Analysis of Connexin 43 and Spermine Co-Localization in Glioblastomas

Abstract

Gap junctions are channels between adjacent cells, contributing to the unhindered exchange of metabolites, second messengers, nucleotides, and other molecules. The functional status of gap junctions in brain tumors is underinvestigated. One avenue of research focuses on exploring the expression of polyamines and their co-localization with the Connexin 43 (Cx43) in the growth zones of glioblastoma multiforme (GBM). The aim of this work was to analyze the expression of Cx43 and spermine in human GBM to reveal their roles in neuro-oncogenesis. Human GBM sample sections were used for the immunochemistry (glial fibrillary acidic protein (GFAP), Cx43, and spermine), confocal laser scanning microscopy, and electron immunohistochemistry. Immunofluorescent analysis revealed that the more extensive processes of GBM cells exhibit GFAP. All GBM samples (n=10) exhibited positive Cx43 signals in the form of variously sized dots and lines. Cx43 formed dotted lines around cell bodies with segmented transformed nuclei, which were also present in the gliovascular complexes. Furthermore, spermine was overexpressed in all tumor samples (cytoplasm, large and thin tumor processes), including the areas of Cx43 localization. Merging the Cx43 and spermine signals showed co-expression in the same regions: the membranes of individual cells and individual points on processes in the tumor tissue. Therefore, we established the staining of the co-localization of Cx43 and the polyamine spermine within glioblastoma, revealing that tumor processes housing the polyamine indeed form gap junctions, suggesting their potential joint interaction. This finding indicates that glioma cells can integrate into the surrounding neural networks, potentially serving as a mechanism to release glycolysis products, relying on gap junction activity facilitated by spermine. Cx43 exhibits sensitivity to polyamines, which play a role in opening gap junctional channels. Furthermore, polyamines have been observed to eliminate the blockades caused by hydrogen ions and calcium, which is crucial for cellular physiology.

1. Introduction

Among glial brain tumors, glioblastoma multiforme (GBM) is the most aggressive and common, representing 50 to 60% of all gliomas, with peak age incidence at 45 to 55 years. There is currently no effective treatment to improve survival for patients with GBM, and the median survival is three months after diagnosis. Significant efforts are directed towards identifying promising targets, the selective modulation of which can enhance the efficacy of treating GBM. Gap junctions 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 tumor expansion into healthy tissue. Gap junctions, composed of docked membrane half-channels, form a pore between adjacent cells, contributing to the unhindered exchange of metabolites, second messengers, nucleotides, and other molecules. In previous studies, we did not find these contacts in oligodendroglioma grade II, but we demonstrated the presence of gap junctions in samples of pleomorphic xanthoastrocytoma grade II [2], hemistocytic astrocytoma grade II [3], in malignant anaplastic astrocytomas grade III [4] and in some samples of glioblastoma grade IV. However, the functional status of gap junction in GBM remains an area of interest requiring investigation. Regulation may occur through small molecules moving through gap junctions, direct control of connexin-encoding genes, oversight of connexin synthesis and traffic, management of connexin degradation, and control over connexin interactions with cytoplasmic proteins. One of the possible options for the Cx43 behavior control could be the interaction with polyamines. Polyamines, multifunctional molecules containing several amine groups, play a vital role in the brain [5]. Spermine biosynthesis is activated in tissues with cell proliferation and is suggested as a marker of various malignancies of gliomas [6]. One avenue of research focuses on exploring the expression of polyamines and their co-localization with the Cx43 protein in the growth zones of glioblastomas. Glial fibrillary acidic protein (GFAP) has proved to be the most specific marker for cells of astrocytic origin under normal and pathological conditions. Gliomas can heterogeneously express GFAP, a signature type III IF protein of glioma cells implicated in tumor migration and invasion. At the same time, biochemical evidence was obtained indicating direct binding of the cytoplasmic domains of integrins to gliofibril proteins at cell-cell contact sites and their substrates. Thus, GFAP may participate in complex cellular processes controlling astrocytoma cell morphology, adhesion, and proliferation. The aim of this work was to analyze the expression of Cx43, GFAP and spermine in human GBM to reveal the roles of these proteins in neurooncogenesis.

