as the third most common malignancy , colorectal carcinoma is the fourth leading cause of cancer - related death worldwide . in china , colorectal carcinoma has been ranked as the fifth most common human malignant disease , accounting for 6.51% of mortality in urban areas and 4.64% in rural areas . the etiology of colorectal cancers is known to be heterogeneous and various mechanisms have been associated with the increased risk for development and progression of colorectal carcinoma , as well as the aggressive behaviors of the tumors [ 35 ] . tumors at the invasive front appear morphologically more poorly differentiated and show different molecular characteristics . previous studies of colorectal carcinoma have suggested the potential role of -tubulin expression in rectal cancer development . a study by portyanko et al . found expression of class iii -tubulin ( iii - tubulin , also known as tubb3 ) at the invasive margin of colorectal cancer . however , the authors mainly focused on the immunoreactivity of iii - tubulin in colorectal cancer patients , and the clinical implication of their findings has not been well investigated . recently , however , the potential effect of iii - tubulin expression at the invasive margin of colorectal cancer was not taken into consideration . therefore , the present investigated the clinical implication of iii - tubulin expression at the invasive margin of colorectal cancer . a total of 111 patients with colorectal carcinoma who underwent radical colorectal resection were recruited at beijing tongren hospital from february 2012 to february 2015 . we excluded patients with tumor history , familial polyposis colon cancer , crohn s disease , malignant transformation of ulcerative colitis , simultaneous multiple primary tumors , or incomplete clinical data . the formalin - fixed and paraffin - embedded tissue samples containing the invasive part of the tumors were obtained and stained with hematoxylin and eosin ( he ) . immunohistochemical staining of iii - tubulin was performed with the method of immunohistochemistry envision - two steps using maxvision2 ( fuzhou maixin biotech . co. , ltd . , fujian , china ) according to the manufacturer s instructions . briefly , the tissue specimen was cut into 4-m - thick sections , and deparaffinized in xylene and later rehydrated in graded alcohol . antigen was retrieved using citrate buffer ( ph 6.0 ) in a microwave oven . after washing 3 times with tris - buffered saline ( tbs ) , the sections were processed with 3% hydrogen peroxide for 510 min to block endogenous peroxidase activity . sections were then incubated with mouse anti - human antibody against iii - tubulin ( clone tuj-1 , mab-0636 , fuzhou maixin biotechnology development co. , ltd . , fuzhou , china ) or cytokeratin ( ae1/ae3 , kit-0009 , fuzhou maixin biotech co. , ltd . , fujian , china ) overnight at 4c , followed by horseradish peroxidase - linked sheep anti - mouse secondary antibody ( kit-5930 - 1 , fuzhou maixin biotech co. , ltd . , expression of iii - tubulin was analyzed under an olympus bx51 microscope ( olympus , tokyo , japan ) by 2 senior professional physicians who were blinded to the clinical data of patients . the proportion of positive tumor cells was scored from 5 randomly selected slides and the results were expressed as the average of data of the 2 physicians . the cytoplasmatic staining intensity was graded as : negative (  , no staining ) , weak positive ( + , pale yellow ) , moderate positive ( + + , claybank ) , and strong positive ( + + + , sepia ) . according to the distribution of iii - tubulin in the tumor tissue , patients were assigned to either the invasive front group or the non - invasive front group . the invasive front group consisted of cases with iii - tubulin - positive tumor cells found only in the invasive front tumor area , while the non - invasive front group consisted of the remaining cases . we recorded clinical data on age , sex , tumor size , differentiation status , tnm stage , and lymphatic metastasis . enrolled patients were followed up from the time of disease diagnosis to the time of death of patients or study deadline ( may 2015 ) . this study was approved by the ethics committee of beijing tongren hospital of the capital medical university and informed consent was obtained from all patients . qualitative data are expressed as proportion and analyzed by  or fisher s exact test . normally distributed quantitative data are expressed as mean sd and analyzed by anova . data with abnormal distribution are expressed as interquartile range and were analyzed by kruskal - wallis test . a total of 111 patients with colorectal carcinoma who underwent radical colorectal resection were recruited at beijing tongren hospital from february 2012 to february 2015 . we excluded patients with tumor history , familial polyposis colon cancer , crohn s disease , malignant transformation of ulcerative colitis , simultaneous multiple primary tumors , or incomplete clinical data . the formalin - fixed and paraffin - embedded tissue samples containing the invasive part of the tumors were obtained and stained with hematoxylin and eosin ( he ) . immunohistochemical staining of iii - tubulin