Abstract
Background
Glioblastoma represents a brain tumor with a notoriously poor prognosis. First-line therapy may include adjunctive Tumor Treating Fields (TTFields) which are electric fields that are continuously delivered to the brain through non-invasive arrays. On a different note, CUSP9v3 represents a drug repurposing strategy that includes 9 repurposed drugs plus metronomic temozolomide. Here, we examined whether TTFields enhance the antineoplastic activity of CUSP9v3 against this disease.
Methods
We performed preclinical testing of a multimodal approach of TTFields and CUSP9v3 in different glioblastoma models.
Results
TTFields had predominantly synergistic inhibitory effects on the cell viability of glioblastoma cells and non-directed movement was significantly impaired when combined with CUSP9v3. TTFields plus CUSP9v3 significantly enhanced apoptosis, which was associated with a decreased mitochondrial outer membrane potential (MOMP), enhanced cleavage of effector caspase 3 and reduced expression of Bcl-2 and Mcl-1. Moreover, oxidative phosphorylation and expression of respiratory chain complexes I, III and IV was markedly reduced.
Conclusion
TTFields strongly enhance the CUSP9v3-mediated anti-glioblastoma activity. TTFields are currently widely used for the treatment of glioblastoma patients and CUSP9v3 was shown to have a favorable safety profile in a phase Ib/IIa trial (NCT02770378) which facilitates transition of this multimodal approach to the clinical setting.
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Introduction
Glioblastoma represents a malignant brain tumor that develops from the intrinsic components of the brain [1]. Despite aggressive first-line therapy that includes maximal safe resection and radio-/chemotherapy with temozolomide, patients with this disease face a dismal prognosis due to therapeutic resistance and recurrence of the tumor [2]. More recently, in a phase III randomized, controlled clinical trial (NCT00916409, EF-14), patients with newly-diagnosed glioblastoma were stratified to either receive radio-/chemotherapy according to the EORTC protocol alone or concomitant with TTFields. The addition of TTFields to maintenance temozolomide was shown to extend the median progression-free survival from 4 to 6.7 months and the median overall survival from 16 to 20.9 months [3]. As a consequence, TTFields joined forces for the first-line therapy of patients with newly-diagnosed glioblastoma.
TTFields involve the transcutaneous delivery of continuous low-intensity (1ā3āV/cm), intermediate-frequency (100ā500ākHz) alternating electric fields that exert biophysical forces on charged and polarizable molecules [4]. In multiple clinical studies, TTFields were shown to have a favorable safety profile and low side effect burden [5]. With respect to the mechanism of action, the anti-mitotic effects of TTFields are most widely recognized [6,7,8]. More recently, interference with DNA repair [9, 10] and pro-immunogenic effects [11,12,13] were observed. Moreover, TTFields were reported to increase the cell permeability of cancer cells [14, 15] and to promote the penetration of small molecules across the blood brain barrier [16].
Intratumoral heterogeneity is regarded as a core feature of glioblastoma and is in great part responsible for the incalcitrant nature of this disease [17]. As a consequence, non-predictable clonal diversion and plasticity of the intracellular signaling pathways in response to changes in the microenvironment and therapeutic pressure render a single-drug/single-target approach futile. To address this, the CUSP9 strategy was developed as a fusion of multitargeting and drug repurposing strategies [18, 19]. A drug repurposing approach was chosen to accelerate clinical transition, minimizing financial costs and safety risks [20]. CUSP9v3 includes nine drugs that are already approved for other indications but also have anti-glioblastoma activity and which are combined with metronomic temozolomide. In vitro, CUSP9v3 was shown to have a strong antineoplastic activity in the setting of glioblastoma [21]. Moreover, in a phase Ib/IIa clinical trial (NCT02770378), a favorable safety profile and good tolerability was found [22].
In this study, we performed preclinical testing of a novel therapeutic strategy combining TTFields and the drug repurposing strategy CUSP9v3 in multiple in vitro models of glioblastoma. Our data show for the first time that this combination treatment leads to predominantly synergistic antineoplastic activity in this setting. TTFields concomitant with CUSP9v3 induce an enhanced pro-apoptotic response which goes along with suppression of oxidative phosphorylation and downregulation of several complexes of the respiratory chain. Both treatment modalities on their own have been safely used in the clinical setting thus facilitating a rapid clinical translation of this combined approach.
