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Cancer Treatment Reviews xxx (2011) xxx–xxx

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Cancer Treatment Reviews

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Antitumor treatment

Cancer stem cells hypothesis and stem cells in head and neck cancers

Giuditta Mannelli ⇑, Oreste GalloFirst University Clinic of Otorhinolaryngology-Head and Neck Surgery, Director Prof. Oreste Gallo, University of Florence, Azienda Ospedaliera Universitaria Careggi,Via Largo Brambilla 3, 50134 Firenze, Italy

a r t i c l e i n f o

Article history:Received 27 September 2011Received in revised form 23 November 2011Accepted 24 November 2011Available online xxxx

Keywords:Cancer stem cellsMarkersTarget therapyHNSCC susceptibility

0305-7372/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.ctrv.2011.11.007

⇑ Corresponding author. Fax: +39 055435649.E-mail address: [email protected] (G. Man

Please cite this article in press as: Mannelli G,doi:10.1016/j.ctrv.2011.11.007

s u m m a r y

There is increasing evidence that the growth and spread of cancer is driven by a small subpopulation ofcancer cells, defined as cancer stem cells (CSCs). Recent data indicate that the initiation, growth, recur-rence and metastasis of cancers are related to the behavior of a small population of malignant cells withproperties of stem cells, and information about them are potentially helpful in identifying the target forthe tumor’s therapeutic elimination. The presence of subpopulation cells with phenotypic and behavioralcharacteristics corresponding to both normal epithelial stem cells and to cells capable of initiating tumorshas been also reported in head and neck squamous cell carcinomas (HNSCCs).

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Introduction

Normal tissue renewal depends on a subpopulation of cells,termed stem cells, that are characterized by an extensive prolifer-ative potential. Stem cells are distributed in vivo in relation to unitsof epithelial structure, and epithelial stem cells typically divideboth to renew themselves and to generate transit amplifying cellsthat undergo a series of amplifying divisions as they differentiate.1

Malignant neoplasia are believed to result from sequential muta-tions, as a consequence of progressive genetic instability and/orenvironmental factors. Reya et al., in 2001, supported the hypoth-esis that cancer may arise from mutations in stem cell popula-tions,2 because, as the only cells permanently resident in thetissue, they appear to form the initial target for the action ofcarcinogens and, the development of epithelial tumors is thoughtto involve initial genetic changes in stem cells themselves.3–5

According to the CSCs theory, only a specific subpopulation ofcancer stem cells have the ability to sustain cancer growth. Fourkey characteristics define CSCs population: (a) only a small por-tion of the cancer stem cells within a tumor have tumorigenicpotential when transplanted into immunodeficient mice; (b) theCSCs subpopulation can be separated from the other cancer cellsby distinctive cell surface markers; (c) tumor, resulting from theCSCs, contains mixed tumorigenic and nontumorigenic cells ofthe original tumors; and (d) the CSCs subpopulation can be seri-ally transplanted through multiple generations, indicating that itis a self-renewing population.6–9

ll rights reserved.

nelli).

Gallo O. Cancer stem cells hyp

To date, human CSCs have been identified and purified in avariety of malignancies, including breast, brain, prostate, ovarian,head and neck cancers; moreover, several studies have demon-strated that drug or radiation treatment of tumor cells can enrichand maintain the CSC subpopulation in vitro and in vivo, sug-gesting a responsibility from CSCs for tumor regeneration. Dueto the difficulty in studying normal cells and CSCs several recentstudies reported empirical models based on the isolation of cellswith stem properties by specific cell surface markers, the charac-terization of their behavior by in vitro cultures and in vivo self-renewal assays. Currently, xenograft assays for different organsites have been established for testing CSCs. As suggested byAACR Workshop on Cancer Stem Cells in 2006, the orthotopicxerograft assay is considered the gold standard for the identifica-tion of CSCs.73

In this review we argue the cancer stem cells hypothesis in headand neck cancers, by comparing the different isolation and purifi-cation techniques of CSCs in solid tumors especially in head andneck tumors, and looking at the its influence in tumor progression,its clinical and therapeutic implications in head and neck patients.We also analyze cancer susceptibility and its relationship withstem cell cancerogenesis.

Head and neck squamous cell carcinoma (HNSCC)

Head and neck squamous cell carcinoma (HNSCC) is among the10th most common cancer worldwide, there are about 780,000 newcases per year over all the world. In the United States, an estimated40,000 new cases of head and neck cancers are diagnosed each year;more than 90% are of squamous cells carcinoma primarily affecting

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Fig. 1. The dependence of normal stem cells on the niche limits their expansion.Interactions between normal stem cells and the tissue stroma (niche). In normaltissues, the niche cells (pale green) provide signals enabling normal stem cells(blue) to self-renew (curved arrow). Transit-amplifying progenitor cells (pink,yellow, orange) do not receive this signal and their proliferation is constrained bycellular mechanisms that count the number of mitotic divisions. With each celldivision, the proliferation capacity of these daughter cells declines (programmeddecline in replication potential), and their degree of differentiation increases.

2 G. Mannelli, O. Gallo / Cancer Treatment Reviews xxx (2011) xxx–xxx

the mucosa of upper aerodigestive tract, and arise from diverseanatomical locations, including the nasal and paranasal sinuses,lip/oral cavity, nasopharynx, orapharynx, larynx and hypopharynx.In 2002 over 500,000 cases of HNSCC were diagnosed and morethan 300,000 deaths from this disease were reported worldwide.10

The American Cancer Society had estimated that approximately35,720 new cases of HNSCC would have been diagnosed in the Uni-ted States in 2009.11

HNSCC is a heterogeneous group of neoplasias, each one charac-terized by its own anatomical, clinical and epidemiological proper-ties. The incidence of this kind of tumor depends on vary factors,one of which is the geographic arrangement: HNSCC reaches the50% of all of the tumors in East Asiatic continent, while in Europeand America reaches the 5%.13,14 These geographic variations re-flect the different distribution of risk tumor factors over differentethnic groups, with particular regard to tobacco, alcohol and sexualuses. About this, the most exemplary data are shown by the in-crease of the incidence of HNSCC in Central and East Europe, duringthe last generation, from 300% to 1000%.15,16 Also the incidence ra-tio men to women has been modified during the last 10 years from5:1 to 3:1, due to the increase of smoking in women17; this valuedepends on the anatomical site which has been considered, andthe maximum distance in ratio men to women is reached withthe larynx carcinoma, 15:1.16 The age acts a role in influencingHNSCC’s incidence too: in Europe patients are more than 40 yearsold in 98% of cases15 and the median age at diagnosis is 60.18

Tobacco use is the principle cause of malignant tumors of headand neck, the risk of developing a HNSCC in smokers is three tofour times more than in non-smokers. The alcohol intakes is thesecond risk factor responsible for cancers of head and neck. Zhanget al.19 have evaluated environmental tobacco smoke (ETS) and itsinteraction with mutagen sensitivity on the risk of head and neckcancer by investigating the relationship between ETS and HNSCCin a case-control study of 173 previously untreated cases withpathologically confirmed diagnosis of squamous cell carcinoma ofthe head and neck and 176 cancer-free controls at MemorialSloan-Kettering Cancer Center between 1992 and 1994. Control-ling for age, sex, race, education, alcohol consumption, pack-yearsof cigarette smoking and marijuana use, the risk of squamous cellcarcinoma of the head and neck was increased in ETS in a dose-dependent manner (adjusted OR, 2.4; 95% CI, 0.9–6.8). In fact,HNSCC could be either caused by multiple genetic alterations accu-mulated by genetic predisposition and chronic inflammationlinked to environmental influences, or by human papilloma virus(HPV) infection persisting, which represent a subgroup of HNSCCs,especially those of the oropharynx; ones induced by toxic sub-stances and others induced by the activity of the viral HPVoncogenes.20

Despite advances in treatment, which have improved quality oflife, survival rates have not improve significantly in more than30 years. Mortality from this disease remains high because ofdevelopment of metastases and the emergent of therapy-resis-tance local and regional recurrences. After receiving standard ther-apy a subset of patients fail to respond the treatment, or theircancer recurs. Nearly 90% of patients with stage I disease can becured, but more than 10% relapse and die. For more advancedstages, the proportion of recurrences and deaths increases to 30%for stage II, 50% for stage III, and more than 70% for stage IV.12

Local–regional relapse after definitive therapy is a major cause ofmorbidity and mortality in patients with head and neck squamouscell carcinoma (HNSCC). Many clinical and pathological prognosticfactors have been described in HNSCC, such as tumor stage, lymphnode involvement, postsurgical margin and histological grade;however these factors lack sensitivity and accuracy in the clinicalsetting and, with the exception of disease stage, are infrequentlyused to guide treatment decisions.21

Please cite this article in press as: Mannelli G, Gallo O. Cancer stem cells hypdoi:10.1016/j.ctrv.2011.11.007

Stem cells

Stem cells have the remarkable potential to develop into manydifferent cell types in the body during early life and growth. Inaddition, in many tissues they serve as a sort of internal repair sys-tem, dividing essentially without limit to replenish other cells aslong as the person or animal is still alive. When a stem cell divides,each new cell has the potential either to remain a stem cell or be-come another type of cell with a more specialized function, such asa muscle cell, a red blood cell, or a brain cell, dividing itself throughan asymmetric way. As stated first from studies of hematopoiesis,two golden standards for defining normal stem cells were firstestablished15: first, they are unspecialized cells capable of renew-ing themselves through cell division, sometimes after long periodsof inactivity; second, under certain physiologic or experimentalconditions, they can be induced to become tissue- or organ-specificcells with special functions. As exemplified by hematopoietic stemcells (HSCs), under normal physiological conditions, adult stemcells can live in a prolonged state of quiescence. Cell cycle regula-tors p21CIP1 and p18INK4C have been shown to regulate the quies-cence of HSCs. Once they have exited from the quiescent state,stem cells either self-renew or differentiate to generate progeniesdepending on the nature of both intrinsic and extrinsic stimulatorysignals. Wnt, Notch and hedgehog signaling pathway have beenshown by genetic models to be essential for promoting stem cellself-renewal in various systems. On the other hand, signals derivedfrom BMP and TGF-b pathways negatively regulate stem cell prolif-eration. Stem cells represent only a minuscule fraction of the cellsthat constitute each tissue bit they are the only cells with self-re-newal capacity. Tipping the delicate balance between positiveand negative regulators of stem cell self-renewal in either directioncan be problematic in vivo.18

The balance between self-renewal and differentiation must bestrictly regulated to maintain the stem cell pool and to generatethe required supply of fully differentiated cells needed for tissuesto carry out their many tasks. Replacement of the mature cells intissues is accomplished by a highly orchestrated process in whicha relatively small population of self-renewing adult stem cellsgives rise to proliferating early and late progenitor cells (some-times called transit-amplifying cells) that undergo limited roundsof mitotic division and then terminally differentiate, losing theirability to proliferate further (Fig. 1).16 One of the main factorswhich contribute to stem cells physiologic number is a specializedmicroenvironment, the so called ‘‘stem cell niche’’.17 Stem-cellpopulations are established in ‘niches’ which are specific anatomiclocations that regulate tissue generation, cells maintenance and



G. Mannelli, O. Gallo / Cancer Treatment Reviews xxx (2011) xxx–xxx 3

repair, by paracrine factors and the role of the extra-cellular ma-trix.22 The niche saves stem cells from depletion, while protectingthe host from over-exuberant stem-cell proliferation. Adult or so-matic stem cells generally have limited function without the niche.The stem cell niche controls stem cell maintenance and the crucialchoice between selfrenewal and the initiation of differentiation. Tomaintain tissue homeostasis, the number of daughter cells that re-tain stem cell identity must be strictly controlled such that differ-entiated cells can be generated in response to, for example,wounding while the stem cell pool is simultaneously replenishedbut not expanded.15

Stem cells and cancer

Generation and regeneration of tissues depends on a subset ofcells described as stem cells; embryonic and somatic stem cells dif-fer in their proliferative potentials and differentiation plasticity,however, stem cells properties may vary quite widely; for example,behavior and marker expression depends on cells’ original site. Theconcept that tumor’s growth depends on a subpopulation of stemcells, like the growth of the normal tissue, was proposed manyyears ago.23 Conventional thinking is that cancer typically arisesfrom alterations in molecules that regulate cellular signaling path-ways involving DNA damage response, and which include tumorsuppressor genes and oncogenes products. This is the traditionalmodel, defined the ‘‘clonal genetic model of cancer’’, where canceris seen as a proliferative disease originating from mutated tumorcells which contribute equally to the tumorigenic activity. Tumorcells progress through a preneoplastic into a neoplastic phaseand subsequently metastasize.24 The greatest part of the existingtherapeutic strategies are still based on this kind of carcinogenesismodel.

Previous research has focused on understanding the geneticchanges directing a cell towards a malignancy and tumor behavior,without looking at into which cells are affected by these mutations.Recent evidences suggest cancer stem cells as sites of thesemutations.

The most recent model for carcinogenesis is the ‘‘stem cellhypothesis’’; here, cancer presents a hierarchy structure wherestem cells are at the top of the pyramid and have the capacity toundergo self-renewal and cell differentiation.

According to the old cancer model, all tumor cells are equallytumorigenic, and tumor growth is carried out by multiple molecu-lar mutations promoting cell proliferation which let cancer appearsas a proliferative disease. Otherwise, a subpopulation of formingtumor cells is the feature of the cancer stem cells hypothesis.

The stem cell origin of cancer hypothesis considers that stemcells or cells that acquired the self-renewal ability tend to accumu-late genetic alterations over long periods of time, evading the strictcontrol of their microenvironment, and giving rise to tumoralevolution.4

The resemblance between stem cells and cancer was observed along time ago. The first register concerning the hypothesis of can-cer origin from a rare population of normal cells with stem cellproperties was proposed almost 150 years ago.18,19 The studiesabout this subject returned over 40 years ago, when some investi-gations confirmed the CSC hypothesis showing that a single tumorcell could generate a heterogeneous progeny and give rise to a newtumor, through investigations performed in tumors derived fromascites fluid in rats, and teratocarcinomas and leukemias inmice.20,25–27 In this way, Park et al.28 observed through a primarycell culture assay some myeloma tumor stem cells in mouse, andHamburger and Salmon 23 corroborated the hypothesis that somecancers could contain a small subpopulation of cells similar tonormal stem cells, because they observed in primary bioassays that


the expansive growth of malignant lesions could suggest the pres-ence of a CSC population with stem cell properties, including indef-inite proliferation.

In animal models, the ability of a small population of cells tooriginate a new malignant neoplasia was demonstrated in a classicexperiment through transplantation of cells from human acutemyeloid leukemia that expressed some cell surface markers associ-ated with normal hematopoietic stem cells. Lapidot et al.29 showedthat these transplanted cells could initiate leukemia in nonobesediabetic/severe combined immunodeficient (NOD/SCID) micewhile other isolated cells could not.28 Since then, this assay has be-come the standard method for determining whether cell popula-tions isolated from solid tumors are CSCs.

Based on the ability of diverse purified populations to form leu-kemia in NOD/SCID mice, investigations started to search for stem-like cells in leukemias. Bonnet and Dick (1997) showed that theinjection of leukemic cells with a primitive hematopoietic progen-itor phenotype resulted in leukemias that could be transplantedinto secondary recipients, and also observed its ability to perpetu-ally self-renew.30 Since then, putative CSCs have been isolatedfrom many other tumors including brain, breast, colon, pancreas,prostate, lung, and head and neck cancer.31–35

Numerous hypothesis exist about the origin of a cancer stemcells. Clarke and Fuller formulated four main mechanisms of theirpossible origin.7

One is that expansion of the stem cell niche allows a corre-sponding expansion of cancer stem cells that have arisen from nor-mal stem cells. The expansion of niche cells may be driven byalterations in the cancer stem cells or in the niche cells themselves.In either case, there is an expansion of the self-renewing cancerstem cell pool that gives rise to aberrantly differentiated butnon-tumorigenic cancer cells, which comprise the bulk of thetumor.

