Combining a noble gas with radiotherapy: Glutamate receptor antagonist xenon acts as a radiosensitizer in glioblastoma

doi:10.21203/rs.3.rs-2752583/v1

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Combining a noble gas with radiotherapy: Glutamate receptor antagonist xenon acts as a radiosensitizer in glioblastoma

https://doi.org/10.21203/rs.3.rs-2752583/v1

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Background

Ionotropic glutamate receptors AMPAR and NMDAR modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as radiosensitizer in GB.

Methods

We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine.

Results

The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Glutamate receptor subunits were present in glioblastoma-derived and to a lesser extent in HeLa cells. Significant radiosensitization by xenon was shown in U251 and U87 at irradiation doses of 4–8 Gy and 2–8 Gy, respectively (p < .05). The effect was further enhanced by the addition of memantine, showing significant reduction in the surviving fraction already at 2 Gy for both glioblastoma cell lines (p < .05). Xenon did not increase the radiosensitivity of HeLa cells until a radiation dose of 8 Gy.

Conclusion

The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled radiosensitizing properties of xenon gas in glioblastoma.

Glioma

Glioblastoma

Glutamate receptors

Xenon

Memantine

NMDA

AMPA

Radiotherapy

Radiation sensitizer

Clonogenic assay

A complex system promoting glioma invasion and growth has been recently discovered in several studies: By forming synapses with astrocytes, glioma cells receive proliferative stimuli that are passed on through tumor cell networks as depolarizing currents (1–5). The excitatory neurotransmitter glutamate and ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) play a key role in neuron–glioma interaction, where glioma cells receive glutamate through unidirectional synaptic connections (1, 3, 5). In addition, glioma cells have been shown to secret large amounts of glutamate in their surroundings acting as an autocrine growth stimulant through AMPARs and N-methyl-D-aspartate receptors (NMDAR), as well as exhibiting toxic effects on adjacent brain tissue and, thus, facilitating tumor invasion (6–11).

In patients suffering from glioblastoma (GB), especially those lacking O6-alkylguanine DNA alkyltransferase (MGMT) promotor methylation, alkylating chemotherapeutic agents have only limited effect on tumor progression and exploration of alternative treatment pathways is imperative (12–16). In light of this, pharmacological targeting of glutamate receptor pathways in GB has been studied, yet, most of the promising in vitro tested agents are not suitable for therapeutic usage in humans due to toxicity and several administrable substances have failed in vivo, possibly because of insufficient accumulation in brain tissue (5, 6, 11, 17–25).

Xenon gas is an established AMPAR and NMDAR antagonist for anaesthesia (26, 27). It is a known neuroprotectant approved for the treatment of perinatal asphyxia and currently under investigation for application in patients after a circulatory arrest (28, 29). As it accumulates in the central nervous system while being well tolerated, xenon is a promising glutamate receptor antagonist for GB therapy.

To enhance xenon’s potential, a combination with radiation therapy (RT) appears sensible as NMDAR antagonists together with ionizing irradiation have been shown to exhibit radiosensitizing effects in glioma in vitro (19, 30).

In radiobiological research, clonogenic survival as determined using the colony formation assay (CFA) represents an established method to evaluate the in vitro effects of RT alone or in combination with additional substances (31). While elements, molecules or pharmaceutical agents in gaseous form (e.g. oxygen (O₂), nitrogen (N₂), nitrous oxide or volatile anaesthetics) can alter radiation effects on cells and therefore exhibit modulating properties in the CFA, their application is more challenging compared to liquids or solids simply dissolved into the cell culture growth medium (32–37).

In the past, the development of methods altering cell culture atmospheric conditions in radiobiological research has mainly been driven by investigations of irradiation and hypo- or hyperoxic conditions, where gas exposure is most commonly performed by purging a chamber containing the cell culture via in- and outlet-valves (38–42). Literature indicates only few studies that have investigated the impact of RT in combination with noble gases (e. g. helium, xenon, or krypton) on tumor cells (43, 44). Despite providing first evidence for gas-mediated radiosensitization, further studies are missing. A reason might be the lack of elaborate methods for gas exposure.

