R2J cells absorbed SS, and an uncomplete recovery suggest the generation of volatiles Se metabolites, as reported in prostate, colon, and Jurkat cell lines [47]. The SS absorption triggered DNA fragmentation and cell cycle arrest in G2, leading to both necrosis and apoptosis via caspase-3 activation, as reported in three GBM cell lines that were SS-treated [26]. These results are essential as R2J cells expressed a high level of HDAC that plays an important role in gene expression associated to cell proliferation (p21/Waf1, p27/kip1, p16/ink4a) and apoptosis via Bad, Trail, and FasL in glioma cells [48].

SS metabolism generated Se intermediates, which are also responsible for SS toxicity, such as selenide, selenocystine, and elemental Se [49]. Oxidative stress via the generation of anion superoxide is considered an essential process by which selenite applies its cytotoxicity [47,49].

To sustain an oxidative stress way of action and subsequent pathways, we showed that SS depleted thiol group concentration, in both R2J (p53 wild–type) and U251 (p53 mutant) cells, contrary to TMZ. Indeed, reactive oxygen species ROS production is dependent on the reduction of selenite to selenide through the consumption of thiols [50]. It was also shown that the changes in the redox status induced cell death via apoptosis and via mitochondria-selective autophagy (mitophagy) in glioma cells [29]. Western-blots experiments showed that SS repressed autophagy in both cell lines and induced reticulum stress only in R2J cells. Interestingly, SS at 2 μM did not cause double strand breaks whereas TMZ did. In parallel, TMZ did not deplete thiol groups, suggesting that SS and TMZ do not have the same mechanism of action and thus could have complementary effects to target GBM cells, if used in combination.

Epigenetic regulation of chromatin organization is a key regulator of gene expression that can significantly impact tumor biology and tumor response to therapies. In many tumor types, mutated or modified epigenetic regulators have been clearly associated to tumorigenesis because of their effects on gene expression (reviewed in [19]), and this has led to the development of pharmacologic inhibitors specific of these actors. At the epigenetic level, we focused on histone acetylation and methylation, both modulated by SS in R2J cells. Indeed, HDAC activity was significantly reduced by SS, as already reported in GBM cell lines [26]. In R2J cells, the inhibition of HDAC concomitant with cell proliferation braking and apoptosis induction is consistent with the role of HDAC in these two processes. Therefore, our results led to the intriguing question: How do both these epigenetic modifications interact and would they be able to re-express suppressor tumor genes? Some recent data have revealed an interplay between histone methylation, DNA methylation, the autophagy process, and apoptosis. An interesting

study [51] showed that the histone methyltransferase, G9a, inhibits the expression of genes involved in the autophagy process. Indeed, the pharmacologic inhibition or genetic depletion of G9a triggers the formation of LC3B-II, the aggregation of p62, and the formation of autophagosomes. This loss of G9a leads to a reduction of H3K9m2 levels. In addition, a recent study [52] showed that BIX01294, an inhibitor of G9a, induced autophagy in glioma cells and stimulated differentiation of glioma stem cells (spheres cultivated without serum), associated with lower levels of H3K9m2 as the promoter of autophagy related genes. Taken together, our data suggest that SS could cause a regional remodeling of chromatin that may trigger apoptosis and cell cycle arrest.

## **4. Materials and Methods**

## *4.1. Clinical Data of the Patient*

In October 2009, the patient (a 49-year old white male without any medical history) felt faint and lost consciousness. Magnetic resonance imaging (MRI) showed a right tempo extensive process compatible with a GBM. In November 2009, the tumor was resected and histological analysis confirmed the diagnosis of GBM with a leptomeningeal extension (Figure 1). After the surgery, hydrocephalia and an unmanageable headache appeared. The patient was unfit to receive any conventional treatment, such as chemotherapy or/and radiotherapy.

