P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro

Glioblastoma is the most aggressive and lethal primary brain malignancy with limited treatment options and poor prognosis. Self-renewing glioblastoma cancer stem cells (GSCs) facilitate tumour progression, resistance to conventional treatment and tumour recurrence. GSCs are resistant to standard treatments. There is a need for novel treatment alternatives that effectively target GSCs. The purinergic P2X receptor 7 (P2X7R) is expressed in glioblastomas and has been implicated in disease pathogenesis. However, the roles of P2X7R have not been comprehensively elucidated in conventional treatment-resistant GSCs. This study characterised P2X7R channel and pore function and investigated the effect of pharmacological P2X7R inhibition in GSCs. Immunofluorescence and live cell fluorescent dye uptake experiments revealed P2X7R expression, and channel and pore function in GSCs. Treatment of GSCs with the P2X7R antagonist, AZ10606120 (AZ), for 72 hours significantly reduced GSC numbers, compared to untreated cells. When compared with the effect of the first-line conventional chemotherapy, temozolomide (TMZ), GSCs treated with AZ had significantly lower cell numbers than TMZ-treated cultures, while TMZ treatment alone did not significantly deplete GSC numbers compared to the control. AZ treatment also induced significant lactate dehydrogenase release by GSCs, indicative of treatment-induced cytotoxic cell death. There were no significant differences in the expression of apoptotic markers, Annexin V and cleaved caspase-3, between AZ-treated cells and the control. Collectively, this study reveals for the first time functional P2X7R channel and pore in GSCs and significant GSC depletion following P2X7R inhibition by AZ. These results indicate that P2X7R inhibition may be a novel therapeutic alternative for glioblastoma, with effectiveness against GSCs resistant to conventional chemotherapy.

self-renewal and differentiation into phenotypically diverse tumour cell populations contributing to tumour heterogeneity (Bjerkvig et al., 2005).The currently available glioblastoma treatments are ineffective in targeting GSCs.Conventional treatment modalities and the natural dynamic shifts in the tumour microenvironment are known to induce GSC differentiation (Ayob and Ramasamy, 2018).The mechanisms of GSC-mediated tumourigenicity and resistance to treatment are complex and multi-faceted.Recent studies have demonstrated GSCs are able to mediate angiogenesis (Xiong et al., 2024), promote immune suppression (Cao et al., 2023) and interact with surrounding immune cells to promote growth (Peng et al., 2022).Generally, a small number of treatment-resistant GSCs remain after most tumour cells have been destroyed by therapy.Residual GSCs subsequently propagate new tumour mass with an increased proportion of treatment-resistant cells, leading to tumour relapse and further treatment resistance (Piper et al., 2021).Moreover, treatment evasion by GSCs occurs due to their ability to remain quiescent, maximise DNA repair capacity, downregulate apoptotic pathways, regulate the release of reactive oxygen and nitrogen species, and manipulate the glioblastoma microenvironment (Li et al., 2021).Studying the effects of novel therapeutic strategies on GSCs is necessary to overcome hurdles associated with treatment resistance.Importantly, targeting GSC populations in glioblastoma might greatly reduce the likelihood of disease recurrence.
In glioblastoma, there is an overexpression of the purinergic P2X7 receptor (Zanoni et al., 2022).P2X7R is a trimeric transmembrane ion channel activated by ATP levels in the 200 micromolar to 1 millimolar range.A hallmark of P2X7R is its ability to transition between two conductance states.With transient ATP stimulation, it serves as a highly selective cation channel allowing Ca 2+ and Na + influx, and K + efflux across the plasma membrane.Alternatively, prolonged ATP stimulation shifts the channel into a non-selective membrane pore that additionally mediates the transport of larger molecules up to 900 Daltons (Di Virgilio et al., 2017).The receptor is expressed on various cell types, including microglia, macrophages and lymphocytes (Volonte et al., 2012).The roles of P2X7R include cell trophism, cytokine release, inflammation and modulation of cellular metabolism (Monif et al., 2016;Savio et al., 2018;Wilkaniec et al., 2020).P2X7R function has been linked to a range of solid organ and haematological malignancies (Giannuzzo et al., 2016;Ledderose et al., 2016;Pegoraro et al., 2021).We have previously demonstrated P2X7R expression, function and upregulation in human gliomas (Drill et al., 2021;Kan et al., 2020;Monif et al., 2014).Additionally, we revealed significant tumour cell death following P2X7R blockade with its specific antagonist, AZ10606120 (AZ), in primary patient-derived high-grade glioma cultures and the human U251 glioblastoma cell line (Kan et al., 2020).Notably, AZ treatment was more effective at tumour cell depletion than TMZ.However, we did not investigate the effects of P2X7R antagonism in the context of treatment resistance.It would be useful to assess the effects of AZ treatment on treatment-resistant GSCs, as overcoming treatment resistance would greatly reduce tumour relapse.
Studies have linked P2X7R stimulation to cell stemness and differentiation.For instance, P2X7R expression and function has been shown to be increased in embryonic stem cells compared to differentiated neural cells, with receptor expression and activity decreasing with decreasing pluripotency marker levels (Glaser et al., 2014).In other cancers, P2X7R activation has been reported to contribute to the maintenance of cancer stem cells (CSCs) by inducing the transition of cancer cells to CSCs (Arnaud-Sampaio et al., 2020).While P2X7R is highly expressed in GSCs, few studies have investigated the role of P2X7R in these cells and report contradictory results (Ziberi et al., 2019;D'Alimonte et al., 2015).Importantly, the P2X7R channel and pore conductance states and the effects of P2X7R antagonism have not yet been clarified in GSCs.
Here, we characterised P2X7R channel and pore function and explored the therapeutic potential of P2X7R inhibition by AZ in GSCs.
We report P2X7R expression, and channel and pore function in GSCs, implicating a functional role of P2X7R in these cells.Notably, treatment with AZ significantly depleted GSC numbers in vitro, superior to conventional TMZ chemotherapy, which showed no therapeutic benefit.Our data sheds further light on the trophic, tumour-promoting role of P2X7R in glioblastoma.Importantly, this study highlights P2X7R inhibition as a potential novel therapeutic alternative for glioblastoma, particularly given its efficacy in a conventional treatment-resistant context.

