, Heidelberg, Germany) discussed a new strategy for identification of normal and malignant brain stem cells in genetically engineered mice (Liu et al. 2010). The neural stem cell-specific overexpression of the nuclear receptor tailless (Tlx) is sufficient to promote normal stem cell expansion and neurogenesis. Genetic inactivation of Tlx leads to the loss of self-renewing tumor cells and induction of cell death, cell cycle arrest and differentiation. This study suggests the significance of targeting stem cell pathways for cancer treatment. Michael H. Muders (Institute of Pathology, Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Germany) discussed the mechanisms of autophagy as important regulators of intracellular recycling, homeostasis and therapy resistance. Recent studies of Muders laboratory demonstrated the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C) and its receptor Neurophilin-2 in inducing autophagy and maintaining cancer cell survival during therapy (Stanton et al. 2013). These studies suggested combining autophagy inhibitors together with conventional chemotherapeutic agents to boost the effectiveness of anti-cancer treatment (Hönscheid et al. 2014). Leoni A. Kunz-Schughart (OncoRay, Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Germany) described an important role of the tumor microenvironment in regulation of functional and phenotypic properties of tumor-initiating cells. A recent discovery of the Kunz-Schughart laboratory revealed a high plasticity of the expression of the CSC marker CD133 in colorectal cancer cell lines in vitro and in vivo (Peickert et al. 2012). This study suggests that cell microenvironment might have an impact on both marker profile and engraftment of tumor cell populations, and targeting of the instructive cues coming to CSC from their microenvironmental niche can be beneficial for tumor control.

Topic 2. Cancer stem cells and radioresistance A major topic of discussion during the second session was the role of cancer stem cell population in prediction of tumor radiocurability and their potential contribution to tumor radioresistance. Michael Baumann (OncoRay, Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, DKTK Dresden and DKFZ Heidelberg, Germany) gave a general overview of current experimental and clinical data regarding the role of CSC for radiotherapy outcome. From the clinical point of view, the CSC hypothesis might provide a potential explanation of the discordance between volume-dependent parameters of tumor response and permanent local tumor control. The proportion of CSC in the individual tumors may be prognostic for outcome of radiotherapy in some tumor entities including glioblastoma, HNSCC, oesophageal, rectal and cervical cancer (Kwon et al. 2007, Murat et al. 2008, Pallini et al. 2008, de Jong et al. 2010, Wang et al. 2009, Sung et al. 2010, Kim et al. 2011, Metellus et al. 2011, Avoranta et al. 2012, Kawamoto et al. 2012, Koukourakis et al. 2012,

3

Smit et al. 2013, Sprenger et al. 2013). However, these studies have several shortcomings, such as a relatively small number of patients that limits the multivariate analysis, using tumor regression or growth delay as an endpoint and a lack of correlation analysis between CSC density and local or loco-regional tumor control, as well as a lack of the functional characterization of biomarker-positive cells. Therefore, further large scale studies, which take into account these limitations, are warranted. In addition, the inter- and intratumoral genetic and epigenetic variability of tumor initiating cells associated with a broad phenotypic diversity has proved challenging for development of predictive biomarkers (Baumann et al. 2008, Brunner et al. 2012, Bütof et al. 2013, Peitzsch et al. 2013). Nevertheless, the use of a panel of CSC-related biomarkers for profiling of individual tumors might be beneficial for more individualized treatment approach. Radiobiological characterization of CSC combining in vitro functional assays as well as in vivo tumor control assays will provide important information about CSC impact on the outcome of radiotherapy. Klaus-Rüdiger Trott (UCL Cancer Institute, University College London, and Poliklinik für Strahlentherapie, Klinikum Rechts der Isar, Technische Universität München) discussed methodological limitations of current radiobiological studies of CSC. The standard method of measuring the radiosensitivity of CSC would be the dilution assay which was devised by Hewitt and Wilson, who used it to build the first in vivo cell survival curve in 1959 (Hewitt and Wilson 1959). This dilution assay was used by other radiobiologists for analysis of the different isogenic mouse models and human cell lines (Hill and Milas 1989, Baumann et al. 1992, Trott 1994). The results of these assays suggested that the proportion of tumorigenic cells in the irradiated tumors decreases exponentially with increasing radiation dose that might indicate a random sterilizing effect of radiation. The straight dose-response relationship suggests that these experimental tumor models did not have significant radio-resistant cell populations. Up to date, no such experiments on primary human cancer cells have been reported, and the vast majority of studies investigating comparative CSC radioresistance have been performed in vitro using endpoints such as colony forming ability, marker expression or cell death, whose relevance for CSC inactivation and tumor cure is questionable. Factors other than CSC radiosensitivity, such as CSC fraction, tumor volume, reoxygenation and repopulation affect cancer cure and might be predictive for individual tumor response to radiotherapy. A host of experimental and clinical studies demonstrated that tumor hypoxia is related to poor response to radiotherapy and unfavorable patient outcome in many types of cancer (Fyles et al. 2002, Evans and Koch 2003, Vaupel and Mayer 2007, Wilson and Hay 2011). Moreover, hypoxic microenvironment positively regulates CSC maintenance through activation of hypoxia-inducible factor (HIF) signaling route and inducing epithelial-mesenchymal transition (EMT) features (Soeda et al. 2009, Yeung et al. 2011, Marie-Egyptienne et al. 2013). The different surrogate