Instead, the patient received weekly intrathecal injections of Trastuzumab because CSF glioma cells overexpressed HER2. In May 2010, symptoms and the leptomeningeal extension decreased, but the patient presented a local tumor progression on the MRI. So, the patient was treated with radiotherapy plus concomitant TMZ. In August 2010, because of tumor progression and leptomeningeal carcinomatosis progression, no more treatment was compatible with the patient's health condition and he received standard palliative care. The patient died in October 2010.

### *4.2. Origin of R2J Cells*

The tumor cells were isolated from the CSF of the patient (November 2009). The analysis of CSF showed numerous tumor cells with a high mitotic index, indicating a leptomeningeal carcinomatosis. These cells were cultured and the derived R2J cell line was successfully cryopreserved at different passages.

## *4.3. Morphological Analysis*

The primary biopsy (original tumor) was processed for histopathologic study using standard methods and sections were stained with hematoxylin and eosin safran (HES).

R2J cells from the CSF were isolated by cytocentrifugation (Cytospin, Shandon, Pittsburgh, PA, USA) and stained using May Grunwald Giemsa (MGG) using a Zeiss microscope (Oberkochen, Germany). Pictures were taken using a Canon Power Shot S50 digital camera (Courbevoie, France).

## *4.4. Cell Culture*

U251 cells were purchased from the American Type Culture Collection ATCC. They were cultured in DMEM medium containing 10% fetal calf serum (FCS) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL, PS) and L-Glutamine (2mM) (Life Technology, Walthma, MA), in humidified normoxia incubator. U251 were harvested using 0.5%-Trypsin-EDTA (10×) (#15400-054, LifeTechnologies). U251 was chosen due to its MGMT negative and p53 mutant status.

R2J were cultured in RPMI 1640 medium enriched as described for U251, in humidified hypoxia incubator (3% O2, 5% CO2, 37 ◦C). R2J were harvested as described for U251.

For sphere formation, the R2J cells were grown in non-adherent 10-cm diameter Petri dishes (Greiner, les Ulis, France) coated with PolyHema (#P3932, Sigma, St Louis, MO, USA,) in DMEM-F12 medium (Life Technologies,) without serum containing 20 ng/mL human EGF, FGF-2, and 1× NeuroBrew-21 (Miltenyi Biotec, Bergirsch Gladbach, Germany), and PS (as above) in hypoxia. To conform with already published methods, the cells were plated at low density (10 per μL) to allow clonal sphere formation [53,54].

PolyHema was prepared at 10 mg/mL in ethanol 95% and sterilized by filtration on a Stericup (Millipore Express PLUS 1070, Temecula, CA, USA). To coat the 10 cm Petri-dish, 4mL of this solution was applied. The dishes were put in a dry incubator overnight, at 60 ◦C to dry the PolyHema.

## *4.5. Immunohistochemistry*

Embedded paraffin tumor was analyzed in 2009 for CD56, Ki67, GFAP, NeuN, NF70, and synaptophysin, according to the current procedure of the laboratory. The Bouin's fluid fixative used at this time is not compatible with the current methods of the laboratory and the immuno-histo staining obtained for some markers (IDH1 and olig2, for example) was unsatisfactory except for EGFR, CD44, CD34, and Nestin, performed after 2009.

R2J cells cultured in 2D or in spheres were fixed in formol 4%, 30 min before being included in HistogelTM (Microm Microtech, Francheville, France) and paraffin embedded. HES and IHC analyses were performed from 3 μm paraffin sections using a Histostain® plus kit (Thermo Fisher Scientific, Waltham, MA) and Vector novaRED™ substrate kit for peroxidase or Ventana Benchmark® XT platform (Roche, Rotkreug, Switzerland). Only nestin was revealed using Fast Red Bond Polymer Red Refine Detection (#DS93390, Leica Biosystems, Newcastle, UK).