Materials and methods
Reagents
Aprepitant (Selleckchem, Houston, TX, USA; stock: 100āmg/mL), auranofin (Sigma Aldrich, St. Louis, MO, USA; stock: 5āmg/mL), celecoxib (Sigma Aldrich; stock: 20āmg/mL), disulfiram (Sigma Aldrich; stock: 5āmg/mL), itraconazole (Abcam, Cambridge, UK; stock: 20āmg/mL) and ritonavir (Sigma Aldrich; stock: 10āmg/mL) were dissolved in dimethyl sulfoxide (DMSO). Captopril (Sigma Aldrich; stock: 50āmg/mL), minocycline (Sigma Aldrich; stock: 25āmg/mL) and sertraline (Sigma Aldrich; stock: 3.8āmg/mL) were dissolved in H2O. All stock solutions were stored at ā20āĀ°C. For all experiments, final concentrations of DMSO were below 0.1% (v/v). TableĀ 1 summarizes the concentrations for each component at the respective dilution used.
In vitro application of TTFields
TTFields were applied using the InovitroTM Lab Bench System (NovocureTM, Haifa, Israel) as described before [23]. For all experiments, the cells were treated with TTFields at an intensity of 1.46āV/cm (RMS) and a frequency of 200ākHz.
Cell cultures and growth conditions
U251 human glioblastoma cells were purchased from Sigma Aldrich in January 2017. The identity of these cells was confirmed by Sigma Aldrich. The initial stocks were expanded, frozen and stored in liquid nitrogen. Fresh aliquots were thawed every 6 weeks. Primary cultured human glioblastoma cells PC38, PC40 and PC128 as well as SC38, SC40 and SC128 stemālike glioblastoma cells were obtained from tumor resections at our institution [24, 25]. The cells were cultured as previously described [26, 27]. The procedures were approved by the institutional review board of the University of Ulm (No.162/10). Consent was granted by the patients or next of kin.
MTT assay
In order to examine effects on cell viability, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were performed. 5āĆā104 cells/well were seeded in 6-well flat-bottomed plates which had a glass coverslip put inside. The cells were left overnight at 37āĀ°C to attach to the coverslips prior to changing the medium to DMEM supplemented with 1.5% FBS and the respective treatments. After 72āh, the medium was aspirated and 800āĀµL MTT solution were added to the wells followed by incubation at 37āĀ°C for 3āh. The reaction was stopped by adding 800āĀµL of 100% isopropanol (Sigma Aldrich) and optical densities were measured at 570ānm using an automated microplate reader.
Spheroid assay
Spheroids were generated to assess effects of the combination treatment in 3 dimensions as described before [26]. Briefly, 3.5āĆā104 U251 or 0.75āĆā104 PC40 and PC128 cells were resuspended in 20āĀµL of a mixture of 80% Matrigel/20% DMEM prior to incubation for 1āh at 37āĀ°C. Then, the cell/Matrigel matrix was gently transferred to 12-well plates containing DMEM supplemented with 10% FBS (20% FBS for PC128 cells) and the spheroids were allowed to form over a period of 5 d. Afterwards, they were transferred again to 12-well plates containing DMEM supplemented with 1.5% FBS and the respective treatments or solvent. Treatments were repeated on day 7 and day 9. On day 12, microphotographs were taken at 4 Ć magnification and maximal sphere expansion (A) was calculated using the formula \(A=\pi {r}^{2}\). In addition, CellTiter-GloĀ® (Roche Diagnostics, Indianapolis, IN, USA) assays were used to assess antiproliferative effects on day 12. To this purpose, spheroids suspended in 100āĀµL of medium were transferred to opaque-walled 96-well plates prior to adding 100āĀµL of the CellTiter-GloĀ® solution and incubation for 10āmin at RT. Afterwards, luminescence was measured.
