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. 2001 Nov;3(6):489–499. doi: 10.1038/sj.neo.7900176

Magnetic Labeling of Activated Microglia in Experimental Gliomas1

Gerrit Fleige *, Christiane Nolte †, Michael Synowitz †, Florian Seeberger *, Helmut Kettenmann ‡, Claus Zimmer †
PMCID: PMC1506563  PMID: 11774031

Abstract

Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that microglia can be labeled magnetically. Labeled microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS.

Keywords: microglia, MRI, glioma, magnetic cell labeling, superparamagnetic iron oxides (USPIO)

Introduction

The rapid development of molecular biology has been paralleled in recent years by dramatic advances in the different imaging modalities for the visualization of cellular and even molecular processes (molecular imaging) [1–3]. Up to this point, functional imaging data have been obtained on gene expression [4–6], gene transfer [7], enzyme activity [8,9], receptor distribution [10], cell migration [11,12], and neuronal plasticity using magnetic resonance imaging (MRI), near-infrared fluorescence [13], bioluminescence, positron emission tomography (PET) [14], and microPET [15,16].

Of these imaging modalities, MRI provides not only the best soft-tissue contrast, but also excellent spatial resolution that comes close to the cellular level, with a voxel size of 10 µm in vitro and 50 µm in vivo [17–19]. The present study was undertaken to determine the extent to which MRI can specifically depict resident microglia of the brain and invading macrophages involved in the pathology of gliomas.

Microglial cells represent one major intrinsic component of the central nervous system (CNS) immune response, and they are extremely sensitive to many pathologic processes. As resident immunomodulating and phagocytosing cells, microglia show an instant reaction and thus participate in the CNS response to several neurologic disorders, including tumors, inflammation, trauma, and neuronal degeneration [20]. On activation, microglial cells transform from a ramified resting state into a reactive amoeboid state. This leads to complex changes in their physiologic and morphologic properties, with a substantial increase in phagocytic capacity [21]. Morphometric investigations revealed a significant increase in macrophages and microglia within malignant gliomas (up to 50% of the viable tumor mass), and it has also been reported that activated microglia are prominent in the border area and adjacent brain tissue [22–24]. Most studies of microglia have been performed with cell cultures or at the histologic level, and only a few PET studies have so far investigated the in vivo visualization of microglia [14,25].

In recent years, numerous studies have been published on the use of different iron oxide preparations for specific magnetic labeling and in vivo tracking of cells by MRI [26,27]. These studies demonstrate that dextran-coated ultrasmall superparamagnetic iron oxides (USPIOs) are predominantly taken up by peripheral macrophages/monocytes, and to a lesser extent by other cell types [28–30]. It has also been shown that cellular uptake occurs after IV injection of the particles, and that MR visualization of experimental gliomas is improved by these methods [31,32]. However, virtually no study to date has been performed to evaluate the role of isolated microglia. Moreover, in prior studies, it remained unclear whether intratumoral USPIO was taken up by glia, peripheral tumor-associated macrophages, or tumor cells [33,34].

This study was designed to test the utility of USPIOs for selectively labeling these cell fractions. Using an experimental glioma model, we investigated the intratumoral distribution of USPIO in the different cell types, and the detection of magnetically labeled microglia/macrophages by MRI. This study has clinical implications for understanding intensity changes of an MR signal in brain tumors following administration of USPIOs, and will shed light on previously open questions regarding the intratumoral distribution of these particles. Because microglia are an important component of the CNS immune response, this technique may also provide in vivo information on the immunologic status of the CNS in the presence of brain tumors as well as on the therapeutic response.

Materials and Methods

Cells

Microglial cells were prepared from the cortex of newborn Naval Medical Research Institute rats, essentially as described previously [35,36]. In brief, cortical tissue was carefully freed from blood vessels and meninges. Tissue was trypsinized for 2 minutes, carefully disintegrated with a fire-polished pipette, and washed twice. The cortical cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, and the medium was changed every third day. After 9 to 12 days, microglia were separated from the underlying astrocytic monolayer by gentle agitation. Microglial cells in the supernatant were washed once and plated onto poly-l-lysine-coated glass coverslips at a density of 105 cells/coverslip for microscopy, and into 96-well plates with 2x104 cells/well for quantitative analysis. Purity of microglial cells was tested by labeling with lectin from Griffonia simplicifolia [Isolectin-B4 (IL-B4); Sigma Chemical Co., Deisenhofen, Germany] and antibodies against glial fibrillary acidic protein (DAKO, Glostrup, Denmark). Cultures typically had a microglial content of more than 95%.

