Flow cytometry of primary cultured cells from our mouse medulloblastoma model was first used to screen for VEGFR-1 overexpression by tumor cells. The results revealed high overexpression of VEGFR-1 (10-fold to 100-fold) in a subpopulation of cells and led to the pursuit of IHC and DAC microscopy experiments to explore the feasibility of a VEGFR-1 probe for optical molecular imaging. The low percentage of cells (~5%) that stained positively for VEGFR-1 through flow cytometry may have been due to the presence of VEGFR-1-positive endothelial cells (GFP-negative) competing for the antibody probe or removal of surface proteins by papain. However, because the experimental conditions for this initial investigation differ significantly from the intended imaging application, additional steps were not taken to optimize the initial flow cytometry screening experiments.
IHC and DAC microscopy of fresh thick tissues were performed to determine if our VEGFR-1 probe could provide adequate molecular image contrast to differentiate between tumor and normal regions in intact tissues. It should be noted that, because our probes are intended for real-time topical application during neurosurgery, we limited our staining durations to a maximum of 10 to 15 minutes to mimic the challenging time constraints of a clinical setting. Other studies have previously demonstrated a similar rapid topical application approach in the literature [22–24
], with reported penetration depths as high as 1 mm [25
] with significant probe binding and/or activation in as little as 1 to 5 minutes [23,24
]. The fact that our VEGFR-1 probe performs well under these short incubation time constraints is a significant finding.
Results indicate that the VEGFR-1 probe is effective in differentiating between tumor and normal tissues at the margins. It is noted that while the VEGFR-1 probe exhibits a significantly higher tumor-to-normal intensity ratio than the isotype control probe (P
< .01), the tumor-to-normal ratios are elevated for both the specific and nonspecific probes. This may be due to an enhanced permeability and retention effect that is seen in many tumors [26
]. In our experience, ambiguity in image contrast during in vivo
molecular imaging can also be present due to nonspecific chemical binding, uneven probe distribution, and/or poor washout of unbound probes. These problems are exacerbated during in vivo
microscopy, because the small fields of view provided by a microscope often prevent one from visualizing differential contrast between tumor and normal tissue regions within a single image frame. By using a calibrated ratiometric imaging method, we are able to distinguish between molecularly specific and nonspecific probe binding and to quantify these levels at the cellular level, thus providing an unambiguous determination of tissue status within a single image. Recently, others have shown that this technique may be used to quantify the binding potential of tumor receptors following the systemic delivery of a targeted and an untargeted imaging agent [27
The ability to resolve individual cells with intraoperative confocal microscopy, while generally limited to a small field of view, may be of great value in cases where tumor cells are loosely distributed, such as at the margins of diffuse gliomas. In addition, certain probes, though targeted, may only label a sparse subset of tumor cells. For example, in recent years, fluorescence image-guided surgery with contrast provided by 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence has been demonstrated to improve outcomes for patients with high-grade gliomas (World Health Organization Grades III and IV) [5,9,28
]. Unfortunately, 5-ALA-induced protoporphyrin IX fluorescence is only generated in sparse proliferative cell populations and is undetectable in low-grade gliomas (World Health Organization Grades I and II) through wide-field techniques [29,30
]. However, it has recently been reported that intraoperative cellular resolution confocal microscopy can be used to visualize the sparse fluorescent cells in low-grade glioma patients treated with 5-ALA [9
]. Therefore, intraoperative confocal microscopy has the potential to serve as a valuable complement to wide-field techniques for the image-guided resection of certain tumors.
Future plans are to investigate the ability of the VEGFR-1 fluorescent probe, used in conjunction with a surgical DAC microscope, to enhance surgical resection in animal models. To achieve this goal, we are developing a miniature multicolor DAC microscope for in vivo ratiometric imaging of VEGFR-1 expression. Use of a transgenic mouse model allows for the minimization of experimental variability while optimizing these imaging technologies. However, our ultimate goal is to translate these synergistic technologies into the clinic for guiding brain tumor resection through the unambiguous real-time delineation of tumor margins in human patients.