human head and neck squamous cell carcinoma ( hnscc ) is the sixth most common cancer worldwide and is also one of the most deadly cancers . the three - year survival rate for patients with advanced - stage hnscc and treated with standard therapy is only 30 to 50% . this deadly disease includes cancers of the lip , oral cavity , pharynx , hypopharynx , larynx , nose , nasal , sinuses , neck , ears , and salivary glands . nearly 40 to 60% of hnscc patients thus , there is currently a great need to clarify the key mechanisms of tumor initiation and progression underlying the clinical behavior of hnscc . accumulating evidence indicates that most tumors contain a small population of cells which persistently initiate tumor growth and promote tumor progression . these  cancer stem cells ( cscs )  [ also called  tumor - initiating cells ( tics )  ] share several of the hallmarks of normal stem cells [ 2 , 3 ] . for example , cscs undergo self - renewal , maintain quiescence , display multipotentiality , and express survival protein / antiapoptosis proteins [ 2 , 3 ] . another well - known property of cscs is their ability to expand the stem cell population by undergoing cell proliferation / survival and/or clone formation and differentiation [ 2 , 3 ] . a number of studies have identified specific molecules expressed in cscs that correlate with both stem cell properties and tumor cell behaviors . among such molecules is cd44 which is a multifunctional transmembrane glycoprotein expressed in many cells and tissues including hnscc cells and other carcinoma tissues [ 2 , 3 ] . cd44 is commonly expressed in various isoforms generated by alternative mrna splicing of variant exons inserted into an extracellular membrane - proximal site . cd44 is expressed in both normal and cancer stem cells ( cscs ) and serves as an important stem cell marker [ 2 , 3 ] . hyaluronan ( ha ) is a major component in the extracellular matrix ( ecm ) of most mammalian tissues . ha is a nonsulfated , unbranched glycosaminoglycan consisting of repeating disaccharide units , d - glucuronic acid , and n - acetyl - d - glucosamine [ 5 , 6 ] . under physiological conditions , ha is synthesized by several ha synthases   and ha fragments of low molecular mass are produced by hyaluronidases or oxidation . one general concept which has emerged from these studies is that ha fragments ( small- versus mid - size - has ) and their larger precursor molecules ( i.e. , intact ha ) may be involved in distinct biological activities [ 9 , 10 ] . in addition , the formation of biologically active ha fragments from the large ha in the ecm occurs during periods of proliferation , migration , differentiation , and development as well as injury - related repairs [ 9 , 10 ] . a number of studies indicate that large size - ha promotes transcriptional activation and differentiation , whereas small - size - ha induces cell proliferation and migration [ 9 , 10 ] . ha is enriched in many types of tumors [ 11 , 12 ] and also has been found to be increased in stem cell niches [ 13 , 14 ] . furthermore , the unique ha - enriched microenvironment appears to be involved in both self - renewal and differentiation of normal human stem cells [ 13 , 14 ] . all cd44 isoforms contain a ha - binding site in their extracellular domain and thereby serve as a major cell surface receptor for ha [ 5 , 6 ] . the fact that both cd44 and ha are overexpressed at tumor attachment sites and that ha binding to cd44 stimulates a variety of tumor cell - specific functions and tumor progression [ 11 , 12 ] suggests that the ha - cd44 interaction is a critical requirement for tumor progression . however , the cellular and molecular mechanisms underlying ha 's ability to regulate cd44-positive cscs by different sizes of ha during hnscc progression remain poorly understood . furthermore , the oncogenic mechanism(s ) occurring during activation of cscs by size - specific has during head and neck cancer progression remain(s ) to be