Arginine Deprivation Therapy: Putative Strategy to Eradicate Glioblastoma Cells by Radiosensitization

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Author Manuscript Published OnlineFirst on August 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0807 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

C. Noreen Hinrichs1*, Mirjam Ingargiola1*, Theresa Käubler1, Steffen Löck1,2, Achim Temme2-4, Alvaro Köhn-Luque5, Andreas Deutsch5, Olena Vovk6, Oleh Stasyk6, Leoni A. Kunz-Schughart1,4,7#

*C. Noreen Hinrichs and Mirjam Ingargiola contributed equally to this work

1OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, TU Dresden, and Helmholtz-Zentrum Dresden – Rossendorf, Germany 2German Cancer Consortium (DKTK), partner site Dresden, and German Cancer Research Center (DKFZ), Heidelberg, Germany 3Department of Neurosurgery, Section Experimental Neurosurgery and Tumor Immunology, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany 4National Center for Tumor Diseases (NCT), partner site Dresden, Germany 5Center for Information Services and High Performance Computing, TU Dresden, Germany 6Department of Cell Signaling, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine 7CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, United Kingdom

Running title: Arginine deprivation radiosensitizes glioblastoma cells

Key words: glioblastoma, metabolic targeting, arginine, enzymotherapy, 3-D culture assay, p53-knockdown, radiosensitization Abbreviations: ADI - arginine deiminase; ADT - arginine deprivation therapy; ASL - argininosuccinate lyase; ASS -argininosuccinate synthase; BBB - blood-brain barrier; BCCAO - bilateral common carotid artery occlusion; CAT - cationic amino acid transporter; Col-I - collagen-type I; DFMO - difluoromethylornithine; DRF - dose reduction factor; FCS - fetal calf serum; FN - fibronectin; GBM - glioblastoma; MEM - Minimum Essential Medium; NO - nitric oxide; ODC - ornithine decarboxylase; PBS - phosphate buffered saline; PE - plating efficiency;rhA - recombinant human arginase I; SCD50 - spheroid control dose 50%; SCP - spheroid control probability; SF -surviving fraction; TCD50 - tumor control dose 50%

Financial support: This work was supported by the European Social Fund (ESF) and the Free State of Saxony (GlioMath DD 100098214 to LA. Kunz-Schughart, A. Deutsch, and A. Temme) and the Else Kröner-Fresenius Foundation (to CN Hinrichs).

#Corresponding author: Prof. Dr. Leoni A. Kunz-Schughart OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden Fetscherstrasse 74, PO box 41 01307 Dresden Germany phone/e-mail: +49-351-4587405 / [email protected]

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Downloaded from mct.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0807 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

ABSTRACT

Tumor cells - even if non-auxotrophic - are often highly sensitive to arginine deficiency. We hypothesized that arginine deprivation therapy (ADT) if combined with irradiation could be a new treatment strategy for glioblastoma (GBM) patients since systemic ADT is independent of local penetration and diffusion limitations. A proof-of-principle in vitro study was performed with ADT been mimicked by application of recombinant human arginase or arginine-free diets.

ADT inhibited 2-D growth and cell cycle progression, and reduced growth recovery after completion of treatment in four different GBM cell line models. Cells were less susceptible to

ADT alone in the presence of citrulline and in a 3-D environment. Migration and 3-D invasion were not unfavourably affected. However, ADT caused a significant radiosensitization which was more pronounced in a GBM cell model with p53 loss of function as compared to its p53- wildtype counterpart. The synergistic effect was independent of basic and induced argininosuccinate synthase (ASS) or argininosuccinate lyase (ASL) protein expression and not abrogated by the presence of citrulline. The radiosensitizing potential was maintained or even more distinguishable in a 3-D environment as verified in p53-knockdown and p53- wildtype U87-MG cells via a 60-day spheroid control probability assay. While the underlying mechanism is still ambiguous, the observation of ADT-induced radiosensitization is of great clinical interest, in particular for patients with GBM showing high radioresistance and/or p53 loss of function.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

1. INTRODUCTION

Arginine is a semi-essential amino acid required for numerous cellular processes such as protein synthesis and arginylation, polyamine production, metabolism of other amino acids and nucleotides, as well as creatine, urea and NO synthesis (1,2). The intracellular arginine pool is usually fueled by arginine uptake from the blood and interstitial fluid through cationic amino acid transporters (CAT), via protein degradation, or by conversion from citrulline catalyzed by the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase

(ASL) (1,3). The latter two processes are sufficient to maintain cell survival in normal tissues in the event of exogenous arginine shortage. Aggressive cancer cells with their high proliferative activity requiring enhanced energy disposal, DNA synthesis and protein metabolism have an extraordinary demand for arginine that cannot be covered by intracellular sources. This is particularly striking for malignancies deficient in one key enzyme of arginine synthesis due to genetic or epigenetic modifications. In most cases, except for hepatocellular carcinoma, the enzyme ASS is lacking in these arginine auxotrophic tumors, and enzymotherapeutic ADT (arginine deprivation therapy) has already proven to effectively inhibit growth of such cancers in preclinical studies and clinical phase I-III trials (1,4–8). ADT combined with radiotherapy is not yet in clinical trial. However, our group has shown recently that ADT can augment the radioresponse even in ASS-positive colorectal cancer cells (9,10) raising hope for improving the treatment outcome also for other arginine non-auxotrophic tumor entities.

ADT is a particularly attractive strategy for brain tumors and metastases as it is a systemic approach based on the catabolic depletion of arginine from the blood supply. It is thus independent of the blood-brain-barrier (BBB), which in spite of its partially enhanced permeability in brain cancer patients (11,12) often impairs adequate drug distribution. The translation of the initial finding that arginine withdrawal induces growth arrest in various tumor entities in vitro via ADT-based animal studies into clinical trials has just commenced and progresses rapidly (1,6,8,9,13–15).

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Recent observations in glioblastoma (GBM) models (16–18), support the idea that ADT might be a treatment option for brain cancer patients. The aim of our project was to systematically extend these previous studies by addressing the impact of ADT not only on growth behavior but on all three major characteristics of GBMs - the high proliferation rate and survival capacity, the strong invasive potential and the severe intrinsic radiation resistance (19).

2. MATERIALS AND METHODS

2.1 2-D & 3-D routine cell culturing

The GBM cell lines U251-MG and U138-MG (both p53mut) as well as isogenic U87-MG- shLuc control cells (p53wt) and their p53-knockdown counterpart U87-MG-shp53 were used.