Live P2X7R channel function assay
To assess P2X7R channel function, GSCs at 80 % confluency in 8-well 0.8 cm 2 glass culture chambers (Sarstedt, cat no.94.6170.802)were treated with Fluo-4 AM, a synthetic fluorescent calcium indicator (1μg/ mL; Thermo Fisher Scientific, cat no.F14201), for 30 minutes.To determine whether changes in fluorescence intensity was P2X7R-specific, half of the wells were also treated with 25μM of AZ for 15 minutes.Cells were subsequently immersed in Dulbecco's PBS (DPBS) containing 1 mM Ca 2+ (DPBS; Thermo Fisher Scientific Australia, cat no.14287080) and imaged live with a Nikon A1r fluorescence confocal microscope equipped with a humidified live cell incubator.Images were captured at 40× magnification across an 8-minute time-series experiment at an approximate rate of one image per second.For each experiment, a baseline fluorescence reading was measured for the first 40 seconds, followed by the addition of 200μM of bzATP, a P2X7R agonist (Merck Australia, cat no.B6396), to stimulate the P2X7R channel.Ionomycin was added to some wells after the time series experiment to confirm that changes in fluorescence intensity were not due to cell death from AZ treatment.P2X7R channel activity was determined as the relative fold change in fluorescence intensity (FI) from the baseline to after the addition of bzATP (ΔFI/FI Baseline ).The relative fold change in fluorescence intensity per well was determined from the average of all cells in the well (20-50 cells).Fluorescence intensity was directly proportional to the amount of calcium influx into the cell, corresponding to P2X7R channel activity.All images were analysed using the ImageJ image processing software (version 2.1.0/1.53c) (Schneider et al., 2012).

Live P2X7R pore function assay
GSCs at 80 % confluency were exposed to 1μM of YOPRO™-1 Iodide (YP; Thermo Fisher Australia, cat no.Y3603), a large nucleic acid stain permeant to cells with P2X7R pore activation, for 30 minutes across three treatment groups: YP only; YP and bzATP (200μM, 30 minutes); and YP, bzATP and AZ (1μM, 15 minutes; Tocris Biosciences, cat no.3323).Cells were subsequently immersed in PBS and imaged live using a Nikon Ti-E inverted motorised fluorescence microscope at 40× objective.For each well, the YP fluorescence intensity was measured from an average of at least 100 cells.Mean YP fluorescence intensity was quantified for each treatment group.All images were analysed via ImageJ (Schneider et al., 2012).