Rat C6 glioma cells, 9L gliosarcoma cells, F98 glioma cell line, and monocytic cell line P-388D1 were obtained from commercial sources and cultured under previously described standard conditions [37]. Rat macrophages were obtained by peritoneal lavage with 11.6% sucrose solution after the rats had been put to death. Cells were washed twice with Hank's balanced salt solution and cultured in a macrophage serum-free medium (Gibco-BRL, Grand Island, NY).

Magnetite Synthesis and Labeling

Dextran-coated USPIO was prepared and kindly provided by M. Kresse (Institut für Diagnostikforschung, Free University Berlin, Germany) using a procedure originally described by Hasegawa et al. [38]. This USPIO preparation has a total iron carboxydextran ratio of 0.92 g/ g, a core diameter of 6.5 nm, and a hydrodynamic diameter of 31.3±15.8 nm. The relaxivities are R1=34.1 l/mmol per second and R2=61.4 l/mmol per second with an R2/R1 ratio of 1.8. The volume of distribution is 0.05±0.00 l/kg and the plasma clearance is 0.81±0.0 ml/min per kilogram with an iron blood half life of 0.64±0.04 hours.

Iron oxides were fluorescein-labeled with either TexasRed-hydrazide or 5-(((2-(carbohydrazino)methyl)thio)acetyl)aminofluorescein (Molecular Probes, Eugene, OR). USPIO was activated for 30 minutes with sodium metaperiodate (NaIO4) dissolved in 0.025 M citrate buffer (pH 5). After purification over a citrate buffer-equilibrated chromatography column (Sephadex G-25, PD-Column; Sigma-Aldrich, Deisenhofen, Germany), the eluent was incubated for 2 hours with one of the fluorescent dyes. Stabilization of the labeling was achieved by twice adding 20 mg sodium cyanoborohydride after 2 hours. Following separation of unbound dye by renewed column chromatography, the fluorescence-labeled USPIO was sterilely filtered and stored at 4°C.

Confocal Fluorescence Microscopy

Immediately before the experiment, excess medium covering the cells was aspirated, and the cells were incubated for different time periods and at varying concentrations of fluorescence-labeled USPIO and culture medium. The medium was then removed, and the cells were washed as before. During the entire procedure, all media were maintained at 37°C.

The coverslips containing the cells were mounted in a specially designed microscopic observation chamber, which allows for continuous rinsing of the cells with buffered solution during in vivo microscopy. The chamber was installed on the stage of a confocal argon laser scanning microscope (Sarastro 2000; Molecular Dynamics, Sunnyvale, CA). The scanner was mounted on an upright microscope (Axioscope; Zeiss, Oberkochen, Germany) equipped with a 40x magnification, numerical aperture 0.75, water immersion objective. Acquisition of the fluorescence data and image analysis was performed using ImageSpace (Molecular Dynamics) and standard PC evaluation software.

Quantification of Cellular Uptake

Microglial cells and peritoneal macrophages were incubated in 96-well plates with fluorescein-labeled USPIO at various concentrations (15, 75, 150, 300, 600, 750 µM, 1.5, 3, 6, 9, 12, 15, 18 mM), and for different time periods (15, 30, 45, 60, 90, and 120 minutes), and were then washed twice with phosphate-buffered saline. Quantitative analysis was conducted with a fluorescence reader (1420 Victor; Wallac Oy, Turku, Finland) at an excitation wavelength of 485 nm and an absorption wavelength of 535 nm. Due to short excitation times and very stable fluorescent compounds, no signal alteration caused by photobleaching was observed. Repeated measurements delivered identical results. USPIO uptake by cell lines (C6, 9L, F98, and P-388D1) and astrocytes (1.5, 3, and 6 mM) was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES) after lysis with concentrated HNO3. The displayed iron uptake data are the total cellular iron after incubation, minus the baseline iron content of control cells.