unknown . in this study we have investigated the effects of different sizes of ha ( ranging from 5  kda to 700  kda ) on csc signaling and function in head and neck cancer cells . our results indicate that 200  kda - ha ( and to a much lesser extent 5  kda - ha , 20  kda - ha , and 700  kda - ha ) plays an important role in the selective activation of pluripotency factor ( stem cell marker ) expression , microrna signaling and csc functions required for tumor cell behaviors lead to head and neck cancer progression . tumor - derived hsc-3 cell line ( from human squamous carcinoma cells of mouth ) , was kindly provided from dr . cells were grown in dmem / f12 medium ( corning , ny ) supplemented with 10% fetal bovine serum . rabbit anti - cd44v3 antibody was obtained from emd chemicals ( gibbstown , nj ) . other immunoreagents such as rabbit anti - ciap1 antibody were from abcam ( cambridge , ma ) and rabbit anti - actin antibody was purchased from cell signaling ( beverly , ma ) , respectively . different sizes of research grade ha fragments ( e.g. , 5  kd , 20  kda , 200  kda , and 700  kda ) were purchased from lifecore biomedical ( chaska , mn ) . the identification of aldehyde dehydrogenase-1 ( aldh1 ) activity from tumor - derived hsc-3 cells was conducted using the aldefluor kit ( stemcell technologies , durham , nc ) . specifically , tumor cells were suspended in aldefluor assay buffer containing aldh1 substrate ( baaa , 1  mol / l per 1  10 cells ) and incubated for 30  min at 37c . as a negative control , hsc-3 cells were treated with a specific aldh1 inhibitor , 50  mmol / l diethylaminobenzaldehyde ( deab ) ( 50  mmol / l ) . next , for labeling cell surface marker , tumor - derived hsc-3 cells were resuspended in 100  l aldefluor buffer followed by incubating with 20  l allophycocyanin- ( apc- ) labeled anti - cd44v3 antibody ( recognizing the v3-specific domain of cd44 ) or apc - labeled normal mouse igg ( as a control ) ( bd bioscience , san jose , ca ) for 15  min at 4c . for facs sorting , tumor cells were resuspended in pbs buffer followed by facs ( bd facs aria llu , bd bioscience , san jose , ca ) sorting using dual - wavelength analysis . subsequently , cd44v3aldh1 tumor cell population was collected and used for various experiments described in this study . nonobese , diabetic / severe combined immunodeficient ( nod / scid ) immunocompromised mice ( 5-week - old female mice ) were purchased from charles river laboratories international , inc . specifically , these nod / scid were injected subcutaneously and/or submucosally in the floor of the mouth with sorted cd44v3aldh1 cells or unsorted hsc-3 cells ( suspended in 0.1  ml matrigel basement membrane matrix ) ranging from 50 , 500 , to 5,000 cells . these mice were then monitored twice weekly for palpable tumor formation and euthanized 4 or 8 weeks after transplantation to assess tumor formation . some tumors were cut into small fragments with sterile scissors and miced with a sterile scalpel , rinsed with hans ' balanced salt solution containing 2% heat - inactivated calf serum ( invitrogen ) , and centrifuged for 5  min at 1,000  rpm . the resulting tissue specimen was placed in a solution of dmed f-12 containing 300  u / ml collagenase and 100  u / ml hyaluronidase ( stemcell technologies , durham , nc ) . the digestion was arrested with the addition of fbs and the cells were filtered through a 40  m nylon sieve . the cells were washed twice with hans ' balanced salt solution plus 2% heat - inactivated calf serum for facs as described above . total rna was isolated from cd44v3aldh1 cells ( pretreated with no ha or 5  kda - ha or 20  kda - ha or 200  kda - ha or 700  kda - ha for 24  h ) using tripure isolation reagent kits ( roche applied science , indianapolis , in ) . first - stranded cdnas were synthesized from rna using superscript first - strand synthesis system ( invitrogen , carlsbad , ca ) . gene expression was quantified using probe - based sybr green pcr