Early metabolic reprogramming and carbonic anhydrase IX-mediated extracellular acidification drive radiotherapy-induced glioblastoma cell dedifferentiation

Perrine Dahan, Tom Maillet, Laure Malric, Caroline Delmas, Vincent Lubrano, Judith Martinez-Gala, Guillaume Flandin, Amélie Aboudaram, Cécile Héliès-Toussaint, Nicolas Skuli, et al.

► To cite this version:

Perrine Dahan, Tom Maillet, Laure Malric, Caroline Delmas, Vincent Lubrano, et al. Early metabolic reprogramming and carbonic anhydrase IX-mediated extracellular acidification drive radiotherapy-induced glioblastoma cell dedifferentiation. Acta Neuropathologica Communications, 2025, 14 (1), pp.1. 10.1186/s40478-025-02161-2. hal-05519377

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# Early metabolic reprogramming and carbonic anhydrase IX-mediated extracellular acidification drive radiotherapy-induced glioblastoma cell dedifferentiation

Perrine Dahan¹†, Tom Maillet¹†, Laure Malric¹, Caroline Delmas¹,², Vincent Lubrano³,⁴, Judith Martinez-Gala¹, Guillaume Flandin¹, Amélie Aboudaram¹, Cécile Héliès-Toussaint⁵, Nicolas Skuli¹, Sylvie Monferran¹,⁶, Yvan Nicaise¹, Clémentine Decamps¹, Christine Toulas¹,⁷, Elizabeth Cohen-Jonathan Moyal¹,⁶,⁸† and Anthony Lemarié¹,⁶*†

## Abstract

**Background** Glioblastomas (GBM) are brain tumors with the worst prognosis despite treatment with surgery and radio/chemotherapy, emphasizing the need for new therapies and improved treatment efficacy. Previously, we showed that clinically relevant ionizing radiation (IR) doses enhance GBM cell dedifferentiation into a stem-like phenotype, increasing stemness markers, self-renewal, and tumorigenic abilities. This work focuses on identifying early mechanisms driving this plasticity, particularly metabolic adaptations, as tumor metabolism may support therapy resistance and recurrence.

**Methods** In this study, primary cell lines were established from GBM biopsies of several patients and cultured either in restrictive medium to form neurospheres (NS) enriched in GBM stem-like cells (GSC) or in normal medium to obtain their differentiated progenies. These differentiated GBM cells were then subjected to a short-term dedifferentiation protocol after IR (24 to 72 h) to characterize their metabolic and phenotypic properties.

**Results** We found that early stem marker increases after IR exposure coincide with higher oxygen consumption rate (OCR), mitochondrial ATP production, and extracellular acidification rate (ECAR). However, lactate production remained unchanged, and glucose uptake showed only a transient, nonspecific increase. This shift to oxidative mitochondrial metabolism, coupled with extracellular acidosis, promotes a stem-like phenotype and is associated with Carbonic Anhydrase IX (CA9) overexpression, an extracellular membrane protein producing H⁺ to buffer intracellular pH. CA9 downregulation via ShRNA reduces extracellular acidification and blocks early IR-induced

Full list of author information is available at the end of the article

<footnote>†Perrine Dahan and Tom Maillet have contributed equally to this work, and should share the first authorship.</footnote>

<footnote>‡Anthony Lemarié and Elizabeth Cohen-Jonathan Moyal have contributed equally to this work, and should share the senior authorship.</footnote>

<footnote>*Correspondence: Anthony.Lemarié anthony.lemarie@inserm.fr</footnote>
dedifferentiation, such as neurosphere (NS) formation and stem marker overexpression. Similarly, increased OCR and ECAR, often linked to CA9 overexpression, are observed in patient-derived GSC compared to their differentiated progenies.

**Conclusions** These findings highlight CA9 as a potential target to block IR-induced early dedifferentiation process as well as the specific metabolic state of GSC. Our study strengthens the therapeutic potential of CA9 inhibition to limit GBM-associated acidic extracellular environment and enhance radiotherapy efficiency by limiting tumor cell plasticity.

**Article highlights**

* Mitochondrial metabolism is enhanced in GSC compared to their differentiated progeny.

* IR-induced dedifferentiation in GBM cells occurs via an early metabolic shift to OXPHOS.

* IR-induced metabolic shift links CA9 induction, extracellular acidification, and stemness.

**Keywords** Metabolic reprogramming, Radiotherapy, Glioblastoma-stem cells, Cell plasticity, Dedifferentiation, CA9, Acidification

**Graphical abstract**

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**Introduction**

Glioblastomas (GBM), classified as grade 4 gliomas by the WHO [1] have a poor prognosis due to high therapy resistance. Despite standard treatment combining surgery, chemo-radiotherapy (RT) with Temozolomide (TMZ) [2] and, recently, Tumor Treating Fields (TTF), recurrence is almost inevitable, with survival ranging from 15 (without TTF) to 21 months (with TTF) [3]. This recurrence is mainly due to clonal selection of heterogeneous resistant subpopulations, particularly Glioblastoma Stem-like Cells (GSC). GSC exhibit high therapy resistance, tumorigenic potential, self-renewal

and intrinsic plasticity [4]. They overexpress stem markers such as Sox2, CD133, Nestin, Olig2, A2B5, or Integrin α6 (ITGA6) [4] and can generate heterogeneous tumors in mouse models mimicking human GBM [5]. GSC therapy resistance involves enhanced various mechanisms such as a better ability to respond to DNA damages [6], to efflux drugs [7] and to activate various pathways such as Notch [8] and Met [9]. Another key factor in GBM recurrence is cellular plasticity, where therapy-resistant, differentiated GBM cells could dedifferentiate into a stem-like state. We and others have shown that dedifferentiated GSC emerge in response to stresses or signals like IR
[10], hypoxia [11], TMZ [12, 13], HGF [14], bFGF [15], EGR/EGFR/EGR1 [16] or CMV-infection [17], expanding the resistant, invasive and tumorigenic cancer stem cell (CSC) pool. Similar dedifferentiation occurs in pancreatic [18], lung [19] or colorectal [20] cancers, in response to different stimuli. However, in GBM, the mechanisms driving and regulating this process remain poorly understood.

Understanding this therapy-induced phenotypic plasticity in GBM could reveal new therapeutic targets. We previously identified the anti-apoptotic protein survivin as a mediator of IR-induced dedifferentiation in vitro [10]. This study explores the early metabolic mechanisms driving IR-induced reprogramming. Since metabolic changes often drive cancer cell adaptation and plasticity [21] and support key GSC traits like self-renewal [22], we investigated the energetic metabolic alterations underlying IR-induced plasticity in GBM cells. Targeting metabolism has shown therapeutic potential in cancers [23], including GBM, where metabolic changes contribute to chemo/radioresistance [24–26]. We previously showed that Metformin, by inhibiting mitochondrial respiration and activating AMPK, shifts metabolism toward glycolysis in GBM cell lines, reducing tumor growth in mice xenografts and enhancing TMZ and IR sensitivity in vitro [27]. IR effects on metabolism are complex. A study on differentiated GBM cell lines showed that IR reduces oxidative phosphorylation (OXPHOS) in favor of glycolysis, and that inhibiting glycolysis improves survival in treated mice treated with IR [26]. Conversely, other studies suggest IR-exposed GSC enhance their repair capacity of DNA breaks and lipid peroxidation via metabolic adaptations, enriching resistant clones which rely less on glucose and can maintain ATP production primarily through OXPHOS [28]. GSC were also shown to use alternative fuels like glutamine [29] and fatty acid [30] to sustain the Tricarboxylic Acid cycle (TCA) and OXPHOS. Notably, glutamine metabolism has been linked to GSC radioresistance [31]. These findings suggest that GSC and differentiated GBM cells exhibit distinct metabolic responses to therapy, both contributing to radioresistance and tumorigenesis. We hypothesize that low-dose IR triggers energetic metabolic reprogramming, driving GBM dedifferentiation into a stem-like phenotype. Studies support this by linking specific metabolic states to GBM cell differentiation and tumorigenicity [32–34]. Additionally, metabolic reprogramming may alter the tumor microenvironment, inducing extracellular acidosis via pH-regulating enzymes and carriers, like Carbonic Anhydrases (CA), Na+/H+ exchangers (NHE) and Vacuolar H+-ATPase (V-ATPase) [35]. This extracellular pH (pHe) drop was shown to promote various cancer processes [36], such as therapy resistance [37, 38], invasion [39] and stemness maintenance in GBM cells [40, 41].

Our data show that IR-induced dedifferentiation in primary GBM cells, previously highlighted by us [10], is driven by an early metabolic shift with increased O₂ consumption and extracellular acidification. This CA9-driven acidosis after clinically relevant IR doses promotes phenotypic plasticity toward a stem-like state in GBM cells. We demonstrate here for the first time that IR, a key GBM therapy, promotes early metabolic and subsequent phenotypic reprogramming, potentially expanding the GSC pool and driving rapid tumor recurrence.

# Materials and methods

## GSC isolation and cell culture

C1, D1, G and I GSC patient cell lines were established as described [10]. SRA5, SRB1 and SRC3 GSC patient cell lines were derived from the STEMRI clinical trial (NCT01872221) [42, 43]. Forced differentiation and IR-induced dedifferentiation (3 Gy) followed our published protocol [10], with a shorter 1-day interval before medium replacement with either FCS-differentiating medium or dedifferentiation-permissive Stem Cell Medium (SCM). See details in Supplementary Methods.

## Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements (Seahorse XF assay)

OCR and ECAR were measured in real time using Seahorse XF24/XF96e (Agilent) as described by us and others [27, 34], using MitoStress (Agilent) kit (see Supplementary Methods).

## ATP measurement

ATP levels were assessed using the Cell Titer-Glo 2.0 assay (Promega) after appropriate treatments to determine total, glycolytic and mitochondrial ATP, as described [27] (see Supplementary Methods).

## Glucose uptake assay

Glucose uptake assay followed a previous study [44] on GDC cells plated in 12-well plates (7.5 × 10³ cells/cm²) before IR and medium change 1 day post-IR. Cells were washed with serum-free DMEM low glucose, pre-incubated for 4h at 37 °C, washed with Krebs-Ringer-Bicarbonate (KRB) buffer and incubated 30min with 100 nM insulin (or cytochalasin B as control, Merck). Glucose uptake was initiated by adding [³H] 2-Deoxy-D-glucose (1 µCi/mL, PerkinElmer) in 0.1 mM D-glucose and incubating 10min at 37 °C. After washing with ice-cold KRB, cells were solubilized in 0.1N NaOH. Half of each well was transferred into scintillation vials with 10mL of scintillation cocktail, Ultima Gold LLT (PerkinElmer). Radioactivity was measured by scintillation counting (Hewlett Packard counter), and protein content normalized.
## Lactate measurement

Lactate levels were measured using the Lactate Colorimetric Assay kit (Sigma-Aldrich) as described [27]. Supernatants from each condition were analyzed in triplicate, with protein normalization.

## shRNA transfection and selection

GDC from D1, G, I and SRC3 patients were plated in 6-well plates (700 000 cells/well) and transfected, using Fugene HD (Promega), with 3µg of plasmids encoding either specific shRNA for CA9 (ShCA9-1 and 4) or unspecific shCTR (Qiagen, Supplementary Table S1). After five days, transfected cells were selected for at least 3 weeks in FCS medium with 0.8 mg/mL G418 (Merck).

## Quantitative real-time RT-PCR (qPCR)

qPCR was performed as described [10] (see Supplementary Methods). Some experiments used commercial RNA (Supplementary Table S2).

## Western blotting and flow cytometry

Western-blot and flow-cytometry were conducted as shown [10] (see Supplementary Methods).

## Neurospheres generation assay

Limiting dilution assays were conducted as described [45] (see Supplementary Methods).

## RNA-sequencing (RNA-seq) analysis

RNA-seq data were downloaded from NCBI Gene Expression Omnibus (GEO) GSE54791, originating from a study by Suva et al. comparing three different human GSC cell lines (MGG4, MGG6 and MGG8) and their matched differentiated progeny (GDC) [46]. Each cell line was analyzed in triplicate condition. Briefly, the counting table (in CPM) has been downloaded from GSE54791 and only genes with sufficient information (more than 10 counts across samples) were retained. The data were then log-normalized (log2(CPM + 1)) and differential analysis was performed with limma [47], taking into account biological replicates and cell types. The p-values were corrected for multiple testing using the method of Benjamini & Hochberg (BH).

Molecular subtype of the GSC cell lines used in the present study was determined according to Wang classification [48], using the Gliovis data portal and the SubtypeME tool (http://gliovis.bioinfo.cnio.es) [49], in order to classify our GSC into three tumor-specific subtypes: proneural (PN), mesenchymal (Mes), and classical (CL). Affymetrix array datasets (for C1, D1 and I GSC) and RNA-seq datasets (for SRA5, SRB1 and SRC3 GSC), obtained from our previous publications ([50] and [51], respectively), were used to subtype our different GSC. For SRA5, SRB1 and SRC3 GSC, we also determined

in a previous publication the molecular subtypes of the patients' biopsies from whom these GSC were derived [42]. These subtypes are described in Supplementary Table S3 and illustrate the molecular heterogeneity of the GSC cell lines used in this study.

## Statistical analysis

Data are means ± S.E.M. ≥ 3 independent experiments. Significance (*P* < 0.05, **P* < 0.01 and ***P* < 0.001) was assessed using the Student *t*-test.

## Results

### Early stem marker expression after IR in GBM Cells subjected to the dedifferentiation protocol

To investigate early mechanisms which could initiate and drive the long-term dedifferentiation induced in GBM cells at 30 days after IR exposition at least [10], we analyzed several stem cell transcription factors (SCTF) and stem markers from 48h to one week post-IR. Olig2, Sox2, and Nestin, highly expressed in parental GSC lines, were specifically upregulated in irradiated cells in stem medium (SCM 3Gy) as early as 48–72h post-IR, with little or no expression in non-irradiated controls (SCM CTR) (Fig. 1). This early SCTF induction, observed in four primary GBM cell lines (C1, G, I and SRA5), correlates with IR-induced long-term dedifferentiation in the same cell lines, enhancing self-renewal and tumorigenesis [10]. As in the long-term protocol [10], IR did not modulate stem marker expression in forced differentiation conditions (FCS). Additionally, survivin, an anti-apoptotic protein mediating IR-induced dedifferentiation [10], was upregulated alongside SCTF as early as 48h post-IR (Fig. 1). These findings suggest that both SCTF and survivin are rapidly induced in permissive SCM conditions and maintained throughout the dedifferentiation process, promoting the phenotypic transition to a stem-like state.

### Metabolic disparity between GSC-enriched NS and their differentiated progeny

To uncover early mechanisms driving short-term adaptation in GBM cells to subtoxic IR dose and SCTF over-expression at 48h post-IR, we analyzed global metabolic pathways supporting ATP production and anaplerotic biosynthesis (lipids, amino acids), crucial for proliferation, stress repair, and survival in GBM [52]. While differentiated GBM cells favor glycolysis and the Warburg effect, GSC rely more on oxidative phosphorylation (OXPHOS), exhibit lower glucose dependency, and display higher oxygen consumption rates (OCR) in CD133+ GSC [32–34]. Using Seahorse system, we measured OCR and ECAR (extracellular acidification rate) in six NS cell lines (C1, D1, G, SRA5, SRB1, SRC3) and their differentiated progeny after at least 15 days of forced
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Early overexpression of stemness markers during irradiation-induced dedifferentiation. Differentiated GBM cells from 4 patient cell lines (C1, G, I and SRA5) treated or not by a 3 Gy irradiation (IR) and placed 1 day after in either FCS or SCM medium for short-term culture (2 to 7 days post-IR) were analyzed by western blotting for stem marker (Olig2, Sox2 and Nestin) and Survivin expression. Protein expression levels in GSC-enriched NS (neurosphere) parental cells were shown as a control for the stem condition. Equal gel loading and transfer efficiency were checked with anti-actin antibody. Blots are representative of at least three independent experiments</figcaption></figure>

differentiation [10]. NS showed significantly higher OCR and ECAR than their differentiated counterparts, reflecting a more energetic phenotype with an increased mobilization of the O₂-consuming/CO₂-producing mitochondrial metabolism for ATP production [53], and a higher maximal respiration (MR) (Fig. 2A–D). Indeed, an elevated ECAR, typically associated with glycolysis via H⁺/lactate extrusion by monocarboxylate transporters (MCT), may also result from a high CO₂ production due to a fast-cycling TCA cycle. This CO₂ is then expelled from the cell and converted into bicarbonates through a H⁺-generating reaction catalyzed by extracellular carbonic anhydrases (CA) [35, 54]. Thus, the increased ECAR in NS likely reflects enhanced mitochondrial metabolism, as indicated by higher OCR and MR levels.

Despite this higher energetic state, total ATP content was similar between GSC (I, SRC3) and their differentiated counterparts (Fig. 2E), suggesting that GSC and GDC may produce a similar ATP level but through distinct energetic pathways. It was indeed shown that this ATP production in NS and FCS-differentiated cells has not the same origin, either through OXPHOS or aerobic glycolysis, respectively [32]. Selective inhibition of these pathways (see Materials & Methods) confirmed that NS cells from I and SRC3 patients rely more on mitochondria and OXPHOS for ATP production than their differentiated progeny (Fig. 2F). These results suggest that metabolic differences are linked to GBM cell differentiation status and may emerge during IR-induced plasticity.

## Early metabolic shift in GBM cells during the first steps of the radio-induced dedifferentiation

To explore the role of metabolic differences between parental NS and their differentiated progeny in early IR-driven dedifferentiation, Seahorse analysis was performed on five primary cell lines exposed to IR for 24–72h in FCS or SCM medium. In all cell lines (D1, G, SRA5, SRB1 and SRC3), OCR and ECAR specifically and significantly increased after IR in SCM at 48h or 72h post-IR (Fig. 3A, B and Supplementary Figure S1). In SRC3 cells, a kinetic analysis showed OCR enhancement in permissive SCM medium at 48h post-IR, further increasing at 72h, compared to non-irradiated cells, along with a similar rise in ECAR. In contrast, no significant OCR or ECAR increase was observed in FCS at any time point (Fig. 3B). Similar expression patterns, illustrated by representative energy phenograms, were observed in the four other cell lines at 48h or 72h post-IR (Supplementary Figure S1). In G cells, increased OCR correlated with higher mitochondrial MR levels in SCM post-IR (Fig. 3C). These findings suggest that IR-induced dedifferentiation occurs specifically in cells undergoing an early and sustained metabolic shift, characterized by increased O₂ consumption and extracellular acidification.