Rat Glioma Model

A rat glioma model with enhanced green fluorescent protein (eGFP)-transfected F98 cells was used for the characterization of USPIO labeling of microglia and invading blood macrophages under pathologic conditions in an in situ environment.

To induce intracerebral tumors, male, 7-week-old Fischer CD 344 rats (Charles River Breeding Laboratories, Wilmington, MA) weighing 150 to 170 g, were anesthetized with an intramuscular injection of 50 mg/kg body weight of ketamine HCl 10%, and 2mg/kg body weight of xylazine HCl 2%. The animals were immobilized in a stereotactic frame. A linear skin incision was made over the bregma, and a 1-mm burr hole was drilled into the skull approximately 2 mm posterior and 4 mm lateral to the bregma. A 10-µl (30 G) gas-tight Hamilton syringe was then used to inject 10 µl of the F98 cell suspension (5x105 cells in serum-free DMEM) into the putamen at a depth of 4 mm from the dural surface. The injection was done slowly over 5 minutes and the needle was withdrawn slowly over another 5 minutes. The burr hole was occluded with bone wax (Ethicon, Somerville, NJ) to prevent leakage of cerebrospinal fluid, and the skin was closed with nonmagnetic sutures.

For improved microscopic differentiation of glioma cells, we conducted our experiments with an eGFP-transfected F98 cell line, kindly provided by S. Kuhn, Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.

Immunohistochemistry and USPIO Labeling of Native Brain Slices

Vibratome brain slices of the glioma-bearing brains were assessed for the uptake of iron oxide by CNS macrophages, tumor cells, and healthy brain tissue.

Ten to 14 days after inoculation with F98 tumor cells, rats were decapitated, the brain was removed, and the forebrain was cut into 120-µm-thick coronal slices, which were kept in bicarbonate-buffered Ringer's solution until further processing. For qualitative analysis of microglia USPIO labeling, brain slices were incubated with TexasRed-labeled USPIOs (3 mM) for 30 minutes, followed by rinsing of the slices and carried out as described above for cultured cells.

To distinguish microglia in glioma brain slices, we performed macrophage- and microglia-specific immunofluorescence staining of the complement 3 receptor (CR3) with OX-42 antibody. The native slices were incubated with OX-42 antibodies (1:150; Serotec, Oxford, UK) for 90 minutes at 37°C. After three washes in bicarbonate-buffered saline solution, the slices were fixed with 4% paraformaldehyde for 1 hour and then permeabilized with blocking buffer (0.1% Triton-X 100, 2% horse serum, and 5% goat serum in 0.1 M phosphate buffer) for 1 hour at room temperature. For visualization, a Cy3-coupled secondary antibody (goat-anti-mouse IgG, 1:250; Dianova, Hamburg, Germany) was added for 1.5 hours. Antibodies were diluted in blocking buffer. After three washes, slices were mounted in moviol and examined by confocal microscopy.

Phantom MRI

MRI was performed with a 1.5-T superconducting magnet (Siemens Vision, Erlangen, Germany) using a human knee coil. The imaging protocol consisted of a coronal T2*-weighted FLASH 3D gradient-echo sequence (TR/TE 34/20 msec, flip angle 15°, FOV 80x80 mm, slice thickness 1 mm). Phantoms were prepared with varying amounts of USPIO-labeled microglial and monocytic P-388D1 cells (0.25, 0.5, 1.0, 1.5, 2.0, and 3.0 million cells), dissolved in 100 µl 4% gelatin following incubation of the cells for 1.5 hours with 3 mM USPIO in culture. The cell solution was embedded in 2% agar-agar wells with 0.02% Magnevist (Schering AG, Berlin, Germany) for a standardized background signal, and covered with an additional 200 µl of 4% gelatin. Standard phantoms were produced in the same way with different concentrations of free USPIO dissolved in 100 µl gelatin. Analysis was performed with NIH image software.

In Vivo MRI and Histology

Tumor-bearing rats (n=6) as described above were imaged with the same MR scanner and T2*-weighted FLASH 3D gradient-echo sequence (TR/TE 34/20 msec, flip angle 15°, FOV 100x100 mm, slice thickness 1 mm).The first series of images was taken 14 days after tumor implantation. Twenty-four hours after administration of 300 µmol USPIO/kg body weight, imaging was repeated. Additionally, a T2-weighted turbo spin echo sequence (TR/TE4500/96 msec, angle 180°, FOV 100x100 mm, slice thickness 2 mm) was conducted. Animals were perfused with formalin, and kryosections (10 µm) were prepared. Corresponding brain slices were stained with HE, DAB-enhanced Pearl's Blue reaction, and OX-42 antibody as described above.

