oral squamous cell carcinoma ( oscc ) is the most common malignant neoplasm of the oral mucosa ; it ranks among the top twelfth most frequently diagnosed cancers worldwide . in china , tongue squamous cell carcinoma ( tscc ) predominates more than 95% of oscc and its incidence is increasing year by year . dysplastic lesions of the oral cavity range from mild to moderate and severe dysplasia , and the presence and degree of dysplasia are often used to predict malignant transformation . moreover , the prognosis for most patients with oscc , of course including tscc , is usually related to clinical stage and histologic grade . therefore , the identification of effective biologic markers that correlate with the stepwise of carcinogenesis and development in tscc is very important to understand this neoplasm . caveolin-1 ( cav-1 ) , a 2124 kda protein is the major structural component of caveolae , vesicular invaginations of the plasma membrane . previous studies have implicated cav-1 in the development of several human cancers , but it appears to play quite different roles in the development of these diseases : down - regulation of cav-1 expression was observed in breast , lung , colon and ovarian cancer ; other studies have reported that cav-1 expression was up - regulated in some human cancers , such as prostate , esophagus , thyroid and pancreas cancers , and that this up - regulation was associated with metastases and poor prognosis . in oscc as well , the role of cav-1 still remains controversial : two research groups found that an increased cav-1 expression plays an important role in carcinogenesis and development of oscc . in contrast , some studies disclosed that the inactivation of cav-1 by a mutation or by reduced expression might play a role in the pathogenesis of oral cancer , which indicated the value to explore cav-1 's biphasic functions in oscc . to our best knowledge whether cav-1 acts as oncogene or tumor suppressor gene in the development of tscc derserves new insights . semiconductor quantum dots ( qds ) are new nanocrystal semiconductor fluorophores consisting of a cadmium selenide core and zinc sulfide or cadmium sulfide shell . as described in previous publications , qds can be used to target malignant tumors with high specificity , when conjugated with biomolecular affinity ligands , such as antibodies , peptides or small molecules . due to their novel optical and electronic properties , such as high brightness , stability and simplified multiple target labeling , qds recently attracted attention for their potential application in advanced biosensors , bioanalytical assays and cell imaging . in this report , using a new method qds - ihc , we sought to investigate the expression of cav-1 in normal tongue mucosa ( ntm ) , hyperplastic tongue mucosa ( htm ) , tongue pre - cancer lesions ( tpl ) and tscc , trying to elucidate the possible role of cav-1 in carcinogenesis and development of tscc . we evaluated 96 cores , including 61 cases of human tscc , 10 cases of ntm , 10 cases of htm and 15 cases of tpl using human tissue microarray ( tma ) technology ( department of oral pathology , school of stomatology , wuhan university ) . histological diagnosis and grades of differentiation were determined in accordance with the world health organization ( who ) criteria for oral tumors . concerning the tscc cases , all the samples were deriving from the tongue of 61 patients , 34 males and 27 females , with an average age of 54.8 years ; the histological differentiation of tscc was classified as the tumor ( t ) stages were : t124 , t234 , t32 , and t41 . the neck ( n ) stages were : n048 , n113 . all the tissues were taken from surgical resection prior to initial topical or systemic therapy . they were fixed in 10% neutral buffered formalin for 24 hours and processed using identical standard operating procedures ( sops ) . tissue samples were arranged in 6 columns of 16 rows for a total of 96 individual cores ( 1.5 mm , 4 m ) . four m - thickness tmas were deparaffined in xylene and rehydrated in a graded series of ethanol . antigen retrieval was performed using citric acid ( 10 mm , ph 6.0 ) at 95c for 10 min and left to cool for 60 min . tmas were incubated with 2% bovine serum albumin ( bsa ) ( sigma , st . the slides were rinsed 3 with tbs - tween 20 ( tbs - t ) ( 5 min ) . sections were incubated with rabbit anti - human cav-1 polyclonal antibody ( 1:300 dilution , santa cruz , ca , usa ) for 2h at 37c , followed by three washes with tbs - t ( 5 min ) , then incubated with biotinylated goat anti - rabbit igg ( zhongshan golden bridge biotechnology co , ltd . , beijing , china ) for 30 min at 37c , and rinsed 3 with tbs - t ( 5 