To analyze glycolysis and OXPHOS processes during early dedifferentiation, ATP sources were examined at 72h post-IR. SRC3 cells showed a significant increase in mitochondrial ATP in SCM 3Gy condition, with a similar trend in C1 cells, but no changes in FCS conditions (Fig. 3D). No changes in total ATP levels were observed
<figure><img src="image_5.png" /><figcaption>**Fig. 2** (See legend on next page.)</figcaption></figure>
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**Fig. 2** Differences in the metabolic state of GSC and their differentiated progeny. GSC-enriched NS cell lines isolated from 7 patient tumors (C1, D1, G, I, SRA5, SRB1, SRC3) were kept in SCM medium (NS) or allowed to differentiate as adherent GBM cells for at least 15 days in FCS medium (FCS). **A–C** Sea-horse analysis of **A** basal Oxygen Consumption Rate (OCR), **B** Extracellular Acidification Rate (ECAR) and **C** energy phenograms of mean OCR and ECAR, after normalization to total protein content for the indicated primary GBM cell lines in FSC or NS culture condition. In **A & B**, results are expressed as ratios to each NS condition. **D** Seahorse analysis of Maximal Respiration in G and SRA5 primary cell lines in FSC or NS culture condition, after normalization to total protein content. Results are expressed as percentages relative to NS condition. **E** Total intracellular ATP content in either NS or FCS condition in the indicated primary GBM cell lines. **F** Percentage of mitochondrial ATP was determined in the indicated primary GBM cell lines. **A–F** Shown are the means ± S.E.M. of at least three independent experiments *P*<0.05, **P**<0.001 compared with the NS condition. ns non-significant

in SRC3, C1, and I cell lines under the same culture conditions, with or without IR (Supplementary Figure S2). This confirms a shift to OXPHOS in response to IR specifically in permissive SCM medium. Lactate, a product of aerobic cytosolic glycolysis, was also quantified in supernatants during in the first dedifferentiation steps. In three cell lines (SRC3, C1, D1), IR did not induce lactate increase in permissive SCM at 48 or 72h post-IR. In FCS medium, only SRC3 cells showed a brief lactate overproduction at 48h post-IR (Fig. 3E). We also checked this lactate production all along the long-term dedifferentiation protocol in G cells and did not observe any lactate increase after IR in either SCM or FCS medium (Supplementary Figure S3). Thus, the significant ECAR increase observed at 48/72h post-IR in SCM does not appear to result from increased lactate production via aerobic glycolysis.

To assess whether IR-induced mitochondrial oxidative metabolism is fueled by glucose via pyruvate synthesis, TCA cycle and subsequent OXPHOS, we quantified glucose uptake at 48h, 72h, and 96h post-IR in SRC3 cells (Fig. 3F). A transient glucose uptake increase was observed at 48h in both FCS and SCM conditions after IR but it disappeared at 72h and 96h. Given the sustained OCR increase at 72h post-IR in SCM but not in FCS condition, this brief glucose uptake rise likely does not drive OXPHOS activation during dedifferentiation. Instead, it may serve as an early defense mechanism for repair and survival following IR-induced damages, independently of the culture condition. This suggests that other substrates, such as glutamine or fatty acids, contribute to IR-induced OXPHOS reprogramming and plasticity.

## Early overexpression of carbonic anhydrase IX (CA9) during IR-induced dedifferentiation

ECAR increased over time after IR specifically in permissive SCM medium (Fig. 3B). Since lactate level remained stable across all conditions (Fig. 3E), lactate/H+ extrusion is likely not the main driver of extracellular acidification. To explore alternative mechanisms behind extracellular acidosis, we examined another source of acidosis in tumor cells, i.e. CO₂ production via TCA cycle and mitochondrial metabolism [35]. Indeed, CO₂ can be expelled into the extracellular microenvironment, generating protons and bicarbonate ions through Carbonic Anhydrases, with HCO₃⁻ re-entering cells to buffer intracellular pH

and maintain cellular function homeostasis. Among these enzymes, Carbonic Anhydrase IX (CA9) is highly expressed in GBM biopsies and linked to poor prognosis in GBM patients [55, 56]. As CA9 is also associated with stemness maintenance in breast and GBM CSC models [57–59], we analyzed it expression in normal adult brain cells (cortex/white matter), Neural Stem cells (NSC), various conventional GBM cell lines (SF763, SF767, U87, U138, U251 & LN18) and our patient-derived NS and their differentiated progenies (Fig. 4A). Normal adult brain cells expressed very low CA9 mRNA levels compared to GBM samples (except SF763/SF767 cell lines). Additionally, a pool of 10 GSC-enriched NS cell lines derived from patient resections exhibited significantly higher CA9 expression than their differentiated progeny. NSC also overexpressed CA9 compared to normal adult brain cells, at levels similar to GSC. Comparing CA9 mRNA expression between NS and FCS-differentiated conditions for each NS cell line, we confirmed that 4/10 NS lines (C1, D1, G, I) significantly overexpressed CA9, with a similar trend in one additional line (K). However, the remaining five NS lines showed similar CA9 levels between differentiation states (Fig. 4B). Since both CA9-overexpressing and non-overexpressing NS cells showed increased ECAR than differentiated cells, basal CA9 expression alone may not explain extracellular acidification in all GSC lines. In some GSC cultures, another extracellular acidifying system may complement or surpass CA9 to stabilize the metabolic stem state.

However, regardless of CA9 overexpression in GSC compared to their differentiated progeny, we observed a specific upregulation of CA9 mRNA during the early dedifferentiation stages in SRC3 at 72h post-IR in permissive SCM, with a similar trend in I cells (Fig. 4C). This was confirmed at the protein level in C1, G, and I cell lines through western-blot or FACS analysis (Fig. 4D, E). Moreover, CA9 overexpression in response to IR in SCM persisted throughout long-term dedifferentiation, as the same expression pattern was observed at both mRNA (I and D1 cells) and protein levels (I cells) 30 days post-IR (Fig. 4F, G). Collectively, these data suggest that IR-induced CA9 overexpression in SCM is linked to (i) an oxidative metabolic shift and (ii) extracellular acidification in GBM cells. This led us to investigate whether CA9 plays a direct role in the dedifferentiation process by
<figure><img src="image_6.png" /><figcaption>**Fig. 3** (See legend on next page.)</figcaption></figure>
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**Fig. 3** Early energetic metabolic shift during IR-induced dedifferentiation. Differentiated GBM cells from the indicated patient cell lines (C1, D1, G, SRC3) treated or not by a 3 Gy irradiation and placed 1 day after in either FCS or SCM medium for short-term culture (24h to 96h post-IR) were analyzed for different metabolic parameters. **A** Representative energy phenogram of OCR and ECAR shift during IR-induced dedifferentiation at 48h post-IR in SRC3 cells either in FSC or SCM condition. Mean OCR and ECAR are shown after normalization to total protein content. **B** Seahorse analysis of basal OCR and basal ECAR of SRC3 cells during the dedifferentiation protocol in either FCS or SCM condition at 24h, 48h and 72h post-IR after normalization to total protein content. NS condition was shown as a control for the stem condition. **C** Seahorse analysis of Maximal Respiration in G primary cell line in FSC or SCM culture condition at 72h post-IR, after normalization to total protein content. Results are expressed as percentages relative to FCS CTR condition. **D** The proportion of mitochondrial ATP (in % compared to total ATP) was determined in SRC3 and C1 cells subjected to the dedifferentiation protocol at 72h post-IR. SRC3 NS condition was shown as control for the stem condition. **E** Lactate content in cell supernatants of SRC3, D1 and C1 cells subjected to the dedifferentiation protocol (48h to 96h post-IR), after normalization to total protein content. **F** Glucose uptake capacity in SRC3 cells subjected to the dedifferentiation protocol at 48h, 72h and 96h post-IR, after normalization to total protein content. **B–F** Shown are the means ± S.E.M. of at least three independent experiments, except for C1 cells in (D) (n=2). \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001 compared with the indicated condition. ns non-significant

promoting phenotypic plasticity toward a stem-like state via CA9-dependent extracellular acidosis.

## Role of early CA9 overexpression in IR-induced dedifferentiation of GBM cells

To investigate CA9's role in IR-driven early dedifferentiation in GBM cells, we stably suppressed its expression in patient-derived differentiated cells. SRC3 cells were transfected with two distinct CA9-targeting shRNAs (Sh1 CA9 and Sh4 CA9) and selected in G418 for at least three weeks. Both shRNAs effectively downregulated CA9 at the mRNA and protein levels, confirmed by western blot and flow cytometry (Fig. 5A, Supplementary Figure S4A), compared to shControl (shCTR). To exclude compensation by CA12, another CA with lower catalytic efficiency and also expressed at the extracellular membrane side [60], we analyzed its expression in CA9-deficient cells and found no significant modulation, thus excluding the possibility of compensation for CA9 loss (Fig. 5A). Since CA9 is linked to stemness in breast and GBM CSC [57–59], we performed Western-Blot analysis of stem markers Olig2, Sox2, and Nestin in SRC3, D1, G, and I GSC following CA9 inhibition via shRNA. All cell lines showed decreased stem-like markers after CA9 inhibition, reinforcing CA9's role in stemness (Fig. 5B). Similar trends were observed at the mRNA level, with decreased Olig2/Sox2 and increased GFAP/TUJ1 differentiation markers in G or SRC3 cells transfected with CA9-targeting shRNA (Supplementary Figure S4B, C). Finally, extreme limiting dilution assays revealed reduced NS formation in CA9-knockdown D1 and G GSC, indicating impaired self-renewal (Fig. 5C). These findings demonstrate that stable CA9 inhibition via shRNA in GSC is not compensated by CA12 and significantly disrupts GSC stemness maintenance.