Measurement of apoptosis
Apoptosis was detected using annexin V/propidium iodide (PI) or PI staining followed by flow cytometry as described before [26, 28].
For PI staining, 5āĆā104 cells were seeded in 6-well plates and allowed to attach overnight. After the respective treatments, the supernatants and enzymatically detached cells (Trypsin/EDTA, Biochrom AG, Berlin, Germany) were centrifuged for 5āmin at 1800ārpm prior to washing twice with PBS. Then, 200āĪ¼l PI staining solution containing 0.05% trisodiumcitrate-dihydrate (Carl Roth, Karlsruhe, Germany), 0.05% triton-X100 and 0.05āmg/ml PI (Sigma Aldrich) was added and cells were incubated for 30āmin at 4āĀ°C prior to flow cytometric analysis.
For Annexin V/PI staining, 5āĆā104 cells were seeded in 6-well plates and allowed to attach overnight. After the respective treatments, the supernatants and enzymatically detached cells (Trypsin/EDTA) were collected. Next, the cells were centrifuged and washed twice with ice-cold annexin V binding buffer containing 140āmM NaCl, 2.5āmM Ca2+ and 1āM 4-(2-hydroxyethyl) piperazine-1- ethane sulfonic acid (pH 7.4) followed by resuspension in 100āĪ¼l binding buffer and incubation with 2.5āĪ¼l Annexin-V-FLUOS (Roche Diagnostics) for 15āmin at RT. Then, the cells were washed once with ice-cold annexin V binding buffer prior to resuspension in 300āĪ¼l buffer, after which PI was added to a final concentration of 2.5āĪ¼M right before performing each single measurement. For each flow cytometric analysis, 10,000 events were recorded using a FACS Cantoā¢ II flow cytometer (BD Biosciences, NJ, USA). FlowJo software version 10.5.3 (Tree Star, Ashland, OR, USA) was used for further quantitative analyses.
To detect intrinsic apoptosis, staining with MitoTrackerTM (Roche Diagnostics) was performed. To this purpose, 5āĆā104 cells were seeded in 6-well plates and allowed to attach overnight. Following the defined treatments and treatment periods, cell culture plates were centrifuged at 1300ārpm for 5āmin prior to aspirating the supernatant and adding 200ānM MitoTrackerTM staining solution. The cells were incubated for 20āmin at 37āĀ°C. Next, the cells were enzymatically detached and centrifuged for 5āmin at 1800ārpm. The supernatants were discarded and the cells were resuspended in 300āĪ¼l PBS followed by flow cytometric analysis. 10,000 events per condition were recorded by a FACS Cantoā¢ II flow cytometer (BD Biosciences) and the data analysis was performed with FlowJo 10.5.3 (Tree Star).
Western blot analysis
Protein expression was determined by Western blot analysis as previously reported [28], using the following primary antibodies: rabbit anti-Mcl-1 (1:1000; #5453āS, clone: D35A5, CST Cell Signaling Technology, Danvers, MA, U.S.A.), rabbit anti-Bcl-2 (1:1000; #2872, CST), rabbit anti-Bcl-xL (1:500; #2764, clone 54H6, CST), rabbit anti-human caspase-3 (1:1000; #9662, CST), total OXPHOS human WB antibody cocktail (1:1000; #ab110411, Abcam, Cambridge, U.K.) and mouse anti-human Ī²-actin (1:2000; clone: AC15; Sigma Aldrich). Secondary HRP-linked antibodies were purchased from CST (#7076āS, #7074āS).
cDNA synthesis and real-time quantitative PCR
Real time PCR experiments were performed as previously described [27] using the primers as outlined in TableĀ 2.
Cell migration assays
Effects on cell migration were analyzed by scratch assays and time-lapse live cell microscopy imaging.
For scratch assays, 5āĆā104 cells/well (PC38 and PC40) or 9āĆā104 cells/well (U251) were seeded on 6-well plates containing glass cover slip inserts. After 24āh, the coverslips were transferred to TTFields ceramic dishes followed by 24āh of the respective treatments. Then, the coverslips were transferred to 12-well plates and a 200āĪ¼l pipet tip was used to induce straight scratches across subconfluent cell layers. Sequential microscopic images were taken at defined time points at 10 Ć magnification (Carl Zeiss, Primovert/AxioCam ICc1, Oberkochen, Germany). The area of the scratch was analyzed using the NIH ImageJ software (http://imagej.nih.gov/ij).
