Dong X F, Zhong J T, Liu T Q, Chen Y Y, Tang Y T, Yang J R
Department of Hepatobiliary, Pancreas and Spleen Surgery, the People's Hospital of Guangxi Zhuang Autonomous Region,Nanning 530021,China.
Department of Hepatobiliary Surgery,Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, China.
Zhonghua Yi Xue Za Zhi. 2021 Mar 9;101(9):654-660. doi: 10.3760/cma.j.cn112137-20200605-01780.
To investigate the molecular mechanism of nest metastasis in blood vessels encapsulated by tumor clusters (VETC) positive hepatocellular carcinoma (HCC). A total of 72 paraffin embedded HCC tissue samples were collected. Immunohistochemistry staining with CD34 (vascular endothelial cell marker protein) was used to observe the morphological manifestations of VETC cancer nests in primary tumors, bile duct cancerous thrombi and portal vein cancerous thrombi, and to study the characteristics of hematogenous metastasis of VETC cancer nests. Bioinformatics was used to predict the key proteins closely related to VETC cancer nest formation. Immunohistochemistry was used to detect the expression of angiogenin-2 (Ang-2), integrin α5, Integrin β1, and cyclooxygenase-2 (COX-2) proteins in HCC. Transwell cell migration assay was used to detect the effect of Ang-2/integrin α5β1 protein on the migration ability of endothelial cells and HCC cells. Western blotting was used to detect the effect of Ang-2/integrin α5β1 protein on the activity of focal adhesion kinase (FAK) protein. Of the collected HCC specimens, 27 cases (27/72) were VETC (+), including 3 cases with biliary duct cancerous thrombus, 5 cases with portal vein cancerous thrombus, and 3 cases with both biliary duct cancerous thrombus and portal vein cancerous thrombus. VETC (+) HCC could metastasize to portal vein, bile duct, and liver in the form of cancer nest, and the nests retain their intact structure. Ang-2, integrin α5 and integrin β1 were overexpressed in tumor cells and endothelial cells of VETC (+) HCC nests, while COX-2 was only overexpressed in tumor cells of VETC (+) HCC nest. Ang-2 could promote the migration of HCC cell [(121±12) vs (186±11), <0.01] and endothelial cells [(81±7) vs (163±14), <0.01]. Integrin α5β1 activation antagonist ATN-161 could significantly block the ability of Ang-2 to promote the migration of HCC cells [(185±10) vs (135±9), <0.05] and endothelial cells [(156±14) vs (103±6), <0.05]. ATN-161 could significantly block the phosphorylation of FAK in HCC and endothelial cells induced by Ang-2. VETC (+) HCC could metastasize as a whole in a nested form, and possesses a specific regulatory protein. Ang-2/α5β1/FAK might be potential protein targets in the treatment of VETC (+) HCC nest-type metastasis.
探讨肿瘤巢包裹血管（VETC）阳性肝细胞癌（HCC）发生巢状转移的分子机制。共收集72例石蜡包埋的HCC组织样本。采用CD34（血管内皮细胞标记蛋白）免疫组化染色观察原发性肿瘤、胆管癌栓及门静脉癌栓中VETC癌巢的形态学表现，研究VETC癌巢血行转移的特点。利用生物信息学预测与VETC癌巢形成密切相关的关键蛋白。采用免疫组化检测HCC中血管生成素-2（Ang-2）、整合素α5、整合素β1和环氧化酶-2（COX-2）蛋白的表达。采用Transwell细胞迁移实验检测Ang-2/整合素α5β1蛋白对内皮细胞和HCC细胞迁移能力的影响。采用蛋白质免疫印迹法检测Ang-2/整合素α5β1蛋白对局灶黏附激酶（FAK）蛋白活性的影响。在收集的HCC标本中，27例（27/72）为VETC（+），其中3例有胆管癌栓，5例有门静脉癌栓，3例既有胆管癌栓又有门静脉癌栓。VETC（+）HCC可呈癌巢形式转移至门静脉、胆管和肝脏，且癌巢结构完整。Ang-2、整合素α5和整合素β在VETC（+）HCC癌巢的肿瘤细胞和内皮细胞中高表达，而COX-2仅在VETC（+）HCC癌巢的肿瘤细胞中高表达。Ang-2可促进HCC细胞[（121±12）对（186±11），<0.01]和内皮细胞[（81±7）对（163±14），<0.01]的迁移。整合素α5β1激活拮抗剂ATN-161可显著阻断Ang-2促进HCC细胞[（185±10）对（135±9），<0.05]和内皮细胞[（156±14）对（103±6），<0.05]迁移的能力。ATN-161可显著阻断Ang-2诱导的HCC和内皮细胞中FAK的磷酸化。VETC（+）HCC可呈巢状整体转移，并具有特定的调节蛋白。Ang-2/α5β1/FAK可能是治疗VETC（+）HCC巢状转移的潜在蛋白靶点。