## Role of CA9 in extracellular acidosis and cell plasticity during IR-induced dedifferentiation

To assess CA9's role in early molecular changes during IR-induced dedifferentiation, transfected differentiated cells underwent the in vitro dedifferentiation protocol in SCM and were analyzed at 72h post-IR. Stem (Olig2)

and differentiation (TUJ1) markers were examined at the mRNA and protein levels to confirm CA9's contribution to stem phenotype acquisition (Fig. 6A, B). CA9-targeting shRNAs effectively reduced basal and IR-induced CA9 expression at 72h post-IR in SRC3 cells, without affecting CA12, which remained unchanged after IR. Notably, CA9 knockdown completely prevented IR-induced Olig2 overexpression and blocked IR-driven TUJ1 reduction in SCM. To further validate CA9's role in IR-driven stem phenotype acquisition, sphere formation assays were performed. In our previous work [10], NS formed in suspension in SCM after 20–30 days. Here, we assessed early changes by monitoring adherent neurospherical structures as an initial NS formation indicator. In D1 and SRC3 cells transfected with shCTR, IR potentiated morphological cellular changes in SCM, leading to the formation of early adherent neurospherical structures at 72h post-IR. However, shCA9-transfected cells did not exhibit these changes in response to IR (Fig. 6C and Supplementary Figure S5). Finally, we investigated CA9's role in early extracellular acidification linked to the mitochondrial metabolic shift induced by IR in cells undergoing dedifferentiation in SCM, in order to connect extracellular acidosis to IR-driven phenotypic plasticity. To assess this, OCR and ECAR were measured in SRC3 cells expressing shCTR or shCA9. Basal analysis and energy maps (Fig. 6D) showed that stable CA9 knockdown with shCA9-1 or shCA9-4 completely prevented the IR-induced ECAR increase at 48h post-IR in SCM, without affecting OCR increase. These results suggest that CA9 contributes to extracellular acidosis induced by IR during early dedifferentiation, with increased mitochondrial O₂ consumption preceding the CA9-associated extracellular acidification.

## Discussion

Despite extensive preclinical and clinical research, no new pharmacologic or antibody-based therapies have significantly improved GBM prognosis since the TMZ/ Radiotherapy (60 Gy total dose in 30 daily fractions of 2 Gy) combination in 2005 [2]. However, adding TTF to TMZ after chemoRT has recently shown improved
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<figure><img src="image_14.png" /><figcaption>**Fig. 4** (See legend on next page.)</figcaption></figure>
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**Fig. 4** Carbonic anhydrase IX (CA9) overexpression in GSC and during IR-induced dedifferentiation. **A** mRNA expression levels of CA9 in NSC (mean of 3 different samples), adult brain cortex (mean of 9 different samples), adult normal white matter (mean of 1 sample analyzed in triplicate), human GSC-enriched NS (mean of 10 different patient GSC cell lines) and their differentiated progeny (FSC, mean of 10 different cell lines) and six conventional human GBM cell lines (SF763, SF767, U87, U138, U251 and LN18, with at least 3 independent samples per cell line, except for SF767 and U138 (n=2)). Shown are the fold amplifications relative to the indicated condition expressed as means ± S.E.M. **B** mRNA expression level of CA9 in GSC-enriched NS cell lines and in their differentiated counterparts kept at least 15 days in FCS medium (FCS). Shown are fold inductions relative to the FCS condition expressed as means ± S.E.M. of at least three independent experiments. **C** mRNA expression level of CA9 at 72h post-IR during the dedifferentiation process in SRC3 and I cell lines. Shown are the fold inductions relative to the indicated condition expressed as means ± S.E.M. of at least three independent experiments. *nd*: not determined. **D** FACS analysis of CA9 at the membrane of C1 cells at 24-48h post-IR (left panel). Representative FACS plot overlay (SCM CTR versus SCM 3 Gy) was depicted (right panel). Shown are the means ± S.E.M. of Specific Fluorescence Index (SFI) values for five independent experiments. **E** Western blotting analysis of CA9 expression in I or G cell lines at the indicated time post-IR during the early dedifferentiation process. NS conditions were shown as controls for the stem condition. Equal gel loading and transfer efficiency were checked with anti-actin antibody. Blots are representative of three independent experiments. **F, G** Expression of CA9 in I or D1 cell lines during the long-term dedifferentiation process. mRNA **F** and protein **G** expression levels of CA9 were analyzed at the end (30 days post-IR) of the dedifferentiation protocol. NS conditions were shown as controls for the stem condition. Quantitative PCR results are expressed as fold inductions relative to the SCM CTR condition and are shown as means ± S.E.M. of at least three independent experiments. For blots, equal gel loading and transfer efficiency were checked with an anti-β2M antibody. Blots are representative of three independent experiments. **A–D, F*P < 0.05, **P < 0.01, ***P < 0.001 compared with the indicated condition. *ns* non-significant

overall (OS) and progression-free survival (PFS) in GBM patients [3]. In combination with chemoRT, several drugs, including Cilengitide (αVβ3/αVβ5-integrin inhibitor), Nivolumab (anti-PD-1 antibody), and Bevacizumab (BVZ, anti-VEGF antibody), failed to enhance OS in phase III trials for newly diagnosed GBM [61], though retrospective analysis suggests BVZ may benefit Proneural GBM patients [61]. The lack of effective treatments highlights the urgent need to understand resistance and adaptive mechanisms in these aggressive tumors. RT is a cornerstone of GBM treatment, but resistant clones, mainly GSC, can survive and be selected under RT-induced stress. This process is a well-known driver of tumor repopulation and the development of a more resistant secondary GBM [5]. Beyond clonal selection, we identified another mechanism, showing in vitro that subtoxic, clinically relevant IR doses trigger a survivin-mediated plasticity process, shifting resistant differentiated GBM cells towards a more stem-like state [10]. This phenotypic shift leads to a sustained increase in their self-renewal and tumorigenic potential, along with heightened expression of multiple stemness markers.

To investigate early dedifferentiation mechanisms induced by IR, we applied our previous methodology [10] to several primary GBM cell lines, focusing on short-term adaptive mechanisms during the first steps of the phenotypic shift (1 to 7 days post-IR). We analyzed early metabolic changes triggered by IR, known to alter energy production and biosynthesis in GBM cells and GSC [25, 26, 28]. This approach helps identify early targets enabling long-term phenotypic adaptation to metabolic stress from therapies like RT, which may drive tumorigenic rebound and recurrence. Additionally, evidence suggests that GBM cell differentiation status is linked to specific metabolic profiles, influencing tumorigenicity and radioresistance [32–34].

## Interplay between mitochondrial metabolism and glioblastoma cell stemness

In this study, we first characterized the basal metabolic state of several GSC cultures and their differentiated progenies. All GSC-enriched NS cell lines, despite marked molecular heterogeneity that can be seen through the presence of different molecular subtypes, exhibited higher OXPHOS-related O₂ consumption and mitochondrial ATP production than differentiated cells. This increased OCR is associated with elevated extracellular acidification, which correlates in most of our GSC lines with CA9 expression, an enzyme that converts CO₂ into H⁺ + HCO₃⁻, as seen in breast and GBM CSC [57–59]. Other mechanisms of extracellular acidosis should be explored, as lactate/H⁺ extrusion or H⁺ export via Na⁺/H⁺ exchangers (NHE) may also contribute to the ECAR increase in GSC. Indeed, MCT4 and NHE9, involved in stemness maintenance, are overexpressed in these cells [62, 63]. Consistent with previous studies, undifferentiated GBM cells favor OXPHOS, while differentiated cells rely almost exclusively on glycolysis [32, 34], similar to breast CSC [64]. This metabolic difference may arise from GSC oxidizing substrates like glutamine, fatty acids or extracellular lactates as observed in GBM cells and GSC in vitro and in vivo [28, 30, 65, 66], potentially enhancing mitochondrial-driven biosynthesis, OXPHOS and tumor repopulation. To support this, we analyzed the GSE54791 Dataset from GEO which contained RNA-seq data from three paired human GSC and GDC cultures [46] and observed a strong tendency to the over-expression of genes linked to Fatty Acid Oxidation (FAO) in GSC compared to their differentiated progeny, as wells as a similar trend for genes involved in glutamine-linked energetic metabolism. Of note, these RNA-seq data also confirmed our observations in GSC, with a significant increase in TCA and OXPHOS-related genes expression in GSC as well as an increase of glycolysis-related genes in paired GDC (Supplementary Figure S6).
A