The original cell lines were purchased in 2010 (CLS Cell Lines Service, Germany). The correct genetic profiles of the frozen stocks derived from originally and genetically modified cells was confirmed prior to use via the multiplex PCR kits Mentype® NonaplexQS Twin

(Biotype AG) and PowerPlex® 16 (Promega Corp.) (Institute of Legal Medicine, TU Dresden,

Germany). Cultures were free of mycoplasms and routinely regrown from the validated frozen stocks for <100 cumulative population doublings using Minimum Essential Medium

(MEM) containing 1.0 g/l D-glucose, 292 mg/l L-glutamine, 25 mM HEPES, and 2.2 g/l

NaHCO3, and supplemented with 10% heat-inactivated fetal calf serum (FCS) as well as 1% penicillin/streptomycin (10,000 U/ml / 10 mg/ml) (all from PAN-Biotech). Exponentially growing cultures were kept at 37°C in a humidified, 5% CO2 in air atmosphere. Single cell suspensions for passaging and for setting up experiments were obtained by enzymatic and mechanic means using a 0.05%/0.02% trypsin/EDTA solution in phosphate buffered saline

(PBS) (PAN-Biotech). A CASY® TTC device (Roche Innovatis AG) was applied for cell counting, cell volume analysis and culture quality assessment.

Spheroid cultures were initiated by seeding defined concentrations of single cell suspensions from exponentially growing monolayer cultures onto 1.5% agarose-coated 96-well plates.

The designated standard spheroid size at day 4 in culture was 370-400 µm. Spheroids were

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas further cultured in liquid overlay and monitored for integrity and volume growth as described earlier (9,23,24) (for details see Suppl. Materials & Methods).

2.2 Therapy implementation

ADT was mimicked by the use of arginine-free culture medium (Pan Biotech) or achieved by exposure to 2 U/ml yeast-derived recombinant human arginase I (rhA, provided by the

Institute of Cell Biology, NASU, Lviv, Ukraine). Successful deprivation of arginine by the enzyme at this concentration was scrutinized before use by a reverse-phase HPLC technique as described in (25). Arginine in culture media is degraded within <10 minutes below detection level and rhA activity remains at maximum level for at least 72 h (O. Chen,

O. Stasyk, personal communication). It was documented earlier that the treatment induces an essential drop in intracellular free arginine within 15-30 minutes; arginine decreases below detection level within 1 h (25,26). In case of amino-acid dietary medium, both control and arginine-deficient media were supplemented with 10% heat-inactivated dialyzed FCS deprived of molecules <10 kDa (Pan Biotech). Spheroids exposed to arginine-free media required the transfer onto 96-well plates coated with 1.5% agarose prepared with the corresponding serum-free amino-acid deprived medium. In some settings, media contained citrulline (Sigma Aldrich) at an arginine-equimolar, hyperphysiological concentration of

0.6 mM or at a concentration of 0.05 mM reflecting the blood/plasma level. Irradiation was performed at room temperature using a single dose X-ray approach (200 kV, 0.5 mm Cu filter, YxlonY.TU 320, Yxlon.international).

2.3 Growth assays

4 For 2-D growth assessment, 1x10 single cells/well in 2 ml culture media were seeded into 6- well plates and allowed to adhere for 24 h; rhA was then added, and cells were counted after

1, 3 and 5 days of treatment as well as 3 days post-treatment, i.e. after retransfer to normal conditions. At least two independent experiments (N=2) were performed each with n=3-4 wells per condition.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Spheroid volume was determined before, during and after exposure to arginine-comprised and arginine-free conditions, respectively, with or without citrulline. For this purpose, spheroids were washed with PBS and carefully transferred in the specific dietary media onto the respective agarose-coated 96-well-plates. After defined time intervals, spheroids were conveyed back into standard liquid-overlay culture and monitored for another 24 d. A minimum of 12 individual spheroids was analyzed per condition, and three independent aliquots of at least 8 spheroids per treatment condition were collected and dissociated to determine the numbers of membrane-intact cells per spheroid. The operating schedule of the

2-D and 3-D growth, regrowth and survival assays is outlined in Suppl. Fig. 1A.

2.4 Static and dynamic cell cycle analyses

For dynamic cell cycle assessment, exponential monolayer cells treated with or without rhA for a total of 24 h were pulse-chased with the thymidine analogue 5-ethynyl-2′-deoxyuridine

(EdU, Invitrogen). The EdU pulse (10 μM, 1 h) and chase periods (0 h and 8 h) were scheduled in a way that all samples could be harvested at the same time. Single cell suspensions were prepared and EdU incorporation was detected using the Click iT EdU Flow

Cytometry Assay Kit (Invitrogen). RNase A (0.1 mg/ml, 37° C, 30 min, Invitrogen) was applied for RNA elimination and propidium iodide (PI, 50 μg/ml, Sigma-Aldrich) for stoichiometric DNA counterstaining. For more details see (27). PI was also applied for static

DNA analyses using the handling protocol from (28). All samples were measured with a BD

FACS Canto II flow cytometer (BD Biosciences, Germany). A minimum of 2×104 single cells were recorded using the PI area and width signals for doublet exclusion. G1/0, S- and G2/M- phase fractions were determined in DNA histograms using the univariate Dean-Jett Fox model implemented in the FlowJo software version 7.6.4 (Tree Star, USA). Gating of particular EdU-positive cell fractions 0 and 8 h after pulse-labeling allowed to detect and follow actively cycling S-phase cells.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

2.5 Colony-forming assay

Clonogenic survival was assessed in colony formation assays. Here, low cell numbers (150-

6000 cells/well), adapted to the individual GBM cell line and to the increasing dose of irradiation (0-10 Gy), were seeded into 6-well plates and allowed to adhere for 1 day. Control cells were then irradiated, while in the other samples, supernatant was exchanged to rhA- containing media with or without either citrulline. Accordingly, arginine-deprived cells which were growth-arrested during treatment were irradiated 24 h later. In a separate set of experiments, rhA-exposure was combined with the endoplasmic reticulum (ER) stress response modifiers salubrinal (20 µM; Sigma Aldrich) or dimethyl sulfoxide (DMSO, 2%;

Sigma Aldrich). A 4 mM stock solution of salubrinal in DMSO was prepared and freshly diluted in medium prior to use; accordingly, control cells were exposed to medium containing

0.5 mM DMSO. 24 h later, treated cells were irradiated (0-10 Gy and 0, 6 & 8 Gy for salubrinal/DMSO assays, respectively). Standard culture conditions in all samples were restored 1 h post-irradiation, and cells were incubated for another 9-13 d depending on the cell-line specific doubling times to allow >5-6 doublings. Colonies were then fixed, stained, and counted manually to determine the plating efficiencies (PE). Surviving fractions (SF) were normalized for cell-line and treatment-dependent differences in PE to quantify radioresponse. Data were reproduced (N=3 with n≥3 wells per experiment and treatment condition), and cell survival curves were fitted employing the linear-quadratic model (D: dose;

2 A,B: variables defining the irradiation dose): SF  e( ADBD )

Cell survival curves after combined treatment were considered distinct from controls

(irradiation only) if values A or B significantly differed.

