CA9 knockdown enhanced ionizing radiation-induced ferroptosis and radiosensitivity of hypoxic glioma cells

Abstract Purpose Ferroptosis is a type of regulatory cell death, caused by excessive lipid peroxidation This study aimed to explore whether ionizing radiation could induce ferroptosis in glioma cells and whether carbonic anhydrase 9 (CA9) knockdown could enhance the killing effect of ionizing radiation on hypoxic glioma cells through ferroptosis. Materials and methods The protein levels of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were detected by Western blot in glioma cells irradiated by different doses of X-ray. The relative mRNA levels of ferroptosis markers and intracellular iron-associated proteins were detected by Real-time qPCR. Lipid peroxidation of glioma cells was detected by oxidation-sensitive probe C11-BODIPY581/591 staining. CCK-8 Assay was used to detect cell viability after X-ray irradiation. Cloning formation assay was used to assess the radiosensitivity of glioma cells. The exposure of cell surface calreticulin was measured by immunofluorescence staining. Results X-ray induced lipid peroxidation and ferroptosis markers expression in U251 and GL261 glioma cells. Knockdown of CA9 in hypoxic glioma cells significantly altered the expression of iron regulation-related proteins and enhanced X-ray-induced ferroptosis and radiosensitivity. The ferroptosis inhibitor significantly improved the survival of cells irradiated by X-ray, while ferroptosis inducers (FINs) enhanced the lethal effect of X-ray on cells. Enhancing ferroptosis in glioma cells promoted the exposure and release of damage-associated molecular patterns (DAMPs). Conclusions Ionizing radiation can induce ferroptosis in glioma cells. CA9 knockdown can enhance the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia. Enhancing ferroptosis will become a new idea to improve the efficacy of radiotherapy for glioma.


Introduction
Glioma is a type of brain tumor with a very poor prognosis.Although the combination treatment of surgery, radiotherapy, and chemotherapy for glioma, the efficacy is always limited (Ostrom et al. 2014).Therefore, it is important to find a new way to enhance the killing effect of ionizing radiation on glioma cells.
Ferroptosis is a non-apoptotic form of regulatory cell death (RCD), which is associated with intracellular free Fe (II) levels and caused by ROS accumulation and lipid peroxidation (Doll et al. 2017).Ferroptosis is regulated by integrated oxidation and antioxidant systems.Unbalanced lipid hydroperoxide accumulation and detoxification may lead to ferroptosis initiation (Stockwell et al. 2017).Ferroptosis is distinct from other RCD with different morphology characteristics, biochemical features and signaling transduction pathways (Gao and Jiang 2018).In general, ferroptotic cells exhibit shrunken mitochondria with condensed membrane, Fe (II)-dependent lethal lipid peroxide accumulation, and a series of gene expression changes in GPX4-dependent and independent systems and ferroptosis-executing systems (Dixon et al. 2012).Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4), Recombinant Solute Carrier Family 7, Member 11 (SLC7A11), and Glutathione Peroxidase 4 (GPX4) are the three main factors that regulate ferroptosis by modulating intracellular lipid peroxidation (Doll and Conrad 2017;Feng and Stockwell 2018).Ionizing radiation (IR) induces the occurrence of ferroptosis by increasing the expression of ACSL4 in cells, so ionizing radiation is also considered as a ferroptosis inducer (FIN) (Lei et al. 2020).When combined with FINs, the killing effect of IR on tumor cells can be greatly improved (Ye et al. 2020), suggesting that inducing ferroptosis be an effective new idea to enhance the radiosensitivity of tumor cells.
Carbonic Anhydrase (CAs) regulates intracellular pH (Supuran 2018).CA9 expression is regulated by oxygen concentration, and is elevated in response to hypoxia (Wykoff et al. 2000;Chiche et al. 2009).Recently, it has been found that inhibitors of CA9-induced ferroptosis in malignant mesothelioma (MM) cells under hypoxia.Whether CA9 is an effective target to eliminate hypoxic glioma cells by inducing ferroptosis is worthy of in-depth study (Li et al. 2019).Moreover, ferroptosis promotes the release of damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), heat shock protein (HSP) and adenosinetriphosphate (ATP), thereby modulating adaptive immune response (Shi et al. 2022).Thus, it is necessary to explore the characteristic of DAMP release during ferroptosis of glioma cells, which would be beneficial for the combination treatment of glioma.
In this study, we used human glioma cells U251 and mouse glioma cells GL261 under normoxic or hypoxic culture to explore whether IR could induce ferroptosis in glioma cells, whether knockdown of CA9 could enhance the killing effect of IR on hypoxic glioma cells through ferroptosis, and the effect of ferroptosis on the exposure and release of DAMPs.

