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Objective To investigate the expression of programmed death ligand 1 (PD-L1) in liver cancer tissues and its clinical significance. Methods The expression levels of PD-L1 in 110 liver cancer tissues, including 95 cases of hepatocellular carcinoma and 15 cases of cholangiocarcinoma were detected by using immunohistochemical staining method, and the relationship between PD-L1 expression and the clinicopathological characteristics of patients with hepatocellular carcinoma was analyzed. Results Immunohistochemistry results showed that the positive rate of PD-L1 in liver cancer tissues was 69.1％(76/110), and the positive rate of membrane and cytoplasm was 46.4％(51/110) and 22.7％(25/110), respectively. The positive rate of PD-L1 expression in hepatocellular carcinoma was higher than that in cholangiocarcinoma [78.9％ (75/95) vs. 6.7％ (1/15)], and the difference was statistically significant (χ2= 31.693, P< 0.01). The positive expression of PD-L1 was closely associated with the depth of invasion and TNM stage of hepatocellular carcinoma (bothχ2= 4.629, both P= 0.031), but there was no relationship with the patients' age, gender and pathological grade (all P> 0.05). Further analysis showed that protein expression localization of PD-L1 was closely related to the pathological grade in hepatocellular carcinoma (P=0.013). Conclusion The positive expression of PD-L1 in hepatocellular carcinoma is closely associated with depth of invasion and TNM stage, which provides a theoretical basis for the immunotherapy of liver cancer targeting PD-L1.
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Objective@#To investigate the expression of programmed death ligand 1 (PD-L1) in liver cancer tissues and its clinical significance.@*Methods@#The expression levels of PD-L1 in 110 liver cancer tissues, including 95 cases of hepatocellular carcinoma and 15 cases of cholangiocarcinoma were detected by using immunohistochemical staining method, and the relationship between PD-L1 expression and the clinicopathological characteristics of patients with hepatocellular carcinoma was analyzed.@*Results@#Immunohistochemistry results showed that the positive rate of PD-L1 in liver cancer tissues was 69.1% (76/110), and the positive rate of membrane and cytoplasm was 46.4% (51/110) and 22.7% (25/110), respectively. The positive rate of PD-L1 expression in hepatocellular carcinoma was higher than that in cholangiocarcinoma [78.9% (75/95) vs. 6.7% (1/15)], and the difference was statistically significant (χ 2 = 31.693, P < 0.01). The positive expression of PD-L1 was closely associated with the depth of invasion and TNM stage of hepatocellular carcinoma (both χ2 = 4.629, both P = 0.031), but there was no relationship with the patients'age, gender and pathological grade (all P > 0.05). Further analysis showed that protein expression localization of PD-L1 was closely related to the pathological grade in hepatocellular carcinoma (P = 0.013).@*Conclusion@#The positive expression of PD-L1 in hepatocellular carcinoma is closely associated with depth of invasion and TNM stage, which provides a theoretical basis for the immunotherapy of liver cancer targeting PD-L1.
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Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer. Methods Hepatic cancer stem cells were enriched by serum-free suspension culture. Immunofluores-cence,cisplatin resistance assay, Real -time qPCR, subcutaneous tumor formation in nude mice, and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies. Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues. The antigen was identified by mass spectrometry. Results MH-CC97L cells were able to form cell spheres in serum -free suspension culture and were labeled with PKH26 dye. Flow cytometry showed that the expression of CD90 + in MHCC97L spheroid cells was 3. 4 times higher than that in the parental cells. In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and in-hibitory rates were 54. 67% ,50. 33% ,45. 73% ,42. 26% ,39. 11% ,and 37. 63% ,respectively. The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells. The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10 + cells was significantly higher than those of MHCC97L 28C10 - cells. Flow cytometry showed that the ratio of 28C10 + in MHCC97L cells and its sphere cells were 7. 98% and 10. 7% ,respectively. The ratio of 28C10 + cells was increased by 1. 34 times. The in vitro globing ability and invasive ability of 28C10 + cells obtained by flow cytometry were significantly higher than those of 28C10 - cells. The results of CCK-8 assay showed that 28C10 + cells were resistance to cispla-tin in 28C10 - cells,which are 1. 96 g/ml and 1. 16 g/ml,respectively. Tumorigenic assay showed that 28C10 + cells were inoculated subcutaneously with 2×104 cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate. Another nude mouse that did not form a tumor had formed a lung metastasis(1/5). Immunohistochemistry showed that the target antigen posi-tive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72. 0% (77/107),while it was lowly expressed in adjacent tissues,and the difference was significant. Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α. Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.