CD-56 (#MAB24081) and Olig2 (#AF2418) antibodies are from R&D Systems (Temecula, CA, USA), MIB1/KI67 (#M7240), p53 (#M7001) and Vimentin (#M0725) were from Dako (Glostrup, Danemark), GFAP (#2202MGF, Eurodiagnostica, Malmö, Sweden), EGFR (#28-0005), E-Cadherin (#18-0223), and MDM2 (#33-7100) were from Zymed Lab, (South San Francisco, CA, USA), IDH1 (#DIA-H09, Histonova, Clinisciences, Nanterre, France), Nestin (#MAB5326), and NeuN (#MAB377) were from Zymed Lab (South San Francisco, CA, USA), ATRX (#HPA001906) and CD44 (#144M-95) were from Sigma Aldrich, P16 (#9511, MTM Roche, France), Neurofilament 70-200 (#Mob080, Diagnostic BioSystems, Pleasanton, CA, USA), and CD34 (#PN IM0786, Beckman Coulter, Brea, CA, USA).

### *4.6. Flow Cytometry Analysis*

R2J-2D cells were seeded at 6000 cell/cm2 in 10 cm Petri-dishes 48 h before treatment with SS (Sigma) for 24 h or 48 h, after which cells and medium were recovered, centrifuged (3 min, 360 g, RT), and cells were rinsed twice with PBS1X.

Apoptosis and the cell-cycle were evaluated with the FITC-Annexin V Apoptosis Detection kit and the Cycle Test Plus DNA reagent kit (BD Biosciences, San Jose, CA) following the manufacturer's instructions. The analysis of cells in the subG1 phase was used to determine DNA fragmentation.

For dimethyl-Histone H3-Lysine-9 (H3K9m2) analysis, 106 cells were fixed 30 min in PFA 1% on ice and then permeabilized with 0.1% Triton × 100 in PBS, 30 min on ice. R2J cells were washed in PBS/BSA 1% twice before adding 1 μL of the rabbit monoclonal antibody, anti-H3K9m2 (#MC554, Millipore), 45 min. The H3K9m2 level was revealed using a (FITC)-conjugated F(ab) 2 fragment goat anti-rabbit IgG(H+L) (Beckman Coulter), 30 min, 4 ◦C.

Cell fluorescence was detected with FACSCantoII (Becton Dickinson) and analysed with FACS Diva Software.

#### *4.7. Chromosome Analysis*

The chromosomes were analyzed according to the International System for Human Cytogenetic Nomenclature. Culture was exposed to 10 μg/mL colchicine for 60 min. Slides were processed for R-binding using heat and the Giemsa-RHG method [55].

## *4.8. Limiting Dilution/Tumor Sphere Initiation Assay (LDA)*

An LDA was performed to quantify in R2J cell lines, the frequency of tumor spheres initiation cells (TS-ICs). R2J parental cells were calibrated to 1000 cells/mL (corresponding to 200 cells/200 μL/well) and then diluted into gradients of cell titers at 500, 250, 125, 62, 100, 50, 25, and 12.5 /mL and transferred into the wells of the 96-well microplate. Clonal spheres (non-adherent, tight, and spherical masses >5 cells) were counted under an inverted microscope (Leica, Wetzlar, Germany), at the end of two weeks. The results are expressed as the percentage of TS-ICs to examine the efficacy of clonogenesis [38].

## *4.9. Intracranial Cell Transplantation into Immunocompromised Mice*

All animal protocols were approved by the GIN Animal Care and Use Committee (number<PHONE_NUMBER>085709). R2J-2D cells (2 × 105) or spheres (2 × 105 or 1000) were implanted into the striatum of 4–6 week male athymicNudeFoxn1nu mice (*n* = 4, Envigo, Gannat, France) to analyze the tumor initiating capacity. Experimental details are available in Appendix A.

Mice were perfused; brains were removed and immediately fixed in formol 4% for subsequent histopathological analyses.