Cell culture
GL261 and U251 glioma cells are cryopreserved in our laboratory.Cells were cultured with DMEM containing 10% fetal bovine serum under 5% CO 2 in a humidified atmosphere.Hypoxic cells were cultured under a gas mixture of 1% O 2 , 5% CO 2 , and 94% N 2 in a hypoxic workstation (Invivo2 1000, Ruskinn, UK).

Irradiation
The source of ionizing radiation is the RS2000 X-ray machine made by the Rad Source Company of the United States.The voltage is 160 Kv, and the dose rate is 1.16 Gy/min.Different doses of X-rays are achieved by adjusting the irradiation time.

Quantitative PCR (qPCR)
Cells were subjected to TRIzol method to extract total RNA, which was reversely transcripted to cDNA using abcam's reverse transcription kit.The qPCR experiments were performed using the fluorescence qPCR kit (Novoprotein, Suzhou) on a 7500 Real-Time PCR System (Applied Biosystems, USA), and the primers (listed in Supplementary 1) were designed and synthesized by Shanghai Sangon Biotech Company.The cycle threshold (Ct) values of the genes were measured for data analysis.

Lipid peroxidation assay
Cells were fixed with 4% paraformaldehyde, followed by staining with 10 lM C11 BODIPY 581/591 at 37 C for 30 min).Then the cells were observed under a laser confocal microscope, and the ratio of the emission fluorescence intensity at 590 nm (Texas Red) and 510 nm (FITC) was measured, which reflected the degree of intracellular lipid peroxidation.

Cell transfection
Cells with stable integration of scramble sequence or CA9 shRNA were generated via gene transfer mediated by lentivirus.Control lentivirus vector (HBLV-sh-Ctrl-ZsGreen-PURO) and recombinant lentivirus vector carrying CA9 shRNA sequence and green fluorescent protein (GFP) (HBLV-sh-CA9-ZsGreen-PURO) were constructed by Hanheng Biotechnology Co., Ltd, Changzhou, China.When the cell fusion rate reached 60%, cells were infected with the viruses (encoding either CA9 shRNA or scramble sequence), then were stably selected for 4 weeks with a medium containing 2 mg/mL puromycin.

CCK-8 assay
Cells were cultured in a 96-well plate.After adding the CCK-8 reagent (Dojino, Japan) to each well, the plate was incubated in an incubator for 2 h.When the color of the culture solution changed from red to orange-yellow, the absorbance at 450 nm per well is determined with a multifunctional microplate reader.

Wound healing assay
Cells were cultured in a six-well plate, then scratched the traces on the bottom of the dish with a sterilized tip.The scratches were observed under a microscope at different time points.Cell migration was evaluated by measuring the width of the wound.

Cloning formation assay
Cells were seeded in six-well plates with different numbers, and cultured in normoxia or hypoxia for 24 h, followed by irradiation with different doses of X-ray.Then the cells were cultured under normoxic conditions for about 2 weeks.Colonies were fixed in 4% paraformaldehyde solution, then stained with crystal violet.Colonies containing more than 50 cells were counted.Cell survival fraction was measured and the mean lethal dose (D0) was calculated by the linear quadratic model.Finally, the sensitizing enhancement ratio (SER) was calculated as a ratio of D0 measured from control and experimental group.

