Cancer Stem Cells as a Predictive Factor in Radiotherapy

Cancer Stem Cells as a Predictive Factor in Radiotherapy Thomas B. Brunner, MD,* Leoni A. Kunz-Schughart, PhD,† Philipp Grosse-Gehling,† and Michael Baumann, MD, PhD†,‡ Cancer stem cell research is one of the most thriving and competitive areas in oncology research because it has the potential to dramatically affect clinical outcomes. Led by progress in hematology, cancer stem cell research has now provided evidence to play an important role for solid cancers as well. Because radiotherapy is only second to surgery in terms of its curative potency, it is very important for radiation oncologists to learn whether progress in cancer stem cell biology can enable them to exploit this knowledge to help cure more patients suffering from cancer. The present article gives an overview about the challenges of the cancer stem cell concept and highlights some important phenomena that are under intense investigation, such as phenotypic plasticity of stemness and impact and dynamics of microenvironmental niches. We discuss the potential and limitations of current experimental and theragnostic tools and end up with an agenda for future research as outlook for translational possibilities in the clinic. Semin Radiat Oncol 22:151-174 © 2012 Elsevier Inc. All rights reserved.

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ne key issue in cancer research and treatment during the past decade has been whether the persistent growth of malignant tumors is driven by substantial proportions of tumor cells or exclusively by rare subpopulations, frequently termed cancer stem cells (CSCs). This question has been at the heart of radiation oncologists and radiation biologists for decades.1,2 Among others, this was spurred by the clinical observation of complete response after radiotherapy followed by local in-field relapse at significant intervals after the end of

*Department of Oncology, Gray Institute for Radiation Oncology and Biology, Oxford, UK. †OncoRay–National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. ‡Department of Radiation Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. Supported in part by the Deutsche Forschungsgemeinschaft (KU971/7-1 to L.A.K-S., BA1433 to M.B.), MRC (grant code G0700730 to T.B.B.), and the NIHR Biomedical Research Centre, Oxford (to T.B.B.). OncoRay is funded by the BMBF in the program “Center for Innovation Competence.” Thomas B. Brunner and Leoni A. Kunz-Schughart contributed equally and share first authorship. Address reprint requests to Michael Baumann, MD, PhD, Department of Radiation Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Dresden University of Technology, Fetscherstraße 74, PO Box 50, 01307 Dresden, Germany. E-mail: [email protected]

1053-4296/12/$-see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.semradonc.2011.12.003

treatment. Such postradiotherapy recurrences can occur up to more than 10 years after therapy in some tumor sites such as breast cancer3 and prostate cancer.4 More frequently and usually much earlier, recurrences may occur in most tumor sites where we apply radical radiotherapy or combined radiochemotherapy (eg, glioma, head and neck cancer, lung cancer, esophageal cancer, cervical cancer, anal cancer; Fig. 1). There are 2 main reasons why radiation oncologists and radiobiologists recognized the clinical relevance of CSCs earlier than oncologists involved exclusively in systemic treatments: (1) (chemo)radiotherapy is much more likely to achieve complete local response than chemotherapy,27 and (2) radiotherapy is much more often used with curative intent, compared with chemotherapy, which is predominant in the palliative and adjuvant setting. Nevertheless, the CSC hypothesis is relevant to both fields as demonstrated by the fact that stem cell markers could be identified, which predict for distant metastasis.15 Indeed, successfully targeting CSCs inducing distant metastasis may eventually strengthen the role of radiotherapy for tumor sites, where distant spread is a significant problem, such as in lung, rectal, or pancreatic cancer. Taken together, the recent developments in the field of CSC research, which has recognized CSCs as a source of local or distant relapse, are of highest importance for radiation oncology with the potential of significant therapeutic benefit to our patients. 151

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Figure 1 Examples of solid tumor types for which cancer stem cells were identified from primary tumors and for which radiotherapy is used for treatment (brain,5,6 head and neck,7 breast,8-10 lung,11 esophagus,12,13 liver,14 pancreas,15,16 bladder,17 cervix,18,19 colorectal,20-22 prostate,23 and melanoma24). This illustrates that on one hand, a few biomarkers (eg, CD44 and CD133) dominate as tools to identify or enrich the putative CSC populations from a broad range of cancers, whereas on the other hand, there is significant variety between tumor types (eg, breast vs pancreatic with CD24⫺ vs CD24⫹). This list is nonexhaustive and continues to grow. At the same time, it became clear in many instances that the classical surface surrogate markers may fail to discriminate cancer stem and non–stem cell subpopulations (details mentioned in the text). For example, the existence of glioma/glioblastoma stem cell populations with CD133⫺ phenotype is now well accepted.25,26

Radiation biology has always attributed CSCs a central place for therapeutic response. Traditionally, several different terms have been used for these cells that may cause recurrences after irradiation, such as tumor stem cells, tumor rescuing units, and in vivo clonogenic cells. In fact, the gold standard assay for tumors in vivo used in radiobiology for decades tests the therapeutic impact on CSCs. As a single surviving CSC can induce recurrent growth, the tumor control probability assay (TCD50, tumor control dose 50%), which determines the rate of local control after different radiation doses and treatments, tests the effect of radiation on CSCs.28 Other frequently used in vivo assays (ie, assessment of tumor volume and regression, or the tumor (re)growth assay) assess proliferation and do not ultimately test the effect of therapy on CSCs. The difference between the 2 assays was recently impressively illustrated by the differences found between 2 strategies to inhibit epidermal growth factor receptor (EGFR) signaling, the use of antibody cetuximab or the tyrosine kinase inhibitor erlotinib.29 Both agents led to a significant tumor regrowth delay when used together with radiotherapy; however, only cetuximab led to a shift of the TCD50 curve to the left indicating that more CSCs are sterilized. This is in line with a host of experiments on combined molecular targeting with radiotherapy approaches that show dissemination of volume-dependent and CSC-dependent endpoints.28,30-34 Clonogenic assays are the keystone radiobiology in vitro experiments, addressing the question of how many colonyforming cells are left after a specific dose of radiation.35 Although colony-forming cells are conceptually not the same as

CSCs, this assay nevertheless is related to the ability of cells to produce progeny over several generations.36 In contrast, proliferation assays, such as the MTT ((3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide based) assay, which are typically used to test the efficacy of chemotherapeutic agents, are not accepted to be of relevance to measure the effects of radiation on cancer cells.37,38 This article will look at CSCs in the perspective of the subpopulations of tumor cells that are responsible for local relapse after radiotherapy. We will discuss the usefulness and limitations as well as functional and predictive value of current surrogate biomarkers and experimental tools in CSC research for experimental and clinical radiotherapy and particularly highlight new insights linking radiotherapeutically relevant microenvironmental constraints to cancer stemness phenotype and plasticity.

At a Glance: The CSC Concept The human CSC model was particularly supported by in vivo transplantation experiments showing that only minor proportions of human malignant cells produce xenografts in immunodeficient mice. Hence, in 2006, a consensus definition resulting from the Cancer Stem Cell Workshop of the American Association for Cancer Research was published describing CSCs as a small subset of cancer cells within the tumor mass, which constitutes a reservoir of self-sustaining cells with exclusive ability of self-renewal and tumor maintenance. Only this small subset of cells was interpreted to have the capacity to divide and expand the CSC reservoir as well as

CSCs as a predictive factor in radiotherapy to differentiate into the heterologous nontumorigenic lineages.39 In analogy to knowledge in normal tissue stem cell biology and hierarchy, it was assumed that the major compartment of the CSC pool was non- or slowly dividing and thus not susceptible to conventional chemotherapeutic strategies directed against the (rapidly) cycling tumor cell fractions. Subsequently, an enormous effort was undertaken to identify, isolate, and characterize the cell population(s) of interest using different putative surrogate markers to convincingly prove the principle concept in various tumor entities and establish new, CSC-directed treatment options. This turned out to be more challenging for various reasons, some of which shall be discussed herein. One of the problems leading to confusion and controversial discussion in the field is just semantics. Another one relates to the rather imperfect tools to detect the putative CSCs. In particular, the specificity of the biomarkers that may or may not have functional relevance and/or play a role in embryogenesis and developmental processes is disputed; the in vivo and in vitro assays and readout systems for human cancer stemness also seem critical. And last but not least, the cancer cells’ potential for phenotype switching and plasticity induced by epigenetic and microenvironmental constraints supposedly leading to loss of a clear cellular hierarchy in tumors has been underestimated and is now subject of intense examination. These issues of the CSC paradigm have been highlighted in numerous review articles.28,36,40-47 As attractive as the concept sounds for establishing new treatments, we will argue in this article that the exclusive targeting of rare populations based on an exclusive steadystate stem-like phenotype is unlikely to sufficiently enhance cancer cure compared with what we can achieve with radio(chemo)therapy today. However, motivated by the contended CSC concept, a concerted effort to link genotypes, phenotypes, and reversible epigenetic mechanisms, including environmental constraints, with in vivo malignant potential and therapy response provides the greatest hope for advancing treatment outcome. Concepts of functional plasticity and clonal evolution driven by the environment must be incorporated into the current promising attempts to identify CSC-selective targets and treatment strategies to be combined with radiotherapy.

