1. Introduction
Glioblastoma multiforme (GBM) is the most aggressive and lethal type of tumor of the central nervous system (CNS), with a median patient survival of 15 months from the time of diagnosis [
1]. Unfortunately, current standard of care, including surgical resection, radiotherapy, and oral administration of the DNA-alkylating drug temozolomide, show limited efficacy in GBM treatment [
2].
The Cancer Genome Atlas (TCGA) proposed four main GBM subtypes based on their genomic features: proneural, mesenchymal, neural, and classical [
3,
4]. Several studies identified the parameters that can be used to characterize various subtypes of the tumor [
4,
5,
6]. Recent data show that a proneural-to-mesenchymal transition (PMT) may occur at different stages of the disease. Tumor transformation into mesenchymal subtype GBM stem cells is associated with more aggressive phenotypes, treatment resistance, and tumor recurrence [
7,
8].
The microenvironment of GBM cells also plays an important role in the development, recurrence, and resistance of brain cancer [
9]. GBM cells undergo dynamic and reversible transitions in response to changes in the microenvironment, which in turn changes the spatial and temporal phenotypic heterogeneity of the tumor [
10]. Numerous studies show that interaction with neurons, as well as direct and indirect consequences of neuronal activity, can influence tumor progression [
11,
12].
It has been shown that neurons affect tumor growth and progression by forming functional synapses with glioma cells [
13,
14]. For instance, GABA-mediated synaptic transmission inhibits GBM growth, whereas active release of glutamate increases cell proliferation [
15]. Other signaling molecules transmitted between glioma cells and normal neurons are brain neurotrophic factor, neuroligin-3, and dopamine [
16]. The cholinergic projections are also known to innervate regions where GBM develops, releasing the neurotransmitter acetylcholine (ACh) [
17].
Several subtypes (mainly containing α1, α7, α9, and α10 subunits) of nicotinic acetylcholine receptors (nAChRs) effectively permeate calcium ions [
18]. It has been confirmed that GBM cells express certain types of nAChRs, and the functionality of these receptors has been demonstrated [
17,
19]. The expression of the majority of nAChR genes in GBM cells is at a lower level compared to control samples. In patient-derived cell lines, the most frequently expressed nAChR subunit mRNAs are
CHRNA5 (which encodes α5 subunit) and
CHRNA9 (α9), and at a lower level,
CHRNA7 (α7) and
CHRNA10 (α10). In the model U87MG line, the expression of muscle nAChR subunits
CHRNA1 (α1) and
CHRNB1 (β1) was also shown [
17,
19].
Intracellular calcium has been implicated in various signaling pathways that affect tumor progression. Several studies suggest that GBM proliferation, migration, and invasion change in the presence of nAChR ligands [
17,
19]. These effects are predominantly mediated by α7 and α9 nAChRs [
17,
19]. Some GBM model lines such as U251 proliferate in the presence of the nAChR agonist acetylcholine [
17]. Complementary to that, 10
−7 M nicotine increases the proliferation of the U87MG and GBM5 lines [
19]. The effect of nicotine is blocked by both α-bungarotoxin (α-Bgt) and methyllycaconitine (MLA), which unselectively inhibit muscle, α7, and α9 nAChRs, as well as by the α7-selective antagonist α-conotoxin analogue ArIB (V11L; V16D) and the α9-selective antagonist α-conotoxin RgIA [
19].
Furthermore, GBM can easily develop radio- and chemoresistance due to the extreme heterogeneity of these tumors [
20]. Different GBM cell subpopulations retain distinct genetic profiles, biomolecular markers, and functional properties [
21]. In this regard, it is crucial to choose the right models to study molecular and biochemical features and to test the effects of potential therapeutic substances. Patient-derived cancer stem cell (CSC)-enriched cultures grown in serum-free media are the most accepted standard for studying the biology of GBM in vitro [
21]. However, the same patient-derived cultures grown under standard in vitro conditions with serum-supplemented media have gene expression profiles that are dramatically different from either CSCs or the original tumors [
22].
