Completeness of resection has been correlated with improved survival in studies of various brain tumor populations [1,2
]. Accurate brain tumor delineation is a challenging task that is crucial to improving the degree of resection while preserving normal tissues. Over the years, a variety of optical agents have been employed to improve this process. The low-molecular-weight fluorochrome fluorescein was employed more than 50 years ago to assess whether it could help differentiate gliomas from normal brain during biopsy [3
]. More recently, indocyanine green was evaluated for its ability to enhance tumor boundaries [4,5
]. Although these fluorochromes provide intraoperative fluorescence in the region of the tumor, they have not been widely accepted as providing accurate margin delineation. These small molecules have limited circulation time and readily diffuse into and out of the interstitial space. A similar pharmacokinetic pattern is obtained with low-molecular-weight gadolinium chelates, which are administered minutes before contrast-enhanced magnetic resonance imaging (MRI) is obtained and do not remain in the same distribution over the hours required for tumor resection. Washout and diffusion of such agents progressively decrease contrast enhancement.
We recently developed a nanoparticle CLIO-Cy5.5, which is both an MRI contrast agent and a near-infrared fluorescent optical probe [6
]. CLIO-Cy5.5 is composed of a superparamagnetic iron oxide core coated with crosslinked dextran to which the fluorochrome Cy5.5 is covalently attached. The nanoparticle has significant advantages over previously used optical agents in terms of brain tumor delineation. Unlike low-molecular-weight fluorescent dyes or gadolinium chelates, the nanoparticle is internalized by cells before surgery commences and does not diffuse out of cells or through the interstitial space during surgery. A comparison of long versus
short circulating iron oxides used clinically showed that only the long circulating nanoparticles accumulated in human brain tumors [7
]. MION, the parent nanoparticle of CLIO, has a blood half-life of about 24 hours in humans [8
], whereas MION and the amino-CLIO nanoparticle have a blood half-life of 10 hours in mice [9
]. Long circulating magnetic nanoparticles may be useful for visualizing some blood-brain barrier disruptions that are not detected by standard gadolinium-enhanced MRI inhuman infarcts[10
]or with some brain tumor metastatic foci [7
]. Over a period of 3 to 7 days, the iron of injected superparamagnetic iron oxides is degraded and used [11
]. Thus, long circulating nanoparticles slowly accumulate in brain tumors, but are even more slowly degraded, yielding a window of approximately 12 to 48 hours postinjection when they are trapped inside cells and can be used to visualize tumor margins. In addition, regions of intraoperative fluorescence can be directly compared with regions of nanoparticle-induced signal loss seen on preoperative MR images. Moreover, the use of a near-infrared fluorochrome (Cy5.5) provides both markedly improved light transmission through tissues [12
] and decreased autofluorescence compared to fluorescein or other visible wavelength-emitting fluorochromes. Finally, near-infrared emission permits simultaneous full-color spectrum white light imaging while acquiring and displaying fluorescence separately [13
], allowing real-time fluorescence-based updates of resected tumor margins without sacrificing color-based cues.
We previously employed the CLIO-Cy5.5 nanoparticle to delineate brain tumor margins in a rat model using a 9L gliosarcoma that is stably transfected to express green fluorescent protein (GFP) [6
]. The goals of the present study were as follows: 1) to examine nanoparticle-based margin delineation with the 9L gliosarcoma cell line implanted in hosts with differing immune responses to the tumor (immunocompetent rats and nude mice); 2) to use a predefined signal intensity-based quantitative method to define tumor margins with fluorescent micrographs, and to assess the accuracy of margin delineation; 3) to determine which cells internalize the CLIO-Cy5.5 nanoparticle by disaggregation of the tumors of injected animals, analysis of cell populations by fluorescence-activated cell sorter (FACS) analysis, and fluorescence microscopy; 4) to examine the distribution of host cells (astrocytes and C11b+
cells) inside and around both tumor models; and, finally, 5) to see if the uptake of CLIO-Cy5.5 by tumor and CD11b+
cells observed with the 9L tumor occurred with other primary and metastatic brain tumor models. CD11b is expressed on both endogenously activated microglia and exogenous host cells such as macrophages [14
] and is thus a useful marker of host response to tumors in the brain. We find that, with the 9L tumor implanted in nude mouse or rat hosts, CLIO-Cy5.5 was effective at delineating tumor margins due to its internalization by both tumor and CD11b+
cells. The nanoparticle was internalized both by tumor and host cells in a model of brain metastasis using a mouse colon tumor line (CT26) and a second primary brain tumor model (Gli36 glioma).