Problems of Semantics/Terminology Viewing cancers from the developmental perspective has clearly led to new insights in cancer biology. However, the implication of the term CSC to define cancers as a disease of stem cells is simplistic and challenged. Indeed, in most cancers, the origin of the putative cancer stem-like cell population remains elusive. There is evidence that they may derive either from transformed tissue stem cells or transformed progenitor cells in some tumor entities, but origination from differentiated organ-specific cells through genetic reprogramming can also not be ruled out.48-50 The finding that both embryonic and adult stem cells not only have the ability

153 for self-renewal and to produce differentiated progeny but also to undergo cell fusion during normal development and regeneration has been comprehensively discussed in several reviews and a recent book series.51 Cell fusion has also been proposed as a mechanism involved in the initiation and progression of cancer and in the generation of recurrent CSC populations because spontaneous fusion between stem cells, and of stem cells with various differentiated cell types, has been observed.52-55 Although still speculative, this clearly adds another degree of complexity to the CSC concept. In any case, irrespective of their cell of origin, cancer cells leading to recurrence of the disease supposedly share some key response elements with stem and progenitor cell populations, intrinsic to their stemness phenotype. These traits may include characteristics as diverse as expression of telomerase, high levels of DNA repair enzymes, or low uptake and exclusion of Hoechst33342. At this point, it is important to also clarify the terminology of the use of the expression “tumor initiating cell” (TIC) and “cancer stem cell” because these 2 terms have not always been clearly separated.5,20,56 However, as the CSC hypothesis and the clonal evolution hypothesis appear to be not mutually exclusive,57 but rather coexisting and interactive, the 2 terms should now be used in separate ways: TIC is preferable for a progenitor during the initiation in the multistep process of tumorigenesis. During the next step of tumorigenesis, the preneoplastic stage (eg, intraductal neoplasias, hyperplasia, or metaplasia), pre– cancerous stem cells represent an early stage of CSC development having the potential of both benign and malignant differentiation.58 The term CSC should be restricted to cancerous lesions, and careful distinction between these terms will help to avoid confusion. This appears also to be a necessity, considering the difference between tumors and cancers. Cancers are defined as malignant disease, whereas tumors can either be benign or malignant. However, these terms are often used interchangeably which can be confusing when describing the cell population of interest.59 Other designations such as “tumor propagating cell” or “cancer propagating cell” (TPC/CPC) are defined from functional aspects, as they describe a cell that is able to induce a tumor specifically in a xenograft model.60 In many experimental studies, use of one of these terms may be advised because xenograft formation is monitored but cancer stemness with all its facets is not proven. Awareness of these subtle differences and proper terminology is recommended as a good compass navigating through the sometimes foggy sea of the CSC research field.

Limitations and Challenges of Functional Assays Principal experimental tools and the workflow to study cancer cell subpopulations are depicted in Figure 2. The gold standard to identify human CSCs still is the in vivo assay of the tumor-propagating potential of tumor cell subpopulations in immunodeficient mice. Here, CSCs are represented by their capability to initiate in vivo tumors with all differen-

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Figure 2 An illustration of the experimental toolbox to identify, isolate, and characterize cancer stem cell populations. The top panel indicates which types of tumor material are available to study cancer stem cells. Primary tumors are regarded to play an essential role in cancer stem cell research. The middle panel illustrates methods for identification and isolation of cancer stem cells, demonstrating techniques for isolation relying on the biomarkers that are illustrated on the right side. The bottom panel provides an overview of in vitro and in vivo assays that are commonly used to test the defining criteria of cancer stem cells (self-renewal and recapitulation of heterogeneity). Whereas the choice of the tumor material is a critical initial step, identification and isolation can proceed or follow assays testing stem cell properties, and this is expressed by the bidirectional arrows between the 2 boxes. ALDH, acetaldehyde dehydrogenase; FACS, fluorescence-activated cell sorting; GEM, genetically engineered mice; RH, recapitulation of heterogeneity; MACS, magnetic cell sorting; SP, side population (Hoechst33342 dye exclusion assay); SR, self-renewal; TCD50 assay, tumor control dose 50% (the radiation dose which is necessary to control 50% of the tumors).

tiated cellular populations of the primary tumor and to be serially transplantable to demonstrate self-renewal. Setup and handling of such assays have to be done with care because interpretation of the data can be quite demanding. As an example, it was initially thought that CSCs were infrequent in solid tumors (0.1%-0.0001%) because large numbers of human cancer cells were required to generate xenografts. Numerous studies were published in which researchers calculated frequencies of human tumorigenic cells in the bulk tumor mass, not taking into account that the nonhuman, mouse environment might be a limiting parameter in this scenario. However, in 2008, Quintana et al showed that tumor formation of malignant melanoma cells differed by several orders of magnitude in common non-obese diabetic/severe combined immune-deficient (NOD/SCID) versus more highly immunocompromised (Il2R␥[⫺/⫺])NOD/SCID mice. Tumor take was further enhanced by optimizing the implantation protocol, that is,

by using Matrigel as a supplement to provide more permissive environmental conditions on injection.61 With this mouse strain and preparation protocol, they were able to engraft unselected single melanoma cells from patients with a frequency of 27%. It was concluded that the NOD/ SCID mouse assay underestimates the frequency of tumorigenic human cancer cells and that malignant melanoma stem cells may not be a rare population.61,62 The data clearly reveal that the estimated frequencies of human CSCs crucially depend on the immune status of the recipient. However, the relationship may not be as simple because inflammatory cells may promote tumor growth by producing conducive cytokines and proangiogenic factors. The finding of remnant immune reactivity to affect human tumor cell engraftment is not new. Accordingly, whole-body irradiation is frequently performed to suppress the extant immune response in nude mice before (human) cell trans-

CSCs as a predictive factor in radiotherapy plantation as initially described and systematically studied by Suit et al in the late 1980s and early 1990s using quantitative orthotopic and subcutaneous transplantation assays.63-67 Immune suppression, however, is sustained only for weeks. As an example, in a human soft-tissue sarcoma xenograft model, TD50 (tumor take dose 50%) values in nude mice that received injection 12 weeks after whole-body irradiation and those which were not irradiated did not differ but were significantly higher than in mice injected 1 day, 4 weeks, or 8 weeks after irradiation.67 Such limitation can be critical for monitoring CSC engraftment where very low cell numbers and extended observation periods may be required (palpable xenografts may develop ⬎70 days after injection). Indeed, the immunogenicity of human cancer cell subpopulations isolated according to putative CSC biomarkers has not been examined vigorously in mouse models. At the same time, it remains rather elusive whether amelioration of intrinsic immune escape mechanisms contributes to the stemness phenotype of human cancer cell subpopulations. Although residual immune reactivity will affect the transplantability of tumors in laboratory animals, successful transplantation could be achieved with one or few tumor cells for some tumors. However, this does not prove that all tumor cells are CSCs or that the stemness of all cancer cells is the same. Important arguments against these assumptions include differences in radiation curability of tumors, which correlated with estimates for CSC number in the tumors treated, with colony-forming ability of cells originating from these tumors or with expression of so-called CSC markers.28,68 The pivotal role of environmental conditions on tumor take is further reflected by differences in site-specific human cancer cell grafting efficiencies (eg, when comparing studies using subcutaneous or intracranial engraftment of brain cancer stem-like cells or tumor formation of selected colon cancer cell subpopulations injected either subcutaneously or into the renal capsule).69 Indeed, it is well known that tumor engraftment and tumor take dose 50% depend on the injection sites and are not identical for heterotopic versus orthotopic implantations.64-66 However, systematic comparative studies on CSC behavior in heterotopic and orthotopic sites are limited, and tumor formation capacity of candidate human cancer cell populations has most frequently been analyzed on subcutaneous injection. This seems reasonable because ectopic location did not prevent the CSCs from generating differentiated progeny resulting in heterogeneous histomorphologies and cellular functions.7,21,69 However, the limitations of such models have to be kept in mind. Even if many characteristics of the primary tumor are reflected in xenografts, there are also important differences. It seems reasonable to assume that the characteristics observed in xenograft tumors may primarily relate to reversible and irreversible phenotype switching induced by local microenvironmental constraints that are, of course, also present in the original tumor and highly relevant in radiotherapy treatment testing. Murine models of cancer overcome some of the problems described earlier in the text and have been applied to study the CSC hypothesis and the biological variability of CSCs in different disease subsets.70 Literature data analysis suggests

155 that the frequency of cancer cells with self-renewing capacity is higher in syngeneic than in xenogeneic mouse assays, again indicating that human CSCs may be underestimated in common nude mouse strains. The basic concept of cancer cell subpopulations with exclusive cancer stemness and propagating potential, however, is supported in murine systems. This is not only true for models of acute myeloid leukemia71,72 but also for solid cancers such as breast tumors developing in p53-null or mouse mammary tumor virus– Wnt-1 mice,73,74 or medulloblastomas observed in Ptc⫹/⫺ mutant mice.75 The fact that CSC compartments can be defined in these immunocompetent models makes them a valuable tool in cancer research. Their usefulness to test new therapeutic strategies directed against human CSC-specific targets and phenotypes may of course be limited. The inability of murine models to reproduce the full range of speciesspecific factors, both paracrine and membrane bound, has to be considered in experimental design and outcome evaluation. Altogether, collective evidence throughout the past years suggests that tumor growth may indeed be sustained by either rare or dominant cancer cell subpopulations that may be defined as CSCs, as they show self-renewal capacity, tumor-propagating potential, and cellular plasticity critically affected by the local microenvironment.45,47 In vivo studies are often combined with in vitro sphereforming assays that have been interpreted as surrogate for the in vivo transplantation assay. Here, the number of spheres formed from single cells is often used to estimate the CSC fraction. The basis of the sphere-forming assays in CSC research is the neurosphere culture method under serum-free conditions and its potential to maintain and expand brain stem and progenitor cells with self-renewal and differentiation potential in vitro.76,77 Sphere cultures of brain tumors were thus among the first to be grown under such conditions. With this approach, in 2004, both Galli et al78 and Yuan et 79 al successfully isolated small subpopulations of glioblastoma multiforme cells with particular self-renewal capacity, potential to differentiate into multilineage progenies, capacity to form spheres and serial subspheres, and potential to produce tumors in athymic mouse models. Many others confirmed the maintenance of a stem-like behavior and enhanced in vivo tumorigenicity both when using the entire cellular material or defined cell subpopulations from tumor biopsies.5,6,56,80-86 The data imply neurosphere formation to be a robust and independent predictor of glioma tumor progression and brain cancer stemness. Meanwhile, sphere formation assays were adapted to various other malignancies often termed according to their tumor tissue of origin (eg, mammospheres,87-90 colo- or colonospheres,20,21,91-94 prostaspheres,95 bronchospheres,96 and even squamospheres for the culture of squamous cell carcinoma (SCC) stem-like cells97 or melanospheres in melanoma stem cell research.98) However, the technology has numerous pitfalls and limitations as highlighted in 2 recent review articles.77,99 Handling is delicate, and the experimental procedure has to be adjusted to obtain sphere formation from single cells only and to avoid artefacts, for example, due to cell-cell adherence. Medium composition, cell density, and therewith medium