2.6 3-D irradiation experiments

Standard sized spheroids were washed with PBS, transferred into arginine-free medium with or without citrulline and irradiated with single doses of 0-30 Gy at day 5 of arginine depletion.

24 h after irradiation arginine was re-supplemented to the standard concentration of 0.6 mM in MEM. Control spheroids were irradiated in the presence of arginine at standard diameter.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Cultures were monitored for a period of 60 d post-treatment. Spheroids which did not regrow to >200 µm and/or did not enlarge at least 3-times in sequence were declared as

"controlled". A minimum of 20 individual spheroids was analyzed per treatment condition.

Spheroid growth delay tSGD represents the different time intervals of treated versus control spheroids to reach 5-times the starting volume (5xV0), with the starting volume (V0) being defined as the spheroid volume directly before irradiation and treatment, respectively. To determine spheroid growth delay, growth and regrowth kinetics were mathematically modelled via the Gompertz function as described earlier (23,24).

Spheroid control probability (SCP) describes the proportion of controlled spheroids as a function of irradiation dose. Spheroid control probability curves were fitted with a sigmoid dose-response model according to the tumor control probability in vivo assays (D: dose; a,b:

1 variables): SCP  1 1 e(ab lnD)

The dose at which SCP amounts to 50% (SCD50) was deduced from the SCP curves; the quotient of SCD50 values (combined treatment/irradiation) yields the dose reduction factor

(DRF).

2.7 Migration and invasion assays

Spheroids at standard size were washed in PBS and either studied under acute arginine- deprivation or pre-exposed to arginine-free medium for a period of 5 days to mimic chronic arginine deficiency. Spheroids were transferred onto flat bottom wells coated with fibronectin or collagen-I (migration), or were embedded into collagen-I gel (invasion) and monitored under acute (no pre-exposure) and chronic (5 d pre-exposure) dietary conditions. The invasion protocol included the utilization of fluorescence-labeled U251-MG-eGFP, U87-MG- shLuc-eGFP and U87-MG-shp53-eGFP spheroids and the combination of z-stacks of about

40 images with a distance of 5 µm. Migration and invasion of spheroid single cells were recorded over a period of 12 h via time-lapse microscopy using an AxioObserver.Z1 system.

Experimental and analytical details are given in Suppl. Fig. 1B. A minimum of 8 individual spheroids was monitored per condition.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

2.8 Western Blotting

Whole cell protein was extracted from exponentially growing 2-D and 3-D cultures untreated or exposed to ADT for 48 h. The basic extraction procedure and protein quantification have been described elsewhere (9). In brief, the lysis buffer for protein extraction contained 2.0 % phosphatase-inhibitor-cocktail 1 + 2 (Sigma Aldrich), 10% complete mini protease inhibitor cocktail

(Roche Applied Science) dissolved in PBS, 1% Na VO , 1% PMSF (100 mM), and 0.2 % NaF in RIPA 3 4 buffer. Protein isolated from HepG2 cells (hepatocellular carcinoma) served as positive standard.

Protein lysates were completed by dithiothreitol-supplemented (1:19) protein loading buffer (1:5) and denatured at 95 °C for 5 min. Equal protein weights were disassembled in 10% SDS-PAGE.

Blotting, antibody incubation and detection were performed as detailed earlier (9) using the antibodies listed in Suppl. Materials & Methods. ASS and ASL signal intensities were semi- quantitatively assessed relative to an -tubulin loading control and normalized to a positive protein standard (HepG2 protein extract). Three independent protein lysates were analyzed for each condition.

2.9 Statistics

Differences in surviving fraction, cell numbers per spheroid and migration & invasion distances were evaluated by independent Mann-Whitney-U tests, while differences in cell- cycle distribution were analyzed by the paired Wilcoxon-rank-sum test. Both tests included

Bonferroni-Holm correction for multiple testing. ANOVA comprising Bonferroni correction was applied for 3-D cell growth assays. Linear regression was used for the analysis of cell survival curves and for the comparison of the parameters of the linear-quadratic model. All tests were performed using SPSS Statistics 21 (IBM Corporation, NY, USA). Logistic regression was employed to compare SCD50 values as well as DRF and to fit the corresponding curves. To determine 95% confidence intervals subsequent bootstrapping with 1000 samples was performed using STATA/SE 11.2 (StataCorp LP, TX, USA). Values are documented according to their significance level as p<0.05 (*), p<0.01 (**), and p<0.001

(***).

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

3. RESULTS

3.1 ADT by rh-Arginase inhibits 2-D GBM cell growth and recovery

Arginine deprivation over a period of 48 h by exposure to a modified recombinant human arginase-I was recently found to be cytotoxic for glioblastoma cells in 2-D culture (17). We examined the impact of ADT in more detail by using different glioblastoma cell line models, variable treatment intervals and by analyzing not only acute growth effects but also the cells’ potential to recover.

We observed a complete growth arrest under acute arginine-deprived conditions for all GBM cell lines tested herein. Furthermore, cells were not capable to recover and show regrowth after completion of ADT, if the treatment interval was ≥5 days (Fig. 1A). Basically, all three

GBM lines included in the initial experimental series express the enzymes involved in citrulline to arginine conversion, i.e. ASS and ASL, but intrinsic protein levels differ (Fig. 1C).

