Three‐dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding

Abstract

Advances in tissue clearing and microscopy make it possible to study human diseases in three dimensions (3D). Highgrade 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 Ecadherin 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.

Tumor budding as a predictor for prognosis and therapeutic response in gastric cancer: A mini review

“…In recent years, tumor budding, a general clinicopathological feature of tumor aggressiveness, invasion, and poor prognosis, has attracted increasing attention. This pathologic phenomenon has also been observed in other tumors, such as extrahepatic cholangiocarcinoma (ECA) ( 2 ), pancreatic ductal carcinoma (PDC) ( 3 ), oral cancer ( 4 ), and cervical squamous cancer (ECSC) ( 5 ); thus, tumor budding is not unique to gastric cancer but is widespread among all tumors.…”

“…In 2020, Yoshizawa et al. ( 2 ) confirmed that high-grade tumor budding had more branch points than low-grade tumor budding (median, 26 vs. 20, p = 0.021) and longer mean protrusion length (median, 53.3 vs 32.1 μm, p < 0.001). Some scholars ( 10 ) have also proposed that tumor nodules within 1 mm of the tumor edge should not be regarded as tumor budding, because tumor nodules may be linked to tumor tissue in deeper sections and should be regarded as “discontinuous diffusion” of tumor tissues.…”

Optical tissue clearing associated with 3D imaging: application in preclinical and clinical studies

“…Nevertheless, the new advances in tissue clearing, antibody penetration, and microscopy offer their potential to obtain detailed 3D images of cancer tissue. For example, cleared formalin-fixed paraffin-embedded biopsy samples from pancreatic, extrahepatic, and prostate tumors were successfully cleared, obtaining detailed 3D images from the cancerous areas (van Royen et al 2016 ; Hong et al 2020 ; Yoshizawa et al 2020 ; Kolesová et al 2021 ) and a visual description of a fatty invasion from a cleared human diabetic pancreas (Tang et al 2018 ).…”

Tissue clearing and 3D reconstruction of digitized, serially sectioned slides provide novel insights into pancreatic cancer

“…First, the connectivity of cancer cells is lost. Cancers that appear to be separate islands of neoplastic ducts embedded in stroma in 2D, are, in fact, often contiguous branching tubes of cancer cells cut in cross section (Yoshizawa et al, 2020). Second, the prevalence of a feature, such as vascular invasion, will be underestimated as focal microscopic features are unlikely to be captured in the minuscule fraction of the cancer sampled.…”