Here we describe a new method of glioma cell visualization in living brain slices that can be used for evaluation of tumor size or visualization of internal tumor structures. Glial cells, as well as glioma cells of glial origin, express high levels of organic cation transporters. We demonstrate that application of a fluorescent substrate for these transporters 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP⁺) to the incubation medium leads to quick accumulation of fluorescence in glioma cells during early developmental stages and in astrocytes, but not in neurons. Stained brain slices can be immediately investigated using confocal or fluorescence microscopy. Glioma and glial cells can be discriminated from each other because of their different morphology. The method described has the advantage of staining living tissue and is simple to perform.

Glioblastoma multiforme (GBM; WHO grade IV tumor) is the most malignant form of cerebral glioma (1). Histopathological features of GBM include cellular polymorphism, nuclear atypia, brisk mitotic activity, vascular thrombosis, microvascular proliferation, and necrosis (2). Although brain imaging and clinical characteristics may suggest the diagnosis of GBM, histopathological analysis of the tumor tissue is mandatory for a definite diagnosis and can be improved only by the use of specific immunologic and molecular markers. On the other hand, it was shown that gliomas possess well expressed non-neuronal type monoamine transporters (3). We suggest that 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP⁺), a well known fluorescent substrate for monoamine transporters, can be a marker of gliomas. Pyridinium derivatives are taken up by both sodium dependent monoamine transporters (4) and by organic cation transporters from the SLC22 family and by some members of the SLC29 transporter family (5-7). Human organic cation transporter OCT type 1 (gene SLC22A1) was first isolated from the human glioma cell line SK-MG-1 (8). Later, the presence of OCT1, 2, and 3 were confirmed in different glioma cell lines (9). Here we report the accumulation of fluorescent ASP⁺ in glioma cells in living brain slices from C57Bl/6 mice at early stages of tumor development. We propose that the ability of glioma cells to accumulate fluorescent ASP⁺ can be the basis for a new method to evaluate glioma tumors in brain slices using fluorescence microscopy.

The method can be utilized to evaluate glioma morphology and size in early stages of tumor development in animal models. In contrast to hematoxylin and eosin (H&E) staining, ASP⁺ staining allows the visualization of 3D tumor structure within living brain slices, without possible tissue deformation caused by fixation and cryostat slicing procedures. It is also a convenient staining method for scientific procedures that require utilization of live tissue, such as electrophysiology.

Our in vitro slice experiments utilized male C57Bl/6 mice (Charles River Laboratories, Wilmington, MA, USA) maintained on a 12 h light/dark schedule with access to food and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee.

GL261 mouse glioma cells were implanted into the right cerebral hemisphere (10) of 12–16 week-old mice weighing 25–30 g, according to the protocol described in Liu et al. (11). Mice were anesthetized with 2.5% avertin (0.02 mg/g, i.p.) mixed with atropine (0.6 mg/kg, i.p.) and a midline incision was made on the scalp. At stereotaxic coordinates of bregma, 2 mm lateral, 1 mm caudal and 3 mm ventral, a small burr hole was drilled in the skull. Two µL of cell suspension (1×10⁵ cells/µL in PBS) was delivered at a depth of 3 mm using a 10 µL Hamilton microsyringe and a 2 pt style needle.

Method summary

Here we describe a new method to visualize glioma cells in living brain slices using a fluorescent compound 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP⁺). The method is based on the propensity of glioma cells to express high levels of organic cation transporters. We demonstrate that application of ASP+, a fluorescent substrate for these transporters, leads to quick accumulation of fluorescence in glioma cells. The method allows visualization of live glioma cells and tumor structures in brain tissue in early stages of glioma tumor development.

In double-staining experiments, we used GL261 cells labeled using the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO, USA). Cells were harvested and a loose pellet was collected in a conical tube. The cells were re-suspended in a final concentration of 2×10⁻⁶ M PKH26 dye and incubated at 25°C for 5 min. After incubation, cells were centrifuged, washed four times with PBS, and prepared for implantation. PKH25 fluorescent dye incorporates into lipid regions of the cell membrane and can be detected in brain slices by fluorescence microscopy (rhodamine red filter, excitation: 570 nm, emission: 590 nm) up to two weeks after implantation. Labeled glioma cells grow substantially after implantation, diluting the fluorescent dye after every division. Because the fluorescent signal is constantly diluted upon tumor growth, it cannot be detected much more than two weeks after implantation.