Accordingly, the cells cannot be considered intrinsically auxotrophic for arginine when the arginine precursor citrulline is bioavailable. We therefore also monitored changes in ASS and

ASL protein upon ADT and found a positive, yet not significant trend of ASS in U87-MG- shLuc and U251-MG while ASL protein seemed rather reduced, a phenomenon that remains ambiguous. Overall, the ASS/ASL high U87-MG-shLuc cells appeared to be less sensitive to

ADT as they more efficiently regenerate after an arginine deprivation of >3 days and upon bioavailability of citrulline than the other GMB cell line models. However, citrulline - even at concentrations far above blood level - neither abolished the ADT-induced growth arrest nor was it sufficient to fully compensate the ADT related loss of regeneration capacity (Fig. 1B).

3.2 ADT by rh-Arginase radiosensitizes GBM cells in 2-D assays

GBM are known for their high intrinsic radioresistance. Our previous study in colorectal cancer models indicated that ADT can enhance radiosensitivity of human epithelial cancer cells (9). This motivated us to examine whether combination of systemically applicable ADT plus irradiation could be a treatment option for GBM patients.

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

24 h ADT reduced the plating efficiency of U251-MG from 44±7% to 26±10% and of U87-

MG-shLuc cells from 10±4% to 6±3%. Normalization of the surviving fractions after irradiation was thus essential to identify a radiosensitizing potential. The respective data shown in Fig. 2 reveal that both U251-MG and U87-MG-shLuc cells are sensitized to irradiation by arginine deprivation in the 2-D assay. However, the effect strongly differed in magnitude. The

ASShigh/ASLlow U251-MG revealed much higher susceptibility with a significant difference in radioresponse for doses ≥2 Gy (p2Gy,4Gy,6Gy,8Gy,10Gy<0.01) in the absence of citrulline and for

high high doses ≥4 Gy (p4Gy,6Gy,8Gy,10Gy<0.01) in the presence of citrulline. The ASS /ASL U87-MG- shLuc cells were sensitized at high doses only (p8Gy,10Gy<0.05 without citrulline, p10Gy<0.05 with citrulline).

3.3 ADT-induced radiosensitization is higher in GBM cells with p53 lack of function

Beside the ASS expression rate, the GBM models used herein differ in their intrinsic p53- status. U138-MG and U251-MG are p53-mutant with loss of functional protein (29,30) while

U87-MG cells are p53-wildtype (p53-wt). Because of the high frequency of TP53 mutations in

GBM, we implemented a p53-knockdown derivative of the U87-MG cell line (U87-MG-shp53) for comparison with its counterpart U87-MG-shLuc to study the role of p53 in ADT response in more detail. In this model, loss of functional p53 (Suppl. Materials & Methods) did not per se alter radioresponse in the clonogenic survival assay (Fig. 2). Also, U87-MG-shp53 cells displayed intrinsic ASS levels at least as high as the U87-MG-shLuc cells and the ASL contents did not differ (Fig. 1D). Putative differences in the response of U87-MG-shLuc and

U87-MG-shp53 to ADT can thus not be attributed to p53-related changes in intrinsic radiosensitivity or ASS/ASL expression.

Indeed, the cell growth and regrowth behavior of U87-MG-shp53 cells upon rhA treatment with and without citrulline supplementation resembled that of its p53-wt ancestor

(Figs. 1A/B). Accordingly, p53 knockdown in U87-MG cells did not overcome ADT-induced growth and cell cycle arrest in 2-D culture. However, the radiosensitizing effect of ADT and arginase treatment, respectively, was enhanced upon p53 knockdown (Fig. 2) with a significant loss in clonogenic cell survival at ≥6 Gy single dose irradiation (p6Gy,8Gy,10Gy<0.01).

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Furthermore, this effect was not at all compensated by citrulline, assuming that p53- knockdown/mutant (i.e. loss of functional p53-protein) accounts for a greater citrulline- independent radiosensitization.

In summary, our data reveal that the radiosensitizing effect of ADT in 2-D culture is more prominent in the p53-deficient U87-MG-shp53 glioblastoma cells and appears autonomous of

ASS and ASL protein expression.

3.4 Mechanism of radiosensitization by ADT in GBM cells remains ambiguous

The relative radioresponse is critically affected by the proliferative activity and cell cycle distribution of cells. Proliferating cells are in general more susceptible than arrested cells. On the other hand, cells in G2/M phase are most sensitive to DNA-damaging factors, while G1- phase cells have a much lower sensitivity (31). We performed flow cytometric cell cycle analyses to evaluate if growth arrest by ADT could relate to radiosensitization due to accumulation of cells in G2/M phase. DNA histograms revealed that all GBM cell cultures show a higher proportion of cells in G1/0 phase while the S-phase and/or G2/M-phase fractions are rather reduced upon ADT (Fig. 3A and Suppl. Fig. 2A).

Dynamic cell cycle analysis using a 1h-EdU pulse was applied because (i) a phase- independent cell cycle arrest by ADT was described recently in colorectal cancer cells (27) and (ii) the changes seen in the DNA histograms of GBM cultures did not fully explain their growth arrest. Fig. 3B representatively documents the flow cytometric DNA dot blot diagrams for U87-MG-shp53 cells, showing that roughly all cells arrest under ADT, i.e. S-phase cells do no longer actively take up EdU (0 h measurement: active S-phase fraction 24.8% for control, 0.7% for ADT and 2.8% for ADT + citrulline). The effect is not sufficiently compensated by hyperphysiological citrulline, because 8 h later <1% of the cells has entered the next G1-phase in both rhA-treated arms in contrast to 18.5% in the arginine-rich control.

This behavior was confirmed for all other cell lines (Suppl. Fig. 2B). Growth inhibition by ADT is thus clearly due to cell cycle arrest but not causally related to radiosensitization.

Arginine deprivation induces endoplasmic reticulum stress in human solid cancer cells including U251-MG (10). Observations in cell lines from other tumor entities (L.A. Kunz-

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Schughart, O. Chen, unpublished data) indicate a causal link between ER stress response and radiosensitization by ADT. In order to prove this hypothesis for GBM cells, we assessed clonogenic survival at 6 Gy and 8 Gy for U87-MG-shp53 treated with and without rhA in the absence and presence of two different ER stress inhibitors. The results documented in Fig.

3C indeed show a reduced radioresponse when ADT is combined with the ER stress response modulators. However, application of both 2% DMSO and 20 µM salubrinal resulted in a pronounced loss of radiosensitivity in U87-MG-shp53 cells even in the presence of arginine. The same was true for U251-MG cells. The data is thus not sufficient to unequivocally prove our hypothesis but rather imply that ER stress response pathways are highly relevant for GBM radioresponse in 2-D culture in general.