Treatment reagents
For cell count experiments, GSC monolayers at 80 % confluency were treated with AZ and/or the conventional chemotherapeutic, TMZ (Merck Australia, cat no.T2577), for 72 hours to ensure adequate time for their effects in culture to be observable, as utilised in a previous in vitro cancer study of AZ (Amoroso et al., 2016).A range of AZ concentrations were used, including 5μM, 15μM, 25μM, 50μM and 100μM, to determine the susceptibility of GSCs at multiple concentrations.50μM of TMZ was used, as it correlates with previously recorded peak physiological TMZ concentrations in both tumour and plasma of glioblastoma patients undergoing TMZ maintenance therapy (Beier et al., 2012).AZ and TMZ were dissolved in dimethyl sulfoxide (DMSO; Merck Australia, cat no.W387509) as per manufacturers' instructions.

GSC quantification
After treatment, GSCs were fixed and stained with 5μM of DAPI for 1 hour at room temperature.Cells were washed in triplicate with PBS and mounted onto glass slides with Dako fluorescence mounting medium.Images were acquired by a Nikon Ti-E microscope at 20× objective.The total number of DAPI+ cells were quantified across 16 random fields.Images were analysed with ImageJ (Schneider et al., 2012).

Annexin V assay
We used the eBioscience™ Annexin V Apoptosis Detection Kit conjugated with PE (Thermo Fisher Scientific Australia, cat no.88-8102-72) to delineate whether AZ treatment was inducing apoptosis in GSCs.Briefly, cells treated with DMSO or AZ (15μM) for 72 hours were dissociated with 0.25 % trypsin (Thermo Fisher Scientific Australia, cat no.25200056), processed into a single-cell suspension and washed with Binding Buffer.Cells were resuspended in Binding Buffer to a concentration of 5×10 6 cells/mL and incubated in the dark for 15 minutes at room temperature with PE-conjugated Annexin V at a 1:20 Annexin V to cell suspension ratio.Cells were subsequently washed and resuspended with 200μL Binding Buffer prior to the addition of 1:40 7aminoactinomycin D (7-AAD; Thermo Fisher Australia, cat no.A1310) to assess cell viability.Cells were analysed via flow cytometry using the Beckman Coulter CytoFLEX flow cytometer at the Alfred Research Alliance Flow Core facility (ARAFlowCore, Melbourne, Australia).Data was analysed using the FlowJo software (version 10.8.1).

Lactate dehydrogenase (LDH) cytotoxicity assay
The Roche colorimetric LDH Cytotoxicity Detection Kit (Roche, cat no.11644793001) was utilised to assess LDH levels released in the GSC culture supernatants, according to the manufacturer's instructions.Briefly, cell-free supernatants were exposed to LDH reaction mixture for 25 minutes at room temperature at a 1:1 ratio.Absorbance at 492 nm was measured using the FLUOstar® Omega Plate Reader (BMG LAB-TECH) and was directly proportional to supernatant LDH levels.

Cleaved caspase-3 staining
GSC cultures at 80 % confluency were treated with 15μM of AZ for 72 hours.Untreated cells served as the control.Cells were subsequently fixed and stained with primary rabbit anti-cleaved caspase-3 antibody (1:100; Cell Signalling Technology®, cat no.9664, RRID: AB_2070042) overnight at 4 • C followed by secondary goat anti-rabbit Alexa Fluor 488 (1:200; Thermo Fisher Scientific Australia, cat no.A-11008, RRID: AB_143165) for 4 hours at room temperature.Cell nuclei were counterstained with DAPI at room temperature for 2 hours.Cells were imaged with the Nikon Ti-E inverted motorised fluorescence microscope at 20× objective.For each replicate, the cleaved caspase-3 fluorescence intensity was measured from an average of at least 100 cells.Mean caspase-3 fluorescence intensity was quantified for AZ-treated cells and untreated cells.All images were analysed via ImageJ (Schneider et al., 2012).

Statistical analysis
We used GraphPad Prism 9 for all statistical analyses.Normality of datasets were assessed via Q-Q plots.In experiments involving three or more groups, we utilised a one-way ANOVA with Brown-Forsythe and Welch's corrections with Tukey's HSD or Dunnett's T3 multiple comparisons post-hoc tests.For experiments comparing two groups, an unpaired t-test with Welch's correction was used.The mean difference and corresponding 95 % confidence interval (CI) were reported where necessary.Data was expressed as mean±SEM.The significance level was set at p<0.05.

