Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA.
Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
J Pathol. 2020 Aug;251(4):400-410. doi: 10.1002/path.5474. Epub 2020 Jul 3.
Advances in tissue clearing and microscopy make it possible to study human diseases in three dimensions (3D). High-grade tumor budding is known to be associated with poor prognosis in various cancers; however, little is known about the 3D architecture of tumor budding. Using tissue clearing, we analyzed the 3D structure of tumor budding and E-cadherin expression in 31 extrahepatic cholangiocarcinomas. A total of 31 thick slabs (up to 5 mm) were harvested from surgically resected tumor tissue, including 27 hilar and 4 distal cholangiocarcinomas. Twenty-eight cases were adenocarcinoma, and three were undifferentiated carcinoma. After clearing, the tissues were immunolabeled with antibodies to cytokeratin 19 and to E-cadherin, and then visualized using light-sheet and confocal laser scanning microscopy. Tumor budding was evaluated in hematoxylin and eosin-stained sections (2D) using standard pathological criteria. Of the 31 cancers, 13 showed low-grade tumor budding and 18 showed high-grade tumor budding. First, 3D analysis revealed that the neoplastic cells in tumor buds of adenocarcinoma were typically not individual islands of cells, but rather tips of attenuated protrusions connected to the main tumor. Second, adenocarcinomas with low-grade tumor budding were composed predominantly of tubules that only focally form cords at the periphery. By contrast, adenocarcinomas with high-grade tumor budding predominantly formed cords in both centers and peripheries of the tumors. Third, adenocarcinoma with low-grade tumor budding was characterized by a few short protrusions with few branches, whereas adenocarcinoma with high-grade tumor budding was characterized by longer protrusions with more branching. Finally, immunolabeling of E-cadherin was stronger in the center of the adenocarcinoma but decreased at the tips of protrusions. E-cadherin loss was more extensive in the protrusions of high-grade tumor budding than in the protrusions of low-grade tumor budding. Our findings suggest that tumor buds as seen in 2D are, in fact, cross-sections of attenuated but contiguous protrusions extending from the main tumor. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
组织透明化和显微镜技术的进步使得在三维（3D）水平研究人类疾病成为可能。已知高级肿瘤芽与各种癌症的不良预后相关；然而，关于肿瘤芽的 3D 结构知之甚少。我们使用组织透明化技术分析了 31 例肝外胆管癌中肿瘤芽和 E-钙黏蛋白的 3D 结构。从手术切除的肿瘤组织中采集了 31 个厚切片（最多 5 毫米），包括 27 例肝门部胆管癌和 4 例远端胆管癌。28 例为腺癌，3 例为未分化癌。透明化后，用抗细胞角蛋白 19 和 E-钙黏蛋白的抗体对组织进行免疫标记，然后用光片和共聚焦激光扫描显微镜进行可视化。使用标准病理标准在苏木精和伊红染色切片（2D）中评估肿瘤芽。在 31 例癌症中，13 例为低级别肿瘤芽，18 例为高级别肿瘤芽。首先，3D 分析显示，腺癌中肿瘤芽的肿瘤细胞通常不是单个细胞岛，而是与主肿瘤相连的变薄突起的尖端。其次，低级别肿瘤芽的腺癌主要由管腔组成，仅在周边偶尔形成索状。相比之下，高级别肿瘤芽的腺癌主要在肿瘤的中心和周边形成索状。第三，低级别肿瘤芽的腺癌的特征是短突起和分支较少，而高级别肿瘤芽的腺癌的特征是更长的突起和更多的分支。最后，E-钙黏蛋白的免疫标记在腺癌的中心更强，但在突起的尖端减弱。高级别肿瘤芽的突起中的 E-钙黏蛋白丢失比低级别肿瘤芽的突起更广泛。我们的研究结果表明，在 2D 中观察到的肿瘤芽实际上是从主肿瘤延伸出来的、变细但连续的突起的横截面。