min ) . slides were incubated with 2% bsa buffer for 10 min at 37c before incubation with streptavidin - conjugated qds605 ( 1:100 , wuhan jiayuan quantum dot co , ltd . , wuhan , hubei , china ) in 2% bsa for 30 min at 37c and rinsed 3 with tbs - t ( 5 min ) , the edges sealed with 90% glycerine ( sigma , st . the qds signals were shown to be target specific , red , bright , and photo stable . cav-1 immunoreactivity was normally localized to fibroblasts and endothelial cells of blood vessels in all tissue specimens examined , which served as an internal quality control in qds - ihc . a reproducible semiquantitative analysis was used to evaluate the expression level of cav-1 in tissue samples . the sections were initially scanned at low power , and areas with high and low staining intensity were selected . at each selected field , cells were classified with respect to staining intensity ranging from 0 to 4 , and the percentage of cells for each staining intensity was estimated by decimal equivalent ranging from 0 to 1 . a numerical value for cav-1 staining in each tissue section was then calculated by multiplying the fraction of cells at each staining intensity by the numerical value of that intensity , resulting in scores ranging from 0 to 4 . expression levels of each cav-1 in tumor samples were graded based on the total score as follows : negative or low = score 1 ; moderate or high score>1 . we defined the cutoff between score and score>1 because score 1 level was the most significant pathologic effect in clinicopathological features . two observers evaluated independently the results of this method , without any knowledge of the clinical data . we evaluated 96 cores , including 61 cases of human tscc , 10 cases of ntm , 10 cases of htm and 15 cases of tpl using human tissue microarray ( tma ) technology ( department of oral pathology , school of stomatology , wuhan university ) . histological diagnosis and grades of differentiation were determined in accordance with the world health organization ( who ) criteria for oral tumors . concerning the tscc cases , all the samples were deriving from the tongue of 61 patients , 34 males and 27 females , with an average age of 54.8 years ; the histological differentiation of tscc was classified as the tumor ( t ) stages were : t124 , t234 , t32 , and t41 . the neck ( n ) stages were : n048 , n113 . all the tissues were taken from surgical resection prior to initial topical or systemic therapy . they were fixed in 10% neutral buffered formalin for 24 hours and processed using identical standard operating procedures ( sops ) . tissue samples were arranged in 6 columns of 16 rows for a total of 96 individual cores ( 1.5 mm , 4 m ) . four m - thickness tmas were deparaffined in xylene and rehydrated in a graded series of ethanol . antigen retrieval was performed using citric acid ( 10 mm , ph 6.0 ) at 95c for 10 min and left to cool for 60 min . tmas were incubated with 2% bovine serum albumin ( bsa ) ( sigma , st . the slides were rinsed 3 with tbs - tween 20 ( tbs - t ) ( 5 min ) . polyclonal antibody ( 1:300 dilution , santa cruz , ca , usa ) for 2h at 37c , followed by three washes with tbs - t ( 5 min ) , then incubated with biotinylated goat anti - rabbit igg ( zhongshan golden bridge biotechnology co , ltd . , beijing , china ) for 30 min at 37c , and rinsed 3 with tbs - t ( 5 min ) . slides were incubated with 2% bsa buffer for 10 min at 37c before incubation with streptavidin - conjugated qds605 ( 1:100 , wuhan jiayuan quantum dot co , ltd . , wuhan , hubei , china ) in 2% bsa for 30 min at 37c and rinsed 3 with tbs - t ( 5 min ) , the edges sealed with 90% glycerine ( sigma , st . the qds signals were shown to be target specific , red , bright , and photo stable . cav-1 immunoreactivity was normally localized to fibroblasts and endothelial cells of blood vessels in all tissue specimens examined , which served as an internal quality control in qds - ihc . a reproducible semiquantitative analysis was used to evaluate the expression level of cav-1 in tissue samples . the sections were initially scanned at low power , and areas with high and low staining intensity were selected . at each selected field , cells were classified with respect to staining intensity ranging from 0 to 4 , and the percentage of cells for each staining intensity was estimated by decimal equivalent ranging from 0 to 1 . a numerical value for cav-1 staining in each tissue section was then calculated by multiplying the fraction of cells at each staining intensity by the numerical value of that intensity , resulting in scores ranging from 0 to 4 . expression levels of each cav-1 in tumor samples were graded based on the total