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B

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C

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**Fig. 5** Role of CA9 in the stemness maintenance. **A–C** SRC3, D1, G or I cells were transfected by either shCTR (non-specific control) or two different shRNA against CA9 (sh1-CA9 and sh4-CA9). Transfected cells were then selected in G418-containing SCM medium in order to check CA9 expression by real-time qPCR and Western blotting **A**, as well as to analyze the effects of CA9 knockdown on stem markers expression **B** and the neurosphere forming ability through limiting dilution assay **C**. **A** mRNA and protein expression levels of CA9 and CA12 in stably transfected SRC3 cells expressing shCTR, sh1-CA9 or sh4-CA9. Shown are the fold inductions relative to the shCTR condition expressed as means ± S.E.M. of at least three independent experiments. **ns** non-significant, \*\*\*P < 0.001. For Western blotting, equal gel loading and transfer efficiency were checked with anti-actin antibody. Blots are representative of at least three independent experiments. **B** Western blotting analysis of CA9, Olig2, Nestin and Sox2 in stably transfected SRC3, D1, G and I cells expressing shCTR, sh1-CA9 or sh4-CA9. Blots are representative of at least three independent experiments for SRC3 and two experiments for D1, G and I cells. **C** Neurosphere (NS) forming assay performed with stably-transfected G (left panel) or D1 (right panel) cells expressing shCTR, sh1-CA9 (G cells) or sh4-CA9 (D1 cells). Assays were performed in limiting dilution conditions as described in *Materials and Methods*. Shown are means ± S.E.M of NS number per well of three independent experiments for G cells and one experiment for D1 cells (N = 16 technical replicates). **A, C** \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 compared with the indicated condition. **ns**: non-significant

## Glioblastoma dedifferentiation via CA9-driven early metabolic shift

We next demonstrated that IR triggers an early metabolic shift from glycolysis to OXPHOS during the initial stages of dedifferentiation in permissive stem medium. This reprogramming, marked by increased OCR and mitochondrial ATP production without lactate accumulation, was sustained over time. As glucose uptake remained unchanged in SCM 3Gy conditions, other substrates like fatty acids or glutamine may be fueling this shift, similar to glutaminolytic adaptations in GBM under mTOR-targeted therapy [65]. From 48h post-IR, OCR increase coincided with extracellular acidification

and overexpression of GSC markers (Olig2, Sox2, Nestin, Survivin). We showed that IR induces an early rise in stem markers, initiating a long-term dedifferentiation process [10]. This increase, along with extracellular acidosis, occurred only in permissive stem medium post-IR, suggesting a link to dedifferentiation. An acidic environment is known to support GSC stemness [40] and enhance self-renewal [41]. To investigate the role of IR-induced acidosis in plasticity, we blocked extracellular acidification.

To identify the source of the ECAR increase, we analyzed extracellular H⁺ production. In tumor cells, lactate overproduction from glycolysis is typically the main
<figure><img src="image_19.png" /></figure>

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<figure><img src="image_24.png" /><figcaption>**Fig. 6** (See legend on next page.)</figcaption></figure>
(See figure on previous page.)

**Fig. 6** Role of CA9 in the early steps of IR-induced dedifferentiation process and metabolic alterations induced by IR in SCM medium. Differentiated stably-transfected SRC3 cells with either shCTR, sh1-CA9 or sh4-CA9 were treated or not by a 3Gy irradiation and subjected to the short-term dedifferentiation protocol in SCM medium. A mRNA expression level of CA9, Olig2 (stem marker), TUJ1 (differentiation marker) and CA12 in stably-transfected SRC3 cells expressing shCTR, sh1-CA9 or sh4-CA9 maintained in SCM medium 72h post-IR. Results are expressed as the means ± S.E.M. fold inductions relative to the shCTR cells in SCM CTR condition of at least three independent experiments. B Western blotting analysis of CA9, CA12, Olig2 in stably transfected SRC3 cells expressing shCTR or sh1-CA9 subjected to the dedifferentiation protocol in SCM medium at 72h post-IR. Equal gel loading and transfer efficiency were checked with anti-actin antibody. Blots are representative of three independent experiments. C Phase-contrast photomicrographs (upper panel) of SRC3 cells stably expressing shCTR or sh1-CA9 subjected to the dedifferentiation protocol in SCM medium at 72h post-IR (Original magnification:×10, scale bar: 6 µm). On the lower panel, shown are the quantifications of the number of neurospherical structures (arrows) counted per well, expressed as means ± S.E.M of at least three independent experiments. D Seahorse analysis of basal OCR (upper panel), basal ECAR (middle panel) and related energy phenograms (lower panel) in stably transfected SRC3 cells expressing shCTR, sh1-CA9 or sh4-CA9 subjected to the dedifferentiation protocol in SCM medium at 48h post-IR. Quantifications of OCR and ECAR variations are expressed as the means ± S.E.M. of at least four independent experiments, after normalization to total protein content. A, C, D *P*<0.05, **P**<0.01, ***P**<0.001 compared with the indicated condition. ns non-significant, *nd* non-determined

driver of pHe reduction [35]. However, our model shows reduced aerobic glycolysis without an increase in extracellular lactate, ruling out MCT transporters in post-IR acidosis. Instead, pHe decrease may result from CO₂ overproduction [67, 68], possibly via enhanced Pentose Phosphate Pathway (PPP) and/or mitochondrial metabolism (TCA cycle and Pyruvate Dehydrogenase), consistent with increased OXPHOS-linked O₂ consumption. This CO₂ is converted into HCO₃⁻ and H⁺ by membrane-bound CA9 and CA12, contributing to acidosis [35]. HCO₃⁻ then re-enter the cell to buffer pH, maintaining optimal functions at physiological pH. We and others have shown that CA9, like CA12, is overexpressed in GBM compared to normal brain tissue [56, 57], and that GSC often express higher CA9 than differentiated cells [59], similar to breast CSC [58]. Both CA9 and CA12 have been linked to poor prognosis in GBM patients [57]. CA9 expression is typically linked to hypoxia via the PI3K/Akt pathway but can also be regulated by the mitogen-activated protein kinase (MAPK) pathway, independently of hypoxia [69]. We showed that, unlike CA12, CA9 is specifically overexpressed by IR in stem medium during early and late dedifferentiation stages. This is among the first evidence that IR can induce CA9 expression also observed in rectal adenocarcinomas post-chemoRT [70]. Additionally, CA9 inhibition has been linked to radio- and chemosensitization in both GBM and colon cells [57, 71–73]. Its downregulation in our model confirmed its role in extracellular acidification and early stem-like features (Olig2 expression, neurospherical structure formation) post-IR. However, OCR increase and CA12 expression remained unchanged after CA9 knockdown, suggesting OCR rise post-IR precedes acidosis and CA12 cannot compensate for CA9 loss.

In summary, our results show that CA9 links IR-induced early metabolic reprogramming to dedifferentiation via extracellular acidification in GBM cells. Two key questions remain: (i) the mechanism behind IR-driven overactivation of the mitochondrial respiratory chain and (ii) the nature of the substrates or metabolites producing enough NADH/FADH2 for increased OXPHOS and

CO₂ generation post-IR. IR has been linked to enhanced OXPHOS activity and ROS production in GBM and other cancers [25, 28, 64, 74]. IR may also activate mitochondrial Complex II [75], a TCA component via its succinate dehydrogenase activity, generating superoxide [76]. Survivin, upregulated early and throughout IR-induced dedifferentiation, was shown to stabilize Complex II, enhancing OXPHOS and ROS production in GBM cells [77]. It would be interesting to determine if IR-induced survivin drives OXPHOS activation via Complex II, mediating post-IR plasticity. Another key question is identifying the substrates sustaining OXPHOS and CO₂ production, triggering acidosis-driven plasticity. FAO is linked to radioresistance in GBM cells and NSC [28, 78, 79], serving as pro-invasive and immune evasion mechanisms in GBM cells [80, 81]. Additionally, glutamine consumption can be activated in GBM cells under therapy [65, 82] or prolonged acidosis in tumor cells [83], potentially creating an auto-amplification loop in our model. Glutamine, explored as a strategy to sensitize cancers to treatment [84], also generate the TCA intermediate α-ketoglutarate, a key factor in maintaining embryonic stem cell pluripotency [85]. Moreover, in glycolysis-impaired cells, glutaminolysis enhances oxidative PPP activity, increasing CO₂ production and extracellular acidification [67]. Understanding the roles of FAO, glutaminolysis, and PPP in IR-induced metabolic adaptation will improve insights into IR-mediated dedifferentiation in GBM.

