The major finding of this study is that PE becomes exposed on the luminal surface of the vascular endothelium of tumors. PE exposure is the most prominent in and around regions of hypoxia and is probably induced by oxidative stresses in the tumor microenvironment.
The PE-binding peptide duramycin tagged with a single molecule of L-biotin was used to assess the presence of PE on the cell surface. Biotinylation seems to have reduced the ability of duramycin to disrupt cell membranes because the hemolytic activity of DLB was four-fold lower than that of duramycin itself. This is likely attributable to the increased hydrophilic character imparted by the water-soluble biotin moiety. This change may explain why DLB stained cell surface structures but not intracellular membranes at the concentrations tested.
DLB staining indicated a fourfold to fivefold increase in exposure of PE on irradiated EC. DLB strongly stained membrane blebs that formed on the surface after irradiation. The pattern of DLB staining in irradiated ABAE closely resembled that of bavituximab, suggesting that both PE and PS preferentially localize to membrane patches and blebs [2
]. Unlike PS, basal levels of PE are exposed on normal (untreated) ABAE cells, either naturally or because ABAE cells do not readily enter a fully resting state in culture. These observations are consistent with data indicating that essentially all the membrane PS and ~80% of the membrane PE are restricted to the cells plasma membrane inner membrane leaflet [27
]. Increased PE exposure has also been shown at the cell surface along the cleavage furrow in Chinese hamster ovary cells during cytokinesis and in a subset of EC in the rat aorta [28,29
]. Although PS and PE are both externalized during apoptosis and in response to stress, PS is more highly restricted to the inner membrane leaflet in healthy cells.
FACS analysis revealed that stress conditions associated with the tumor microenvironment induced significant PE exposure when applied to in vitro
-cultivated EC. Tumors are known to contain high levels of ROS owing to a number of dysregulated metabolic processes. These include growth factor-mediated activation of mitochondria, inflammatory responses from tumor-infiltrating leukocytes, and activation/overexpression of enzymes such as NADPH-oxidase [30–32
]. Transient hypoxia in tumors can lead to ROS generation by this last mechanism (i.e., activation of NADPH-oxidase) [13
]. In addition to high ROS levels, the tumor microenvironment is also often characterized by low pH relative to normal tissues. Tumor cells can undergo high rates of glycolysis irrespective of oxygen tension (the Warburg effect) and generate lactic acid as a byproduct [33
]. We found that the fraction of ABAE that expressed PE at the cell surface after treatment with ROS (H2
), hypoxia, or acidic medium significantly increased (). These stresses also induced PS exposure. These observations suggest that ROS, hypoxia, and acidic pH, possibly in concert with other factors (e.g., inflammatory cytokines and thrombin), promote the exposure of PE and PS in tumors.
Without exception, increased PE exposure was found on the tumor endothelium in all the tumors examined. These models included syngeneic, human xenografts, subcutaneous, orthotopic, and transgenic tumors. This suggests that, much like PS, PE functions as a broad tumor marker common to many malignancies. The percentage of PE-positive vessels varied between models but corresponded to the fraction of PS-positive vessels (determined by bavituximab binding) for each type of tumor. Moreover, the exposure of PE and PS occurred on the same tumor vessels, suggesting that the same mechanism is responsible for the redistribution of both PE and PS. Tumor-bearing mice were coinjected with DLB and pimonidazole HCl to assess hypoxia in areas immediately surrounding PE-positive blood vessels. A general correlation between DLB binding and pimonidazole staining showed that PE-positive vessels were concentrated in hypoxic portions of the tumor. RM-9 tumors, which have abundant PE-positive vessels, were markedly hypoxic, whereas 4T1 tumors, which have relatively few PE-positive vessels, were largely not hypoxic. These results further indicate that hypoxia and other stresses in hypoxic tumor regions drive PE exposure on tumor vasculature.
Despite evidence from in vitro
experiments that normal EC contain basal levels of PE on their surface, DLB did not localize to endothelium in heart, lung, liver, spleen, stomach, intestine, muscle, fat, brain, or testis. This difference might be because EC grown in vitro
are activated because of sustained low levels of chronic stress that promote higher levels of exposed PE, whereas EC in normal tissues in vivo
attain true quiescence. The transport enzymes that maintain membrane lipid asymmetry have a higher affinity for PS, and its rate of PS transport is approximately 10-fold higher than its rate of PE transport [27,34
]. This could explain why stress has a more profound effect on the redistribution of PE.
The selective exposure of PE on tumor vascular endothelium suggested that it might serve as a marker for imaging tumor vasculature. Being luminally exposed and in direct contact with the blood, we reasoned that fluorescently labeled derivatives of duramycin would localize rapidly and specifically to tumor endothelium. To test this hypothesis, we labeled duramycin with a near-infrared dye, 800CW, and injected it into mice bearing RM-9 prostate tumors. The biodistribution of injected 800CW-DUR confirmed that the levels of exposed PE are indeed high in tumors. 800CW-DUR binding to RM-9 tumors allowed them to be clearly distinguished above background fluorescence, suggesting that PE-specific probes may be a powerful tool for tumor imaging. However, both 800CW-DUR and DLB showed high uptake in the kidneys, as observed by Zhao and Bugenhagen [19
] with 99m
Tc-duramycin. Immunohistochemical analysis revealed that DLB localized strongly to the distal kidney tubules and less strongly to the proximal kidney tubules. DLB also stained intertubular blood vessels between distal tubules, although it did not bind endothelium in larger renal vessels or the glomeruli. Other studies have shown that duramycin forms ion channels in artificial membranes and can disrupt mitochondrial membranes at concentrations greater than 5 µM [35,36
]. Cinnamycin has also been shown to be toxic and promote its own binding by inducing flipping of membrane lipids [37
]. It is possible that, as the kidneys filter DLB or 800CW-DUR from the blood, local concentrations of the drug in the collecting ducts and the associated vasculature increase to the extent that it disrupts membranes and exposes PE.
In conclusion, the data presented in this report demonstrate that PE becomes exposed on the luminal surface of tumor vascular endothelium. PE seems to be a broad marker found in a number of mouse models of solid malignancies including spontaneously developing tumors. We demonstrate that PE on tumor vasculature can be imaged with PE-targeting probes. These probes are small compared with PS-targeting antibodies and annexins, and clear rapidly from the bloodstream making them particularly suitable for imaging purposes. In addition, PE is more abundant than PS, giving the potential for stronger signals. Indeed, duramycin labeled with 99m
Tc is being developed for imaging exposed PE in cardiac ischemia [19
]. The results reported herein suggest that PE also has potential as a marker for imaging human malignancies.