For time-lapse live cell microscopy imaging, 5āĆā104 (PC128 and PC40) or 9āĆā104 (U251) cells were seeded on glass cover inserts placed in 6-well plates and allowed to attach overnight. Then, the cover slips were transferred to TTFields ceramic dishes and treated for 24āh. Afterwards, the cells were enzymatically detached and 3āĆā103 cells/well were seeded on 96-well plates, and microscopic images were taken with an IncucyteĀ® S3 live-cell analysis system (Sartorius/Essen BioScience) every 30āmin for a total observation time of 24āh. During this period, cells were kept at standard culture conditions (37āĀ°C, 5% CO2, water-saturated atmosphere). Single-cell tracking was perfomed with the MtrackJ plugin (www.imagescience.org/meijering/software/mtrackj/) for the NIH ImageJ software. Normalized āwind-roseā plots were generated with the chemotaxis and migration tool from Integrated BioDiagnostics (Martinsried, Germany, www.ibidi.com).
Extracellular flux analysis
5āĆā104 PC128 or 9āĆā104 U251 cells were seeded on glass cover inserts placed in 6-well plates and allowed to attach overnight. Then, the cover slips were transferred to TTFields ceramic dishes and treated for 24āh. Afterwards, the cells were enzymatically detached and 1āĆā104 cells/well were seeded on XF96 V3 PS cell culture microplates (Agilent Technologies Inc., Wilmington, DE, USA) and allowed to attach for 24āh followed by one wash with XF assay medium containing 5āmM glucose (pH adjusted to 7.5). Afterwards, the mito stress test kit (Agilent Technologies Inc.) was used as described by the manufacturer applying serial injections of oligomycin at a final concentration of 2āĀµM, FCCP at a final concentration of 2āĀµM and rotenone/antimycin A at a final concentration of 0.5āĀµM. All analyses were performed on an Agilent Seahorse XFe96 analyzer.
Statistical analysis
Statistical significance was determined by one-way ANOVA followed by NewmanāKeuls post hoc analysis using PRISM version 5.04 (GraphPad, La Jolla, CA, U.S.A.). Statistical significance was assumed for pā<ā0.05. BLISS analysis was performed to characterize the interaction between the different treatment modalities [29, 30]. The total response was calculated as a fractional response to treatment modality A (Fa) + fractional response to treatment modality B (Fb) - Fa Ć Fb. A ratio of the actual total response and the expected total response of 0.9 to 1.1 was considered as additive, a ratio <0.9 as antagonistic and a ratio >1.1 as synergistic.
Results
TTFields concomitant with CUSP9v3 reduce the viability of glioblastoma cells in a predominantly synergistic manner
Both TTFields and CUSP9v3 have been previously shown to affect the viability of glioblastoma cells when applied alone. We hypothesized that an application of both treatment modalities results in a synergistic inhibitory effect on the viability of glioblastoma cells. In order to test this hypothesis, U251 cells (Fig.Ā 1a), PC38, PC40 and PC128 glioblastoma primary cultures (Fig.Ā 1bād) as well as SC38, SC40 and SC128 stem-like glioblastoma cells (Fig.Ā 1eāg) were treated with solvents, TTFields, CUSP9v3 or both modalities. After 72āh of treatment MTT assays were performed. In all glioblastoma models tested, treatment with either TTFields or CUSP9v3 alone led to reduced cell viability (Fig.Ā 1aāg). However, when applied together, the inhibitory effect on cell viability was significantly enhanced across all cells tested including those with stem-like features (Fig.Ā 1aāg). To further characterize the nature of the interaction of the two treatment modalities, Bliss analysis was performed. As shown in Fig.Ā 1h, for the majority of the cells, a synergistic interaction between TTFields and CUSP9v3 was found. In PC128 cells, an additive interaction was found and only in SC128 cells, an antagonistic effect of the combination was observed (Fig.Ā 1h).