3.5 ADT induces acute growth arrest but does not limit 3-D regrowth capacity

We described earlier, that a 3-D culture environment renders epithelial cancer cells profoundly less susceptible to single amino acid starvation (32). It was therefore of utmost relevance to validate the effect of combined treatment in a 3-D cell context. We applied a sophisticated long-term spheroid assay which was designed to assess the putative curative potential of new combination treatment strategies in the 3-D environment before turning into whole animal studies.

The two p53-mutated GBM cell lines U251-MG and U138-MG form regular, round-shaped and membrane-intact multicellular spheroids but turned out to show no intrinsic 3-D volume growth under standard culture conditions even in the presence of arginine (Suppl. Fig. 3A). In contrast, U87-MG-shLuc and U87-MG-shp53 grow well under these conditions indicating that the proliferation arrest is not causally related to the p53 status. As a further prerequisite, we compared ASS and ASL protein levels in 2-D and 3-D cultures. ASS but not ASL level in

GBM cells tends to change from 2-D to 3-D conditions; however, the differences were neither systematic nor significant. Nonetheless, ADT-related alterations in protein expression were qualitatively comparable in the 2-D and 3-D environment (Suppl. Figs. 3B/C).

We next analyzed the impact of 1, 5 and 10 days of ADT on 3-D volume growth and regrowth capacity for U87-MG-shLuc and U87-MG-shp53 spheroids. ADT led to a clear spheroid

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas growth arrest in both models (Figs. 4A/C). This observation was confirmed by counting the viable cell number in spheroids grown either in the absence or presence of arginine for 5 and

10 days (Figs. 4B/D). In contrast to the situation in 2-D culture, all spheroids completely restored growth upon arginine re-supplementation. The regrowth kinetics was exiguously slowed only after long-term (10 d) ADT, i.e. the time to reach the analytical end volume

(5xV0) after arginine supplementation increased from 4 (control) to 5 days.

Note that the application of rhA to therapeutically eliminate arginine in the supernatant of

U87-MG-shp53 spheroids resulted in an acute growth arrest analogous to arginine-free media. Spheroid assays were therefore performed using dietary ADT (Suppl. Fig. 4A).

3.6 ADT-induced radiosensitization manifests in the 3-D environment

For the evaluation of radioresponse in 3-D, we adapted our well-established growth delay and spheroid control probability (SCP) assays to the U87-MG models. The approach allows the estimation of SCD50 as analytical endpoint reflecting the clinically relevant readout of tumor control dose 50% (TCD50) determined in xenograft models after irradiation.

We first applied the p53-knockdown U87-MG-shp53 cells due to their higher susceptibility to

ADT in the 2-D radioresponse assays. In the SCD50 approach, ADT combined with single dose irradiation up to 12.5 Gy could not prevent U87-MG-shp53 spheroid regrowth but led to a massive growth delay (Fig. 5A and Suppl. Fig. 4B). This growth delay effect was less than additive at lower doses but increased exponentially to become supra-additive at intermediate doses of 12.5-15 Gy (Fig. 5B and Suppl. Table 1). Citrulline at physiological concentrations during ADT did not abolish this effect. First signs of controlled (non-regrown) spheroids over a period of 60 days post-irradiation were seen at a dose of 15 Gy only under ADT. Controlled spheroids in all groups, yet different proportions, were documented at 17.5 Gy shown in Fig.

5C as function of time after treatment. Here, an advantage but not full compensation of the

ADT effect in the presence of citrulline became obvious.

The complete spheroid control probability curves finally revealed that U87-MG-shp53 spheroids are significantly and strongly radiosensitized in the absence of arginine (Fig. 5D).

The SCD50 for U87-MG-shp53 control spheroids was 20.8 Gy (95% CI: 20.2-21.4) and

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas differed significantly (p<0.001) from the values determined under arginine-free conditions in the absence (SCD50: 16.5 Gy; 95% CI: 16.0-17.0) and presence of citrulline (SCD50:17.3 Gy;

95% CI: 16.7-17.9). The therapeutic benefit of ADT is reflected by a DRF of 1.27 (95% CI:

1.21-1.32, p<0.001). Note that bioavailability of the arginine precursor citrulline did not abrogate the ADT-induced radiosensitization in this model resulting in a DRF of 1.21 (95%

CI: 1.15–1.27, p<0.001).

3.7 3-D assay better distinguishes radiosensitization by ADT in p53-wt GBM cells

ADT-induced radiosensitization of p53-wildtype U87-MG-shLuc cells was quite weak in the 2-

D assay. It was unclear if the effect would be entirely lost in the 3-D environment or more pronounced due to the fact that higher doses are applied. The assessment of spheroid volume growth delay and monitoring of spheroid control probability revealed that (i) U87-MG- shLuc cells in spheroids in contrast to the 2-D assay are more sensitive to radiotherapy than their p53-knockdown counterparts, (ii) ADT-induced radiosensitization is clearly visible in the

3-D environment and (iii) the cells’ response to treatment is qualitatively but not quantitatively similar to the U87-MG-shp53.

Again, the growth delay effect became supra-additive at intermediate doses i.e. 10-12.5 Gy for this spheroid type (Fig. 5E/F, Suppl. Fig. 4B and Suppl. Table 1). Controlled U87-MG- shLuc spheroids in all treatment arms were seen at doses ≥15 Gy (Fig. 5G) and the SCD50 values calculated from the spheroid control probability curves (Fig. 5H) were 16.4 Gy (95%

CI: 15.8-16.9) under arginine-rich, 13.8 Gy (95% CI: 13.3-14.4) under arginine-free and 15.0

Gy (95% CI 14.5-15.4) under arginine-free, citrulline-supplemented conditions. As for the

U87-MG-shp53, SCD50 values of U87-MG-shLuc spheroids in the treatment arms significantly differed (p<0.001) from the controls. However, the DRF calculated for arginine- free conditions was 1.18 (95% CI: 1.12-1.24, p<0.001) and only 1.09 (95% CI: 1.05-1.14, p<0.001) in the presence of citrulline. This reflects a lower therapeutic effect of the ADT treatment and a higher compensating potential of citrulline bioavailability in the p53-wt U87-

MG-shLuc compared to the U87-MG-shp53 model.