Results

Microglia/Macrophages are Labeled by Iron Particles in a Rat Glioma Model

After USPIO incubation (3 mM for 30 minutes) of the freshly prepared glioma brain slices, confocal microscopy revealed a massive uptake of TexasRed-labeled USPIOs by microglia and macrophages, and almost no detectable uptake by glioma cells. Gliomas could be detected by labeled microglia/macrophages surrounding and infiltrating the tumor (Figure 1C and D). USPIO was predominantly taken up by amoeboid microglia. Ramified resting microglia showed no evident USPIO incorporation in contrast to activated microglia/macrophages, which have a high phagocytic capacity (Figure 1B). It was even possible to detect single infiltrating tumor cells that were encircled by labeled microglia and macrophages (Figure 1D, inset). As an indicator for the activated state of microglia in the tumor region, phagocytic cells migrated rapidly to the surface of the slice and took up USPIOs during incubation.

Figure 1.

Figure 1

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Activated microglia and invading macrophages infiltrate the tumor region in rat gliomas and show rapid uptake of USPIO in a slice model. Brain slices (150 µm thick) of the tumor region were incubated with 3 mM TexasRed USPIO for 30 minutes, and investigated in the confocal microscope with varying magnifications. (A) OX-42 staining (blue) reveals the proportion of microglia and invading macrophages within the tumor. The inset displays green eGFP-transfected tumor cells in an unlabeled slice. (B) After incubation of slices with TexasRed-labeled USPIO, the magnetite was clearly localized intracellularly, and predominantly within OX-42-positive ameboid cells (inset in B; USPIO red, OX-42 blue). OX-42-positive microglia with a resting ramified appearance (blue) showed no significant USPIO uptake. (C) Visualization of microglia and macrophages by intracellular labeling with TexasRed-stained USPIO showed diffuse infiltration of the tumor, as seen in (A). (D) Formation of a border around the tumor, differentiating the tumor from healthy brain tissue, was also detectable by USPIO labeling. A single tumor cell encircled by USPIO-labeled macrophages could be detected at a distance from the main tumor mass (inset in D).

Distribution of magnetically labeled microglia/macrophages could be confirmed with immunohistochemistry: labeling of microglia and invading blood macrophages with the OX-42 antibody, which recognizes CR3, demonstrates a diffuse distribution of OX-42-positive cells within the tumor (blue in Figure 1A). A native slice of the tumor region shows the eGFP-labeled tumor cells (green in Figure 1A, inset). The amount of CR3-positive cells within a tumor varied depending on its individual morphology. The tumors were encircled at the border by a more or less dense zone of microglia and macrophages.

These observations suggest that labeling of microglia/macrophages with USPIOs reproduces the distribution of these cells within and around a tumor, and that labeled microglia/macrophages can be used to identify and determine the extent of gliomas.

USPIO is Preferentially Taken Up by Microglia in Mixed Culture

Because we could detect almost no USPIO uptake by other cells of the CNS and tumor cells in the slice experiments, we studied uptake capacity in comparison to microglia under cell culture conditions.

Astrocytes are involved in the CNS pathology and may potentially also perform endocytosis. Therefore, astrocytes were directly compared to microglia regarding their capacity to incorporate USPIOs. Cells were assessed in a co-culture with microglial cells growing on a monolayer of astrocytes. Under such identical conditions, uptake by microglia is evident, whereas there is almost no detectable uptake by astrocytes. This underlines the importance of microglia as phagocytic cells in the CNS. All photomicrographs in Figure 2 show the same field of view at different cell levels. A and B document the confocal cell level of microglia, and C and D the level of astrocytes. B and D were taken after incubation of the co-culture with 3 mM USPIO for 30 minutes.

Figure 2.