#### *4.10. Magnetic Resonance Imaging*

MRI sessions were performed at 4.7 T (Avance III console; Bruker, Ettlingen, Germany; IRMaGe MRI facility, Grenoble, France) using an actively decoupled cross-coil setup (volume coil for radiofrequency transmission and surface coil for signal reception). All MR experiments were performed under anaesthesia: 5% isoflurane for induction and 2% for maintenance in air. Mice were maintained at 37.0 ◦C and breath rate was cared for 60 breath/min throughout the acquisition. After shimming, MRI sequences were designed for anatomical T2-weighted (T2W) images acquisition using a spin-echo MRI sequence (TR /TE = 2500/33 ms, NA = 14, 21 slices with field of view (FOV) 20 × 20 mm2, matrix = 256 × 256 and voxel size = 156 × 156 × 500 μm3). Acquisition duration was 9 min 20 s. Tumor volume was calculated by adding each tumor surface × slice thickness.

### *4.11. Selenium Absorption*

R2J-2D cells were seeded at 6000 cell/cm2 in 10 cm Petri-dishes, 24 h before SS supplementation (2.5, 5, and 10 μM).

Se concentration was determined both in cell lysates and medium using an ICP-MS (X series II, Thermo Fisher Scientific, Waltham, Massachusetts, USA) equipped with Collision Cell interference elimination Technology (CCT) after dilution in 1% nitric acid containing Gallium (Ga) as an internal standard. The gas introduced in the CTT was a mix of He and H2. The isotope ratio 78Se/71Ga and an external calibration curve (16, 80, 160 nmol Se/L) allowed the determination of the Se concentration. The results (mean ± SD) were calculated in nmoles of Se per gram of protein and expressed as a percentage of the Se measured vs. Se added. The Se recovery was calculated by adding the quantity of Se both measured in the medium and in the lysates and compared to the Se quantity added (referred as 100%).

## *4.12. MTT Assay*

A MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay (Sigma) was performed to determine R2J-2D cell survival after SS treatment at 2.5 to 12 μM for 24 h, 48 h, or 72 h. 1 × 105 cells/well were seeded in 96-wells plate 24h before the addition of SS. The results (mean ± SD) are reported as the cell survival percentage versus the control (not treated). IC50 (concentrations inducing 50% cell death) values were determined by curve-fitting (using Prism 5, GraphPad).

In parallel, TMZ (Temodal, MSD, France) was dissolved in dimethyl sulfoxide (DMSO) and R2J cells were treated for 24 h, 48 h, and 72 h with 1, 10, 100, 500, and 1000 μM of TMZ. Cells were also treated with DMSO alone at the corresponding volume. A long term treatment was also performed by exposing R2J-2D cells to 400 μM TMZ for 72 h followed by a wash-out of 72 h. Results are expressed as the percentage of cell survival versus control (DMSO concentrations used were not cytotoxic).

## *4.13. Confocal Microscopy Analysis of Cell Viability*

R2J-2D cells were set on a Lab-Tek-Chamber Slide system (Nalge Nunc International, Rochester, NY, USA) and were incubated with 5 μM of Fluorescein Diacetate (FDA, Thermofisher Scientific, Courtaboeuf, France), 1μM Hoechst 33342 (Interchim, Montluçon, France), and 10mg/mL Propidium Iodide (PI) (Interchim) in a 5% CO2 incubator, 37 ◦C, 20 min. After staining, cells were installed on the microscope incubation chamber with a controlled atmosphere at 37 ◦C and 5% CO2 (POC Chamber, Pecom, Erbach, Germany). Images were collected with a Leica TCS SP2 AOBS (Acoustico Optical Beam Splitter) inverted laser scanning confocal microscope equipped with an ×63 oil immersion objective (HCX PL APO 63.0× 1.40). Laser excitation and emission (adjusted with AOBS) were, respectively, 351–364/425–485 nm for Hoechst, 488/500–540 nm for FDA, and 543/600–650 nm for PI. Confocal pinhole (Airy units) was 1 for all channels. Each experiment was performed on a randomly chosen field. Raw image merging was obtained by LCS Lite software (Leica LCS, version 2.61, Overlay).