was performed with the method of immunohistochemistry envision - two steps using maxvision2 ( fuzhou maixin biotech . co. , ltd . , fujian , china ) according to the manufacturer s instructions . briefly , the tissue specimen was cut into 4-m - thick sections , and deparaffinized in xylene and later rehydrated in graded alcohol . antigen was retrieved using citrate buffer ( ph 6.0 ) in a microwave oven . after washing 3 times with tris - buffered saline ( tbs ) , the sections were processed with 3% hydrogen peroxide for 510 min to block endogenous peroxidase activity . sections were then incubated with mouse anti - human antibody against iii - tubulin ( clone tuj-1 , mab-0636 , fuzhou maixin biotechnology development co. , ltd . , fuzhou , china ) or cytokeratin ( ae1/ae3 , kit-0009 , fuzhou maixin biotech co. , ltd . , fujian , china ) overnight at 4c , followed by horseradish peroxidase - linked sheep anti - mouse secondary antibody ( kit-5930 - 1 , fuzhou maixin biotech co. , ltd . , fujian , china ) for 60 min . the slides were then counterstained with hematoxylin . the same procedure with the primary antibody omitted was used as a negative control . expression of iii - tubulin was analyzed under an olympus bx51 microscope ( olympus , tokyo , japan ) by 2 senior professional physicians who were blinded to the clinical data of patients . the proportion of positive tumor cells was scored from 5 randomly selected slides and the results were expressed as the average of data of the 2 physicians . the cytoplasmatic staining intensity was graded as : negative (  , no staining ) , weak positive ( + , pale yellow ) , moderate positive ( + + , claybank ) , and strong positive ( + + + , sepia ) . according to the distribution of iii - tubulin in the tumor tissue , patients were assigned to either the invasive front group or the non - invasive front group . the invasive front group consisted of cases with iii - tubulin - positive tumor cells found only in the invasive front tumor area , while the non - invasive front group consisted of the remaining cases . we recorded clinical data on age , sex , tumor size , differentiation status , tnm stage , and lymphatic metastasis . enrolled patients were followed up from the time of disease diagnosis to the time of death of patients or study deadline ( may 2015 ) . this study was approved by the ethics committee of beijing tongren hospital of the capital medical university and informed consent was obtained from all patients . qualitative data are expressed as proportion and analyzed by  or fisher s exact test . normally distributed quantitative data are expressed as mean sd and analyzed by anova . data with abnormal distribution are expressed as interquartile range and were analyzed by kruskal - wallis test . a total of 111 patients who underwent radical colorectal resection were included in this study . the staining intensity was negative in 6 cases ( 5.41% ) , weakly positive in 10 cases ( 9.01% ) , moderately positive in 65 cases ( 58.56% ) , and strongly positive in 30 cases ( 27.03% ) . the invasive front group had 72 patients with iii - tubulin expressed only in the invasive front of tumor tissue , and the non - invasive front group had the remaining 39 patients . he staining results of colorectal cancer in the non - invasive front group and the invasive front group are shown in figure 1a and 1b . immunohistochemical analysis showed positive staining of cytokeratin ( ae1/ae3 ) in the non - invasive front group ( figure 1c ) and the invasive front group ( figure 1d ) . immunohistochemical analysis of iii - tubulin showed heterogeneous labeling in both the non - invasive front group and invasive front group , with weakly , moderately , and strongly stained areas observed ( figure 1e1j ) . the rate of positive staining of iii - tubulin in colorectal carcinoma patients was significantly correlated with staining intensity and tissue distribution of iii - tubulin expression ( p=0.018 and p=0.001 , respectively , table 2 ) . the positive staining rate of iii - tubulin was significantly higher in tumor tissues with higher staining intensity ( p<0.05 ) . the rate of iii - tubulin positive staining in the non - invasive front group was also significantly higher when compared with the invasive front group . the other data ( age , sex , lymphatic metastasis , tnm stage , local recurrence , distant metastasis , and mortality ) were not found to be significantly associated with positive staining of iii - tubulin . there was no significant difference in the clinical data among the groups with different iii - tubulin staining intensity ( table 3 ) the patients were further divided into invasive front and non - invasive front groups according to the tissue distribution of iii - tubulin in colorectal carcinoma . the rate of positive staining and staining intensity of iii - tubulin were significantly different between the invasive and non - invasive front groups ( p=0.001 and p=0.006 , respectively , table 4 , figure 2a , 2b ) , and there was a significant difference in tumor differentiation between the 2 groups ( p=0.032 , figure 2c ) . however , the differences in other data between the invasive and non - invasive front