We have demonstrated the feasibility of using PGN635 F(ab′)2
labeled with the NIR dye, 800CW, for optical imaging of exposed PS in both subcutaneous and orthotopic mouse models of U87 glioma. Histologic studies showed that the PS signals detected in nonirradiated gliomas by in vivo
NIR imaging were due to exposed PS on tumor vascular endothelial cells. This is in good agreement with previous studies performed in various tumor models: prostate R3327-AT1 tumors [30
], breast cancer MDA-MB231, 4T1 cells [31
], lung cancer H460 and A549, and F98 gliomas [12,13
]. Irradiation-induced increase in exposed PS was successfully visualized by 800CW-PGN635 optical imaging in mice. Fluorescence microscopy confirmed that both PGN635 and 800CW-PGN635 localized more strongly to the irradiated tumors. In irradiated tumors, both the vessels and the tumor cells were PS positive. The antigen-binding specificity of PGN635 was verified by competition experiments with unconjugated PGN635 and by the lack of staining with the irrelevant control 800CW-Aurexis.
Noninvasive molecular imaging of apoptosis is of clinical importance because it could give an early indication of the responsiveness of a patient's tumor to therapy, allowing alterations in the therapy to be made if the responses were not as good as expected [5,6,20
]. In the past several years, much effort has been made in developing molecular imaging agents to monitor tumor responses. Radiolabeled and fluorescent annexin V preparations have been extensively tested in animal studies [21–23
]. More recently, clinical studies of PET or SPECT imaging with radiolabeled annexin V have shown that a higher uptake of annexin V by tumors during or after treatment correlates positively with a better prognosis in patients with breast, head and neck, or lung cancer [24–26
]. However, not all PS-expressing tumor cells in responding tumors are apoptotic. Hammill et al. [27
] found that many of the cells that stain positively for annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the therapy is discontinued. Their results indicate that exposed PS, and thus loss of membrane asymmetry, precedes commitment to apoptotic death.
PGN635, bavituximab, and related murine monoclonal antibodies recognize PS and other anionic phospholipids in a β2
GP1-dependent fashion. The antibodies have a more restricted specificity for PS than does annexin V, which recognizes PE in addition to PS and other anionic phospholipids [7,28,29
]. We previously radiolabeled bavituximab with 74
As, which is a long-lived positron emitter having a radioactive half-life of 17.8 days, and used it to image the vasculature of Dunning prostate R3227-AT1 tumors in rats. The long half-life of 74
As allowed the intact antibody to reach its optimal target to background selectivity at 72 hours without appreciable radioactive decay [30
]. Tumor-selective targeting was observed by PET imaging. A maximum tumor-to-liver ratio of 22 was achieved.
In the present study, we used the F(ab′)2
fragment of PGN635 for optical imaging to ensure its rapid clearance from the bloodstream and the achievement of high tumor to background ratios by 24 hours after injection. Because the target on vascular endothelial cells is directly accessible to the blood, PGN635 localizes rapidly to tumor vessels. We have previously demonstrated that exposed PS on tumor vessels in orthotopic gliomas is freely accessible to PS-targeting antibodies [12
]. Even tumor cells having exposed PS were labeled, probably because local disruption of blood brain barrier occurs in brain tumors. PGN635 is not internalized to any significant extent by cells and so remains for 2 to 3 days on the endothelial cell surface, giving plenty of time for the unbound PGN635 F(ab′)2
in the blood to be cleared. We found in the present study that PGN635 labels about 27% of the vessels in nonirradiated U87 gliomas. Different tumors vary in the percentage of their vessels that have exposed PS [7,8,12,13,30
]. In Dunning R3227-AT1 prostate tumors, 40% of vessels have exposed PS [30
]. In F98 gliomas, the percentage is only 11% [12
]. Exposed PS on viable endothelial cells is induced by hypoxia, acidity, and other stresses known to be present in the tumor microenvironment [7
]. Differences in PS positivity in different tumors probably relate to the levels of oxidative stresses in the tumor microenvironment [31
Irradiation of U87 tumors with 12 Gy increased the percentage of tumor vessels that had exposed PS from 27% to 64% and increased the TNR from 2.8 to 4.0. These findings accord with our earlier finding that irradiation of A549 NSCLC xenografts and F98 gliomas increase vascular expression of PS [12,13
]. We have previously applied fractionated radiation (2 Gy x 5) to treat H460 lung tumor xenografts. Similar to our current study, the fractionated radiation sufficiently induced PS exposure [13
]. Endothelial cells in tumors are highly sensitive to irradiation and expose PS after as little as 5 Gy. Our previous studies have shown that endothelial cells in tumors irradiated with 5 Gy seem to remain viable. They remain morphologically intact and lack markers of apoptosis for several days [13
]. In the present study, 12 Gy of irradiation also increased PS exposure on the tumor cells in U87 tumors, thus contributing to the increased localization of 800CW-PGN635 in irradiated tumors. Further studies are needed to determine whether the PS-expressing tumor cells are apoptotic or not.
Optical imaging is increasingly being used in preclinical cancer research [32,33
]. It is being used in particular to study cancer-specific markers and drug pharmacokinetics and to monitor drugs' effects in small animals [17,34,35
]. The attraction of the technique is that it is inexpensive, is simple to conduct, gives real-time results, and does not require the handling and disposal of radioactive isotopes. In the clinic, optical imaging by visualizing fluorescently labeled tumor cells has recently emerged as an attractive approach to facilitate identification of tumor margins or sentinel lymph node metastases [36,37
]. Several previous studies have demonstrated in vivo
optical imaging of apoptosis with annexin V labeled with Cy5.5, a red excitable dye [22
]. However, the NIR dye IRDye800CW seems to be superior to Cy5.5 for in vivo
]. NIR fluorescence penetrates more deeply into tissues because it has lower tissue absorption and scattering of light and causes relatively little autofluorescence. Our previous study showed that it is possible to image deep-seated orthotopic gliomas in mice with IRDye800-labeled 2-deoxyglucose [18
]. Here, we have demonstrated the ability of NIR optical imaging to detect PS translocation in both subcutaneous and orthotopic glioma models.
Clinical applications of optical imaging are currently limited to the detection of tumor margins or deposits during surgery, to the detection of superficial tumors, and to the detection of deep-seated tumors by endoscopy. The current study is also a proof-of-principle study, indicating the potential of this unique antibody for detecting deep-seated tumors using PET, SPECT, or MRI. Other labels besides optical dyes should be considered for attachment to PGN635 F(ab′)2 fragments. PGN635 is particularly impressive as a targeting ligand because of its high specificity, lack of uptake by the liver or any other organs, rapid acquisition by its vascular target, and its persistence on the vascular target for days. It is an excellent candidate, for example, for labeling with DOTA and 64Cu for PET or 111In for SPECT. It should have applications not only in tumor imaging but also in the imaging of thrombi or sites of ischemia for cardiovascular investigations. It will also be interesting to correlate PGN635 optical imaging data with functional MRI studies of apoptosis by diffusion-weighted MRI and vascular perfusion or permeability by dynamic susceptibility contrast MRI.
In summary, we have combined NIR optical imaging with PGN635, a novel monoclonal antibody that binds to PS, to monitor in vivo dynamics of exposed PS on mouse gliomas. We show that irradiation increases the PS signal, predictive of the response to therapy. The high tumor specificity of PGN635 in the present study underscores the prospects of using PGN635 and related antibodies to treat cancer in humans.