Figure 2

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Visual comparison of USPIO uptake by microglia and astrocytes under identical conditions. Confocal microscopy of a microglia-astrocyte co-culture. All photomicrographs display the same field of view, but are focused on two different cell levels. (A) and (B) are focused on microglia attached to the astrocytes. (C) and (D) are focused on the astrocytic monolayer beneath the microglia. Phase-contrast photographs focusing on each cell type show cell morphology (A and C). After incubation with 3 mM TexasRed-labeled USPIO for 30 minutes, (B) and (D) display intracellular uptake predominantly by microglia.

A co-culture study of microglia with F98 tumor cells again revealed massive incorporation of USPIOs by microglia, and no detectable uptake by the tumor cells after incubation with 3 mM USPIO for 30 minutes (Figure 3). Microglial cells were counterstained with IL-B4 to distinguish them from the tumor cells (Figure 3C).

Figure 3.

Figure 3

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Labeling of co-cultured microglia and F98 tumor cells with TexasRed-USPIO confirmed the results of glioma slice experiments. (A) Due to the incorporated iron oxide, microglial cells can be detected with confocal microscopy after incubation with 3 mM USPIO for 30 minutes. F98 cells do not incorporate USPIO. (B) Corresponding phase-contrast photograph showing the F98 cells (arrows) between the microglia. (C) Labeling with Isolectin-B4 after incubation identifies phagocytosing cells to be microglia.

Thus, microglia can be magnetically labeled with iron oxides and, through their intracellular USPIO content, can be distinguished from other cells involved in glioma pathology under in vitro conditions, as well.

Characterization of the intracellular distribution of iron oxides with confocal microscopy revealed that USPIOs labeled with TexasRed fluorescence dye are arranged in a typical endosome-like pattern within the microglia cells after incubation with 3 mM USPIO for 30 minutes (Figure 4), similar to what has been previously described for USPIO internalization in other cell types [31,39]. Cellular vacuoles depicted by the phase-contrast technique can be identified as mainly USPIO-containing organelles of the cells, and there seems to be no free USPIO in the cytoplasm. Microscopy studies also revealed that not all microglial cells internalize equal amounts of USPIO, which is in agreement with other investigations of microglia phagocytosis [40].

Figure 4.

Figure 4

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Intracellular distribution of TexasRed-labeled USPIO incorporated by microglia. After incubation of cultured microglia with 3 mM TexasRed-labeled USPIO for 30 minutes, an endosome-like distribution pattern of the internalized contrast agent can be observed by confocal microscopy. (A) Phase-contrast, (B) fluorescent USPIO. Control cells not incubated with USPIO showed weak autofluorescence (insets).

Quantification of USPIO Uptake by Microglia and Tumor Cells In Vitro

Quantitative analysis of USPIO incorporation by cultured microglia shows both concentration and time dependencies. Cellular uptake after 30 minutes of incubation is characterized by a typical logarithmic concentration-dose response. The concentration range between 1.5 and 12 mM is associated with the highest rate of incorporation by the cells, and saturation seems to occur at a level of 15 to 18 mM USPIO (Figure 5A). Invading blood macrophages are another important phagocytic cell population involved in CNS pathology. Their specific capacity to phagocyte USPIOs in comparison to microglia was likewise assessed under in vitro conditions. Incubation of cultured peritoneal macrophages as an equivalent cell type also revealed a concentration-dependent uptake of USPIOs similar to that seen in microglia (Figure 5A).

Figure 5.

Figure 5

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USPIO incorporation characteristics of cultured microglia. (A) Concentration dependency: Microglia: cells were incubated at 37°C in different concentrations (15 µM to 18 mM) of TexasRed-labeled USPIO for 30 minutes, and cellular uptake was determined by fluorescence scanning. Inset displays uptake by identically treated peritoneal macrophages. (B) Time dependency: incorporation was quantified after microglia had been incubated for varying time periods (15 to 120 minutes) in 3 mM TexasRed-labeled USPIO.

Assessment of USPIO uptake by microglial cells at a constant concentration of 3 mM, but for different time periods (15 to 120 minutes), showed an almost linear correlation within this time interval (Figure 5B). As the exact amount of USPIO uptake fluctuates among different experiments, depending on many factors that influence cell activation, the results of USPIO uptake by microglia are presented in a semiquantitative manner. Because microglial cells seem to incorporate huge amounts of iron oxides, the influence of USPIO phagocytosis on cell viability was assessed using a commercial assay. Cell survival depended on magnetite concentration and incubation time, which was also confirmed by microscopic time-lapse video observation (not shown here). USPIO — in the concentrations used in this study — was not toxic for cells, but further increases in concentration or incubation times led to uninhibited USPIO incorporation, with swelling of the cells and consecutive cell death as observed by microscopy.