groups did not reach statistical significance . staining intensity of iii - tubulin in patients in the invasive and non as shown in table 5 , in the non - invasive front group , staining intensity of iii - tubulin was significantly associated with the rates of iii - tubulin - positive staining and lymphatic metastasis of tumors ( p<0.001 and p=0.048 , respectively , figure 2d ) , but there were no significant differences in any of the clinical data with respect to the iii - tubulin staining intensity in patients in the invasive front group . a total of 111 patients who underwent radical colorectal resection were included in this study . the staining intensity was negative in 6 cases ( 5.41% ) , weakly positive in 10 cases ( 9.01% ) , moderately positive in 65 cases ( 58.56% ) , and strongly positive in 30 cases ( 27.03% ) . the invasive front group had 72 patients with iii - tubulin expressed only in the invasive front of tumor tissue , and the non - invasive front group had the remaining 39 patients . he staining results of colorectal cancer in the non - invasive front group and the invasive front group are shown in figure 1a and 1b . immunohistochemical analysis showed positive staining of cytokeratin ( ae1/ae3 ) in the non - invasive front group ( figure 1c ) and the invasive front group ( figure 1d ) . immunohistochemical analysis of iii - tubulin showed heterogeneous labeling in both the non - invasive front group and invasive front group , with weakly , moderately , and strongly stained areas observed ( figure 1e1j ) . the rate of positive staining of iii - tubulin in colorectal carcinoma patients was significantly correlated with staining intensity and tissue distribution of iii - tubulin expression ( p=0.018 and p=0.001 , respectively , table 2 ) . the positive staining rate of iii - tubulin was significantly higher in tumor tissues with higher staining intensity ( p<0.05 ) . the rate of iii - tubulin positive staining in the non - invasive front group was also significantly higher when compared with the invasive front group . the other data ( age , sex , lymphatic metastasis , tnm stage , local recurrence , distant metastasis , and mortality ) were not found to be significantly associated with positive staining of iii - tubulin . there was no significant difference in the clinical data among the groups with different iii - tubulin staining intensity ( table 3 ) the patients were further divided into invasive front and non - invasive front groups according to the tissue distribution of iii - tubulin in colorectal carcinoma . the rate of positive staining and staining intensity of iii - tubulin were significantly different between the invasive and non - invasive front groups ( p=0.001 and p=0.006 , respectively , table 4 , figure 2a , 2b ) , and there was a significant difference in tumor differentiation between the 2 groups ( p=0.032 , figure 2c ) . however , the differences in other data between the invasive and non - invasive front groups did not reach statistical significance . staining intensity of iii - tubulin in patients in the invasive and non - invasive front groups was further analyzed . as shown in table 5 , in the non - invasive front group , staining intensity of iii - tubulin was significantly associated with the rates of iii - tubulin - positive staining and lymphatic metastasis of tumors ( p<0.001 and p=0.048 , respectively , figure 2d ) , but there were no significant differences in any of the clinical data with respect to the iii - tubulin staining intensity in patients in the invasive front group . iii - tubulin is one of 9 -isoforms that participate in formation of microtubules ; it is known to play a critical role in cell growth , division , motility , signaling development , and cell shape maintenance . overexpression of iii - tubulin was originally identified to be a prominent factor contributing to resistance to taxane drugs [ 1318 ] . down - regulation of iii - tubulin expression has been reported to contribute to favorable clinical outcome following anti - tubulin therapy . recently , however , increasing studies have shown that iii - tubulin is involved more in tumor development and progression than as a predictor of response to chemotherapy in various types of tumors [ 1823 ] . iii - tubulin has been reported to be expressed in a variety of tumors , especially in those with aggressive behavior that are likely to metastasize [ 2426 ] . in the present study , the tissue distribution of class iii -tubulin expression at the invasive margin or diffuse distribution was investigated in colorectal cancer . our study showed that iii - tubulin was expressed at the invasive margin in 64.78% of our colorectal cancer cases . the expression of iii - tubulin may be involved with tumor differentiation and lymphatic metastasis , suggesting a potential role of iii - tubulin in tumor differentiation and metastasis . in colorectal cancer , aberrant iii - tubulin expression has been suggested to be involved with tumor development and poor survival of the patients . however , a previous study of iii - tubulin expression in colorectal cancer indicated that expression of iii - tubulin was relatively uncommon in colorectal carcinomas . a study