In this paper, we explore nAChR subtypes in patient-derived and in model U87MG GBM cell lines using a range of methods, including real-time PCR, fluorescent calcium imaging, electrophysiology, and AlamarBlue proliferation assay. In our work, we studied the effect of cultivation conditions of patient-derived cultures on the expression and functional activity of different nAChR subtypes, as well as the effect of nAChR ligands on cell proliferation under different growing conditions. To distinguish the effects of different nAChRs, a variety of ligands was used in the current study (
Figure 1). Among them, epibatidine activates all nAChR subtypes except α9, for which it is an inhibitor. In order to determine which specific nAChR subtype activation is associated with changes in GBM cell proliferation, the α1, α7, and α9 subunit-selective antagonists azemiopsin, α-conotoxin [A10L]PnIA, and α-conotoxin RgIA, respectively, were added to cells, along with epibatidine, acetylcholine, or choline or without any externally added agonists. To our knowledge, this is the first report on the use of several nAChR subtype-selective peptide neurotoxins in studying primary GBM.
3. Discussion
In recent years, a wide range of methods has been applied to study GBM, which made it possible to understand certain mechanisms of tumor development [
32]. Cancer cell lines have been used as the standard both for studying the biology of human tumors and as preclinical screening models of potential therapeutic agents [
32]. However, it is becoming increasingly clear that phenotypic characteristics and a variety of genetic aberrations found in cancer cell lines, which repeatedly occurs in vitro, for instance, often bear little resemblance to those found in the corresponding human patient-derived tumor. This may have led to some significant misinterpretations regarding the significance of aberrant signaling pathways within cell lines compared to patient-derived tumors. GBM tumor cell populations have a variety of functional and molecular biological features, which makes this type of tumor extremely heterogeneous [
20]. In particular, glioblastoma stem cells (GSCs), which are characterized by increased resistance to chemotherapy and radiotherapy, are present in cell populations [
33].
The immortalized U87MG cell line has been used in many laboratories worldwide, but due to genetic drift in FBS-supplemented medium and successive passages, the cells with the highest proliferative potential are selected, reducing the genetic heterogeneity inherent to the original tumors, which can affect the reproducibility of studies [
34]. The cell cultures obtained from patients are more representative models, but the use of media containing fetal bovine serum (FBS) can lead to loss of the GSC subpopulation [
34], making the tumor homogeneous. Among other things, the presence of GSCs is closely linked to the formation of spheroids, called neurospheres, in tumor cell cultures of neural-originating tumors [
35]. It was previously shown that glioma cells cultured in either serum-containing or serum-free medium showed profound biological differences, particularly, in terms of the increased differentiation of CSCs and the loss of tumor heterogeneity [
22]. One approach to the cultivation of cells as microspheres to prevent the loss of tumor heterogeneity is to use serum-free medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and a neuronal viability supplement (B27 or NS-21) [
36].
In the current work, the effects of the cultivation conditions on the expression of several nAChR subtypes in different patient-derived GBM cell lines were investigated. Using nAChR subtype-selective ligands, we also studied the roles played by these receptors in the proliferation of GBM cell lines. The presence of nAChR gene expression of several nAChR subunits in patient-derived cultures of samples 011, 019, 067, and 022 at the mRNA level was demonstrated. Under different cell culture conditions (FBS-supplemented vs. NS-21/EGF/bFGF-containing), patient-derived GBM cultures from different human sources showed distinct profiles of nAChR gene expression (
Figure 4). Of note, α5 and α9 nAChR mRNA were detected in all GBM lines tested. GBM line 011 expressed the β2 nAChR subunit gene exclusively when cultured on FBS-supplemented medium, and mRNA of the α9 nAChR subunit was significantly increased in FBS-supplemented medium in contrast to serum-free medium. Lines 011, 019, and 022 showed a significant increase in muscle nAChR α1 subunit gene expression in serum-free medium in contrast to FBS-supplemented medium. Gene-encoding α7 nAChR was reduced in serum-free medium compared to FBS-supplemented medium in the U87MG model cell line, but in the 019 line, the expression of the same nAChR gene was observed only on FBS-supplemented medium, whereas other lines expressed this receptor independently of culturing conditions. Overall, mesenchymal lines 022 and 067 showed less dependence on nAChR gene expression on the composition of the medium (FBS-supplemented or serum-free medium) than proneural lines 011 and 019. The presence of nAChR in mesenchymal line 067 was confirmed by specific binding of fluorescent ligand Alexa Fluor 555 α-bungarotoxin (
Figure 5), showing that nAChR gene expression led to the respective protein production by GBM cells.