Figure 6 summarizes the quantitative uptake of USPIO by different CNS tumor cell lines (9L, C6, and F98), a monocytic cell line (P-388D1), and isolated astrocytes. The amount of USPIO incorporated was determined by direct measurement of intracellular iron levels using ICP-AES. Uptake of iron oxides by all cell types was concentration-dependent (1.5, 3, and 6 mM), and ranged from 26 ng to nearly 1000 ng Fe/106 cells. As expected, uptake was highest for the monocytic cell line, which is known to have a high phagocytic activity. In comparison, isolated astrocytes incorporated substantially lower amounts of USPIO, but significantly more than the tumor cell lines studied. These results are consistent with previous investigations using slightly different paramagnetic particles and other cell types [27,32].

Figure 6.

Figure 6

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Concentration dependency of USPIO incorporation in different cultured cell types involved in glioma pathology. Cultured cell lines [C6, 9L, F98 (glioma), and P-388D1 (monocytic)] and primary astrocytes were incubated in unlabeled USPIO for 30 minutes at varying concentrations (1.5 to 6 mM), and cellular uptake was determined as the intracellular iron level by means of ICP-AES.

Cultured, Iron-Labeled Microglia/Macrophages Can be Detected by MRI in a Phantom Model

To assess if USPIO-labeled microglia and macrophages can be detected by MRI, gel phantoms containing magnetically labeled cells were prepared and examined with a conventional 1.5-T scanner. Cell phantoms were arranged in such a way as to simulate a tumor-like condition in terms of the amount of microglial cells and macrophages within the tumor mass. Even with a conventional human imaging system, it was possible to detect the labeled primary microglia, and to correlate the received signal intensity with varying cell concentrations. The received signal intensity within the cell phantom decreased with increasing concentrations of embedded cells per well (0.25, 0.5, 1.0, 1.5, 2.0, and 3.0 million cells/100 µl), representing higher amounts of USPIO per well. Labeled microglia could be detected by MRI by altering the received signal intensity starting at the lowest concentration of 0.25x106 cells, corresponding to 0.8 µg of pure USPIO in the standard phantom (Figure 7A). Imaging of an identically prepared P-388D1 cell phantom yielded similar results, with signal intensity also correlating to the concentration of labeled cells. Quantitative analyses revealed an analogous signal intensity in both cell types at identical cell concentrations (Figure 7B).

Figure 7.

Figure 7

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Effect of microglia USPIO labeling on MRI signal intensity, visualized in phantom models. Signal intensity correlates with intracellular USPIO concentration in cell and standard phantoms. 3D FLASH sequence (TR/TE 34/20 msec, flip angle 15°) provides high spatial resolution. (A) Varying concentrations (0.25x106, 0.5x106, 1x106, 1.5x106, 2x106, and 3x106) of microglia and P-388D1 cells were incubated for 1.5 hours with 3 mM USPIO, and embedded in 100 µl of 4% gelatin. (B) Analysis of the standardized MRI signal revealed similar USPIO uptake by cultured microglia and macrophages (P-388D1).

USPIO Distribution and MRI After In Vivo Administration

MRI of a glioma-bearing rat displays a huge tumor with a size more than the right hemisphere (Figure 8). After administration of USPIOs, a significant signal intensity decrease in T2*-weighted pictures can be seen, which reflects the extent of the tumor and correlates quite well to the perifocal edema [A(II)]. An inhomogeneous intratumoral signal pattern occurs due to necrosis and varying distribution of microglia and macrophages within the tumor. The tumor border, demarcated by microglia, can be precisely differentiated from the healthy brain tissue.

Figure 8.