Immunofluorescence staining
Cells were fixed in 4% paraformaldehyde 24 h after irradiation (10 Gy in normoxia; 25 Gy in hypoxia).Then, the cells were incubated with a primary anti-CRT antibody (1:1000, Cell Signaling) overnight at 4 C.After washing three times, samples were incubated with a goat anti-mouse secondary antibody (Alexa fluor 555, 1:200, Abcam) at room temperature for 2 h, and counterstained with DAPI.Fluorescent images were acquired by an LSM 510 Meta microscope (Carl Zeiss, Germany).

Enzyme-linked immunosorbent assay (ELISA)
After irradiation (10 Gy in normoxia; 25 Gy in hypoxia), glioma cells were cultured in normoxia or hypoxia for 24 h, then the supernatants were centrifuged at 500 g for 5 min at room temperature and stored at À80 C. The extracellular HSP70 protein contents were detected using a HSP70 ELISA kit (Shanghai sig Biotechnology Co., Ltd.) according to the corresponding manufacturer's instructions.

Statistical analyses
All samples were carried out in three independent experiments and data were presented as mean ± SD using GraphPad Prism 8 software.Student's t test was used for pair comparisons.One-way ANOVA with Tukey's test was used for multiple comparisons.p < .05 was considered statistically significant.

X-ray-induced ferroptosis marker ACSL4 expression in glioma cells
In order to explore whether ionizing radiation affects the occurrence of ferroptosis in glioma cells, X-ray irradiation of 0 Gy, 4 Gy, 8 Gy, 12 Gy and 16 Gy were carried out with U251 cells and GL261 cells, respectively, and proteins were extracted at different time points (2 h, 24 h, 48 h), then the expression changes of ACSL4 in cells were detected by Western Blot.The results showed that the expression of ACSL4 in the two cell lines increased significantly at 2 h, 24 h and 48 h after irradiation, and the most significant increase was induced by 8 Gy X-ray (Figure 1(A, B)).Next, U251 cells and GL261 cells were irradiated with 8 Gy X-ray, then ACSL4 expressions were detected by Western Blot experiments at 6 h, 12 h, 24 h, and 48 h after irradiation, respectively.The results showed that ACSL4 expressions in both cell lines increased with time after irradiation (Figure 1 Effect of X-ray on mRNA levels of ferroptosis-related markers in glioma cells ACSL4, SLC7A11, and GPX4 mRNA levels in glioma cells were detected by qPCR at different time points (2 h, 24 h and 48 h) after 0 Gy, 4 Gy, 8 Gy, 12 Gy and 16 Gy X-ray irradiation.The results showed that mRNA levels of ACSL4, a ferroptosis promoter modulating polyunsaturated fatty acids (PUFAs)-phospholipids (PLs) biosynthesis, increased at 2 h after irradiation, while mRNA levels of SLC7A11 and GPX4, both functioned as ferroptosis inhibitors, decreased (Figure 2), which were consistent with the characteristic changes of ferroptosis (Lei et al. 2020).Moreover, ACSL4 mRNA levels increased at 24 h and 48 h after irradiation, and the mRNA levels of SLC7A11 and GPX4 also increased (Figure 2), which might be a cellular adaptive response and act as a negative feedback loop to restore cell survival upon IR induced ferroptosis (Lei et al. 2020).

X-ray induced lipid peroxidation and morphologic feature of ferroptosis in glioma cells
Next, we examined lipid peroxidation of glioma cells irradiated by 8 Gy X-ray.The results showed that the fluorescence intensity at 510 nm increased, while the intensity of 590 nm emission fluorescence decreased after irradiation of U251 and GL261 cells (Figure 3(A,B)), suggesting that IR can induce lipid peroxidation, one of the significant markers of ferroptosis, in glioma cells.In addition, IR also induced characteristic morphologic changes of ferroptosis, such as cell membrane broken, shrunken mitochondria, mitochondrial crista reduction and even disappearance (Figure 3(C)).