master mix kits , abi prism 7900ht sequence detection system , and sds software ( applied biosystems , foster city , ca ) . a cycle threshold ( minimal pcr cycles required for generating a fluorescent signal exceeding a preset threshold ) was determined for each gene of interest and normalized to a cycle threshold for a housekeeping gene ( 36b4 ) determined in parallel . the 36b4 is a human acidic ribosomal phosphoprotein po whose expression was not changed in tumor cells . the q - pcr primers used for detecting gene expression of oct4 , sox2 , nanog , and klf-4 were as follows : specifically , two oct4-specific primers ( the sense primer 5-ggtattcagccaaacgacca-3 and the antisense primer 5-cacactcggaccacatcctt-3 ) ; two sox2-specific primers ( the sense primer 5-gacagttacgcgcacatgaa-3 and the antisense primer 5-taggtctgcgagctggtcat-3 ) ; and two nanog - specific primers ( the sense primer 5-gtgatttgtgggcctgaaga-3 and the antisense primer 5-acacagctgggtggaagaga-3 ) ; two klf4-specific primers ( the sense primer 5-caccatggacccgggcgtggctgccagaaa and the antisense primer 5-ttaggctgttcttttccggggccacga ) were used . the q - pcr primers used for detecting gene expression of various mirnas were as follows : specifically , two mir-10b - specific primers ( the sense primer 5-ggataccctgtagaaccgaa and the antisense primer 5-cagtgcgtgtcgtggagt ) ; two 23b-27b - specific primers ( the sense primer 5-tcacattgccagggattacca and the antisense primer 5-tgcacctgttctccaatctgc ) ; two mir-373 primers ( the sense primer 5-ccttcaacagctcatcaagggct and the antisense primer 5-tacccgccccctcacccaatcaa ) ; two mir-34a primers ( the sense primer 5-tggcagtgtcttagctggttg and the antisense primer 5-ggcagtatacttgctgattgctt ) ; two mir-145 primers ( the sense primer 5-ggtccagttttcccagg and the antisense primer 5-cagtgcgtgtcgtggagt ) ; two mir-181a ( the sense primer 5-aacattcaacgctgtcggt and the antisense primer 5cagtcaacggtcagtggttt ) . finally , for detecting 36b4 gene expression , two 36b4-specific primers ( the sense primer 5-gcgacctggaagtccaactac-3 and the antisense primer 5-atctgctgcatctgcttgg-3 ) were used . ripa buffer - solubilized cell lysate of untransfected cd44v3aldh1 cells ( pretreated with no ha or 5  kda - ha or 20  kda - ha or 200  kda - ha for 3 days ) or cd44v3aldh1 cells transfected with anti - mir-10b inhibitor or mirna - negative control followed by 200  kda - ha ( 50  g / ml ) addition ( or no ha addition ) for 3 days at 37c were immunoblotted using various immunoreagents [ e.g. , mouse anti - ciap-1 ( 2  g / ml ) or goat anti - actin ( 2  g / ml ) ( as a loading control ) , resp . ] . cd44v3aldh1 cells were analyzed for spheroid formation and self - renewal using the methods described previously . for analyzing the clone formation ( differentiation ) properties , cd44v3aldh1 cells ( treated with 200 kda - ha or no ha ) were dissociated from spheres and inoculated to 6-well plates at a density of 200 cells per well in six - well plates , and cultured with dmem / f12 plus 10% serum for ~21 days . after most cell clones expand to > 50100 cells , they were fixed with methanol followed by staining with crystal violet . the clone formation efficiency ( cfe ) was expressed as the ratio of the clone number to the planted cell number . sphere - derived cd44v3aldh1 cells ( 5  10 cells / well ) were incubated in basal medium with b27 plus 200  kda - ha ( 50  g / ml ) or no ha . medium were replenished every 3 days . twenty - on ( 21 ) days after plating , total cell growth was then counted under a microscope at low magnification . in some cases , these sphere - derived cd44v3aldh1 cells ( transfected with anti-10b inhibitor or mirna - negative control ) were also incubated with various concentrations of cisplatin ( 020  m ) with no ha or with 200  kda - ha ( 50  ug / ml ) . after 7-day incubation at 37c , celltiter - glo luminescent cell viability assays ( promega , madison , wi ) were analyzed as described previously . the percentage of absorbance relative to