Openings between defective endothelial cells explain tumor vessel leakiness

Abstract

Leakiness of blood vessels in tumors may contribute to disease progression and is key to certain forms of cancer therapy, but the structural basis of the leakiness is unclear. We sought to determine whether endothelial gaps or transcellular holes, similar to those found in leaky vessels in inflammation, could explain the leakiness of tumor vessels. Blood vessels in MCa-IV mouse mammary carcinomas, which are known to be unusually leaky (functional pore size 1.2-2 microm), were compared to vessels in three less leaky tumors and normal mammary glands. Vessels were identified by their binding of intravascularly injected fluorescent cationic liposomes and Lycopersicon esculentum lectin and by CD31 (PECAM) immunoreactivity. The luminal surface of vessels in all four tumors had a defective endothelial monolayer as revealed by scanning electron microscopy. In MCa-IV tumors, 14% of the vessel surface was lined by poorly connected, overlapping cells. The most superficial lining cells, like endothelial cells, had CD31 immunoreactivity and fenestrae with diaphragms, but they had a branched phenotype with cytoplasmic projections as long as 50 microm. Some branched cells were separated by intercellular openings (mean diameter 1.7 microm; range, 0.3-4.7 microm). Transcellular holes (mean diameter 0.6 microm) were also present but were only 8% as numerous as intercellular openings. Some CD31-positive cells protruded into the vessel lumen; others sprouted into perivascular tumor tissue. Tumors in RIP-Tag2 mice had, in addition, tumor cell-lined lakes of extravasated erythrocytes. We conclude that some tumor vessels have a defective cellular lining composed of disorganized, loosely connected, branched, overlapping or sprouting endothelial cells. Openings between these cells contribute to tumor vessel leakiness and may permit access of macromolecular therapeutic agents to tumor cells.

Figure 1.

Diverse size and morphology of blood vessels in MCa-IV tumors. A: Hematoxylin-and-eosin-stained 2-μm methacrylate section showing irregularly shaped blood vessels (arrowheads) emptied of blood by vascular perfusion and surrounded by sleeves of tumor cells (arrows) interspersed by necrotic tissue (*). Region in box is shown at higher magnification in B. B: Irregular luminal lining layer (arrowheads) of vessel surrounded by tumor cells. Necrotic tissue is present at the bottom (*). C: Scanning EM view of 100-μm Vibratome section of a subcutaneous MCa-IV tumor with skin at the top. Blood vessels appear as black holes (arrows). Necrotic tissue is pale (*). D: FITC-labeled L. esculentum lectin (green) marks blood vessels in 100-μm section of tumor with overlying skin at the top. Extravasation of lectin, which makes vessel borders appear fuzzy (arrowheads), is most conspicuous in the tumor’s highly vascular capsule (arrows). E: Fluorescent cationic liposomes (red) mark the same blood vessels as those labeled with lectin in D. Vessel borders (arrowheads) are more clearly defined than with the lectin because of the lack of extravasation. Capsule, arrows. F: Brightfield image showing CD31 immunoreactivity in 100-μm section of tumor. Both blood vessels (arrows) and tiny sprouts (arrowheads) have CD31 immunoreactivity. Densely vascular capsule (double arrows). G and H: Region of tumor with a wide range of vessel size (arrows) shown by green lectin colocalized with red cationic liposomes. I: Tumor with CD31-immunofluorescent blood vessels (arrows), tiny sprouts (arrowheads), and densely vascular capsule (double arrows). Necrotic region (*) has nonspecific fluorescence. Scale bar in I applies to all figures; bar length represents 300 μm in A, D, E, and G–I; 80 μm in B; and 400 μm in C and F.

Figure 2.

Bar graph showing size distribution of vessels in MCa-IV tumors. Diameters of 50 blood vessel profiles were measured in hematoxylin-and-eosin-stained, 2-μm methacrylate sections of each of 6 tumors.

Figure 3.