TTFields concomitant with CUSP9v3 inhibit the growth of spheroids
Considering that patientsā tumors grow spatially in 3 dimensions, we sought to determine whether TTFields and CUSP9v3 would be able to impair the proliferation of tumor cells in a more representative setting by using 3-dimensional spheroid assays. PC40, PC128 and U251 spheroids were allowed to form over a period of 5 d prior to starting with the treatments for another 7 d. Both TTFields and CUSP9v3 alone caused a slight inhibition of spheroid growth (Fig.Ā 1i, j, lān and Supplementary Fig.Ā 1). In spheroids that were derived from U251 and PC128, this inhibitory effect was more pronounced when TTFields and CUSP9v3 were used together as shown in Fig.Ā 1j and Supplementary Fig.Ā 1. While the combination treatment did not lead to a reduction in size of spheroids derived from PC40 cells (Supplementary Fig.Ā 1), we did observe a reduced cellular density and also a decreased cohesion of the cells within the spheroids (Fig.Ā 1i). These observations were confirmed by quantitative analysis showing a significantly reduced ATP content when compared to treatment with either TTFields or CUSP9v3 alone in PC40 and PC128 cells (Fig.Ā 1l, m). In U251 cells, a marked decrease of the ATP content following the dual treatment was also noticed (Fig.Ā 1n). However, statistical significance was not reached.
TTFields together with CUSP9v3 lead to enhanced apoptosis
To address the question of whether the inhibitory effect of the combination treatment on cell viability and the growth of spheroids is due to induction of cell death, we stained U251 spheroids with PI (Fig.Ā 1k). Spheroids that were subjected to treatment with TTFields and CUSP9v3 showed a strong PI staining which was markedly less pronounced in those spheroids that were treated with either TTFields or CUSP9v3 alone. In line with this finding, the subG1 fraction of U251 and PC38 cells that were treated with PI and analyzed by flow cytometry was significantly increased following dual treatment with TTFields and CUSP9v3 (Fig.Ā 2aād). In order to further dissect whether this observation can be interpreted as a result of apoptosis, PC40 and PC128 cells were stained with Annexin V and PI followed by flow cytometric analysis. As shown in Fig.Ā 2eāh, glioblastoma cells subjected to treatment with the combination of CUSP9v3 and TTFields showed a significant increase of the fraction of Annexin Vāpositive (apoptotic) cells when compared to either treatment alone.
The pro-apoptotic effect of dual treatment with TTFields and CUSP9v3 is caspase-dependent
Since we observed a TTFields/CUSP9v3-induced pro-apoptotic response as outlined by an increased fraction of Annexin V-positive cells (Fig.Ā 2e, f), we decided next to examine whether this effect is caspase-dependent. While treatment with TTFields or CUSP9v3 alone did not induce cleavage of caspase-3 beyond baseline levels, the dual treatment led to a markedly enhanced cleavage of effector caspase-3 (Fig.Ā 2i). Along this line, the pro-apoptotic response of the combination was significantly reduced when PC40 and PC128 cells were treated with TTFields/CUSP9v3 in the presence of the pan-caspase inhibitor zVAD (Fig.Ā 2jāl). Next, we investigated whether the pro-apoptotic effect is in part mediated by the intrinsic pathway. To this end, PC40 and PC128 glioblastoma cells were treated with CUSP9v3, TTFields or both. Staining with MitoTrackerTM was performed to assess effects on the mitochondrial membrane potential (Fig.Ā 3aād). In both primary cultures tested, the dual treatment led to a significant increase in the fraction of cells with a reduced mitochondrial outer membrane potential.