Ca9, CA12, and iron-regulation-related protein expressions in glioma cells were affected by hypoxia
CAs provide a guarantee for the survival and proliferation of most tumor cells under hypoxic, acidic, and other conditions (Supuran 2018).To explore whether CAs in glioma cells could respond to hypoxic microenvironment, U251 cells and GL261 cells were cultured in different oxygen concentrations for 24 h, followed by RT-qPCR detection of CA9 and CA12 mRNA levels.The results showed that hypoxia induced the expression of CA9 and CA12 in glioma cells and the increase of CA9 mRNA levels was much more significant than that of CA12 mRNA levels (Figure 4(A)).Therefore, in subsequent experiments, we will try to knock down the expression of CA9 in glioma cells.In addition, the mRNA levels of some iron-related proteins are altered under hypoxia.The results showed that the mRNA levels of iron storage protein FTL and FTH, and iron regulatory proteins IRP1 and IRP2 increased in hypoxic U251 or GL261 cells.Iron transporter FPN1 mRNA decreased in hypoxic U251 or GL261 cells.Iron transporter TFR1 mRNA increased in hypoxic U251 cells, while it decreased in hypoxic GL261 cells (Figure 4(B)).

Effects of CA9 knockdown on normoxic glioma cell migration and viability
Knockdown of CA9 was verified by real-time PCR and western blot in cells with stable integration of CA9 shRNA.The results showed that both mRNA and protein levels of CA9 in U251 or GL261 cells with stable integration of CA9 shRNA decreased significantly comparing to those with stable integration of scramble sequence (Figure 5(A)).
Migration and proliferation were important malignant behaviors of tumor cells.Cell migration and viability were detected by wound healing assay and CCK-8 assay, respectively.The results showed that both the migration and the proliferation capacity of glioma cells were inhibited after CA9 knockdown (Figure 5(B,C)).

Effects of CA9 knockdown on mRNA levels of intracellular iron-associated proteins
To explore the effects of CA9 knockdown on mRNA levels of intracellular iron-associated proteins in glioma cells, whose changes were detected by RT-qPCR.The results showed that TFR1, IRP1, FTL and FTH mRNA decreased, while IRP2 and FPN1 mRNA increased after CA9 knockdown in normoxic U251 cells.IRP2, FTL, and FTH mRNA decreased, while TFR1, IRP1 and FPN1 mRNA increased after CA9 knockdown in normoxic GL261 cells.Although these results were statistically significant, the changing amplitude was moderate (Figure 6(A)).Under hypoxia, CA9 knockdown significantly upregulated iron transporter TFR1 and FPN1 mRNA, and iron regulatory proteins IRP1 and IRP2 mRNA, while downregulated iron storage protein FTL and FTH mRNA (Figure 6(B)), which might result in a significant increase in free Fe (II) levels in hypoxic glioma cells.

CA9 knockdown combined with FINs enhanced X-ray induced ACSL4 expression
In order to explore whether CA9 knockdown and FINs enhanced the killing of glioma cells by X-ray, the changes of ACSL4 expression in cells were detected by Western Blot.The results showed that ionizing radiation could induce ACSL4 expression in glioma cells.Erastin (10 lM) alone also increased ACSL4 expression but showed no significant difference compared with NC group.Notablely, it can be observed that ACSL4 expression in cells of CA9 knockdown group was higher than that of NC group under either normoxic or hypoxic conditions.When combined with Erastin, CA9 knockdown enhanced X-ray-induced ACSL4 expression in normoxic or hypoxic glioma cells (p < .05)(Figure 7).The above results suggested that CA9 knockdown might enhance ACSL4 expression, thereby inducing ferroptosis in glioma cells.