156 volume, culture dish surface area, and the period of observation are just some of the critical parameters.77 Also, the dissociation and preparation of high-quality single cell suspensions from primary tumor tissue, xenografts, and 3-dimensional sphere cultures without loss of cell quality can be challenging even if cells are not further processed for magnetic or fluorescenceactivated cell sorting for the isolation of subpopulations of interest. Last but not least, there is increasing evidence that the sphere assay culture conditions may be selective for a particular (cancer) stem cell phenotype and not reflect all facets of niche conditions promoting cell renewal. Thus, the key element of the neurosphere culture paradigm that nonadherent plating and serum-free conditions are required to inhibit cell differentiation and maintain stemness has recently been called into question because of the development of normal and malignant neuronal stem cell cultures from primary tissues that could be successfully isolated and propagated on adherent laminin-coated surfaces.99-101 These approaches will be further evaluated and may substitute for the primary cell sphere assay, which is fragile and not expected to become routine in experimental radiotherapy any time soon. The field of CSC research is dominated by the use of primary cancer cells because the biological properties of established cell lines may have changed throughout culture. Among others, loss of gene amplification102,103 as well as alterations in the ability to migrate and metastasize,104 to maintain the distinct sense of a stem-like cell population,104 and to preserve the dependency on embryonic signaling pathways105 have been described. Most importantly, these properties are often not restored when established cell lines are grown as xenografts. One example of the biological consequence of the differences between primary xenografts and matched cell lines is the marked difference in sensitivity to standard chemotherapeutic agents in colon cancer.106 Of course, the response to chemotherapeutic agents is not specifically related to CSCs.106 However, it demonstrates that caution is necessary when comparing patients’ responses to therapy with matched established cell lines and that similar observations can be expected in the field of CSCs. This was recently shown in a small-cell lung cancer study, in which 3 scenarios were compared: primary xenografts, matched offspring cell lines, and xenografts obtained from these cell lines. Gene expression revealed that a group of tumor-specific genes was only expressed in the primary cancer and xenografts, but lost during transition to tissue culture. Reinjecting the cell line to form xenografts did not lead to reexpression of these genes.107 In this context, it is also debated whether primary tumor fragments implanted and serially passaged in mice without any intermediate step of culture in vitro may be more suitable for therapy testing and maintenance of human CSC pheno- and genotypes.108 Despite of this criticism of the use of established cell lines for CSC research, cell lines are a very useful tool because they are readily available, and cultures are reproducible and not contaminated with stromal cells. Although they do not reflect the heterogeneity of tumor tissue, they provide a place to start to test a hypothesis, which can later be validated using primary cells and tissues. The lack of the CSC phenotype uni-

T.B. Brunner et al formity in CSCs by using surface markers in established cell lines has often been regarded as a proof that cell lines are inadequate for CSC research. However, it is now becoming accepted that phenotypic heterogeneity within CSC subpopulations is highly likely to exist.57 For instance, it was possible to propagate glioblastomas from CD133⫺ cells in a study in which the cells were cultured as adherent spheres and cell lines before transplantation109 although CD133⫺ tumor cells appeared to have a distinct molecular profile compared with the CD133⫹ cells. Similarly, sorting the established breast cancer cell lines MDA-MB231 and MDA-MB468 for CD24⫺/endothelial-specific antigen (ESA⫹) cells led to a similar fraction of CSCs (0.48% and 0.7%, respectively), but functionally only MDA-MB231 CD24⫺/ESA⫹ cells had a higher clonogenic survival after radiotherapy and not MDAMB468.110 This observation supports the view that the CSC phenotype may not be uniform between cancer subtypes or even tumors of the same subtype. In a patient with a comedo type adenocarcinoma, for instance, the tumor-propagating subfraction was CD44⫹CD24⫹EpCAM⫹ (epithelial cellular adhesion molecule) as opposed to cancer cells that were CD24⫺ and positive for the other 2 surface markers (Note: ESA, endothelial-specific antigen; EpCam, epithelial cellular adhesion molecule; CD326). A recent study compared colorectal cancer cell lines using the cell surface markers CD44 and CD24 as well as a Matrigel-based cell differentiation assay to investigate CSCs.111 Megacolonies from SW1222 in Matrigel had complex 3-dimensional structures resembling colonic crypts differentiating into enterocytes, enteroendocrine, and goblet cell lineages. After desegregation of putative CSCs from megacolonies, single cells were able to self-renew and differentiate. Xenograft tumors from CD44⫹CD24⫹ megacolonies developed in NOD/SCID mice, showing a histology similar to that of a well-differentiated primary human lumen-forming tumor. These observations were compared with HCT116, a highly aggressive cell line with almost no capacity to differentiate, and with HT29, which possesses an intermediate capacity to differentiate. In line with the morphology, HCT116 did not express the differentiation marker CDX1, whereas only few cells in HT29 colonies expressed this marker. Forced expression of CDX1 in HCT116, on the other hand, reduced HCT116 clonogenicity and induced lumen formation within the colonies. This study suggests that HCT116 cells contain mainly CSCs, which is in line with the high tumorigenic potential of this cell line in vivo,112 whereas SW1222 cells had only 0.5%-1% of CD44⫹CD24⫹ cells at flow cytometric analysis. In conclusion, certain cell lines appear to be well suited for CSC experiments, whereas other cell lines fail current functional tests to discriminate CSC subfractions from non-CSC fractions. Indeed, it is reasonable that some of the cell lines have maintained or even selected for an eligible CSC fraction under standard culture conditions as indicated by their distinct tumorigenic potential in vivo. This may be in line with earlier studies in experimental radiotherapy that have identified the number of clonogens as a parameter reflecting in vivo tumor establishment and growth and resistance to therapy (re-

CSCs as a predictive factor in radiotherapy viewed by Milas and Hittelman).113 Although cancer cell clonogens and CSCs are functionally defined by different endpoints (vide supra) and do not necessarily reflect identical cells, it is likely that there is a link between clonogenic survival capacity and stemness phenotype in cell lines under certain (patho)physiological constraints and that the ability to propagate a solid or metastatic tumor is not exclusive to cells exposed to serum-free conditions in vitro. Therefore, one important task for the future is to identify suitable cell line models for in vitro and in vivo use to test CSC-relevant questions in solid tumors. On the other hand, functional assays (eg, Matrigel differentiation assays or CSC enrichment assays after chemotherapy or radiotherapy) could be useful to identify novel biomarkers. Serum-free sphere culture protocols have been applied as one approach to in vitro enrich putative CSC from a variety of established cell lines that were propagated a priori as monolayers in classical media for extended periods.114 However, the sphere-forming assay has clear limitations as discussed earlier. Ultimately, biomarker candidates will have to be validated in human tissue and/or primary tumor cells.

CSC Surface Biomarkers: Current Controversies and Relevance in Radiotherapy The identification and isolation of cancer cells with stem (-like) characteristics often relies on surrogate biomarkers. Various biomarkers ranging from surface antigens to enzyme and membrane transporter activities have been used in research related to the CSC hypothesis and are critically discussed as tools to identify cancer cell populations and/or phenotypes with exclusive tumor-propagating potential or stem-like features. Comprehensive lists of biomarkers used for the identification or isolation of putative CSCs in different tumor entities are given in several recent review articles.40,70,115-118 The expression or positivity for such markers, however, may greatly differ between samples, and they may not be present in all tumors even of 1 entity. The most frequently used markers and marker profiles to identify the CSC pool in various cancer tissues are summarized in Figure 1. In this chapter, we will exemplify some of the challenges when using such markers and take them further into the context of radioresponse. CD133 (prominin-1) is a 92- to 110-kDa pentaspan membrane glycoprotein, and its use as a putative CSC biomarker was primarily based on work in glioblastomas followed by 2 reports in 2007 that independently showed CD133 to enrich for colorectal cancer cells that are capable of promoting and sustaining tumor growth in murine xenograft models.20,21 However, this was challenged by contradictory observations from Shmelkov et al119 who reported CD133-negative cells from colon cancer metastases to also be capable of inducing xenograft tumors in NOD/SCID mice. Also, there is clear evidence for the existence of CD133-negative glioma/glioblastoma stem cell (GSC) subpopulations.25,26 The functional relationship between CD133 protein and putative cancer cell

157 stemness and plasticity remains elusive.116,119,120-124 Detection of CD133 expression turned out to be quite demanding because CD133 profiling depends on posttranslational modifications of the protein.125 Furthermore, the most frequently used antibody to select for CD133 positivity was recently found to detect a different epitope than initially thought, and antibody binding was critically affected by the glycosylation status and folding of the protein. Positive staining thus reflects the status of the protein but not its expression per se and may also be affected by various treatments.116,125 Today, it is hypothesized that particular antibody-binding epitopes are lost on colorectal CSC differentiation.125,126 Several recent studies showed a correlation between the presence of CD133-expressing cells in primary colorectal cancer or residual rectal cancers after radiochemotherapy and recurrence of disease and/or poor survival, indicating that a cell fraction that stained positive with a particular antibody somehow relates to or reflects response to treatment.127-132 This could argue for further investigating the relevance of CD133 in antitumor therapy response. However, such experiments would have to be designed with great caution. Based on our current knowledge with colorectal cancer cell lines (unpublished data from Kunz-Schughart et al. and Ref. 112), it seems unlikely that the molecule per se has any functional relevance in radioresistance. CD24 is a glycosylphosphatidylinositole-anchored protein that binds to the ligand P-selectin (CD62P). It is highly and differentially glycosylated in different cell types, with a molecular weight ranging from 25 to 75 kDa; in normal epithelial cells, CD24 is about 35-45 kDa.133 The molecule is relevant for the maturation of hematopoietic cells and during embryonic development. In breast cancer, lack of CD24 is implicated in invasiveness, metastasis, and cancer cell stemness,134,135 whereas it seems of minor importance for identifying CSCs in other tumor entities. In colorectal cancer and in other malignancies, CD24 was frequently overexpressed and is considered a potential oncogene and therapeutic target.136,137 Therefore, it is difficult to relate CD24 to radioresponse in general without taking the particular tumor entity into account. Despite its use as a marker to identify breast CSC enrichment after treatment, there is no evidence that CD24 protein directly affects radioresponse. CD49f is the ␣ chain of very late antigen 6 or ␣6 subunit of integrins and primarily forms complexes with CD29 (very late antigen ␤1 chain ¡ ␣6ß1) and ␤4 integrin (¡ ␣6ß4). The ␣6␤1 complex is the main receptor for laminin-induced outside-in signaling (haptotaxis). The complexes play a role in reproduction, epithelial integrity, and organogenesis. CD49f was found to be coexpressed with conventional glioma stem cells markers and to enrich for glioma cell population, and targeting of CD49f inhibited self-renewal, proliferation, and tumor formation capacity.138 CD49fhigh cell populations were reported to be enhanced during prostate cancer initiation and progression,139 and both CD29 as well as CD49f have been discussed as putative mammary gland stem cell biomarkers.140 In colorectal cancer cells, CD49f was identified as one of the integrin subunits (in addition to ␣2 and ␤4) involved in extravasation because functional block-