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

4

A. Dubrovska

markers for tumor hypoxia, such as expression of hypoxia-related endogenous proteins including HIF-1alpha, glucose transporter type 1 (GLUT-1), carbonic anhydrase 9 (CA9) or exogenous bioreductive probes (e.g., pimonidazole, CCI-103F, EF5 and F-misonidazole [FMISO]) can help predict local tumor control after radiotherapy (Peitzsch et al. 2014). Richard P. Hill (Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network and Campbell Family Institute for Cancer Research, Toronto, Canada) presented results of a recent study that analyzed tumor initiating cells in the hypoxic fraction of primary cervix cancer xenografts. This study demonstrated that CA9 is a marker of hypoxia in this xenograft model that was confirmed by using other hypoxia markers such as EF5 and GLUT-1 (Chaudary and Hill, 2009). Tumor initiating cells were significantly enriched in the CA9 ⫹ fraction of the xenograft tumors suggesting that the hypoxic microenvironment of early passage orthotropic xenografts of cervix cancer plays a role in the maintenance of tumorigenic cancer cell population. Daniel Zips (Radiation Oncology, Eberhard Karls University Tübingen) discussed the results from recent translational and clinical studies of novel approaches to overcome cancer cell radioresistance by modifying hypoxia including targeting of the HIF-1 signaling route and individualization of radiotherapy based on hypoxia FMISO-positron emission tomography (PET)/computerized tomography (CT) imaging. These studies demonstrated that selective inhibition of HIF-1 signaling reduces hypoxia tolerance of tumor cells which led to the eradication of radioresistant hypoxic tumor cells and, therefore, increased tumor radiocurability (Helbig et al. 2014). The prognostic value of hypoxia imaging before and during radio-chemotherapy was analyzed in patients with locally advanced HNSCC (Zips et al. 2012). This study demonstrated that FMISO-PET/CT imaging parameters during the initial (1 or 2 weeks) phase of treatment have an association with the local-progression-free-survival (LPFS) endpoint and strong prognostic value. Features of CSC such as attachment-independent survival, invasion properties and ability to seed the tumor at the secondary site suggest their involvement in metastatic dissemination, which is traditionally considered as migration of single cells (Baccelli and Trumpp 2012, Trautmann et al. 2014). However, cancer cell invasion is not restricted to individually migrating cells, and many invasive solid tumors are characterized by invasion of groups of cells maintaining cell-cell contacts (Friedl et al. 2012). Peter Friedl (Radboud University Nijmegen Medical Centre, The Netherlands and The University of Texas, MD Anderson Cancer Center, Houston, TX, USA) described an intravital infrared multiphoton imaging model for the multi-parameter visualization of collective cancer cell invasion. The collective invasion strands were resistant to high dose (20Gy) of hypofractionated irradiation, which was sufficient for regression of tumor volume (Alexander et al. 2013). Targeting of a stroma-dependent cancer cell survival signaling with RNA interference (RNAi) of β1 and β3 integrins or with anti-β1/aV integrin antibody treatment

was sufficient to overcome resistance to radiation therapy and ablate both tumor lesions and invasive niches. This study suggests an important role of the tumor microenvironment in cancer invasion and radioresistance.

Topic 3. Discovery of new biomarkers and therapeutic approaches Cancer heterogeneity is a reflection of CSC heterogeneity and can be resolved by identifying a panel of reliable biological indicators of cancer cells and their functional properties. Development of powerful proteomic and genomic technologies in conjunction with bioinformatic analysis allows the simultaneous identification and measurement of thousands of biological molecules (Lundholm et al. 2014, Mäbert et al. 2014, Skvortsov et al. 2014a). Ira-Ida Skvortsova (Laboratory for Translational Research on Radiation Oncology, Innsbruck Medical University, Innsbruck, Austria) discussed the methods of CSC isolation and proteomic analysis for the dissection of signaling pathways that control CSC properties and susceptibility to radiotherapy, and can guide development of new predictive biomarkers and CSC-targeted treatment (Skvortsov et al. 2014b). Anna Dubrovska (OncoRay, Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Germany) discussed the results of recent studies suggesting that radioresistant derivatives of the established prostate cancer cell lines share many properties with tumor progenitor cells including an enhanced expression of CSC markers, induction of the epithelial-mesenchymal transition and increases in DNA repair capacity. Whole genome gene expression profiling revealed the signaling pathways commonly deregulated in CSC and radioresistant cells, including tumor growth factor β (TGFβ), integrin, phosphoinositol-3 kinase (PI3K)/AKT and G protein-coupled receptor signaling pathways, whose targeting can be beneficial for the enhancement of tumor radiocurability (Cojoc et al. 2013, Peitzsch et al. 2013, Mäbert et al. 2014, Trautmann et al. 2014). Frank Buchholz (University Cancer Center Dresden (UCC), Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Germany) presented studies utilizing RNAi screens, large-scale protein-tagging (TransgeneOmics) and genome-wide pooled short hairpin RNA (shRNA) screens in patient-derived leukemic cells to investigate stem cells, cancer relevant processes and identify novel therapeutic targets (Poser et al. 2008, Ding et al. 2012, Wermke et al. 2013). Laure C. Bouchez (Novartis Pharma AG, Basel, Switzerland) presented data from a chemical screen for the regulators of leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4). G protein-coupled receptors play a central role in signal transduction, thereby controlling different cellular functions (Cojoc et al. 2013, O’Hayre et al. 2014, Trautmann et al. 2014). Recently, LGR4 has been identified as one of the key regulators of embryonic development and survival of adult stem cells (Nakata et al. 2014). Finding LGR4 agonists might have an impact on the development of potential therapeutics for cancer treatment. An unbiased cell-based phenotypic screen on the irradiated three dimensional (3D)-intestinal