by portyanko et al . showed the preferential localization of iii - tubulin at the invasive margin in colorectal cancer , suggesting a potential role of iii - tubulin in modulation of the invading activity of cancer cells . however , those studies had relatively small sample sizes and the clinical implications of iii - tubulin at the invasive margin in colorectal cancer had not yet been defined . therefore , in the present study , 111 patients with surgically resected colorectal cancer were recruited , and the expression of iii - tubulin at the invasive margin was investigated . the findings of our study showed that iii - tubulin was expressed at the invasive margin in 72 of 111 cases of colorectal cancer , accounting for 64.87% of all cases . these data dramatically differed from the results reported by portyanko et al . , which showed iii - tubulin expression at the invasive margin in 28 of 29 cases of colorectal cancer . these conflicting results can be partially explained by differences in race and number of patients recruited , as well as the lack of consistency in study criteria . however , despite these differences , our study showed a significant difference in tumor differentiation between the invasive front and non - invasive front groups , with many more poorly and moderately differentiated tumors identified in the invasive front group . this finding was partially corroborated with the results of a previous study reporting more iii - tubulin immunoreactivity in poorly differentiated colorectal carcinomas . sex has been recently suggested to play a role in the ability of iii - tubulin to predict poor outcome in colorectal cancer . in our study , however , no such sex - related difference was observed . the upregulated expression of iii - tubulin has been reported in several types of cancers and was associated with aggressive behavior . a previous papillary thyroid carcinoma study showed strong staining of iii - tubulin in widely infiltrating ptcs , particularly at the invasive margin , or in moderately differentiated ptcs , and most cases with lymphatic metastasis showed strong iii - tubulin immunoreactivity . in an esophageal squamous cell carcinoma study , yu et al . found that iii - tubulin expression was associated with the lymphatic metastasis of tumors . the results of the present study showed that the staining intensity of iii - tubulin was associated with the lymphatic metastasis in the non - invasive front group , suggesting the potential role of iii - tubulin in tumor invasion or metastasis . however , our study showed no significant association of iii - tubulin with tumor recurrence , metastasis , or survival . further research is needed to clarify the potential role of iii - tubulin in colorectal cancer and patient prognosis . therefore , only the patients with iii - tubulin expressed in the invasive front of tumor tissue were included in the invasive front group , and all remaining patients were included in the non - invasive front group . however , despite these limitations , our study did show the association of iii - tubulin expression with tumor differentiation and lymphatic metastasis in colorectal cancer . studies with larger samples are needed to further clarify the clinical implications of iii - tubulin expression at the invasive margin in colorectal cancer . our data showed the tissue distribution of class iii -tubulin expression at the invasive margin or diffuse distribution , and that iii - tubulin expression may be involved in tumor differentiation and metastasis . this study may shed light on iii - tubulin as a novel potential molecular target for new anti - cancer drugs .
backgroundclass iii -tubulin ( iii - tubulin ) has been reported to express at the invasive margin of colorectal cancer . 
 the present study aimed to investigate the clinical implication of iii - tubulin expression at the invasive margin of colorectal cancer.material/methodswe recruited 111 patients with surgically resected colorectal carcinoma for iii - tubulin expression analysis . 
 the cases with iii - tubulin - positive tumor cells found only in the invasive front tumor area were assigned to the invasive front group , while the remaining cases were all assigned to the non - invasive front group . 
 clinical analysis of iii - tubulin and other clinical data was performed.resultsthe positive staining rates and staining intensity of iii - tubulin were significantly different between the invasive and non - invasive front groups ( p=0.001 and p=0.006 ) , and there was a significant difference in tumor differentiation between the 2 groups ( p=0.032 ) . in the non - invasive front group , 
 staining intensity of iii - tubulin was significantly associated with positive staining rates and lymphatic metastasis ( p<0.001 and p=0.048).conclusionsour data showed the tissue distribution of iii - tubulin expression at invasive margin or diffuse distribution . 
 expression of iii - tubulin was correlated with tumor differentiation and lymphatic metastasis , suggesting a potential role of iii - tubulin in tumor differentiation and metastasis . 
 this study may shed light on iii - tubulin as a novel potential molecular target for a new anti - cancer drug .