158 ade resulted in reduced cancer cell extravasation and colon cancer migration in an animal model system.141 One study revealed that ␣6 integrin cleavage can sensitize human prostate cancer cells to ionizing radiation.142 CD29 has been implicated in resistance to cytotoxic chemotherapy and radiation by mediating key survival signals after irradiation through various integrin signaling pathways, and ␤1 integrin inhibition has thus recently been proposed as a promising strategy for radiosensitization.143 CD166 (activated leukocyte cell adhesion molecule) belongs to the immunoglobulin superfamily of cell adhesion molecules and plays a role in the maintenance of tissue architecture. It is described as a mesenchymal stem cell marker that is expressed in cells of all 3 embryonic lineages, but expression is limited to subsets of cells in most adult tissues (epithelium, endothelium). In normal epithelium, the N-glycosylated CD166 (110 kDa) is localized at intracellular junctions presumably as part of the adhesive complex. CD166 has been implicated in the progression of various cancers, but the data are contradictory and seem not only to depend on tumor entity but also on CD166 location (membranous vs cytoplasmic). In colorectal cancer, upregulation of CD166 was thought to be an early event in malignant transformation, as significantly enhanced protein level was already seen in adenomatous glands.144 Dalerba et al145 were the first to describe CD166 as a putative biomarker for colorectal cancer stem-like cells independent of and synergistic to CD44. However, although membranous expression was initially found to correlate with poor clinical outcome in colorectal cancer patients,146 Lugli et al147 reported in a more recent colorectal cancer tissue microarray study that loss of membranous CD166 and standard CD44 (CD44s) seems to be linked to aggressiveness and worse survival. Here, CD166 and CD44s were particularly lowered at the invasion front. The authors also showed the CD44⫺CD166⫺ sorted cell subpopulations from 3 established colorectal cell lines to exhibit a higher invasive potential in vitro than their positive counterparts and concluded that loss rather than overexpression of CD44s and CD166 is linked to tumor progression and that membranous expression is more or less linked to cell adhesion pattern. There is no experimental hint so far that CD166 affects radioresponse. However, CD44 as one of the most commonly used biomarkers in CSC research has to be discussed in more detail. In a recent study, CD44 expression was shown to be predictive for local recurrence after radiotherapy in laryngeal cancer.148 A cohort of 19 patients with local recurrence was matched with 33 controls, and gene expression array of CD44 was found to correlate with recurrence. This result could be confirmed by immunohistochemical analysis of CD44 expression in an independent validation series. Finally, several laryngeal carcinoma cell lines were investigated to test clonogenic survival in relation to the presence of CD44, and, most interestingly, CD44 presentation did not correlate with intrinsic radiosensitivity. However, CD44 mRNA levels correlated significantly with plating efficiency. The authors concluded that CD44 expression does not control intrinsic radiosensitivity but rather the fraction of CSCs.

T.B. Brunner et al According to these data, CD44 expression in laryngeal cancer cells appears to indicate a constant subfraction with CSC potential. This finding has important implications for other observations reporting that CSC markers do not correlate with intrinsic radiosensitivity, which focused on relative clonogenic survival and not absolute plating efficiencies and therefore might have missed the effect of CSC load as a predictor of radiocurability.110 In fact, it was shown much earlier that transplantability of different tumor models correlated with the TCD50 after single-dose irradiation.2,149 The relevance of the finding on CD44 positivity as correlate for treatment outcome was discussed earlier.68 In the following paragraph, we intend to go beyond this initial discussion to give mechanistic insights on CD44 as an integral molecule in several survival strategies of cancer cells that may indirectly affect radioresponse or stemness plasticity.

CD44 and Other Functional CSC Surrogate Markers: Key Elements in Cancer Cell Survival CD44 is a transmembrane protein with clear functional relevance in cancer progression and the major hyaluronan (HA) receptor. The CD44s is an 80- to 95-kDa transmembrane protein that not only binds to HA but also to other extracellular matrix (ECM) molecules such as fibronectin, laminin, and collagen as well as to CD62E/L.117,150 Its major physiological role is to maintain organ and tissue structures through cell-cell and cell-matrix adhesion. Therefore, CD44 does not seem to belong to those genes known as master genes for maintaining pluripotency, such as NANOG or OCT4. However, there is some relation because CD44 plays an essential role in the cross talk between normal (hematopoietic) stem cells and their niche, which is rich in HA and indirectly supports stemness behavior.117 On the other hand, interaction between HA and CD44 concomitantly stimulates the production of urokinase-type plasminogen activator receptor, matrix metalloproteinase 2, and matrix metalloproteinase 9, leading to a local accumulation of these proteases at the cell surface to support migratory activity.151,152 CD44 has also been described as a target of the Wnt pathway, which is central in colorectal cancer development. Increased expression of CD44s was reported to appear throughout the adenoma-carcinoma sequence, that is, from normal through adenomatous to carcinoma tissue.147,153,154 CD44 has long been proposed as a marker of tumor invasiveness and metastasis and has only recently been described as a putative CSC marker in various epithelial malignancies, including colorectal cancer.57 However, although many early studies on gene expression of CD44 or its splice variants reported a correlation between high expression levels with poor survival,155,156 more recent data in the same tumor entity are conflicting, suggesting either no role for CD44 or worse clinical outcome with loss of protein presentation.147,153,157-160 A major challenge in this field is that CD44 is an abundantly expressed molecule that not only associates with a wide panel of cytoplasmic and intracellular interaction

CSCs as a predictive factor in radiotherapy partners but also makes use of alternative splicing. Accordingly, different CD44 splice variants may be present or absent in individual tumors or at particular tumor sites, supporting various processes associated with tumor development, progression, dissemination, and recurrence of disease after treatment. CD44 variants have specific posttranslational modifications that include binding sites for various glycosaminoglycan-associated proteins, such as basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor, or osteopontin, leading to local concentration of these ligands and enhancing their capacity to bind and signal through their receptors. CD44v6 is a major splice variant engaged in matrix remodeling through induction of HA synthase 3, was associated with a malignant phenotype in many tumor types, and has been considered a therapeutic target.161 In addition, CD44v6 supports the secretion of high molecular mass HA leading to the capture of various growth factors, complement components, and proteases involved in tumor cell migration and adhesion. CD44 and this particular CD44v6 variant contributes to the remodeling of premetastatic niches in lymph nodes, lungs, and bone marrow in the absence of tumor cells by creating a soluble matrix and preactivated resident cells that support the recruitment of cancer cells.151 In addition, CD44v6 expresses coreceptor functions by interacting with the scatter factor/hepatocyte growth factor receptor Met, whereas CD44v3 is known to contribute to the activation and presentation of various other growth factors. Such functions are particularly interesting with respect to the increasing knowledge of stem cell niches as described later in the text and the impact of microenvironment and epigenetics on cancer stemness and tumor cell plasticity. It is beyond the scope of this review to fully discuss the knowledge on all CD44 splice variants. For this, we refer to 2 comprehensive reviews,117,162 which are the basis for Figure 2. However, it has to be emphasized that, although the relevance of CD44 splice variants in tumor progression has been clearly demonstrated in different tumor entities,163 most researchers have applied pan-CD44 antibodies that do not discriminate standard and splice variants, and the particular CD44v expression pattern and plasticity related to a cancer stemness phenotype has not been explored in detail. CD44 RNA expression and protein profiling is of relevance for radiotherapy research because extracellular and cytoplasmic domains of CD44 variants associate and interact with a large panel of molecules in addition to the ones described earlier in the text, which may in concert result in enhanced cancer cell survival, reduced response to treatment, metabolic plasticity, and potential to undergo epithelial-mesenchymal transition (EMT). CD44 mediates several cell signaling pathways through protein interactions despite its lack of an intrinsic kinase domain.164 It is involved in the activation and presentation of growth factors and may act as coreceptor (eg, for receptor tyrosine kinases)117,162 to affect intracellular signaling as depicted in Figures 3A, B. An interplay of CD44 with various EGFR family members was documented earlier, and colocalization with EGFR in some tumor entities was thought to coregulate the signaling of potential CSC popula-

159 tions166 and also to lead to increased tumor cell motility.167 Preclinical and clinical observations imply that EGFR can mediate radioresistance in various solid malignancies, and combination of radiotherapy with EGFR inhibitors was shown to improve local tumor control, indicating that a higher fraction of the CSC pool in these cancer models could be eradicated. Mechanisms identified in experimental studies to contribute to the benefit of EGFR inhibition include cellular radiosensitization through modified signal transduction, inhibition of DNA damage repair, and reduced repopulation as well as improved reoxygenation during fractionated radiotherapy; however, direct killing of CSCs by EGFR inhibitors was also proposed.168 The role of CD44 in this scenario is still speculative. However, CD44 variants may be important coplayers that modulate the diverse and heterogeneous effects and mechanisms of different classes of EGFR inhibitors on radioresponse in different tumors and normal tissues. CD147 (extracellular matrix metalloproteinase inducer [EMMPRIN]) was identified recently as an interaction partner linking HA and CD44 to ATP-binding cassette (ABC) and monocarboxylate transporters (MCTs), that is, molecules involved in drug resistance on one hand and metabolic behavior on the other hand.169 The interaction of CD44, CD147, and ABC transporters (Fig. 3D), a family of intracellular enzymes shown to contribute to drug resistance in many cancers by pumping chemotherapeutics out of cells, seems of particular interest in CSC research. Evidence for high expression of ABC transporters comes from normal stem cells in which they are responsible for the extrusion of harmful agents.170 The respective “side population” technique to identify such cell populations is based on the enhanced efflux of lipophilic dyes like Hoechst33342171 and has been applied as another functional nonsurface biomarker approach to isolate stem cells from both normal tissues172 and various tumors.173-177 Chemoresistance and side population phenotype in cancers were found to be mediated by ABC transporter proteins such as ABCG2/breast cancer resistance protein, ABCB2/MDR-1 (P-glycoprotein; multidrug resistance), or ABCC1/MDR-related protein 1. ABC transporter blockade is thus considered as an approach to chemosensitize side population stem-like cells in cancers. In an interesting preclinical study on SCC, Loebinger et al178 found purified SCC-side population (SP) cells to exhibit enhanced tumorigenicity in vivo, to show increased clonogenic potential in secondary cultures, and to remain chemotherapeutically sensitive on ABC transporter inhibition. Future treatment strategies may thus consider coupling targeting of this particular cancer stem-like cell subpopulation and phenotype, respectively, with standard treatment methodologies, including radiotherapy, and also with other putative CSC-targeting strategies (eg, those directed against interaction partners in this network, such as CD44 variants).179 ABC transporters are also integral to the efflux of certain radiopharmaceuticals and radiosensitizers,180 and a CSC fraction with side population phenotype may be predictive for the success of such approaches, for example, when combined with external irradiation.