Concomitant treatment with TTFields and CUSP9v3 suppresses the expression of Bcl-2 and Mcl-1
Given that the dual treatment reduces the mitochondrial outer membrane potential, we sought to examine the effect on the expression of anti-apoptotic Bcl-2 family proteins which are important regulators of the intrinsic pathway of apoptosis. While in PC128 cells treatment with either TTFields or CUSP9v3 alone led to decreased expression of Bcl-2, only a slight decrease in Bcl-2 levels was observed in U251 cells (Fig.Ā 3e, g, j). However, in both PC128 and U251 cells treatment with either TTFields or CUSP9v3 alone led to downregulation of Mcl-1 (Fig.Ā 3e, h, k). Notably, treatment with TTFields and CUSP9v3 together markedly reduced the expression of Bcl-2 and Mcl-1 when compared to treatment with either modality alone (Fig.Ā 3e, g, h, j, k). The anti-apoptotic Bcl-2 family protein Bcl-xL was not affected by TTFields or CUSP9v3 alone or when applied together (Fig.Ā 3f, i, l).
Concomitant treatment with TTFields and CUSP9v3 enhances the turnover of Mcl-1 protein and downregulates Bcl-2 mRNA transcripts
We next assessed the underlying mechanism of Mcl-1 and Bcl-2 downregulation by TTFields and CUSP9v3. In order to examine whether the protein stability of Mcl-1 or Bcl-2 is affected, glioblastoma cells were treated with cycloheximide in the presence or absence of the dual treatment. A comprehensive analysis was then conducted of the changes in Mcl-1 and Bcl-2 protein levels over time. Under the condition of blocked translation, simultaneous treatment with TTFields and CUSP9v3 accelerated the decay of Mcl-1 when compared to cells treated with solvents (Fig.Ā 4a, b). Since the protein stability of Bcl-2 did not seem to be affected by the combination treatment, we next examined whether mRNA levels vary among cells treated with TTFields/CUSP9v3 and solvents. As shown in Fig.Ā 4c, the dual treatment led to a marked decrease in Bcl-2 mRNA expression as early as after 6āh of treatment and lasting for at least 24āh of treatment.
Dual treatment with TTFields and CUSP9v3 suppresses oxidative phosphorylation
Since concomitant treatment with TTFields and CUSP9v3 significantly reduced the ATP content in glioblastoma spheroids (Fig.Ā 1lān), we next assessed whether this treatment also affects tumor cell metabolism. To this end, we performed extracellular flux analyses. In both, PC128 and U251 glioblastoma cells, oxidative consumption rates (OCR) were suppressed at baseline when compared to TTFields or CUSP9v3 alone (Fig.Ā 5a, c, d, f). Moreover, during the course of mitochondrial stress tests OCRs remained at markedly reduced levels (Fig.Ā 5a, d). In contrast, extracellular acidification rates were not altered in a consistent manner when compared to the single treatment modalities (Fig.Ā 5b, c, e, f).
Treatment with TTFields plus CUSP9v3 downregulates respiratory chain complexes I, III and IV
In order to further examine on a molecular level how the dual treatment inhibits the oxidative phosphorylation in glioblastoma cells, the expression of respiratory chain complexes was analyzed by Western blot (Fig.Ā 5gāj). While the single therapeutic approaches led to some reduction of the expression of the proteins of the respiratory chain, the combination treatment induced significantly enhanced downregulation which was most consistently seen for complexes I, III and IV (Fig.Ā 5gāj). For complex II, a similar trend was observed, however, statistical significance was not reached.
The dual treatment inhibits the migration of glioblastoma cells
Dysregulated migratory activity on one hand represents a typical feature of cancer cells and is highly energy-dependent on the other hand. We therefore tested whether the metabolic effects of the combination treatment are associated with reduced migration of glioblastoma cells. For this purpose, time lapse analyses were performed and wind rose plots were generated. As shown in Fig.Ā 6aāc, the cellular tracks were less spread-out following treatment with the single therapeutic modalities while a strong restriction of the migratory paths was observed when the cells were treated with TTFields and CUSP9v3 together. Quantitative analysis showed a slight reduction of the total migratory distance following treatment with the single approaches which, however, was significantly more pronounced when the cells were subjected to the combination treatment (Fig.Ā 6dāf). In order to examine whether this finding also holds true in other settings, scratch assays were performed (Fig.Ā 6gāk). In line with the results of the time lapse analyses, treatment with TTFields or CUSP9v3 alone led to some inhibition of the closure of the scratch (Fig.Ā 6gāk). However, when both treatment modalities were combined, the anti-migratory effects were significantly increased (Fig.Ā 6gāk).