### *4.14. Caspase-3 Activity Evaluation*

The caspase-3 Fluorometric Assay (BioVision, Mountain View, CA, USA) was used. R2J-2D cells were harvested and lysed into 50 μL of chilled Cell Lysis Buffer at 4 ◦C, 10 min, and centrifuged for 10 min, 10000 g at 4 ◦C. Protein concentration was determined (see below) and 50μL of supernatant containing 50 μg to 200 μg of protein was mixed with 50 μL of 2× reaction buffer and 5 μL of DEVD-AFC substrate (1 mM), in a 96-wells plate. Reaction was achieved for 1h at 37 ◦C. The plate was read on a fluorometer (Ex/Em = 400/505 nm). Caspase-3 activity is expressed in AU/g prot.

## *4.15. Determination of HDAC Activity*

R2J-2D cells were treated for 24 h, 48 h, and 72 h with 2.5 and 5 μM SS and harvested. Pellets were rinsed twice in PBS and lysed in 400 μL sterile water, 10 min. HDAC activity was investigated in cell lysates from 50 μg total protein using a Fluorometric Assay (BioVision, Mountain View, CA, USA) as described by the manufacturer. HDAC activity is expressed in relative fluorescence units (RFU) per μg protein.

## *4.16. Real Time PCR*

Total RNA extraction was performed with an RNeasy Mini Kit, following the manufacturer's recommendations, with RNase-free DNase I treatment (Qiagen, Courtaboeuf, France). cDNA was reverse transcribed from 1 μg of total RNA with the SuperScriptIII First-Strand Synthesis (Life Technologies) followed by RNaseH (1 μL) treatment. Real time PCR was conducted using the QuantiTect SYBR Green RT-PCR kit (Qiagen) and the Stratagene 3005MxPro (Santa Clara, CA, USA). The primers (sequences in Supplementary Material Table S1) (Life Technologies) were all used at 400 nM, Tm at 60 ◦C, with the following qPCR program: 1 cycle: 15 min-95 ◦C, 40 cycles: 15 s-94 ◦C, 30 s-60 ◦C, 30 s-72 ◦C, 1 cycle: 1 min-95 ◦C, 30 s-60 ◦C, 30 s-95 ◦C.

Gene expression was quantified using the comparative threshold cycle (Ct) method [56] with a normalization against HPRT1, RPL27, and RPL32.

## *4.17. Thiol Group Determination*

Thiol groups were determined by a colorimetric method using the reducing properties of the SH groups as previously described [25]. The thiol group levels were determined as micromoles per gram of total cell proteins and expressed in percentage versus the control.

#### *4.18. Western Blot Analysis*

U251 and R2J, after 72 h treatment followed by 72 h of wash-out with TMZ or SS or chloroquine (Cq, used to block autophagy 1 h before SS or TMZ treatment at 20 μM, Sanofi-Aventis), were harvested in protein extraction buffer (Tris-HCl 20 mM, pH 7.4, NaCl 137 mM, EDTA 2 mM, Triton X-100 1%, supplemented with proteases' and phosphatases' inhibitors (1/100, Sigma-Aldrich). After pipetting up and down with a P1000, supernatants were collected after centrifugation at 10,000 g for 10 min, at 4 ◦C. Protein concentration was measured by the BCA method. Proteins (20 μg total) were treated with SDS-PAGE sample buffer [6× concentrated: 350 mM Tris, 10% (w/v) SDS, 30% (v/v) glycerol, 0.6 M DTT, 0.06% (w/v) bromophenol blue], boiled for 5 min at 95 ◦C, and loaded on 15% (for LC3, p62, and phospho-H2Ax) and 8% (for Grp78) acrylamide/bisacrylamide gels. Proteins were transferred onto nitrocellulose membranes, blocked with 5% bovine serum albumin containing 0.5% Tween (Euromedex) (PBST) for 1 h at room temperature, and probed overnight at 4 ◦C with the following primary antibodies diluted in 5% bovine serum albumin-PBST: 1:1000 rabbit anti-p62 antibody (Sigma-Aldrich), 1:200 goat anti-Grp78 antibody (Santa Cruz Biotechnology, Dallas, TX), 1:1000 anti-LC3-II antibody, 1:1000 rabbit anti-phospho-H2Ax antibody (both from Cell Signalling Technology), and 1:5000 mouse anti-alpha tubulin (Sigma-Aldrich). Membranes were washed with PBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (from Jackson Immunoresearch), and visualized using electrochemiluminescence (ECL) western blotting substrate (ThermoFisher Scientific) on a Chemidoc imaging system (BioRad, Hercules, CA).