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Figure 3 CD44 splice variants can contribute to cancer cell survival and tumor progression through different interaction partners and mechanisms, some of which may be integral to cancer stemness and plasticity in epithelial malignancies. CD44 interdigitates mechanisms of survival, growth, invasion, drug resistance, and metabolism (Compiled and modified from Zöller,117 Ponta et al,162 Morrison et al165). (A) CD44 variants in concert with hyaluronan modulate the dimerization and activity/autophosphorylation of receptor tyrosine kinases such as members of the epidermal growth factor receptor family and insulin-like growth factor 1 receptor (IGF1R) and modify PI3K-AKT (phosphoinositide 3-kinase/AKT) as well as Ras-ERK (Ras/extracellular signal-regulated protein kinase) pathways. CD44 can act as activator and suppressor of the Ras-ERK pathway dependent on the absence (left) or presence of Merlin (Nf2) (right). (B) CD44v3 interaction with CD147 (EMMPRIN) leads to matrix metalloproteinase 7– driven release/activation of growth factors, which can then bind to their respective receptors on cancer and stromal cells. (C) Hyaluronan/CD44 interacts with matrix metalloproteinase 9 to induce extracellular matrix degradation leading to release and activation of matrix-bound factors and an invasive phenotype. (D) CD147 stimulates hyaluronan expression and colocalizes with hyaluronan-CD44 to affect ATP-binding cassette transporter expression and activity, promoting drug resistance and an invasive phenotype. (E) Interaction of CD44 variants with CD147 is required for the stabilization of monocarboxylate transporters, leading to proton-dependent lactate efflux. The hyaluronan/CD44 axis also signals through serine/ threonine Rho kinase and phosphorylates an Na⫹/H⫹ exchanger to create an acidic extracellular environment that supports extracellular matrix degradation and invasion.

The CD44/CD147/MCT interplay (Fig. 3E) is less well recognized in the CSC field but is of great interest in tumor metabolomics and radiotherapy. The interaction with the major proton-dependent transmembrane lactate transporters supposedly contributes to its inside-out transport.169,181 This will, in concert with other proton pumps, lead to local extracellular tissue acidosis in tumor areas by avoiding lethal intracellular acidification of cancer cells in hypoxic tumor areas due to increased glycolytic flux (Pasteur effect) or as a result of abnormal aerobic energy production under normoxic con-

ditions (Warburg effect), for example, through aerobic glycolysis or glutaminolysis.182,183 It is unclear whether the CD44/CD147/MCT interaction is also relevant for the uptake of lactate reported in various oxygenated cell types to fuel respiration. This seems reasonable because both MCT1 and MCT4 were found to be coexpressed and to interact with CD147 and CD44 variants. MCT1 is highly affine for lactate, ubiquitously expressed, and supposed to mainly support outside-in transport, whereas MCT4 has a lower affinity, is expressed in cells with high glycolytic activity and/or under

CSCs as a predictive factor in radiotherapy hypoxia as seen in many cancers, and plays a major role in the disposal of cellular lactate.184-186 Both mechanisms are beneficial to the survival of cancer cell subpopulations, and the potential for metabolic switching at the CD44/CD147/MCT axis may thus be integral to cancer (stem) cell plasticity. The link to radioresponse comes from preclinical data showing high pretreatment lactate content to strongly, and independently of hypoxia, correlate with lower tumor control probability after clinically relevant fractionated irradiation in a panel of human SCC xenografts.187,188 It is unclear whether lactate can directly contribute to intrinsic radioresistance or is a biomarker of high turnover rates in the glycolytic pathway reflecting radioresistance due to other environmental constraints such as limited perfusion.189 However, lactate and tissue acidosis may provide a pro-CSC environmental (niche) condition or could be a surrogate for the cancer cells’ metabolic flexibility. pH is involved in normal stem cell fate, and acidic stress was described to promote a stem cell phenotype in glioma.190 It is thus likely that not only hypoxia (as will be discussed later) but also other milieu conditions and gradients affect the putative CSC pool. This is in line with the experimental SCC study and also with the clinical observation of lactate accumulation in various solid human cancers to correlate with poor prognosis and advanced progressive and metastatic disease.183,191-193 Lactate-induced cancer cell migration as shown in in vitro assays194,195 may reflect a different aspect in this context. A CD44-independent functional marker is ALDH1 (aldehyde dehydrogenase 1). ALDH1 is a cytosolic detoxifying system and enzyme family that oxidizes various intracellular aldehydes to carboxylic acids. The ALDH1 family belongs to a superfamily of aldehyde dehyrogenases that consist of 19 known functional genes in 11 families and 4 subfamilies.196,197 The majority of the isoforms are widely distributed in tissues, with highest expression levels in liver and kidney tissue. High expression of ALDHs can provide a route for chemoresistance. The protein can be detected with antibody approaches in tissue sections. However, the important parameter in stem cell fate is not ALDH1 RNA expression or presence of the protein but its enzymatic activity. ALDH1 activity can be determined by flow cytometry by comparing the Aldefluor reaction (conversion of a weakly fluorescent into a bright fluorescent substrate) in the cell sample of interest with a respective control exposed to an enzyme inhibitor.198 ALDH1 activity was thought to have capacity as a more universal means for the identification and isolation of normal and CSCs from various sources. This has not been proven in detail, but ALDH1 activity has clearly been shown to enrich for cancer stem-like cells in various cancers and was documented to correlate with poor prognosis in prostate, breast, pancreatic, non–small-cell lung, and head and neck squamous cell cancers.196,199,200 First indication for a relevance of ALDH1 to radioresponse comes from a very recent in vitro study. Here, ALDHlowCD44⫺ and ALDHhighCD44⫹ populations from 2 cell lines (MDA-MB-468, MDA-MB-231) were sorted, and clonogenic survival after 2 ⫻ 3-Gy and 2 ⫻ 5-Gy doses, respectively, was evaluated. The ALDHhighCD44⫹ population was more radioresistant and could be sensitized by ALDH1 inhibition.201

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Microenvironment: The Yin and Yang With all the controversy and lack of consensus on the usefulness of surrogate biomarkers for the identification of cancer stem (like) cells, it is worth noting that the cancer cell population of interest is more or less defined by its biological functions, in particular, its ability for self-renewal and the potential to initiate and propagate a tumor (tissue) on transplantation. It is therefore thought that such CSC populations share functional and regulatory similarities with normal stem or progenitor cells. It is well accepted that the functional features of stem cells are regulated by the local environment. Particular niches, that is, specific cellular signals in these niches, are thought to be critical determinants for stem cell survival and maintenance as well as induction of proliferation or differentiation processes. The microenvironment in most normal adult tissues is supreme for differentiated cells to fulfill their biological function. It is to some extent also permissive for differentiation of tissue-specific stem or progenitor cells. However, uncommitted stem cells are often found in a unique, yet dynamic, microenvironment that is considered as an entity of action202 where particular factors modulate gene expression to control cellular phenotype and plasticity and to maintain a balance between stem cell quiescence and proliferative activity. These niche factors include ECM components, stromal cell compartments (eg, vascular elements), and secreted biological modifiers such as bone morphogenic proteins and Wnt proteins, which are known to counteract. In addition, micromilieu conditions such as pH, oxygen tension, and ionic strength are also crucial elements in the CSC scenario. With the recent progress in the characterization of such stem cell niches and the observation that cell fate and homeostasis of normal stem cell compartments is affected by both external environmental cues and intrinsic gene regulation,202,203 focus in CSC research turned to the importance of the microenvironment and the localization and regulation of the putative cancer stem(-like) cell populations.204-207 Neural cancers have probably been studied most extensively in this context during the past years, and the interplay of neural CSCs and the tumor microenvironment has recently been designated as the “deadly teamwork.”208 Today, 3 potential niches for cancer stemness maintenance are proposed in brain tumors but are also considered in other tumor entities (Fig. 4). The perivascular niche is a microanatomical vascular unit dominated by the cross talk of cancer cells with growing microvascular structures. It is defined by proliferating endothelium, its cellular interaction partners, including other stromal cells such as pericytes and macrophages, and the respective paracrine and ECM components.210-213 It is still unclear, whether and how acute spatiotemporal pathophysiological changes in blood supply and perfusion affect cancer stemness phenotype in the perivascular area. The impact of these concerted niche conditions on the radioresponse of the

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Figure 4 The central role of microenvironment. There is evidence that stem-like phenotype(s) are maintained in particular microenvironmental niches. Three different niches are hypothesized in tumor tissue, in particular based on work in brain cancer models: A hypoxic niche, a perivascular niche, and a niche at the invasion front (right panel). The role of acute spatiotemporal modulations in the local milieu (eg, acute intermittent hypoxia) as well as the effect of pathophysiological gradients and other metabolic constraints is still elusive. It has been proposed that stem-like cells might migrate between niches. This requires further proof. However, there is clear evidence for environmentally driven phenotype switching from stemness to nonstemness characteristics (left panel) (Modified from Lathia208 and Cruz209).