Figure 8

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In vivo distribution of USPIO in glioma-bearing rats. MRI (A) before (I, II) and after (III, IV) administration of USPIO reveals a massive accumulation of iron oxides within the tumor, seen as a signal intensity decrease. Immunohistology [B(III and IV)] confirms predominant USPIO uptake (red) by macrophages and microglia (blue), also under in vivo conditions. T2*-weighted 3D FLASH sequence (TR/TE 34/20 msec, flip angle 15°) is displayed in I and III; T2-weighted turbo spin echo sequence (TR/TE 4500/96) in II and IV. Imaging was performed before and 24 hours after administration of 300 µmol/kg USPIO. Through DAB staining, image [B(II)] confirms iron deposition within and around the tumor. HE staining of corresponding slice [B(I)]. OX-42 staining detects high proportion of microglia and macrophages (red) within the tumor (green) [B(III)]. Also after in vivo administration, USPIO (red) can predominantly be found in the macrophages and microglia [B(IV)].

Confocal microscopy analysis reveals that the intravenously administered TexasRed-labeled USPIOs (red) can be found in microglia and macrophages (blue), and hardly at all in tumor cells (green) [B(III)]. On exact observation, an identical intracellular perinuclear distribution pattern, as seen in cultured microglia (Figure 4), can be detected. Again, the tumor mass consists of a high amount of these immunologic cells. After DAB-enhanced iron staining, USPIOs can be found within and around the tumor in a border-like pattern corresponding to the fluorescent pictures and MRI [B(II)].

In conclusion, detection of gliomas with MRI and iron oxides is predominantly based on the visualization of the labeled microglia and macrophages, and not the tumor cells themselves.

Discussion

Nearly any kind of pathologic CNS process leads to an activation of microglia and a change of their cellular properties. Making use of the markedly increased phagocytic capacity as the characteristic immune function of activated microglia, magnetic labeling of this cell type with a sensitive MR marker may have the potential to improve detection of CNS pathology by MRI. We have demonstrated for the first time that: 1) microglia can be labeled with magnetic particles, 2) microglia possess the highest labeling capacity in comparison to other cell types involved in glioma pathology, 3) labeled activated microglia very precisely represent the tumor morphology, and 4) labeled microglia can be detected with MRI in vitro and in vivo.

It is well known, and has been described by other groups using C6 and RG2 glioma models, that gliomas are infiltrated by macrophages and differ from each other only in the intratumoral distribution patterns of these macrophages [41–44]. Morphometric studies of phagocytic infiltration in human gliomas identified amounts ranging between 20% and 40% of the tumor cell mass [45,46]. Our investigation confirms that rat F98 gliomas are infiltrated by a huge proportion of OX-42-positive microglia/macrophages. Although microglia, as the main intrinsic immunoeffector cells of the CNS, are primarily thought to be involved in immune defense, recent investigations suggest that microglia promote rather than inhibit the invasive and proliferative properties of gliomas by cytokine secretion (IL-10) [47–49]. For this reason, glioma cells seem to even secrete cytokines that attract microglia in a kind of symbiotic reaction [41,50]. Although it is not possible to differentiate clearly between microglia and invading macrophages, both cell types seem to participate in the CNS immune response.

Whereas preliminary investigations suggested that cultured microglial cells are able to incorporate USPIOs [51], this has never been demonstrated. In contrast, labeling of other immune cells, such as macrophages and lymphocytes, with slightly different types of USPIO has been shown by different groups to be possible [27,39,52]. Cells other than professional phagocytes and immune cells, such as tumor cells, are also able to incorporate iron oxides [31,32]. Our study shows that cultured microglial cells can be labeled very effectively with dextran-covered iron oxides. The observed intracellular uptake of such USPIO particles with an endosome- like distribution pattern, as well as the centripetal movement of the vesicles toward the perinuclear space after plasma membrane invagination, corresponds to the aforementioned observations made in other cell types.

Recent work by A. Moore describes USPIO uptake in tumor cells and tumor-associated macrophages. Uptake in tumor cells appeared to be directly proportional to cellular proliferation rates [34]. Though we could also prove uptake by tumor cells in culture, it was much higher in microglia/macrophages. Confocal microscopy of co-cultured microglia and astrocytes showed that USPIO is predominantly taken up by microglia. The fact that USPIO uptake by astrocytes is still higher than incorporation by the investigated tumor cell lines (F98, 9L, and C6), underlines the extraordinary position of microglia regarding their phagocytic capacity, and thus their potential for USPIO labeling among the cell types involved in glioma pathology.