Blood vessels in MCa-IV tumors marked by green fluorescent lectin staining (A, D, and G) or CD31 immunoreactivity viewed by Cy3 fluorescence (B, E, and H) and ABC-DAB histochemistry (F and I) in 100-μm Vibratome sections. Necrotic tissue (*). A and B: Like the lectin, CD31 immunoreactivity defines the luminal surface of tumor vessels, but, unlike the lectin, it is also present on sprouts (arrows) radiating from the vessel lining into the tumor. C: Scanning EM showing lumenless sprouts (arrowheads), similar to those in A and B, radiating into sleeves of tumor tissue (arrows). Necrotic tissue surrounds the tumor sleeves. D and E: Tiny sprouts (arrows), which apparently have no lumen because they have CD31 immunoreactivity but no lectin staining, are about 1 μm in diameter and interconnect tumor vessels. F: CD31-immunoreactive sprouts (arrows), made visible by ABC-DAB histochemistry, extend from the vessel surface into the tumor. G and H: Lumenless CD31-immunoreactive sprouts (arrows) without lectin staining penetrate the sleeve of tumor tissue. I: CD31-immunoreactive sprouts (arrows) radiate from the perimeter of a vessel into the tumor. Scale bar in I applies to all figures; bar length represents 150 μm in A, B, D, E, G, and H; 100 μm in C; 75 μm in F and I.

Figure 4.

Scanning EM view of luminal surface of normal endothelial cells in mammary gland compared to a range of abnormalities in vessel lining cells in MCa-IV tumors. A: Endothelial cells in this normal venule have a relatively uniform size and shape and are flat except for the region of the nucleus (arrows). Cells form a monolayer, and cell borders (arrowheads) have little overlap. B: Unbranched endothelial cells in a tumor vessel are irregularly shaped and overlap one another (arrows). Some cell borders are clearly visible (arrowheads); others are not. C and D: More severely deformed, branched endothelial cells in tumor vessels. These cells overlap one another, are abnormally thick, have multiple cell projections (arrows), and do not have normal connections with other cells. E: Luminal surface of a tumor vessel showing branched lining cells with extensive cellular overlap, bridges and tunnels (arrows), and cellular projections as long as 50 μm (double arrows). F: Abnormal lining cells that partition (arrowheads) the lumen of a tumor vessel. Scale bar in F applies to all figures; bar length represents 10 μm in A−D and 15 μm in E and F.

Figure 5.

A−F: Blood vessel lining cells (arrows) that protrude into the lumen of vessels in MCa-IV tumors. Vasculature emptied of blood by perfusion of fixative. A: Protruding cells in a large, thin-walled vessel in a toluidine blue-stained 0.5-μm epoxy section photographed at low magnification. B and C: Protruding cells (arrow) in a tumor vessel (black lumen) labeled with FITC-lectin (green) and rhodamine-cationic liposomes (red). D: Scanning EM view of a vessel with lining cells that protrude into the lumen as in A−C. Extensive cellular overlap, numerous cytoplasmic projections (arrows), and openings between cells (arrowheads) are present. White box marks region shown at higher magnification in Figure 7, B and C ▶ . E: Lining cell with a thread-like process that spans the vessel lumen. F: Overlapping lining cells in tumor vessel. One region (*) of the cell marked 1 is located above cell 2, which in turn is superficial to cells 3 and 4, but another region (**) of cell 1 is beneath cells 2 and 3. Projections from cell 3 appear to penetrate cells 1 and 4 (arrows). Inset shows outline of endothelial cell borders. G: Lining cells with numerous transluminal cytoplasmic processes (arrows) in a blood vessel of a Shionogi male mammary carcinoma. H: Openings (arrowheads) and other defects in the vessel lining of a LS174T human colon carcinoma grown in a SCID mouse. I: Openings (arrowheads) and other defects in the vessel lining of an islet cell tumor in a transgenic RIP-Tag2 mouse. Scale bar in I applies to all figures; bar length represents 25 μm in A and G−I; 50 μm in B and C; 20 μm in D; 10 μm in E; 5 μm in F.

Figure 6.