Discussion
DrugĀ repurposing has emerged as a promising translational approach to bypass the downsides of common drug development pipelines. Major advantages of repurposing older drugs are the lower costs of already existing drugs and a well-characterized safety profile [31]. Therefore, transition to clinical application is greatly facilitated. CUSP9v3 represents such an example which was conceptualized in 2013 [18], validated in the preclinical setting [21] and taken to application in patients, first in a compassionate use setting [21] and followed by a phase Ib/IIa clinical trial [22] in less than ten years and within a purely academic setting.
Expanding on one of the most important intrinsic principles of CUSP9v3, i.e., multitargeting, we have explored ways of enhancing the anti-glioblastoma activity of CUSP9v3 before. In a previous study, our data showed that CUSP9v3 combined with Bcl-2/Bcl-xL inhibition using Navitoclax (ABT-263) resulted in predominantly synergistic antineoplastic activity among different glioblastoma models [32]. In the present study, we observed that TTFields lower Bcl-2 and Mcl-1 levels, which may add to the biological effect when combined with CUSP9v3 similar to the mechanism seen when inhibiting Bcl-2/Bcl-xL by Navitoclax along with CUSP9v3 treatment. However, Bcl-xL levels were unaffected by TTFields or CUSP9v3 alone and by the combination which differs from the mechanism seen in the previous study. At this point, our data indicate that a reduction of Bcl-xL levels does not seem to be necessary to induce the biological effects of TTFields/CUSP9v3 in this setting. However, what we have not yet explored are the effects of the combination on the interaction with the natural pro-apoptotic counterplayers such as Bad, which may result in functional impairment of Bcl-xL. If Bcl-xL is not part of the mechanism, addition of a selective Bcl-xL inhibitor might further increase the efficacy of the TTFields/CUSP9v3 approach.
Notably, we observed that the antiglioblastoma activity following treatment with TTFields and CUSP9v3 was associated with suppression of oxidative phosphorylation. Alteration of the tumor cell metabolism as a prominent feature of cancer cells has first been described by Otto Warburg in the 1920s [33]. More recently, interest has grown in developing therapeutic strategies directly targeting tumor cell metabolism or approaches that take advantage of specific tumor cellsā metabolic vulnerabilities [34, 35]. Patel and colleagues recently reported that TTFields cause a shift away from the aberrant glycolytic metabolism in glioblastoma [36]. In that study, the authors used the radiotracer [18F]DASA-23 to measure pyruvate kinase M2 expression as a surrogate for the glycolytic activity in glioblastoma cells. In U87 cells, a 35% and in GBM39 cells, an 81% reduction in [18F]DASA-23 uptake was found following exposure to TTFields for 6 days. Our analyses also showed effects of TTFields alone or concomitant with CUSP9v3 on extracellular acidification rates as read-out for the glycolytic rate. However, we observed that these metabolic changes in response to TTFields were not consistent and varied among different cell models. The discrepancy between our work and the study of Patel et al. might in part be due to differences in timing. In this study, we examined earlier time points (after 24āh and 48āh of treatment), whereas Patel et al. examined the metabolic effects of TTFields after 72āh and 96āh. The varying results may also be attributed to differences in the surrogate markers, the different genetic background of the glioblastoma cells used and frequence specificity. In contrast, our analyses showed a very strong and consistent downregulation of OXPHOS when the cells were subjected to the combination treatment. To our knowledge, these are the first data showing suppression of OXPHOS following a treatment that includes TTFields. While we were not able to completely decipher the cause of this response, we noticed a significant decrease in the expression of respiratory chain complexes. Further studies are planned to gain more insight into the mechanism. Along this line, another tempting question is whether inhibition of additional metabolic pathways that are involved in the progression of glioblastoma, such as glycolysis, the pentose phosphate pathway or Ī²-oxidation, might allow for further enhancement of TTFields/CUSPv3.
