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Characterization of the Stem Cell Niche and ItsImportance in Radiobiological ResponseFrank Pajonk, MD, PhD,*,† and Erina Vlashi, PhD*

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Normal tissues are organized hierarchically with a small number of stem cells, able to self-renew and give rise to all the differentiated cells found in the respective specialized tissues.The undifferentiated, multipotent state of normal stem cells is codetermined by theconstituents of a specific anatomical space that hosts the normal stem cell population, calledthe “stem cell niche.” Radiation interferes not only with the stem cell population but also withthe stem cell niche, thus modulating a complex regulatory network. There is now mountingexperimental evidence that many solid cancers share this hierarchical organization with theirtissueof origin,with thecancer stemcells alsooccupyingspecializedniches. In this review,wehighlight some of the best-characterized aspects of normal tissue stem cells, cancer stemcells, and their niches in the bonemarrow, gut, and brain, aswell as their responses to ionizingradiation.Semin Radiat Oncol 23:237-241 C 2013 Elsevier Inc. All rights reserved.

Introduction

The radiation responses of protozoa and complex multi-cellular organisms share many aspects of the deoxyribo-

nucleic acid (DNA) damage repair process. However, inprotozoa, the stem cell is the organism itself and its survivalafter exposure to radiation only depends on the ability of thecell to cope with the DNA damage.The functional integrity of higher multicellular organisms

depends on compartmentalization, specialization, and hier-archy among cells. While the majority of cells in a tissue aredispensable and unable to reconstitute the tissue after damage,stem cells can produce all types of differentiated progeny foundin a tissue. Thus, stem cells have to be protected and supportedso as to guarantee their function during the lifetime of theorganism. The anatomical space in which stem cells aremaintained and protected is the stem cell niche.In this paper, we review (1) recent advances in the under-

standing of normal tissue stem cells, (2) characteristics of theirniche, and (3) their malignant counterparts and also

ont matter & 2013 Elsevier Inc. All rights reserved.16/j.semradonc.2013.05.007

by grants from the National Cancer Institutend 1R01CA161294) and the Army Medical Researchandʼs Breast Cancer Research Program (W81XWH-11-

ionOncology, DavidGeffen School ofMedicine, UCLA,

ive Cancer Center, UCLA, Los Angeles, CA.

ts to Frank Pajonk, MD, PhD, Department of Radiationeffen School of Medicine, UCLA, 10833 Le Conte Ave,0095-1714. E-mail: fpajonk@mednet.ucla.edu

(4) summarize the current knowledge of the stem cell responseto ionizing radiation.

The Normal Stem Cell NicheSchofield was the first to introduce the concept of a stem cellniche as a stem cell–specific microenvironment. He suggestedthat such a niche would provide an anatomical space thatdetermines the number of stem cells that can be supported,would be responsible for the maintenance of the stem cellphenotype, and would affect the mobility of stem cells.1

One of the simplest and best-understood examples of a stemcell niche is found in the ovary of the fruitfly, Drosophilamelanogaster, in which the so-called “cap cells” provide thephysical space for a defined number of germinal stem cells andthe signals to keep them in an undifferentiated state.2 In higherorganisms, niche components are less defined. In the followingparagraphs, we discuss 3 mammalian stem cell niches thathave been extensively studied in the past and which havesignificant relevance for the radiation responses of tissuesduring and after cancer treatment.

The Bone Marrow Stem Cell NicheSchofield originally developed the niche concept while study-ing hematopoietic stem cells (HSCs).1 Although all compo-nents of the HSC niche have not been defined, growingevidence suggests that the bone marrow contains at least2 different types of niches: (1) one niche is found periosteally

237

F. Pajonk and E. Vlashi238

and provides a hypoxic environment and (2) the second isfound in the perivascular location. However, in bothlocations, nestinþ mesenchymal stem cells with adipo-genic, chondrogenic, and osteogenic potential seem to playa major role in HSC maintenance and homing.3 Interest-ingly, HSCs in the hypoxic niche can be eliminated bytreatment with tirapazamine, a prodrug that specificallytargets hypoxic cells.4 This indicates that hypoxia levels inthis niche are severe enough to be physiologically mean-ingful. This raises 2 questions: (1) if there is a differentialsensitivity of normal HSCs to radiation depending on theirlocation in the bone marrow and (2) if hypoxic cellsensitizers have a yet-to-be-studied, late toxicity to thehematopoietic system. In general, quiescent HSCs werefound to be radioresistant compared with their lineage-committed progeny that rely on error-prone DNA repairand are thus susceptible to leukemogenesis.5

Figure (A) The neural stem cell niche in the subventricular zone.