The second mechanism is that alterations in cancer stem cellsenable them to commandeer alternative niche cells to providethem with self-renewal signals. This mechanism could result inthe invasion of local tissues or may facilitate cancer stem cellgrowth at sites of metastasis. Again, the resultant tumors are amixture of cancer stem cells and their non-tumorigenic progeny.

Another possible origin is from genetic or epigenetic alterationsin cancer stem cells which enable them to become niche-indepen-dent such that they undergo cell-autonomous self-renewal gener-ating tumors containing self-renewing stem cells and their non-tumorigenic progeny, which make up the bulk of the tumor. It ispossible that progression from niche dependence to independenceis common during the progression of a neoplasm to a more aggres-sive tumor.

Another pathogenetic mechanism concerns mutations that im-bue transit-amplifying progenitor cells with the stem cell propertyof selfrenewal. Further genetic events in this pool of abnormallyselfrenewing cells culminates in a malignancy. In this example ofselfrenewal, the cancer stem cell is derived not from a normal stemcell but from one of its partially differentiated progeny.

A different hypothesis about a cancer stem cell activation con-siders an extremely active process: cellular fusion that brings totransdifferentiation. Fusion between a stem and a cancer cell, withreassembling of genotype, may be the first step of neoplastic trans-formation. Bone marrow derived mesenchymal stem cells (BMSC)have exhibited the ability to leave the bone marrow, circulate inthe blood, and home in injured tissues and inflammation.36 Re-cently have been shown that BMSC are capable of directed migra-tion towards the tumors of various types and origins; Takaishiet al.,37 described selective migration of hematopoietic stem cellinto the gastric epithelia during Helicobacter gastric-infection,observing that the potentiality to develop a gastric carcinoma isowned only by this bone marrow population. These observations



https://www.researchgate.net/publication/14007548_Bonnet_D_Dick_JE.._Human_acute_myeloid_leukemia_is_organized_as_a_hierarchy_that_originates_from_a_primitive_hematopoietic_cell._Nat_Med_3_730-737?el=1_x_8&enrichId=rgreq-783f2213-eef1-4370-93d5-819f39b8a7d4&enrichSource=Y292ZXJQYWdlOzUxOTIzNjEyO0FTOjIzMDc4MDY2MDA4ODgzOEAxNDMyMDMzODA1OTc2

Fig. 2. In the stem cell model, only the stem cells or their progenitor cells have theability to form tumours. Tumour characteristics vary depending on which cellundergoes the malignant transformation. DTL, definitive tissue line; EP, earlyprogenitor; LP, late progenitor; SC, stem cell.


consider the possibility that fusion between a migrated stem cellsand an adult mutated cell within the tissue may lead to carcinomaformation.38 Indeed, BMSC are a source of circulating stem cells re-cruited from the blood into peripheral solid organs in times of tissuestress or injuries.39 Similar to wound healing, it is thought thattumor expansion also requires BMSC for angiogenesis and growth.

Several studies point to the ability of BMSC to produce growthfactors, such as vascular endothelial growth factor (VEGF), insulinlike growth factor (IGF-1) or transforming growth factor (TGFb)in a paracrine manner.40 BMSC participate in the formation oftumor-associated stroma. This may have an influence on the tumorsurvival.

The microenvironment of CSCs, also known as ‘‘niche’’, protectsCSCs against differentiation and apoptosis and maintains the statusof self-renewing,22 including cell-cell interaction, cell-non-cell ma-trix interaction, cytokines and blood vessels surrounding CSCs. Todate, accumulating evidences have demonstrated that the nichesplay a role in other aspects of CSC’s properties such as metastasis,therapeutic resistance and genetic instability. Hypoxic stress is animportant factor of niche, cancer cells express hypoxia induciblefactors (HIFs) to accomplish the adaptation to changes microenvi-ronment. Hypoxic stress also selects competent CSCs whichachieve more invasive properties through genetic instability.41

Sun et al.42 assert that fluctuating hypoxia induce malignantprogression and maintain CSCs.

As previously described, deregulation of the stem cells signal-ling pathways may lead to neoplastic proliferation with the devel-opment of a cancer stem cell.5 Numerous signaling pathways havebeen implicated in this process including Notch, Wnt, LIF (leuke-mia inhibitory factor), PTEN (phosphatase and tensin homologuedeleted from chromosome 10), SHH (sonic hedgehog) and BMI.The Notch pathway is important in haematopoietic and mammaryepithelial stem cells and its mutations have been observed in lym-phoblastic leukaemia and breast cancer.6 The Wnt pathway isimplicated in colorectal cancer, lymphoblastic leukemia, pilomatri-coma, medulloblastoma and in prostate, ovarian and breasttumors.43

Tumors are heterogeneous, but the mechanisms underlying thisare still unclear. Heterogeneity may result from mutations occur-ring early in a stem cell’s maturation. For example, chronic myeloidleukaemia is believed to derive from an early stem cell progenitorbecause its cytogenetic marker (BCR-ABL) is present in several celllineages, for example lymphoid, myeloid and platelet cells. How-ever, acute promyelocitic leukaemia may result from an abnormal-ity in a late stem cell progenitor in the myeloid lineage at thepromyelocitic stage. Tumors derived from an early stem cell maydevelop a more heterogeneous phenotype and have an increasedmetastatic potential. Mutations in late progenitor stem cells maylead to tumors of a single cell type with reduced metastatic poten-tial43 (Fig. 2).

These evidences have been supported for solid tumors too. A di-verse range of breast cancer may develop depending on where amutation occurs in this pathway.44 Consequently, a stem cell mod-el for estrogen receptor (ER) expression in breast cancer has beenproposed, dividing breast cancer into three types, in an attemptto explain how ER-positive, ER-negative or heterogeneous receptorstatus tumors can be created by mutations in the stem cell or pro-genitor cell populations. In early fetal life, stem cells are ER-nega-tive, but presumably under the influence of environmental factorsincluding estrogen, progenitor cells that are both ER positive andER negative can be identified at various times during growth, inparticular during puberty and pregnancy.44

Type 1 tumors develop from mutations in ER-negative stem/progenitor cells, blocking differentiation and preventing the devel-opment of ER-positive progenitors. These tumors are poorly differ-entiated and appear to be more aggressive with a poorer prognosis.


Less than 10% of these tumors are ER positive. Type 2 tumors arealso derived from mutations in the ER-negative stem/progenitorcells. However, a variable percentage of the tumor will differenti-ate into ER-positive cells. Anti-estrogen therapy can produce a de-crease in tumor size. Type 3 tumors are well differentiate andresult from mutations in ER-positive progenitor cells.

The main hallmarks of CSCs are their properties of self-renewal,their ability to generate tumors from very few cells, their slow celldivision, their ability to give rise to a phenotypically diverse prog-eny, and their selective resistance to radio- and chemotherapy.2

The self-renewal and differentiation characteristics lead to the pro-duction of all cell types in a tumor, generating heterogeneity.2,45,46

The differentiated cells constitute the tumor bulk, but they are notusually tumorigenic, because of lacking of self-renewal capacityand limited proliferation potential. However it has been shown be-fore, through several tissue systems, that the switch to carcinogen-esis can occur in either stem cells or progenitor cells which thenacquire stem properties. Additional confirmation that stem cellscan play a role in carcinogenesis are the homologies between nor-mal and cancer stem cells. In fact, by the analogy to normal stemcells, CSCs will be inherently resistance to chemotherapy andradiotherapy through mechanism able to protect cells from DNAdamages, including in addition to self-renewal, production of dif-ferentiated cells, activation of anti-apoptotic pathways such asangiogenesis, and the ability to migrate and spread in metastasis.

Stem cell markers

To facilitate the identification and purification of normal stemcells and CSCs expression, some specific cell surface markers hasbeen investigated, and several stem cell markers could be sharedby CSCs in multiple human tumor types. The standard proceduresfor the isolation of CSCs have been similar in many investigations.Among the most used in vivo models is the fractionation of tumorcells using cell-surface markers with stem cell characteristics fol-lowed by their implantation into NOD-SCID mice to let xenograftgrowth. The main surface marker phenotypes associated with stemcell characteristics include CD133, CD44, and CD24.

CD133, also called prominin 1 (PROM1), it is a glycoprotein ofcell’s surface, with five trans-membrane domains and two bigglycosilate extra-cellular rings. It was discovered as a marker ofnormal hematopoietic stem cells and was later used to purify puta-tive CSCs in several tumor types. Its power to identify cell with stemproperties it has been confirmed by recent studies, one of whom47

proposed the transplantation of autologous progenitor cell (EPC) asan alternative treatment to promote the revascularization of criticallimb ischemis (CLI). EPCs (CD34+ and CD133+) were implanted inthe ischemic limb by intramuscular injections and Kaplan–Meier




analysis revealed a limb salvage rate of 74.4% after 1 year. Thisstudy showed the angiogenesis capacity, a stem property, belong-ing to cell CD133+. Also, intracoronary infusion of CD133+ endothe-lial progenitor cells improves heart function and quality of life inpatients with chronic post-infarct heart insufficiency.48 To test vas-culogenic functionality of CD133+ cells NOD/SCID mice underwentligation of the right femoral artery followed immediately by cellinjection. Cells were tested for potential mechanisms mediatingthe in vivo effects, including migration, cytokine secretion andangiogenic augmentation (Matrigel assays); blood flow recovery,necrosis, bone marrow (BM) engraftment of human cells and histo-logic capillary density were determined. Surface expression analy-sis showed that CD31 (PECAM) expression was greatly increased inumbilical cord blood (UCB) CD133+ cells compared with BM MNC.At 28 days, perfusion ratios were highest in animals receivingUCB CD133+ cells, while animals receiving BM CD133+ cells. Ani-mals receiving CD133+ cells showed a statistically higher capillarydensity, reduced severe digit necrosis and increased engraftmentin the BM than animals treated with unselected BM MNC. CD133+

cells exhibit robust vasculogenic functionality.Here, we are interest in CD133+ not hematopoietic cells but in

CD133+ cancer cells, showing same stem properties anyway.49

About prominin’s family includes the prominin 2 which binds cho-lesterol and it is on the superficial protrusion of epithelial cytoplas-mic membrane. Anyway, high levels of CD133 expression are at themoment accepted as important marker on several cancer cellularlines. That is the reason why Mizrak et al.50 defined prominin‘‘molecule of the moment’’. Thanks to CD133’s selectiveness, ithas been started to use it, not only for allogenic transplants of hu-man hematopoietic stem cells, but also in order to identify stemcells’ niche in some solid tumors. Indeed, its principal characteris-tic is its sudden disappearing from cellular surface of differentiatedcells, this fact let CD133 be a unique and unequivocal superficialmarker of stem cells and their progenitor cells. In accordance withcancer stem cells theory, which specks about the existence of tu-mor initiating cancer stem cells, these clones should present notonly similar stem behavior, but also similar stem phenotype,CD133 expression included.

CD133’s display has been found in several solid tumors ofdifferent anatomical sites, such as: central nervous system can-cer,51–54 prostate cancer,31,55 pancreas cancer,56 colon cancer,57,58

liver cancer,59,60 melanoma,61–63 breast cancer,64 lung cancer,65

laryngeal cancer66 and osteosarcoma.67

Cheng et al.68 studied the accuracy of CD133 as marker for iden-tification and isolation of cancer stem cells in central nervous can-cers system. Yin et al.69 produced a new monoclonal antibody ableto identify the antigen AC133, CD133’s component. Singh et al.51

found that CD133+ cells could successfully grow under unattachedconditions, with neurosphere-like formations, whereas CD133�

cells could not. Only the CD133+ cell fraction, isolated from humanmedulloblastomas and glioblastomas and injected into the brainsof NOD SCID mice, contained cells capable of initiating tumors,with phenotypic similarity between engrafted and original tumor.According to other studies, CD133 has been shown to play a role inmigration and asymmetric division of stem cells.70

Some authors expressed doubts about using CD133 as singleand selective marker of cellular stemness,71–74 because they havedemonstrated the existence of CD133-negative colonies of centralnervous cancer cells able to generate in vivo tumors of the centralnervous system. A study on medulloblastoma cells75 showedCD133-posite and CD133-negative cancer cells with same cancero-genesis abilities. Also Meng et al.76 spoke about the possibility touse CD133 as a co-marker and not as the only one phenotypic char-acteristic of stem properties; indeed, by testing lung cancer celllines (A549 and H446), they proved that both CD133-positiveand CD133-positive colonies were able to forming new colonies,


to self-renewing, to proliferate, to differentiate, able to invade localand distant tissue, and with drug-resistance capacity.

Nevertheless, many literature’s results agree with identifyingCD133 as stemness’ marker. Indeed, the subcutaneous implanta-tion of hepatic cancer cell lines (Hun-7) CD133-positive intoNOD/SCID mouse made tumor growing, while CD133-negativeimplantation did not achieved the same results.59 This indicatesthe chance of a CD133-positive subpopulation, less than 1% ofthe implanted cancer cells, able to let tumor growing up insidean immunodeficient system.

In conclusion, CD133 is able to identify and isolate cancer stemcells.

CD44 is a cell surface glycoprotein receptor for hyaluronanacid, and it seems to be involved in cell adhesion, migration,and metastasis of cancer cells.77 Hyaluronic acid (HA), one ofthe main components of extra-cellular matrix and most notablyof the fundamental substance of conjunctive tissues. It displaysimportant biological properties and plays a significant role in cru-cial physiological processes especially when cellular plasticity isinvolved such as inflammation, immune reactions, angiogenesisand would healing. It is also strongly involved in neoplastic cellsmigration and therefore in metastatic spreading of malignant tu-mors. These varied physiological functions are related either tointeractions with specific or less specific cellular membranereceptors or to the production of HA fragments of different sizes,generated for example by local traumatism or inflammation, frag-ments displaying specific properties according to their size. TheCD44 cell-surface marker has been used to identify putative CSCsin many tumor types, such as breast tumors,77 prostate,31 pancre-atic,34 and head and neck carcinomas.35 It is a single gene 60 kbweight product containing 20 exons, which mapped on chromo-some locus 11p13. Up to 1–17 exons codify extra-cellular do-mains, the 18th exon codify a short trans-membrane domain,while 19 and 20 codify a cytoplasmatic domain. Up to 1–5 andto 16–20 produce standard the isoform of CD44 (CD44s) or its he-matic isoforms (CD44H). The last 10 exons are variable, primarilyexons 6–14, they are may be alternatively spliced giving rise tomultiple variant CD44. Alternative splicing and post-translationmodifications are tightly regulated and permit expression of mul-tiple different CD44 isoforms. Various CD44 variant isoforms aredifferentially expressed in normal and malignant cells. CD44 isa surface glycoprotein which acts as inter-cellular receptor or ma-trix-receptor, as a signal carrying and grow factor presenting.CD44 includes many types of trans-membrane glycoproteinsbelonging to several tissues (hemopoietic, endothelial, mesenchy-mal and epithelial ones). The CD44 protein has got extracellular,trans-membrane and cytoplasmatic domains, which differentiateeach other by a glycosaminoglycan change or a specific N- or O-glycosilate type-specific. Therefore, CD44 isoforms are differentin dimensions, going from 90 to 250 kDa, and in their tissue dis-tribution. CD44 isoforms are: CD44s (standard one), CD44e (epi-thelial one) and CD44v (splice-variant), all of these does codifyproximal portion of extra-cellular domains.78,79 Overexpressionof several CD44 variant isoforms has been associated with tumorprogression, suggesting that these isoforms may have unique sig-naling properties. Several human tumors show CD44 alteredexpression and translation, with CD44 changing during tumorprogression. In lung cancer, CD44v5 and v6 are the preferentialvariant expression promoting metastasis in squamous cell carci-noma and bronchioalveolar carcinoma80,81; in colon cancer,CD44v3 seems to promote invasion and resistance to apoptosis,while CD44v6 is associated with metastasis and decreased dis-ease-free survival82–84; in breast cancer CD44v3 promotes metas-tasis and cancer progression.85–87 These observations couldindicate an important role of CD44 in tumor progression, cellulartransforming, tumor diagnosis and prognosis.