From a translational perspective, when applying drugs to target brain tumors, as in the CUSP9 approach, two important pharmacokinetic attributes need to be considered: First, the transfer of the drugs from the blood to the tumor and second, the distribution of the drugs within the tumor. Recently, Salvador et al. reported that TTFields may facilitate penetration across the blood-brain barrier [16]. In this study, the authors showed that application of TTFields at a frequency of 100ākHz for 72āh led to enhanced accumulation of Evans Blue and TRITC-dextran in rat brain tissue indicating increased permeability of the cerebral vasculature. Moreover, in an orthotopic rat glioblastoma model, treatment with paclitaxel, a drug that is known to poorly pass the blood-brain barrier, led to a significant reduction of tumor volume when combined with TTFields. Admittedly, other potential mechanisms besides a facilitated opening of the blood-brain barrier cannot be fully excluded. Along this line, another group observed that TTFields affect the structure of cancer cell membranes [15]. In this work, Chang et al. showed that exposure of U87-MG cells to TTFields at 200ākHz led to significantly enhanced cellular uptakes of ethidium D, 5-ALA and FITC-labeled dextran with sizes of 4 and 20ākDa. Moreover, electron microscopy showed that exposure to TTFields for 72āh led to a significantly increased number of holes in the membranes of U87MG cells. The results of these studies provide additional support of benefit from adding TTFields to treatment with CUSP9v3 in patients by enhancing exposure of glioblastoma cells to the CUSP9v3 drugs.
In conclusion, we provided proof of principle that a multimodal, multitargeting approach that includes TTFields and CUSP9v3 results in mutually enhanced activity against glioblastoma. Both treatment modalities on their own are mostly well-tolerated by patients and carry low risk for serious adverse events. These results encourage further studies and transition to the clinical setting.
Data availability
The data supporting the results reported in the article are deposited at the translational brain tumor research laboratory, Department of Neurological Surgery, Ulm University Medical Center, Albert-Einstein-Allee 23, D-89081 Ulm, Germany.
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Acknowledgements
We are very grateful to Dr. Yaara Porat and Dr. Moshe Giladi (NovocureTM, Haifa, Israel) for technical assistance and guidance with the InovitroTM system. We thank Dr. Daniel Tews (Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany) and the Core Facility āExtracellular Flux Analyzerā at Ulm University for excellent help and assistance.
Funding
This study was supported by a research grant from NovocureTM to M.E.H. and G.K.M. as well as National Institutes of Health, National Institute of Neurological Disorders and Stroke grants (K08 347 NS083732, R01 NS095848 and R01 NS102366) to M.D.S.; P.F.P. received funding from Deutsche Forschungsgemeinschaft (DFG, Heisenberg professorship; project number 497387083). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Open Access funding enabled and organized by Projekt DEAL.
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Conceptualization, M.E.H., G.K.M.; methodology, Q.C., A.H., P.F.P., G.K.M. and M.A.W.; formal analysis, Q.C., A.H. and G.K.M.; investigation, Q.C., A.H. and G.K.M.; resources, G.K.M. and C.R.W.; data curation, A.H. and G.K.M.; writingāoriginal draft preparation, Q.C. and G.K.M.; writingāreview and editing, Q.C., A.H., M.E.H.,Ā M.H., G.K.M., C.L., P.F.P.,Ā M.D.S., H.S., M.A.W. and C.R.W.; supervision, G.K.M.; project administration, G.K.M. All authors have read and agreed to the published version of the manuscript.
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M.E.H. is the inventor on patent DE 10 2020 205 076 and holds patent ZL2018800727756.
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Patientās or next of kinās consent was obtained, and procedures were done following internal review board approval (ethics proposal No.162/10, University of Ulm). The study was performed in accordance with the Declaration of Helsinki.
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Cao, Q., Hajosch, A., Kast, R.E. et al. Tumor Treating Fields (TTFields) combined with the drugĀ repurposing approach CUSP9v3 induce metabolic reprogramming and synergistic anti-glioblastoma activity in vitro. Br J Cancer 130, 1365ā1376 (2024). https://doi.org/10.1038/s41416-024-02608-8
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DOI: https://doi.org/10.1038/s41416-024-02608-8