The functions of nAChRs in GBM cell cultures were studied using selective antagonists—α-conotoxins [A10L]PnIA and RgIA, which bind preferably to α7 and α9 nAChRs, respectively [
37]. It should be noted that RgIA can also bind to GABA
B receptors, which might be expressed by GBM cells. However, the use of RgIA with the respective nAChR agonists choline and acetylcholine allowed for the effects to be monitored selectively. The neurotoxin azemiopsin from
Azemiops feae viper venom is known to selectively block muscle α1β1δε nAChR [
38]. The activity of the receptors was monitored using fluorescent Ca
2+ imaging. Acetylcholine application to cells evoked an increase in intracellular Ca
2+, which was detected as a fluorescence intensity increase (
Figure 6). If this increase in calcium was depressed by a selective antagonist, it was interpreted as mediated (at least in part) by the respective nAChR subtype. Interestingly, the U87MG cell line showed a significant depression in the acetylcholine-evoked calcium increase by α7 nAChR-targeting [A10L]PnIA only in cells cultured on FBS-supplemented medium (
Figure 7A), which is in a good agreement with RT-PCR data showing a decrease in α7 nAChR gene expression under serum-free conditions. However, no significant evidence of functional muscle or α9 nAChR was found in U87MG cells grown on the medium containing either FBS or serum.
The proneural cell line 011 did not show signs of an acetylcholine-evoked calcium increase in either serum-free or serum-supplemented medium (
Figure 7B). The other proneural GBM line 019 demonstrated strong evidence of muscle, α7, and α9 nAChRs when cultured on FBS-supplemented medium (
Figure 7C). The presence of α7 nAChR was further supported by patch-clamp recordings of whole-cell currents elicited by the selective α7 nAChR agonist in combination with the selective α7 nAChR positive allosteric modulator (
Figure 7D). Despite nAChRs being detected by RT-PCR in 019 cells grown on both types of medium, no significant effects of selective inhibitors were detected in calcium-imaging experiments on cells cultured in serum-free conditions (
Figure 7C, right panel), suggesting the significant influence of culture conditions on the receptors’ translation or membrane transport. In contrast to proneural GBM lines 011 and 019, mesenchymal lines 022 and 067 both showed strong evidence of α7 nAChR functioning only when cultured on serum-free medium (
Figure 7E and
Figure 7F, respectively). These results demonstrate the variability of the GBM cell lines in respect to functional nAChR expression.
It was recently reported by Pucci et al. that nicotine and choline increased the proliferation of glioblastoma model cell lines U87MG and GBM5 [
19]. In this report, the U87MG line was cultured in FBS-supplemented medium and GBM5 was kept in serum-free medium supplemented with Neurobasal and B27 additives. Interestingly, Neurobasal supplement contains choline at a concentration of 28.6 μM. The concentration of choline in blood serum is estimated to be in the range of 7.1–28.9 μM [
30]. Some reports used LC-MS/MS to estimate the choline concentration in blood plasma at a level of 11.4 ng/μL, which corresponds to ~100 μM [
29], suggesting that in medium supplemented with 10% FBS, the choline levels can reach concentrations of up to 10 μM. A similar choline concentration was obtained in 1:1 DMEM/F12 with a Neurobasal mixture. Thus, GBM cells under such experimental conditions may be affected by constant muscle, α7, and α9 nAChR activation.
