Confocal laser and electron microscopic investigation of gap junctions in anaplastic astrocytomas

Abstract

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localization and ultrastructure of gap junctions in samples of malignant brain tumor (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumors fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated, and embedded in epoxy resin by a plane-parallel method. Ultrathin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localized around or at the intersection of small processes containing GFAP. Electron microscopy of the tumor samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumor cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100 μm². Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

1. Introduction

Anaplastic astrocytoma is a malignant brain tumor that arises from astrocytes. Gap junctions (GJ) and connexins (Cx) are considered crucial pathogenetic mechanisms of malignant tumor growth and promising targets for targeted therapy [1]. Cx43 is a multifunction protein that forms gap junction channels and hemichannels and thus is suggested to play a key role in oxygen-glucose deprivation, induced by neuroinflammation during the process of astrocytoma expansion into healthy tissue. On the one hand, it has been shown that interaction of intracellular C-domain Cx43 with the pro-oncogenic kinase Src leads to a decrease in the proliferative activity of tumor cells, and the restoration of intercellular interactions through GJ promotes the spread of apoptosis signals in tumor cell population and suppression of tumor growth [2]. On the other hand, the interaction of Cx43 extracellular domains with extracellular matrix and proteins of neighbor cells, as well as intercellular exchange through the GJ, enhance the ability of tumor cells to migrate and promote the transformation of surrounding non-malignant tissue into a tumor one [3, 4]. However, understanding of the association between astrocytoma and Cx43 along with gap junctions remains unclear. It is uncertain whether the positive expression of connexins is associated with the formation of functional GJ (nexuses) or the accumulation of protein in the cytosol in astrocytic tumors of various degrees of malignancy.

In previous studies, we demonstrated the presence of GJs in samples of pleomorphic xanthoastrocytoma grade II [5], hemistocytic astrocytoma grade II, glioblastoma grade IV but did not find these contacts in oligodendroglioma grade II [6]. However, the information regarding cell-cell interactions in malignant anaplastic astrocytomas, i.e., diffusely infiltrating grade III astrocytomas that tend to transform into grade IV glioblastomas, is absent in the available literature.

The aim of this work was to study connexin 43 expression, localization, and ultrastructure of gap junctions in samples of anaplastic astrocytoma grade III.

2. Methods

The studies were carried out with accompanied written informed consent of patients and their relatives. Fragments of surgically resected human brain glial tumors with a confirmed histological diagnosis of anaplastic astrocytoma grade III were used as material for research. Tumor samples (n=8) were fixed in 4% paraformaldehyde. Vibratome (VT 1000E, Leica, Germany) was used to cut 40 μm sections from the samples. Sections were cryoprotected and instantly frozen over liquid nitrogen vapor. For immunohistochemistry, sections were thawed in a phosphate buffer at room temperature. After that, sections of tumor fragments were incubated in a mixture of rabbit polyclonal antibodies to Cx43 (Spring Bioscience, USA) and mouse monoclonal antibodies to glial fibrillar acidic protein (GFAP, Dako, Denmark) with the addition of 0.1% sodium azide for seven days at 10°C. Sections were washed in phosphate buffer and incubated for 24 hours at 10°C in a mixture of secondary antibodies conjugated with a fluorescent label: goat anti-Rabbit CF488A antibodies (Sigma-Aldrich, USA) and goat anti-mouse CF555 antibodies (Sigma - Aldrich, USA). After incubation, sections were washed in phosphate buffer, mounted on glass slides in an anti-fading medium Fluorescence Mounting Medium (Dako, Denmark), covered with a cover glass, and examined using a laser scanning confocal microscope (Zeiss LSM880, Germany). After fluorescence study, the cover glass was carefully removed from a part of tumor sections (n=6). They were washed in phosphate buffer and additionally postfixed in 1% OsO4, dehydrated and embedded in epoxy resin by a plane-parallel method. After polymerization, 1 mm tissue fragments were excised and polymerized to the top of the epoxy resin block of. Single and serial ultrathin sections obtained from the tumor sections were cut using EM UC 7 ultramicrotome (Leica, Germany) and an ultra 45° diamond knife (Diatome, Switzerland), contrasted with uranyl acetate and lead citrate. Images were taken under an electron microscope Jem 1011 (Jeol, Japan) with an accelerating voltage of 80 kV.

3. Results and Discussion

Confocal microscopy of fluorescently-labeled 40 μm tumors sections showed that GFAP is present in the cytoplasm, as well as in large and small chaotically branching processes of malignant cells (Fig.1A). A fluorescence study of Cx43 in astrocytoma tissue showed a reaction pattern of expression: positive signals have the form of fluorescent points of various sizes (Fig. 1B). A double fluorescence study made it possible to demonstrate that the majority of Cx43 points are localized around or at the intersection of small processes containing GFAP (Fig. 1C, arrows). Serial optical imaging (0,2 um step size along the z-direction) revealed that some of the Cx43+ signals could be detected in deeper sections than others, reflecting the heterogeneous size of Cx43+ along the z-axis. Such positive Cx43 + extended areas in the studied volume were also associated with GFAP-positive structures of astrocytomas. Tumor’s regions containing the most significant number of positive labels for Cx43 were further examined by electron microscopy.