## Study considerations

This study has several limitations that will need to be addressed in our future work in order to increase its impact and confirm the CA9 target at the clinical level. First, it is necessary to confirm at the preclinical level that targeting CA9 could block the IR-induced metabolic shift and dedifferentiation of GB cells leading to faster recurrence. To this end, we plan to conduct experiments on 3D cerebroid models implanted with primary GSC lines [86]. We also plan to perform orthotopic xenografts of irradiated GB cells from patients in nude mouse models, as we
routinely do in the laboratory [10, 45], with and without CA9 inhibitors, some of which are already in clinical use [87]. Next, although we reproduced our results in 7 primary GB cell lines from 7 different patients, and although the molecular subtypes are quite heterogeneous among these cell lines (Supplementary Table 3), it must nevertheless be considered that this process of metabolic and phenotypic plasticity induced by IR may vary depending on certain types of GB. Particular attention should be paid to this molecular heterogeneity of GB, and the genetic and epigenetic characterization of the GB lines used needs to be correlated with the analysis of this radio-induced plasticity. Finally, from a more mechanistic point of view, as mentioned earlier in the discussion, it is essential to identify the nature of the substrates (glutamine, fatty acids, etc.) that fuel the post-IR increase in OXPHOS and the subsequent dedifferentiation process, in the absence of a proven increase in glucose utilization. Several studies have shown that, unlike GDC, GSC can use these alternative substrates [29, 30], and we have also been able to confirm, through published RNA-seq data analysis of three paired GSC and GDC lines (GSE54791, [46]) that many genes linked to FAO pathway are overexpressed in GSC (Supplementary Figure S6). Our next investigations will therefore focus on determining these metabolic pathways, particularly FAO, during the process of IR-induced dedifferentiation and, if possible, identifying a key player among these pathways whose inhibition could constitute a new therapeutic approach in the radiosensitization of GB.

## Conclusions

From a clinical standpoint, this study highlights the therapeutic potential of targeting molecular mediators, like CA9 and survivin, involved in the early adaptive response to IR. Inhibiting these pathways may prevent long-term phenotypic shifts toward a stem-like state, hinder GSC repopulation, reduce tumor recurrence and ultimately enhance RT effectiveness in GBM patients.

### Abbreviations

<table><tr><td>BVZ</td><td>Bevacizumab</td></tr><tr><td>CA</td><td>Carbonic anhydrases</td></tr><tr><td>CA9</td><td>Carbonic anhydrase IX</td></tr><tr><td>CA12</td><td>Carbonic anhydrase XII</td></tr><tr><td>CMV</td><td>Cytomegalovirus</td></tr><tr><td>CSC</td><td>Cancer stem cell</td></tr><tr><td>ECAR</td><td>Extracellular acidification rate</td></tr><tr><td>FAO</td><td>Fatty acid oxidation</td></tr><tr><td>FCS</td><td>Fetal Calf Serum</td></tr><tr><td>GBM</td><td>Glioblastoma</td></tr><tr><td>GDC</td><td>Glioblastoma differentiated cell</td></tr><tr><td>GSC</td><td>Glioblastoma stem cell</td></tr><tr><td>IR</td><td>Ionizing radiation</td></tr><tr><td>MCT</td><td>Monocarboxylate transporter</td></tr><tr><td>NHE</td><td>Na+/H+ exchanger</td></tr><tr><td>NS</td><td>Neurosphere</td></tr><tr><td>OCR</td><td>Oxygen consumption rate</td></tr><tr><td>OS</td><td>Overall survival</td></tr></table>

<table><tr><td>OXPHOS</td><td>Oxidative phosphorylation</td></tr><tr><td>PFS</td><td>Progression free survival</td></tr><tr><td>PPP</td><td>Pentose phosphate pathway</td></tr><tr><td>ROS</td><td>Reactive oxygen species</td></tr><tr><td>RT</td><td>TMZ-Radiotherapy</td></tr><tr><td>SCM</td><td>Stem cell medium</td></tr><tr><td>SCTF</td><td>Stem cell transcription factor</td></tr><tr><td>TCA</td><td>Tricarboxylic acid cycle</td></tr><tr><td>TMZ</td><td>Temozolomide</td></tr><tr><td>TTF</td><td>Tumor treating fields</td></tr><tr><td>WHO</td><td>World Health Organization</td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40478-025-02161-2.

Supplementary Material 1.

Supplementary Material 2.

### Acknowledgements

We thank Jean-José Maoret and Frédéric Martins at the Toulouse GeT Platform, Jean-Emmanuel Sarry and Laurence Huc for scientific discussion, Manon Baraut for Seahorse expertise and the CRCT Technology Cluster. We are also grateful to the patients who participated in this study.

### Author contributions

Conceptualization and scientific design: A.L., P.D., C.T. and E.C-J.M. Design of the experiments: A.L. and P.D. Data acquisition and analysis: P.D., T.M., L.M., G.F., A.A., C.H-T., C.D., Y.N. and A.L. Data interpretation: A.L., P.D., T.M., N.S. and E.C-J.M. Tumor biopsies & primary cell lines generation & characterization: C.D., A.L., L.M., S.M., J.M-G., V.L. and P.D. Figures design: A.L., C.D., T.M. and P.D. Manuscript writing and review: A.L., P.D., E.C-J.M. and T.M., with comments from all authors. All authors have read and approved the manuscript.

### Funding

P.D was supported by a PhD fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche, L.M by Plan Cancer 2014-2019/ITMO and T.M by la Ligue Contre le Cancer. This research was funded by la Ligue Contre le Cancer (Hautes-Pyrénées, Lot and Haute-Garonne committees) (to A.L.), by the Association pour la Recherche sur les Tumeurs Cérébrales (ARTC 31, to A.L.), by the Groupe de Recherche de l'Institut Claudius Regaud (GRICR IUCT, to C.T. and E.C.J.M), by the RITC foundation & Région Midi-Pyrénées, by the Fondation ARC PGA 2022 (ARCPGA2021120004247_4869) and by the Institut National de la Santé et de la Recherche Médicale (INSERM).

### Data availability

Not applicable.

### Declarations

#### Ethics approval and consent to participate

The samples were all obtained after written-informed consent from patients admitted to the Neurosurgery Department at Toulouse University Hospital and processed in accordance with the Institution's Human research Ethics Committee. As mentioned, three samples were notably derived from the STEMRI clinical trial (NCT01872221), which was reviewed and approved by the institutional review board, the French ethics committee on 30 May 2012 (registration number 2012-A00585-38), and the French Drug Administration (ANSM) on 15 June 2012 (registration number B120639-40).

#### Consent for publication

Not applicable.

#### Competing interests

The authors declare no competing interests in relation to this work.

#### Author details

¹Université de Toulouse, INSERM, CNRS, Centre de Recherches en Cancérologie de Toulouse, Toulouse, France
²Oncopole Claudius Regaud, IUCT-Oncopole, Interface Department, Toulouse, France

³Université de Toulouse, INSERM, CNRS, Toulouse Neuro Imaging Center, Toulouse, France

⁴Neurosurgery Department, CHU de Toulouse, Toulouse, France

⁵Université de Toulouse, INRAE, ENVT, INP-Purpan, Toxalim (Research Centre in Food Toxicology), Toulouse, France

⁶Faculty of Health, Université de Toulouse, Toulouse, France

⁷Oncopole Claudius Regaud, IUCT-Oncopole, Cancer Biology Department, Molecular Oncology Division, Toulouse, France

⁸Radiation Oncology Department, IUCT-Oncopole, Oncopole Claudius Regaud, Toulouse, France

Received: 20 May 2025 / Accepted: 14 October 2025

Published online: 28 November 2025

## References

1. Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D et al (2021) The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol 23:1231–1251. https://doi.org/10.1093/neuonc/noab106

2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996

3. Stupp R, Taillibert S, Kanner A, Read W, Steinberg DM, Lhermitte B et al (2017) Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: a randomized clinical trial. JAMA 318:2306. https://doi.org/10.1001/jama.2017.18718

4. Gimple RC, Yang K, Halbert ME, Agnihotri S, Rich JN (2022) Brain cancer stem cells: resilience through adaptive plasticity and hierarchical heterogeneity. Nat Rev Cancer 22:497–514. https://doi.org/10.1038/s41568-022-00486-x

5. Biserova K, Jakovlevs A, Uljanovs R, Strumfa I (2021) Cancer stem cells: significance in origin, pathogenesis and treatment of glioblastoma. Cells 10:621. https://doi.org/10.3390/cells10030621

6. Annovazzi L, Mellai M, Schiffer D (2017) Chemotherapeutic drugs: DNA damage and repair in glioblastoma. Cancers (Basel) 9:57. https://doi.org/10.3390/cancers9060057

7. Lee CAA, Banerjee P, Wilson BJ, Wu S, Guo Q, Berg G et al (2020) Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism. J Biol Chem 295:7774–7788. https://doi.org/10.1074/jbc.RA120.013778

8. Ulasov IV, Nandi S, Dey M, Sonabend AM, Lesniak MS (2011) Inhibition of sonic hedgehog and notch pathways enhances sensitivity of CD133(+)-glioma stem cells to temozolomide therapy. Mol Med 17:103–112. https://doi.org/10.2119/molmed.2010.00062

9. Joo KM, Jin J, Kim E, Ho Kim K, Kim Y, Gu Kang B et al (2012) MET signaling regulates glioblastoma stem cells. Cancer Res 72:3828–3838. https://doi.org/10.1158/0008-5472.CAN-11-3760

10. Dahan P, Martinez Gala J, Delmas C, Monferran S, Malric L, Zentkowski D et al (2014) Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance. Cell Death Dis 5:e1543. https://doi.org/10.1038/ccdis.2014.509

11. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN (2009) The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle 8:3274–3284

12. Gao X-Y, Zang J, Zheng M-H, Zhang Y-F, Yue K-Y, Cao X-L et al (2021) Temozolomide treatment induces HMGB1 to promote the formation of glioma stem cells via the TLR2/NEAT1/Wnt pathway in glioblastoma. Front Cell Dev Biol 9:620883. https://doi.org/10.3389/fcell.2021.620883