## *4.19. Protein Concentration Determination*

The concentration of protein in cell lysates (obtained as mentioned above) was determined using the BCA assay (Interchim, Montluçon, France) as previously described [24]. The absorption at 562 nm was measured using the Varioskan Flash (Thermo Fisher Scientific) associated to the SkanIt Software for the quantification.

## *4.20. Statistical Analysis*

Results are expressed as means ± SD for the number of experiments indicated. All statistical analysis of data was computed using StatView® (SAS Institute, CA, USA). The sources of variation for multiple comparisons were assessed by analysis of variance ANOVA. The differences were considered statistically significant at *p* < 0.05 versus the control.

## **5. Conclusions**

In conclusion, we described a newly established glioblastoma cell line (R2J), from a CSF from a patient affected by a metastatic GBM. R2J were MGMT+ and resistant to TMZ. In the presence of FGFb and EGF, the R2J forming spheres were phenocopied on 2D cultured R2J cells except for CD56, a neuronal marker, suggesting that R2J may differentiate into neuronal-like cells. The cell line could form a tumor when intracranially implanted into nude mice even at 1000 spheres, providing conclusions on their CSC properties. In vitro, SS could exert its anti-cancer properties in R2J cells, and is thus relevant for testing in this new GBM bearing mouse model.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/11/1/12/s1, Figure S1: A representative G-banded karyotype of R2J cells. The R2J cell line was identified as near-triploid (hypotriploid) with 64 chromosomes, Table S1: primer sequences used for RT-q-PCR.

**Author Contributions:** Conceptualization: P.C., L.L. and F.H.-P..; Methodology, S.B., P.C., E.C., C.L., C.C., C.C.-R., J.A., C.G., L.L. and F.H.-P.; Validation, S.B., P.C., C.C.-R., J.A., F.L., J.B., L.L. and F.H.-P.; Formal Analysis, P.C., C.C.-R., J.A. and F.H.-P.; Resources, F.L., P.F. and F.H.-P.; Data Curation, S.B. and F.H.-P.; Writing-Original Draft Preparation, F.H.-P.; Writing-Review & Editing, S.B., P.C., C.C.-R., J.A., F.L., J.B., P.F. and F.H.-P.; Visualization, F.H.-P.; Supervision, L.L. and F.H.-P.; Project Administration, S.B., P.C., J.B., L.L. and F.H.-P.; Funding Acquisition, L.L. and F.H.-P.

**Funding:** This research was funded by grants from the Direction de la Recherche Clinique (DRC) of the Centre Hospitalier Universitaire Grenoble Alpes (CHUGA, France) and by the Groupement des Entreprises Françaises de Lutte contre le Cancer (GEFLUC).

**Acknowledgments:** We express our thanks to Laurence David-Boudet, Yveline Maffren, Aurélie Soldini, Martine Micheloni and Véronique Curri for technical assistance. We are grateful to Fabienne Agasse for the careful reading of the manuscript, Emmanuelle Grillon and Emmanuel Barbier for MRI assistance.