untreated controls ( i.e. , cells treated with neither ha nor chemotherapeutic drugs ) was plotted as a linear function of drug concentration . the 50% inhibitory concentration ( ic50 ) was identified as a concentration of drug required to achieve a 50% growth inhibition relative to untreated controls . overexpression of cd44v3 has been shown to be closely associated with hnscc development and progression . aldehyde dehydrogenase-1 ( aldh1 ) , a detoxifying enzyme responsible for the oxidation of intracellular aldehydes , is also considered to be a common marker for both normal stem cells and malignant cancer stem cells ( cscs ) from hnscc [ 1517 ] . in this study we isolated cd44v3aldh1 subpopulations from tumor - derived human hnscc cells ( hsc-3 ) using facs - fluorescence - activated cell sorting procedures ( figure 1 ) . our results indicate that cd44v3aldh1 cells ( to a much lesser extent cd44v3aldh1 , cd44v3aldh1 , cd44v3aldh1 , or unsorted cells ) were capable of forming large size tumors in nod / scid mice injected with as few as 50 cd44v3aldh cells ( table 1 ) . these findings indicate that cd44v3aldh1 cells display cancer stem cell- ( csc- ) like properties by exhibiting very high tumor initiation potential . however , the cellular and molecular mechanisms that produce csc - like properties of cd44v3aldh1 cells were not known and , therefore , they are the focus of this investigation . identification of the extracellular matrix ( ecm ) components [ e.g. , hyaluronan ( ha ) ] contributing to csc - like properties will enable us to gain a better understanding regarding the role of the microenvironment in initiating and maintaining csc properties . the question of whether different sizes of ha fragments ( ranging from 5  kda to 700  kda ) regulate csc signaling and function in head and neck cancer cells has not been fully addressed and therefore is the focus of this study . there is compelling evidence showing that certain stem cell markers such as nanog , oct , sox2 , and klf-4 are known to form a self - organized core of transcription factors that maintain pluripotency and self - renewal of human embryonic stem cells [ 1821 ] . in this study we found that the expressions of nanog , oct , sox2 , and klf-4 are significantly increased in cd44v3aldh1 cells treated with 200  kda - ha based on q - pcr analyses ( figure 2 ) . there is only a low level of stem cell marker expression detected with other sizes of has ( e.g. , 5  kda - ha , 20  kda - ha or 700  kda - ha , or no ha treatment ) ( figure 2 ) . these findings clearly indicate that the stem cell markers ( nanog , oct4 , sox2 , and klf-4 ) are upregulated in the cd44v3aldh1 cell subpopulation isolated from hnscc ( hsc-3 ) following 200  kda - ha treatment . to determine whether 200  kda - ha - treated cd44v3aldh1 cells ( overexpressing stem cell markers , nanog , oct4 , sox2 , and klf-4 ) are capable of undergoing self - renewal and long - term tumor cell growth , we assessed the ability of these tumorigenic cd44v3aldh1 cells to grow in a  sphere forming  culture by incubating them in serum - free spheroid medium containing 200  kda - ha ( or no ha ) . after 14 days of incubating these cells in the serum - free medium , we observed that the 200  kda - ha - treated cd44v3aldh1 cells form large numbers of spheres ( figure 3(a)-(b ) ) , ranging from 50 to 100 cells per spheroid ( figure 3(a)-(b ) ) . in contrast , only a very small number of spheres were detected in those cells that were not treated with ha ( figure 3(a)-(b ) ) . therefore , it appears that sphere formation with cd44v3aldh1 cells involves the binding of 200  kda - ha . to further test the ability of these cd44v3aldh1 cells ( untreated or pretreated with 200  kda - ha followed by dissociation from spheres after a serial passage of 1st , 2nd , and 3rd generation of sphere formation ) to undergo clone formation and differentiation , we conducted clone formation assay ( figure 3(b ) ) . our data showed that the level of