malignant cell population with self-renewal capacity will thus clearly be multifaceted. Adult stem cells of various normal tissues are located in hypoxic niches and functionally influenced by the local oxygen concentration.214,215 Accordingly, hypoxia was recently proposed to support cancer stemness and CSC characteristics.208,216,217 In glioblastoma, hypoxia is known to play an important role and has been critically discussed with respect to treatment strategies tested in vitro to compare CD133⫹ and CD133⫺ subpopulations.218 Interestingly, there have been several reports describing the enrichment of CD133⫹ tumor cell populations under oxygen-deprived culture conditions. CD133 phenotype in vitro, however, was not only affected by hypoxic conditions but also by the switch from culture normoxia (⬃20%) to tissue normoxic conditions, which relates to an oxygen concentration of 4%-7% in most tissues but can be as low as 2%-4% in the brain.219,220 This may be accompanied by an intrinsically different radiosensitivity of CD133⫹ and CD133⫺ populations as shown for a bipotent (CD133⫹/⫺ mixed) medulloblastoma cell line. However, because CD133 as a biomarker for cancer stemness is increasingly disputed, other stemness surrogate characteristics have been used to verify the role of local oxygen availability on the cancer/glioma stem cell pool. Based on mechanistic in vitro studies and molecular analyses of tumor stem cell gene signatures, hypoxia-inducible factors, in particular HIF2␣ (hypoxia-inducible factor 2␣), a molecule in the oxygen-sensing machinery, has been proposed as a critical regulatory factor that may promote stemness in glioblastomas.221-223 Heddleston et al described an enhanced selfrenewal capacity, upregulation of neuronal stem cell factors such as Oct4, Nanog and c-myc, as well as increased neurosphere formation under 2% versus 21% oxygen. They also reported that glioblastoma cells lacking the classical glioma stem cell markers produced larger xenografts in BALB/c nu/nu mice on HIF-2␣ overexpression.223,224 The studies de-

mand verification using limiting dilution experimental designs to quantify tumor take rates as a basis to state that HIF2␣ indeed is able to reprogram stemness and does not just promote tumor growth. In addition, different oxygen concentration levels are to be considered to accommodate the fact that tissue normoxia differs in various tissues (it can be as low as 2% in normal brain areas) and to implement radiotherapeutically relevant oxygen concentration of ⬍1%. Here, we have to point to another problem of semantics in the literature—the definition of the term hypoxia in in vitro studies. Hypoxia is often referred to culture normoxia of ⬃20%; thus, many studies turn out to have used tissue normoxic conditions as hypoxic correlates. The interpretation of the data whether the changes are physiological or pathophysiological reactions to hypoxia will thus clearly differ. Despite this limitation, the findings of Heddleston et al are interesting in light of a recent series of studies related to HIF2␣.225,226 In one approach using HIF2␣ RNA interference, HIF2␣ silencing prevented the proliferation of various human tumor cell lines in vivo and autonomous growth in vitro. HIF2␣ was found to regulate receptor tyrosine kinase expression and signaling (EGFR and insulin-like growth factor 1 receptor [IGF1R]), and it was concluded that human cancers converge at a HIF2␣ oncogenic axis.227 Another set of reports using a renal cancer model showed that HIF2␣ not only promotes hypoxic cell proliferation by enhanced c-myc transcription, but its inhibition and deficiency, respectively, induced p53 activation, apoptosis, and cell cycle arrest in G2/M phase and led to high levels of reactive oxygen species (ROS) as well as radiosensitization of the tumor cells by accumulation of DNA damage. Indeed, oxygen deficiency accompanied by reduced production of ROS is a “classical” pathophysiological radioresistance factor. Locoregional acute and chronic hypoxia are well-known phenomena and have been studied with respect to treatment and clinical outcome for more than 2 de-

CSCs as a predictive factor in radiotherapy cades.228-233 There is no doubt that cancer cell phenotype and expression profiles are altered under such conditions, but enhanced rates of mutation and genetic alterations of cancer cells and selection of more aggressive populations at very low concentrations have also been shown. A subfraction of the phenotypically adapted cancer cells may indeed show stemness characteristics as defined by functional and/or surrogate biomarkers combined with enhanced survival capacity. Treatment strategies to overcome hypoxia-driven radioresistance have thus always aimed at enhanced killing of this cancer cell population. This is also challenging if microenvironmental cues cause phenotypic heterogeneity and plasticity with potential of cancer cells for reprogramming or shifting from a putative nonstemness to stemness behavior and has to be taken into account as indicated in Figure 4.209 Here, we want to emphasize that in a recent convincing in vivo study, phenotypic heterogeneity of tumorigenic melanoma cells from patients was revealed and shown to be reversible and not hierarchically organized.234 The stem-like hierarchy, at least for this tumor entity, was challenged, and a phenotype switching concept was proposed because of the high tumor take rates of melanoma cells, with as few as 1 injected cell producing a tumor in several studies.61,235,236 Such phenotype switching must not be driven by genetic but epigenetic and complex environmental constraints even beyond hypoxia. Therefore, the niches described herein are also important for this alternative or amendatory model. Last but not least, an invasive niche has been hypothesized because putative CSC populations and cancer cells undergoing EMT at the invasion front show some overlapping phenotypic characteristics (marker expression, microRNA).237-239 It has been proposed that only the fittest CSCs will succeed to metastasize and that EMT programs are important regulators of phenotypic plasticity. This seems reasonable because to give rise to distant metastases, cancer cells have to survive and adapt to different environmental conditions during migration and invasion and finally have to settle and proliferate in the local (obviously metastatic) niche of the remote tissue. Factors defining the invasive and metastatic niches and driving the EMT–mesenchymal-epithelial transition programs are under intense investigation. Just as a reminder, CD44v6 and its effect on the local stromal cell and ECM is considered to support the recruitment of cancer cells into premetastatic niches in lymph nodes, lungs, and bone marrow.151 Another factor in metastatic and invasive niches may be paracrine stromal cell-derived factor 1 (CXCL12), which is highly expressed in stromal cell compartments and at some organ sites of preferential distant metastasis of epithelial malignancies, such as bone, lungs, liver, and also in lymph nodes. Indeed, CD184 or CXCR4, the main receptor for CXCL12, has been designated as a CSC biomarker in various cancers, including breast and pancreas carcinomas. Here, it seems to define particularly aggressive, metastasizing subpopulations with self-renewing potential, probably directed by CXCL12 gradients according to normal hematopoietic stem cells.240-242 Targeting the CXCR4-CXCL12 axis may thus be a promising approach to treat metastatic disease.243,244

163 At any given moment, cells within a tumor exhibit differentiated, proliferative, and invasive phenotypes, and today neither an ultimate or exclusive trigger nor the speed of the shift between cancer stemness and nonstemness can be predicted. Cell fate decisions and activity of cancer cells are clearly driven by their location inside a tissue.245 Location and microenvironment also modulate the cancer cell’s bioenergetics. Thus, one may speculate that bioenergetic variability and remodeling is likely to be a characteristic feature of cells with high plasticity to support their survival and propagation in different environmental niches (perivascular, hypoxic, invasive, metastatic) and after treatment. Schieke et al246 recently showed that mitochondrial energy metabolism modifies not only the differentiation of mouse embryonic cells but also their tumor formation capacity. Oxidative phosphorylation and aerobic glycolysis have been shown to cooperate during cancer progression, and metabolomic analyses in xenograft models suggest the existence of bioenergetic remodeling throughout tumor growth.247,248 In a given tumor, energy production can vary widely from glycolytic to oxidative pathways according to the genetic background, the local microenvironment (niche), and the proliferative or invasive status.249,250 Rapidly proliferating cancer cells may be advantaged by aerobic glycolysis and may depend on angiogenesis. However, this may be less relevant for quiescent tumor cells with self-renewing potential and stem cells that are not actively replicating their DNA.251,252 Metabolic flexibility of some cancer cell subpopulations to switch between glycolysis and oxidative phosphorylation and to recruit other substrates for energy production, such as glutamine or ketones and lactate,185,253 may thus turn out to be a phenomenon but also dilemma of cancer stemness plasticity.

CSCs and EMT One of the classical 6 “hallmarks of cancer” is the activation of invasion and metastasis, which may be attributed to the activation of a developmental regulatory EMT program.59,80 This process involves epithelial cells to change their characteristic morphology and gene expression pattern toward transcriptional program characteristics of mesenchymal cells. Much of the knowledge on EMT–mesenchymal-epithelial transition has been learned from gastrulation in early embryogenesis, where individual cells peel away from the ectoderm and migrate to the center of the embryo to form the mesoderm. Another physiological process in which EMT plays a role is wound healing. In both cases, taking a mesenchymal tumor phenotype appears to be necessary to allow epithelial cells to reshape and become flexible. In cancers, heterotopic interactions of cancer cells with adjacent tumorassociated stromal cells have been described to activate transcriptional regulators of EMT.254,255 Cancer cells showing EMT characteristic features are primarily found at the invasion front of tumors, and the phenotypic switch can be attributed to microenvironmental stimuli.256 Recently, EMT was reported to enhance expression of stem cell markers in immortalized human mammary epithelial cells.257 These cells were found to have an increased ability to form mam-

164 mospheres and also to form tumors more efficiently.257 Aberrant activation of the Ras/MAPK (mitogen-activated protein kinase) pathway enhanced the CD44⫹CD24⫺/low cell frequency in human mammary epithelial cells, and the number of CD24⫺ cells within a primary tumor reflected the sensitivity of the cancer cells to EMT-inducing signals.258 An interesting concept of the interrelation between CSC and EMT as the migrating CSCs concept was proposed by Brabletz et al.259 According to this concept, mobile CSCs transiently develop from stationary CSCs by the combination of stemness and EMT with genetic alterations and tumor microenvironment producing a combined driving force for malignant progression. Reciprocal interactions of E-cadherin and ␤-catenin with EMT-inducing transcriptional repressors such as zincfinger enhancer– binding transcription factors and their feedback loop with specific, epigenetically controlled microRNAs have been proposed to play a role in the phenotypic plasticity of cancer cells.260-263 Very recently, E-cadherin expression and mesenchymal conversion were found to be associated with higher survival in clonogenic assays of breast and lung cancer cells,264 indirectly pointing to an enhanced CSC presence. In line with this, exposure of pancreatic cancer cell lines to synchronous gemcitabine and radiotherapy led to an enrichment of the cells defined by putative CSC markers Oct4, ABCG2, CD24, and CD133 and at the same time of markers of EMT. Injection of these cells revealed a higher tumorigenicity in BALB/c nude mice.265 Another report describes an enhanced radiation resistance in clonogenic assays related to immune-induced EMT and a parallel enrichment of CD44⫹CD24⫺/low phenotype as well as enhanced engraftment.266 In summary, there is accumulating evidence pointing to an interrelation of EMT and an enhanced CSC phenotype, including observations of enhanced resistance to radiotherapy.