In conclusion, CD44 could have several functions linked toswelling, advancing and metastasizing of the cancer. It is not onlyhyaluronan acid’s receptor, but also it is able to interact with manymatrix proteins such as fibronectina, collage type I and IV, sergly-cina and osteopontyne, which is the basal mechanism for tumor’sproliferation, migration and angiogenesis.88 CD44 can link itselfto proteins of the cytoscheletro. It can interact with molecular sig-nals of the oncogenesis such as p56Ick and p185HER2. Severalgrowth factors (bFGF, HB-FGF, HGF, PDGF) and cytokines (IL-8) in-duce CD44 expression by linking its extra-cellular eparan-solfatechains.89

About squamous-tissue CD44 expression, normal squamousepithelia are strongly positive at CD44s-antibody and CD44v-anti-body, especially for CD44v4, v6 and v9. CD44 v2–v10 are expressedby normal epithelium cells and cancer squamous cell lines, whilethey are not present on malignant cells’ surface.

CD24 is a mucin-like adhesion molecule expressed by neutro-phils, pre B lymphocytes and a large variety of solid tumors.Functionally, CD24 enhances the metastatic potential of malig-nant-cells, because it has been identified as a ligand of P-selectin,an adhesion receptor on activated endothelial cells and platelets.90

Lim and Oh investigated the role of CD24 in various human epithe-lial neoplasias, and demonstrated that intracytoplasmic CD24expression was found to be highly associated with adenocarci-noma of the colon, stomach, gallbladder, and ovary.

These investigations suggest that diverse stem cell markers canbe expressed in different tumors by the CSCs, and the significanceof these observations in most human cancers remains to be deter-mined. Therefore, it is possible that each tumor could have a pre-valent and specific CSC phenotype (Table 1).91

CD133 and CD44 and metastases

The role of CSCs in metastasis is not clear yet, but it is likely theyare the cells responsible for the development of metastasis. CSCswould migrate and attach a new location, and local conditions

Table 1Cancer stem cell phenotypes according to stem cell markers in different organs(modified from Ref. [91]).

Stem cellmarker

Organ Cancer stemcell phenotype

References

CD44 Breast CD44+/CD24� Al-Hajj et al.24

CD44+/CD24�/CD133+ Wright et al.64

Pancreas CD44+/CD24+/ESA+ Li et al.101

Olempska et al.56

Head and neck CD44+ Pries et al. 176

Prince et al.35

Prostate CD44+/CD24� Hurt el al.225

Miki et al.55

CD133 Brain CD133+ Singh et al. 226

Taylor et al.52

Calabrese et al.53

Wu et al.54

Chang et al.122

Prostate CD133/CD44/a1b2 Collins et al.31

Melanoma CD133+ Marzani et al. 91

Head and neck CD133+ Zhou et al.66

Zhang et al.140

Bone CD133+ Tirino et al.67

Colon CD133+ O’Brien et al. 91

Ricci-Vitiani et al.58

O’Brien et al.57

Li et al.101

CD24 Colon, stomach,gallbladder andovary

CD24+ Lim and Oh90

Pancreas CD44+/CD24+/ESA Li et al.101

Olempska et al.56


should able to stimulate and support CSCs and the production oftheir progenitors.

In 2006 Hill et al.92 spoke about the emerging role of CD44 incontrolling bone’s metastasis both in hematopoietic cancer and so-lid tumors. It was evidenced that the interaction between CD44and hyaluronan acid would help the entrance of cancer circulatingcells into bone marrow and its colonization.

Shmelkov et al.93 studied the CD133’s contribute in inducingmetastasizing process in colon cancer. By using CD133lacZ mouse,where the endogenous CD133 expression controls the expressionof lacZ gene, and where chronic colic inflammation induces cancerprogression, it has been demonstrated that CD133 was presentedby cells expressing adhesion molecules (EpCAM). Neverthelessthey concluded that CD133 was not able to identify cancer stemcells in metastatic cancers, because CD133-positive and CD133-negative populations demonstrated same colonies-forming capa-bility in vitro, and same tumorigenesis ability in vivo. Moreover,CD133-negative population formed more aggressive colonies.

On the other hand, some Italian searchers proposed CD133 as amarker able to identify cancer stem cells both in primitive tumorsand in their metastasis.94 They studied 17 primitive tumors andtheir correlated eight cases of hepatic metastasis. Their results con-firmed the existence of a CD133-positive population in both prim-itive and metastatic tumors, in this last case CD133-positive cellswere more numerous than in the primitive site. And CD133-posi-tive cells showed a strong capacity on forming colonies much morethan CD133-negative ones. A study on cancer colon metastatic celllines (HTC116) evidenced a more clonogenic and tumorigenicproperties belonging to cancer cells co-expressing CD133 andCD44 than CD44�/CD133� cancer cells.95

It has been demonstrated the role of CD133 in inducing metas-tases also in ovarian tumor96 and prostatic cancer.97

In breast tumors CD44+/CD24�/low population has showntumorigenic ability. The prognostic value of CD44+/CD24�/low pop-ulation’s prevalence has been analyzed in 136 patients with breastcancer, with and without local recurrence. In normal mammary tis-sue the presence of CD44+/CD24�/low population was between 0%and 40%, and it increased until 80% in cancer tissue. CD44+/CD24�/low was expressed in less than 10% of the total cancer cellsin 122 cases, while the remaining tumor expressed it with morepercentage.98 Here CD44 and CD133 expression were not corre-lated with metastases’s incidence.

Ling et al.99 made a mouse model in order to identify the phe-notypic aspect of metastasizing cancer cells in breast tumors. Theyused CD44+/CD24�/low cancer human breast cell populations andthey injected into NOD/SCID mice transplanted with marrow bonefour different concentrations of these cancer populations: group Awith 1 � 105, group B with 1 � 106, group C with 1 � 106 of cancerhuman breast cancer cell lines (MDA-MB-231) and group D with1 � 106 of MDA-MB-231 but into NOD/SCID mouse without mar-row bone transplanted. In conclusion, group B was stronglyCD44-positive, with weak CD24 expression and with high inci-dence of distant bone metastasis suggesting a role of CD44 in guid-ing the metastasizing process.

Sheridan et al.100 studied the invasive and proliferative abilitiesof CD44+/CD24� cell populations coming from metastasis sites.They made intra-cardiac injection in NOD/SCID mice of cancerstem cells taken from five different breast cancer cell lines(MDA-MB-231, MDA-MB-436, SUM1315, Hs578T and HBL-100).Results demonstrated a high percentage of CD44+/CD24�, morethan 30% in the primitive tumor, but a reduction of their prolifer-ative and expansive capabilities in metastatic sites, suggesting thatCD44 is necessary but sufficient to identify metastatic cells withstem properties.

Recent studies confirm the metastatic potential of these cancerstem cells. In fact, Li et al.101 tested tumorigenic potential of cancer




cell line SW480 CD133+, and orthotopic transplantation experi-ments showed that the CD133 possessed heterogeneity in intesti-nal wall invasion, lymph node and liver metastases. The CD133positive-single cell progenies and expression of cancer stem cellsmarkers (CD133, CD44, and CXCR4) associated with metastatic po-tential. On the other hand, CD133 negative-single cell progeniesdid not produce secondary transplanted tumor, intestinal invasionand metastasis. Another study102 examined the correlation be-tween CD133 expression in childhood melanoma and lymph nodeand/or visceral metastasis. Double immunostaining with CD133and Ki-67 was performed in the cases showing CD133 positivity.Three of 12 melanoma patients had lymph node metastasis andonly one had multivisceral metastases; CD133 was positive onlyin these four patients.

An animal model trial investigated transcript isoform changesin exons of breast cancer cells that characterize tumors of differentabilities to form growing metastasis. Significant expressionchanges has been identified and gene pathways analysis showedthat alternative isoforms of CD44 were involved in cell growth, cellinteractions, cell proliferation, cell migration and cell death.103

Recent results confirmed that high level of CD133 is associatedwith higher risk of dissemination in glioblastoma.104

An evaluation of the CSCs’ role in metastasis will produce newinsights into this process and will lead to new treatments in orderto prevent or eliminate metastatic disease.

CD133 and CD44 and clinical outcome

Choi et al.105 studied the relationship between CD markersexpression (CD133, CD44 and CD24) and, invasiveness and differ-entiation of colorectal adenocarcinoma. In colorectal adenocarci-noma 128 of 253 (24.5%) were CD133-positive and 395 (75.5%)CD133-negative; 264 (50.5%) were CD24-positive and 259(49.5%) were CD24-negative. Five hundred and two (96%) wereCD44-positive and 21 (4%) were CD44-negative. CD133 was pres-ent more in advanced T stage cancer. CD24 expression was seenin the degree of differentiation and correlation between CD44expression and clinicopathological factors was in tumor size. Atthe end authors concluded that markers expression was not closelyrelated to survival. Nevertheless, Wang et al.106 by investigatingCD133 expression in a series of 73 patients with rectal cancer ofvarious type of TNM stages, after preoperative radiation and cura-tive resection, found that the proportion of CD133+ cells was a sig-nificant prognostic factor (p < 0.05) for adverse disease-freesurvival, suggesting CD133 expression as an independent markerof patient survival in rectal cancer. Another relationship betweencancer cells CD133-positive and patient’s 5-years-survival, wasanalyzed by Chun-Yan et al.107 who studied CD133 expression in104 stage IIIB colon cancer who had underwent radical resectionof the tumor. Results spoke of CD133+ cancer cells’ percentageand invasive depth of tumor as independently prognostic factors.Indeed, tumors with CD133 low expression (<5%) were stronglyassociated with a higher 5-years-survival rate than those with ahigher percentage of CD133+ cancer cells (>55%). This fact ofCD133 expression linked to poorer prognosis in patient with lo-cally advanced colon cancer suggested a CD133+ cells contributeto tumor progression.

An immunohistochemistry test studied, between 1999 and2004,108 the expression of p53, p21, PNNA and CD44v6 in 259 colo-rectal cancers. The correlation between clinicopathological ormolecular variables and clinical outcomes, including local recur-rence, metastasis, disease-free survival and overall survival, wasanalyzed. TNM staging, preoperative CEA and CD44v6 level wereindependent risk factors predicting overall survival or disease-freesurvival. But, TNM staging was the only risk factor predicting localrecurrence.


CD133 is an independent prognostic marker in glioma, too. Acombined detection of Nestin/CD133 co-expression predict theaggressive nature of the tumor109; in fact, the low expression ofthe two markers significantly correlated with long survival of gli-oma patients. In vitro presence of CD133+/Ki67+ cells is a indexof disease progression and poor clinical outcome.110

In gastrointestinal stromal tumors (GISTs) loss of CD44s positiv-ity is correlated with poor clinical outcome.111 Thirty-three GIST-affected patients followed a 17.5-months long control and theoverall median survival was of 25 months. CD44s and its variants(CD44v3–6 and v9) expression was evaluated by immunohisto-chemistry test in order to check a correlation between CD44 pres-ence and clinical outcome. Nine of the 33 (27%) patients hadmetastasis, 9 (27%) had recurrent disease, and 9 (27%) died for dis-ease. More than half (53%) of patients with GIST CD44s expressiondied. Only the expression of CD44s correlates with clinicaloutcome.

Pacifico et al.,112 demonstrated a high significant correlation be-tween CD44v3 expression, Breslow’s thickness, Clark’s levels andpatient age in primary cutaneous melanoma. Furthermore,CD44v3 expression was showed to be significantly associated withbetter outcome.

Diaz et al.,113 showed an increased-free survival for patientswith tumor breast cells high CD44s positivity, suggesting a favor-able prognosis in patients with node-negative invasive breast car-cinoma and CD44s-positive. Another study,114 proposed thedominance of the CD44+/CD24�/low tumor cells inversely associ-ated with lymph node metastasis, and tended to inversely associ-ated with the stage of the disease.

A recent meta-analysis conducted by Zhou et al.,115 about theprognostic role of cancer stem cells in breast cancer, showed thatcancer stem cells, in particularly those positive for aldehyde dehy-drogenase 1 (ALDH1), were significantly associated with high his-tological grade, estrogen receptor negativity, progesteronereceptor negativity, human epidermal growth factor receptor 2(HER2) positivity and associated with poor overall survival. How-ever, the presence of cancer stem cells was not associated with tu-mor size or nodal status.

In chondrosarcoma patients a study highlighted the CD44abnormal expression in 56.7% as the CD44v5 in 43.3% as theCD44v6 in 6.7% of tumors. Further the major overexpression wasfound in grade III and II chondrosarcoma. The multivariate analysissuggested the prognostic role of CD44 for chondroid bone tumorsindependent of grading and other covariates.116

A multicentre study regarding on incidental prostate cancerevaluated the prognostic role of the expression of CD44s. Theauthors recorded that a CD44s expression was stronger (inverse)correlated with Gleason scores than did other conventional prog-nostic variables and, therefore, might become a valuable adjunctto better predict outcome in incidental prostate cancer prior toradical prostatectomy.117

Recent study examinedthe prognostic impact of CD133 expres-sion in gastric carcinoma.118 The CD133 expression positively cor-related with tumor extension and the degree of nodal involvement.A multivariate analysis, furthermore, revealed CD133 positivity asan independent prognostic factor superior to the depth of invasionand similar to nodal involvement in gastric cancer (p < 0.05).

CD133 and CD44 and resistance to radiotherapy and chemotherapy

The CSC theory of carcinogenesis requires to rethink how toevaluate the efficacy of therapies. Responses to chemotherapyand radiotherapy in models have relied on tumor shrinkage andpercentage of cells killed as a marker of treatment effectiveness,but in patient the response to therapy is classified in term of tumorsize reduction. Indeed the reduction in tumor size does not mean




that CSCs elimination have been completed. Several studies havebeen reported at testifying the difficulty in killing not the tumorbulk but the cancer initiating population.

Tumor initiating cells result be resistant to chemotherapy61,119

and to radiotherapy,120,121 thanks to mechanisms such as: mem-brane transporters (ABC glycoproteins) able to pull drugs out ofthe cell, and DNA-damage checkpoint repair systems.

CD133-expressing glioma cells play a critical role in tumor pro-gression and present a radio-resistant phenotype122; Chang et al.demonstrated that CD133+/SirT1 co-expression significantly en-hanced the sensitivity of glioblastoma-derived-CD133-positivecells (GBM-CD133+) to radiation and increased the level of radia-tion apoptosis, proposing SirT1 as a potential therapeutic target.

This does not confirm the role of CD133 as unique marker ofcancer’s radiotherapy-resistance, but suggests CD133 could con-tribute to induce and maintain radio-resistance.

Several studies spoke about the role of cancer stem cells markersin stopping or reducing chemotherapy efficacy. Although chemo-therapy with temozolomide may contain tumor growth because itinduces a dose- and time-dependent decline of the stem cell popu-lations, recurrences suggest that these subpopulations maintain tu-mor persist. Beier et al.,123 investigated the effect of temozolomideon CD133+ and CD133� glioblastoma cancer stem cell lines (GBMCSC lines). Incubation with sub lethal concentration of the drugfor two days completely depleted clonogenic tumor cells in vitro,and substantially reduced tumorigenicity in vivo. Cancer stem celllines (CSC lines) expressing O6-methylguanine-DNA-methyltrans-ferase (MGMT) needed a 10-fold higher dose to obtain the sameresult that with MGMT-negative CSC lines. Glioblastomas arenotorious for resistance to therapy, which has been attributed toDNA-repair proficiency and to the particular biologic behavior oftumor stem-like cells. A study published on the Journal ClinicalOncology,124 aimed to identify the specific molecular cancer profilefor treatment resistance. Authors interrogated 80 glioblastomas forassociation with resistance to therapy and patients were treatedwith radiotherapy and adjuvant temozolomide. A dominatingexpression of HOX genes, comprising prominin-1 (CD133),emerged as independent predictor of poor survival. This confirmedthe implication role of CD133, cancer stem cell marker, in reducingradio- and chemo-therapy tumor response.

Several studies evidenced the over-expression of the ATP-pomp(ABCG5), both in CD133-positive cells and progenitors of epider-mal human melanocytes and in a malignant melanoma cell sub-population.61 CD133+/ABCG5 melanoma cells were resistance todoxorubicina. In conclusion, many cases report CD133-expressionstrongly closed to chemo- and radio-resistant cancer cells.

