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.

Figure 1

Gross image of cleared human pancreatic parenchyma. In this 5-mm–thick cleared section of pancreatic tissue, the refractive index matching reduces light scattering and produces cleared tissue that enables fluorescent microscopy of deeper structures. Original magnification, ×2.5.

Figure 2

Three-dimensional imaging of normal and neoplastic human pancreatic parenchyma. Sections of grossly normal and neoplastic human pancreas were immunolabeled with antibodies for cytokeratin 19 and then cleared. A: In the normal pancreas, the branching morphology at the edge of a lobule can be seen, including one of the ducts with looping (arrow). B: PanIN precursor lesions are identified in sections of grossly normal human pancreas—these lesions have intraluminal papillary projections of columnar epithelial cells. C: Imaging of grossly identified pancreatic ductal adenocarcinoma shows multiple patterns of invasion, including growth as invasive long and thin ductal structures. D: Analysis of pancreatic ductal adenocarcinoma shows a unique pattern of perivascular spread of malignant cells. The blood vessel passing from the upper left to the lower right is identifiable by its autofluorescence. In this tumor, invasive carcinoma grows in the perivascular space, parallel to the vessel. Scale bars: 200 μm (A, C, and D); 150 μm (B).

Supplemental Figure S1

Validation of the three-dimensional imaging of human pancreatic ductal adenocarcinoma and its relationship to blood vessels by hematoxylin and eosin (H&E) staining. After immunolabeling with antibodies for cytokeratin 19, clearing, and imaging, the tissue was formalin-fixed, paraffin-embedded and sectioned for routine H&E staining. A: Three-dimensional imaging with a Zeiss LSM800 confocal laser scanning microscope of human pancreatic ductal adenocarcinoma and its relationship to blood vessels. B: A 4-μm thick section of the same area, confirming the presence of malignant ducts in the interstitium around a blood vessel. Scale bars: 200 μm (A); 100 μm (B). Original magnification, ×5 (A).