Blood lakes in pancreatic islet cell tumors in transgenic RIP-Tag2 mice. Brightfield (A) and fluorescence (B) micrographs showing blood lakes (arrows) in a whole mount of a small (∼1 mm) RIP-Tag2 tumor. In A, red blood lakes stand out from the remainder of the tumor washed free of blood by vascular perfusion of fixative. In B, blood lakes appear black, whereas blood vessels are marked by green fluorescent lectin and orange-red fluorescent cationic liposomes injected intravenously before fixation. Cationic liposome fluorescence predominates in the tumor; lectin fluorescence predominates in the surrounding normal acinar pancreas. C: Histological section of RIP-Tag tumor showing blood lakes. Also shown are tumor vessels (*), which are much smaller than the lakes and were emptied of blood by perfusion of fixative. Hematoxylin-and-eosin-stained 2-μm methacrylate section. D and E: Transmission EM of blood lakes showing erythrocytes (RBC) next to tumor cells. D illustrates the proximity of RBC and tumors (arrowheads). F and G: Transmission EM showing endothelial cells (arrows) of tumor blood vessels, emptied of blood by perfusion fixation, surrounded by extravasated erythrocytes (RBC). In G, the blood vessel and cluster of tumor cells are enveloped by multiple layers of basement membrane (arrowheads). At this site, an erythrocyte and tumor cells are separated by basement membrane (apposing arrowheads). Scale bar in G applies to all figures; bar length represents 150 μm in A and B; 30 μm in C; 3 μm in D and F; 1 μm in E and G.

Figure 7.

A−C: Scanning EM comparison of blood vessel (A) and extravascular blood lakes (B and C) in pancreatic islet cell tumors of transgenic RIP-Tag2 mice. A: Tumor vessel, emptied of blood by perfusion of fixative, has a well defined lining layer (arrowheads) with scattered defects (arrows). B and C: Blood lakes, which contain extravasated erythrocytes, lined by tumor cells (arrowheads). Few of the erythrocytes remain in C, showing the tumor surface and multiple holes between the tumor cells (arrows). D−G: Transmission EM view of abnormal lining cells in MCa-IV tumor vessels. The vessel lumen is at the top of each figure. D: Multiple layers of loosely connected cells (arrows 1–3) line a tumor vessel. E: Tall, rounded cells (arrows) with large nuclei, morphologically quite different from those in D, form the lining of this tumor vessel. One cell (arrowhead) is connected to others by a stalk. F: Three lining cells (arrows 1–3) are layered on top of one another in the wall of a tumor vessel. Parts of each are in contact with the vessel lumen. The abluminal surface of cell 3 is bordered by dense extracellular matrix (arrowheads). G: Basement membrane (arrowheads) of variable thickness on the abluminal surface of cells (arrows) lining a tumor vessel as well as on the surface of multiple cells beneath. Scale bar in G applies to all figures; bar length represents 15 μm in A and C; 20 μm in B; 4 μm in D−F; 2 μm in G.

Figure 8.

Potential pathways for leakage from blood vessels in MCa-IV tumors. A: Three intercellular openings (arrows) between lining cells of a tumor vessel viewed by scanning EM. Basement membrane is visible through the largest opening (double arrows). A large hole through one lining cell exposes the plasma membrane of an underlying cell (*). B and C: Multiple intercellular openings (arrows) and three transcellular holes (arrowheads) in branched lining cells of a tumor vessel. The intercellular openings are much larger than the holes. Region in box in B is shown at higher magnification in C. D: Intercellular opening (between arrows) in lining of a tumor vessel viewed by transmission EM. E and F: Fenestrae (arrowheads) in lining cells of MCa-IV blood vessels as seen by transmission EM (E), which reveals the fenestral diaphragms, and scanning EM (F), which shows clusters of fenestrae in a thin region of a horizontally oriented cytoplasmic projection (arrows). Scale bar in F applies to all figures; bar length represents 2.5 μm in A and C; 10 μm in B; 2 μm in D; 0.5 μm in E; 0.75 μm in F.

Figure 9.

Bar graph showing size distribution of 100 intercellular openings and 8 transcellular holes found in a sample of 700 lining cells in blood vessels of MCa-IV tumors viewed by scanning EM. Diameters were calculated from area measurements of the openings with the assumption that they were circular. Transcellular holes in the sample were treated similarly.