Yasuda et al.,125 studied the CD133 role in residual colon cancercells after chemoradiotherapy (CRT). CD133 in residual cancer cellswas higher than in stromal cells in post-CRT specimens. Patientswho developed distant recurrence had a higher post-CRT CD133compared with those without recurrence. So, elevated post-CRTCD133 was associated with poor disease-free survival and reducetherapy response. Saigusa et al.,126 investigated the role of threestem cell markers (CD133, OCT4, and SOX2) in rectal cancer andtheir level association with clinical outcome post-CRT. Each markerand their co-expression presented a poor prognosis predictionpost-CRT.

CD44+/CD24�/low cancer initiating cells has been isolated frombreast cancer cell lines (MCF-7 and MDA-MB-231) and their re-sponse to radiation was investigated by measuring reactive oxygenspecies (ROS) levels.127 This experiment evidenced a relativelyradio-resistance subpopulation which increase in number aftershort courses of fractionated irradiation.

CD44s and hyaluronate (HA) receptor play a role in acquiredresistance to cisplatine (CDDP) in non-small cell lung cancer(NSCLC).128 In fact, NSCLC cell lines transfected with the CD44s


gene (H322/CD44s) cultured in HA coated plates were more resis-tance to CDDP than that on bovine serum albumin. Multidrugresistance protein 2 (MRP2) was induced in H322/CD44s-positivecells. CD44 demonstrated its chemotherapy-resistance role alsoin human ovarian cancer.129 Chemotherapy eliminates the bulkof the tumor but it leaves a core of cancer cells with high capacityfor repair and renewal. Understanding how cancer initiating cellsreact to cancer treatment will facilitate improvement of cancertreatment in the future.

There are likely multiple downstream signaling pathwaysthrough which HA-CD44-mediated chemoresistance occurs. Incolorectal cancer, CD44 was reported to mediate chemoresistancethrough the activation of the PI-3 kinase/AKT pathway35 IncreasedAKT phosphorylation is known to suppress the activation of proa-poptotic signals generated as a result of the DNA damage caused bytreatment with cisplatin and other chemotherapeutic drugs.130–132

Because activation of the PI-3 kinase/AKT signaling pathway isthought to promote cisplatin resistance, some authors studiedwhether PI-3 kinase might also mediate HA-CD44 promotion ofcisplatin resistance in HNSCC. Because Rho kinase has been re-ported to interact with the PI-3 kinase/AKT pathway, they alsoinvestigated the role of this signaling protein in HA-mediated cis-platin resistance. They observed that simultaneous inhibition ofRho kinase and PI-3 kinase reduced cisplatin resistance in HNSCCto a greater degree than was observed with inhibition of either en-zyme alone. These findings suggested that both Rho kinase and PI-3 kinase are involved in HA-CD44-mediated cisplatin resistance inHNSCC. The last study133 suggested the importance of Rho kinaseand PI-3 kinase signaling in HA-CD44-mediated cisplatin resis-tance in HNSCC. It also illustrated how inhibition of these enzymesdiminishes the capacity of HA and CD44 to promote malignant tu-mor phenotypes such as abnormal proliferation, migration, andinvasion in a single HNSCC cell line; and that CD44 and its associ-ated signaling molecules (i.e., Rho kinase and PI-3 kinase) may beimportant targets for the future development of novel therapiesfor the treatment of head and neck cancer.

A trial investigated the behavior of cancer initiating cells (CICs)after ionizing radiation treatment.134 The authors found that irra-diated CICs survived and retained their self-renewal capacity forat least four generations and they demonstrated that fractionatedradiation not only spared CICs but also mobilized them from a qui-escent/G0 phase of the cell cycle into actively cycling cells, whilethe surviving non-tumorigenic cells were driven into senescence.

In a study was evaluated the correlation of CD133, OCT4 andSOX2 expression with distance recurrence in colorectal patientsafter chemoradioterapy. The authors found a significant correla-tions among these three genes after chemoradiotherapy, infact pa-tients, who showed high levels of these three genes, developeddistant metastases and had a poor disease-free survival.126

Isolation and purification of CSCs

The increasing evidence that the growth and spread of cancersis driven by a small subpopulation of cancer stem cells (CSCs),which are capable of long-term self-renewal and generation ofthe phenotypically diverse tumor cell population, had led towardsnew treatment strategies for the elimination of cancer by consider-ing the presence of this kind of cancer cells. However these newand hoped CSCs-targeted strategies are currently hindered by thepoor understanding of their behavior and by the lack of specificand unique markers for their identification. The concept that can-cers arise from stem cells was first proposed more than 150 yearsago, but only recently advances in biology have allowed for moredirect validation of the CSC hypothesis existence. It is well knownthat CSCs and normal stem cells share some properties, both form




small self-renewing subpopulations but they differ in that self-re-newal is highly regulated in normal stem cells but poorly regulatedin CSCs. They share organogenic capacity but while the normalones generate normal tissue, the CSCs typically generate tissuewith aberrant differentiation patterns.135 Despite these differences,the parallels existing between normal somatic stem cells and CSCs,suggest that the principles of normal stem-cell biology may beusefully applied to studies of CSC identification and their roles intumor development and progression.9 Current methods for deter-mining whether cells isolated from solid tumor are CSCs, consistof purification of these one based on such stem properties as theirability to form spheres in culture,1,136,137 membrane effluxactivity,138,139 specific cell surface molecule expression,140,141 andenzymatic activity detection of aldehyde dehydrogenase 1(ALDH1).142,143 Purified cells are then tested for the capacity tooriginate tumors into immunodeficient mice models.144

In vitro culture of spheres cells

The patterns of behavior are not easily studied in vivo, and cellinteractions are not well modeled standard in vitro conditions asthese fail to re-establish in vivo-like patterns of normal epithelialgrowth. However, advances in tissue culture techniques have al-lowed the development of in vitro organotypical models that canregenerate patterns of cell behavior and differentiation similar tothose found in vivo.145,146 These cultures are created by plating epi-thelial cells onto collagen matrices populated with fibroblasts andthen maintaining the cultures at the air/medium interface to allowdifferentiation.147 Using such cultures systems, Javaherian andco-workers148,149 have demonstrated an important role of untrans-formed keratinocytes for cell interactions in the growth and sur-vival of the transformed cells. The use of this method have beenextended to investigate the behavior of cells derived from naturallyoccurring tumors. Mackenzie,150 by analyzing the patterns ofgrowth and clonal behavior of pure malignant cell lines of oralsquamous cell carcinoma, has shown their interaction with the nor-mal cells and also provided some evidence for the persistent of stemand amplifying subpopulations in malignant tumors. Interestinglyhe found that, even at early stages, tumors cells were found in clus-ters within the normal epithelium appearing formed by focal prolif-eration of malignant cells, and the number of clusters indicated thatonly a subpopulation of the tumors cells had participated in the for-mation of the expanding clones. Results suggest that clonogenicand expansive properties belong to the primitive stage of cells.The emerging proliferative hierarchy of stem and amplifying cellsresults having a range of proliferative potentials and showing cellkinetic heterogeneity.151 Estimates for the number of cells actingas clonogens suggest fractions as low as 0.01–1% of the total popu-lation,152 but it is recognized that the identity of clonogenic cellswith stem cells in questionable.153 The primary aim of Locke andMeckenzie’s study with carcinoma-derived cell lines,1 was to deter-mine whether this morphologic heterogeneity, typically found inmalignant epithelial cell lines, was mainly the result of an underly-ing stem and amplifying cell pattern and, if so, how differing cellu-lar properties by such morphologic differences. Subpopulation withself-renewal property, its generation of amplification hierarchy andtheir production of differentiated cells were the three criteria cho-sen to indicate the presence in malignant cells of stem patterns.Then, all the cell lines examined showed marked clonal heterogene-ity and developed a range of colony morphologies paralleling toholoclone, meroclone and paraclone morphologies produced bynormal keratinocytes, and the colonies produced by malignant celllines indicated that holoclone morphology predicts higher levels ofexpression of stem cell-related molecules.

Clonal assays thus seem to provide a robust and reliable methodfor the identification and isolation of cells with stem cell properties


from both normal and neoplastic oral mucosa and can provide sys-tems for the characterization of CSC responses to various factorsand therapeutic agents.154

Flow cytometry using Hoechst dye exclusion

In 1996, Goodell et al. described a side population (SP) of bonemarrow-derived cells that were able to efflux Hoechst dye 33342and were enriched in hematopoietic stem cells (HSCs).155 This re-sults in a characteristic flow cytometric profile with dye-excludingcells distinct from the main cell population and they are referred toas side population (SP) cells. The cellular capacity for Hoechst dyeefflux is determined by the concentration of ATP-binding cassette(ABC) transporter superfamily efflux pumps in the plasma mem-brane, including multidrug resistance 1 (Mdr1a/1b, mouse; MDR1,human)14 and breast cancer resistance protein 1 (Bcrp1)/ATP bind-ing cassette, subfamily G member 2 (ABCG2). SP cells express highlevels of stem-associated genes and possess multipotent differenti-ation potential, suggesting that they have stem cell behaviors. SPcells have also been identified in a large variety of cancer cell linesand primary tumor tissue samples.

Zhang et al.,140 in order to characterize the biological features ofside population, they sorted and compared SP and non-SP in oralsquamous cell carcinoma (OCC). They demonstrated the presenceof SP in most of OCC cell lines and primary tumors, suggestingthe SP may play a role in tumorigenesis of OCC. Conclusions werethat SP phenotype from OCC showed stronger capacity to form col-onies; in nude mice, only 10,000 SP cells were needed for tumordevelopment compared to 1,000,000 non-SP cells and the SP cellsgenerated SP and non-SP populations. These findings providedthe tumor stem cell phenotype of SP in OCC and their role inOCC tumorigenesis.

Wan et al.156 examined the role of SP cells in human laryngealcancer cell lines by flow cytometry. They identified cancer stem-like properties of SP such as self-regeneration, high proliferativecapacity, radiotherapy resistance, and tumorigenicity. However,not all SPs are CSCs. Despite this, the SP is a good source of en-riched resident stem cells in tissues when the CSC-specific surfacemarkers are unknown. A study of June 2010,141 investigated theexistence of stem-like cells in established head and neck squamouscell carcinoma (HNSCC) lines, HSC3 and HSC4. Higher expression ofstem cell markers was detected in SP than in main population (MP)cells, suggesting the existence of cancer stem-like cells in HNSCCs.

Flow cytometry using cell surface markers

Since their discovery in hematopoietic cancer, CSCs have beenisolated from many solid tumors. Jones and colleagues were thefirst to identify normal epithelial stem cells by using cell surfacemarkers and flow cytometric techniques.157

Sorting cells based on specific surface markers expression, hadsupported the isolation of small subpopulation of cells with relativecell-cycle quiescence and high long-proliferative potential. Inter-estingly, CD44 was the primary marker used to isolate tumor-initiating cells from breast cancers.1,24

About normal oral epithelium few reliable stem cells markershave been found, and most of them, being intracellular markers,are unsuitable for flow sorting. Studies on OSCC-derived cell lineshave identified additional markers which could be useful for theisolation of stem cells from neoplastic mucosa. Locke et al.1

showed high levels of stem-cell related molecules such as b1-inte-grin, E-cadherin, b-catenin, epithelial specific antigen (ESA) andCD44. Then Prince et al.35 demonstrated the isolation of a tumori-genic subpopulation of cancer cells from HNSCC specimens iso-lated by fluorescence-activated cell sorting (FACS) analysis usingantibody against CD44. These CD44+ cancer cells were comprised




of less than 10% of the bulk tumor, were able to give rise to tumorformation in immunodeficient mouse, showing their tumor-initiat-ing capacity. There are several reports regarding isolation of CSCsfrom HNSCC most of them look at CD44 as the most common usedstem cell surface marker, while some others have established CSCsusing FACS analysis with antibody against another cell surface pro-tein, which is CD133.

Enzymatic activity detection of ALDH1

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be amarker for cancer stem cells. However, the role of ALDH1 in headand neck squamous cell carcinoma (HNSCC) has yet to be deter-mined. The aldehyde dehydrogenase (ALDH) families of enzymesare cytosolic isoenzymes that are responsible for oxidizing intra-cellular aldehydes and contributing to the oxidation of retinol toretinoic acid in early stem cell differentiation. Furthermore, activ-ity of the ALDH1 enzyme has been identified as being responsiblefor the resistance of progenitor cells to chemotherapeutic agents152

and can be used to select for a highly enriched population of pro-genitor cells in bone marrow153 and umbilical cord sources.154

ALDH1 could be a useful marker for sorting cancer stem-like cellsand ALDH1+-lineage cells, which have the highest self-renewalability, tumorigenicity, and radio-resistance. Visus et al. suggestedthat ALDH1A1 is a marker in HNSCC for distinguishing premalig-nant cells and is also an essential epitope for developing ALD-H1A1-based vaccines for HNSCC therapy.158 Recent studies haveshown that ALDH1 is a CSCs marker and that its presence stronglycorrelates with tumor malignancy as well as self-renewal proper-ties of stem cells in different tumors, including breast cancer,hepatoma, colon cancer, and lung cancer.159–162 However, whetherALDH1 can be a useful marker of CSC and/or aid cancer diagnosis inHNSCC is still an open question. ALDH1 may have a role in earlydifferentiation of stem cells through its function in oxidizingretinol to retinoic acid. Retinoic acid signaling is linked to cellulardifferentiation during development and plays a role in stem cellself-protection throughout an organism’s lifespan.163 ALDH1 activ-ity can provide a common marker for both normal and malignantstem cells. Cells with high ALDH1 activity have been associatedwith several types of human hematopoietic and neural stemcells,153 and the ALDEFLUOR assay was also successfully used toisolate CSCs from leukemia and multiple myeloma.164 The ALDE-FLOUR assay is a simple method for identify CSCs and it is basedon enzymatic activity of ALDH1, responsible of the intracellularoxidation of aldehydes. Therefore, in agreement with Crokeret al.,163 the use of ALDH1 activity detection as a purification strat-egy allows an efficient isolation of normal and malignant humanstem cells based on a developmentally conserved stem cellfunction.

In mammary gland ALDH1 is a marker of stem/progenitor cellsof normal human breast and breast carcinomas.159 Furthermore,ALDH1+ cells had the ability to generate tumors in NOD/SCID mice.Liu et al.165 also demonstrated the relationship betweenALDH1-positive breast cancer cells and high risk of recurrence.

Chen et al.133 confirmed that ALDH1+ cell lines evidenced epi-thelial–mesenchymal transition (EMT) shifting, which is a processby which epithelial cells lose their polarity and are converted to amesenchymal phenotype. This process has been regarded as thecritical event to induce morphogenetic changes during embryonicdevelopment, organ fibrosis, and tumor metastasis.166 Snail, amember of the zinc-finger transcription factor family, is one ofthe master regulators in promoting EMT and mediates invasive-ness and metastasis in many different types of malignant can-cers.167,168 Chen et al.,133 first showed that the increasedincidence of ALDH1 expression correlated positively with the stag-ing of 226 HNSCC patients. Furthermore, they demonstrated that


HNSCC-ALDH1+-lineage cells (ALDH1+ and CD44+ CD24_ALDH1+)were involved in tumor malignancy and invasiveness and alsoexhibited refractory properties for radiotherapy. Then, the role ofSnail in maintaining cancer stem-like properties of HNSCC-ALDH1+-lineage cells was further investigated. A higher expressionof embryonic stem cell-related genes, drug-resistant genes, andEMT-related genes in ALDH1+ and CD44+ CD24_ALDH1+ cells wasdetected, and the knockdown of Snail expression showed a signif-icantly decreasing of the expression of ALDH1, inhibition of cancerstem-like properties, and blocked the tumorigenic abilities ofCD44+ CD24_ALDH1+ cells. Finally, in a xenotransplanted tumori-genicity study, the treatment effect of chemoradiotherapy forALDH1+ improved by Snail siRNA was confirmed.