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3.8 ADT does not adversely affect motility and invasion of 3-D GBM cells

Pavlyk et al. (16) demonstrated recently that arginine deprivation reduces the migration and invasion capacity of U87-MG and U251-MG in various 2-D assays. We therefore hypothesized that ADT - best-case - can have a triple benefit for GBM treatment outcome by

(i) inhibiting growth, (ii) inducing radiosensitivity and (iii) diminishing invasion. Accordingly, we analyzed the migration of U87-MG-shp53 spheroid cells on fibronectin (FN) and collagen- type I (Col) surfaces and their invasion when embedded into a 3-D collagen-type I gel under acute and chronic ADT (0 and 5 day pre-exposure).

A significant reduction in migration distance on FN was observed for both acute and chronic

ADT conditions (p<0.02) (Fig 6A and Suppl. Fig. 5). Yet, the effect mainly manifests as parallel shift but not as reduced slope in the curves describing migration distance as function of time. This suggests that not migration rate per se is impacted. In culture dishes, we observed reduced adherence of single cells in arginine-free medium. The reduced migration radius on FN may thus also be the consequence of a delayed adherence of spheroids and spheroid cells, respectively, to the ECM-coated surface under arginine dietary conditions. A similarly effect is not seen on Col; here, ADT caused no difference in adherence or migration rate (Fig. 6A and Suppl. Fig. 5). We further applied a spheroid 3-D invasion assay, which requires degradation of the ECM for cells to migrate out of the spheroid. Suppl. Videos 1-3 provide representative time-lapse microscopic videos, which indicate that the invasion capacity of U87-MG-shp53 spheroid cells is not modified by ADT. Fig. 6B documents the corresponding average invasion distances as function of time. While we could not prove the hypothesized triple effect in our 3-D model, the data insistently demonstrate that ADT- induced growth arrest and radiosensitization are unlikely to be accompanied by an adversely stimulated invasion.

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4. DISCUSSION

Over the past decades various therapy strategies to target GBM have been invented, but none of them evidently advanced patients’ outcome. Since the establishment of the current standard of care radiochemotherapy with temozolomide about 10 years ago, the prognosis of newly diagnosed GBM patients has not further improved with an overall survival of only

14.6 months (19,33,34). Large epigenetic and genetic heterogeneities contribute to the challenges of this disease (19) with the tumor suppressor gene TP53 as the most common gene altered in approximately 28% of primary and 65% of secondary GBMs (35–37). The increasingly recognized potential of ADT as new metabolic targeting strategy for numerous solid cancers (1,6,9), and its systemic, blood-brain-barrier independent mode of action, motivated us to systematically examine the impact of ADT in various GBM models. By using conventional 2-D and advanced 3-D survival, growth, migration and invasion assays combined with specially formulated media and an enzymotherapeutic approach with recombinant human arginase I, we could show for the first time that p53-mutated/knockdown

GBM cells are particularly sensitive to ADT and ADT-induced radiosensitization. Our data further reveal that this effect is unlikely to be impaired by an enhanced citrulline-to-arginine conversion, which is a pre-requisite for selective in-vivo effectiveness.

We found all GBM cell models to express the enzymes relevant for intrinsic arginine synthesis. However, the protein levels were quite variable and those that were most sensitive to ADT-induced growth arrest and reduced recovery in the 2-D assays were extremely poor in either ASS1 (U138-MG) or ASL (U251-MG) and did not upregulate these proteins in the absence of arginine. This effect might be due to epigenetic silencing via methylated CpG islands in the ASS1 or ASL gene, respectively, which was recently shown by Syed et al. (38)

(i) to occur in 8/22 and 5/22 primary GBM cell cultures, (ii) to correlate with reduced median overall survival time and (iii) to functionally alter the response to arginine deiminase treatment. As described for other ASS/ASL positive cancer cells (16–18), all GBM models studied herein showed reduced regrowth capacity after long-term ADT reflecting some loss of reproductive survival. The effect clearly attenuates in the presence of citrulline which is in

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas line with findings in other, previously studied tumor entities (4,7,13,32,39). The loss of efficacy was most evident in the ASS/ASL high expressing models, indicating that ASS/ASL low GBM cells might indeed be more sensitive to ADT due to a limited capability of intrinsic de novo arginine synthesis.

The observations are also relevant in the context of the two therapeutic enzymes in clinical trial, i.e. arginine deiminase (ADI) and arginase. ADI degrades arginine via production of ammonia and citrulline resulting in enhanced serum citrulline levels. It is unclear, however, if the increased serum citrulline detected after ADI treatment (40) leads to a critical augmentation of intratumoral citrulline levels which could reduce the efficacy upon long-term treatment. The primary product of arginase-dependent arginine degradation is ornithine.

Ornithine is converted via intermediates to arginine only in cells with a highly active urea cycle, i.e. liver cells, and the respective negative loop can thus be ignored in GBM treatment.

However, ornithine also serves as precursor for various aliphatic polyamines involved in multiple regulatory processes such as ion channel function, DNA stabilization, regulation of gene expression as well as cell proliferation, and might therefore adversely promote tumor growth and survival (41). In the present study, both arginine-free media and recombinant human arginase were applied to achieve ADT in vitro with no evidence for a detrimental impact of catabolites on treatment outcome. Furthermore - as for citrulline -, the increase in mean ornithine blood level may not be accompanied by intratumoral accumulation of ornithine, e.g. due to the locoregionally and spatiotemporally poor perfusion and blood supply. Yet, it might be speculated that patients with cancers showing high levels and activity of ornithine decarboxylase (ODC), the enzyme catalyzing the conversion of ornithine to putrescine as the first polyamine in the putrescine - spermidine - spermine axis, might profit from combining ADT with an ODC inhibitor. In a previous clinical phase III trials, combination of the ODC inhibitor -difluoromethylornithine (DFMO) with radiotherapy and/or various chemotherapeutics did not result in a survival benefit for newly diagnosed GBM patients even if their tumors overexpressed ODC (42,43). The proposed multi-metabolic targeting approach may thus only be considered in the case of critically enhanced intratumoral

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas ornithine levels during ADT and if DFMO does not as also indicated critically interfere with the arginine-depriving enzymes.