score as follows : negative or low = score 1 ; moderate or high score>1 . we defined the cutoff between score and score>1 because score 1 level was the most significant pathologic effect in clinicopathological features . two observers evaluated independently the results of this method , without any knowledge of the clinical data . immunofluorescence by qds analysis revealed focal positive immunoreactivity for cav-1 in normal squamous epithelium ( figure 1a ) and hyperplastic squamous epithelium ( figure 1b ) . among 15 dysplastic tissues , 5 showed moderate to high levels of cav-1 expression ( score>1 ) , characterized by intensive immunoreactivity in the full thickness of epithelium and all the left 10 tissues showed cav-1 expression confined to basal or parabasal cells ( figure 1d ) . in all sections examined , a strong , diffuse , cytoplasmic staining pattern in the endothelial cells in the stroma vessels was detected , as described previously . figure 1cav-1 expression by qds - ihc in normal , hyperplastic and displastic tongue mucosa . ( a ) focal expression of cav-1 in normal tongue mucosa ( qds - ihc200 ) . ( b ) focal expression of cav-1 in hyperplastic tongue mucosa and internal control showed a strong , diffuse , cytoplasmic staining pattern in the endothelial cells in the stroma vessels ( qds - ihc200 ) . ( c ) almost full thickness of epithelium in severely dysplasitic tongue mucosa is positive for cav-1(qds - ihc400 ) . ( d ) cav-1 expression confined to basal or parabasal cells in mild or moderate dysplastic tongue mucosa ( qds - ihc200 ) . ( a ) focal expression of cav-1 in normal tongue mucosa ( qds - ihc200 ) . ( b ) focal expression of cav-1 in hyperplastic tongue mucosa and internal control showed a strong , diffuse , cytoplasmic staining pattern in the endothelial cells in the stroma vessels ( qds - ihc200 ) . ( c ) almost full thickness of epithelium in severely dysplasitic tongue mucosa is positive for cav-1(qds - ihc400 ) . ( d ) cav-1 expression confined to basal or parabasal cells in mild or moderate dysplastic tongue mucosa ( qds - ihc200 ) . among the primary tumors examined , moderate to high levels of cav-1 expression ( score>1 ) were noted in 45 tumors ( 74% ) . tumor cells positive for cav-1 were clearly detected and brighter images were obtained in a fine , granular pattern at the surface membrane and the cytoplasm ( figure 2a ) . interestingly , we observed the stronger positive signals in the peripheral cancer nests than in the middle cancer nests ( figure 2a ) and in the same section the cav-1 expression in tumor nests was more intensive than in dysplastic mucosa ( figure 2b ) . even single cell out of cancer nests positive for cav-1 could be clearly detected ( figure 2c ) . the poorly differentiated cells of these tumors with high levels ( score>2 ) of cav-1 expression showed stronger diffuse cytoplasmic and cell surface staining than that showed by well differentiated tumor cells with low levels of expression ( score1 ) ( figure 3a  c ) . ( a ) tumor cells in ptscc positive for cav-1 were obtained in a fine , granular pattern at the surface membrane and the cytoplasm ; in the peripheral cancer nests were observed stronger positive signals than in the middle cancer nests ( qds - ihc400 ) . ( b ) in the same section , the cav-1 expression in tumor nests was more intensive than in dysplastic mucosa ( qds - ihc200 ) . ( c ) single tumor cell in ptscc positive for cav-1 could be clearly detected ( qds - ihc400 ) . ( d ) negative control showed negative reactivity for cav-1 ( qds - ihc100 ) . cav-1 expression by qds - ihc in ptscc . ( a ) tumor cells in ptscc positive for cav-1 were obtained in a fine , granular pattern at the surface membrane and the cytoplasm ; in the peripheral cancer nests were observed stronger positive signals than in the middle cancer nests ( qds - ihc400 ) . ( b ) in the same section , the cav-1 expression in tumor nests was more intensive than in dysplastic mucosa ( qds - ihc200 ) . ( c ) single tumor cell in ptscc positive for cav-1 could be clearly detected ( qds - ihc400 ) . ( d ) negative control showed negative reactivity for cav-1 ( qds - ihc100 ) . ( a ) tumor cells in ptscc with high degree of histological differentiation showed weak positivity for cav-1 ( qds - ihc100 ) . ( b ) tumor cells in ptscc with moderate degree of histological differentiation showed moderate positivity for cav-1 ( qds - ihc100 ) . ( c ) tumor cells in ptscc with low degree of histological differentiation showed intensive positivity for cav-1 ( qds - ihc100 ) . ( a ) tumor cells in ptscc with high degree of histological differentiation showed weak positivity for cav-1 ( qds - ihc100 ) . ( b ) tumor cells