2. Methods

Surgically resected human glioblastoma samples were used as material for research. The informed consent was obtained from patients or their relatives. The sample group included tumor fragments from patients with ages ranging from 45 to 66 years (3 women and 7 men) with a pathomorphological diagnosis of glioblastoma grade 4. Samples (n=10) were fixed in 4% paraformaldehyde. Vibratome (VT 1000E, Leica, Germany) was used to cut 40 μm sections from the samples. Sections were cryoprotected in 15% and 30% sucrose and instantly frozen over liquid nitrogen vapor. For immunohistochemistry, sections were thawed in a phosphate buffer (PBS). The sections were then subjected to a 7-day incubation using the following mixtures: 1) rabbit polyclonal to Cx43 (Elabscience, USA)/mouse monoclonal to GFAP (Sigma-Aldrich, USA), 2) rabbit polyclonal to spermine (Cloud-Clone, USA)/mouse monoclonal to GFAP (Sigma-Aldrich, USA) 3) rabbit polyclonal to spermine (Cloud-Clone, USA)/mouse monoclonal to Cx43 (Thermo Fisher Scientific, USA). Sections were washed in PBS and incubated for 24 hours at 10°C in a mixture of secondary antibodies conjugated with a fluorescent label: 1) goat anti-rabbit conjugated with Abberior STAR ORANGE (Abberior, Germany), 2) goat anti-mouse conjugated with Abberior STAR RED (Abberior, Germany), and 3) with Sytox green stain (Thermo Fisher Scientific, USA). Finally, the sections were mounted in an anti-fade solution and examined using a confocal laser scanning microscope (Abberior Facility Line, Abberior Instruments GmbH, Germany). After confocal microscopy, the cover glass was carefully removed from a part of the tumor sections (n=3). Sections were incubated in secondary antibodies RTU Envision Flex/HRP (Dako, Denmark) for 24 hours at 20°C. Immune complexes were detected using the EnVision HRP + Peroxidase imaging system (Dako, Denmark). For electron microscopy, the sections were washed in PBS and postfixed in 1% OsO4, dehydrated, and embedded in epoxy resin. 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 a Jem 1011 electron microscope (Jeol, Japan) with an accelerating voltage of 80 kV.

3. Results and Discussion

Confocal microscopy demonstrated that GBM forms clusters of polymorphic cells with branching processes of various diameters and directions, containing GFAP (Fig. 1A). All GBM samples exhibited positive Cx43 signals in the form of dots of different sizes located on the cell processes. Figure 1B shows the nucleus of a tumor cell and Cx43 points located along its periphery, apparently on the processes of the cells. Co-localization analysis demonstrated that most Cx43 points are localized in processes containing GFAP in GBM and around microcapillaries. In some cases, the Cx43 signals were observed as narrow lines associated with the cell bodies (Figure 1C, 1D, arrow). Serial optical imaging (0.2 μm step size along the Z-axis) unveiled variations in the depth at which Cx43 signals were detectable, indicating the heterogeneous size of Cx43. The Cx43-positive signals within the examined volume were also correlated with GFAP-positive structures in GBM. We observed Cx43+ reaction in the form of dots in all tumor samples. In each sample, we performed a separate count of dots (elements) around tumor cells (n=20). The median of the numerical series was determined to be equal to 11. In this case, the results ranged from 5–20 elements per sample. Spermine was found to be overexpressed in all tumor samples. Spermine was localized in the cytoplasm, and the large and thin tumor processes were in the intercellular space of GBM.

Figure 1.