CA9 knockdown enhanced glioma radiosensitivity
In order to explore the effect of CA9 knockdown on the radiosensitivity of glioma cells in normoxia or hypoxia, we  detected the radiobiological parameters by cloning formation assay.The experimental results showed that normoxic U251 cells with CA9 knockdown (D0 ¼ 2.727 Gy) were more radiosensitive than normoxic U251 cells with stable integration of scramble sequence (D0 ¼ 3.768 Gy) and the SER was 1.382.Similarly, normoxic GL261 cells with CA9 knockdown (D0 ¼ 2.522 Gy) were more radiosensitive than normoxic GL261 cells with stable integration of scramble  sequence (D0 ¼ 3.736 Gy) and the SER was 1.481.Hypoxic U251 cells with CA9 knockdown (D0 ¼ 4.489 Gy) were more radiosensitive than hypoxic U251 cells with stable integration of scramble sequence (D0 ¼ 6.266 Gy) and the SER was 1.396.Similarly, hypoxic GL261 cells with CA9 knockdown (D0 ¼ 4.231 Gy) were more radiosensitive than hypoxic GL261 cells with stable integration of scramble sequence (D0 ¼ 6.415 Gy) and the SER was 1.516 (Figure 8).These data indicated that CA9 knockdown enhanced the radiosensitivity of glioma cells under both normoxia and hypoxia.Moreover, we combined CA9 knockdown with Erastin to further enhance the radiosensitivity of glioma cells.The SER of U251 and GL261 cells was 1.685 and 1.679 under normoxia, respectively.Under hypoxia, the SER of U251 and GL261 cells was 1.796 and 1.796, respectively (Figure 8).

Effect of IR combined with ferroptosis inhibitors or inducers on glioma cell viability
In order to explore the effect of IR in combination with FINs on cell viability, we employed CCK-8 assay to detect the viability changes of glioma cells in different treatment groups.The results showed that X-ray reduced cell viability and Erastin enhanced the killing effect of X-ray under both normoxia and hypoxia.In cells with CA9 knockdown, the effects of X-ray and/or Erastin on cell viability were further enhanced, especially under hypoxia.The addition of ferroptosis inhibitor Ferrostatin-1, apoptosis inhibitor Z-VAD-FMK, or necrosis inhibitor Necrostatin-1s restored the survival of glioma cells to a certain extent, and the effects of Ferrostatin-1 or Z-VAD-FMK were more significant than Necrostatin-1s (Figure 9).These results suggested that ferroptosis played an important role in glioma cell death induced by IR.

Effect of ferroptosis on the exposure or release of DAMPs
One of the common DAMPs released by immunogenic cell death (ICD) is calreticulin, whose exposure to the surface of the cell membrane is a 'eat me' signal that guides dendritic cells to phagocytose tumor cells (Obeid et al. 2007).Cell surface exposure of calreticulin was detected by immunofluorescence staining.The results showed that X-ray increased calreticulin exposure on the surface of the cell membrane.Treating cells with the combination of X-ray and Erastin further enhanced calreticulin exposure.In addition, CA9 knockdown also promoted calreticulin exposure.When cells were treated with X-ray and Ferrostatin-1, calreticulin exposure decreased compared with X-ray alone (Figure 10).The results suggested that ferroptosis in glioma cells could promote calreticulin exposure.
HSP70 is also a common DAMPs, and there are few reports about the effect of ferroptosis on the release of HSP70.We found that X-rays reduced the intracellular HSP70 levels, suggesting that HSP70 was released outside the cell.X-rays combined with Erastin further reduced the intracellular HSP70 levels under both normoxia and hypoxia, while Ferrostatin-1 showed the opposite effect (Figure 11(A)).Further, we assessed the release of HSP70 in U251 cell supernatants at 24 h after X-ray irradiation by ELISA.Compared with NC group, the extracellular HSP70 protein contents were increased in the NC þ IR group and NC þ IR þ Erastin group under normoxic or hypoxic conditions.Similarly, the extracellular HSP70 protein contents were increased in the shCA9 þ IR group and shCA9 þ IR þ Erastin group compared with shCA9 group (Figure 11(B)).These results suggested that the levels of ferroptosis could modulate the release of HSP70 and enhanced ferroptosis could increase HSP70 release.