We designed and validated a system to conduct CFAs with gas exposure before, during or after RT and, for the first time, investigated the radiosensitizing effect of xenon gas (Xe) exposure on GB cells.

Vented cap T25 flasks

T25 flasks (FALCON® 25 cm² Rectangular Canted Neck Cell Culture Flask with Vented Cap, Corning Inc., Corning, USA) contain liquid cell medium and residual air/gas, with a total capacity of 70.0 ml (FALCON® technical data sheet). Additionally, the volume displaced by a capped T25 was determined via submersion in water to be 96.0 ml. The flasks’ caps allow for gas exchange with a hydrophobic membrane consisting of polytetrafluoroethylene (PTFE) with pores of 0.2 µm.

Gas Exposure

Our gas exposure system consists of a chamber made from acrylic glass with inner dimensions of 15.0 x 18.0 x 14.0 cm defining a volume of 3780.0 cm³ and a detachable lid containing a round hole (Ø 3.0 cm). Designed to be used in combination with vented cap T25 cell culture flasks, up to 15 flasks can be stockpiled inside. First, a polyethylene (PE) bag is placed into the chamber with its open side facing the upwards and flasks are inserted. The open side of the PE-bag is then fitted through the hole in the lid and connected to the ending of a flexible silicon tube in a gas-tight manner. The lid is attached and a vacuum pump (Mityvac® Silverline No. 04010, Lincoln Industrial, St. Louis, USA) is attached to the gas tubing, subsequently removing air until the PE-bag is empty and abuts the stack of T25 flasks. Now, vessels containing prepared gas mixtures can be connected to the gas tubing and the PE-bag can be refilled until its expansion reaches the chambers inside limits, thus defining an exact volume of the filling. Simple clamping of the gas tubing afterwards preserves the atmosphere inside (Supplementary Fig. 1).

Validation

Stability of gas levels was tested by monitoring O₂ as tracer gas with an O₂-sensor (GOX 100, GHM Group Greisinger GmbH, Regenstauf, Germany) inside the PE-bag during exposure with 100% O₂ (Nippon Gases Deutschland GmbH, Düsseldorf, Germany).

Redistribution of gases within the flasks was examined with O₂ and carbon dioxide (CO₂) as tracers. According to the exposure method described above, T25 flasks with vented caps were exposed to 100% O₂ with the O₂ sensor inside one of the flasks. Additionally, diffusion of larger molecules through the vented cap was tested by exposing a flask to an atmosphere comprised of 5% CO₂ in ambient air and tracking CO₂ inside the flask with a CO₂ sensor (GM70, Vaisala Oyj, Vantaa, Finland).

Lastly, dose aberration by Xe inside T25 flasks was measured. With an ionization chamber mounted inside a flask, a Xe-containing atmosphere (50% Xe, 25% N₂, 20% O₂, and 5% CO₂) was established in the flask as described above. Then, irradiation was performed with the measurement flask and the surrounding dummy flasks between acrylic glass plates to balance secondary electrons from photons of 6 MV energy (Elekta Synergy, Elekta AB, Stockholm, Sweden).

Cell culture

U87 and U251 were selected as glioblastoma derived cell lines. HeLa cells, derived from squamous cell carcinoma, were chosen as non-glial derived control. Cells were cultivated in Dulbecco’s modified eagle’s medium (DMEM, Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum, stored in a 37° C incubator gassed with additional 5% CO₂. Cells were subcultured regularly before confluence.