Choline is a metabolic precursor of the major endogenous cholinergic neurotransmitter acetylcholine and plays an important role in nAChR function [
39]. It has been shown to act as an agonist of muscle, α7, and α9 nAChRs and as a modulator of α3β4 nAChR [
26,
40,
41]. Additionally, it has recently been shown that choline promotes glioblastoma cell proliferation [
19]. In our work, the effects of nAChR agonists on various GBM cell lines were investigated in relation to cell-culturing conditions. To test the possibility of choline influence on the results, serum-free medium was based on the Neuro Brew
TM NS-21 supplement (reformulated variant of B27), which does not contain choline.
In the current study, no significant effect of epibatidine (which is an agonist of all nAChR subtypes except α9) on U87MG or patient-derived GBM microsphere cultures was detected in a wide range of concentrations (3 nM–1 μM) (
Figure 6). Surprisingly, the application of nAChR antagonists along with epibatidine in some experiments stimulated proliferation: α-conotoxin [A10L]PnIA, which selectively inhibits α7 nAChR, stimulated the proliferation of proneural cell line 011 grown on FBS-supplemented medium (
Figure 8A), but no effects of [A10L]PnIA or azemiopsin were detected in serum-free medium (
Figure 8B). No effect of nAChR ligands (antagonists or agonists) was detected on the proliferation of proneural cell line 019 (
Figure 8C,D) despite this line clearly showing the presence of functional nAChRs in calcium-imaging and patch-clamp experiments. It is widely accepted that nAChR-mediated effects are dependent on protein kinase C activation and subsequent mitogen-activated protein kinase signal cascades [
16]. Thus, exact reactions of the GBM to nAChR ligands should depend not only on nAChR subtype surface expression but also on the pre-existence of such mechanisms in GBM cells.
The proliferation of mesenchymal cell lines 022 and 067 and model line U87MG cultured on FBS-supplemented and serum-free media was stimulated by [A10L]PnIA, suggesting the possible role of α7 nAChR, which is in a good agreement with calcium-imaging and RT-PCR results (
Figure 8E–J). Azemiopsin, the selective muscle nAChR inhibitor, stimulated proliferation of mesenchymal line 022 and model line U87MG in both types of medium (FBS and serum-free, see
Figure 8E,F,I,J), but for mesenchymal line 067 its effect was detected solely in FBS-supplemented medium (
Figure 8H).
In contrast to data reported by Pucci et al., the activation of nAChR by epibatidine did not stimulate the proliferation of GBM cells (apart from a subtle effect on the proneural 011 line in serum-free medium, see
Figure 8B) [
19]. At the same time, the inhibition of α7, α9, and muscle nAChRs stimulated cell proliferation. A similar effect of α7 and α9 nAChR-selective α-conotoxins on glioma cells was previously shown [
42]. In our study, the influence of agonists on proliferation was studied in the absence of externally added agonists (
Figure 9). Strikingly, on the FBS-supplemented medium, the U87MG model line and proneural GBM patient-derived cultures 011 and 019 demonstrated a significant increase in proliferation in response to α9 nAChR-selective antagonist RgIA (
Figure 9A–C). The U87MG and 019 lines also increased proliferation in response to muscle and α7 nAChR-selective antagonists in FBS-supplemented medium (
Figure 9A,C). No significant nAChR antagonist-stimulated proliferation was detected on serum-free medium for these lines. Mesenchymal GBM spheroid patient-derived cultures 022 and 067 did not increase proliferation in response to nAChR antagonists on either FBS-supplemented or serum-free medium (
Figure 9D,E). In these experiments, the response to antagonists was higher in cells grown in FBS-supplemented medium. Two explanations for these results have been proposed: (i) nAChR agonist choline normally contained in blood serum increases cell surface expression of the respective nAChRs, which partially correlates with RT-PCR results (
Figure 4), e.g., line 011 showed an increased expression of the α9 nAChR gene under FBS conditions in comparison with serum-free medium. In the same line, proliferation increased in the FBS presence in response to α9 nAChR-selective antagonist RgIA. (ii) Cell cultivation in serum-supplemented medium leads to an increase in cell differentiation due to a lower concentration of growth factors in serum-supplemented medium than in serum-free medium supplemented with EGF and bFGF [
22]. Cells differentiation leads to more stable proliferation rates, i.e., a lack of response to external stimuli (nAChR antagonists).