For the preparation of brain slices for ASP⁺ staining, 13–21 days after implantation of GL261 cells, mice were decapitated and the brain removed from the skull in ice-cold (2–4°C) artificial cerebrospinal fluid (ACSF) composed of 126 mM NaCl; 2.5 mM KCl; 1.2 mM NaH₂PO₄; 1.0 mM MgCl₂; 2.0 mM CaCl₂; 25 mM Glucose; 25 mM NaHCO₃; saturated with 95% O₂ and 5% CO₂. 250 µm slices were cut from the tumor-containing tissue using a vibroslicer (Leica VT1000S, Leica Microsystems, Wetzlar, Germany). Adjacent slices were used for ASP⁺ or H&E staining. Slices intended for ASP⁺ staining were placed in an incubation chamber containing ACSF at 35°C. ASP⁺ stock solution (2 mM, dissolved in water) was added to the incubation chamber at a final concentration of 1 µM, for direct staining for 10 min.

H&E staining of sections adjacent to ASP+ stained sections were performed to identify the morphology of tumor cells detected by ASP+ fluorescence. 10µm-thick cryostat sections were obtained from the 250 µm slice. Sections were stained using Mayer's hematoxylin, counterstained with eosin Y solution, and visualized using an Olympus BX51WI microscope (Olympus, Shinjuku, Tokyo, Japan).

For the fluorescent confocal microscopy, live ASP⁺ stained brain slices were placed on microscope slides and covered with glass coverslips (#1.5; Warner Instruments, Hamden, CT, USA). Slices were immediately visualized using an Olympus Fluoview FV1000 confocal microscope with 4×–10× objectives or 40×–60× oil immersion objectives. Fluorescent cells that had accumulated ASP⁺ were identified using a FITC excitation – emission filter set (absorption maximum at 494 nm and emission maximum of 521 nm). Images were captured starting from a depth of 50 µm to avoid surface areas damaged during slice preparation and finishing at a depth of 200 µm (1.5 µm step size, 100 steps).

ASP⁺ is metabolized in living cells, so the intensity of the fluorescence gradually decreases, disappearing approximately one hour after staining. In the presented experiments we visualized ASP⁺ fluorescence in live brain slices within 30 min after ASP⁺ application. For double staining experiments, a rhodamine red filter set (absorption maximum at 570 nm and emission maximum of 590 nm) was used to visualize the GL261 cells labeled with PKH26 Red Fluorescent Linker. There is no cross-detection between the FITC and rodamine red filter sets used for ASP⁺ and PKH26 detection. Images were captured with a black and white camera and ASP⁺ fluorescence was pseudo-colored green, whereas PKH26 fluorescence was pseudo-colored in red for better visual discrimination of the two signals. The fluorescent images were processed using Image J (NIH, http://rsbweb.nih.gov/ij/download.html).

To verify that ASP⁺ staining is not toxic to glioma cells and does not affect their morphology during the time of the experiment, we performed viability tests using the Live/Dead Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). GL261 mouse and U87 and HS683 human glioma cells were incubated for 4 h prior to assessing viability in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and ASP⁺ (1µM) and maintained in a humidified atmosphere of 5% C0₂ at 37°C. The media were then removed and replaced with PBS containing 4 µM calcein and 2 µM Ethidium homodimer-1 for 5 min. Fluorescent images were captured and analyzed (Olympus BX51WI fluorescent microscope). Live cells acquire green fluorescence by taking up calcein, whereas dead cells acquire red fluorescence by taking up Ethidium. ASP⁺ has orange fluorescence, therefore live cells that have accumulated ASP⁺ and calcein may appear yellow (see Supplementary Figure S3). (Unless otherwise specified, all chemicals were purchased from Sigma Chemical Company, St. Louis, MO, USA).

Double staining confirms that ASP⁺ targeted cells of glioma origin. Figure 1 A1 demonstrates a 14-day old tumor originating from the injected GL261cells labeled with PKH26 Red Fluorescent Cell Linker. At this time, all tumor cells retain their red marker. In addition, accumulation of ASP⁺ fluorescence was detected in these same cells (green; Figure 1A2 and 1A3). In the overlay figure (Figure 1A3), ASP⁺ (green) and PKH26 (red) fluorescence are detected in the same cells yielding a cumulative yellow color. These results confirm that the cells originating from the implanted GL261 cells accumulated ASP⁺. The identity of ASP⁺ stained cells and tumor cells was also confirmed with H&E staining of adjacent slices (supplemental Figure 1) that reveal similar tumor size, shape, location and morphology. PKH26 Red labeling has a time restriction because it gets diluted and faint in the membranes of glioma cells upon cell division. ASP⁺ staining, in contrast, allows the bright fluorescence of glioma cells in brain slices to be visualized at any time during the early development of a tumor.