clone formation appears to be significantly higher in these 200  kda - ha - treated cd44v3aldh1 cells ( figure 3(b)-(b ) ) as compared to those detected in cd44v3aldh1 cells with no ha treatment ( figure 3(b)-(a ) ) . these observations suggest that cd44v3aldh1 cells are capable of displaying cancer stem cell - like properties ( e.g. , sphere formation , cell growth / self - renewal , and clone formation / differentiation ) in a 200  kda - ha - specific manner . accumulating evidence now indicates that noncoding micrornas ( mirnas , approximately 22 nucleotides ) are involved in head and neck cancer development [ 2225 ] . our previous study demonstrated that certain oncogenic micrornas promote the cancer stem cell functions of cd44v3aldh1 ( csc - like ) subpopulation from hnscc ( hsc-3 ) . in this study we found that a panel of stem cell - related mirnas including mir-10b , mir-27b , mir-373 , mir-181 , mir-34a , and mirna-145 is preferentially upregulated by 200  kd - ha . in contrast , the other sizes of ha ( e.g. , 5  kd - ha , 20  kd - ha , and 700  kda - ha ) fail to induce various mirna productions in the cd44v3aldh1 ( csc - like ) cell subpopulation ( figure 4 ) . most noticeably , mir-10 appears to undergo the highest level of stimulation by 200  kda - ha ( figure 5 ) . in order to verify whether the 200  kda - ha - induced mirna-10b contributes to malignancy in the head and neck cancer cells , survival proteins such as inhibitors of apoptosis proteins ( iaps ) are frequently upregulated in cscs following ha treatment . importantly , high levels of iaps in cscs increase cell survival due to the binding of iaps to caspases and the suppression of apoptosis . here , we found that the expression of iaps such as c - iap-1 significantly increased in cd44v3aldh1 cells treated with 200  kda - ha based on anti - c - iap-1-mediated immunoblot analyses ( figure 6 ) . there is only a relatively low level of c - iap-1 expression detected with other sizes of has ( e.g. , 5  kda - ha , 20  kda - ha , or no ha treatment ) ( figure 6(a ) ) . these findings clearly indicate that the survival proteins such as c - iap-1 are upregulated in the cd44v3aldh cell subpopulation isolated from hnscc ( hsc-3 ) following 200  kda - ha treatment . our present data also demonstrate that downregulation of mir-10b by treating cd44v3aldh1 cells with an anti - mir-10b inhibitor ( but not a negative - control mirna ) results in the downregulation of ciap-1 ( figure 6(b ) , lane 3 and lane 4 versus lane 1 and 2 ) in the presence of 200  kda - ha . together , these results indicate that the signaling network containing mir-10b is functionally coupled to the stimulation of survival protein production in cscs . further analyses indicate that the addition of 200  kda - ha in negative mirna - treated cd44v3aldh1 cells significantly decreases the ability of cisplatin to induce tumor cell death ( figure 7(a ) versus figure 7(b ) ; table 2 ) . these observations strongly suggest that 200  kda - ha causes both a decrease in tumor cell death and an increase in tumor cell survival leading to the enhancement of chemoresistance ( figure 7(a ) versus figure 7(b ) ; table 2 ) . moreover , downregulation of mir-10b by treating cd44v3aldh1 hnscc cells with an anti - mir-10b inhibitor ( but not negative mirna control - treated samples ) effectively attenuates 200  kda - ha - mediated tumor cd44v3aldh1 cell survival ( figure 7(c ) versus figure 7(d ) ; table 2 ) and enhances cisplatin sensitivity in cd44v3aldh1 cells ( table 2 ) . these findings clearly indicate that downregulation of the 200  kda - ha - induced mir-10b function ( by anti - mir-10b treatment ) may represent a new target for therapeutic agents designed to cause head and neck cancer cscs to undergo cell death and remain chemotherapy sensitive . advanced head and neck squamous cell carcinoma ( hnscc ) is an aggressive disease and a deadly cancer . thus , clarification of key aspects of tumor cell functions underlying the