A recent study, published by Clay et al. in September 2010 169 hasidentified the tumorigenic capacity of ALDH-high HNSCC subpopula-tion by in vivo experiment, confirming ALDH1 as possible highly selec-tive marker for cancer stem-like cells in HNSCC.

Head and neck cancer stem cells

Cancer stem cells (CSCs) are tumor cells which have stem fea-tures such as self-renewal, high migration capacity, drug resis-tance, high proliferation abilities. A large number of articles havebeen written about their role in carcinogenesis of solid tumors,2

whereas to date, only few studies have been reported about HNSCCCSCs.12 In the literature there are only six principal review articleswhich discuss the role of CSC in HNSCC,12,20,170–174 and only one ofthem12 introduces own results about cancer stem cells in vivoexperiments. If we have a look, in the literature, at the articleswhich treat the theme of CSCs in HNSCC, numbers are very limited,too. Studies on HNSCC cell lines are more widespread instead ofthose on primary tumors, this could be referred to the difficultyin culturing fresh specimens, and to the costs of this assay.

In fact, about ten main studies on HNSCC cell lines have beenwritten during the last 6–7 years,1,19,42,133,136–138,153,175,176 whileonly four articles present experiments on primary tumors of headand neck, one of which is a review article.12,35,169,177

Head and neck cancer is the sixth most common cancer andabout 300,000 new cases of it are diagnosed each year, despite ad-vances in treatment, 5-year survival rate for this cancer has notbeen improved in more than 30 years.172 Progress in treatmentand prognosis for HNSCC have been limited by understanding tu-mor growth and progression.

Human tumors, including HNSCC, present heterogeneous his-tology, and additional concept is that cancers contain subpopula-tions of cancer cells with highly tumorigenic capacity, so-calledcancer stem cells (CSCs). This heterogeneity has been attributedto a process of clonal expansion in which various clones are contin-uously generated, and has been interpreted as not the result of ge-netic alterations, but it is due to different abilities to proliferate of asingle tumor clone.170 In his review, Costea et al.170 described thepossible origins of CSCs that could support the hierarchical prolif-erative structure of an oral squamous cell carcinoma (OSCC). Themore accepted hypothesis is that CSCs may originate from normalsomatic stem cells. As normal oral epithelia have a rate of renewalestimated to be about 14–24 days, most epithelial cells do not existenough to accumulate the genetic changes necessary for the devel-opment of an OSCC. The hierarchical stem cell structure present inhuman oral epithelia, indicates that stem cells are the only long-time residents of oral epithelia and, consequently, the only cellsable to accumulate the necessary number of genetic changes forthe development of a malignancy.53 It has been estimated thatthree to six genetic events are required to transform a normalhuman cell into a cancer cell. Califano et al.178 published datawho found a direct relationship between a built-up of genetic



https://www.researchgate.net/publication/41014346_Clay_MR_Tabor_M_Owen_JH_Carey_TE_Bradford_CR_Wolf_GT_et_al.._Single-marker_identification_of_head_and_neck_squamous_cell_carcinoma_cancer_stem_cells_with_aldehyde_dehydrogenase._Head_Neck_32_1195-1201?el=1_x_8&enrichId=rgreq-783f2213-eef1-4370-93d5-819f39b8a7d4&enrichSource=Y292ZXJQYWdlOzUxOTIzNjEyO0FTOjIzMDc4MDY2MDA4ODgzOEAxNDMyMDMzODA1OTc2


alterations and malignant phenotypic progression of oral squa-mous cells carcinomas, and he resumed them into a genetic pro-gression model. This model is supported by in vivo experimentalanimal studies of skin carcinogenesis that indicate that exposureto initiating agents produce cellular changes which are retainedwithin the tissue for extended periods of time.54 The early observa-tion that only a slowly cycling subpopulation of adult murine epi-dermal cells retains carcinogenesis in skin, also sustains theconcept that epithelial CSCs are derived from their normal tissuecounterparts. Another hypothesis, supported by both in vitro andin vivo experiments suggests that oncogenic events can occur inkeratinocyte that are downstream from the primitive stem cell,and can induce stem-cell renewal capacity and reduce terminaldifferentiation.60

Over 90% of all human neoplasia are derived from epithelia.179

In normal epithelial tissues stem cells are usually located in the ba-sal layers, and they perform asymmetrical division which allowsstem cells self-renewal and differentiating in terminal cells. Whenthey divide they renewal themselves and also produce cells whichenter the differentiation pathways to amplify cell differentiatedpopulation. The degree of differentiation of a carcinoma dependson the proportion of undifferentiated tumor stem cells, the stageof maturation of the majority of cells of the bulk, and on the abilityof some cells to escape arrest and to differentiate.180 The hierarchi-cal stem cell structure present in human epithelia indicates thatstem cells are the unique long-term residents cells and the onlycells capable of accumulate necessary number of genetic changesfor malignant development.170 It becoming apparent that thegrowth of tumors is associated with stem and amplifying patternssimilar to those of normal tissues.

Six acquired hallmarks of cancer have been suggested.181 limit-less cell replication, self-sufficient growth, avoidance of apoptosis,insensitivity to antigrowth signals, sustained angiogenesis, inva-sion and metastasis.

The identification of cancer stem cells in head and neck tumorsrepresents a fundamental goal in the agenda of stem cell biologistsas well as the detection of the key factors involved in self-renewaland differentiation pathways. Nevertheless, it is to keep in consid-eration that there is still an open controversy on the role of cancerstem cells in tumor development and progression. In addition, thebiomolecular markers defining cancer stem cell minor subpopula-tions show a large variance that makes difficult their characteriza-tion and the study of the tumor biology.

The difficulty in studying both normal and cancer stem cellsfrom human tissue has been related to the lack of experimentalmodels able to assess their growth and function. The patterns ofbehavior of tumor cells are not easily studied in vivo, and cell inter-actions are not well modeled by standard in vitro conditions. How-ever, development of in vitro ‘‘organotypical’’ models is able toregenerate pattern of cell behavior and differentiation similar tothose found in vivo.150 Garlik and co-workers148,149 incorporatedcells an in vitro transformed cell line into populations of untrans-formed keratinocytes and demonstrated the important role ofcell-interactions in growth and survival of the transformed cells.Mackenzie,150 extended this system to provide information aboutthe behavior of cells derived from primary oral squamous cell car-cinomas. He demonstrated the possibility to isolate and subse-quently generate expanding cell colonies from the majority oftumor samples collected, where the main cause of failure to estab-lish cell lines was the inability to suppress contaminating organ-isms, despite the presence of antibiotics in the culture medium.This is a snag with which we also faced to in our preliminary expe-rience. In fact, about one-third of the cell isolated from primary tu-mor samples developed bacterial or fungal infections which led todeath of the culture (data not published). Mackenzie150 found thateven if at early stages, tumor cells were found in clusters within


the normal epithelium, only a subpopulation of the tumor cellshad participated in the formation of expanding clones. The epithe-lium appeared to contain fewer tumor cells than would have beeninitially present, and this discrepancy became clearer with furthergrowth. Interestingly, each of the tumor lines maintained a rangeof morphological differences which reflected those differencesexisting between individual tumors. Finally, tumors showedpatterns of kinetic heterogeneity. Locke et al.1 proposed an investi-gation in order to determinate whether the morphologic heteroge-neity, typically found in malignant epithelial cell lines, is mainlythe result of an underlying and amplifying cell pattern, and if sohow differing cellular properties are predictable from such mor-phologic differences. Three criteria were chosen, based on thoseof normal epithelia, as able to indicate the functional presence ofstem cell patterns in malignant cell lines: presence of a subpopula-tion with self-renewal ability, presence of their amplification hier-archy, and their asymmetric cell division. For the normal humanepithelia stem cell properties have been assessed by the differingpatterns of keratinocyte clonogenity. Colonies are divided into:holoclones, meroclones and paraclones, where the first one con-sisting in tightly packed small cells with high-proliferative patterncorresponding to stem cells, the second one contain larger cellswith less proliferative potential and not able to self-renewal, whichcorrespond to transit amplifying cells, the third one with larger andflattened cells with low proliferative potential and correspondingto early differentiated cells. Locke et al.1 demonstrated that cellsisolated from holoclones were able to generate all colony typesand were thus identified as the source of the rest of the cell popu-lation that was undergoing progressive loss of regeneration ability.Evidence of differentiation markers for human oral mucosa, cyto-keratins (CK) 6 and 16, were shown by staining techniques inmalignant paraclones. Further, normal epithelial stem cells andholoclones share expression of molecules such as b-catenin, b1integrin and E-cadherin, while lower level of these markers werefound in meroclones and paraclones. Malignant holoclones sharepatterns of marker expression with both normal epithelial stemcells and tumor-initiating cells. Taken together these findings indi-cate the presence of a malignant epithelial subpopulation withincell lines able of self-renewal, produce proliferative and differenti-ating progeny, and that its proliferative capabilities are maintaineduntil cells begin to differentiate.

By showing that clonogenicity is restricted to a subpopulationof the total cells, authors have demonstrated that heterogeneityof cells within HNSCC cell lines reflects a stem cell pattern.1,175 Clo-nogenicity was associated with a particular cellular morphologyand expression pattern, indicating that most of cell and colony het-erogeneity was due to the presence of hierarchies of cells at differ-ent stages of maturation. Several observations indicate that cancercell lines fulfill the three criteria, mentioned before, which indicatethe persistence of stem cell pattern in vitro. For example, stainingfor differentiation markers such as CK6 and 16 indicates the persis-tence of asymmetric cell division which is the key of the originaltumor heterogeneity. The production of differentiating cells indi-cates that the structure of malignant tissue reflects the structureof the tissue of origin.

Ones the maintenance of a subpopulation of stem cells duringpassage of carcinoma cell lines in vitro has been shown, additionalcharacteristics of malignant stem cells have been described.

Mackenzie150 observed that in organotypical cultures all the tu-mors stratified and showed some differences between basal andsuprabasal cells, where tumor cells tended to remain basal, andthose leaving the basal layer entered into the differentiation path-way. Locke et al.1 investigated how the holoclones morphology,considered predictive of the stem cell phenotype, correlated withthe expression of CD44 and ESA (epithelial-specific-antigen) whereCD44 stained the peripheries of cells in holoclones, and ESA was




less tightly restricted to meroclones. Also Prince et Ailles12 evi-denced the developmental hierarchy of HNSCC by demonstratinga cellular organization with differentiation from basal layer towardapical one. Immunohistochemistry on sections from primaryHNSCC showed that the tumorigenic population of cancer cells cor-related with the more basal-appearing cells, where the cytologicand architectural tumor features were similar to normal squamousepithelium. CD44 co-staining CK5 and 14, markers of basal regionin normal squamous epithelium, were limited at the basal layer, asinvolucrin, differentiation marker, stained areas of the tumorCD44-negative belonging to apical layer.

The preliminary immunohistochemical results (Fig. 3) of ourongoing study, on sections from primary HNSCCs, has providedfurther evidence for a developmental hierarchy in HNSCC, similarto that expected in normal squamous mucosa. Well- to moderatelydifferentiated HNSCC demonstrate a cellular organization with dif-ferentiation from a basal layer with cells of immature morphologytoward an atypical layer with a more mature appearing squamousmorphology. The analysis of the distribution of CD44 in well- ormoderately differentiated tumors, suggested a clear topographicalredistribution in favor of a strong positive basal cell layers, unlikethe more keratinized and differentiated cells in which this markerwas absent (Fig. 4). These findings provide further evidence thatHNSCC is organized into a developmental hierarchy, as is predictedby the CSC theory of carcinogenesis and suggest a possible value ofCD44 in HNSCC CSCs characterization.

Goodell et al.155,182 described primitive stem cells with long-term proliferating ability in the murine haematopoietic systemable to exclude the DNA-binding dye Hoechst 33342, this popula-tion differs from the main cell population and it is referred as side-population (SP). However the characteristics of this population arenot yet entirely clear and it also appears to vary for different tis-sues and morphological states; for example, for normal epidermis

Fig. 3. Immuhistochemistry for CD133(a) and CD44(b) marker stem cells of tumorbiopsies.

Fig. 4. Analysis of the distribution of CD44 in moderately or highly differentiatedtumors, suggesting a clear topographical distribution of CD44-positive cells of thebasal layers, while in more differentiated and keratinized tissue this marker isabsent.


there is no correspondence with label-retaining cells or other prop-erties expected for stem cells.175 Otherwise, Zhang et al.140 demon-strated the presence of SP in most of oral squamous cell carcinoma(OSCC), by analyzing OSC cell lines and primary tumors. Theynoted that SP was highly variable among the OSC samples from0.1% to 10% of the cellular population. Their proliferation rate gaveminimal contribution to tumor growth. More SP cells were accu-mulated in G1 phase, whereas more non-SP cells were in S phase.SP cells demonstrated less squamous differentiation pattern bylower expression of CK13 and involucrin, and higher expressionof CK19, a stem cell marker. These results indicated that SP possessstem cell features and phenotypes, because also able to regeneratea population of both SP and non-SP cells in vitro and in vivo. At thesame time the heterogeneous distribution of the CD44 and Bmi-1suggested the existence of a subpopulation in SP cells. IdentifyingSP cells may help in isolate CSC, Wang et al.183 underlined thatwhereas SP cells possess many stem cells properties, particularsurface markers are required to distinguish kinds of CSC in differ-ent tissues. They observed the presence of a ‘‘side population’’(SP) with stem-like properties, studying cancer-cell lines purifiedfrom 5 nasopharyngeal carcinomas. Immunofluorescence analysisallowed to define a cellular subpopulation (2.6%) which showedradioresistance, chemoresistance, and the peculiar exclusion fromthe dye Hoechst 33342, a property shared with normal stem-cells.From in vitro cultures, these cells were able to develop tumors inNOD/SCID mice.148 Tabor et al.184 have recently published a studywhich adds evidence to the cancer stem cells theory, by showinghow side population are able to produce tumors in animal model,whereas non-side populations were not; they underlined that SPrepresents cells with cancer stem cell properties and provided amethod by which CSCs can be isolated and studied.

A well known property of normal stem cells is their dependenceon their microenvironment, or ‘‘niche’’, to maintain their quiescentand undifferentiated state, while maintaining their proliferationand differentiation potential.185 Cell-to-cell interactions throughdirect contact or secreted factors support the survival and maintainthe stemness of stem cells in cancer and normal tissues. Severalstudies indicate an important role of the stroma in epithelial stemcell survival and in controlling cell behavior; in fact, recent insightsshow that cancer is not only a disease of the transformed epithe-lium, but it is also influenced and dependent on its stromal envi-ronment. The malignant progression of oral epithelial cellsappears to be accompanied by microenvironmental alterationsassociated with fibroblast activation or conversion to a myofibro-blast phenotype.57 Activated myofibroblasts can produce pro-inva-sive signals for transformed oral keratinocytes mediated either bydiffusible or solid matrix molecules, by direct cell-to-cell contact orby a combination of both.31

De Boeck et al.186 suggested in their review that, resident andbone marrow-derived mesenchymal stem cells are precursors ofthe stroma associated with HNSCC and contribute to blood andlymph angiogenesis. Perivascular niches have been identified inneural tumor stem cells187 however, it is not known if head andneck cancer stem cells are localized in close proximity to bloodvassels; Krishnamurhy et al.188 have recently demonstrated, byin vitro analysis, that endothelial cell-secreted factors promotedself-renewal of head and neck CSCs and that about 80% of thesecells were located next to blood vassels in human tumors, suggest-ing both the enhancing stem cells survival and self-renewal byendothelial cells, and the existence of perivascular niches inHNSCC.

