Three-dimensional visualization of cleared human pancreas cancer reveals that sustained epithelial-to-mesenchymal transition is not required for venous invasion

Abstract

Venous invasion is three times more common in pancreatic cancer than it is in other major cancers of the gastrointestinal tract, and venous invasion may explain why pancreatic cancer is so deadly. To characterize the patterns of venous invasion in pancreatic cancer, 52 thick slabs (up to 5 mm) of tissue were harvested from 52 surgically resected human ductal adenocarcinomas, cleared with a modified iDISCO method, and labeled with fluorescent-conjugated antibodies to cytokeratin 19, desmin, CD31, p53 and/or e-cadherin. Labeled three-dimensional (3D) pancreas cancer tissues were visualized with confocal laser scanning or light sheet microscopy. Multiple foci of venous and even arterial invasion were visualized. Venous invasion was detected more often in 3D (88%, 30/34 cases) than in conventional 2D slide evaluation (75%, 25/34 cases, P < 0.001). 3D visualization revealed pancreatic cancer cells crossing the walls of veins at multiple points, often at points where preexisting capillary structures bridge the blood vessels. The neoplastic cells often retained a ductal morphology (cohesive cells forming tubes) as they progressed from a stromal to intravenous location. Although immunolabeling with antibodies to e-cadherin revealed focal loss of expression at the leading edges of the cancers, the neoplastic cells within veins expressed e-cadherin and formed well-oriented glands. We conclude that venous invasion is almost universal in pancreatic cancer, suggesting that even surgically resectable PDAC has access to the venous spaces and thus the ability to disseminate widely. Furthermore, we observe that sustained epithelial-mesenchymal transition is not required for venous invasion in pancreatic cancer.

Desmin and CD31 immunolabeling for detecting venous invasion of the pancreatobiliary tract cancers

“…Cancer cells formed tube-like structures (which can be interpreted as INlike patterns) and were found to grow within the venous wall. Single or clustered cancer cells were detached at the end of the tube-like growth of cancer cells (which can be interpreted as conventional pattern in the present study) [18]. When E-cadherin immunolabeling was performed, the neoplastic cells crossed the media of veins and formed tube-like structures within the venous wall with intact E-cadherin labeling in the large cohesive cluster of neoplastic cells, and loss of E-cadherin was observed only in the tips of small tongues of cells extending from these clusters [18].…”

“…Furthermore, the destructive pattern of VI disappeared after several additional consecutive cut sections. In addition, by 3-dimensional (3D) reconstruction of the cleared pancreatic cancer tissue, we previously observed that neoplastic cells crossed the media of the involved veins as cords and tube-like growths of cancer cells (which can be translated as destructive pattern in the present study) rather than individual cells crossing the media of veins [18]. Cancer cells formed tube-like structures (which can be interpreted as INlike patterns) and were found to grow within the venous wall.…”

“…When E-cadherin immunolabeling was performed, the neoplastic cells crossed the media of veins and formed tube-like structures within the venous wall with intact E-cadherin labeling in the large cohesive cluster of neoplastic cells, and loss of E-cadherin was observed only in the tips of small tongues of cells extending from these clusters [18]. Combining our present and previous observations, we hypothesized that these patterns may not be independent but may be related, and our hypothesized sequence of venous invasion patterns is depicted in Fig 9. Recent 3D imaging of VI of pancreas cancers revealed that neoplastic cells that covered the vascular endothelial spaces extended for a distance with a mean length of approximately 560 μm [18]. The length of conventional pattern might be short.…”

The genetics of ductal adenocarcinoma of the pancreas in the year 2020: dramatic progress, but far to go

“…Even if a perfect test were developed, it might never be possible to detect curable pancreatic cancers, as the window of opportunity for the detection of curable pancreatic cancer is likely extremely short in some patients. For example, several studies have shown that even early pancreatic cancers invade veins within the pancreas [202,217,218]. These veins drain directly into the liver, suggesting that early liver metastases may be almost universal in pancreatic cancer [202,217,218].…”

The Metastatic Cascade as the Basis for Liquid Biopsy Development

“…Indeed, Gkountela et al ( 53 ) demonstrated a specific methylation pattern that promotes expression of stemness-related genes, when CTCs are clustered, but they did not find any change in the methylation profile of EMT-related genes. Other studies also suggested that EMT might not have a role in CTC clusters ( 55 , 56 ).…”