P2X7R is highly expressed in GSCs
We first performed immunofluorescent staining on GSC monolayers to assess expression levels of P2X7R, common stem cell markers, SOX2 and NANOG, and the glioma cell marker, GFAP.Cells were stained with the following antibodies: primary goat anti-P2X7R with secondary donkey anti-goat Alexa Fluor 488, primary rabbit anti-SOX2 with secondary goat anti-rabbit Texas Red-X, primary rabbit anti-NANOG with secondary goat anti-rabbit Texas Red-X, and primary rabbit anti-GFAP with secondary goat anti-rabbit Texas Red-X.All cells were counterstained with DAPI.Slides were imaged with the Nikon A1r confocal fluorescence microscope.Our findings demonstrate P2X7R expression and moderate SOX2 expression in GSCs (Fig. 1).However, no NANOG or GFAP was visible.

The P2X7R channel and pore is functional in GSCs
P2X7R channel function was assessed by quantifying the change in intracellular calcium levels following channel stimulation with transient exposure to bzATP.GSCs were incubated with Fluo-4, a synthetic fluorescent calcium indicator, and the fold change in fluorescence intensity following bzATP treatment was recorded live over an 8-minute timeseries experiment.The magnitude of calcium influx into the cell is proportional to the level of Fluo-4 fluorescence intensity and thus the amount of P2X7R channel activity.To confirm that the calcium influxes were P2X7R-mediated, some the cells were also pre-treated transiently with the specific P2X7R antagonist, AZ.Pre-treatment with 25μM of AZ significantly inhibited P2X7R channel activity, with a significant reduction in relative fold change in Fluo-4 intensity upon bzATP exposure following AZ treatment (mean relative fold change in Fluo-4 intensity to baseline of 0.61±0.16)compared to untreated cells (mean relative fold change in Fluo-4 intensity to baseline of 4.72±0.45)(Fig. 2).Treatment with 25μM of AZ did not induce cell death, as confirmed with ionomycin administration after the timeseries experiment (Supplementary Figure 1).Based on these data, the P2X7R channel is functional in human GSCs and is significantly inhibited with 25μM of AZ.
To measure P2X7R pore function, GSCs were incubated with either YP (baseline pore activity); YP and bzATP (pore stimulation); or YP, bzATP and 1μM of AZ (pore inhibition).YP is a large fluorescent dye that enters the cell only upon P2X7R pore activation and can thus be utilised to measure P2X7R pore function.For each group, P2X7R pore activity was quantified by the total YP fluorescence intensity.There was a trend showing more intense YP fluorescence intensity in GSCs treated with bzATP (mean YP intensity of 703.6±71.34),compared to GSCs treated with YP only (mean YP intensity of 462.1±66.42),although this was not statistically significant (Fig. 3).Moreover, YP fluorescence intensity was significantly decreased in GSCs treated with AZ (mean YP intensity of 255.4±31.55),compared to both the YP-only and YP + bzATP groups (Fig. 3).These results indicate a functional P2X7R pore, which was inhibited by AZ.Taken together, both the P2X7R channel and pore are functional in GSCs, with AZ treatment inhibiting channel and pore activity.

P2X7R inhibition by AZ depletes GSCs in a concentration-dependent manner
Given that there was widespread P2X7R expression and P2X7R channel and pore function in GSCs, we next characterised the effects of prolonged P2X7R antagonism by AZ in these cells.We have previously demonstrated a significant decrease in tumour cell numbers in primary human high-grade glioma cultures and the U251 human glioblastoma cell line (Kan et al., 2020), but the effects of AZ treatment on GSCs have not yet been investigated in the literature.We first wanted to determine the minimum effective concentration of AZ required for anti-tumour activity in GSCs.GSC monolayers at 80 % confluency were treated with either 5μM, 15μM, 25μM, 50μM or 100μM of AZ for 72 hours.Untreated cells served as the control.Cells were subsequently fixed, stained with DAPI and visualised with fluorescence microscopy.The total number of DAPI+ cells were quantified over 16 fields.We observed lower GSC numbers with increasing AZ concentration, with significantly decreased GSC counts in groups treated with 15μM of AZ and greater, compared to the control (Fig. 4A).There was no significant GSC inhibition with 5μM of AZ, compared to untreated cells (Fig. 4A).Hence, 15μM of AZ was selected as the lowest effective concentration for subsequent experiments.