13. Lee G, Auffinger B, Guo D, Hasan T, Deheeger M, Tobias AL et al (2016) Dedifferentiation of glioma cells to glioma stem-like cells by therapeutic stress-induced HIF signaling in the recurrent GBM model. Mol Cancer Ther 15:3064–3076. https://doi.org/10.1158/1535-7163.MCT-15-0675

14. Li Y, Li A, Glas M, Lal B, Ying M, Sang Y et al (2011) c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype. Proc Natl Acad Sci U S A 108:9951–9956

15. Fessler E, Borovski T, Medema JP (2015) Endothelial cells induce cancer stem cell features in differentiated glioblastoma cells via bFGF. Mol Cancer 14:157

16. Almairac F, Turchi L, Sakakini N, Debruyne DN, Elkeurti S, Gjernes E et al (2020) ERK-Mediated Loss of miR-199a-3p and Induction of EGR1 Act as a “Toggle Switch” of GBM Cell Dedifferentiation into NANOG- and OCT4-Positive Cells. Cancer Res 80:3236–3250. https://doi.org/10.1158/0008-5472.CAN-19-0855

17. Fornara O, Bartek J, Rahbar A, Odeberg J, Khan Z, Peredo I et al (2016) Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact. Cell Death Differ 23:261–269. https://doi.org/10.1038/cdd.2015.91

18. Zhang L, Shi H, Chen H, Gong A, Liu Y, Song L et al (2019) Dedifferentiation process driven by radiotherapy-induced HMGB1/TLR2/YAP/HIF-1α signaling enhances pancreatic cancer stemness. Cell Death Dis 10:724. https://doi.org/10.1038/s41419-019-1956-8

19. Wang P, Wan W, Xiong S-L, Feng H, Wu N (2017) Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer. Cell Death Discov 3:16105. https://doi.org/10.1038/cddiscovery.2016.105

20. Nakano M, Kikushige Y, Miyawaki K, Kunisaki Y, Mizuno S, Takenaka K et al (2019) Dedifferentiation process driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer. Oncogene 38:780–793. https://doi.org/10.1038/s41388-018-0480-0

21. Ali MY, Oliva CR, Noman ASM, Allen BG, Goswami PC, Zakharia Y et al (2020) Radioresistance in glioblastoma and the development of radiosensitizers. Cancers (Basel) 12:2511. https://doi.org/10.3390/cancers12029511

22. Oliva CR, Markert T, Gillespie GY, Griguer CE (2015) Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas. Oncotarget 6:4330–4344. https://doi.org/10.18632/oncotarget.3015

23. Yu Y, Yu J, Ge S, Su Y, Fan X (2023) Novel insight into metabolic reprogramming in cancer radioresistance: A promising therapeutic target in radiotherapy. Int J Biol Sci 19:811–828. https://doi.org/10.7150/ijbs.79928

24. Tsai Y-T, Lo W-L, Chen P-Y, Ko C-Y, Chuang J-Y, Kao T-J et al (2022) Reprogramming of arachidonate metabolism confers temozolomide resistance to glioblastoma through enhancing mitochondrial activity in fatty acid oxidation. J Biomed Sci 29:21. https://doi.org/10.1186/s12929-022-00804-3

25. Lu C-L, Qin L, Liu H-C, Candas D, Fan M, Li JJ (2015) Tumor cells switch to mitochondrial oxidative phosphorylation under radiation via mTOR-mediated hexokinase II inhibition - a Warburg-reversing effect. PLoS ONE 10:e0121046. https://doi.org/10.1371/journal.pone.0121046

26. Shen H, Hau E, Joshi S, Dilda PJ, McDonald KL (2015) Sensitization of glioblastoma cells to irradiation by modulating the glucose metabolism. Mol Cancer Ther. https://doi.org/10.1158/1535-7163.mct-15-0247

27. Sesen J, Dahan P, Scotland SJ, Saland E, Dang VT, Lemarie A et al (2015) Metformin inhibits growth of human glioblastoma cells and enhances therapeutic response. PLoS ONE 10:e0123721. https://doi.org/10.1371/journal.pone.0123721

28. Ye F, Zhang Y, Liu Y, Yamada K, Tso JL, Menjivar JC et al (2013) Protective properties of radio-chemoresistant glioblastoma stem cell clones are associated with metabolic adaptation to reduced glucose dependence. PLoS ONE 8:e80397. https://doi.org/10.1371/journal.pone.0080397

29. Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L et al (2015) Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma. Nat Cell Biol 17:1556–1568. https://doi.org/10.1038/nccb3272

30. Lin H, Patel S, Affleck VS, Wilson I, Turnbull DM, Joshi AR et al (2017) Fatty acid oxidation is required for the respiration and proliferation of malignant glioma cells. Neuro-oncol 19:43–54. https://doi.org/10.1093/neuonc/now128

31. Garnier D, Renoult O, Alves-Guerra MC, Paris F, Pecqueur C (2019) Glioblastoma stem-like cells, metabolic strategy to kill a challenging target. Front Oncol 9:118. https://doi.org/10.3389/fonc.2019.00118

32. Janiszewska M, Suva ML, Riggi N, Houtkooper RH, Auwerx J, Clement-Schatzo V et al (2012) Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells. Genes Dev 26:1926–1944

33. Kuramoto K, Yamamoto M, Suzuki S, Sanomachi T, Togashi K, Seino S et al (2020) Verteporfin inhibits oxidative phosphorylation and induces cell death specifically in glioma stem cells. FEBS J 287:2023–2036. https://doi.org/10.1111/febs.15187

34. Vlashi E, Lagadec C, Vergnes L, Matsutani T, Masui K, Poulou M et al (2011) Metabolic state of glioma stem cells and nontumorigenic cells. Proc Natl Acad Sci U S A 108:16062–16067

35. Parks SK, Chiche J, Pouyssegur J (2013) Disrupting proton dynamics and energy metabolism for cancer therapy. Nat Rev Cancer 13:611–623
36. Boedtkjer E, Pedersen SF (2020) The acidic tumor microenvironment as a driver of cancer. Annu Rev Physiol 82:103–126. https://doi.org/10.1146/annur ev-physiol-021119-034627

37. McIntyre A, Patiar S, Wigfield S, Li J, Ledaki I, Turley H et al (2012) Carbonic anhydrase IX promotes tumor growth and necrosis in vivo and inhibition enhances anti-VEGF therapy. Clin Cancer Res 18:3100–3111. https://doi.org/1 0.1158/1078-0432.ccr-11-1877

38. Reichert M, Steinbach JP, Supra P, Weller M (2002) Modulation of growth and radiochemosensitivity of human malignant glioma cells by acidosis. Cancer 95:1113–1119. https://doi.org/10.1002/cncr.10767

39. Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornell HH, Ibrahim-Hashim A et al (2013) Acidity generated by the tumor microenvironment drives local inva- sion. Cancer Res 73:1524–1535. https://doi.org/10.1158/0008-5472.can-12-27 96

40. Hjelmeland AB, Wu Q, Heddleston JM, Choudhary GS, MacSwords J, Lathia JD et al (2011) Acidic stress promotes a glioma stem cell phenotype. Cell Death Differ 18:829–840

41. Hu P, Li S, Tian N, Wu F, Hu Y, Li D et al (2019) Acidosis enhances the self- renewal and mitochondrial respiration of stem cell-like glioma cells through CYP24A1-mediated reduction of vitamin D. Cell Death Dis 10:25. https://doi.o rg/10.1038/s41419-018-1242-1

42. Lemarié A, Lubrano V, Delmas C, Lusque A, Cerapio J-P, Perrier M et al (2023) The STEMRI trial: magnetic resonance spectroscopy imaging can define tumor areas enriched in glioblastoma stem-like cells. Sci Adv 9:eai0114. http s://doi.org/10.1126/sciadv.ad10114

43. Duval T, Lotterie J-A, Lemarie A, Delmas C, Tensaouti F, Moyal EC-J et al (2022) Glioblastoma stem-like cell detection using perfusion and diffusion MRI. Cancers 14:2803. https://doi.org/10.3390/cancers14112803

44. Helies-Toussaint C, Peyre L, Costanzo C, Chagnon MC, Rahmani R (2014) Is bisphenol S a safe substitute for bisphenol A in terms of metabolic function? An in vitro study. Toxicol Appl Pharmacol 280:224–235

45. Malric L, Monferran S, Delmas C, Arnauduc F, Dahan P, Boyrie S et al (2019) Inhibiting integrin β8 to differentiate and radiosensitize glioblastoma-initiat- ing cells. Mol Cancer Res 17:384–397. https://doi.org/10.1158/1541-7786.MC R-18-0386

46. Suva ML, Rheinbay E, Gillespie SM, Patel AP, Wakimoto H, Rabkin SD et al (2014) Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells. Cell 157:580–594. https://doi.org/10.1016/j.cel l.2014.02.030

47. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W et al (2015) Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43:e47–e47. https://doi.org/10.1093/nar/gkv007

48. Wang Q, Hu B, Hu X, Kim H, Squatrito M, Scarpace L et al (2017) Tumor evolu- tion of glioma-intrinsic gene expression subtypes associates with immuno- logical changes in the microenvironment. Cancer Cell 32(42–56):e6. https://d oi.org/10.1016/j.ccell.2017.06.003