Figure 1.  ASP⁺ uptake and electrophysiological characteristics of GL261 glioma cells implanted in the cortical region of a C57Bl/6 mouse brain. (Click to enlarge)

Our data indicate that glioma tumors have different morphology and cell composition in periods of time during the first two weeks after implantation as compared with later periods (Figure 1A2 and Figure 1B, respectively). During the first two weeks of tumor development, all glioma cells have similar sizes and shapes and all cells have a high capacity to take up ASP⁺. These tumors acquire bright fluorescence upon ASP⁺ application and the method described allows evaluation of size, morphology, and blood vessel development in the tumor (Figure 1A2, Supplementary Figure 2).

In mature tumors, cell morphology changes dramatically. Three weeks after implantation, we observed tumor cell polymorphism, microvascular proliferation, and necrosis (Figure 1B). Examples of tumor cell polymorphism can be observed by comparing the uniform and round shaped glioma cells seen in the tumor 14 days after implantation (as in Figure 1A1–1A2) with the large heterogeneity in the shapes and diameters of the glioma cell bodies (╛15–60 µm) in the tumor 21 days after implantation (Figure 1B). Glioma cells in mature tumors change their ability to take up ASP⁺. Figure 1B demonstrates that cells in the core of the mature tumor have different sizes and shapes and also different intensities of fluorescence, ranging from relatively bright to a complete lack of fluorescence.

Normal astrocytes also take up ASP⁺ (Figure 1A2, arrows). Astrocytes differ morphologically (smaller body size, long processes) from glioma cells, therefore they can be easily distinguished from glioma cells. Despite the heterogeneity in glioblastoma cell size they all are much bigger (up to 60 µm) than normal astrocyte cell bodies (around 10 µm in diameter) and do not have long branched processes.

ASP⁺ staining reveals the internal tumor structure. Glioblastoma tumors characteristically have internal cavities (Figure 1A2 and 1A3) caused by degeneration and necrosis in the central part of the tumor (2). ASP⁺ staining allows reliable 3D visualization of the blood vessel configuration (Figure 1C, Supplementary Figure 2) and internal tumor cavities up to two weeks after tumor implantation. At longer times after implantation, the size of the central cavity cannot be unambiguously evaluated using ASP⁺ staining since the uptake of ASP⁺ in glioma cells within these tumors can be reduced or absent.

Additionally, we examined the ability of cultured mouse GL261 and human U87 and HS683 glioma cells to accumulate ASP⁺ (Supplementary Figure 3A). Our results demonstrate that ASP⁺ was taken up into all of these cells suggesting that ASP⁺ uptake is a common feature of different glioma lines and can be used for glioma visualization. Furthermore, viability tests performed with GL261, U87, and HS683 glioma cells demonstrate that application of ASP⁺ for 4 h did not change the ratio of live/dead cells in all tested glioma cultures (Supplementary Figure 3A and B).

It was shown earlier that the SLC22 transporter gene, as well as some other solute carrier families of transporters under control of SOX2 (12), are overexpressed in some malignant gliomas, while displaying minimal expression in normal tissues (12, 13). Gene expression analyses identified a high expression of SOX2 in high-grade glioma cultures (14), except in glioma cultures of mesenchymal origin. Some reports indicate that during tumor development, glioma cells may lose or translocate some of their surface transporters (15, 16) and our data demonstrating strong ASP⁺ uptake in cultured and implanted GL261 glioma cells during the first two weeks after implantation in mouse brain and significant reduction of ASP⁺ uptake in older tumors support these findings. Thus OCT family transporters are initially overexpressed in many gliomas and ASP⁺ can be used as a fast and effective glioma marker.

We conclude that ASP⁺ staining allows the visualization of GBM cells in living brain slices in early stages of tumor development, revealing most specific features of these malignant tumors. The method may be used for tumor size evaluation and distinguishing the internal 3D structure of tumors and blood vessels.

Acknowledgments

The project described was supported by NIH grants G12 MD007583 and G11 HD052352 and by the UCC Pilot Project Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This paper is subject to the NIH Public Access Policy.

Competing interests

Authors have no competing interests

Correspondence
Address correspondence to Michael Inyushin, Ph.D., Department of Physiology, Universidad Central del Caribe, P.O. Box 60327, Bayamón, PR. E-mail: [email protected]

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