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

Report on the Cancer Stem Cell Workshop organoid cultures was performed with using a preselected library of LGR4 effectors and led to the identification of the compounds regulating 3D organoid survival. Michael Bachmann (Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Center for Regenerative Therapies Dresden (CRTD), Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Germany) discussed a new approach for redirecting of immune effector T cells using intracellular La antigen as an inducible surface target. The La antigen was detected on the surface of ultra-violet (UV)-irradiated and apoptotic cells. Moreover, La antigen is released from dying cells and can tightly bind to the neighboring intact viable cells inducing antibodydependent cell-mediated cytotoxicity (ADCC). Bispecific recombinant antibodies, which on one hand are directed to the CD3 complex of T cells and on the other hand recognize La epitope are efficient activators of the anti-tumor immunity. This approach can potentially be used in combination with conventional therapy, such as radiotherapy, to target tumor cells including tumor-initiating cell population (Cartellieri et al. 2010, Koristka et al. 2012, 2013). Recent studies have indicated that new types of cancer irradiation with heavy ions might be more efficient in targeting CSC than commonly used photon radiotherapy because of a higher linear energy transfer (LET) resulting in decreased cell cycle-dependent radiosensitivity, reduced oxygen enhancement ratio and enhanced DNA damage of cancer cells (Cui et al. 2011). Sara Chiblak and Amir Abdollahi (DKTK, Heidelberg Ion Therapy Center, Heidelberg Institute of Radiation Oncology, Germany and Tufts School of Medicine, Boston, USA) presented data from their study of the sensitivity of AC133 positive and negative glioblastoma cells to photon, proton and carbon irradiation using classical radiobiological assays. A soft agar colony formation analysis for glioblastoma cells showed a higher relative biological effectiveness (RBE) values for carbon irradiation as compared to photons. The relevance of this observation for CSC is awaiting in vivo validation using functional endpoints such as local tumor control. A growing body of evidence suggests that integrin signaling is an essential regulator of tumor radioresistance (Mantoni et al. 2011, Eke et al. 2012a, Ahmed et al. 2013, Goel et al. 2013). Furthermore, recent data demonstrate the important role of the integrin-stimulated focal adhesion kinase (FAK) signaling route in CSC maintenance, survival, motility, and invasion (Schober and Fuchs 2011, BianchiSmiraglia et al. 2013, Golubovskaya 2013, Rentala et al. 2013). Iris Eke and Nils Cordes (OncoRay, Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Germany) presented a recent study dissecting a role of integrin/FAK in HNSCC radiotherapy resistance. This study demonstrated that inhibition of β1 integrin by AIIB2 monoclonal antibody reduced cellular radiation survival in vitro and in vivo, and that the FAK-cortactin complex is an important mediator of integrin-associated radioresistance (Eke et al. 2012a, 2012b). Tumor vascularity and hypoxia play an important role in the maintenance and survival of CSC populations and tumor radiation response. Therefore, targeting of the tumor

5

microenvironment is a promising strategy to improve radiation therapy outcome (Fyles et al. 2002, Evans and Koch 2003, Vaupel and Mayer 2007, Wilson and Hay 2011, Fokas et al. 2012a). Ruth J. Muschel (University of Oxford, Department of Oncology, UK) discussed an approach for altering the tumor microvascular environment to improve radiation therapy. Inhibition of the RAS/PI3K pathway before irradiation leads to a remodeling of the tumor vessels. These changes in vasculature lead to increased perfusion and decreased hypoxia as well as to improved drug delivery and better response to radiotherapy (Fokas et al. 2012b, Kelly et al. 2014). Inhibition of angiogenesis after irradiation has been suggested as a potential strategy to inhibit tumor regrowth (Harada et al. 2012). The post-radiation administration of neutralizing antibody to the Notch ligand DLL4 (delta ligand like-4), which is critical for angiogenesis, led to massive necrosis of irradiated tumors and enhanced tumor growth delay in mouse HNSCC xenograft models (Liu et al. 2011). This study suggests that coordinated delivery of irradiation and anti-vascular therapy holds promise as a way to improve radiation therapy. Andreas Androutsellis-Theotokis (Medical Faculty Carl Gustav Carus, University Hospital, TU Dresden, Center for Regenerative Therapies Dresden (CRTD), Germany) presented the discovery of a novel signaling pathway and biomarker of glioma cancer stem cells. This study revealed the transcription factor Hes3 (Hairy and Enhancer of Split 3) as an effector of a pro-survival JAK/ STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway and a regulator of proliferation of glioblastoma stem-like cells. Knockdown of Hes3 expression with RNAi inhibits the growth of glioblastoma cells (Park et al. 2013, Poser et al. 2014). In addition, recent findings suggest that JAK/STAT/Hes3 pathway also regulates neural stem cells, and Hes3 is expressed in the ocular surface, ciliary body, and in pterygium. Furthermore, radiation reduces the number of Hes3⫹ cells in the subconjunctival space and strongly promotes the nuclear localization of Hes3 in the ciliary body epithelium (Economopoulou et al. 2014). Taken together, these studies can suggest that Hes3 is a putative marker of glioblastoma and normal stem cells and JAK/STAT/Hes3 pathway plays a critical role in CSC maintenance suggesting a novel therapeutic strategy. The recent development of new techniques of ex vivo culturing of patient-derived tumor cells in conditions that closely resemble the tissue microenvironment has opened a new avenue for the improvement of diagnostics and prediction of therapy response. Serhiy Souchelnytskyi (Karolinska Institutet, Stockholm, Sweden) presented an approach for a functional molecular diagnostics (FMDx) which has been developed for use in the clinic. FMDx allows assessment of the responsiveness of individual patient’s tumors to the different treatment including chemo-and radiotherapy by testing responsiveness of living tumor samples in organ culture, analysis of circulating tumor cells, directed testing of the molecular mechanisms of treatment action, and unbiased analysis of the tumor proteome profile (Lomnytska et al. 2010, Mints and Souchelnytskyi 2011, Attarha et al. 2013, Mäbert et al. 2014).