The Intestinal Stem Cell NicheDecades ago, the normal stem cell niche in the small intestinewas found to be localized to the base of the crypts. It wasgenerally accepted that the stem cells reside in position þ4(þ2 toþ7) of the crypt base and that they cycle infrequently.6

Subsequently, theywere found to be positive for the polycombgroup protein BMI1 and the atypical homeobox proteinHopx.7 This relatively rare type of BMI1þ cells is mostabundant in the first 5 cm of the duodenum.8 More recently,the Lgr5þ crypt base columnar cells have been identified as asecond type of intestinal stem cell.9 This group of cells isintersected by Paneth cells, which are derived from Lgr5þ cellsand form the niche for the latter. However, Lgr5þ werereported to be dispensable for cryptmaintenance and appearedto be progeny of BMI1þ cells.8 Conversely, Lgr5þ cells werereported to generateHopxþ cells in positionþ4 suggesting theexistence of 2 different intestinal stem cell niches. Further, itwas found that these populations can dynamically interconvertinto each other.10

The intestinal stem cell in positionþ4 has long been knownto be extremely radiosensitive, with almost all cells undergoingrapid apoptosis after irradiation with doses of 0.5-1 Gy.11

Potten suggested that the more radiation-resistant progeny ofthese cells in positionþ5,þ6, andþ7would retain some stemcell traits, enabling them to repopulate crypts after higher dosesof radiation. However, a more recent report suggested thatLGR5þ crypt base columnar cells are radioresistant intestinalstem cells and the source of repopulation after radiationinjury.12

Neural stem cells (NSCs) or type B1 cells (blue) are in contactwith the endothelial cells of nearby blood vessel but also havecontact to the ependymal cells (brown) and give rise to transientlyamplifying or neural progenitor cells type C cells (dividing cell inlight blue). Neural progenitor cells divide and produce neuroblastor type A cells (orange), which migrate into the periphery.Supporting astrocytes are illustrated in purple. (B) The cancerstem cell niche of brain tumors. The cancer stem cell niche ofbrain tumors recapitulates features of the normal neural stem cellniche. Cancer stem cells are found most frequently in proximityto endothelial cells.

The Neural Stem Cell NicheUntil the late 1960s,13 the adult central nervous system (CNS)had been considered postmitotic but it is now clear that neuralstem cells (NSCs) continuously contribute to tissue repair andplasticity in the adult brain.The subgranular zone of hippocampal area and the sub-

ventricular zone (SVZ) of the lateral ventricles are the mostprominent areas of adult neurogenesis. However, cells that

fulfill the criteria of NSCs by operational means can be foundthroughout the entire CNS, including the cerebellum, retina,and spinal cord,14 suggesting that the CNS contains multipleNSC niches that may serve different functions.Our understanding of the anatomy of the NSC niche in the

SVZ is quite advanced.14-16 It appears that NSCs in the SVZ arelocated in a perivascular and an ependymal niche (Fig. part A).The exact phenotype of NCSs is still under debate but there isevidence that a nestinþ/GFAPþ astrocyte population withdirect contact to the vasculature contains the NSC popula-tion.15,16 However, this might be specific for the SVZ and notnecessarily the universal phenotype for NSCs in all niches inthe CNS.14

Assessment of radiation responses of NSCs in situ, for themost part, did rely on neurogenesis as an end point. As such,most of these studies have investigated radiation responses ofNSCs and committed progenitor cells. However, the generalinterpretation of the data is that NSCs are highly radiosensitiveand that radiation inhibits neurogenesis.17 Interestingly, overtime, different structures of the brain recover with different

Characterization of the stem cell niche 239

kinetics. Although NSCs in the hippocampal area are depletedby radiation, neurogenesis in the SVZ can recover to somedegree.18 This indicates that there is either differential radio-sensitivity of NSCs in different niches in the brain, differentnumbers of NSCs are present at the time of irradiation, orincreased attraction of surviving NSCs into the stem cell nichein the SVZ.