49. Bowman RL, Wang Q, Carro A, Verhaak RGW, Squatrito M (2017) GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol 19:139–141. https://doi.org/10.1093/neuonc/now247

50. Boyrie S, Delmas C, Lemarie A, Lubrano V, Dahan P, Malric L et al (2018) RND1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma. Oncotarget 9:33788–33803. https://doi.org/10.18632/oncotarget.26082

51. Bentaberry-Rosa A, Nicaise Y, Delmas C, Gouazé-Andersson V, Cohen-Jona- than-Moyal E, Seva C (2023) Overexpression of growth differentiation factor 15 in glioblastoma stem cells promotes their radioresistance. Cancers (Basel) 16:27. https://doi.org/10.3390/cancers16010027

52. Bernhard C, Reita D, Martin S, Entz-Werle N, Donwenll M (2023) Glioblas- toma metabolism: insights and therapeutic strategies. IJMS 24:9137. https://d oi.org/10.3390/ijms24119137

53. Ferrick DA, Neilson A, Beeson C (2008) Advances in measuring cellular bioen- ergetics using extracellular flux. Drug Discovery Today 13:268–274

54. Becker HM, Deitmer JW (2021) Proton transport in cancer cells: the role of carbonic anhydrases. IJMS 22:3171. https://doi.org/10.3390/ijms22063171

55. Cetin B, Gonul II, Gumusay O, Bilgetekin I, Algin E, Ozet A et al (2018) Carbonic anhydrase IX is a prognostic biomarker in glioblastoma multiforme. Neuropa- thology 38:457–462. https://doi.org/10.1111/neu.12485

56. Proeschold MA, Merrill MJ, Stoerr EM, Lohmeier A, Pohl F, Brawanski A (2012) Function of carbonic anhydrase IX in glioblastoma multiforme. Neuro Oncol 14:1357–1366

57. Boyd NH, Walker K, Fried J, Hackney JR, McDonald PC, Benavides GA et al (2017) Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide

treatment delays glioblastoma growth in vivo. JCI Insight. https://doi.org/10.1 172/jci.insight.92928

58. Lock FE, McDonald PC, Lou Y, Serrano I, Chafe SC, Ostlund C et al (2013) Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche. Oncogene 32:5210–5219

59. Tatari N, Zhang X, Chafe SC, McKenna D, Lawson KA, Subapanditha M et al (2022) Dual antigen T cell engagers targeting CA9 as an effective immu- notherapeutic modality for targeting CA9 in solid tumors. Front Immunol 13:905768. https://doi.org/10.3389/fimmu.2022.905768

60. Becker HM (2020) Carbonic anhydrase IX and acid transport in cancer. Br J Cancer 122:157–167. https://doi.org/10.1038/s41416-019-0642-z

61. Wang Y, Li S, Peng Y, Ma W, Wang Y, Li W (2023) Progress in phase III clinical trials of molecular targeted therapy and immunotherapy for glioblastoma. Cancer Innovation 2:114–130. https://doi.org/10.1002/cai2.59

62. Kondapalli KC, Llongueras JP, Capilla-Gonzalez V, Prasad H, Hack A, Smith C et al (2015) A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma. Nat Commun 6:6289

63. Lim KS, Lim KJ, Price AC, Orr BA, Eberhart CG, Bar EE (2013) Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner. Onco- gene 33:4433–4441

64. Vlashi E, Lagadec C, Vergnes L, Reue K, Frohnen P, Chan M et al (2014) Metabolic differences in breast cancer stem cells and differentiated progeny. Breast Cancer Res Treat 146:525–534. https://doi.org/10.1007/s10549-014-30 51-2

65. Tanaka K, Sasayama T, Irino Y, Takata K, Nagashima H, Satoh N et al (2015) Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment. J Clin Invest 125:1591–1602

66. Guyon J, Fernandez-Moncada I, Larrieu CM, Bouche CL, Pagano Zottola AC, Galvis J et al (2022) Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis. EMBO Mol Med 14:e15343. https://do i.org/10.15252/emmm.202115343

67. Helmlinger G, Sckell A, Dellian M, Forbes NS, Jain RK (2002) Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism. Clin Cancer Res 8:1284–1291

68. Yamagata M, Hasuda K, Stamato T, Tannock IF (1998) The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase. Br J Cancer 77:1726–1731

69. Kopacek J, Barathova M, Dequiedt F, Sepelakova J, Kettmann R, Pastorek J et al (2005) Mapk pathway contributes to density- and hypoxia-induced expres- sion of the tumor-associated carbonic anhydrase IX. Biochim Biophys Acta 1729:41–49. https://doi.org/10.1016/j.bbaexp.2005.03.003

70. Bádon ES, Beke L, Mokánszki A, András C, Méhes G (2023) Carbonic anhy- drase IX expression and treatment response measured in rectal adenocarci- noma following neoadjuvant chemo-radiotherapy. Int J Mol Sci 24:2581. http s://doi.org/10.3390/ijms24032581

71. Doyen J, Parks SK, Marcié S, Pouysségur J, Chiche J. Knock-down of hypoxia- induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis. Front Oncol. 2013;2. https://doi.org/10.3389/f onc.2012.00199

72. Huang W, He Y, Yang S, Xue X, Qin H, Sun T et al (2023) CA9 knockdown enhanced ionizing radiation-induced ferroptosis and radiosensitivity of hypoxic glioma cells. Int J Radiat Biol 99:1908–1924. https://doi.org/10.1080/0 9553002.2023.2235433

73. Amirı A, Le PU, Moquin A, Machkalyan G, Petrecca K, Gillard JW et al (2016) Inhibition of carbonic anhydrase IX in glioblastoma multiforme. European J Pharmaceutics Biopharmaceutic 109:81–92. https://doi.org/10.1016/j.ejpb.20 16.09.018

74. Lynam-Lennon N, Maher SG, Maguire A, Phelan J, Muldoon C, Reynolds JV et al (2014) Altered mitochondrial function and energy metabolism is associ- ated with a radioresistant phenotype in oesophageal adenocarcinoma. PLoS ONE 9:e100738

75. Yamamori T, Yasui H, Yamazumi M, Wada Y, Nakamura Y, Nakamura H et al (2012) Ionizing radiation induces mitochondrial reactive oxygen species pro- duction accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint. Free Radical Biol Med 53:260–270

76. Lemarie A, Grimm S (2011) Mitochondrial respiratory chain complexes: apoptosis sensors mutated in cancer? Oncogene 30:3985–4003

77. Rivadeneira DB, Caino MC, Seo JH, Angelin A, Wallace DC, Languino LR et al (2015) Survivin promotes oxidative phosphorylation, subcellular
mitochondrial repositioning, and tumor cell invasion. *Sci Signaling.* 8:ra80. https://doi.org/10.1126/scisignal.aab1624

78. Pike LS, Smift AL, Croteau NJ, Ferrick DA, Wu M (2011) Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. *Biochimica et Biophysica Acta (BBA)* 1807:726-734

79. Stoll EA, Makin R, Sweet IR, Trevelyan AJ, Miwa S, Horner PJ et al (2015) Neural stem cells in the adult subventricular zone oxidize fatty acids to produce energy and support neurogenic activity. *Stem Cells* 33:2306-2319. https://doi.org/10.1002/stem.2042

80. Duman C, Di Marco B, Nevedomskaya E, Ulug B, Lesche R, Christian S et al (2023) Targeting fatty acid oxidation via Acyl-CoA binding protein hinders glioblastoma invasion. *Cell Death Dis* 14:296. https://doi.org/10.1038/s41419-023-05813-0

81. Jiang N, Xie B, Xiao W, Fan M, Xu S, Duan Y et al (2022) Fatty acid oxidation fuels glioblastoma radioresistance with CD47-mediated immune evasion. *Nat Commun* 13:1511. https://doi.org/10.1038/s41467-022-29137-3

82. Pike Winer LS, Wu M (2014) Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate. *PLoS ONE* 9:e109916

83. Corbet C, Draoui N, Polet F, Pinto A, Drozak X, Riant O et al (2014) The SIRT1/HIF2alpha axis drives reductive glutamine metabolism under chronic acidosis and alters tumor response to therapy. *Cancer Res* 74:5507-5519

84. Fan Y, Xue H, Li Z, Huo M, Gao H, Guan X (2024) Exploiting the Achilles' heel of cancer: disrupting glutamine metabolism for effective cancer treatment. *Front Pharmacol* 15:1345522. https://doi.org/10.3389/fphar.2024.1345522

85. Carey BW, Finley LWS, Cross JR, Allis CD, Thompson CB (2015) Intracellular [agr]-ketoglutarate maintains the pluripotency of embryonic stem cells. *Nature* 518:413-416. https://doi.org/10.1038/nature13981

86. Linkous A, Fine HA (2020) Generating patient-derived gliomas within cerebral organoids. *STAR Protoc* 1(1):100008. https://doi.org/10.1016/j.xpro.2019.100008

87. Ronca R, Supuran CT (2024) Carbonic anhydrase IX: an atypical target for innovative therapies in cancer. *Biochimica et Biophysica Acta (BBA) - Rev on Cancer* 1879:189120. https://doi.org/10.1016/j.bbcan.2024.189120

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