Three-dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding.

Advances in tissue clearing and microscopy make it possible to study human diseases in 3-dimensions (3-D). High-grade tumor budding (TB) is known to be associated with poor prognosis in various cancers; however, little is known about the 3-D architecture of TB. Using tissue clearing, we analyzed the 3-D structure of TB 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. TB was evaluated in hematoxylin and eosin stained sections (2-D) using standard pathological criteria. Of the 31 cancers, 13 showed low-grade TB and 18 showed high-grade TB. First, 3-D 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 TB were predominantly composed of tubules that only focally form cords at the periphery. By contrast, adenocarcinomas with high-grade TB predominantly formed cords in both centers and peripheries of the tumors. Third, adenocarcinoma with low-grade TB was characterized by few short protrusions with few branches, while adenocarcinoma with high-grade TB 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 TB than in the protrusions of low-grade TB. Our findings suggest that tumor buds as seen in 2-D are, in fact, cross-sections of attenuated but contiguous protrusions extending from the main tumor. This article is protected by copyright. All rights reserved.