Signaling in the Stem Cell NicheThe normal stem cell niche not only provides an anatomicalspace to host stem cells but also provides signals to maintainthe stem cell state. In general, signaling involves soluble factorsas well as cell-bound receptor ligands and adhesion moleculesexpressed on the surface of stem cells and niche cells. Thesemultiple points of regulation allow the niche to integrate long-and short-range signals to fine-tune stem cell fate decisions.We are only beginning to understand this complex network ofpositive- and negative-feedback regulation that underlies tissuehomeostasis. Developmental signaling pathways, like theNotch, Wnt, and Hedgehog pathways, are involved in tissuehomeostasis mediated by the transforming growth factor-βprotein family and cadherin- and integrin-mediated adhesiveinteractions. Importantly, the signals required to maintain thestem cell statemight vary widely for different tissues. However,in the context of tissue radiation responses, it is remarkable thatmany of the pathways involved, including theNotch19 and theWnt20,21 pathways are activated. The expression of adhesionmolecules like integrins22 is also upregulated in response toionizing radiation. The resulting radiation-induced dangersignal integrates the stem cell niche and the stem cells into atissue response that triggers wound healing and restores tissueintegrity.23

Cancer Stem Cells (CSCs) andTheir Cell of OriginThe CSC hypothesis24 is an old concept25 that gained newmomentum after the first successful prospective identificationof breast CSCs.26 In many solid cancers, cell populationsenriched for CSCs can now be prospectively identified by cellsurface marker profiles,26,27 aldehyde dehydrogenase activ-ity,28 or lack of proteasome function.29 As seen in normaltissues, this model assumes hierarchy amongst the heteroge-neous cell populations in solid cancers. It postulates theexistence of a small population of CSCs, which are able toregrow the tumor after sublethal treatment, that are capable ofproducing all lines of progeny found in the original tumor. Theterm “cancer stem cell” does not necessarily imply that CSCsderive from normal tissue stem cells but instead indicates stemcell traits with regard to tumor initiation and maintenance. Toavoid this confusion, CSCs are sometimes also called “tumor-initiating cells.” According to the CSC hypothesis, cancer curecan only be achieved if all CSCs are eliminated. This hasimportant implications for anticancer treatments. Specifically,the CSC hypothesis explains why most anticancer drugs have

failed and why, despite enormous financial efforts, progressagainst cancer has been slow andmainly driven by prevention.Efficacy assessment of anticancer treatments is currently basedon bulk tumor responses and not on the response of CSCs. Assuch, these bulk assessments may not be reliable indicators toselect one drug over another.Even though the term CSCs does not imply a specific cell of

origin, experimental evidence fromelegant animal experimentssupports that normal tissue stem cells or progenitor cells arethe cell or origin for CSCs. This was determined by experi-ments in which tumor suppressor genes or oncogenes weretargeted in or to normal tissue stem cells and their differ-entiated progeny. In the gut,30 the brain,31 or the bonemarrow,32 only stem cells or progenitor cells were capable offorming tumors after deletion of tumor suppressor genes orintroduction of oncogenes. The same manipulations in differ-entiated progeny did not lead to tumor formation. Further-more, more recently, formal proof was provided on this. Atleast in mouse tumor, CSCs exist and maintain tumorgrowth.33-35

An alternative model of tumor organization is the clonalevolution model,24 which has been and continues to befavored by many in the field of oncology. It attributes theability to gain features of a tumor-initiating cell to every cell in atumor. It is appealing for many clinicians because it shows thatthe current clinical practice of treatment response assessment isindeed a valid approach. Themost recent study to support thismodel, which gained broad attention, came from theMorrisonLab showing that in very advancedmelanoma cases, 1 in 4 cellscould initiate a xenograft if coinjected with Matrigel intoseverely immune-deficient NOD scid gamma (NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Further, melanoma CSCmarkers did not identify cell populations with increasedtumorigenicity.36 However, these data have not been repro-duced using CSCs from other solid tumors. In our hands, theuse of NSG mice does increase tumor take in general, but itdoes not change the fact that prospectively identified CSCsfrombreast cancer or glioblastomamultiforme (GBM)have 1-2magnitudes higher tumorigenicity than their differentiatedprogeny.In essence, theCSCshypothesis has gainedbroad acceptance

since the first prospective identification of breast CSCs a decadeago, and a growing body of evidence shows that undisturbedgrowing tumors are indeed organized hierarchically.