Figure 1.

Immunofluorescence and electron microscopic examination of GJs in grade III anaplastic astrocytomas. A - expression of GFAP, B - GJ protein of astroglia Cx43 (arrows) is located in the form of points in the tumor tissue, C – a combination of GFAP and Cx43. Scale bar 20 microns. D - GJs between the processes of tumor cells, D* - typical ultrastructure of GJ, E - GJ connecting the tumor cell soma and processes: GJ between the soma and the process and another two GJ, formed on the same process from opposite sides, F – ultrastructure of GJ, white arrows indicate bridges penetrating two bilipid membranes, G - GJ formed inside the tumor cell, G * - curved GJ inside the cell soma. Legend: Gf - gliofibrils, V - vesicles, N – nucleus, GFAP- expression of glial fibrillary acidic protein, Cx43 expression of connexin 43, arrow indicate gap junction (GJ). Magnification: D - 40,000, D* - 200,000, E - 50,000, F - 400,000, G-40,000, G * - 200,000.

Sequential examination of ultrathin sections, selected from anaplastic astrocytoma samples revealed numerous single and closely spaced GJ. Fig. 1D shows two tumor cells, the cytoplasm, and processes filled with gliofibrils. The process of cell 1 is larger than the process of cell 2. These processes are adjacent to each other and are in contact at several points through extended GJ. Ultrastructural details of the detected GJ were visualized at high magnifications and rotation of the sample using a goniometer (Fig. 1D, D*). Electron microscopic examination showed that GJ of anaplastic astrocytomas did not have visible perforations. In cases where the GJ nexuses were in the cut plane, they were seen as “stuck together” bilipid membranes with an osmiophilic gap between them, which corresponds to a classic seven-layer structure of the GJ. Higher magnification revealed thin electron-transparent bridges between the two membranes crossing the osmiophilic dark contact gap. These structures are, apparently, intercellular pores, providing the transition of ions and micromolecules from the process of one tumor cell to the process of another (Fig 1G). In addition, we found areas where networks of GJ were formed both between thin processes and between processes of a large area. Moreover, most of the GJ junctions were located in the immediate vicinity of tumor cell bodies. With a magnification of 40,000 in one field of view, up to 6 GJs can be detected. They predominantly have an even or slightly curved shape, short and elongated nexuses, connecting along or across the cut processes of different cells. Besides GJ formation between processes of various diameters, we found numerous GJs on the soma of tumor cells. Such contacts can be conditionally divided into two types: GJ between the soma and the process of another cell and GJ between the own compartments of the cell. Fig. 1E shows 3 GJs formed on three sides of one process (Fig. 1E). The first GJ is formed between the tumor cell soma and the thinnest process. That process has two more GJs are located on opposite sides: one extended and curved, another one with extended elongated nexus. Another type of the GJ location is shown in Fig. 1F. An electronogram shows a vacuolated tumor cell with an eccentrically located nucleus. A curved intracytoplasmic GJ was revealed at higher magnification in the peripheral part of its cytoplasm (Fig. 1F, F*). In order to analyze the total number of gap junctions in areas of astrocytomas selected for the study, we performed the morphometric analysis. Sequential imaging of the visual fields revealed 62 GJs in an area of 100 μm², which significantly exceeds the number of GJs in other previously studied tumors of glial origin.

The obtained results show that positive fluorescence signals of connexin 43 correspond to the formation of GJs with a typical ultrastructure in anaplastic astrocytomas grade III. Identification of the significant number of GJs in gliomas supports the hypothetical tumor glial cells’ ability to integrate into the surrounding neuronal and neuroglial networks of the brain. This hypothesis was based on the discovery of glutamatergic synapses between glioma cells and the presynaptic terminals of neurons [7,8]. The existence of such synapses promotes tumor proliferation, growth, and stability. Presumably, an excitatory signal in the synapse between a glutamatergic neuron and glial tumor cell is transmitted to other tumor cells through GJs. This phenomenon was found not only in glioma cells but also in some types of breast cancer that metastasize to the brain [9].

The numerous gap junctions in anaplastic astrocytoma shown in our study may indicate electrotonic and metabolic transmission between glioma cells, promoting its progression. GJs can promote the diffuse transfer of lactate and bicarbonate (end-product of glycolytic metabolism) from more hypoxic tumor cells to normoxic ones by providing metabolic cooperation with low resistance. One possible method to block the glycolysis products release by tumor cells is to inhibit massive intercellular transmission via gap junctions identified in this study.

4. Conclusions

In this work, we have demonstrated for the first time a significant number of GJs in anaplastic astrocytomas. These numerous containing connexin 43 contacts are formed both between processes of different cells and between the soma of one cell and the process of another cell, as well as between the soma and the process of the same cell.