Our in situ experiments in glioma slice preparations confirmed the cell culture findings of extensive USPIO incorporation by activated microglia and macrophages, and almost no detectable uptake by tumor cells. USPIO labeling of microglia and macrophages occurred only in areas of CNS pathology, whereas resting microglia and other cell types in healthy brain tissue showed no USPIO uptake. By labeling of microglia and macrophages with fluorescence-stained USPIOs, it was thus possible to detect gliomas and to differentiate them from unaffected surrounding tissue. Even solitary invading tumor cells encircled by labeled microglia but far away from the main tumor mass could be identified microscopically.

Regarding the question as to which kind of macrophages was labeled with USPIOs [33], we could exclude predominant labeling of endothelial cells. Nevertheless, an exact differentiation between invaded blood macrophages and activated microglia remains difficult. Because both cell types show similar USPIO uptake patterns, the exact determination of their origin is of minor importance for our investigations.

Most of the commonly used MRI contrast agents based on gadolinium conjugates are not specifically taken up by any kind of cell, and signal enhancement is mainly caused by interstitial accumulation or increased vascularization. Thus, MRI is still limited in terms of exact delineation, early detection, and tissue differentiation. Our investigations demonstrate that the distribution of incorporated USPIOs within glioma slices represents the region of activated microglia and immigrated macrophages. As described by Roggendorf et al. [23], gliomas are frequently surrounded by microglia and macrophages in a border-like pattern. This property, extremely helpful for localizing the exact tumor border, was also detected in the tumors studied here. Our studies of magnetically labeled microglial cells embedded in gelatin phantom models demonstrated that it is possible to visualize these cells by MRI. Furthermore, the received signal intensity displayed the incorporated amount of contrast medium or the amount of cells within the phantom. Gradient-echo sequences, having an extremely high sensitivity for iron, offer the possibility to detect iron amounts as low as 62.2 ng/mm3 as shown in previous studies [53]. After in vivo administration, MRI of magnetically labeled microglia and macrophages depicted the glioma extent very precisely.

Intravenous injection in human adults of a normal dose of 2.5 mg (45 µmol) Fe/kg body weight results in a blood concentration of 70 µg/ml (1.4 mM), assuming a 70-kg subject (7% blood volume and 46% hematocrit). At this dosage and a given blood half-life of 40 to 80 minutes, exposure of activated microglia and invaded macrophages to USPIOs should be sufficient for their effective labeling in vivo, as seen here. Under in vivo conditions, there exists no risk of significant cell damage when such a physiologic dosage is administered [54].

Precise visualization of gliomas is the most important prerequisite for initiating and performing any kind of therapy. Early detection of disease would prevent an unnecessary delay in intervention and, as demonstrated here, it might even be feasible to visualize single infiltrating cells far away from the main tumor. Because such minimal lesions are not necessarily associated with a blood-brain barrier disruption, a mechanism must be introduced to transfer USPIOs across this natural barricade. One possibility is to conjugate USPIOs with a ligand, permitting specific transport and incorporation. This procedure additionally increases cell labeling specificity and uptake rates, as demonstrated by previous investigations [55]. The fact that predominantly highly activated microglial cells have phagocytic activity could make it possible to detect even minimal lesions.

Microglia activation and invasion of macrophages also play an important role in several other CNS diseases. Acute and chronic inflammation induces a pronounced immune response including microglial activation [56]. Magnetic labeling of microglia/macrophages provides the potential for detection of any kind of inflammatory focus, or for staging of disorders such as multiple sclerosis [57]. Furthermore, immunologic information could be gained for therapeutic follow-up, and possibly for new treatment strategies. Specific labeling of peripheral cells such as T lymphocytes could complement such immunologic interaction [58].

Based on these promising data, one should focus on microglia and invading macrophages when labeling cells with intracellular contrast agents for MRI of CNS pathology.

Acknowledgements

The authors thank Maik Kresse for providing USPIOs, Susanne Kuhn for the transfection of F98 cells with eGFP, and Daniela Laux for the histologic processing of the in vivo experiments.

Abbreviations

USPIO

ultrasmall superparamagnetic iron oxide

Footnotes

1

This work was supported by grants from the German Research Foundation (GRK 238).

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