Preliminary Study On The Effect And Mechanism Of HLA-DR Mediated Macrophages In Invasion And Metastasis Of Kazakh Esophageal Carcinoma

Part 1 The distribution of macrophages in Kazakh esophageal squamous cell carcinoma and its clinicopathological significanceObjective:To investigate distribution of different phenotype macrophages and the expression of metastasis related proteins in Kazakh esophageal squamous cell carcinoma(ESCCs)and its correlation with clinicopathological features and prognosis.Methods:(1)280 cases of ESCCs and cancer adjacent normal(CANs)paraffin embedded tissues were collected and tissue slices and tissue microarray were prepared.(2)CD68,HLA-DR double staining and CD 163 immunohistochemical SP method were used to detect the distribution of M1 and M2 macrophages in tumors and its relationship with the clinical data and prognosis of patients with Kazakh ESCCs.(3)CD34 and D2-40 were used to detect the distribution of microvessels and lymphatic vessels in ESCCs.Immunohistochemical method was used to detect the expression of HLA-DR,VEGF and MMP9,and its correlation with clinicopathological features.(4)The relationship between the distribution of macrophages,microvessels,lymphatic vessels,the expression of HLA-DR,VEGF and MMP-9 in Kazakh ESCCs was analyzed comprehensively.Results:(1)The macrophages of M1 and M2 macrophages were found in the tumor nests and perinatal tumor stroma of ESCCs.The density of macrophages in tumor stroma was significantly higher than that in tumor nests.The density of M1 and M2 macrophages in the ESCCs was significantly higher than that in the CANs(M1 macrophages in tumor nests 3.28�2.23 vs 1.04� 0.86,tumor stroma 11.67 � 5.8 vs 6.41 � 2.05;M2 macrophages in tumor nests 13.18 � 11.24 vs 1.73�1.73,tumor stroma 55.21 � 25.71 vs 20.90 � 9.60;P<0.001).The ratio of M2/M1 macrophages in Kazakh ESCCs was also significantly higher than that in CAN tissues(in tumor nests 4.96 � 4.47 vs 1.57 � 1.67;tumor stroma 6.44 � 5.81 vs 3.53 �1.75;P<0.001).(2)Combined with clinicopathological parameters,we found that the density of M1 macrophages in tumors was negatively correlated with lymph node metastasis of ESCCs(P<0.05).There was a significant positive correlation between M2 type macrophage density and M2/M1 ratio in the tumor nests,tumor stroma,and the vascular invasion,lymph node metastasis and clinical stage of ESCCs(P<0.05).It was also found that the ratio of M2/M1 macrophages was positively correlated with the depth of invasion of tumor(P<0.05).The high density M2 type macrophages and high M2/M1 ratio predicts poor prognosis in patients with Kazakh esophageal squamous cell carcinoma(P<0.05).It was also found that the density of M2 macrophages could be used as an independent prognostic factor for ESCCs.The higher the density,the worse the prognosis of patients with ESCCs.(3)Macrophages was one of the important HLA-DR expression cells.The expression of HLA-DR was negatively correlated with the depth of invasion,lymph node metastasis and clinical stage of Xinjiang Kazakh ESCCs(P<0.05).The distribution of microvessels and lymphatic vessels,and the expression of VEGF and MMP9 were significantly higher in ESCCs than those of cancer adjacent normal tissues(CANs),and which were close correlated with high invasion and clinical staging of tumors(all P<0.05).(4)Combined with the distribution of M2 macrophages in ESCCs,found the density of M2 macrophages in the stroma was negative correlated with HLA-DR expression,while the density of M2 macrophages in the stroma and the ratio of M2/M1 in nest and stroma were positively correlated with the microvessels and lymphatic vessels density,and the expression of VEGF and MMP9(P all<0.05).Conclusion:(1)The increased of M2 macrophages was positively correlated with the density of microvessels,lymphatic vessels and the vascular invasion,lymph node metastasis and clinical stage in Kazakh esophageal carcinoma tissues.M2 macrophages may be used as an important independent reference index for the invasion and prognosis of ESCCs.