Immunofluorescence and electron microscopic examination of Cx43 and spermin in glioblastomas. A – localization of GFAP in GBM cells, scale bar 20 μm; B – localization of Cx43+ dots around the tumor cell, scale bar 5 μm; C – Cx43+ dots in the GFAP branches around the microcapillary and Cx43+ lines on membranes of single cells (arrow), scale bar 10 μm; D – Cx43+ signal in GBM, scale bar 10 μm; E – GFAP+ signal, indicate the glial fibrills, Sytox green indicate nuclei, scale bar 10 μm; F – Localization of spermine in GBM, scale bar 10 μm; G – Merge of GFAP, Sytox green and spermine, scale bar 10 μm; H – Electron immunohistochemistry with antibodies to spermine. Two cells of GBM accumulated spermine (sper). Magnification - 12 000; I – Gap junction formed by the spermine-containing branch. Legend: GF-glial fibrils, MB- myelinated branch, arrows-gap junction. Magnification - 80 000; J – Cx43+ signals in GBM, scale bar 10 μm; K – Spermine+ signals in GBM, scale bar 10 μm; L – Co-localization of Cx43 and spermine as yellow dots in tissue and yellow membrane reactivity of cells, scale bar 10 μm.

Moreover, a positive spermine reactivity was identified in regions where GFAP response was weak or negative (Fig. 1E–G). Furthermore, we observed heterogeneity in the expression of polyamines in the specific areas of the glioblastoma. In addition, heterogeneity in the expression of polyamines in some areas of glioblastoma was noted. GBM regions containing the most significant number of spermine were further examined by electron immunohistochemistry.

Electron immunohistochemistry showed that polyamine mainly accumulated in the cytoplasm of GBM cells, located amidst a network of glial filaments and remnants of myelinated fibers of neuron axons (Figure 1H) — these large bizarre cells with pleomorphic atypical nuclei often contained inclusions. The nuclei were frequently segmented and did not contain spermine. Multiple Golgi complexes and centrioles indicated the process of endomitosis. The polyamine-containing cell processes formed contact with gap junctions (Figure 1I). They were seen as “stuck together” bilipid membranes with an osmophilic gap between them. Co-expression of polyamine and Cх43 was done by double fluorescent labeling. The analysis showed that both Cx43 and spermine had the exact intracellular localization. However, connexin had membrane reactivity, while spermine had both membrane and cytoplasmic reactivity (Figure 1J, K). Merging the two signals showed co-expression in the same areas: the membranes of individual cells and individual points on processes in the tumor tissue (1L, yellow color). Therefore, we established the staining of the co-localization of Cx43 and the polyamine spermine within glioblastoma, revealing that tumor processes housing the polyamine indeed form gap junctions, suggesting their potential joint interaction. The obtained results show that GBM contains numerous positive fluorescence signals of Cx43 in the form of dots and lines on cell membranes that indicate hemichannels and gap junctions. The multiple gap junctions in GBM may support the hypothesis of forming syncytium for electrotonic and metabolic transmission between glioma cells, promoting its progression. Gap junctions can facilitate 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. Therefore, it is essential to identify molecules that influence the closed and open states of these contacts.

It has been demonstrated the joint staining of Cx43 and polyamines, which play a role in opening the gap junctional channelsxFurthermore, polyamines have been shown to eliminate the blockades caused by hydrogen ions and calcium [7], which is crucial for cellular physiology. The detection of both gap junction networks and spermine overexpression in the same areas of glioblastomas suggests that glioma cells may be able to integrate into the surrounding neural networks of the brain, potentially serving as a mechanism to release glycolysis products [8]. Implementing these functions in syncytium relies on the opening of gap junctions, which is facilitated by spermine.

4. Conclusions

We have shown, for the first time, a significant number of Cx43 and polyamines in GBM cells. In glial syncytia, gap junctions are crucial for regulating ion concentrations, neurotransmitters, metabolic processes, and homeostasis. Polyamines, like spermine, facilitate potassium and calcium wave propagation in brain tumor syncytia. Gap junctions and hemichannels can directly control the activity of individual neurons and neural networks surrounding the tumor. To carry out these functions in the syncytium, a necessary condition is opening the gap junctions associated with the presence of polyamines (spermine, spermidine, putrescine, agmatine).