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Objective To identify-and study a monoclonal antibody (McAb) against pancreatic cancer stem cell in vitro,as well as to provide candidate antibody-drug for cancer stem cell-targeted therapy of pancreatic cancer.Methods Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cell in PANC-1 cell line.Flow cytometry was used to detect the expression of CD24 and CD44 in PANC-1 cells and sphere cells,Immunofluorescence was used to detect the expression of CD24 and antigen recognized by 15D2.The effects of 15D2 on self-renewal,proliferation and chemosensitivity to gemcitabine of PANC-1 parent or sphere cells were identified by serum-free suspension culture and CCK-8 assay,Immunohistochemistry was applied to detect the level of antigen recognized by 15D2 in cancer and adjacent tissues.Results PANC-1 cells could survive,proliferate and form sphere cells in serum-free medium.The sphere-forming rate was (2.5±0.5) ％.The percentage of CD44+ CD24+ cells population in sphere cells increased by 11.4 folds compared to PANC-1 cells,in which single nearly 97 ％ CD24+ cells was CD44+ CD24+ cells.Therefore,CD24+ was selected for cancer stem cell marker in PANC-1 in this study.The two-color immunofluorescence assay showed that 15D2 could recognize cells which was also stained by CD24.In vitro functional experiments demonstrated that 15D2 significantly suppressed the sphere formation of PANC-1 cells,with the inhibitory rate being 30.4 ％.Meanwhile,the combination of 15D2 and gemcitabine can significantly attenuate the growth of PANC-1 sphere cells.The IC50 was 0.10 μmol/L in 15D2+gemeitabine group,and 0.39 μmol/L in mlgM+gemcitabine group,Immunohistochemical results showed that the antigen recognized by 15D2 was greatly expressed in about 76.9 ％ (11/13) human pancreatic cancer tissues and hardly detected in adjacent normal tissues (10.0 ％,1/10).Conclusion McAb 15D2 can inhibit self-renewal and drug-resistance of pancreatic cancer stem cell in PANC-1 cell line,and it might become a candidate drug for target pancreatic cancer stem cell treatment.
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Objective To evaluate the clinical value of autoantibody spectrum against ovarian cancer associated antigens combine CA125 in detecting and monitoring ovarian cancer. Methods Circulating IgG, IgM autoantibodies against ovarian cancer associated antigens which included TM4SF1, C1D,TIZ, OV-142,FXR1 and OV-189 were measured by indirect ELISA in serum from 126 patients with ovarian cancer (prior treatment), 42 patients with benign ovarian masses, 142 healthy women. Cut off value of IgG, IgM autoantibodies were determined by receive operating characteristic (ROC) curve. CA125 was measured in serum by immunoradiometric assay (IRMA). We evaluated the clinical value of combining multiple autoantibodies (autoantibody spectrum ), combining autoantibody spectrum with CA125 by binary logistic regresion. The positive ratio of autoantibody spectrum in serum (prior and post treatment ) of 24 synchronization patients with ovarian cancer was analyzed to evaluate the value in monitoring state of illness.Results Our data indicated that serum contains IgG, IgM autoantibodies against ovarian cancer associated antigens. The positive ratio of IgG autoantibodies in serum from ovarian cancer patients and cancer-free patients were 34. 1% - 47. 6% and 13.0% - 19. 0%, respectively ( P ＜ 0. 05 ). The positive ratio of IgM autoantibodies in serum from ovarian cancer patients and cancer-free patients were 39. 7% - 53.2% and 12. 0% -33.2%, respectively (P ＜0. 05). The positive ratio of IgG autoantibodies against FXR1 and IgM autoantibodies against TIZ,FXR1 and OV-189 in early stage ( Ⅰ - Ⅱ ) ovarian cancer(55.3% ,63.8%,61.7% and 66. 0% ) were significantly higher than those in advanced ( Ⅲ - Ⅳ )ovarian cancer( 34. 2%,39. 2% ,26. 6% ,45.6%; all P ＜ 0. 05 ). Combining five autoantibodies ( TM4SF1 IgG, TM4SF1 IgM, C1D IgG, FXR1 IgG and TIZ IgM ) showed significantly improved sensitivity (75.4%, P ＜ 0. 05 ), lower specificity (78. 3% ,P ＜ 0. 05 ) and similar accuracy (77. 1%, P ＞ 0. 05 ) in detecting ovarian cancer compared to those of CA125 (61.1% ,88.0% ,77. 1% ). But the autoantibody spectrum showed significantly improved sensitivity in classifying early stage (76. 6% ), compared to those of CA125 (51.1% ,P ＜ 0. 05 ).Combining autoantibody spectrum with CA125 showed significantly improved sensitivity ( 85.7% ), specificity (90. 8% )and accuracy (88.7%) in detecting ovarian cancer compared to those of autoantibody spectrum alone ( all P ＜ 0. 05 ), while CA125 ( 61.1%, P ＜ 0. 05; 88. 0%, P ＞ 0. 05; 77. 1%, P ＜ 0. 05 ). The positive ratio of combine the autoantibody spectrum with CA125 was significantly lower in 24 post-treatment serum (42%) compared to the pairing prior treatment serum ( 88%, P ＜ 0. 05 ). Conclusion Combining the autoantibody spectrum against ovarian cancer associated antigens with CA125 can improve sensitivity,specificity and accuracy in detecting early ovarian cancer and may be used to monitoring state of illness.