*Cancers* **2019**, *11*, 12

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**

## **Appendix A**

Cells were harvested using Accutase (Sigma), counted, and suspended in 10 mL PBS. Cell viability and counting were assessed before and after the procedure using Scepter® (Millipore). All surgery was performed using sterile techniques within a tissue culture hood. Mice were anesthetized with isoflurane 5% and maintained under 2.5% during the surgery. NaCl 0.9% was put on eyes throughout the procedure and mice were kept on a warm pad throughout. Cells were injected into the right striatum using a stereotaxic apparatus. Injection coordinates were 2.6 mm lateral to Bregma and a

3.6mm depth. A small bore hole was made using a 25 g needle. The cell suspension was mixed and loaded into the syringe just prior to injection. 3 μL of cells was delivered using a 26 g Hamilton microsyringe (701RN, 10 μL). The syringe was stabilized for 1 min before injection and 0.5 μL of cell suspension was injected each 30 s. The needle was kept in place for a further 4 min to prevent backflow and was then removed slowly (1 μL/min). After cell injection, the skin incisions were closed. To avoid suffering, Buprecare® was intracutaneously injected (0.1 mg/kg) to each mouse. The animals were then revived under an infrared lamp and then returned with their congeners.

## **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article*

## **Dysregulation of Macropinocytosis Processes in Glioblastomas May Be Exploited to Increase Intracellular Anti-Cancer Drug Levels: The Example of Temozolomide**

## **Margaux Colin 1, Cédric Delporte 2 , Rekin's Janky 3, Anne-Sophie Lechon 1, Gwendoline Renard 1, Pierre Van Antwerpen 2 , William A. Maltese 4 and Véronique Mathieu 1,5,\***

Received: 27 December 2018; Accepted: 20 March 2019; Published: 22 March 2019

**Abstract:** Macropinocytosis is a clathrin-independent endocytosis of extracellular fluid that may contribute to cancer aggressiveness through nutrient supply, recycling of plasma membrane and receptors, and exosome internalization. Macropinocytosis may be notably triggered by epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), two well-known markers for glioblastoma aggressiveness. Therefore, we studied whether the expression of key actors of macropinocytosis is modified in human glioma datasets. Strong deregulation has been evidenced at the mRNA level according to the grade of the tumor, and 38 macropinocytosis-related gene signatures allowed discrimination of the glioblastoma (GBM) samples. Honokiol-induced vacuolization was then compared to vacquinol-1 and MOMIPP, two known macropinocytosis inducers. Despite high phase-contrast morphological similarities, honokiol-induced vacuoles appeared to originate from both endocytosis and ER. Also, acridine orange staining suggested differences in the macropinosomes' fate: their fusion with lysosomes appeared very limited in 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP)-treated cells. Nevertheless, each of the compounds markedly increased temozolomide uptake by glioma cells, as evidenced by LC-MS. In conclusion, the observed deregulation of macropinocytosis in GBM makes them prone to respond to various compounds affecting their formation and/or intracellular fate. Considering that sustained macropinocytosis may also trigger cell death of both sensitive and resistant GBM cells, we propose to envisage macropinocytosis inducers in combination approaches to obtain dual benefits: increased drug uptake and additive/synergistic effects.

**Keywords:** glioma; macropinocytosis; methuosis; honokiol; vacquinol-1; MOMIPP; temozolomide

## **1. Introduction**

Glioblastoma (GBM) remains associated with a dismal prognosis, partly due to a high level of resistance to proapoptotic stimuli and a propensity for cell migration and invasion of normal brain

tissue [1,2]. Although the majority of chemotherapeutic drugs operate by triggering caspase-dependent apoptotic cell death, 17 cell death types have been described to date [3–5], including autophagy. The latter is a programmed pathway that can either promote or hinder tumor development. It is triggered by several anti-tumor agents, including temozolomide (TMZ), particularly in cancer cells lacking essential apoptotic modulators, such as GBM [6]. The autophagosomes arise under the control of the PI3K-Akt pathway—probably from diverse sources, including endoplasmic reticulum (ER), Golgi and plasma membrane—and further fuse with lysosomes to digest their contents [5]. To date, autophagy remains the best way to combat GBM, since it is induced by the standard-of-care drug TMZ [7]. Other forms of non-apoptotic cell death include methuosis, paraptosis, necroptosis, oncosis and lysosomal membrane permeabilization plus senescence. All these forms of cell death are associated with accumulation of cytoplasmic vacuoles (vacuolization) [3,5]. Irreversible vacuolization marks cytopathological conditions leading to cell death; however, the size, content, function and origin of the vacuoles may differ markedly from one type of cell death to another.