in ptscc with moderate degree of histological differentiation showed moderate positivity for cav-1 ( qds - ihc100 ) . ( c ) tumor cells in ptscc with low degree of histological differentiation showed intensive positivity for cav-1 ( qds - ihc100 ) . statistically , cav-1 expression in the tpl and ptscc showed significant increase compared with that of normal mucosa and hyperplastic mucosa respectively , ( p<0.01 , table 1 ) . cav-1 expression of ptscc was also significantly higher compared with tpl ( p<0.01 , table 1 ) . in ptscc , the clinicopathologic variables are listed in table 2 . statistical correlation was found between the enhanced cav-1 expression and the degree of histological grades (  well  ,  moderate  and  poor  , p<0.05 ) . in addition , altered expression of cav-1 was associated with the t stage and n classification . in contrast , there was no correlation between the positivity of cav-1 expression and gender and age . table 1correlations of cav-1 expression among normal tongue mucosa , hyperplastic tongue mucosa , tongue premalignant lesion and tongue squamous cell carcinoma.groupscav-1 scorep1.0>1normal and hyperplastic tongue mucosa200p<0.01premalignant lesion105p<0.01tongue squamous cell carcinoma1645p<0.01p , normal and hyperplastic tongue mucosa vs premalignant lesion;p , premalignant lesion vs tongue squamous cell carcinoma;p , normal and hyperplastic tongue mucosa vs tongue squamous cell carcinoma . 
  , normal and hyperplastic tongue mucosa vs premalignant lesion ; , premalignant lesion vs tongue squamous cell carcinoma ; , normal and hyperplastic tongue mucosa vs tongue squamous cell carcinoma . table 2relationship between clinicopathologic features and caveolin-1 expression in primary tongue squamous cell carcinoma by qds-ihc.variablecav-1 scorep1.0>1gender  male727p>0.05  female918age  60618p>0.05  < 601027 t status *  t11410p<0.05 t2+t3+t4235n status  n01632p<0.05  n1013differentiation  well1518p<0.05  moderate116  poor011*tumor stage , size or direct extent of the primary tumor;neck stage , degree of spread to neck lymph nodes . tumor stage , size or direct extent of the primary tumor ; neck stage , degree of spread to neck lymph nodes . determination of the expression and spatial distribution of molecular epitopes , or antigens , in patient tissue specimens has substantially improved the pathologist 's ability to classify disease processes . the primary goals of tumor research are to identify these molecular factors , among which cav-1 has attracted attention because of its altered expression patterns in tumour cells , which contribute to tumorigenesis and tumor progression . in this report , we have used a new qds - ihc method to observe the expression of cav-1 in stepwise carcinogenesis and development of tscc on basic fluorescence microscope . qds , a novel inorganic fluorophores , may be cross - linked to biomolecules such antibodies , oligonucleotides or small molecule ligands to render them specific to biological targets . due to their novel optical and electronic properties , the use of multicolor qds probes is considered one of the most important and clinically relevant applications . they have been received as technological marvels with such characteristics able to greatly improve biological imaging and detection . a previous study revealed that qds can be applied to formalin - fixed , paraffinembedded tissues ; this technique achieves sensitivity and specificity levels that are sufficient for the potential application of known expression signatures to biopsy specimens in a semi - quantitative way , while the semi - automated nature of this method enables application to high - throughput studies . some researchers demonstrated that qds probe could be used for multiplexed profiling of molecular biomarkers , and ultimately for correlation with disease progression . our previous study also indicated qds - ihc is sensitive and specific in detecting the protein in paraffin - embedded lung tissue . our present data indicate that cav-1 expression with moderate to high levels using qds - ihc in 74% of tscc ; positive signals were strong and clear with clean background , indicating qds - ihc might be an efficient method for protein detection in tongue samples . cav-1 is the principal component of caveolae , which are special invaginated micro - domains of the plasma membrane present in most mammalian cells . it plays a key role in the regulation of several signal transduction molecules and attracts more and more attention due to its paradoxical biological functions in malignant tumors . in this study , we noted for the first time a progressive increase of cav-1 expression from ntm , htm , tpl to ptscc ( a