Discussion
Ferroptosis is a form of regulatory cell death (Dixon et al. 2012), mainly caused by the oxidation of phospholipids (PL) containing Polyunsaturated fatty acids (PUFA) (Stockwell et al. 2017), and the morphology and mechanism of ferroptosis are different from apoptosis or other RCD (Gao and Jiang 2018).Ferroptosis is inseparable from the accumulation of a large number of free Fe (II) in cells, resulting in the occurrence of the Fenton reaction, which produces a large number of highly reactive oxygen radicals, which quickly oxidize the surrounding biomolecules, thereby indirectly damaging the cells (Dixon et al. 2012;Shi et al. 2018;Zhu et al. 2019).ACSL4 is a key marker that regulates ferroptosis, and lowering ACSL4 significantly reduces ferroptosis by dampening biosynthesis of PUFA-PL (Yang et al. 2016;Yuan et al. 2016;Kagan et al. 2017).IR induced ACSL4 expression and ferroptosis in lung cancer cell lines and was considered as a FIN (Lei et al. 2020).This was also confirmed with glioma cells under both normoxia or hypoxia conditions in this study.At present, FINs are generally divided into three categories.Type I FINs cause ferroptosis by inhibiting SLC7A11-mediated cystine transport (Koppula et al. 2018); Type II FINs cause ferroptosis by inhibiting GPX4 activity; Type III FINs work by inhibiting the GPX4 and coenzyme Q 10 (Yang et al. 2014).In this study, when combined with FINs, the killing effect of IR on glioma cells was significantly enhanced.
Iron ions are critical in the formation of iron-sulfur clusters and in the synthesis of heme, which is essential for maintaining the mitochondrial respiratory chain and citric acid cycles (Schultz et al. 2010;Subramanian et al. 2011).Due to the Fenton reaction, excess free Fe (II) can cause severe oxidative damage (Zhou et al. 2018).Hypoxia is a distinguishing feature of the tumor microenvironment, which regulates the expression of hypoxic inducible factor (HIF), which controls ironassociated proteins.HIF-regulated proteins include the transferrin receptor (TFR1) and iron transporter (FPN1), whose expression levels are associated with the transport of iron ions (Tacchini et al. 2008;Taylor et al. 2011;Matak et al. 2013;Ma et al. 2019).The storage of iron is also important, which can prevent cells from oxidative stress caused by an overload of free iron.FTH and FTL are proteins associated with the storage of iron in cells (Arosio and Levi 2002;Arosio et al. 2017).Most proteins that regulate free the iron ions levels are regulated by hypoxia.In the present study, we found that the mRNA levels of iron transport protein decreased and the iron storage protein increased, which suggested that intracellular free Fe (II) would be reduced, thereby reducing the occurrence of ferroptosis.This might be one of the reasons why glioma cells developed strong radiation resistance under hypoxia.
CAs is ubiquitous in the biological world and act as a balancer between CO2, bicarbonate, and H þ in regulating intracellular pH (Supuran 2018).CAs are divided into eight separate enzyme families, in which are presented in human beings belong to the a class, which consists of 15 members, classified according to their cell location (intracellular or extracellular) or enzyme activity (Sly and Hu 1995;Hilvo et al. 2008).Five active family members (CA1, CA2, CA3, CA7, and CA13) are located in the cytoplasm, four are membrane- associated CAs (CA4, CA9, CA12, and CA14), two mitochondria CAs (CA5A and CA5B), and one is secretory CA (CA6) (Pastorekova et al. 2006;Aspatwar et al. 2013;2014).CA9 is a membrane-associated a CA that is widely found in mammals, particularly primates (Banerjee and Deshpande 2016), and is a key drug target in tumor therapy owing to its key role in the survival and proliferation of tumor cells under adverse conditions such as hypoxia and acidity (Supuran 2008;Becker 2020).In this study, the knockdown of CA9 significantly changed the expression of iron-related proteins in glioma cells under hypoxia and increased ACSL4 expression upon IR and/or FINs.These results suggested that the killing of glioma cells by IR could be enhanced by inducing ferroptosis and CA9 might be a key target for inducing ferroptosis under hypoxia.With this target, we may effectively overcome the radiation resistance of hypoxic gliomas and enhance the efficacy of radiotherapy for gliomas.However, there is still a lack of support for in vivo experiments or clinical trials, which will be explored in the near future.
DAMPs, including CRT (Kroemer et al. 2013;Fucikova et al. 2018), ATP (Garg et al. 2012;Martins et al. 2014), HSP (Salimu et al. 2015), and high mobility group protein 1 (HMGB1) (Scaffidi et al. 2002), are common accompaniments to ICD.DAMPs exposure or release triggers an immune response (Inoue and Tani 2014).Exposure of calreticulin to the surface of cell membranes represents a 'eat me' signal that induces attack from immune cells, which occurs early in ICD (Obeid et al. 2007).Exposed calreticulin sends a strong phagocytic signal to antigen-presenting cells (APCs) such as phagocytes, which subsequently present antigens to the immune system, initiating anti-tumor immunity (Obeid et al. 2007) and attacking cancer cells (Wolff et al. 1991).HSP70 is also a DAMP associated with ICD (Dudek et al. 2013).During the process of ICD, intracellular HSP70 may be exposed to the cell surface and released into the extracellular environment, which helps APCs to present tumor antigens to the immune system and activate cytotoxic T cells (Swaminath et al. 2017).In this study, ferroptosis levels of glioma cells irradiated by IR were regulated by using Erastin or Ferrostatin-1, which affected the exposure or release of DAMPs.The increase of ferroptosis could promote DAMPs exposure or release, suggesting a feasible new idea to improve the immunosuppressive microenvironment of glioma through ferroptosis.
In conclusion, IR could induce ferroptosis in glioma cells under normoxic or hypoxic conditions.CA9 knockdown enhanced the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia.Enhancing ferroptosis might be an effective way to improve the efficacy of radiotherapy for glioma.