Colony Formation Assay

The CFA was performed in triplicates of T25-flasks for each irradiation dose of 0, 2, 4, 6 and 8 Gy in three independent experiments. In preparation of CFA, cells were harvested, counted, and distributed into T25 flasks starting with 100 (U251), 200 (HeLa) or 1000 cells per flask (U87) for non-irradiated groups increasing the cell count with irradiation dose to 200, 600, 800, 2000 (U251), 400, 800, 2000, 4000 (HeLa) or 1000, 2000, 5000, 10000 (U87). Cells were allowed to attach overnight and 25 µM of either memantine-hydrochloride 0.25 mg/ml (Sigma-Aldrich Corp., St. Louis, USA) or distilled water were added one hour prior to irradiation.

Irradiation

For irradiation, T25 flasks triplicates were placed on top of of eight acrylic glass plates (thickness 1.0 cm) with two plates further added on top for buildup of secondary electrons (Supplementary Fig. 2). Vertical gantry position of a conventional linear accelerator (Elekta Synergy, Elekta AB, Stockholm, Sweden) was allocated by wall-mounted lasers marking the centre of the flasks’ caps membrane, horizontal position by opening the linear accelerators light field (32.0 x 32.0 cm) and positioning the plates centrally. The triplicate from the 0 Gy dose group was removed immediately afterwards and exchanged with dummy T25-flasks filled with 5 ml of water. After delivery of 6 MV photons, equivalent to an effective dose of 2 Gy, the 2 Gy triplicate was removed and replaced by dummy flasks. This process was repeated until a cumulative dose of 8 Gy for the last triplicate was reached.

Gas Exposure

Gas exposure was performed immediately after irradiation. The flasks were exposed to a preformed gas mixture for 60 minutes comprised of either 75% Xe, 20% O₂ and 5% CO₂ (Air Liquide S.A., Paris, France) or 75% N₂, 20% O₂ and 5% CO₂ (Nippon Gases Deutschland GmbH, Düsseldorf, Germany). This resulted in an atmosphere inside of the flasks of either 50% Xe, 25% N₂, 20% O₂, and 5% CO₂ for xenon groups or 75% N₂, 20% O₂, and 5% CO₂ for memantine or control groups. After exposure, the flasks vented caps were sealed and stored at 37° C for 24 hours. Finally, the vented caps were unsealed and flasks were transferred into the 5% CO₂-incubator.

Evaluation

Eleven (U251, HeLa) or 14 (U87) days after irradiation and gas exposure, cells were fixed, stained and colonies of more than 50 clonal cells were counted. Surviving fraction (SF) was calculated and fitted by the linear-quadratic model (ln(SF) = -(αD + βD²)) by nonlinear regression using SigmaPlot v11 (SigmaPlot, Systat Software Inc., San Jose, USA).

Indirect immunofluorescence staining

A method for glutamate receptor staining was performed as described before (45). Thousand U87, U251, and HeLa cells were transferred onto an 8-well chamber slide (FALCON® 8 well sultureslide, Corning Inc., Corning, USA). Primary antibodies: rabbit-derived GluN1 (Ab109182, Abcam, Cambridge, UK) and GluA1 (13185S, Cell Signaling Technology, Cambridge, UK)). Secondary antibody: Goat anti-rabbit IgG Fluor (AP307F, Merck KGaA, Darmstadt, Germany). Results were assessed with fluorescence microscopy.

Statistical analysis

All experiments, except for immunostaining, were repeated at least three times. Mean, standard deviation, standard error and 95% confidence interval were calculated unless otherwise mentioned. An unpaired, two-tailed t-test was used to test statistical significance (p < 0.05).

Gas exposure Method

Handling characteristics

Our gas exposure method provided simple handling with accessible and affordable components. Exposure could be scheduled at any time prior to or after irradiation. Gas consumption was dependent on the desired concentration, the prepared gas (mixtures) and the amount and kind of cell culture vessels. In our setting, 2340 ml of gas mixture per 15 flasks was required No microbiological contamination was observed in context of our experiments.

Validation

The PE-bag filled with 100% O₂ provided sufficient stability for gas exposure with a decrease of O₂ concentration to 96.8% (range: 96.7–97%) within 60 min.