6

A. Dubrovska

All together, the research work presented at the International Workshop ‘Cancer stem cells: The mechanisms of radioresistance and biomarker discovery’ demonstrated that implications of the CSC concept for radiobiology and radiotherapy is dynamically emerging and brings a promise to improve cancer treatment. The future translation of CSCrelated predictive biomarkers and CSC-targeted therapy into clinical application is only possible if based on a broad dialogue and close collaboration between basic and clinical scientists.

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

Declaration of interest The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.

References Ahmed KM, Zhang H, Park CC. 2013. NF-κB regulates radioresistance mediated by β1-integrin in three-dimensional culture of breast cancer cells. Cancer Res 73:3737–3748. Alexander S, Weigelin B, Winkler F, Friedl P. 2013. Preclinical intravital microscopy of the tumour-stroma interface: invasion, metastasis, and therapy response. Curr Opin Cell Biol 5:659–671. Armstrong AJ, Marengo MS, Oltean S, Kemeny G, Bitting RL , Turnbull JD, Herold CI, Marcom PK , George DJ, Garcia-Blanco MA . 2011. Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol Cancer Res 9:997–1007. Attarha S, Andersson S, Mints M, Souchelnytskyi S. 2013. Individualised proteome profiling of human endometrial tumours improves detection of new prognostic markers. Br J Cancer 109:704–713. Avoranta ST, Korkeila EA , Syrjänen KJ, Pyrhönen SO, Sundström JT. 2012. Lack of CD44 variant 6 expression in rectal cancer invasive front associates with early recurrence. World J Gastroenterol 18:4549–4556. Baccelli I, Schneeweiss A , Riethdorf S, Stenzinger A , Schillert A , Vogel V, Klein C , Saini M, Bäuerle T, Wallwiener M, Holland-Letz T, Höfner T, Sprick M, Scharpff M, Marmé F, Sinn HP, Pantel K , Weichert W, Trumpp A . 2013. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat Biotechnol 31: 539–544. Baccelli I, Trumpp A . 2012. The evolving concept of cancer and metastasis stem cells. J Cell Biol 198:281–293. Badeaux MA , Jeter CR, Gong S, Liu B, Suraneni MV, Rundhaug J, Fischer SM, Yang T, Kusewitt D, Tang DG. 2013. In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals. Cell Cycle 12:2395–2408. Baumann M, DuBois W, Pu A , Freeman J, Suit HD. 1992. Response of xenografts of human malignant gliomas and squamous cell carcinomas to fractionated irradiation. Int J Radiat Oncol Biol Phys 23:803–809. Baumann M, Krause M, Hill R. 2008. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8:545–554. Bianchi-Smiraglia A , Paesante S, Bakin AV. 2013. Integrin β5 contributes to the tumorigenic potential of breast cancer cells through the SrcFAK and MEK-ERK signaling pathways. Oncogene 32:3049–3058. Bidard FC, Peeters DJ, Fehm T, Nolé F, Gisbert-Criado R, Mavroudis D, Grisanti S, Generali D, Garcia-Saenz JA , Stebbing J, Caldas C, Gazzaniga P, Manso L , Zamarchi R, de Lascoiti AF, De Mattos-Arruda L , Ignatiadis M, Lebofsky R, van Laere SJ, Meier-Stiegen F, Sandri MT, Vidal-Martinez J, Politaki E, Consoli F, Bottini A , Diaz-Rubio E, Krell J, Dawson SJ, Raimondi C, Rutten A , Janni W, Munzone E, Carañana V, Agelaki S, Almici C, Dirix L , Solomayer EF, Zorzino L , Johannes H, Reis-Filho JS, Pantel K , Pierga JY, Michiels S. 2014. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: A pooled analysis of individual patient data. Lancet Oncol 15: 406–414. Brunner TB, Kunz-Schughart LA , Grosse-Gehling P, Baumann M. 2012. Cancer stem cells as a predictive factor in radiotherapy. Semin Radiat Oncol 22:151–174.