Since nAChR inhibitor-provoked proliferation tends to emerge (although not exclusively) in FBS-supplemented medium, which by design contains choline as a component of blood serum, the possible role of choline was studied via external addition n varying concentrations to the serum-free medium (
Figure 10). No strong evidence of choline provoking cell proliferation was detected on serum-free medium for the U87MG, 011, 019, or 022 line (
Figure 10A–D). There was some indication of 100 nM of choline stimulating proliferation of the 067 mesenchymal line (
Figure 10E). However, similar to the results of the epibatidine experiment (
Figure 8), the addition of selective antagonists along with choline provoked proliferation of the 011, 019, and 067 lines (
Figure 10B,C,E). Note that no antagonist-stimulated proliferation was detected on the same serum-free medium when no choline was added (
Figure 9), suggesting that some basal nAChR activation by choline is needed for nAChR antagonist-stimulated proliferation.
The serum-free medium used in current study contained EGF and bFGF. EGF has previously been shown to support growth and tumor spread [
43]. In particular, EGF is secreted by tumor-associated macrophages and microglia. EGF secretion plays a role in the mechanisms of healing and regeneration of brain tissues, determining the migration of microglia to GBM lesions [
44]. EGF influences glial-mesenchymal transition and promotes the microevolution of GBM malignancy and enhances the invasive potential of GBM cells and their ability to penetrate healthy tissues [
45]. Thus, the conditions for growing cell cultures determine the heterogeneity of the cell population and their microevolution even in the absence of an active microenvironment (e.g., tumor-associated macrophages). To account for the possible influence of growth factor combinations on nAChR antagonist-stimulated proliferation, the 067 line was grown on NS-21-supplemented serum-free medium, bFGF-supplemented serum-free medium (NS-21/bFGF), or plain serum-free DMEM/F12 (
Figure 9). Notably, on medium supplemented solely with NS-21, no significant effects of epibatidine or nAChR antagonists in the presence of epibatidine were detected (
Figure 11A). Cell proliferation in the presence of bFGF was stimulated by α7 nAChR antagonist [A10L]PnIA (in mixture with epibatidine), but no effects of epibatidine itself were detected (
Figure 11B). Interestingly, on plain serum-free DMEM/F12, epibatidine significantly reduced cell proliferation, and its effect was suppressed by muscle nAChR-selective inhibitor azemiopsin and by α7 antagonist [A10L]PnIA (
Figure 11C).