CSCs: Die Hard After Radiotherapy? As outlined earlier in the text, CSCs are expected to mediate radioresistance based on functional radiobiological tests such as clonogenic survival and TCD50. These suggest that the last CSC has to be killed to permanently cure a tumor locally. A growing body of evidence supports this view; basically, these studies consist of combinations between available CSC markers such as surface markers or functional markers (eg, ALDH1 and SP) with radiobiological endpoints. This hypothesis has been tested mostly in glioblastoma,6,110,219,267,268 breast cancer,3,269-272 head and neck cancer,148,273 colorectal cancer, and pancreatic cancer.110,265 The first study in brain tumors testing the role of CD133⫹ cancer cells on radiation sensitivity used primary glioblastomas and reported an enrichment of the CD133⫹ CSC fraction after fractionated radiotherapy in vitro and in vivo as well as reduced sensitivity to radiation-induced apoptosis.6 These observations were in line with enhanced DNA repair in the respective subpopulation. Additionally, CD133⫹ cells were equally successful in inducing orthotopic

T.B. Brunner et al tumors in nude mice after 2- or 3-Gy irradiation compared with nonirradiated controls. Similarly, CD133⫹ cells of the medulloblastoma cell line Daoy were already resistant in a clonogenic assay compared with their CD133⫺ counterparts.219 The CD133⫹ subpopulation was found to be larger in hypoxic conditions, which has important implications for radiation treatment. The second most frequently investigated tumor site for the role of CSC in radiotherapy is breast cancer. This work has relied predominantly on established cell lines. One group tested MCF-7, MDA-MB-231, and T47D cells using CD24⫺/lowCD44⫹ surface markers269 and the lack of proteasome activity272,274 to isolate CSCs. Clonogenic survival was compared between single cells from mammospheres, which are supposedly enriched for CSCs, versus monolayer cells with a low CSC subpopulation and showed enhanced survival of cells from mammospheres and a lower rate of ␥H2AX induction 1 hour after irradiation. The CSC population was enriched after fractionated radiotherapy (5 ⫻ 3 Gy) in the floating fraction of monolayer cells that was regarded to contain CSCs. In a follow-up study, the same group investigated the same surface markers combined with the absence of proteasome activity to select CSC in the 2 cell lines, MCF-7 and T47D.272 Selfrenewal capacity was maintained for up to 4 generations of mammospheres in T47D cells but not in MCF-7 cells after fractionated radiation. Also, a combined measurement of DNA and RNA content was used to quantify mobilization of CSCs from the G0 cell cycle phase to nonresting cell cycle phases, and the putative T47D CSC fraction decreased from 25% to 6.5%. The radioresistance effect of CSC was also described in a report that investigated several solid tumor types, including breast cancer.110 The cell line MDA-MB-231 was sorted for the breast CSC surrogate markers CD24 and ESA, and the radioresistant phenotype of the CD24⫺ESA⫹ subfraction was confirmed by reduced ␥H2AX foci. The same subpopulation showed higher number and larger size of spheres compared with unsorted cells in a mammospheres assay and enhanced xenograft formation. Further confirmation of the radioresistant phenotype of breast cancer stem-like cells comes from another group showing an increase in the lin⫺CD24⫹CD29⫹ population of MCF-7 cells on irradiation.270 This was confirmed by an increased fraction of cells being Sca1⫹ (stem cell antigen 1) and an increase of MCF-7 cells with side population characteristics. Interesting additional insight into the radiation response of breast CSCs comes from a recent report using early passage patient-derived primary cells in a xenograft model.3 Hormone-positive UM2 xenografts showed an enrichment of the CD44⫹CD24⫺lin⫺ subpopulation, an increase of the fraction of ALDH1⫹ cells 2 weeks after 8-Gy single-dose radiotherapy, and an increased mammosphere formation capability. Head and neck cancer is one of the most important tumor sites for radiotherapy. A recent convincing study has shown that the putative head and neck CSC marker CD44 predicts recurrence of laryngeal cancer.148 This is one of the most important studies of CSCs in radiation oncology reported so far; it did not set out to test the CSC question in the first step,

CSCs as a predictive factor in radiotherapy but CD44 mRNA was identified in a gene expression data analysis from 52 laryngeal cancer patients. CD44, which correlated with recurrence, was analyzed immunohistochemically in an independent validation series confirming the predictive potential of the marker. This was then taken further to investigate 8 laryngeal cancer cell lines for the expression of CD44. Although CD44 did not correlate with radiosensitivity in clonogenic assays, it was significantly correlated with plating efficiency. In conclusion, this could signify that the overall initial load of CSCs can predict for recurrence in laryngeal cancer. This observation is also in agreement with the old truism of oncology that tumor volume is one of the strongest predictors of local control, and obviously, the total number of CSCs is in this entity directly related to tumor volume. More highly translational evidence for the role of CSCs in radiotherapy came from a Japanese group investigating resection specimens from patients who underwent neoadjuvant chemoradiotherapy for rectal cancer.130,132,275 Patients with distant recurrence after chemoradiotherapy had higher levels of CD133, Oct4, and Sox2 (sex-determining region Y-box 2) in residual cancer cells compared with those without recurrence. Another interesting aspect of their observations is that radiation caused upregulation of all 3 genes in LoVo and SW480 colon cancer cell lines. They then extended the findings to report that CD133 positivity was detected at a much higher percentage in resection specimens from 50 preoperatively treated patients (70% of specimens) compared with 40 patients who had undergone primary surgery (27.5%).130 They described 4 histopathological staining patterns for CD133, and patients with cancer sections showing both luminal surface and cytoplasmic staining had a significantly lower chance to respond compared with patients with other staining patterns. In pancreatic cancer, 4 pancreatic cancer cell lines (SW1990, BxPC3, JF305, pc3) were exposed to gemcitabine and concomitant radiotherapy to obtain resistant subpopulations276 that were then analyzed for colony and tumor sphere formation as well as engraftment in BALB/c nude mice. The resistant clones were positive for the CSC surrogate markers Oct4, ABCG2, CD24, and CD133, and they showed enhanced clonogenic and tumorigenic capacity and were more invasive and migratory than their ancestors. Despite this substantial evidence that CSCs mediate the radioresistant phenotype, there are substantial weaknesses in some of the presented studies and there have been contradictory reports. One difficulty of CSC assays is that primary cancer cells are commonly accepted to be the best model. However, primary cancer cells are difficult to obtain, and results are difficult, if not impossible, to be replicated because of the limited lifespan of these models. Therefore, one of the concerns is that some reports have so far exclusively used established cell lines.110,112,219,269,270,272,277,278 Other weaknesses relate to the nature of the survival assays used in the context of radiotherapy. For instance, apoptosis assays (used by Bao et al6) have not been accepted to be of relevance to predict clonogenic survival accurately after radiotherapy.278 Measuring the level of DNA damage and DNA damage repair using foci such as ␥H2AX was in disagreement with clono-

165 genic survival assays. One study, for example, described dramatically reduced residual ␥H2AX levels in putative pancreatic CSCs without any difference in clonogenic survival.110 Another example is the use of the alkaline6 and not the neutral comet assay as readout system, which measures rather DNA single- than double-strand breaks (DSBs); however, only the latter are regarded to be of relevance to radiation survival. Other possible factors are the use of different media in comparison groups, for example, between monolayer and mammosphere breast-cancer cells,269 or the lack of proof that differences relate to stemness and are not just a reflection of differences between 2- and 3-dimensional cell-to-cell and cell-to-matrix interactions. Beyond these methodological problems, there are a number of reports describing either the absence or presence of a radioresistant phenotype of CSCs. The latter was found by a group testing primary glioblastoma cells and established cell lines after sorting for CD133⫹.268 In line with the survival results, authors reported reduced DSB repair capacity as defined by the neutral comet assay and ␥H2AX and Rad51 foci. More recently, an immunohistochemical analysis of 88 patients with glioblastoma could not find any impact of the CSC markers CD133, nestin, and CD15 in a homogeneously treated retrospective patient cohort.267 Nonetheless, there is more evidence pointing to a radiation-resistant phenotype of cancer cells with cancer stem-like properties despite the contradictory reports. Generally, cell surface markers only appear to identify treatmentresistant subpopulations in a subset of the tested tumors both from established cell lines110 and from primary cancers.3 Therefore, it is very important for the future to refine surface markers and to use them alongside functional markers.

Potential Mechanisms of Radioresistance of CSCs From the perspective of cancer treatment, we are interested in finding new ways to target CSCs to enhance the efficacy of our therapy. In this context, CSCs are to be regarded as the “upper crust” among cancer cells: they will always strive to find surprising mechanisms of escape and have exclusive access to these. The very first steps to identify pathways that play a role in the radioresistance of CSCs that can be targeted have been made. Autophagy is a catabolic survival mechanism of cells during starvation, by which a cell’s components are degraded by the lysosomal machinery. This process was recently linked to CSC radiation resistance, when it was described to be induced by radiation in glioma cells in general and to a large degree in CD133⫹ cells within 1-2 days.279 The inhibition of autophagy sensitized the glioma CSCs to radiotherapy and reduced their ability to form neurospheres. This observation is in line with others reporting an enhancement of the cytotoxic effect of radiotherapy on inhibition of autophagy.280-282 Nevertheless, there are also studies reporting the opposite such as induction of autophagy by rapamycin and by inhibition of the catalytic subunit of DNA-dependent protein konase (DNA-PKcs) leading to sensitization of glioma stem cells

