Immunolabeling of Cleared Human Pancreata Provides Insights into Three-Dimensional Pancreatic Anatomy and Pathology

Abstract

Visualizing pathologies in three dimensions can provide unique insights into the biology of human diseases. A rapid and easy-to-implement dibenzyl ether-based technique was used to clear thick sections of surgically resected human pancreatic parenchyma. Protocols were applicable to both fresh and formalin-fixed, paraffin-embedded tissue. The penetration of antibodies into dense pancreatic parenchyma was optimized using both gradually increasing antibody concentrations and centrifugal flow. Immunolabeling with antibodies against cytokeratin 19 was visualized using both light sheet and confocal laser scanning microscopy. The technique was applied successfully to 26 sections of pancreas, providing three-dimensional (3D) images of normal pancreatic tissue, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and infiltrating pancreatic ductal adenocarcinomas. 3D visualization highlighted processes that are hard to conceptualize in two dimensions, such as invasive carcinoma growing into what appeared to be pre-existing pancreatic ducts and within venules, and the tracking of long cords of neoplastic cells parallel to blood vessels. Expanding this technique to formalin-fixed, paraffin-embedded tissue opens pathology archives to 3D visualization of unique biosamples and rare diseases. The application of immunolabeling and clearing to human pancreatic parenchyma provides detailed visualization of normal pancreatic anatomy, and can be used to characterize the 3D architecture of diseases including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and pancreatic ductal adenocarcinomas.

The importance of circulating and disseminated tumor cells in pancreatic cancer

“…In the Johns Hopkins series of patients with resected PDAC, 78% exhibit regional lymph node metastases, 85% have perineural sheath invasion, and 64% have microvascular invasion (unpublished results). The ability of these cells to invade the microvasculature is nicely demonstrated in this clear mount photomicrograph published by Hruban ( Fig 2 ) [ 26 , 27 ].

Fig 2 Invasion of microvasculature and pancreatic cancer: This figure depicts a “clear mount,” in which the green-labeled cancer cells can be seen invading a small vessel.

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Three‐dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding

“…Ten fresh cholangiocarcinoma tissues were processed as described previously [29,30]. In brief, the fresh tissues were fixed in 80% methanol and 20% dimethyl sulfoxide (DMSO).…”

“…Recently, advances in tissue clearing, antibody penetration, and microscopy offer the potential to obtain detailed 3D images of cancer tissue. We have previously demonstrated the utility of this new technique in obtaining 3D images of invasive pancreatic cancers [29,30]. Here, using clearing, we analyzed the 3D architecture of extrahepatic cholangiocarcinoma and TB, and we couple this with an analysis of the patterns of E-cadherin expression, a marker of EMT.…”

Improving Outcomes in Pancreatic Cancer

“…[21][22][23] In addition, based on 3D models, pancreatic cancer appears to metastasize early, often before it is even diagnosed. 24 This may be based on simple pathophysiology, as early-stage pancreatic cancer invades its surrounding vasculature, draining into the hepatic circulation, and thus results in early metastatic spread. 11 There is no standard diagnostic tool or method for early detection of pancreatic cancer, and the benefit of early detection may be questionable.…”