Somewhat unexpectedly, azemiopsin significantly stimulated proliferation of 067 cells grown on NS-21-supplemented serum-free medium without EGF or bFGF (
Figure 11D). The addition of bFGF to the medium diminished azemiopsin-stimulated proliferation and promoted the effects of [A10L]PnIA and RgIA, which might indicate that bFGF enhances α7 and α9 nAChR expression or function but inhibits the expression of muscle nAChR (
Figure 11E). No pro-proliferative effect of antagonists was detected on plain DMEM/F12 serum-free medium (
Figure 11F), suggesting that despite the functional expression of muscle and α7 nAChRs (compare
Figure 11C,F), antagonist-stimulated effects are dependent on the activation of the receptor by an agonist. NS-21, on which the Neuro Brew
TM used in the current study is based, contains ethanolamine [
46]. To our knowledge, ethanolamine does not activate human nAChRs but is a predecessor of the choline and acetylcholine in the biosynthetic pathways, which might explain the significant stimulation of cell growth by α7 and α9 nAChR antagonists on serum-free medium containing Neuro Brew
TM (
Figure 11). Additionally, the distinct anti-proliferative effect of nAChR agonist epibatidine on plain medium might be explained by the increase in intracellular Ca
2+ levels above a critical toxic threshold due to chronic nAChR activation. This effect might be suppressed by nAChR inhibition. However, no cytotoxic effect of nAChR activation has been shown in medium-containing growth factors. In this light, it should be mentioned that both EGF and bFGF show neuroprotective effects against calcium-induced excitotoxicity in vitro [
47].
5. Materials and Methods
5.1. Media for the Cultivation of GBM Cells
Medium I (FBS-supplemented medium): DMEM/F12 (Sigma–Aldrich, Taufkirchen, Germany) containing 2 mM glutamine, 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin–streptomycin solution (Sigma–Aldrich, Germany).
Medium II (serum-free medium): DMEM/F12 (Sigma–Aldrich) containing 2 mM glutamine, 2% NS-21 (MACS NeuroBrew-21 supplement (Miltenyi Biotec, Waltham, MA, USA)), 20 ng mL−1 basic fibroblast growth factor (bFGF; Sigma–Aldrich), 20 ng mL−1 epidermal growth factor (EGF; Sigma–Aldrich), 1% penicillin–streptomycin solution (Sigma–Aldrich).
Medium III: DMEM/F12 (Sigma–Aldrich) containing 2 mM glutamine, 2% NS-21 (MACS NeuroBrew-21 supplement (Miltenyi Biotec)), 20 ng ml−1 basic fibroblast growth factor (bFGF; Sigma–Aldrich), 1% penicillin–streptomycin solution (Sigma–Aldrich).
Medium IV: DMEM/F12 (Sigma–Aldrich) containing 2 mM glutamine, 2% NS-21 (MACS NeuroBrew-21 supplement (Miltenyi Biotec)), 1% penicillin–streptomycin solution (Sigma–Aldrich).
Medium V: DMEM/F12 (Sigma–Aldrich) containing 2 mM glutamine, 2%, 1% penicillin–streptomycin solution (Sigma–Aldrich).
5.2. Cell Culture
All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. bFGF and EGF were added twice weekly, and the culture medium was changed every 5–10 days. To attach the cells to the glass or plastic, the surface was preincubated overnight at room temperature with a solution of laminin in PBS (1:200). Patient-derived culture microspheres were dissociated using StemPro Accutase (Thermo Fisher Scientific, Waltham, MA, USA). U87MG cells were dissociated using Trypsin-Versene solution (PanEco, Moscow, Russia).
5.3. RNA Isolation and RT-qPCR
RNA was isolated using the ExtractRNA kit (Evrogen, Moscow, Russia). RNA concentration was determined using a Nanodrop One C spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using the MMLV reverse transcription kit (Evrogen) according to the manufacturer’s protocol. qPCR was performed on a LightCycler 96 (Roche, Basel, Switzerland) with qPCRmix-HS SYBR reagent (Evrogen). The cycling conditions were 95 °C for 150 s, followed by 45 cycles of 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. Data were collected using LightCycler software (version 4.1). The 18S RNA was used as an intermediate control. The primer sequences are provided in
Supplementary Table S1.