series of tissues ranging from normal to neoplastic ) , which is a trend of increased expression of cav-1 in the stepwise tongue carcinogenesis . moreover , in the same sample the cav-1 expression in tumor nests was more intensive than that in dysplasia mucosa . the up - regulated expression of cav-1 observed in the present study using qds - ihc was similar to that reported in studies of esophageal squamous cell carcinoma , prostate cancer and pancreatic cancer . it has been suggested that the secreted fraction of cav-1 is associated with cell released micro - vesicles , which are able to confer tumorigenicity in vitro as well as in vivo . our study showed that up - regulated cav-1 expression was associated with advanced clinical stage and high - grade malignancy of tscc , while nakatani et al . found that the level of cav-1 expression did not differ among stages or other clinical parameters , except for the level of cav-1 expression between the well - differentiated and poorly differentiated groups . the reasons might be as follows : i ) different methods ; our group just used a different method in the present study . ii ) different samples ; nakatani et al . used oscc samples in different sites including tongue , maxilla , mandibular and floor of mouth , while all of our samples arised from the tongue . iii ) a different score system for the immunoevaluation used in the present study . in addition , in the present research , in ptscc were observed stronger positive signals in the peripheral cancer nests than in the middle cancer nests ; this is in agreemetn with the results of previous studies , showing that cav-1 is preferentially expressed in tumor cells with basal - like immunophenotype , as defined by cdna microarrays or immunohistochemistry . on the other hand , a previous study disclosed that the inactivation of cav-1 by a mutation or by reduced expression might play a role in the pathogenesis of oral cancer . some authors also found that cav-1 might be a tumor suppressor , because of its decreased expression in a variety of cancer such as breast carcinoma , colon carcinoma , uterine cervical carcinoma , sarcoma of head and neck . lee and colleagues suggested that the diverse effects of cav-1 might be simply mediated by the different regions of cav-1 molecule and might be dependent on the levels of other molecules that are co - expressed with cav-1 . although the sample size for tongue epithelial dysplasia in the current study is insufficient for statistical analysis on cav-1 expression , 10  mild  to  moderate tongue epithelial dysplasia revealed basal or parabasal cells staining , whereas 5  severe  tongue epithelial dysplasia samples revealed the whole epithelial layer staining . this staining pattern of upward extension of cav-1 protein may suggest that the cav-1 expression was consistent with the severity of oral epithelial dysplasia . in conclusion , our results may suggest that cav-1 protein is one of the oncogenes that contribute to the carcinogenesis and development of tscc . whether cav-1 is an important predictor or prognosis for survival
quantum dots ( qds ) are a new class of fluorescent probes to detect biomarker expression . 
 the role of caveolin-1 ( cav-1 ) in tongue squamous cell carcinoma ( tscc ) is still unknown . 
 this study aimed to investigate the expression profile of cav-1 in carcinogenesis and development of tscc by qds immunofluorescence histochemistry ( qds - ihc ) and discuss the relationship between the cav-1 expression and the clinicopathological outcomes . 
 qds - ihc was used to detect cav-1 expression in tissue microarrays including normal tongue mucosa ( ntm ; n=10 ) , hyperplastic tongue mucosa ( htm ; n=10 ) , tongue pre - cancer lesions ( tpl ; n=15 ) and primary tongue squamous cell carcinoma ( ptscc ; n=61 ) . 
 correlations between the cav-1 expression and clinicopathologic variables were evaluated statistically . 
 cells positive for cav-1 were clearly detected and bright images were obtained in a fine , granular pattern at the cell membrane and cytoplasm using qds - ihc . 
 the rate of cav-1 immunoreactivity increased progressively from ntm ( 0% ) , htm ( 0% ) , tpl ( 36% ) to ptscc ( 74% ) . when compared with each other , there was statistical significance among ptscc , tpl and ntm as well as among ptscc , tpl and htm . 
 moreover , cav-1 expression level in ptscc was correlated positively with clinical stage and histologic grade . 
 qds - ihc could accurately detect protein location in tongue mucosa . 
 an increased expression of cav-1 in the stepwise carcinogenesis from ntm , htm , tpl to ptscc suggested that cav-1 might be an oncogene in the development of tongue squamous cell carcinoma .