Bütof R, Dubrovska A , Baumann M. 2013. Clinical perspectives of cancer stem cell research in radiation oncology. Radiother Oncol 108:388–396. Cartellieri M, Bachmann M, Feldmann A , Bippes C, Stamova S, Wehner R, Temme A , Schmitz M. 2010. Chimeric antigen receptorengineered T cells for immunotherapy of cancer. J Biomed Biotechnol 956304. Chaudary N, Hill RP. 2009. Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors. Lab Invest 89:587–596. Chen X, Rycaj K , Liu X, Tang DG. 2013. New insights into prostate cancer stem cells. Cell Cycle 12:579–586. Cheng L , Ramesh AV, Flesken-Nikitin A , Choi J, Nikitin AY. 2010. Mouse models for cancer stem cell research. Toxicol Pathol 38:62–71. Cojoc M, Peitzsch C, Trautmann F, Polishchuk L , Telegeev GD, Dubrovska A . 2013. Emerging targets in cancer management: Role of the CXCL12/CXCR4 axis. Onco Targets Ther. 6:1347–1361. Cui X, Oonishi K , Tsujii H, Yasuda T, Matsumoto Y, Furusawa Y, Akashi M, Kamada T, Okayasu R. 2011. Effects of carbon ion beam on putative colon cancer stem cells and its comparison with X-rays. Cancer Res ;71:3676–3687. Ding L , Poser I, Paszkowski-Rogacz M, Buchholz F. 2012. From RNAi screens to molecular function in embryonic stem cells. Stem Cell Rev 8:32–42. Economopoulou M, Masjkur J, Raiskup F, Ebermann D, Saha S, Karl MO, Funk R, Jaszai J, Chavakis T, Ehrhart-Bornstein M, Pillunat LE, Kunz-Schughart L , Kurth I, Dubrovska A , Androutsellis-Theotokis A . 2014. Expression of the transcription factor Hes3 in the mouse and human ocular surface, and in pterygium. Int J Radiat Biol PMID:24512568. Eke I, Deuse Y, Hehlgans S, Gurtner K , Krause M, Baumann M, Shevchenko A , Sandfort V, Cordes N. 2012a. β1Integrin/FAK/cortactin signaling is essential for human head and neck cancer resistance to radiotherapy. J Clin Invest 122:1529–1540. Eke I, Dickreuter E, Cordes N. 2012b. Enhanced radiosensitivity of head and neck squamous cell carcinoma cells by β1 integrin inhibition. Radiother Oncol 104:235–242. Evans SM, Koch CJ. 2003. Prognostic significance of tumor oxygenation in humans. Cancer Lett 195:1–16. Fokas E, Im JH, Hill S, Yameen S, Stratford M, Beech J, Hackl W, Maira SM, Bernhard EJ, McKenna WG, Muschel RJ. 2012b. Dual inhibition of the PI3K/mTOR pathway increases tumor radiosensitivity by normalizing tumor vasculature. Cancer Res 72:239–248. Fokas E, McKenna WG, Muschel RJ. 2012a. The impact of tumor microenvironment on cancer treatment and its modulation by direct and indirect antivascular strategies. Cancer Metastasis Rev 31:823–842. Friedl P, Locker J, Sahai E, Segall JE. 2012. Classifying collective cancer cell invasion. Nat Cell Biol 14:777–783. Fyles A, Milosevic M, Hedley D, Pintilie M, Levin W, Manchul L, Hill RP. 2002. Tumor hypoxia has independent predictor impact only in patients with node-negative cervix cancer. J Clin Oncol 20:680–687. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A , Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A , Jones D, Raine K , Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L , Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA , Swanton C. 2012. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–892. Goel HL , Sayeed A , Breen M, Zarif MJ, Garlick DS, Leav I, Davis RJ, Fitzgerald TJ, Morrione A , Hsieh CC, Liu Q, Dicker AP, Altieri DC, Languino LR. 2013. β1 integrins mediate resistance to ionizing radiation in vivo by inhibiting c-Jun amino terminal kinase 1. J Cell Physiol 228:1601–1609. Golubovskaya VM. 2013. FAK and Nanog cross talk with p53 in cancer stem cells. Anticancer Agents Med Chem 13:576–580. Gorges TM, Tinhofer I, Drosch M, Röse L , Zollner TM, Krahn T, von Ahsen O. 2012. Circulating tumour cells escape from EpCAMbased detection due to epithelial-to-mesenchymal transition. BMC Cancer 12:178. Harada H, Inoue M, Itasaka S, Hirota K , Morinibu A , Shinomiya K , Zeng L , Ou G, Zhu Y, Yoshimura M, McKenna WG, Muschel RJ, Hiraoka M. 2012. Cancer cells that survive radiation therapy acquire HIF-1 activity and translocate towards tumour blood vessels. Nat Commun 3:783. Harkness L , Novikov SM, Beermann J, Bozhevolnyi SI, Kassem M. 2012. Identification of abnormal stem cells using Raman spectroscopy. Stem Cells Dev 21:2152–2159.