The growth of normal and malignant stem cells in suspensionwas initially shown for neural cells and subsequently for cellsfreshly isolated from both normal and malignant stem cells; thisability to growth in suspension appears to be a generalized prop-erty of malignant cells and each of the HNSCC cell lines examined



Fig. 5. Epithelial nature of cells confirmed by the presence of cells cytokeratine-positive, in order to exclude inflammatory and fibroblastic cells replication.


in the three main studies1,150,175 contained cells with the ability toproliferate in suspension to form ‘‘tumor spheres’’. Expansion insuspension, therefore, did not appear to alter their prior in vitrogrowth characteristics. For all cell lines the overall patterns of clo-nogenicity, markers of expression, side population distribution andgrowth in suspension were similar but some differences betweencell lines did exist. Such minor differences are probably due to theirrandom acquisition of different genetic changes during malignantprogression, anyway they do not seem to be correlated withchanges in mechanisms controlling stem cell behavior. Isolationand prolonged culture of malignant cells could be associated withadaptive changes to in vitro conditions, where cells lack of stromalinteractions able to influence malignant cells behavior. Demonstra-tion of CSC existence is accomplished through experimental strat-egies which combine sorting of tumor subpopulation identified bydiffering surface markers expression with functional transplanta-tion into animal model.

Currently, various markers and methodologies are performed toidentify CSCs; however, no single surface marker or method canprovide unequivocal identification of CSCs in HNSCCs. Therefore,the combination of surface markers and proliferation rate wouldallow a clearer definition of CSCs.

It is known that the in vitro tumor sphere method for the enrich-ment and isolation of CSCs may not imitate well the in vivo situa-tion, since maintaining a small population of cancer stem cells ina cancer require a specific microenvironment189; so far, the serialtransplantation method is generally considered the gold standardfor the isolation of the small population of high malignant cellsfrom a solid tumor. Chen et al.190 found an increased expressionof stem cell markers between malignant stem cells of establishedcell lines and cancer stem cells in original tumors. It could be dueto the enrichment of all highly malignant cancer stem cells of estab-lished cell lines in sphere assay, while the serial transplantationmethod may enrich only the original cancer stem cells that producetumors in vivo. Interestingly, the xenograft-derived cells also dis-played increased capability for metastasis, which suggests thatthe enriched subpopulation may also present metastatic capability.

Prince et al.12 suggested the CD44 surface protein as anotherputative marker of CSC in HNSCC. In their study, nine primaryHNSCCs tumoral specimens were analyzed and were subjected toFACS analysis with the aim to identify two distinct cellular popula-tions CD44� and CD44+. The latter ones, once injected in NOD/SCIDmice, showed, in vivo, much more capacity to develop tumors, thanCD44� cells. Although the CD44+ HNSCC cells were able to producenew tumors, the relatively number which must be implanted to in-duce tumor growth led to hypothesize that this population did notrepresent the ‘‘pure’’ CSC population of HNSCC. In fact, in HNSCCexpression of CD44 or epithelial specific antigen does not seemto able to isolate CSC population, however it is possible to refinethe ability to isolate HNSCC CSCs using additional cell surfacemarkers. A CD44+ population was also reported by Okamotoet al. to characterize HNSCC CSC-like cells from HNSCC cell line(Gun-1).138 It was found that CD44+ cells possessed not only acapacity for forming tumor spheres, proliferation, migration, andinvasion in vitro, but also a resistance to chemotherapeutic agents.Supporting these observations, four relevant chemoresistant genes,ABCB1, ABCG2, CYP2C8, and TERT, were upregulated in the CD44+

population.Pries et al.176 studied the CD44 expression in both HNSCC prim-

itive tumors and permanent HNSCC cell lines, and demonstratedthat CD44 was constitutively expressed in permanent HNSCC celllines. This result suggests the potential role of CD44-positive cellsin the establishment of permanent cell lines.

Otherwise, CD44 is ubiquitously expressed and its expressionmeaning should be correlated to the expression of other stem cellmarkers. For example, we have investigated CD44s expression, by


using immunohistochemistry assay in 29 different primary tumorspecimens, reporting a percentage of expression of 93.1% of theglobal cells examined, being 27 on 29 cases CD44-positive(Fig. 3b). We have to underline that we did not use a CD44 vari-ant-specific antibody, ours was able to recognize every kind ofCD44 antigens so, we need more tests in order to affirm that ourCD44 positivity indicates stem properties.

The most of studies in the literature investigate CD44 expres-sion in correlation with cancer stem cell properties, both in vitroand in vivo systems, but it should be reminded the importance ofanalyzing additional cell surface markers in order to identify thespecific phenotype of tumor-initiating population and its tumori-genic attitude in subsequent transplanted models.

A report of Wei et al.137, using immunohystochemical and cyto-fluorimetric analysis, revealed CD133 expression as a CSC marker,in a laryngeal carcinoma derived cell-line termed Hep-2. In thisstudy the percentage of CD133+ cells resulted less than 5% of thetotal cell population. A similar percentage of CD133-positive can-cer cells has been documented by us in an ongoing preliminarystudy (data not published). In 29 primary tumor specimens ana-lyzed by immunohistochemistry only 3 specimens were CD133+

(10.34%) (Fig 3a). The percentage of CD133-positive tumor cellswas variable, indicating that only a fraction of the total neoplasticcells expressed this marker in our limited series of 29 cases stud-ied. In relation to our limited analysis, we can report that CD133+

cells were identified in cancer biopsies from 2 of 19 (10.5%) of N+

cases studied. Accordingly Wei et al.137 showed an in vitro prolifer-ating and differentiating rate capacity of such CD133+ cellular sub-populations being faster than those of CD133� cells. Moreover,they found that CD133+ cells possessed not only a marked capacityfor self-renewal, extensive proliferation and multilineal differenti-ation potency in vitro, but also a stronger tumor-forming abilityin vivo. Furthermore, Wei et al., using the same techniques, ob-served that with the decreasing of the CD133+ cells, proliferatingactivity was impaired too (from 90.88% to 4.53% in 12 days). Suchdata indicate that CD133+ cells, instead of those CD133�, form asubpopulation of high-proliferating cancer cells with stem potenti-ality. So far, CD133 has yet to be validated as a potential marker forCSC in other HNSCC cell-lines and it has not been reported to iden-tify CSCs in primary HNSCC.

During our preliminary ongoing experimental study, we suc-ceeded in obtaining primary cultures from tumor biopsies takenfrom primary tumors and from their lymph node metastasis, firstwe proved their epithelial origin (Fig. 5) then, by FACS analysis(fluorescence activated cell sorting),we documented the increasingexpression of CD44+ subpopulations through several in vitro cul-tural steps which confirmed their clonogenic property. We also




identified that clonogenicity was more evident in cultures obtainedfrom metastatic lymph node cells than that from the primitive tu-mor, suggesting a more aggressive attitude of cancer stem cells(Fig. 6a–b). However, in our preliminary analysis we did not iden-tify CD133-positive cells.

To date, there are only two studies in the literature which pro-pose the double staining for the markers CD44 and CD133 inHNSCC. Harper et al.175 tested stem cell patterns in HNSCC celllines trough in vitro analysis. They performed FACS analysis of po-tential stem cell markers (CD44, CD133 and CD29) in six HNSCCcell lines. They obtained a subpopulation with high expression ofCD44 for each cell line, while the CD133 expression was less in-tense, with low levels anyway consistent with levels reported inthe literature for neuronal and prostate tumor cells,31,191 even ifa small fraction of cells was usually identified and restricted tothe central region of holoclones. They also made analysis of doublestaining which confirmed a percentage of both markers CD44 andCD133 of about 0.1%, and for CD44 and CD29 (b1-integrin) of about0.8–3.6%. However, they found technically difficulties associatedwith the preparation of cells for CD133 staining; we also in ourpreliminary study had to face up to this kind of problems to pre-pare CD133 FACS analysis due to high CD133 antibody sensitivityto proteolytic digestion. Harper et al.175 found a co-expression ofhigh levels of CD44 and CD29, while a very small fraction of highlyexpressing CD133 cells showed high co-expression of CD44 orCD29. At the end, how such expression patterns relate to clonoge-nicity remains to be determined.

Yu et al.177 tried to identify the highly tumorigenic cell popula-tion in laryngeal carcinoma cells obtained from primary tumors by

Fig. 6. The comparison between FACS-analysis results of primary tumor and lymph nodtissues at To (time-zero) than in primary tumor at To. Conversely, CD133-positive cells haof CD133 expression does not completely exclude the possibility of its own real co-e(Allophyicocianin, specific phluorochrome for CD44) and PE (phyicoeritrin, specific phluoafter few culture passages. (b) Primary culture from N (pt T2N2b): control vs. APC and


using CD44/CD133 cell population. This is the only study whichcompare four kind of cell population: CD44+ CD133+, CD44�CD133-,CD44+ CD133� and CD44�CD133+. They found that the same dose of1 � 106 CD44+ CD133+ cells was injected into mice, the weight andvolume of the tumor were higher than those generated from thethree cell populations. They also analyzed the cell population inva-sion capability and that one of the CD44+ CD133+ population wassignificantly higher than that of other cell subsets. Moreover,immunohistochemistry analysis and semi-quantitative RT-PCRand Western blot analysis highlighted an abundant expression ofstem cell antigens such as SCA-1 and b1-integrin, and Bmi-1expression in CD44+ CD133+ than other cell populations (p < 0.01).

Okamoto et al.138 tried to study the CD44 and CD133 co-expres-sion in HNSCC lines (Gun-1), in order to define the correlation be-tween the presence of CSCs and tumor behavior. But this studyshowed the independent proliferative properties of cells CD44+

and CD133+, confirmed that tumor cells CD44+ let cancer stem cellsbe permanent in cancer cell lines, and found that the expression ofCD133 and ABCG2 (dye-excluding property) on CD44+ cells washigher than on CD44� cells.

Our ongoing study showed positivity at the immunohistochem-istry assay for CD133 in only 3 of the 29 cases analyzed (10.34%);while the marker CD44 resulted over-expressed in 27 of the 29cases examined (93.1%). In particular, all the three tumorsCD133-positive were co-expressing CD44. In conclusion, we can-not exclude the hypothesis that the co-expression of these twomarkers could help in selecting the initiating-cancer population.

Other possible CSC markers expressed in HNSCC are ALDH1, orgenes such as Bmi-1, implicated in self-renewal and considered a

e metastasis cultures, tends to show a more intensive signal for CD44 in metastaticve not been identified due to low sensitivity of FACS analysis. However, the absencexpression in CD44+ cells. (a) Primary culture from T (pt T1N1): control vs. APC

rochrome for CD133). CD44 expression is 25.7% at time-zero, while it becomes 99.5%PE. Here CD44 expression is 98.2% at the beginning.




stem cell-related gene.191,192 Zhou Chen in his review.172 demon-strated a statistically increase in ALDH1 expression in HNSC tu-mors with lymph node metastasis (LNM) compared to tumorswithout LNM (p < 0.0003). This result suggests that ALDH1 couldbe used as a potential CSC marker in HNSCC. About Bmi-1, Princeet al.35 found that it was differentially expressed in the tumori-genic population of HNSCC suggesting its potential role in this tu-mor growth. Through the combination of CD44+ cells and Bmi-1nuclear expression they highlighted their use in the diagnosis ofHNSCC with or without LNM or distant metastasis. Moreover,Krishnamurhy et al.188 have recently demonstrated that the combi-nation of CD44 and ALDH1 expression is able to select a subpopu-lation of cells with properties of CSCs highly tumorigenic, betterthan if used as single markers.

Furthermore, Chen et al.193 have tried to analyze the coexpres-sion of BMI1 and CD133 in laryngeal CSCs. They discovered thatBMI1 maintained CD133+ cell proliferations and prevented apopto-sis. Later,194 they underlined how BMI1 expression is associatedwith proliferation and tumor progression in laryngeal cancers.

It has been demonstrated the possibility of identify genes withpotentially important biological activities differentially expressedbetween subpopulations of tumor cells, thus emphasizing theimportance of identifying and isolating the appropriate subpopula-tions of tumor. Because CSCs represent a small portion (610%) ofthe tumor bulk and the genes of interest may be expressed atlow levels, analysis of the entire tumor may help in detecting theetiology of cancer.

The literature reports that HNSCC ALDH1+/CD44+ cells displayhigh tumor-initiating and radioresistant properties, and differen-tially express the Bmi1 gene, which is a promoter of the epithelial–mesenchymal transition133,195; Lo et al.196 reported the presence ofan inverse correlation in head and neck cancers between Bmi1 andmiR200c, this molecule belongs to a MicroRNAs (miRNA) class ofsmall RNA molecules acting as oncogenes but also as tumor suppres-sors; their syudy was addressed towards the understanding of tu-mor regulating properties and stemness properties of miR200c,and whether it can regulate the radioresistance properties ofHNSCC-CSCs by controlling the expression levels of Bmi1; moreover,the role of miR200c in regulating metastatic capabilities in HNSCC-CSCs was clarified. Lo et al. obtained that miR200c is inverselycorrelated with Bmi1 expression in HNSCC pathogenesis, and thatit maintains cancer stemness characteristics of HNSCC-CSCs bynegatively modulating Bmi1; furthermore, the overexpression ofmiR200c in ALDH1+/CD44+ cells let them be more sensitive to che-motherapy. Therefore, miR200c may represent a new therapeuticapproach for HNSCC-derived CSC populations treatment.

Clinical significance of cancer stem cell markers in head and neckcarcinoma

Accumulating evidence suggest that CSCs contribute not only totumor initiation, but also to aggressive tumor behaviors such asmetastasis, chemoresistance and radioresistance. There are severalstudies in the literature who show same and concordant resultsabout the main CSCs surface markers expression in head and necksquamous cell cancers (HNSCCs).

Pries et al.,176 by analyzing at the flow cytometry the expressionof different putative stem cell marker proteins in both solid HNSCCtumors as well as permanent HNSCC cell lines, noticed that distinctpopulations of CD44 expressing potential stem cells could be iden-tified in solid tumor of HNSCC patients. And the potential stem cellmarker CD44 was found constitutionally expressed on the surfaceof all the permanent HNSCC cell lines analyzed. This suggestedcancer stem cell CD44-positive may play a role in the establish-ment of permanent HNSCC cell lines, by selecting cell entities ableto drive progression and metastasis of HNSCC.


CD44 has an un ubiquitous expression, but its isoforms presenta tissue-specific distribution. CD44 splice variants are long-knownas being associated with cell transformation, and CD44s wasshown to be part of the signature of CSCs in colon, breast and inHNSCCs, too. The literature reports CD44v3 as promoter of tumorspreading and stopper of apoptosis in colon cancer, whileCD44v6 showed metastasizing properties. In lung tumor CD44v5and v6 seem to be correlated with metastasizing phenotype.CD44v3 let breast cancer progression and metastasis. There areseveral studies who confirm CD44 and its variants as cancer stemcell markers in HNSCCs. In fact, the CD44 family of receptors in-cludes multiple variant isoforms, several of which have been linkedto malignant properties including migration, invasion andmetastasis.

In order to identify CD44 expression both in tumor and normaltissue immunohistochemistry, immunofluorescence and immuno-blotting tests use antibodies against CD44. Monoclonal antibodiesagainst CD44 may be divided into two different classes:

(1) Antibody against cytoplasmic domain (pan-CD44).(2) Antibody against CD44 tissue-specific.

Studies through RT-PCR and Northern-blot analysis, have con-firmed the over-expression of CD44s and some of its isoforms innormal and tumor head and neck samples. In fact, normal oral,pharyngeal and laryngeal epithelium express high levels ofCD44s and multiple CD44-variant isoforms. About HNSCC, CD44sand CD44v splice variants expression is limited to cancer cells withstem properties, in fact not all the tumor cells present these mem-brane antigens. Their distribution is heterogeneous among the en-tire tumor mass. Several studies tried to analyze the possiblecorrelation between CD44 isoforms expression and clinical patho-logical and prognostic parameters (Table 2).

