Pseudo-hemorrhagic region formation in pancreatic neuroendocrine tumors is a result of blood vessel dilation followed by endothelial cell detachment

Abstract

Aberrant blood vessel formation and hemorrhage may contribute to tumor progression and are potential targets in the treatment of several types of cancer. Pancreatic neuroendocrine tumors (PNETs) are highly vascularized, particularly when they are well-differentiated. However, the process of vascularization and endothelial cell detachment in PNETs is poorly understood. In the present study, 132 PNET clinical samples were examined and a special type of hemorrhagic region was observed in ~30% of the samples regardless of tumor subtype. These hemorrhagic regions were presented as blood-filled caverns with a smooth boundary and were unlined by endothelial cells. Based on the extensive endothelial cell detachment observed in the clinical samples, the formation process of these blood-filled caverns was hypothesized. Blood vessel dilation followed by detachment of endothelial cells from the surrounding tumor tissue was speculated. This was further supported using an INS-1 xenograft insulinoma model. As the formation process was distinct from the typical diffusive hemorrhage, it was named 'pseudo-hemorrhage'. Furthermore, it was demonstrated that epithelial (E-) cadherin and β-catenin were overexpressed in tumor cells surrounding these pseudo-hemorrhagic regions. Therefore, even though no statistically significant association of pseudo-hemorrhage with clinical features (metastasis or disease recurrence) was identified, the high levels of E-cadherin and β-catenin expression may suggest that a number of features of normal islet cells are retained.

Keywords: INS-1 cells; endothelial cells; epithelial cadherin; hemorrhage; pancreatic neuroendocrine tumor; β-catenin.

Figure 1.

Blood-filled caverns unlined by endothelial cells are observed in PNET clinical samples. Hematoxylin and eosin staining images of several PNET samples: (A and E) Insulinoma, (B and F) glucagonoma, (C and G) gastrinoma and (D and H) non-functional PNET. (E-H) Higher magnification of the boxed areas in (A-D). Scale bars for (A) to (D), 1 mm; and for (E) to (H), 50 µm. PNET, pancreatic neuroendocrine tumors.

Figure 2.

Pancreatic neuroendocrine tumors are characterized by endothelial cell detachment. (A-C) H&E staining of an insulinoma sample with blood-filled caverns unlined and lined by endothelial cells. (B and C) Higher magnification of blood-filled caverns presented in (A). a, Blood-filled cavern lined by endothelial cells; b, blood-filled cavern partially lined by endothelial cells; c, blood-filled cavern unlined by endothelial cells linked to normal vasculature (white arrows). (D and E) H&E staining of two different insulinoma samples with endothelial cells detached from the surrounding tumor cells. (F) H&E staining of a gastrinoma sample with endothelial cells detached from the surrounding tumor cells. The boxed area is presented at a 2-fold higher magnification in the lower left corner. Endothelial cells are indicated by arrows and the filamentous cell-cell connections are indicated by arrowheads. Scale bar, 25 µm. H&E, hematoxylin and eosin.

Figure 3.

Blood-filled caverns unlined by endothelial cells generated in an INS-1 xenograft insulinoma model. A total of 10⁶ INS-1 cells were subcutaneously injected into nude mice. Xenograft insulinoma tumors were harvested 2–3 weeks later. (A and B) An insulinoma tumor observed under a stereomicroscope. (A) The tumor has a bloody appearance and intact envelope. (B) Multiple hemorrhagic spots are observed under higher magnification. Scale bar, 500 µm. (C and D) Hematoxylin and eosin staining of paraffin slides of INS-1 tumors. (C) Numerous blood-filled caverns can be observed in the slide. (D) The blood-filled caverns are unlined by endothelial cells as presented in higher magnification. Scale bar, 50 µm.

Figure 4.

Blood-filled caverns unlined by endothelial cells in INS-1 tumors are distinct from vasculogenic mimicry. (A) PAS staining of INS-1 tumors. (B) CD31 staining of INS-1 tumors. PAS and CD31 staining was positive for normal blood vessels and negative for tumor cells lining the blood-filled caverns unlined by endothelial cells (marked by asterisks). Signals of endothelial cells are marked by black arrows. Certain blood-filled caverns were directly linked to normal vasculature (white arrows). (C) Laminin staining of INS-1 tumors. Laminin staining was negative in the internal face of the lining tumor cells. Only a fragment of laminin-positive staining was able to be observed in a specific blood-filled cavern (arrow). Scale bar, 50 µm. PAS, periodic acid-Schiff.

Figure 5.

Blood-filled caverns unlined by endothelial cells are generated from blood vessel dilation followed by endothelial detachment as demonstrated by Sirius red staining of INS-1 tumors. (A-C) Represent different sites in the same slide. (A) Certain endothelial cells were not connected (arrows) with adjacent tumor cells in dilated blood vessels or blood-filled caverns (marked as a). (B) Detached endothelial cells formed a cluster (arrows) in blood-filled caverns unlined by endothelial cells (marked as b). (C) In specific blood-filled caverns (marked as c), collagen deposition was observed in a particular site of the lakeside (arrowheads). Endothelial cell detachment (arrow) was also observed. Scale bar, 50 µm.

Figure 6.

Expression of epithelial markers in normal islets and INS-1 xenograft tumors. (A-F) Expression of E-cadherin (red) and insulin (green) examined by immunofluorescence. (A and B) Normal human pancreas, (C and D) rat pancreas and (E and F) INS-1 xenograft tumors. Insulin and DAPI (blue) staining serves as an indication of islets and insulinoma cells, respectively. (G-L) Expression of β-catenin (red) and insulin (green) examined by immunofluorescence. (G and H) Normal human pancreas, (I and J) rat pancreas and (K and L) INS-1 xenograft tumors. Insulin and DAPI (blue) staining serves as an indication of islets and insulinoma cells, respectively. Blood-filled caverns unlined by endothelial cells are indicated by asterisks in INS-1 tumors. Scale bar, 50 µm. E-cadherin, epithelial cadherin.