In contrast to GBM cell growth and regrowth, radiosensitization upon ADT appears to be rather independent of ASS/ASL protein levels, and citrulline did not impair the synergistic effect sine qua non for in vivo effectiveness. Vice versa, it is a matter of debate whether high citrulline concentrations - if indeed not interfering with ADT-induced tumor cell radiosensitization – might at the same time protect normal cells. Citrulline and reduced citrulline plasma levels, respectively, are mainly considered as biomarker for intestinal failure and mucosal injury after radio-/chemotherapy (44–46). A recent study using a chemically induced murine mucositis model revealed that pretreatment with L-citrulline positively affected mucosal architecture and function of the small intestine (47). Also, orally administered L-citrulline was found to improve memory deficits by inhibiting neuronal death and cerebrovascular injury in a bilateral common carotid artery occlusion (BCCAO) mouse model (48). The proposed mechanism of cerebrovascular protection was associated with increased eNOS expression and required the recycling of L-Arg via the ASS/ASL enzyme system followed by production of nitric oxide (NO). These data indicate a protective potential of citrulline, an interesting phenomenon to be further assessed in the context of combinatorial treatment strategies with ADT.

Independent of any treatment, radioresistance of U87-MG-shp53 spheroids was found to be higher than of p53-wt U87-MG-shLuc spheroids at standard size (360-400 µm) and comparable cell numbers per spheroid. It is noted though, that the U87-MG-shLuc spheroids were on average slightly smaller at the time of irradiation, i.e. the difference at absolutely identical size might be less pronounced. The discrepancy in radioresponse of U87-MG-shLuc and U87-MG-shp53 cells was not seen in our monolayer assays but is indeed in line with earlier studies using several glioblastoma cell lines with distinct p53 mutational status as well as U87-MG models with and without inactivation of the TP53 gene (49,50). Differences in cell numbers during irradiation are of course an important aspect for data interpretation in the 3-D assay. This has been addressed in our experimental design by attempts to irradiate treated

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas and untreated spheroids at similar size, i.e. by exposing the controls at different time points to radiation than ADT-deprived cultures. Although the mean number of viable cells/spheroid was not absolutely identical, differences were always <15% and cannot explain the highly significant effects seen in the spheroid control probability assays. From independent experiments using various colorectal, HNSCC and pancreatic cancer cell spheroids of different size ranges between 250 µm and 650 µm we know that the cell number/spheroid has to change considerably to result in a significant change in SCD50. An increase in cell number by a factor of 5 and 7, respectively, resulted in an increase in SCD50 of 6 Gy and 7

Gy in two HNSCC models independent of their intrinsic radiosensitivity (unpublished data). A reduction in cell number by about 14% as seen in the U87-MG-shp53 spheroids after 5 days of ADT is therefore unlikely to translate into an SCD50 change of >0.2 Gy.

An important outcome of our work is that TP53-mutated and -knockdown cells appear exceedingly responsive to the combined therapy design of ADT and irradiation. ADT-induced radiosensitization was proven in our sophisticated 3-D radioresponse assay which was particularly useful to also substantiate the clear, yet less pronounced radiosensitizing effect of ADT in the p53-wildtype model. Conceivably, the particularly high efficiency of ADT in the p53-lacking cells might be linked to decreased p53-associated DNA repair or lack of p53- induced cell cycle arrest as previously shown in colorectal cell models (26), leading to enhanced mitotic catastrophe. However, p53 also plays a regulatory role in various fundamental metabolic pathways. Amongst others - and in contrast to its proapoptotic function during DNA-damage stress, p53 is known to support cell survival under metabolic stress conditions such as lack of glucose, glutamine or serine (51–54). We also have first indications that functional p53 is relevant for metabolic stress response and survival during

ADT in epithelial cancer cells (26).

Cumulative evidence suggests that caspase-dependent apoptotic pathways play a major role in cell death after ADT in different tumor cell types (55). However, tumor cells may evade apoptosis through the onset of autophagy, which is frequently described as an early consequence and a possible pro-survival mechanism after amino acid starvation. On the

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas other hand, induction of extensive autophagy may lead to cell death through activation of stress-related pathways, mainly due to the accumulation of reactive oxygen species (55,56).

In colorectal cancer cells, arginine starvation was found to induce prolonged endoplasmic reticulum stress resulting in an upregulation of the AKT/MAPK-pathway but not massive apoptosis (10). In the respective study, 2% DMSO significantly reduced the arginine deprivation-induced ER stress. DMSO is not a specific ER stress inhibitor, but clearly alleviates UPR pathways and correspondent ER stress gene activation. It is known to reduce protein aggregation and accumulation in the ER lumen and to trigger all UPR pathways (57).

In contrast to DMSO, salubrinal is a specific inhibitor of eIF2α phosphatase enzymes and indirectly inhibits eIF2 as a result of reduced dephosphorylation. It thus enhances eIF2α phosphorylation and ATF4 expression, shuts down protein translation when the ER lumen is overloaded with unfolded protein, and reduces UPR activation (58,59). Today, salubrinal is widely used experimentally to study ER stress response and has also shown earlier to inhibit drug-related ER stress-induced apoptosis in neuroblastoma cells (60,61) and T98G glioblastoma cells (62) at concentrations of 10-40 µM. Nonetheless, our initial attempt to causally link ER stress response to ADT-induced radiosensitization by simultaneous application of either DMSO or salubrinal remained inconclusive.

About one-third of GBM show mutations or deletions of TP53 and these cancers appear to be more resistant to the current standard-of-care chemotherapeutic agent temozolomide

(35,63,64). While the causal relation and underlying mechanistic link for GBM still needs to be unravelled, the observation of ADT-induced radiosensitization is of great clinical interest, in particular in GBM cells showing p53 loss of function. Mechanistic studies and transfer into appropriate GBM in vivo models are thus envisioned to further explore the potential and challenges of ADT in combination with radiotherapy and standard-of-care therapeutics to allow rapid implementation of this promising approach into a clinical trial.

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5. ACKNOWLEDGEMENTS

We thank Mrs. Melanie Hüther, Mrs. Marit Wondrak and Mr. Alexander Krüger for excellent technical assistance and gratefully acknowledge Drs. Yaroslav Bobak, Yuliya Kurlishchuk,

Oleg Chen, Barbara Klink and Ralf Wiedemuth for constructive discussions and methodological support.

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FIGURE LEGENDS

Figure 1

GBM cells differ in their 2-D growth response to ADT and in their enzyme equipment for intracellular arginine synthesis; Citrulline partially compensates the arginase- induced loss of regrowth capacity but not the growth arrest. Knockdown of TP53 per se neither affects the enzyme pattern nor the growth response to ADT.