Referring to data from Table 2, many possible correlations be-tween CD44 expression and clinical-pathological and prognosis as-pects in HNSCCs emerge. A loss of CD44v3 expression was shownin all the poor differentiated tumors and its reduction of 50% wasseen in all the mild and total differentiated cancers.197 Down-reg-ulation of CD44 isoforms, specially of CD44 v4–5 and v6, correlatedwith the degree of differentiation of tumor cells and an increasedfrequency of regional lymph node metastasis, and loss ofCD44v4–5 expression was more evident in metastasis than in theprimitive tumor.197

A decrease of CD44v7 and v9 isoforms was observed to be cor-relate negatively with a shorter survival time and a shorter time toonset of relapse; CD44v9 expression was found reduced in 19/40(47.5%) of primary SCC of the oral cavity tumors, and this corre-lated with cell differentiation and tumor with secondary lymphnode metastases, suggesting a role of CD44v9 in metastases.198

Any change in CD44s and CD44v6 expression was observed in tu-mor tissues, but a significant correlation between CD44v2 anddown-regulation of less differentiated tumor cells was foundaccompanied by a lower survival time.199 An Iranian study200 hadtried to evaluate the expression rate of CD44 and cervical lymphnode metastasis in squamous cell carcinoma of the tongue. Atthe time of the diagnosis about 51% of the patients had cervicalmetastasis; there was no statistically relationship between histo-pathologic grading and cervical metastasis, but in 19 patients(95%) of patients with lymph node metastasis CD44 was statisti-cally significantly expressed. They deduced that CD44 could beused as a predict marker for cervical lymph node metastasis in pa-tients with squamous cell carcinoma of the oral tongue. Wanget al.136 investigated the role of CD44v3, v6 and v10 in HNSCC tu-mor progression behaviors, and they demonstrated that CD44 vari-ants expression was correlated with advanced T stage (v3 and v6),regional (v3) and distant (v10) metastasis, perineural invasion (v6),



Table 2CD44 and its splice variants expression in HNSCC (modified from D. Assimakopoulos et al., Review, The role of CD44 in the development and prognosis of head and neck squamouscell carcinoma, Histology and Histopathology, 2002).

Cd44 Tissue Method Expression and comments References

CD44 13 oral SCC (tonsil, tongue,antrum)

Im, FS, PE pan-CD44 Strong to moderate in 12/13 Hudson et al.197

v3 (3G5) Reduced in 13/13 with total loss in all poorly differentiatedtumours and in 50% of moderate and well-differentiated

v4–5 3D2 Absent or very weak in 7/13v6 2F10 Absent or very weak in 5/13v8 1.00E+08 Absent or very weak in 9/13

CD44s 38 primary tongue cancers(T1/T2N0)

Im, PE pan-CD44 Decreased, particularly in the group with late nodalmetastases

Masuda et al. 227

v3 56 SCC of the border of thetongue

Im Downregulation of v3 in 37.5% of cases, of v4 in 67.9% of casesand of v6 in 33.9% of cases which correlated with celldifferentiation, tumor grade and invasion

Fonseca et al. 228

BBA11v4–5 BBA25v6 BBA13

v4–5 11 primary oral SCCswithout metastases 9primary carcinomas with 19metastases

Im, FS, PE Loss of CD44v4–5 in all but more marked in metastases. Nocorrelation with behavior or grade

Oliveira et al. 229

3D2

v5 55 oral SCCs and 29 lymphmetastases from tongue(12), oral cavity (5), pharynx(35), larynx (31)

Im, FS Reduction of CD44v7, v8 and v10 but not of v5 and v6. Herold-Mende et al. 230

v6 VFF8v7 VFF7v8 VFF9 Total loss of v7, v8, and v10 in lymph node metastasesv10 VFF17

VFF16

v5 62 oral SCCs Im Reduction of CD44v7 in 22/62(35.5%) which correlatednegatively with shorter survival time

Kuo et al. 231

v6 Anti splice variantv7–8

v6 100 oral SCCs Im, FS Reduction or loss of CD44v6 which correlated with tumourcell differentation and increased frequency of regional lymphnode metastases

Bahar et al. 232

2F10 or Var3.1 Kunishi et al. 233

Soukka et al. 234

v4 99 primary oral ororopharyngeal SCCs

Im, PE Reduction of one or more isoforms in 39/99 (39.4%).Reduction of v7 and v9 correlated negatively with shortersurvival and recurrence-free interval

Stoll et al. 235

v6 Anti-splice variantv7 mAbs Stoll et al. 236

v9

v9 40 primary oral SCC Im Reduction of v9 in 19/40 (47.5%) cases which correlated withtumour cell differentation, primary and secondary metastasisto lymph node

Ue et al.198

Anti-v9

v3 82 HNSCCs Im Strong expression of v3 in 14/24 lymph metastases and 38/82cases, of v6 in 18/24 lymph metastases and 26/82 cases, ofv10 in 14/24 lymph metastases and 16/82 cases

Wang et al.136

v6v10CD44s 89 head and neck SCCs Im, PE No change in CD44/intron 9 but no correlation with tumour

stage or metastasisKanke et al.199

v2v6

v6 9 oral SCCs RT-PCR/southern blot Increase of v6 and CD44/intron 9 but no correlation withtumour stage or metastasis

Higashikawa et al. 237

CD44 withintron 9

v4 100 oral SCCs Im, PE No change in v5 and v6 but decrease of v4 and v9 Piffkò et al.202

v5v6 Piffkò et al.203

v9

v6 277 HNSCCs Im No, or marginal downregulation of CD44v6 in 268/277tumours

Van Hat et al. 238

U36, U39VFF18

CD44s Sinonasal invertedpapillomas(SIPs) andassociated SCCs

Im, PE 76 SIPs expressed CD 44s, 2 SIPs with SCC in situ showedstrong expression. No expression in 6/10 (60%) SIPs. With SCCand weak expression in 4/10 (40%) SIPs with SCC

Ingle et al. 239

A3D8

CD44s 70 LSCCs Im Decrease of CD44 and v6 correlated with an increase inmetastasis and a decrease in survival

Spafford et al. 240

2C5v6 2F10

v3 12LSCCs Im Reduction of v3 irrespective of TNM stage Repassy et al. 241

v5 28 LSCCs withoutmetastases and 25 LSCCswith metastases

Im, PE Reduction of v5 and v6 in the inner proliferative tumour area,but no correlation to metastatic ability

Ostwald et al. 242


Please cite this article in press as: Mannelli G, Gallo O. Cancer stem cells hypothesis and stem cells in head and neck cancers. Cancer Treat Rev (2011),doi:10.1016/j.ctrv.2011.11.007


Table 2 (continued)


VFF8v6 VFF7

CD44 27 dysplastic laryngealepithelium

Im, PE No change in dysplastic epithelium compared to normal.Focal reduction in LSCCs. Loss of CD44 was associated withpoor differentiation, increased mitotic index and nodal ormetastatic spread

Hirvikoski et al.204

172 LSCCs Hermes 3

CD44 66 LSCCs Im, PE Progressive overexpression of CD44 in laryngealcarcinogenesis

Sugar et al. 243

67 keratosis pan-CD4497 carcinomas

CD44 34 LSCCs Im, PE Progressive overexpression of CD44 in laryngealcarcinogenesis

Ioachim et al. 244

13 in situ carcinomas pan-CD4435 dysplastic tissue10 papillomas17 keratosis

CD44 Oral SCC Im Strong to moderate in 12/13 Hudson et al.197

CD44s Im Decrease in 38/38 cases Masuda et al. 227

No change in 89/89 cases Kanke et al.2002

v2 Im Reduction in 89/89 cases Kanke et al.199

v3 Im Reduction in 34/69 cases Hudson et al.197

Fonseca et al. 228

v4 Im Reduction in most of 199 cases Piffkò et al.202

Stoll et al. 235,236

v4–5 Im Reduction in 65/89 cases and in 19/19 metastases Hudson et al.197

Kanke et al.199

Fonseca et al. 228

v5 Im No change in 200 oral SCCs and in 29 lymph metastases Herold-Mende et al. 230

Piffkò et al.202

Kuo et al. 231

v6 Im Reduction in 163/268 cases Hudson et al.197

Bahar et al. 232

Kunichi et al. 233

Stoll et al. 235,236

No changes in 306 cases and 29 metastases Herold-Mende et al. 230

Piffkò et al.202

Kuo et al. 231

Kanke et al.199

RT-PCR Increase in 9/9 cases Higashikawa et al. 237

v7 Oral SCC Im Reduction or loss in 94/154 cases and in 29 metastases Herold-Mende et al. 230

Stoll et al. 235

v7–8 Im Reduction in 22/62 cases Kuo et al. 231

v8 Im Reduction in 64/68 cases, loss in 29 metastases Herold-Mende et al. 230

Hudson et al.197

v9 Im Reduction in 152/239 cases Piffkò et al.202

Ue et al.198,

v10 Im Reduction in 55/55 cases, loss in 29 metastases Herold-Mende et al. 230

CD44 Im Reduction in poorly differentiated. No correlation withmalignancy

Mack and Cires135

v6

CD44 LSCC Im Progressive overexpression of CD44 (100 LSCCs, 84 keratosis,10 papillomas, 35 dysplastic tissue and 110 carcinomas usingpan-CD44)

Ioachim et al. 244

Sugar et al. 243

No changes in 27 dysplastic tissues. Focal reduction in 172LSCCs associated with poor differentation

Hirvikoski et al.204

CD44s Im Reduction in 70 LSCCs correlated with increased metastasesand decreased survival

Spafford et al. 240

v3 Im Reduction in 12/12 Repassay et al. 241

v5 Im Reduction in 54 cases. Ostwald et al. 242

No correlation with metastases

v6 Im Reduction in 124 cases Spafford et al. 240

Ostwald et al. 242

Im Markers of recurrence and aggressiveness in laryngealintraepithelial neoplasia

Staibano et al. 245

CD44s Im CD44 expression correlates with local recurrence of laryngealcarcinoma after radiotherapy treatment

De Jong et al.206

v3 HNSCC Im CD44v3 expression decrease in hippharyngeal carcinoma Franzmann et al.209

(continued on next page)


Please cite this article in press as: Mannelli G, Gallo O. Cancer stem cells hypothesis and stem cells in head and neck cancers. Cancer Treat Rev (2011),doi:10.1016/j.ctrv.2011.11.007


Table 2 (continued)


CD44s Im Play a key role in establishment of permanent HNSCC celllines, and might drive progression and metastasis of HNSCC

Pries et al.176

CD44s Oral SCC Im CD44 predict cervical lymph node metastasis Mostaan et al.200

CD44 Cell lines HNSCC FACS-analysis In vitrro and in vivo: CD44 and metastatic potential Davis et al.207

CD44 HNSCC FACS-analysis CD44 squamospheres and chemoresistant, with increasedlevels of ABCG2 and tumorigenesis in in vivo models

Lim et al. 246

CD44 Lin-CD44 FACS-analysis CD44 and poor prognosis Joshua et al.208

Im, immunohistochemestry; RT-PCR, reverse transcription-polymerase chain reaction; SCC, squamous cell carcinoma; LSCC, laryngeal SCC; PE, paraffin-embedded tissues; FS,frozen sections.


and radiation failure (v10). CD44v6 and v10 were significantlyassociated with short disease-free survival, too. Recently, Faberand collegues201 underlined the CD44’s role as a diagnostic andprognostic factor in HNSCCs, and its possible use as therapeutictarget.

Overall, these studies show that the reduction or loss of expres-sion of one or more exons of the CD44 variants, can be used as bio-logical markers during malignant transformation of squamousepithelia of the oral cavity. Otherwise, some studies showed theopposite concept. Piffkò et al.202,203 showed that the expressionof both CD44v5 and v6 isoforms, during development and progres-sion of squamous cell carcinomas (SCC) were strongly expressed innormal and dysplastic mucosa and all primary tumors or meta-static SCC, suggesting that CD44v5 and v6 expression is not alteredduring development and progression of oral cancer. However, thesame authors showed that the expression of CD44v4-and CD449-variant isoforms was significantly decreased from normal oral epi-thelium.202,203 Also Mack and Gires135 tried to clarify the pattern ofCD44 expression in HNSCC. They analyzed CD44s and CD44v6expression, by immunohistochemistry, in primary head and necktissues. They presented results about a CD44s and CD44v6 expres-sion in normal epithelia of 60–95% and 50–80%, respectively; inoral leukoplakja and in moderately differentiated carcinomas theirlevels were slightly increased. Carcinomas in situ displayed un-changed levels of both proteins whereas poorly differentiated car-cinomas consistently expressed diminished CD44s and CD44v6levels. The study concluded that these two markers do not helpin distinguish normal from benign or malignant epithelia. Fromhere, some suspects arose on CD44 ability in identify cancer cellswith stem properties.

Normal squamous epithelium of head and neck district presentshigh expression of CD44s and its splice-variants. NeverthelessCD44 isoforms seem to be able to modulate interactions betweencells and matrix, their functions in both normal and cancer tissueis still not known indeed.

Even if some studies, like those above, produce doubts on con-sider this superficial proteins as markers of stem cell properties,there are other many authors who confirm these markers as bio-markers, able to isolate cancer cells with stem behavior andcapacities.

CD44 expression was analyzed also in laryngeal cancer, whereCD44v5 and v6 have been proposed as markers of tumor prolifer-ation.204 Irregular expression of hyaluronan acid and its receptorCD44, was associated with the metastatic phenotype in laryngealSCC. The immunohistochemistry (IHC) analysis of CD44 expressionin tissue sections from 27 samples of dysplastic laryngeal epithe-lium and 127 laryngeal squamous cell cancer (LSCC) showed thathyaluronic acid (HA) and CD44 (Hermes 3) were expressed in90% of dysplasia and tumor sections compared with normal tis-sue.204 However, whereas their distribution in normal epitheliumwas homogeneous, the malignant transformation was associatedwith focal reductions of hyaluronan acid (HA) and its receptor dis-tribution. Moreover, the loss of HA and CD44 expression in LSCC


was associated with poor differentiation, an increased mitotic in-dex and nodal or metastatic spread. This focal reduction was pre-dominant in poor differentiated cancers.204 Staibano et al.205 byexamining osteopontina (OPN) and its functional receptor(CD44v6) expression in several type of laryngeal dysplasia, foundthat OPN expression levels correlated positively with degree ofdysplasia (P = 0.0094) and negatively with disease-free survival(p < 0.0001). OPN expression was paralleled by cell surface reactiv-ity for CD44v6. CD44v6 expression correlated negatively with dis-ease-free survival, as well (p = 0.0007). These findings identify OPNand CD44v6 as predictive markers of recurrence or aggressivenessin laryngeal intraepithelial neoplasia by pointing out an importantsignaling complex in the evolution of the laryngeal dysplasia. AlsoDe Jong et al.206 analyzed 52 larynx cancer patients, matchingthose with a local recurrence for T-stage, subsite, treatment, gen-der and age with non-recurrence patients. Nineteen patients witha local recurrence were matched with 33 controls. Gene sets forhypoxia, proliferation and intrinsic radiosensitivity did not corre-late with recurrence, whereas expression of the putative stem cellmarker CD44 did immunohistochemical analysis of CD44 expres-sion on the independent validation series confirmed CD44’s predic-tive potential. CD44 was the only biological factor tested whichsignificantly correlated with response to radiotherapy in earlystage larynx cancer patients, both at the mRNA and protein levels.Further studies are needed to confirm this and to assess how gen-eral these findings are for other head and neck tumor stages andsites.

All of these reported examples show the important role of CD44and its variants expression as molecular markers for HNSCC pro-gression. From here the importance of their investigation as poten-tial therapeutic targets. CD44v6 is a tumor associated antigenabundantly expressed in HNSCC and in normal squamous epithe-lium. Because of the frequent and homogeneous expression ofCD44v6 isoforms in squamous cell carcinoma, antibodies recogniz-ing these proteins were used in clinical trials for patients sufferingfrom head and neck squamous cell carcinoma (HNSCC). Further-more, the expression of several CD44 proteins correlates withaggressive stages of various human cancers. A recent study of Daviswith Prince et al.207 succeeded in designed in vitro and in vivomodels of metastatic behavior of CD44-positive HNSCC CSCs, andthey evidenced a metastatic phenomenon in in vivo assay. Re-cently, Joshua et al.208 analysed the main frequency of CD44-posi-tive cells in 31 primary HNSCCs and obtained a successfulxenograft implantation in 53% of cases, then they highlighted thecorrelation between a high frequency of CD44-positive cells andpoor prognostic factors, and by these findings they supported thestem cell concept in HNSCC and pointed out CD44 as HNSCC bio-logical marker.