P2X7R inhibition by AZ significantly reduced GSC count and was more effective than TMZ
To compare the effects of AZ treatment to conventional TMZ  chemotherapy, we treated GSC monolayers at 80 % confluency with either AZ 15μM, TMZ 50μM, or with AZ and TMZ combined for 72 hours.Untreated cells served as the control.GSCs were subsequently fixed, stained with DAPI and imaged with a fluorescence microscope.The total number of DAPI+ cells were counted across 16 fields.There was a significant reduction in GSCs following treatment with AZ 15μM (7220±520.8cells), compared to untreated cells (11,809±700.4cells) (Fig. 4B-C).TMZ treatment did not lower GSC count (9087±471.3cells), compared to the control.Notably, AZ treatment was more effective at GSC depletion than TMZ.However, when co-administered with TMZ, there was no synergistic anti-tumour effect -AZ 15μM (7220±520.8cells) versus AZ + TMZ (7164±289.4cells) (Fig. 4B-C).Moreover, the anti-tumour activity of AZ-TMZ combined was similar to that of AZ treatment alone and was more effective than TMZ (Fig. 4B-C).Collectively, AZ treatment induced significant anti-tumour activity in GSCs, whereas conventional TMZ chemotherapy was ineffective in this assay.This shows promise for AZ as a therapeutic option for glioblastoma.

Inhibition of P2X7R with AZ induces cytotoxic cell death in GSCs
To delineate whether AZ treatment induced a cytotoxic anti-tumour effect on GSCs, culture supernatants from GSCs treated with various concentrations of AZ (5μM, 15μM, 25μM and 100μM) were assessed in a LDH cytotoxicity assay.Treatment with AZ significantly increased LDH levels, compared to untreated cells (Fig. 5).Interestingly, there was a trend for increasing LDH release with increasing concentrations of AZ from 25 to 100μM of AZ.However, GSCs treated with 15μM of AZ released similar quantities of LDH to cells treated with 100μM of AZ, suggestive of a biphasic cytotoxic response between the two concentrations.This should be further explored.Nonetheless, administration of AZ increased LDH release in GSCs, indicative of a cytotoxic anti-tumour effect.

Inhibition of P2X7R with AZ does not induce apoptosis in GSCs
To determine whether AZ-mediated cytotoxicity in GSCs is due to apoptosis, GSC monolayers treated with 15μM of AZ were harvested and incubated with Annexin V. GSCs were also incubated with 7-AAD to assess cell viability.Annexin V levels were quantified via flow cytometry, with cells gated into the following categories: viable (Annexin V-/ 7-AAD-), early apoptotic (Annexin V+ / 7-AAD-) and necrotic (7-AAD+).There was no significant change in the proportion of early apoptotic cells in GSC cultures treated with AZ, relative to untreated cells (Fig. 6B).Notably, we observed a significant mean 1.7±0.14-foldincrease in the proportion of necrotic cells following treatment with AZ (Fig. 6C).No changes in viable cell proportions were observed between each group (Fig. 6A).
We also performed cleaved caspase-3 staining on GSC cultures treated with 15μM of AZ to further elucidate the effect of AZ treatment on apoptosis.There was no significant difference in cleaved caspase-3 expression between untreated cells and AZ-treated cells (Fig. 7).Taken together, data from both Annexin V and cleaved caspase-3 staining suggests that AZ treatment does not induce apoptosis in human GSCs.Future experiments should investigate alternative cell death pathways induced by AZ.