Under exposure conditions, the final gas concentration inside of the 15 flasks can be calculated assuming an ideal gas distribution:

$$\frac{V\left(flasks\right)*c\left(flasks\right)+V\left(bag\right)*c\left(gas mixture\right)}{V\left(chamber\right)}$$

For our specific validation setting, this resulted in a calculated value of 81% O₂. After 60 min of exposure, we measured a mean O₂ concentration of 80.6% (range: 79.8–81.2%), approximating our calculated values and indicating a near-ideal gas distribution.

By observing an increase in CO₂ concentration inside the flasks reaching 3.246% after 60 minutes, smooth transition of molecules larger than O₂ through the cap’s vented membranes was demonstrated. Irradiation of Xe-filled flasks yielded significant change in effective radiation dosage with 2022 mGy (95% confidence interval (CI): [2020.6-2023.4] compared to ambient air with 1993 mGy [1990.5-1995.4].

Clonogenic survival

For U87, differences in mean survival fractions of unirradiated cells treated with Xe, memantine or both remained nonsignificant from the control group. After irradiation, significant differences in SF were observed in the cells treated with Xe at 4 Gy (p = .037, 6 Gy (p = .033), and 8 Gy (p = .020), respectively. With memantine alone, U87 mean SF in relation to the control group were not significantly decreased. After both Xe exposure and added memantine, significant differences in SF were found at 2 Gy (p = .040), 4 Gy (p = .010), 6 Gy (p = .016), and 8 Gy (p = .009), respectively. Results are summarized in Fig. 1, data is provided as appendix.

For U251, differences in mean survival fractions of unirradiated cells treated with Xe, memantine or both remained nonsignificant from the control group. After irradiation, significant differences in SF were observed in the cells exposed to Xe or treated with memantine alone as well as the combination of both at 2 Gy (p < .001), 4 Gy (p < .001), 6 Gy (p < .001), and 8 Gy (p < .001), respectively. Results are summarized in Fig. 2, data is provided as appendix.

For non-glia derived HeLa, differences in mean survival fractions of unirradiated cells treated with Xe remained nonsignificant from control group. After irradiation, significant differences in SF were only observed in the cells treated with Xe at 8 Gy (p < .004). Results are summarized in Fig. 3, data is provided as appendix.

AMPAR and NMDAR expression

Representative images of the immunofluorescence slides obtained by microscopy are presented in Fig. 4. A strong signal could be detected for AMPAR (GluA1) and NMDAR (GluN1) receptor subunits in U87 and U251. In HeLa, signals for both receptor subunits were also detectable, but comparatively weak.

Despite intensive research efforts, glioblastoma remains an often fatal disease with limited therapeutic options (46). Whereas targeted therapies have achieved relevant advances in numerous entities, this approach does not yet seem to be successful in GB and there is an urgent need for more effective treatments to improve outcomes (16, 47).

Targeting ionotropic glutamate receptors to reduce proliferation and invasion of GB, promising preclinical data exist inter-alia for AMPAR- and NMDAR-inhibitors including memantine (3, 6, 8, 11, 18, 19, 23, 48). As radiotherapy plays a central role in GB treatment, testing for radiosensitizing potential of glutamate receptor targeted therapy to enhance irradiation’s effectiveness appears sensible especially from a clinical perspective. In vitro data have, so far, shown radiosensitizing potential for the NMDAR-inhibitors ifenprodil, dizocilpine, riluzole and memantine (17, 19).