(A/B) Membrane intact monolayer cells per surface area after different periods of ADT (0-5 d) and 3 days after termination of treatment (3 d recovery); all experiments and results documented here have been reproduced (N=2, n≥3); the experimental series in (B) was performed in the presence of arginine-equimolar citrulline concentrations.

(C) Intrinsic ASS (top) and ASL levels (bottom) in GBM monolayer cells treated with and without rhA (recombinant human arginase). Shown are representative Western blots and semi-quantitative densitometric analyses of N=3 independent experiments (mean±SD).

(D) Intrinsic ASS (top) and ASL levels (bottom) in p53-wildtype U87-MG-shLuc and its p53- knockdown counterpart U87-MG-shp53 treated with and without rhA in monolayer culture.

Data are presented according to (C).

Note that we added U87-MG-shp53 cells later in our study. For adequate comparison, all figures exclusively show data obtained with the corresponding vector control U87-MG-shLuc described in Suppl. Materials & Methods. Growth behavior and response to ADT of original and vector control cells did not differ.

Figure 2

ADT by recombinant human arginase (rhA) radiosensitizes GBM monolayer cells; radiosensitization is independent of citrulline.

Graphs show the mean surviving fractions (±SD) of three different GBM cell lines irradiated under either standard culture conditions or upon 24 h pre-treatment with rhA in the absence or presence of citrulline (N=3, n≥3). Data were fitted with a linear-quadratic model as depicted in Materials and Methods. Survival curves upon ADT significantly differed from controls for all cell lines (p<0.05).

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Figure 3

ADT induces cell cycle arrest in GBM 2-D cultures; ADT-induced radiosensitization is neither due to accumulation of cells in G2/M-phase nor unambiguously relates to ER stress response.

(A) Cell cycle distributions determined from flow cytometric DNA histograms (see Suppl. Fig. 2A) of GBM cells exposed for 24 h to arginine-rich or arginine-deprived conditions. Mean values with standard deviation from N=5 independent experiments are shown. ADT was achieved by exposure to rhA enzymotherapy (N=2) or arginine-free media (N=3).

(B) Flow cytometric dot blot diagrams of U87-MG-shp53 cells cultured for 24 h in the presence or absence (rhA treatment) of arginine with or without 0.6 mM citrulline. Cells were pulse-chased for

1 h with EdU and then immediately (0 h) or 8 h later harvested and counterstained with PI. The gate in the 0 h samples illustrates the S-phase fraction that actively incorporated EdU while the gate in the 8 h samples highlights the proportion of EdU-labeled cells that has entered the next

G1-phase as sign of active cycling (N=1).

Gy or 8 Gy single dose irradiation (N=3, n≥3). Cells were irradiated under standard conditions

(+Arg) or pre-treated with rhA in the absence or presence of either 2% DMSO or 20 µM salubrinal, two different inhibitors of ER stress response. p<0.05 (*), p<0.01 (**), and p<0.001 (***)

Figure 4

ADT inhibits 3-D cell growth but not regrowth capacity; citrulline only partially restores growth under ADT after an initial delay.

(A/C) 3-D volume growth and regrowth of U87-MG-shp53 and U87-MG-shLuc spheroids during and after various periods (1, 5, 10 d) of ADT (n≥20; mean±SD).

(B/D) Numbers of membrane intact U87-MG-shp53 and U87-MG-shLuc cells per spheroid

(mean±SD; N=3, n≥2) and representative phase contrast images of spheroids before and directly after 5 and 10 days of ADT

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Figure 5

Combined ADT and irradiation postpone 3-D growth. ADT leads to a clear radiosensitization reflected by enhanced spheroid control probability (SCP) and reduced spheroid control dose 50% (SCD50) in both the p53-knockdown and the p53-wt

U87-MG models.

(A-D) show U87-MG-shp53 spheroid data, (E-H) document the observations in U87-MG- shLuc (vector control) spheroids (n≥29 for each cell line and treatment group).

(A/E) Spheroid volume growth kinetics upon combined ADT (5 days) and up to 12.5 Gy

(U87-MG-shp53) or 10 Gy (U87-MG-shLuc) single dose irradiation. The complete data sets are documented in Suppl. Fig. 4B. Control spheroids and spheroids under ADT were irradiated at similar size (360-400 µm), i.e. irradiation of controls at day 4 in culture and of arginine-deprived spheroids after 5 days of ADT.

(B/F) Spheroid growth delay as function of time estimated from the volume growth data documented in (A/E) and Suppl. Fig. 4B. The underlying values and analytical details are given in Suppl. Table 1. Values representing an additive effect were calculated from the spheroid volume growth delay induced by the respective mono-treatments and are shown in parallel to visualize the enhanced efficacy of combined treatment with increasing dose.

(C/G) Proportion of controlled spheroids upon combined ADT (5 days) and 17.5 Gy (U87-

MG-shp53) or 15 Gy (U87-MG-shLuc) single dose irradiation; data are shown as function of time post-treatment.

(D/H) Spheroid control probability (SCP) of U87-MG-shp53 and U87-MG-shLuc spheroids as a function of irradiation dose (0-30 Gy); SCD50 values were calculated from the SCP curves via a sigmoid dose-response model. Experimental set-up and ADT were according to (A/E).

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Hinrichs et al. Arginine deprivation radiosensitizes glioblastomas

Figure 6

ADT may delay spheroid outgrowth on specific surfaces but does not critically alter invasive capacity of U87-MG-shp53 cells. Migration and invasion were monitored under standard culture conditions (+Arg) and acute ADT(-Arg) as well as during chronic arginine deficiency with a pre-treatment of 5 days (-Arg, 5 d); all results were reproduced (N=2)

(A) Outward migration of U87-MG-shp53 spheroid cells on fibronectin (FN) and collagen type

I (Col-I) coated surfaces; shown are mean migration distances (±SD) of ≥5 individual spheroids.

(B) Invasion distance of cells from treated and untreated U87-MG-shp53 spheroids into a

3-D collagen type I gel; mean values (±SD) from ≥8 individual spheroids are documented in the graph (left) and representative phase-contrast images of spheroids at different time points are depicted (right).

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Arginine Deprivation Therapy: Putative Strategy to Eradicate Glioblastoma Cells by Radiosensitization

C. Noreen Hinrichs, Mirjam Ingargiola, Theresa Käubler, et al.

Mol Cancer Ther Published OnlineFirst August 22, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-16-0807

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2017/08/22/1535-7163.MCT-16-0807.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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