pleomorphic adenoma ( pa ) is the most common salivary gland tumor classified as benign epithelial tumors 1 .
various cell types can be seen in the tumor indicating a high occurrence of cell differentiation .
the results suggested that wnt is involved in cell differentiation in pa . in the present study
, we considered that notch might also be involved in cell proliferation and differentiation in the same manner that okuda et al .
, we focused on notch expression in pa since it has been hypothesized to be strongly associated with neoplastic cell differentiation .
tissue samples from department of oral pathology , school of dentistry , aichi gakuin university , diagnosed histologically as pa were further re - evaluated for the purpose of this research . a total of 30 cases of pa based on who classification were used in this experiment .
the specimens used in this study were the same as those used by okuda et al . 2 , described in table 1 .
specimens were fixed in neutral buffered formalin solution , subjected into series of alcohol for dehydration and then embedded in paraffin . then after , the specimens were serially sectioned into 4 m stained with hematoxylin and eosin and examined under a light microscope .
briefly , deparaffinized sections were pre - treated for antigen retrieval in citrate buffer ( mitsubishi chemical medience , tokyo , japan ) with a ph of 6.0 and placed in autoclave at 120 c for 15 min . then after , sections were covered with serum - free protein block ( dako japan co. , ltd . ,
notch1 rabbit polyclonal antibody ( anti - notch-1 intracellular domain antibody , ab83232 , abcam , cambridge , uk ) was used as the primary antibody .
immunofluorescent staining was also performed using notch1 rabbit polyclonal antibody , ck7 mouse monoclonal antibody ( 1:100 ; abcam ) and ck13 mouse monoclonal antibody ( ae8 ; 1:100 ; abcam ) .
double staining was done by combining notch1 and ck7 as well as notch1 and ck13 .
after deparaffinization , notch1-ck7 and notch1-ck13 were pre - treated in citric acid buffer ( mitsubishi chemical medience , tokyo , japan ) with a ph of 6.0 and placed in microwave for 1 min . then after , sections were covered with serum - free protein block ( dako ) , incubated at room temperature for 30 min .
the primary antibodies notch1 and ck7 ( 1:100 ; can get signal , toyobo co. , , osaka , japan ) were allowed to react at 4 c for 16 hours . for the secondary antibody , donkey anti - rabbit igg h&l ( 1:200 ; alexa fluor 594 ; abcam ) and donkey anti - mouse igg h&l ( 1:200 ; alexa fluor 488 ; abcam ) were carried out after reaction with can get signal ( toyobo ) at 1:200 at room temperature for 60 min .
the present study was approved by the ethics committee of aichi gakuin university , school of dentistry under the title  diagnosis and clinicopathological study on the elucidation of salivary gland tumors ' ( no .
generally , the tumor consists of various tissue types with small and/or large duct - like / cystic spaces in the tissues . the tumor is round in shape and covered with relatively thin fibrous connective tissue .
the substantial portion of the tumor consists of glandular structures of tumor nests surrounded by fibrous tissue .
the tumor stroma consists of duct - like structures predominantly ductal and myoepithelial cells forming cystic cavities .
the mesenchymal part consists of myoepithelial cells , spindle shaped cells and myxoma - like tissue formed by round or oval cells dissociated from the tumor growth .
the myoepithelial cells have sparsely formed cord - like structures and the presence of mucous was confirmed .
cartilage - like tissues can be seen in a wide range possibly due to the presence of cartilage - like matrix and cartilage - like cells .
neoplastic myoepithelial cells are dissociated from the mucous or cartilage - like substrate by their own secretions .
the tumor parenchyma might have been formed by myxomatous cells , which migrated to seep into the structure of mesenchymal and cartilage - like cells .
thus , the epithelial component is dissociated and mixed into the mesenchymal - like tissue , giving rise to a ' mixed appearance ' .
notch1 expression was observed in the cytoplasm of ductal epithelial cells as well as in some of the nuclei .
notch expression was also observed in the nucleus of tumor cells surrounding solid tumor nests ( fig .
notch was expressed in the cytoplasm of plasmacytoid myoepithelial cells but not in the nucleus . in areas undergoing squamous metaplasia ,
notch was strongly expressed in the cytoplasm and nucleus of basal cells . however , the expression became weak towards the outer squamous cells ( fig .