5.4. Single-Cell Calcium Imaging
Patient-derived cultures of GBM cells, as well as U87MG cells, were grown in 96-well plates in media I and II at 37 ° C, 5% CO2 atmosphere, 100% humidity. Before calcium imaging began, the growing medium was replaced with extracellular buffer (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4), and then each well was loaded with a cell-permeant 2,5 μM Fluo-4AM solution (ex/em = 494/506 nm; Thermo Fisher Scientific) for 40 min. The fluorescent dye solution was further removed, and cells were kept in an extracellular buffer for 1 h. Ca2+ dynamics were recorded using an Olympus IX71 epifluorescent microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Tokyo, Japan). The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently. Videos were recorded and processed using CellA imaging software version 3.1 build 1274 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Data analysis was performed using open-source ImageJ Fiji software (version 1.54f), where changes in fluorescent intensity per cell before and after the nAChR ligand exposure were calculated. The response of at least 5 cells was measured.
5.5. Patch-Clamp
Cells were grown on laminin-covered glass placed on 24-well plates under the same conditions described in
Section 2.4. To conduct the electrophysiological recording of the α7 nAChR-mediated macroscopic currents, a whole-cell patch clamp was set up as follows. Cells attached to the glass were transferred to a bath filled with extracellular electrophysiology solution (140 mM NaCl, 2 mM CaCl
2, 2.8 mM KCl, 4 mM MgCl
2, 20 mM HEPES, 10 mM glucose, pH 7.4). A microelectrode pipette was filled with intracellular buffer solution (140 mm CsCl, 6 mm CaCl
2, 2 mm MgCl
2, 2 mm MgATP, 0.4 mm NaGTP, 10 mm HEPES/CsOH, 20 mm BAPTA/KOH). A microelectrode was attached to the cell membrane until a resistance of at least 1 GOhm was reached. After establishing the gigaseal, the cell membrane was ruptured using suction and the recordings were created. A typical experiment consisted of 1 s cell wash with control extracellular buffer, then the bath solution was changed to an extracellular solution with 1, 10, or 100 μM of α7 nAChR-selective agonist PNU-282987 (Tocris, Bristol, UK) supplemented with 10 μM of α7 nAChR-selective positive allosteric modulator PNU-120596 (Tocris) for 5 s followed by 5 s wash-out with control extracellular buffer.
5.6. Fluorescent α-Bungarotoxin Binding Assay
GBM cells were grown on laminin-covered glass placed on 24-well plates under the same conditions described in
Section 2.4. To assess the cell expression of α7, α9, and muscle-type nAChRs, the cells were fixed with 4% PFA and then stained with Alexa-Fluor 555-conjugated α-bungarotoxin (50 nM, α-Bgt-Alexa 555, Thermo Fisher Scientific) overnight at 37 °C. The cells were washed extensively with extracellular buffer (140 mM NaCl, 2 mM CaCl2, 2.8 mM KCl, 4 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH 7.4) to remove any unbound α-bungarotoxin. Controls were run simultaneously with 1 μM of unlabeled d-tubocurarine (Tocris). Twelve-bit digital images were obtained using a DuoScanMeta LSM510 laser scanning microscope (Carl Zeiss, Weimar, Germany) equipped with a Plan-Apochromat 63×/1.40 objective (numerical aperture). Image acquisition parameters were as follows: for green fluorescence—excitation at 488 nm and emission at 505–550 nm, for red fluorescence—excitation at 561 nm and emission at 575 nm. Pictures were processed with open-source ImageJ Fiji software version 1.54f.
5.7. Cell Proliferation Assay
Cells were plated on a 96-well plate at a density of 6000 cells per well in 150 μL medium. The number of cells was assessed using AlamarBlue reagent (Thermo Fisher Scientific). The fluorescence was measured using a Fusion α-FP HT Universal Microplate Analyzer (PerkinElmer, Waltham, MA, USA) with an excitation filter for 535 nm and an emission filter for 620 nm. The measurements were taken on day 5.
5.8. Data and Statistical Analysis
The non-parametric Kruskal–Wallis test and Dunn’s post-hoc test were used to study the significance of the effects in the calcium-imaging assay due to the obvious deviation of response amplitudes from normal distribution. One-way or two-way ANOVA with Tukey’s or Dunnett’s post-hoc test was used to analyze cell viability assays.