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

Report on the Cancer Stem Cell Workshop Helbig L , Koi L , Brüchner K , Gurtner K , Hess-Stumpp H, Unterschemmann K , Pruschy M, Baumann M, Yaromina A , Zips D. 2014. Hypoxia-inducible factor pathway inhibition resolves tumor hypoxia and improves local tumor control after single-dose irradiation. Int J Radiat Oncol Biol Phys 88:159–166. Hewitt HB, Wilson CW. 1959. A survival curve for mammalian cells irradiated in vivo. Nature 183:1060–1061. Hill RP, Milas L . 1989. The proportion of stem cells in murine tumors. Int J Radiat Oncol Biol Phys 16:513–518. Hwang-Verslues WW, Kuo WH, Chang PH, Pan CC, Wang HH, Tsai ST, Jeng YM, Shew JY, Kung JT, Chen CH, Lee EY, Chang KJ, Lee WH. 2009. Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. PLoS One 4(12):e8377. Hönscheid P, Datta K , Muders MH. 2014. Autophagy: Detection, regulation and its role in cancer and therapy response. Int J Radiat Biol, PMID: 24678799. de Jong MC, Pramana J, van der Wal JE, Lacko M, Peutz-Kootstra CJ, de Jong JM, Takes RP, Kaanders JH, van der Laan BF, Wachters J, Jansen JC, Rasch CR, van Velthuysen ML , Grénman R, Hoebers FJ, Schuuring E, van den Brekel MW, Begg AC. 2010. CD44 expression predicts local recurrence after radiotherapy in larynx cancer. Clin Cancer Res 16:5329–5338. Kasimir-Bauer S, Hoffmann O, Wallwiener D, Kimmig R, Fehm T. 2012. Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells. Breast Cancer Res 14: R15. Kawamoto A , Tanaka K , Saigusa S, Toiyama Y, Morimoto Y, Fujikawa H, Iwata T, Matsushita K , Yokoe T, Yasuda H, Inoue Y, Miki C, Kusunoki M. 2012. Clinical significance of radiation-induced CD133 expression in residual rectal cancer cells after chemoradiotherapy. Exp Ther Med 3:403–409. Kelly CJ, Hussien K , Fokas E, Kannan P, Shipley RJ, Ashton TM, Stratford M, Pearson N, Muschel RJ. 2014. Regulation of O2 consumption by the PI3K and mTOR pathways contributes to tumor hypoxia. Radiother Oncol (DOI: 10.1016/j.radonc.2014.02.007). Kim KJ, Lee KH, Kim HS, Moon KS, Jung TY, Jung S, Lee MC. 2011. The presence of stem cell marker-expressing cells is not prognostically significant in glioblastomas. Neuropathology 31:494–502. Klein CA . 2009. Parallel progression of primary tumours and metastases. Nat Rev Cancer 9:302–312. Koristka S, Cartellieri M, Arndt C, Bippes CC, Feldmann A , Michalk I, Wiefel K , Stamova S, Schmitz M, Ehninger G, Bornhäuser M, Bachmann M. 2013. Retargeting of regulatory T cells to surface-inducible autoantigen La/SS-B. J Autoimmun 42: 105–116. Koristka S, Cartellieri M, Theil A , Feldmann A , Arndt C, Stamova S, Michalk I, Töpfer K , Temme A , Kretschmer K , Bornhäuser M, Ehninger G, Schmitz M, Bachmann M. 2012. Retargeting of human regulatory T cells by single-chain bispecific antibodies. J Immunol 188:1551–1558. Koukourakis MI, Giatromanolaki A , Tsakmaki V, Danielidis V, Sivridis E. 2012. Cancer stem cell phenotype relates to radiochemotherapy outcome in locally advanced squamous cell headneck cancer. Br J Cancer 106:846–853. Kreso A , Dick JE. 2014. Evolution of the Cancer Stem Cell Model. Cell Stem Cell 14:275–291. Kwon GY, Ha H, Ahn G, Park SY, Huh SJ, Park W. 2007. Role of CD24 protein in predicting metastatic potential of uterine cervical squamous cell carcinoma in patients treated with radiotherapy. Int J Radiat Oncol Biol Phys 69:1150–1156. Kurth I, Peitzsch C, Baumann M, Dubrovska A . 2014. The role of cancer stem cells in tumor radioresistance. In: Rajasekhar VK , editor. Cancer stem cells. Hoboken, NJ: John Wiley & Sons. Li H, Tang DG. 2011. Prostate cancer stem cells and their potential roles in metastasis. J Surg Oncol 103:558–562. Liu SK , Bham SA , Fokas E, Beech J, Im J, Cho S, Harris AL , Muschel RJ. 2011. Delta-like ligand 4-notch blockade and tumor radiation response. J Natl Cancer Inst 103:1778–1798. Liu HK , Wang Y, Belz T, Bock D, Takacs A , Radlwimmer B, Barbus S, Reifenberger G, Lichter P, Schütz G. 2010. The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation. Genes Dev 24:683–695. Lomnytska MI, Becker S, Hellman K , Hellström AC, Souchelnytskyi S, Mints M, Hellman U, Andersson S, Auer G. 2010. Diagnostic protein marker patterns in squamous cervical cancer. Proteomics Clin Appl 4:17–31. Lundholm L , Hååg P, Juntti T, Lewensohn R, Viktorsson K . 2014. Phosphoprotein analysis reveals MEK inhibition as a way to target