the cartilage - like cells representing the characteristic feature of pa showed strong cytoplasmic expression but not nuclear reaction ( fig .
likewise , the myoepithelial cells in myxomatous area showed faint expression on the cell membrane but no cytoplasmic or nuclear reaction were detected ( fig .
double immunofluorescent staining revealed that notch and ck7 were co - expressed by tumor cells in ductal structures .
notch expression was observed in most of the nucleus of tumor cells in solid nests as well as those in ductal structures ( fig .
on the other hand , notch was observed in the nucleus from basal cell to squamous cells ( fig .
double immunofluorescent staining of notch and ck13 revealed that most basal cells expressed notch although expression decreased towards the squamous cells .
furthermore , nuclear expression was observed in basal cells and the expression became weak on the prickle cell layer ( fig .
generally , the tumor consists of various tissue types with small and/or large duct - like / cystic spaces in the tissues . the tumor is round in shape and covered with relatively thin fibrous connective tissue .
the substantial portion of the tumor consists of glandular structures of tumor nests surrounded by fibrous tissue .
the tumor stroma consists of duct - like structures predominantly ductal and myoepithelial cells forming cystic cavities .
the mesenchymal part consists of myoepithelial cells , spindle shaped cells and myxoma - like tissue formed by round or oval cells dissociated from the tumor growth .
the myoepithelial cells have sparsely formed cord - like structures and the presence of mucous was confirmed .
cartilage - like tissues can be seen in a wide range possibly due to the presence of cartilage - like matrix and cartilage - like cells .
neoplastic myoepithelial cells are dissociated from the mucous or cartilage - like substrate by their own secretions .
the tumor parenchyma might have been formed by myxomatous cells , which migrated to seep into the structure of mesenchymal and cartilage - like cells .
thus , the epithelial component is dissociated and mixed into the mesenchymal - like tissue , giving rise to a ' mixed appearance ' .
notch1 expression was observed in the cytoplasm of ductal epithelial cells as well as in some of the nuclei .
notch expression was also observed in the nucleus of tumor cells surrounding solid tumor nests ( fig .
notch was expressed in the cytoplasm of plasmacytoid myoepithelial cells but not in the nucleus . in areas undergoing squamous metaplasia ,
notch was strongly expressed in the cytoplasm and nucleus of basal cells . however , the expression became weak towards the outer squamous cells ( fig .
the cartilage - like cells representing the characteristic feature of pa showed strong cytoplasmic expression but not nuclear reaction ( fig .
likewise , the myoepithelial cells in myxomatous area showed faint expression on the cell membrane but no cytoplasmic or nuclear reaction were detected ( fig .
double immunofluorescent staining revealed that notch and ck7 were co - expressed by tumor cells in ductal structures .
notch expression was observed in most of the nucleus of tumor cells in solid nests as well as those in ductal structures ( fig .
, notch was observed in the nucleus from basal cell to squamous cells ( fig .
double immunofluorescent staining of notch and ck13 revealed that most basal cells expressed notch although expression decreased towards the squamous cells .
furthermore , nuclear expression was observed in basal cells and the expression became weak on the prickle cell layer ( fig .
pa is the most common benign epithelial tumor of salivary glands based on who classification 1 .
the tumor shows a wide variety of tumor development brought about by cell proliferation and differentiation as mentioned in previous studies 4 - 6 .
the results showed that tumor cells in ductal structure and in solid tumor nests expressed wnt .
it was inferred that -catenin pathway was involved in cell differentiation indicated by wnt expressions in cells undergoing squamous metaplasia 2 .
for this reason , we believed that notch would have a role in tumor cell differentiation considering its association with other tumors 7 , 8 .
notch appears as a typical signal that controls the growth of tissues responsible for the fate of cells 9 .
notch is a single - pass transmembrane receptor with a domain outside and inside the cell membrane .
notch intracellular domain ( nicd ) is cleaved and binds to a ligand such as jagged .
cleaved nicd is moved from the cell membrane into the cytoplasm , binds with suppressor of hair - less ( su(h ) ) to form a complex and acts on downstream target gene in the nucleus thus activating the expression 10 .