However, transfer to clinical application is difficult as unacceptable toxicity or bioavailability in the central nervous system (CNS) can be limiting factors (49). Although the BBB is considered locally disrupted in GB, the disease occultly infiltrates the brain tissue (5, 50). Therefore, therapeutic agents must be also capable of targeting tumor cells located in areas with an intact BBB. A further essential characteristic of a useful agent is adequate tolerability at therapeutic doses, which, due to the frequently occurring and accumulating side effects of surgery plus irradiation, is a major concern for GB patients (51, 52). Here, the AMPAR- and NMDAR-antagonist xenon represents a long-tested and approved drug, which readily crosses the blood-brain barrier. (53, 54). Due to its favourable toxicity profile good tolerability in patients is expected even at high concentrations of up to 50% xenon (55). To explore whether xenon does exhibit anti-tumoral and, possibly, radiosensitizing effects, we have designed and validated a method for combining xenon gas exposure with a conventional CFA, the gold standard for determining effects on cellular clonogenic survival.

As trace gas in the atmosphere, commercial extraction of xenon is expensive (56). The entry into our research was thus marked by the lack of a method that would allow for an economical use of gaseous agents while being compatible with the requirements of a CFA. Hence, as other authors have previously experienced, we were compelled to develop a proprietary system (42).With our method, we were able to reduce xenon consumption to about a quarter of the amount required by purging with inlet and outlet valves (57). In the subsequent validation experiments, we were able to maintain a stable gas atmosphere in the exposure system and found a gas distribution that showed adequate approximation to our calculated target values. Since the physical behaviour of the larger xenon molecules cannot be fully depicted by O₂, gas distribution was additionally investigated using CO₂. Carbon dioxide resembles xenon in collision diameter and Boltzmann constant as the relevant parameters according to Chapman-Enskog theory while xenon, vice versa, is used as an established tracer gas for CO₂ (58, 59). Hence, we consider our CO₂ diffusion measurements across the PTFE membrane to be applicable for xenon. Detecting a significant radiation dose increase due to the presence of high-Z element xenon within the T25 flasks resembled qualitative evidence of the altered atmospheric composition and validated our approach for the subsequent cell culture experiments.

As a subsequent proof of concept, we could show for the first time that xenon applied after irradiation mediates a radiosensitizing effect on AMPAR- and/or NMDAR-positive glioblastoma cell lines U87 and U251 in the CFA. The radiosensitizing effect was further enhanced by the addition of memantine, while a stand-alone effect of memantine has already been described before and was observed again in our study (19).

Previous research has revealed that NMDAR-mediated calcium influx with downstream signalling pathways represents a key factor in repairing irradiation-induced DSBs and accounts for radiation resistance in GB (19). Presumably, the results obtained demonstrate impaired DNA repair after irradiation mediated by the inhibition of the calcium permeable glutamate receptors. Memantine inhibits calcium influx through NMDAR, whereas xenon modulates calcium permeability of NMDAR as well as AMPAR (60, 61). Our hypothesis is supported by immunofluorescence staining, demonstrating expression of the corresponding receptors subunits in both cell lines as described in earlier reports (8, 9, 30, 62). The hypothesis is further corroborated by the low impact of xenon, memantine, or a combination of both on cell survival in the sham-irradiated groups lacking irradiation-induced DNA damage. Because xenon and memantine act at different binding sites at the NMDAR, namely the glycine binding site for xenon and the Mg²⁺ binding site for memantine, there is no competitive but possibly an additive effect when both substances are coupled (63). The enhanced radiosensitizing effect observed in groups treated with both substances consorts with a twofold receptor modulation.

Overall, however, the mechanism of action of xenon in GB has not been completely understood and it cannot be entirely ruled out whether additional signalling pathways are involved, yet. The detection of a considerably weaker but eventually significant radiosensitization of HeLa cells at high irradiation doses indicates that glutamate receptors might exhibit different downstream effects in non-glial cell lines.

Our study aimed to explore xenon’s potential as glutamate receptor antagonist and the gas was therefore applied only after irradiation. Effective radiation dose was altered when irradiation was delivered under xenon gas atmosphere, likely because of xenon’s high atomic number. As an altered effective radiation dose not only affects tumor cells but also surrounding healthy tissue possibly increasing side effects in a clinical setting, xenon gas use during irradiation does not seem feasible. Yet, the immediate application of xenon after irradiation is desirable as DNA damage repair in tumor cells starts within minutes after DNA damage is inflicted (64).