Discussion
Here, we assessed the efficacy of pharmacological P2X7R inhibition by AZ in GSCs and further characterised P2X7R expression and function in these cells.To our knowledge, this is the first investigation of AZ treatment and P2X7R channel and pore function in GSCs.Our results demonstrate P2X7R expression with intact channel and pore function in GSCs.Importantly, inhibition of P2X7R by AZ led to a significant reduction in GSC number.These findings reveal future therapeutic implications of P2X7R inhibition in glioblastoma, particularly in cases of recurrent disease where current treatments are ineffective.The prognosis for patients with glioblastoma remains bleak.The median survival is 14-18 months, with current treatment limited to surgical resection, radiotherapy and chemotherapy (Alexander and Cloughesy, 2017).GSCs within the glioblastoma microenvironment are believed to be major culprits behind treatment failure as these cells contribute to extensive tumour infiltration, heterogeneity, drug resistance and subsequent tumour relapse (Dymova et al., 2021).Hence, development of therapies effective against GSCs is crucial to prevent glioblastoma recurrence.
We used a previously validated human GSC line for this study (Smiley et al., 2021).As expected, our data demonstrating P2X7R expression in GSCs (Fig. 1) aligns with the current literature (Ziberi et al., 2019).P2X7R upregulation has been previously demonstrated in gliomas (Zanoni et al., 2022;Monif et al., 2014).Interestingly, immunocytochemical staining revealed moderate SOX2 expression and no NANOG expression in GSCs (Fig. 1).Both SOX2 and NANOG are considered GSC markers (Lathia et al., 2015), but given the heterogeneity of GSCs, expression of proteins can differ.Studies have previously identified GSCs using CD133, Nestin, CD34, CD90, CD44, L1CAM, A2B5 and GPD1 (Tang et al., 2021;Jin et al., 2013); however, their expression in stem cell lineages is not standardised and so reproducibility may be difficult.Further examination of stem cell marker expression both at the protein and mRNA level is required.
Live cell imaging assays revealed functional P2X7R channel and pore in GSCs (Figs. 2 and 3).Few studies have examined specific roles of the P2X7R channel and pore states on tumour cell growth.Importantly, differential activation of the P2X7R channel and pore states may play a role in the degree of cell trophism.Perhaps the most discussed model in the current literature is the existence of various P2X7R splice variants, which are associated with distinct channel-and pore-forming abilities.P2X7A is the most defined P2X7R isoform, characterised by its ability to function as both an ion channel and a highly permeable pore due to its fully formed C-terminal (Rabelo et al., 2021).In contrast, P2X7B possesses a truncated C-terminal, hindering its pore-forming ability (Rabelo et al., 2021).Previous studies have speculated that activation of P2X7R pore function leads to cell death by dysregulation of cellular homoeostasis (Rotondo et al., 2022).However, studies conducted in exogenous settings involving excessive receptor stimulation are likely not representative of P2X7R pore function in the tumour microenvironment in vivo.Alternatively, P2X7R channel activation is thought to facilitate cellular growth (Rotondo et al., 2022).In this aspect, the tumour-promoting roles of P2X7R activation in glioma might be the result of differential expression of P2X7R splice variants in favour of P2X7B expression.Different P2X7R splice variants likely have altered channel/pore forming capacity and thus have differing functional consequences in the cell.Our study did not specifically focus on P2X7R variants, but given both channel and pore functionality were maintained, we presume no functionally relevant P2X7R truncations in GSCs.Rather, both the P2X7R channel and pore state may contribute to cell growth in glioblastoma.However, future studies should focus not only on the analysis of P2X7R splice variants, but also the presence of single nucleotide polymorphisms which may have relevance to receptor function in the glioblastoma setting.
Interestingly, in the P2X7R pore function assay, there was no significant increase in YP intensity following P2X7R stimulation with bzATP.Of note, the p-value was 0.05, which was not statistically significant (95 % CI of − 483.1-0.028).However, there was an observable trend supporting increased YP uptake following P2X7R stimulation, which may be validated with an increased sample size.Alternatively, the P2X7R pore might be maximally functioning in GSCs.In this aspect, the P2X7R pore plays a key role in NOD-like receptor protein 3 (NLRP3) inflammasome activation, crucial for IL-1β release (Franceschini et al., 2015).IL-1β release is dependent on the P2X7R pore (Monif et al., 2016).The cytokine has been widely characterised as a driver of immunosuppression and tumourigenesis in gliomas (Fathima Hurmath et al., 2014;Wang et al., 2012;Yeung et al., 2013).In fact, IL-1β exposure has been shown to induce stem-like self-renewal properties in glioma cells, promoting neurosphere formation in vitro (Wang et al., 2012).Hence, constitutive P2X7R pore activation in GSCs might play a role in GSC tumourigenesis and cancer cell stemness, whereby continuous P2X7R pore opening leads to IL-1β release that further promotes tumour cell stemness in an autocrine feedback loop.P2X7R inhibition by AZ for 72 hours reduced the number of GSCs in a concentration-dependent manner, with 15μM of AZ being the lowest effective concentration required for significant GSC inhibition (Fig. 4A).Importantly, 15μM of AZ was more effective than conventional TMZ chemotherapy (Fig. 4B).TMZ did not significantly deplete GSCs in this study.GSCs are strongly treatment resistant and GSC resistance to TMZ is well-reported in the literature (Auffinger et al., 2014;Fu et al., 2009;Zheng et al., 2021).No synergistic effect was observed with AZ and TMZ co-treatment (Fig. 4B).Hence, AZ treatment alone was sufficient to induce GSC depletion.We have previously demonstrated significant tumour cell reduction following antagonism of P2X7R with AZ in primary patient-derived glioblastoma cultures and U251 cells (Kan et al., 2020).Studies have not yet investigated the effects of AZ on GSCs, although links between P2X7R and cancer cell stemness have been demonstrated.For instance, P2X7R inhibition with antagonist, Brilliant Blue G, downregulated the expression of CD133 and β-catenin (Matyśniak et al., 2022).β-catenin enhances tumour invasion by promoting EMT and modulates stem cell pluripotency, self-renewal and differentiation capacity (Kalluri and Weinberg, 2009;Kim et al., 2019;Zhang and Wang, 2020).P2X7R activation has been further speculated to induce the phenotypic transition of cancer cells to CSCs (Arnaud--Sampaio et al., 2020).In this regard, P2X7R expression profiles have been linked to the degree of cellular differentiation, with increased receptor expression and activity in embryonic stem cells compared to differentiated neural cells (Glaser et al., 2014).Decreased expression of markers of pluripotency also correlated to decreased P2X7R expression and activity (Arnaud-Sampaio et al., 2020).Taken together, AZ treatment might also indirectly inhibit glioma progression by downregulating the expression of markers of pluripotency and preventing the development of GSCs.