in addition to the role of transmitting instructions for various morphogenesis and tissue differentiation during development , notch plays an important role in cell - cell signaling and is involved in stem cell maintenance , differentiation and neuronal function in adult . when there is a disturbance in the activity of notch signaling pathway , it becomes oncogenic detected in several cancers such as esophageal cancer , breast cancer and lymphoblastic acute leukemia ( t - all ) 11 , 12 .
in addition , notch has been the focus of researches on the metastasis of malignant tumors such as adenoid cystic carcinoma and malignant ameloblastoma 13 , 14 , believed to be a huge factor in the progress of malignant tumors .
studies in notch have been done in odontogenic tumors such as ameloblastoma 15 - 17 .
notch is expressed in solid nests and in squamous metaplasia in ameloblastoma , implicating its contribution in cell differentiation and morphogenesis . moreover , in calcifying cystic odontogenic tumor , calcifying epitheioma ( pilomatrixoma ) , odontoma and craniopharyngioma , notch expression in ghost or shadow cells was observed 18 - 20 .
this means that notch inhibits the production of a ligand in receiving cell signal between notch ligand and notch receptor to adjacent cells through a negative feedback mechanism .
in contrast , during binary cell fate determination , numb acts an inhibitor of notch signaling cycle causing asymmetric distribution of the notch pathway .
this led to overexpression suggesting that notch - jagged1 is the primary mechanism in determining the fate of ghost cell 21 .
squamous metaplasia depicted as having a variety of cell differentiation is commonly observed in odontogenic tumors such as ameloblastoma .
muraki et al suggested a close relationship between notch and ameloblastoma shown by the localization of notch in peripheral tumor nests 15 . from this aspect
, we suspected that notch might also be involved in cell differentiation in pa . comparing the results of our study with previous reports , from the partial ck7 expression and substantial notch expression in ductal epithelial cells as well as the notch expression in solid tumor nests , it can be inferred that notch is involved in cell differentiation 2 , 22 .
ck13 expression was observed in cells undergoing squamous metaplasia and notch expression was seen in the nucleus of basal and squamous cells 23 .
the intense notch expression in basal cells and weak expression in squamous cells suggests that notch is involved in the differentiation from basal to squamous cell .
moreover , the loss of nuclear expression on the surface layer would signify that differentiation is about to end or has been terminated .
notch was expressed in the cytoplasm of cartilage cells and in the cell membrane of mucous cells but not in the nucleus indicating that differentiation has been concluded .
notch involvement is suspected in cell differentiation in areas showing ductal structures and squamous metaplasia . in the study by okuda et al ,
small cuboidal cells forming ductal structure expressed wnt and the expression was also confirmed in the basal cells surrounding those cells undergoing squamous metaplasia .
the results coincided with our present study implicating the function of notch in the same sites causing cell differentiation 2 . in summary
moreover , notch is expressed by basal cells undergoing squamous metaplasia suggesting the participation of notch in cell differentiation in pa .
 the expression of notch in 30 cases of pleomorphic adenoma was examined by immunohistochemistry . comparing the results of our study with previous literatures , from the partial ck7 expression and substantial notch expression in ductal epithelial cells as well as the notch expression in solid tumor nests 
 , it can be inferred that notch is involved in cell differentiation . 
 ck13 expression was observed in cells undergoing squamous metaplasia and notch expression was seen in the nucleus of basal and squamous cells . 
 the intense notch expression in basal cells and weak expression in squamous cells suggests that notch is involved in the differentiation from basal to squamous cell . 
 moreover , the loss of nuclear expression on the inner layer would signify that differentiation is about to end or has been terminated . 
 notch was expressed in the cytoplasm of cartilage cells and in the cell membrane of mucous cells but not in the nucleus indicating that differentiation has been concluded . 
 notch involvement is suspected in cell differentiation in areas showing ductal structures and squamous metaplasia . in summary , 
 notch is involved in cell differentiation of ductal cells in pa . 
 nuclear expression was shown in tumor cells in solid nests and surrounding structures . 
 moreover , notch is expressed by basal cells undergoing squamous metaplasia suggesting the participation of notch in cell differentiation in pa . 
Introduction
Materials and Methods
Results
Histopathological examination
Immunohistochemical findings
Discussion