Limitations and prospects

Several limitations restrict the conclusions of our work. First and foremost, the study was conducted in vitro and its effects need to be confirmed in vivo. Furthermore, even with very robust results, the investigation was carried out on no more than three cell lines. The method of gas exposure employed allows for an economical and simple use of the expensive noble gas xenon but has not yet been described before and can potentially lead to biased results. Despite its limitations, our data provide a strong rationale for in vivo exploration. Unlike other substances that have demonstrated radiosensitization of glioblastoma cell lines, xenon and memantine are easily available and approved agents that have few side effects and thus allow for a transfer into a preliminary clinical setting.

In summary, the ionotropic glutamate receptor pathway in GB that recently moved into the centre of research continues to be a promising target for new treatment options. With our work, we gathered first evidence suggesting xenon, an already approved drug with excellent CNS accumulation a low toxicity profile, could function as a radiosensitizer in GB patients.

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and materials: The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: AMR and FAG are co-inventors in the patents pending EP4119191A1 and WO2023285170A1, “Xenon gas for use in the treatment of gliomas”. AMR has received or receives personal fees and/or travel expenses from Carl Zeiss Meditec AG. FAG has received or receives research grants, personal fees and/or travel expenses from Carl Zeiss Meditec AG, personal fees from Roche Pharma AG and Medac, grants and personal fees from AstraZeneca, Bristol-Myers Squibb, Cureteq AG, Elekta AB, FoMF GmbH, Guerbet SA, MSD Sharp and Dohme GmbH and Opasca GmbH, stocks, grants and personal fees from TME Pharma AG (formerly Noxxon Pharma AG), compensation for advisory boards from the Federal Joint Committee (G-BA) of the Federal Republic of Germany and of the German Cancer Aid and non-financial support from Oncare GmbH and Opasca GmbH. The remaining authors declare that they have no competing interests.

Funding: The work was not financially supported

Authors' contributions: TB designed the work, acquired, analysed and interpreted data and drafted the manuscript. MKE acquired and analysed data. FAG concepted the work and revised it substantially. MRV concepted the work and revised it substantially. CH concepted the work and revised it substantially. AMR: concepted and designed the work, interpreted data, drafted the manuscript and revised it substantially. All authors read and approved the final manuscript.

Acknowledgements: Not applicable

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Competing interest reported. A.M.R. and F.A.G. are co-inventors in the patents pending EP4119191A1 and WO2023285170A1, “Xenon gas for use in the treatment of gliomas”. A.M.R. has received or receives personal fees and/or travel expenses from Carl Zeiss Meditec AG. F.A.G. has received or receives research grants, personal fees and/or travel expenses from Carl Zeiss Meditec AG, personal fees from Roche Pharma AG and Medac, grants and personal fees from AstraZeneca, Bristol-Myers Squibb, Cureteq AG, Elekta AB, FoMF GmbH, Guerbet SA, MSD Sharp and Dohme GmbH and Opasca GmbH, stocks, grants and personal fees from TME Pharma AG (formerly Noxxon Pharma AG), compensation for advisory boards from the Federal Joint Committee (G-BA) of the Federal Republic of Germany and of the German Cancer Aid and non-financial support from Oncare GmbH and Opasca GmbH. The remaining authors declare that they have no competing interests.

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Combining a noble gas with radiotherapy: Glutamate receptor antagonist xenon acts as a radiosensitizer in glioblastoma

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Vented cap T25 flasks

Gas Exposure

Validation

Cell culture

Colony Formation Assay

Irradiation

Gas Exposure

Evaluation

Indirect immunofluorescence staining

Statistical analysis

Results

Gas exposure Method

Handling characteristics

Validation

Clonogenic survival

AMPAR and NMDAR expression

Discussion

Limitations and prospects

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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