(2)The increased density of M2 macrophages is positively correlated with down-regulated of HLA-DR expression,the overexpression of VEGF and MMP9,which may be involved in the invasion and metastasis of ESCCs.Part 2 The effect HLA-mediated of macrophages on the biological behavior of esophageal carcinoma and its mechanism.Objective:To investigate the effect of HLA-mediated transformation of macrophage phenotype on the biological behavior of esophageal squamous cell carcinoma(ESCCs)and its mechanism.Methods:(1)Macrophages and ESCC cells were observed by non-contact co-culture.The phenotype of macrophages(HLA-DR/CD68,HLA-DR,TNF-a and IL-10)between non-coculture and co-culture were detected by immunofluorescence and real-time quantitative PCR(qRT-PCR).The morphological changes of cancer cells between non-coculture and co-culture were observed by inverted microscope.(2)The expression of VEGF and MMP9 was detected by qRT-PCR and westernblot in co-culture of macrophages.The proliferation,migration and invasion of cancer cells were detected by CCK8,colony cloning assay,Transwell migration and invasion assay separately.(3)The expression of HLA-DR in macrophages was down-regulated by lentiviral shRNA transfection.Immunoprecipitation,qRT-PCR and western blot were used to detect the changes of macrophage phenotype after transfection.qRT-PCR and westernblot were used to detect the expression changes of invasion and metastasis related genes VEGF and MMP9 in HLA-DR macrophages.The effects of HLA-DR macrophages on the proliferation,migration and invasion of esophageal squamous cell carcinoma were examined by CCK8 proliferation,Transwell migration and invasion assay.Results:(1)Immunofluorescence showed that the number of CD 163 positive macrophages(M2)was significantly increased and the number of macrophages in CD68/HLA-DR double staining(M1)was significantly decreased after co-culture with macrophages for 48 h.The expression of HLA-DR and TNF-a(M1 phenotype)was significantly decreased and IL-10(M2 phenotype)was significantly increased by qRT-PCR detected(P<0.05).Westernblot detection from the protein level also confirmed the co-culture of M2 phenotype macrophages increased(P<0.05).(2)After co-culture with macrophages,the morphology of most ESCC cells was changed from the characteristics of circular epithelial cells to the characteristics of spindle mesenchymal cells.The proliferation of cancer cells was significantly lower than that of the control group(P<0.05).The expression of MMP9 and VEGF in macrophages increased significantly after co-culture(P<0.05).Transwell migration and invasion experiments showed that the migration and invasion of ESCCs cells were significantly increased after co-culture(P<0.05).(3)After downregulated HLA-DR in macrophages,the expression of HLA-DR and TNF-a in macrophages was significantly decreased and the expression of IL-10 was significantly increased(P<0.05).Indicating that more macrophages to M2 phenotype of macrophages.(4)After co-culture with down-regulation of HLA-DR macrophages and ESCC cells,The expression of MMP-9 and VEGF in macrophages was significantly increased(P<0.05).And the proliferation of esophageal squamous cell carcinoma cells increased significantly,the migration and invasion of tumor cells were significantly increased(P<0.05).Conclusion:(1)Macrophages and esophageal cancer cells co-culture to promote the M2 type macrophage phenotype transformation and esophageal cancer cell invasion and metastasis,HLA-DR has the role of promoting the transformation of M2 macrophages(2)Interference with HLA-DR can affect the expression of VEGF and MMP9 and the proliferation,migration and invasion of esophageal cancer cells.The mechanism may be related to the transformation of HLA-DR to promote the transformation of M2 phenotype macrophages and increase the secretion of VEGF and MMP9.Molecular mechanism still need further in vivo and in vitro studies.