Lastly, AZ treatment significantly enhanced LDH release in GSC cultures, indicative of cytotoxic cell death (Fig. 5).Annexin V and cleaved caspase-3 staining revealed that apoptosis was not induced by AZ treatment (Figs. 6 and 7).Apoptotic cells express phosphatidylserine on the cell surface to which Annexin V binds (Bendall and Green, 2014).Moreover, cleaved caspase-3 is a primary mediator of proteolysis during apoptosis and is therefore a well-defined apoptotic marker (Crowley and Waterhouse, 2016).Caspase cleavage is also the most common supporting evidence for apoptosis (Bendall and Green, 2014).General trophic P2X7R signalling has been reported to prevent apoptosis by activating NFATC1, a known promoter of tumour cell migration (Adinolfi et al., 2009(Adinolfi et al., , 2012;;Mancini and Toker, 2009).In colorectal cancer, P2X7R antagonism with A438079 promoted apoptosis via the Bcl-2, caspase 9 and caspase 3 pathways, both in vitro and in vivo (Zhang et al., 2021).Links between P2X7R inhibition and apoptosis in glioblastomas are not fully understood.Given that our data revealed (Chamberlain, 2010) significantly increased LDH release and increased proportion of necrotic cells following AZ treatment, (Walcher et al., 2020) no differences between cleaved caspase-3 between control and AZ-treated cells, and (Bjerkvig et al., 2005) a reduction in GSC count following AZ treatment, we speculate that the AZ treatment-induced cell death was not caused by apoptosis.Alternatively, P2X7R activation has been linked to increased mitochondrial number and mitochondrial potential (Matyśniak et al., 2022;Sarti et al., 2021).Lack of P2X7R decreased basal respiratory rate, ATP-coupled respiration, resting mitochondrial potential and mitochondrial matrix calcium level (Sarti et al., 2021), suggesting an important role of P2X7R in cellular energy biogenesis.Whether P2X7R inhibition with AZ induces alterations in mitochondrial number and function in GSCs is unknown and warrants further study.Other potential modes of cell death induced by AZ treatment, including autophagy, ferroptosis and pyroptosis, should also be investigated.
This study is not without its limitations.Whilst it focuses on the role of GSCs, a critically important target in the context of glioblastoma, it is limited to investigations conducted in a single in vitro GSC cell line.Future studies should corroborate the findings in multiple patient derived glioblastoma stem cell lines, in addition to in vivo animal models.Nevertheless, our findings are very important and novel demonstrating that AZ can induce cellular toxicity in the GSCs assessed, Fig. 6.Annexin V staining in human glioblastoma stem cells (GSCs) following P2X7 receptor (P2X7R) antagonism with AZ10606120 (AZ).GSCs were treated with 15μM of AZ for 72 hours at 80 % confluency.Untreated cells were the controls.Cells were subsequently harvested and stained with Annexin V conjugated to phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD).Data were quantified via flow cytometry.Data are presented as mean±SEM.Annexin V results were yielded from an unpaired t-test with Welch's correction, N=6 replicates.A) Fold change (FC) in the proportion of viable cells in GSCs treated with AZ relative to untreated cells.The difference was not statistically significant: t(8.05)=0.22,mean difference=-0.01-fold,95 % CI=-0.17-0.14, p=0.83.B) FC in the proportion of early apoptotic cells in GSCs treated with AZ relative to untreated cells.The difference was not statistically significant: T(8.8)=0.67,mean difference=-0.03-fold,95 % CI=-0.14-0.08,p=0.52.C) FC in the proportion of necrotic cells in GSCs treated with AZ relative to untreated cells.t(5.3)=5.05,mean difference=0.7-fold,95 % CI=0.35-1.05,**p=0.003.Data are presented as mean±SEM.
whereas TMZ did not.One of the main reasons for glioblastoma recurrence is lack of efficacy of TMZ and its impact on tumour associated stem cells.The fact that we have demonstrated an anti-tumour impact of AZ on these usually hard to target cells is noteworthy for future development of this therapy to combat not only de novo glioblastoma but also recurrent cases of disease.
Overall, this study demonstrated P2X7R channel and pore function in human GSCs for the first time and confirmed trophic P2X7R activity in glioblastoma.Notably, inhibiting the receptor with AZ depleted GSC count with higher efficacy than TMZ, likely via cellular cytotoxicity independent of apoptosis.More clarification is required on the mode of cell death with a focus on necroptosis and pyroptosis.Also detained analysis of mitochondrial function and GSC energy metabolism in response in response to AZ exposure is warranted.No synergism was observed between AZ and TMZ.Given previous associations between P2X7R and cancer cell stemness, P2X7R might facilitate GSC development and treatment resistance in glioma, which can be prevented by AZ.These findings should be confirmed in other human GSC lines.Correlation and RNA-sequencing analyses between P2X7R and the expression of various GSC markers would provide more insight into the receptor's role in GSC development.It would be interesting to assess the potential synergistic relationship of AZ with radiotherapy, as well as characterise the expression and function of various P2X7R isoforms in GSCs.P2X7Rsilencing experiments on treatment-resistant GSCs would also be useful in delineating the role of the receptor in treatment resistance.Importantly, despite significant associations with treatment resistance, GSCs