The CSC NicheA stem cell niche specific for CSCs has not been identified; butif CSCs derive from normal tissue stem cells, then it is verylikely that signals that support and maintain a stem cellphenotype in normal tissue stem cells would also supportCSCs. For brain tumors, the Gilbertson Lab was able todemonstrate that CD133þ CSCs are preferentially found inclose proximity with endothelial cells,37 thus resembling nichepreferences similar to those of NSCs, the most likely cell oforigin for CSCs in these types of tumors (Fig.part B). As aconsequence, normal tissue stem cell nichesmay serve as a safe

F. Pajonk and E. Vlashi240

harbor for quiescent CSCs, which may cause recurrence if leftuntreated. Early evidence supporting this hypothesis camefrom our laboratory when we reported that higher radiationdoses given to the SVZ of patients with glioma correlated withsignificantly improved survival.38 So far, this observation hasbeen independently confirmed by at least 3 other groups39-41

and it may offer a promising approach to improve outcome forpatients with GBM. However, our findings are in conflict withhistorical clinical data from trials in which whole-brainirradiation, where the SVZ was included in the target volume,did not lead to improved treatment outcome.42 Therefore, thisprovocative hypothesis needs validation in a prospective trial.An additional opportunity to improve radiation treatment

outcomemay lie in the tropism of CSCs to certain niches. Firstreported by Paget and leading to his “seed and soil” hypoth-esis,25 it is generally accepted that metastatic spread does notfollow a random distribution throughout the body or could beeasily explained by differences in perfusion between differentorgans. In other words, although both breast cancer andprostate cancer, frequently spread to the bone, the former alsofrequently forms brain metastases whereas spread of prostatecancer to the CNS is extremely rare.43 In addition, even thoughglioblastomas are highly vascularized, they almost never initiatevisceral metastases. A deeper understanding of the signals thatallow for homing of CSCs into specific niches, permissive formetastatic growth, could lead to novel anticancer therapies thatrestrict cancers to their primary sites, which can often becontrolled by radiation therapy.

Radiation Response of CSCs andthe CSC NicheOne important aspect of CSCs is their relative resistance tochemotherapeutic agents and radiation. First described forGBM by the Rich Lab44 and for breast cancer by ourlaboratory,19 this radioresistance is based in the increasedability to scavenge free radicals formed in response toradiation19 and differences in how DNA double-strand breaksare processed and repaired.19,44 These observations have beenreproducedbymanyother laboratories and for several differentsolid cancers. It is possible that these features are notnecessarily acquired during carcinogenesis but rather inheritedfrom their nonmalignant stem cell of origin.5,12

A consequence of the relative radioresistance of CSCs is thatradiation enriches for CSCs. A general explanation for thisobservation has been selective killing of radiosensitive non-tumorigenic cells.19,44,45 However, a closer look at the absolutenumbers of breast and glioma CSCs before and 5 days aftersingle doses of radiation and consideration of cell cycleduration and intrinsic radioresistance of CSCs indicated thatradiation generates CSCs from previously nontumorigeniccells.46 This process was preferentially observed in a polyploidcell population and was dependent on re-expression ofepigenetically silenced transcription factors Klf4, Sox2, Nanog,andOct4,46,47 the same factors used to reprogram somatic cellsinto induced pluripotent stem cells.48,49 Importantly, thesecells not only expressed CSC markers but also were 32-fold

more tumorigenic than the nonirradiated cells they originatedfrom.46

Irradiation of tissues leads to a proinflammatory micro-environment23 and chronic inflammation has long beenknown to cause cancer. A recent report demonstrated thatactivation of nuclear factor-κB signaling, the master regulatorypathway of inflammation,50 can dedifferentiate postmitoticintestinal epithelial cells into CSCs.51 Furthermore, a hypoxicmicroenvironment, frequently found in human cancers andpreviously thought to protect cancer cells from radiation, alsoseems to promote acquisition of a CSCs phenotype bypreviously nontumorigenic cells.52 Even though we were notable to detect hypoxia-mediated radioprotection of CSC,53

areas of low oxygen tension could be the birthplace of de novogenerated CSCs that led to tumor recurrence. Remarkably,radiation-induced reprogramming seems to be under tightnegative-feedback control by preexistingCSCs46 andmay holda key to improve radiation treatment outcome.Taken together, the current data suggest that neither the

CSC hypothesis nor the clonal evolutionmodel are exclusivelycorrect but may rather describe tumor organization at differentstages of a disease and under different microenvironmentalconditions.

Concluding RemarksOur understanding of normal and CSCs and their respectiveniches is far from complete. However, from a radiobiologicalpoint of view, it is common sense that the interaction of thestem cell with its niche codetermines the response to radiation.Dissecting these interactions is challenging but it may holdgreat promise to alter radiation treatment outcome in the clinic.

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