clinical behavior of hnscc is greatly needed . cancer stem cells ( cscs ) found in head and neck cancer have been implicated in the initiation / development of malignancy . because little is known regarding the molecular basis underlying csc signaling and function , it is important to identify molecule(s ) which can be used for predicting the oncogenic potential and possible drug targets for head and neck cancer - derived cscs . hyaluronan ( ha ) is well - known as one of the major components in extracellular matrices ( ecm ) . ha is often bound to cd44 , a ubiquitous , abundant , and functionally important cell surface receptor . different sizes of ha are known to bind to cd44 ; and our studies show that they influence a variety of cellular functions . however , our understanding of cd44 interaction with different sizes of ha fragments in cancer stem cells ( cscs ) from hnscc is lacking . in this study we found that size - specific ha ( in particular , 200  kda - ha ) plays a pivotal role in the selective activation of cd44v3aldh1 ( csc - like ) functions and microrna signaling ( in particular , mir-10b ) required for tumor cell survival , chemoresistance , and head and neck cancer progression . thus , silencing or inhibiting 200  kda - ha - activated mirna-10b may provide important new therapeutic targets to block matrix ha - associated csc behaviors ( e.g. , tumor cell survival and chemoresistance ) in head and neck cancer . as summarized in figure 8 , we propose that the binding of 200  kda - ha ( step  1 ) to cd44v3aldh1 cells promotes specific target gene expression ( step  2 ) , including stem cell marker ( nanog , oct4 , sox2 , and klf-4 ) expression ( step  3-a ) . the resultant stem cell marker expression then induces spheres / self - renewal properties and clone formation ( differentiation ) ( step  4-a ) contributing to cancer stem cell functions and highly tumorigenic properties ( step  5-a ) . at the same time , the binding of 200  kda - ha to cd44v3aldh1 cells also stimulates mir-10b gene expression / mature mir-10b production ( step  3-b ) which then stimulates survival protein , iap ( c - iap1 ) expression ( step  4-b ) , and hnscc cell anti - apoptosis / survival as well as chemoresistance ( step  5-b ) . taken together , these findings suggest that ha- ( in particular , 200  kda - ha- ) mediated cancer stem cell ( csc ) pathways and mir-10b function play a critical role in promoting tumor formation and chemoresistance leading to head and neck cancer progression ( step  6 ) .
we determined that human head and neck cancer cells ( hsc-3 cell line ) contain a subpopulation displaying cancer stem cell ( csc ) properties and are very tumorigenic . 
 specifically , we investigated whether different sizes of hyaluronan ( ha ) ( e.g. , 5  kda , 20  kda , 200  kda , or 700  kda - ha - sizes ) play a role in regulating these cscs . 
 first , we observed that 200  kda - ha ( but not other sizes of ha ) preferentially induces certain stem cell marker expression resulting in self - renewal and clonal formation of these cells . 
 further analyses indicate that 200  kda - ha selectively stimulates the expression of a panel of micrornas ( most noticeably mir-10b ) in these cscs . 
 survival protein ( ciap-1 ) expression was also stimulated by 200  kda - ha in these cscs leading to cisplatin resistance . 
 furthermore , our results indicate that the anti - mir-10 inhibitor not only decreases survival protein expression , but also increases chemosensitivity of the 200  kda - ha - treated cscs . 
 these findings strongly support the contention that 200  kda - ha plays a pivotal role in mir-10 production leading to survival protein upregulation and chemoresistance in cscs . 
 together , our findings suggest that selective activation of oncogenic signaling by certain sizes of ha ( e.g. , 200  kda - ha ) may be instrumental in the formation of csc functions leading to tumor cell survival and chemoresistance in head and neck cancer progression .