166 to radiotherapy.283,284 However, currently available literature supports the idea that autophagy is a survival mechanism for CSCs.285 Oxidative stress, which is commonly encountered in cancer, leads to the production of ROS as inducer of autophagy.286 Long before ROS were recognized to induce autophagy, the oxidation of bases in the DNA by ROS leading to products such as 8-oxo-deoxyguanosine and deoxythymidine glycol was described and shown to have the potential to lead to mutations. In line with findings in normal stem cells, breast CSCs were found to have reduced levels of ROS to protect them from DNA damage mediated by elevated levels of freeradical scavengers.287 Furthermore, putative breast CSCs were shown to have lower levels of DNA damage as measured by the alkaline comet assay and ␥H2AX foci and to express higher clonogenic survival after radiation than their non-CSC counterparts. Pharmacological depletion of glutathione led to an inhibition of colony formation of CSC after radiotherapy. Because DNA is the principal target for radiation-induced cell killing, DNA damage repair has been intensively studied in CSCs. The number of residual ␥H2AX foci measured 24 hours after radiation in general corresponds well with clonogenic survival288,289 and is commonly used as a surrogate marker of radiation cytotoxicity even if this correlation could not always be confirmed.110 It is believed that ␥H2AX forms 1 immunohistochemically detectable focus per DSB.290 A number of studies investigated the induction and resolution of ␥H2AX foci in CSCs; a lower number of induced ␥H2AX foci was reported in human breast CSCs,269,291 and other studies have documented a faster resolution of ␥H2AX foci as compared with the bulk tumor cells or putative non-CSCs.6,110,270,292 No such differences could be found in another study, which, however, showed a radiation-dependent increase in the activation of Chek1 and Chek2.293 As there are many different repair mechanisms playing a role after radiation-induced DNA damage, their importance has been investigated. DSBs are particularly important after radiotherapy. They are predominantly repaired by homologous recombination (HR) and nonhomologous end joining. Rad51, a protein involved in HR, was reported to be upregulated in the CSC signature of 33 cell lines294 and to be related to radiation resistance.271 This observation is interesting because the mechanism could protect the genome of CSCs, as HR is the high-fidelity version of DSB repair compared with nonhomologous end joining, which is more error prone. The accumulating evidence appears to confirm a role of CSCs as a subset of tumor cells with specific (radio)biological characteristics for the long-term control of tumors treated with radiotherapy. Many researchers have focused on identifying specific pathways such as PI3K (phosphoinositide 3-kinase), RANK (receptor activator of NF-kB), notch, ␤-catenin, or heat shock protein 90, which have been found to be upregulated in CSCs and are therefore expected to present druggable targets with or without radiotherapy.76,213,265,292,295-297 However, it should be emphasized that not only direct targeting of these pathways is promising but

T.B. Brunner et al also targeting of specific niches of CSCs such as the perivascular niche of brain tumors298 or the hypoxic niche.299

Does CSC Biology Matter for the Clinician in Radiation Oncology? All of what has been said so far will ultimately have to stand the trial of usefulness for treatment of cancer patients with radiotherapy. Table 1 summarizes the potential consequences of our understanding of CSC biology for radiation treatment. It is important to stress that this is rather an agenda for future research and that we may never be able to achieve some of the aims mentioned in this table. The table follows the typical treatment chain starting with diagnosis, followed by radiotherapy treatment planning. After the start of therapy, in-treatment imaging and biomarker analysis have recently been added to the treatment chain. After completion, therapy response is evaluated with reimaging and histopathological analysis. Finally, outcome is evaluated using endpoints such as local and distant tumor control, overall survival, and late toxicity. As illustrated in this table, all of the mentioned steps can potentially be influenced by CSC biology, provided that our current indications of its impact on radiation cytotoxicity hold true. There is a large discrepancy between certain items summarized in this table placing validated factors next to highly hypothetical ones. Total tumor volume is one of the first recognized prognostic factors in radiotherapy and may be directly proportional to the total number of CSCs.68,148 Provided that we can image the spatial distribution of CSCs and of CSC niches such as hypoxia, perivascular niche, or invasive niche (Fig. 4), we can use this information directly for treatment planning, prescribing a higher dose using modern intensity-modulated radiotherapy techniques. Anatomical knowledge about the predominance of neural stem cells in the subventricular zone (SVZ) and the subgranular layer was recently translated into a retrospective clinical study in 55 patients, hypothesizing that brain CSCs might be recruited from these areas.304 Patients treated with a dose higher than the median to the SVZ lived significantly longer than those with lower doses, whereas total prescription dose had no significant impact on survival. If these results can be validated, the SVZ could represent a clinical target volume in the future. Histopathologic analysis of pretherapeutic biopsies or resection specimens should allow screening for CSCs to determine the ratio of CSCs relative to the total tumor volume in conjunction with imaging to predict radiocurability and total dose required. These approaches may also be useful to determine whether there are any tumor cells with a metastasisspecific CSC phenotype (eg, CXCR4 in pancreatic cancer).15 These data could then be used to decide whether treatment should start with chemotherapy or (chemo-)radiotherapy first. Further information on activated pathways in CSCs (Fig. 3) would allow one to select a specific CSC-targeting agent (or set of agents) because most molecular pathways

CSCs as a predictive factor in radiotherapy

167

Table 1 Overview on the Theragnostic Relevance and Consequences of Cancer Stem Cell (CSC) Populations in Radiotherapy Step in the Treatment Chain Image

Readout/Work List Item Total tumor volume Spatial distribution of CSCs CSC niches: hypoxia

Pathology

Histologic (sub)typing

Ratio of CSCs/tumor volume68 Metastasis-specific CSCs Activated molecular CSC pathways CSC-based treatment plan

Fractionation of RT Scheduling of RT and systemic therapy

CTV margins

Quality of particles In-treatment CSC imaging

Induction chemo-/hormonal therapy Induction MTT Total dose of RT Fractionation of RT Combinatorial approach CSC-IGRT–guided dose painting

Acute in-treatment toxicity Response

Interpret classical imaging

CSC-specific imaging274,302 Tumor response grading303 CSC response grading131

Outcome

Local recurrence Metastasis Survival Late toxicity

CSC-Related Consequence Inversely corresponds with radiocurability because of number of CSCs Dose painting to tumor subvolumes Dose painting to tumor subvolumes Identify CSC markers (eg, squamous cell vs adenocarcinoma in lung or esophageal cancer) to target related activated pathways Prediction of radiocurability after correction for the tumor volume 1. Selection between RT and Cx/MTT 2. Time scheduling of the above Select specific CSC targeting agent Tailor fractionation according to individual CSC pattern6 (eg, avoid hypofractionation for hypoxic tumors) 1. Determine optimal timing for combined therapy with RT 2. Consider induction chemotherapy before RT if metastasis-specific CSC prevail 1. Choose highly conformal vs margin increased technique according to distance of CSC to tumor edges 2. Include CSC recruitment areas (eg, subventricular zone in glioma) Consider heavy particles Adjust length depending on CSC induction Target hypoxic niche before RT300,301 Prescribe total dose depending on early tumor response of CSC Adjust total treatment time and number of fractions Introduce new CSC-targeting agent during RT after CSC imaging Complete or abandon neoadjuvant treatment 1. Dose paint according to in-treatment changes of number and spatial distribution of CSC 2. Dose paint according to changes in hypoxia Report acute toxicity of CSC targeting agents especially on normal stem cells Correlate RECIST and PERCIST with CSCs before and after RT (poor correlation expected as standard therapy mainly targets non-CSCs) Predict tumor control and decide on additive treatment Relate TRG to pretherapeutic CSCs after neoadjuvant therapy or Bx after RT Compare the number, profile, and spatial distribution of CSCs before and after treatment and match with dose delivered Validate CSC markers to predict curability after RT or combination with CSC-targeting agent(s) Validate CSC markers to predict distant failure Confirm effect of RT ⴙ CSC-targeting agent on cancerspecific survival Compare late toxicity after RT ⴙ CSC-targeting agent(s) vs RT only

Challenges for translation into the clinic relate to (i) the limited availability and fragmentary characterization of functionally relevant useful biomarkers and the respective detection techniques and analytical tools, respectively; (ii) the presence of more than 1 CSC phenotype in individual tumors; (iii) the existence of various CSC niches beyond hypoxia; and (iv) the potential for spatiotemporal phenotypic stemness plasticity. All of these aspects are discussed in the text. RT, radiotherapy; Cx/MTT, chemotherapy/molecular targeted therapy; CTV, clinical tumor volume; IGRT, image-guided radiotherapy; RESIST, response evaluation criteria in solid tumors; PERSIST, PET (positron emission tomography) response criteria in solid tumors; TRG, tumor regression grade; Bx, biopsy.

168 have become druggable. Generic factors of radiotherapy treatment planning could be influenced by knowledge on CSCs; the choice between fractionation schedules is of particularly high interest. Hypoxia would indicate that conventionally fractionated schedules are more appropriate to allow reoxygenation and mobilization of CSCs out of the hypoxic niche. Alternatively, a specific hypoxia-targeting induction therapy could be chosen to precede radiotherapy for tumors in which hypoxia plays an important role.300,301 In-treatment CSC-specific imaging274 may be especially useful for treatment factors such as total dose and total treatment time, and it could foster alterations in the total dose of radiotherapy, adjustment of the total treatment time and number of fractions after assessment of early CSC response, introduction of new CSC-targeting agents during radiotherapy, and/or CSCimage guided radiotherapy dose painting. It may facilitate the decision whether neoadjuvant treatment should be continued or abandoned in favor of surgery or whether chemotherapy should be replaced by chemoradiotherapy.305 The feasibility of such adaptive radiotherapy was recently reported for head and neck cancer and lung cancer.306,307 Studies of radiation combined with CSC-targeting agents will have to look into acute toxicity especially in respect to normal stem cells. On completion of therapy response assessment, adding CSC-specific imaging is predicted to dramatically improve the prognostic power of restaging and could dramatically shorten the time required to reach clinical trial endpoints. RECIST308 and PERCIST309 criteria heavily depend on the response of the bulk of the tumor mass consisting in non-CSCs and therefore correlate poorly with local tumor control. Tumor response grading, a valuable tool after neoadjuvant treatment, can integrate CSC response grading as recently shown for rectal cancer treatment.131 Finally, the long-term outcome parameters of local recurrence, metastasis, and survival will allow us to validate whether molecularly targeted therapies combined with radiotherapy achieve their purposes, while not leading to increased late toxicity by also affecting the survival of normal stem cells. In conclusion, the field of CSC biology—which was born in radiobiology and radiotherapy, developed first in hematologic tumors and by now has made dramatic progress in the field of solid cancer types—is of extremely high interest for radiotherapy. The authors of this article believe that the currently available evidence points to the existence and biological importance of CSC phenotypes despite many open questions and conflicting data, and the methodological problems to study the stem cell question. Much further progress will have to be made before the translational aspects envisioned earlier in the text can be applied in the clinic; however, encouraging first steps toward that aim have been made, and these are expected to contribute to higher cure rates for our patients treated with radiotherapy.

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