7

non-small cell lung cancer tumor initiating cells. Int J Radiat Biol, PMID: 24646078. Mantoni TS, Lunardi S, Al-Assar O, Masamune A , Brunner TB. 2011. Pancreatic stellate cells radioprotect pancreatic cancer cells through β1-integrin signaling. Cancer Res 71:3453–3458. Marie-Egyptienne DT, Lohse I, Hill RP. 2013. Cancer stem cells, the epithelial to mesenchymal transition (EMT) and radioresistance: potential role of hypoxia. Cancer Lett 341:63–72. Metellus P, Nanni-Metellus I, Delfino C, Colin C, Tchogandjian A , Coulibaly B, Fina F, Loundou A , Barrie M, Chinot O, Ouafik L , Figarella-Branger D. 2011. Prognostic impact of CD133 mRNA expression in 48 glioblastoma patients treated with concomitant radiochemotherapy: A prospective patient cohort at a single institution. Ann Surg Oncol 18:2937–2945. Mints M, Souchelnytskyi S. 2011. Proteomic strategy for detection of circulating tumor cell surface antigens. Exp Oncol 33:193–195. Murat A , Migliavacca E, Gorlia T, Lambiv WL , Shay T, Hamou MF, de Tribolet N, Regli L , Wick W, Kouwenhoven MC, Hainfellner JA , Heppner FL , Dietrich PY, Zimmer Y, Cairncross JG, Janzer RC , Domany E, Delorenzi M, Stupp R, Hegi ME. 2008. Stem cell-related ‘self-renewal’ signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J Clin Oncol 26:3015–3024. Mäbert K , Cojoc M, Peitzsch C, Kurth I, Souchelnytskyi S, Dubrovska A . 2014. Cancer biomarker discovery: Current status and future perspectives. Int J Radiat Biol, PMID: 24524284. Nakata S, Phillips E, Goidts V. 2014. Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells. Cancer Manag Res 6:171–180. O’Hayre M, Degese MS, Gutkind JS. 2014. Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr Opin Cell Biol 27C :126–135. Pajonk F, Vlashi E, McBride WH. 2010. Radiation resistance of cancer stem cells: The 4 R’s of radiobiology revisited. Stem Cells 28:639– 648. Pallini R, Ricci-Vitiani L , Banna GL , Signore M, Lombardi D, Todaro M, Stassi G, Martini M, Maira G, Larocca LM, De Maria R. 2008. Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme. Clin Cancer Res. 24:8205–8212. Park DM, Jung J, Masjkur J, Makrogkikas S, Ebermann D, Saha S, Rogliano R, Paolillo N, Pacioni S, McKay RD, Poser S, Androutsellis-Theotokis A . 2013. Hes3 regulates cell number in cultures from glioblastoma multiforme with stem cell characteristics. Sci Rep 3:1095. Peickert S, Waurig J, Dittfeld C, Dietrich A , Garbe Y, Kabus L , Baumann M, Grade M, Ried T, Kunz-Schughart LA . 2012. Rapid re-expression of CD133 protein in colorectal cancer cell lines in vitro and in vivo. Lab Invest 92:1607–1622. Peitzsch C, Kurth I, Kunz-Schughart L , Baumann M, Dubrovska A . 2013. Discovery of the cancer stem cell related determinants of radioresistance. Radiother Oncol 108:378–387. Peitzsch C, Perrin R, Hill RP, Dubrovska A , Kurth I. 2014. Hypoxia as a Biomarker for Radioresistant Cancer Stem Cells. Int J Radiat Biol in press. Pilati P, Mocellin S, Bertazza L , Galdi F, Briarava M, Mammano E, Tessari E, Zavagno G, Nitti D. 2012. Prognostic value of putative circulating cancer stem cells in patients undergoing hepatic resection for colorectal liver metastasis. Ann Surg Oncol 19: 402–408. Poser SW, Park DM, Androutsellis-Theotokis A . 2014. The STAT3Ser/Hes3 signaling axis in cancer. Front Biosci (Landmark Ed) 19:718–726. Poser I, Sarov M, Hutchins JR, Hériché JK , Toyoda Y, Pozniakovsky A , Weigl D, Nitzsche A , Hegemann B, Bird AW, Pelletier L , Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K , White KP, Dietzel S, Mechtler K , Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA . 2008. BAC TransgeneOmics: A high-throughput method for exploration of protein function in mammals. Nat Methods 5:409–415. Qin J, Liu X, Laffin B, Chen X, Choy G, Jeter CR, Calhoun-Davis T, Li H, Palapattu GS, Pang S, Lin K , Huang J, Ivanov I, Li W, Suraneni MV, Tang DG. 2012. The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration. Cell Stem Cell 10:556–569. Rentala S, Chintala R, Guda M, Chintala M, Komarraju AL , Mangamoori LN. 2013. Atorvastatin inhibited Rho-associated kinase 1 (ROCK1) and focal adhesion kinase (FAK) mediated adhesion and differentiation of CD133 ⫹ CD44⫹ prostate cancer stem cells. Biochem Biophys Res Commun 441:586–592.

Int J Radiat Biol Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/15/14 For personal use only.

