RT is a standard treatment for GBM as well as other brain and head and neck cancers. Until the development of commercially available small animal image-guided microirradiators like the SARRP, preclinical research in these disease sites was limited by small animal radiation techniques that lagged decades behind current clinical practice in the precise targeting of tumor volumes and the sparing of normal tissue [2,3
]. Previously, only superficial tumors could be targeted with any degree of precision using crude lead blocking to shield the surrounding normal tissue. The development of the SARRP has opened up new avenues for preclinical RT research in these disease sites. The novel stereotactic head restraint and fiducial marker targeting techniques that we describe in this article offer an improvement on this technology, with potential applications not only for preclinical brain irradiation studies but also for studies involving any disease sites of the head and neck.
The SARRP's onboard CT scanner is limited in its ability to resolve intracranial tumors for targeting purposes. While the bony anatomy of the skull is well defined, allowing for easy identification of landmarks, the xenografts themselves are not well demarcated (). Use of a contrast agent can somewhat improve delineation of tumors, but our early experimentation with this technique has so far been unsatisfying. To supplement the limitations of the onboard CT scanner to visualize brain tumors, we believe it is valuable to use MRI scans with long echo time/long repetition time using thin slices to determine the tumor's location and dimensions for accurate targeting of the tumor.
While MRI is very good at defining tumor location and dimensions, the absence of treatment planning software that allows CT/MRI fusion somewhat limits the utility of MRI. BLI, although it cannot characterize the tumor's dimensions with the accuracy of MRI, is useful because the area of maximal BLI, which can be readily identified and marked on the animal in real time, is a very good surrogate marker for the center of the tumor as defined by MRI as we have demonstrated in our experiments. Placement of a fiducial marker over the area of maximal BLI signal can serve to confirm the location of the appropriate isocenter immediately before delivering radiation treatments using an appropriately sized collimator selected on the basis of the pretreatment MRI. The development of an onboard bioluminescent imager for real-time imaging when using the SARRP would greatly improve the efficiency of treatment planning, tumor targeting, and treatment setup by obviating the need to place fiducial markers on the scalp corresponding to the area of highest BLI signal.
As an illustration of the many potential applications of our SARRP-based, image-guided RT delivery techniques using our mouse model of GBM, we performed a pilot study measuring the effect of stereotactic cranial RT to disrupt the GBM tumor BBB. Several preclinical and clinical studies have demonstrated that RT can cause focal disruption of the tumor BBB [12,13
]. In our GBM mouse model, we previously confirmed that cranial RT effectively disrupted the GBM tumor BBB by showing that RT resulted in significantly greater IgG extravasation into tumors compared with unirradiated tumors or irradiated normal brain tissue. The effects were durable, with increased extravasation of IgG in the tumor still detectable by 35 days after RT [11
]. Using in vivo
near-infrared fluorescent imaging to assess endothelial permeability, we found that focal radiation of the tumor resulted in significantly more extravasation of pegylated near-infrared contrast agent into the tumor compared with unirradiated control tumors of equal size, a finding that was confirmed on ex vivo
fluorescent imaging. This finding has implications for the use of targeted radiation to enhance delivery of cytotoxic agents that currently do not cross the tumor BBB in sufficient concentration to produce a therapeutic effect. To our knowledge, this pilot study is the first to use near-infrared in vivo
imaging using a pegylated fluorescent probe to assay tumor BBB permeability in response to a treatment intervention. We think this technique may be a promising avenue for additional research and validation because in vivo
fluorescent imaging using pegylated contrast agents is more cost-effective, is more rapid, and requires less expertise than do preclinical perfusion MRI studies such as dynamic contrast-enhanced MRI. A major limitation to the use of the in vivo
fluorescent probe technique is that repeat injections of the fluorescent probe cannot be performed at less than 10-day intervals because of the long half-life of the agent in the circulation.
Our method of stereotactic irradiation of brain tumors applied to our animal model system of GBM represents an improvement in the ability of investigators to deliver precise radiation to carefully defined targets in preclinical experiments. Future advances in the technology of small animal microirradiator imaging and treatment planning, including the ability not only to define an isocenter but also to contour tumor volumes accurately using onboard imaging, will result in further improvements. The future of clinically relevant preclinical irradiation studies has never looked brighter.