Already in 2001, Franzmann et al.209 proposed CD44v3 isoformsas an effective tumor markers and targets for HNSCC therapy. Rie-chelmann et al.139 tested the immunoconjugate bivatuzumab mer-tansine (BIWI 1), a highly potent antimicrotubule agent coupled toa monoclonal antibody against CD44v6, in a clinical phase I trial




adult patients with recurrent or metastatic HNSCC. They confirmedthe capacity of BIWI 1 in controlling CD44 tumor expression.Unfortunately there was a fatal outcome due to a serious skin tox-icity which led to the termination of the study, but this remains anexample for future researches with new targets. Some authors143

investigated whether hyaluronan (HA) and CD44, which actsthrough multiple signaling pathways to influence cellular behav-ior, promoted Rho kinase- and phos-phatidylinositol 3 (PI-3) ki-nase-mediated oncogenic signaling to alter cisplatin sensitivityand stimulate tumor cell proliferation, migration, and matrixmetalloproteinase secretion in HNSCC. They measured Rho kinaseand PI-3 kinase activity, myosin phosphatase and AKT phosphory-lation, tumor cell growth, migration, and matrix metalloproteinasesecretion in the presence or the absence of HA, cisplatin, and inhib-itors of Rho kinase and PI-3 kinase. The addition of HA, but not HAplus anti-CD44 antibody, resulted in increased Rho kinase and Pl-3kinase activity. Immunoblotting studies demonstrated that HApromotes Rho kinase-mediated myosin phosphatase phosphoryla-tion and Pl-3 kinase-mediated AKT phosphorylation. Hyaluronanwas shown to promote migration and increased matrix metallo-proteinase secretion through Rho kinase-mediated signaling.Hyaluronan treatment promoted increased tumor proliferationand resulted in a 12-fold reduced ability of cisplatin to causeHNSCC cells death. These results suggest that HA and CD44 pro-mole Rho kinase- and PI-3 kinase-mediated oncogenic signalingand cisplatin resistance. Perturbation of HA-CD44-mediated Rhokinase and PI-3 kinase signaling pathways may be a novel strategyto treat HNSCC.

CD133 is less expressed than CD44, and its sensibility of identifycancer stem cells is still object of discussion. Anyway, someauthors confirm CD133 as CSCs marker. Wei et al.137 identifiedCD133-positive cancer cells with stem properties in laryngeal can-cer cell lines (Hep-2), where CD133+ was expressed in 3.15% ±0.83% of tumor cell mass. Here CD133-positive cells demonstratedto possess self-renewal property, extensive proliferation, tumor-forming ability in vivo. Also Okamoto et al.138 proposed CD133antigen as a co-marker together with CD44 and ABCG2. A studyof September 2010, analyzed the CSCs CD133-positive role indetermining chemo-resistance properties140; authors identifiedthe presence of cancer cells CD133+ (1–2%) in human oral squa-mous cell carcinomas (OSCCs), and for the first time they demon-strated CD133+ tumor cells stem properties by showing theirclonogenic potential in vitro, tumorigenic potential in vivo, andtheir evident chemo-resistance capacities.

All the last literature’s results propose CD44 as marker ofidentification of cancer stem cells in head and neck squamous cellcarcinomas, and present CD133 as a co-expressed marker of stemproperties. At the end we could question about the possible exis-tence of a CD44 variant-antigen more specific in identifying cancercells with stem properties, but the last data do not focus on an onlyCD44 isoform but analyze all the variants sight without presentingone CD44 variant more significant than others. Moreover, if wehave a look at recent study on breast cancer progression210 thestandard isoforms of CD44 antigen (CD44s) seems to be criticalfor regulating epithelial–mesenchymal transition (EMT) which isabnormally activated during cancer metastasis and recurrence.Together these data suggest that regulation of CD44 alternativesplicing could contribute to EMT and cancer breast progression,and these findings could be found in head and neck cancers, too.

HNSCC susceptibility

There are two main models which have tried to explain headand neck squamous cell tumor progression: the stochastic clonalevolution model and the cancer stem hierarchy model. According


to the first one, all tumor cells have equal ability to propagatethe tumor and its consequent morphological heterogeneity is ex-plained by aberrant cell differentiation due to genetic/epigeneticinstability of the tumor cells. The cancer stem cell model, whichapplies the stem cell biological principles, proposes CSCs maintain-ing the tumor proliferation through generation of both undifferen-tiated and more differentiated cells which form the tumor mass.

Consequently, each tumor forming model suggests differenttherapeutic approaches.211

Cameron et al.,212 gave support to the stochastic clonal evolu-tion model by isolating and analyzing stem cell surfacing markersand clonal cells in combination with a xenotransplant approach. Infact, ones tumor cells were isolated from HNSCC cell lines, theywere propagated for two to five passages in vitro before theimplantation; this concerns with somewhat and somehow have al-tered tumor cells. Moreover, they proposed, on the basis of boththe existence of a dynamic equilibrium between CSCs and non-CSCs and that interactions of tumor cells with their microenviron-ment could lead to altered growth and differentiation, that thetumor-initiating ability is influenced by the neighboringpermissive stroma.

Looking at history, in 1953 the term of ‘‘field cancerization’’ wasproposed by Slaughter213 in order to explain the propensity to de-velop local recurrence after treatment of HNSCC; he and his col-leges, observed the dysplastic changes surrounding these tumorsand they linked these conditions to the high occurrence of localrecurrences and/or multiple primary tumors. This process of fieldcancerization found molecular terms of definition in the geneticmulti-step progression model for HNSCC, which was postulatedin 1996.214

We can conclude that a clonal relationship exists between thegenetic profiles of carcinoma and their surrounding fields.

In general, cancer arises through genetic/epigenetic changesaccumulated in genes acting in cancer cells, and signaling path-ways together with genetic predisposition are involved.

It is known that head and neck squamous cell carcinomas arisein the pharynx, oral cavity and larynx, they are the sixth leadingcancer worldwide and their most important risk factors are tobac-co use, alcohol consumption and, particularly those tumors of theoropharynx, are caused by human papillomavirus (HPV) infec-tion.215 Tobacco use and alcohol consumption are well-establishedrisk factors for these tumors. For tobacco smoking, a dose–re-sponse trend has been reported. Relative risks of developing laryn-geal and oropharyngeal cancers are 1.8 and 1.3, respectively, forpersons who smoke 630 cigarettes per day are 7.7 and 2.9, respec-tively, for persons who smoke >30 cigarettes per day comparedwith non-smokers15,18 Alcohol consumption is also linked to in-creased risk of HNCs (Head and Neck Carcinomas). For personswho consume >4 drinks (=47.5 g of pure ethanol) per day, the rel-ative risks of developing laryngeal and oropharyngeal cancers are4.5 and 7.2, respectively, compared with non-drinkers.15,18 A syn-ergistic effect was observed in persons who both smoke tobaccoand drink alcohol. The relative risks of developing laryngeal andoropharyngeal cancers are 34.6 and 21.2, respectively, amongthose who smoke >30 cigarettes a day and consume >4 drinksper week.

HPV-positive and HPV-negative tumors represent different clin-icalpathological and molecular entities215; the first group of tu-mors presents increasing incidence in under 60 years population,presents infrequent p53 mutations, which is very frequent inHPV-negative cancers, HPV infection shows oropharynx as predi-lection site and could find a link with oral sexual habits, andHPV-positive tumors present better prognosis than HPV-negativegroup.

Then, also diet, poor oral health, exposure to environmental car-cinogens and genetic polymorphisms in carcinogen metabolizing




enzymes (ALDH, GST) and DNA repair genes, represent potentialrisk factors for HNSCC.

In conclusion, on the basis of environmental agents exposure,HPV status and high/low chromosome instability DNA profile, thescientific community could propose an initial genetic tumor classi-fication model. In fact, a defective DNA damage repair mechanismcould result in genomic instability, followed by unregulated cellgrowth and increased cancer risk.216 Phenotypically normal indi-viduals with reduced DNA repair capacity may have higher cancerrisk, and these kind of people could be treated with target thera-peutic program. Flores-Obando et al.,217 tried to evidence the pos-sible link of the effect of smoking, alcohol consumption, HPVinfection and race, and the role of DNA repair gene polymorphismsin head and neck cancer risk. In conclusion, their meta-analysissupported that the polymorphisms of DNA repair genes (XRCC1 co-don 194, XPD codon 156 and Asp312Asn) could be associated withoral, pharyngeal and laryngeal cancer risk.

The ARCAGE project, which is one of the largest multicenter ge-netic epidemiologic studies on cancers of the upper aerodigestivetract (UADT) in Europe, published on Cancer Research in 2009,218

had as its major objective the investigation of the role of geneticvariations with regard to the metabolism of alcohol and carcino-genesis from tobacco smoke in the development of UADT cancers.This study was born from the idea that there are gene polymor-phisms involved in the metabolism of carcinogens from tobaccoand alcohol in DNA repair and that the study of single nucleotidepolymorphisms (SNPs) in these cancers could help in better under-standing the pathways leading to cancers. For example, Sabithaet al.219 identified an association between pack-years of smokingand risk of HNSCC among cases with CYP1A1 polymorphism; infact, heavy smokers (consumers of >15 years) showed increasedrisk for HNSCC in association with both m1 and m2 mutations. Itis evident that biometabolism genes may play a significant rolein the transformation of benign lesions to malignant ones.

In conclusion, DNA repair capacity and cell cycle control are thepotential actors of the interindividual variability in relation to thedevelopment of cancers.

The consequent question could be if we can apply distinct epi-genetic profiling to the cancer stem cell hypothesis and if the chro-mosome instability leads to the maintaining of the stem properties.

Furusawa et al.220 tried to identify epigenetic signature in CSCsin HNSCC by performing molecular and microarray analysis. Oneshave sorted CD44+ and CD44� populations from HNSCC lines, theirlooked for stemness gene expression and their response to chemo-therapeutic agents; they obtained that CSCs with CD44 highestexpression showed a unique epigenetic profile critically requiredin maintaining the stemness or pluripotency of CSCs.

Gammon et al.221 studied the high risk of developing HNSCC inFanconi anemia (FA) patients; FA is an autosomal recessive disor-der associated with deficiencies of DNA repair, chromosomal insta-bility and cellular hypersensitivity to DNA cross-linking agentssuch as mytomicin C, and it presents a clinical spectrum includingcongenital predisposition to hematopoietic malignancies andHNSCCs.222 In order to assess the effects of FA gene defects onthe expression of stem cell properties, they compared CSC patternsin cell lines derived from FA-related and sporadic HNSCCs, becauseif the CSC patterns persist in FA tumors, better therapies for FA pa-tients could act through such stem cell targeting. In fact, Gammonet al.221 found colony morphologies mirrored the stem cell patternsin FA cell lines, and combining CD44 expression and FA cells, CD44higher expression cells showed lower rates of apoptosis and agreater DNA damage induced block in the G2 phase of the cell cyclethe CD44 low expression cells. Finally, fluorescence activated cellsorting, immunohistochemistry and QPCR analysis showed differ-ent patterns of gene expression of CD44 cell in both sporadic andFA cell lines.


A recent review of Sayed et al.174 tried to understand the cancerstem cell biology in HNSCC. Even if genomic instability and epige-netic errors lead to the tumor heterogeneity, the complex mecha-nism underlying cancer stem cell genomic plasticity is not yet fullyunderstood and described.

At the end, a better understanding of the mechanisms involvedin tumor biology will show the way for prevention and early detec-tion of the tumor.

Clinical and therapeutic implications of cancer stem cells inhead and neck cancer

Therapeutic modalities such as surgery, radiation, chemother-apy and combinations of each are used in the management of headand neck cancer disease, but at the same time the scientific com-munity is ongoing throughout the improvement of early detectionand prevention of HNSCC. One of the key in determining the treat-ment failure could be represented by the presence of CSCs that canescape currently therapeutic strategies. Actually, specific CSCmarkers such as ALDH1, CD44 and Bmi1 showed promising resultsin detection and new therapeutic protocol.

Solid epithelial tumors are the major cause of cancer deaths andit has now been demonstrated that they are driven by a small sub-population of malignant stem cells.24 In normal epithelia, stem andamplifying cells divide in different ways, present different apopto-tic sensibility and different drug resistance transporters expres-sion; and, the persistence of such differences between malignantstem and amplifying cells could influence therapeutic outcomes.Identification of self-renewal and differential properties is there-fore required in order to monitoring or better, targeting, therapeu-tic interventions on cancer stem cells.

The existence of a hierarchical cancer stem cell pattern suggeststhe possible achievement of a shifting asymmetric division to pro-duce tumor stem cell loss. It has been suggested that normal andmalignant stem cells present similar self-renewal mechanisms fur-ther, focus on mechanism controlling asymmetric division inmalignant stem cells, might assist the development of new strate-gies for their therapeutic manipulation.

The ability in identifying cancer stem cells should lead towardsa more specific tumor treatment. In fact, by comparing geneexpression profiles of cancer stem cells, the bulk tumor cell popu-lation, normal stem cells and normal tissue, it may be possible toidentify therapeutic targets preferentially attacking cancer stemcells only.

The first successful targeted therapy (EGFR-specific antibodies)demonstrates that improved understanding of the molecularpathways underlying HNSCC will yield valuable new treatmentprotocols.

Scherzed et al.223 focused on the role of tumor-bone marrowstem cell (BMSC)-interactions in development and manifestationof chemoresistance in HNSCC; they found a reduction of theapoptosis and an enhancement in the viability of squamous cellcarcinoma cell lines treated with paclitaxel and cultured simulta-neously with bone-marrow derived mesenchymal stem cells. Theauthors propose a paracrine secretion of various cytokines withwhom BMSC could have influence tumor invasion and HNSCCresistance to treatment with paclitaxel. In fact, CSCs grow in aniche which protect them from the exogenous agents and thisenvironment has also been implicated to protect cells from the ef-fects of hypoxia and radiation.

Recently, Krishnamurthy et al.224 observed that ALDH+ cells areconsistently localized within close proximity of blood vessels;studies in hematopoietic stem cells suggest that the vascularniche can promote cell survival signals and could make themresistant to chemotherapies; moreover, antiangiogenic agents such




as bevacizumab, have been shown to mediate depletion in the CSC,and these data could suggest therapeutic strategies including anti-angiogenic agents, too.

In conclusion, clinical implications of cancer stem cells couldlead to targeting of CSC tumor population and molecular pathwaysinvolved in CSC immortality, to the early detection of CSC from thebulk cell tumor and preventing normal stem cells differentiatinginto cancer stem cells. Moreover, CSC therapeutic implicationscould lead to the understanding of therapeutic resistance and tu-mor recurrence, by analyzing

We must not forget that precancerous fields should be exam-ined with more attention in order to prevent local recurrenceand/or multiple primary tumors. The discovery of specific markersets for cancer stem cells is still in its infancy, and the targetedtherapeutic destruction of these cells remains a challenge. Currentanticancer treatments usually do not eradicate clones of such cells,and instead do favor the expansion of the cancer stem cell pool, orthe selection of resistant clones, or both, which eventually lead tothe failure of treatment. To be curative, new cancer treatmentsshould include agents that eradicate these cells and prevent the re-growth of neoplastic cell populations. There is continuing need todiscover more specific markers and to explore CSCs physiologicalroles. Improved understanding of transition process from pluripo-tency to various stage of tissue would aid innovations to noveltherapeutic strategies. More predictive biomarkers will be de-manded by target therapy, and personalized treatment will bethe future goal cancer treatment.

Conclusions

Cancer stem cells is still an enigma in oncology, in recent yearsthey appeared as the possible key to explain the tumor treatmentfailure. Recently the role of CSCs in tumor neo-angiogenesis hasbeen documented, but we are still long way from understandingthe molecular mechanisms that guide carcinogenesis, but we hopethat the develop of in vitro and in vivo models of CSCs might pro-vide a means of devising more effective cancer preventing, diag-nostic, prognostic and therapeutic strategies especially in headand neck squamous cell carcinomas.

Conflict of interest statement

Authors disclose any financial and personal relationships withother people or organisms that could inappropriately influencetheir work.

There was no conflict of interest between the authors in theconception or in the content of this article.

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