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Objective To investigate the value of autoantibody of breast cancer susceptibility 1 associated RING domain (BARD1) splice variant (OV-142) in detection of ovarian cancer.Methods We cloned OV-142 gene into plasmid pET-30b(+).The recombinant protein of OV-142 was expressed in pET30b(+) system and purified. The autoantibody of OV-142 was detected by indirect enzyme-linked immunosorbent assay (ELISA).Results We successfully constructed the recombinant plasmid of OV-142.The recombinant protein was expressed in pET-30b(+) system and purified.The purification rate of the recombinant protein was up to 90%.The relative amount of autoantibody of OV-142 detected by indirect ELISA was analyzed by receiver operating characteristic curve (ROC) and the cutoff value was determined.Combination of the autoantibody IgG of OV-142 and CA125 was analyzed by logistic regression. The sensitivity,specificity and accuracy was 71.4%,89.1%,and 81.9%,respectively,which were higher than IgG (41.3%,84.2%,66.8% ) and CA125( 61.1%,88.0%,77.1% ) when used alone each.Conclusions OV-142 is a splice variant of BARD1.It may be a potential immunotherapy target of ovarian cancer.Detection of autoantibody of OV-142 is a potent complementary tool of CA125 in ovarian cancerdiagnosis.
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<p><b>OBJECTIVE</b>To investigate the the expression and hypoxic regulation of vascular endothelial growth factor(VEGF) and matrix metalloproteinase-9.</p><p><b>METHODS</b>VEGF mRNA and MMP-9 mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR) in 43 esophageal carcinoma specimens including 18 para-tumorous esophageal tissues. The expression of VEGF protein and mean microvessel density (MVD) in 56 specimens were examined by immunohistochemical stain. The effect of hypoxia on VEGF and MMP-9 expression in esophageal cancer cell lines was quantitatively determined by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The VEGF expression in the tumorous tissue, being significantly correlated with MVD in the tumor, was remarkably higher than that in the para-tumorous tissue. VEGF and MVD expression in the tumor was significantly associated with stage and metastasis of esophageal carcinoma. The MMP-9 expression in the tumorous tissue, being uncorrelated with vessel count and clinicopathologic features in esophageal carcinoma, was significantly higher than that in the para-tumorous tissue. Hypoxia significantly increased the VEGF expression in esophageal cancer cell lines but did not affect the MMP-9 expression.</p><p><b>CONCLUSIONS</b>The expression of VEGF plays an important role in the angiogenesis and metastasis of esophageal cancer, which is regulated by hypoxia. VEGF may serve as a predictor of progression in esophageal carcinoma and a potential target for antiangiogenic therapy of esophageal carcinoma.</p>
Subject(s)
Female , Humans , Male , Middle Aged , Endothelial Growth Factors , Genetics , Esophageal Neoplasms , Metabolism , Gene Expression Regulation , Hypoxia , Lymphokines , Genetics , Matrix Metalloproteinase 9 , Genetics , Oxygen , Metabolism , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Objective To evaluate the expression of MAGE-1 gene in esophageal carcinoma and determine whether esophageal carcinoma patients should be eligible for MAGE-1 antigen-based vaccine therapies． Methods MAGE-1 mRNA expression in esophageal carcinoma was assessed by reverse transcription and polymerase chain reaction amplification． The PCR products were then digested by restriction endonucleases and inserted into the pET-30a(+) vector． The sequence of the inserted gene fragment was confirmed by DNA sequence analysis． Results Out of the 30 esophageal carcinomas studied, 43% of the esophageal carcinomas tissues expressed MAGE-1 gene and no expression was found in matched adjacent normal esophageal mucosae． The entire cDNA of the gene was cloned． Conclusion Owing to the high frequency of MAGE-1 gene expression in esophageal carcinoma and MAGE-1 antigen can be recognized by cytolytic T lymphocytes when presented by class-I HLA molecular, a large proportion of these patients might be suitable candidates for immune therapy involving tumor specific antigens encoded by MAGE-1 gene．