8

A. Dubrovska

Rycaj K, Tang DG. 2014. Cancer stem cells and radioresistance. Int J Radiat Biol, PMID: 24527669. Schober M, Fuchs E. 2011.Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-β and integrin/focal adhesion kinase (FAK) signaling. Proc Natl Acad Sci USA 108:10544–10549. Skvortsov S, Debbage P, Cho WC, Lukas P, Skvortsova I. 2014a. Putative biomarkers and therapeutic targets associated with radiation resistance. Expert Rev Proteomics 11:207–214. Skvortsov S, Debbage P, Skvortsova II. 2014b. Proteomics of cancer stem cells. Int J Radiat Biol PMID:24350919. Sinha N, Mukhopadhyay S, Das DN, Panda PK, Bhutia SK. 2013. Relevance of cancer initiating/stem cells in carcinogenesis and therapy resistance in oral cancer. Oral Oncol 49:854–862. Smit JK, Faber H, Niemantsverdriet M, Baanstra M, Bussink J, Hollema H, van Os RP, Plukker JT, Coppes RP. 2013. Prediction of response to radiotherapy in the treatment of esophageal cancer using stem cell markers. Radiother Oncol 107:434–441. Stanton MJ, Dutta S, Zhang H, Polavaram NS, Leontovich AA, Hönscheid P, Sinicrope FA , Tindall DJ, Muders MH, Datta K. 2013. Autophagy control by the VEGF-C/NRP-2 axis in cancer and its implication for treatment resistance. Cancer Res 73:160–171. Soeda A, Park M, Lee D, Mintz A , Androutsellis-Theotokis A , McKay RD, Engh J, Iwama T, Kunisada T, Kassam AB, Pollack IF, Park DM. 2009. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene 28: 3949–3959. Sottoriva A, Spiteri I, Piccirillo SG, Touloumis A , Collins VP, Marioni JC, Curtis C, Watts C, Tavaré S. 2013. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci USA 110:4009–4014. Sprenger T, Conradi LC, Beissbarth T, Ermert H, Homayounfar K, Middel P, Rüschoff J, Wolff HA, Schüler P, Ghadimi BM, Rödel C, Becker H, Rödel F, Liersch T. 2013. Enrichment of CD133-expressing cells in rectal cancers treated with preoperative radiochemotherapy is an independent marker for metastasis and survival. Cancer 119:26–35. Sun YF, Xu Y, Yang XR, Guo W, Zhang X, Qiu SJ, Shi RY, Hu B, Zhou J, Fan J. 2013. Circulating stem cell-like epithelial cell adhesion moleculepositive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology 57:1458–1468. Sung CO, Park W, Choi YL, Ahn G, Song SY, Huh SJ, Bae DS, Kim BG, Lee JH. 2010. Prognostic significance of CD24 protein expression in patients treated with adjuvant radiotherapy after radical hysterectomy for cervical squamous cell carcinoma. Radiother Oncol 95:359–364.

Tang DG. 2012. Understanding cancer stem cell heterogeneity and plasticity. Cell Res 22:457–472. Tinhofer I, Saki M, Niehr F, Keilholz U, Budach V. 2014. Cancer stem cell characteristics of circulating tumor cells. Int J Radiat Biol, PMID:24460132. Trautmann F, Cojoc M, Kurth I, Melin N, Bouchez LC, Dubrovska A , Peitzsch C. 2014. CXCR4 as biomarker for radioresistant cancer stem cells. Int J Radiat Biol, PMID:24650104. Trott KR. 1994. Tumour stem cells: The biological concept and its application in cancer treatment. Radiother Oncol 30:1–5. Uckermann O, Galli R, Anger M, Herold-Mende C , Koch E, Schackert G, Steiner G, Kirsch M. 2014. Label-free identification of the glioma stem-like cell fraction using Fourier-transform infrared spectroscopy. Int J Radiat Biol, PMID: 24597751. Vaupel P, Mayer A . 2007. Hypoxia in cancer: Significance and impact on clinical outcome. Cancer Metastasis Rev 26:225–239. Wang Q, Chen ZG, Du CZ, Wang HW, Yan L , Gu J. 2009. Cancer stem cell marker CD133 ⫹ tumour cells and clinical outcome in rectal cancer. Histopathology 55:284–293. Wermke M, Camgoz A , Paszkowski-Rogacz M, Thieme S, von Bonin M, Dahl A , Theis M, Ehninger G, Brenner S, Bornhäuser M, Buchholz F. 2013. A genome wide shRNA screen in primary human AML cells identifies ROCK1 as a novel therapeutic target. Blood 122:170. Wilson WR, Hay MP. 2011. Targeting hypoxia in cancer therapy. Nat Rev Cancer 11:393–410. Wurdak H. 2012. Exploring the cancer stem cell phenotype with high-throughput screening applications. Future Med Chem 4: 1229–1241. Wurdak H, Zhu S, Romero A , Lorger M, Watson J, Chiang CY, Zhang J, Natu VS, Lairson LL , Walker JR, Trussell CM, Harsh GR, Vogel H, Felding-Habermann B, Orth AP, Miraglia LJ, Rines DR, Skirboll SL , Schultz PG. 2010. An RNAi screen identifies TRRAP as a regulator of brain tumor-initiating cell differentiation. Cell Stem Cell 6:37–47. Yeung TM, Gandhi SC, Bodmer WF. 2011. Hypoxia and lineage specification of cell line-derived colorectal cancer stem cells. Proc Natl Acad Sci USA 108:4382–4387. Zips D, Zöphel K , Abolmaali N, Perrin R, Abramyuk A , Haase R, Appold S, Steinbach J, Kotzerke J, Baumann M. 2012. Exploratory prospective trial of hypoxia-specific PET imaging during radiochemotherapy in patients with locally advanced head-and-neck cancer. Radiother Oncol 105:21–28.