Figure 1 .
Figure 1.Effect of X-ray irradiation on the expression of ACSL4 in glioma cells.(A) The protein expressions of ACSL4 in U251 cells were detected by Western Blot at 2 h, 24 h and 48 h after different doses of X-ray irradiation; Ã p<.05, ÃÃ p<.01, ÃÃÃ p<.001 vs 0 Gy.(B) The protein expressions of ACSL4 of GL261 cells were detected by Western Blot at 2 h, 24 h and 48 h after different doses of X-ray irradiation; Ã p<.05, ÃÃ p<.01, ÃÃÃ p<.001 vs 0 Gy.(C) The protein expressions of ACSL4 in glioma cells were detected by Western Blot at different time points after 8 Gy X-ray irradiation.Ã p<.05, ÃÃ p<.01, ÃÃÃ p<.001 vs Control.

Figure 4 .
Figure 4.The mRNA levels of CA9, CA12 and iron-regulation related proteins in hypoxic glioma cells.(A) Hypoxia induced increased expression of CA9 and CA12 in glioma cells; (B) The effect of hypoxia on the expression of iron-regulation related proteins.

Figure 5 .
Figure 5. Effects of CA9 knockdown on normoxic glioma cell migration and viability.(A) Changes of CA9 protein level and mRNA level in glioma cells after stable transfection of CA9 shRNA; (B) Migration of glioma cells after CA9 knockdown; Ã p<.05, ÃÃ p<.01, ÃÃÃ p<.001 vs NC.

Figure 6 .
Figure 6.Effects of CA9 knockdown on mRNA levels of intracellular iron associated proteins.(A) Iron-regulation related proteins mRNA levels in glioma cells after CA9 knockdown under normoxia; (B) Iron-regulation related proteins mRNA levels in glioma cells after CA9 knockdown under hypoxia.Ã p<.05, ÃÃ p<.01, ÃÃÃ p<.001 vs NC.

Figure 8 .
Figure 8. Knockdown of CA9 enhanced the radiosensitivity of glioma cells.(A) Pictures of cloning formation of glioma cells irradiated by different doses of X-ray; (B) Survival fraction curve and radiobiological parameters.

Figure 10 .
Figure 10.Immunofluorescence staining of calreticulin exposure on the surface of glioma cells.