Fig. 2 .
Fig. 2. The P2X7 receptor (P2X7R) channel is functional in human glioblastoma cancer stem cells (GSCs).GSCs at 80 % confluency were treated with Fluo-4 AM, a synthetic fluorescent indicator, for 30 minutes.To confirm a P2X7R-specific response, some cells were also treated for 15 minutes with 25μM of the specific P2X7R antagonist, AZ10606120 (AZ).Cells were immersed in Dulbecco's PBS and imaged live with a Nikon A1r fluorescence confocal microscope at 40× magnification over an 8-minute time-series experiment.BzATP (200μM), a P2X7R agonist, was added at approximately 40 seconds in.P2X7R channel activity was determined as the relative fold change in fluorescence intensity (FI) from the baseline after the addition of bzATP (ΔFI/FI Baseline ).A) BzATP stimulation induced P2X7R channel responses.There was a significant decrease in Fluo-4 FI in GSCs treated with AZ. Results were yielded from an unpaired t-test with Welch's correction; n=12 replicates, t(21.23)=8.569,mean difference=-4.12,95 % CI=-5.114 to − 3.118, ****p<0.0001.Data are presented as mean±SEM.B) Graphical representation of the change in FI across 8 minutes in cells treated with Fluo-4 and cells treated with Fluo-4 + AZ.C) Representative timeseries images of FI across 8 minutes in a sample exposed to Fluo-4.D) Representative timeseries images of FI across 8 minutes in a sample exposed to Fluo-4 and pre-treated with AZ.

Fig. 7 .
Fig. 7. Cleaved caspase-3 staining in human glioblastoma cancer stem cells (GSCs) following P2X7 receptor (P2X7R) inhibition by AZ10606120 (AZ).GSCs at 80 % confluency were fixed and stained with primary rabbit anti-cleaved caspase-3 antibody and secondary goat anti-rabbit Alexa Fluor 488.Cell nuclei were subsequently stained with DAPI.Cells were imaged with a Nikon Ti-E fluorescence microscope at 20× magnification.Mean cleaved caspase-3 fluorescence intensity (FI) was quantified for each treatment group across an average of at least 100 cells.A) There was no significant difference in cleaved caspase-3 FI between GSCs treated with AZ and untreated GSCs: t(5.25)=0.22,mean difference=-12.88,95 % CI=-164.7-138.9,p=0.84.Results were yielded from an unpaired t-test with Welch's correction.N=6 replicates.Data are presented as mean±SEM.B) Image representation of cleaved caspase-3 FI in untreated GSCs.C) Image representation of cleaved caspase-3 FI in GSCs treated with AZ 15μM.