One mechanism that can lead to cellular vacuolization is dysregulation of macropinocytosis. Macropinocytosis is a clathrin-independent endocytic mechanism that was first described by Warren Lewis in 1931 [8]. This process is characterized by the nonselective uptake of extracellular fluid [9]. Macropinosomes consist of large vesicles (0.2 μm < diameter < 5 μm), formed by actin-mediated ruffling of the plasma membrane. The nascent macropinosomes migrate in a centripetal manner and rapidly acquire markers of late endosomes, such as Rab7, before their fusion with the lysosomal compartment [9]. Cancer cells generally employ macropinocytosis to internalize cell surface receptors and certain nutrients [10]. Starving tumors by inhibiting macropinocytosis was recently suggested as a potential therapeutic strategy to combat Ras-driven cancers [11]. Deregulation of macropinocytosis in cancer cells seems to be correlated with oncogenic *RAS* [12], and more specifically *K-RAS* in clinical samples [13,14], even if induction of Src may also trigger macropinocytosis [15,16]. With respect to glioma, high levels of active wild-type Ras have been reported in high grade astrocytomas, but oncogenic mutations in *H-RAS*, *K-RAS* or *N-RAS* are rare (occurring in less than 2% of the cases) [17]. Another major pathway known to stimulate macropinosome formation is under the control of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) [18]. Amplification of wild-type or mutant *EGFR* is relatively common (43% of GBM cases), with the most frequent mutation being *EGFRvIII*. Mutation and/or overexpression of *EGFR* or *PDGFR* are very well known markers of glioma malignancy [19,20]. Importantly, EGF-mediated macropinocytosis appeared dependent on c-Src activity [16].

Despite the potential importance of macropinocytosis for survival of glioma cells, little information is currently available about the possible deregulation of macropinocytosis in these tumors. The possible relevance of macropinocytosis in gliomas is underscored when considering that it contributes, at least partly, to the uptake of extracellular vesicles (particularly exosomes) produced by glioma cells to enhance cancer progression and promote angiogenesis, metastasis, immunosuppression and chemoresistance [21,22]. While active macropinocytosis may represent a survival strategy for gliomas, it may also be exploited to increase the intracellular delivery of therapeutics (soluble as well as vesicular-formulated ones) [22–25]. Moreover, there is strong evidence that hyperstimulation of macropinocytosis by Ras overexpression or by treatment with small molecules can be detrimental to glioma cells, leading to a form of cell death termed as methuosis [26]. Normally, macropinosomes are formed from plasma membrane ruffling via Rac1-dependent rapid polymerization/depolymerisation of branching actin filaments. The macropinosomes then undergo a maturation process wherein they are either degraded via a late endosome/lysosome process or recycled back to the plasma membrane [23]. During abnormal macropinocytosis, as observed in methuosis, macropinosomes do not fuse with lysosomes or recycle to the plasma membrane. Instead, they fuse with each other to form multiple larger vacuoles that exhibit properties of non-functional late endosomes [27]. Through mechanisms that are yet to be elucidated, the extreme vacuolization ultimately leads to death and rupture of the cell. While Ras-induced methuosis is accompanied by caspase activation, cell death cannot be

prevented by caspase inhibitors. Methuosis is therefore considered to be caspase-independent [28]. Although early studies demonstrated that methuosis can be induced in glioma by overexpressing active forms of Ras or Rac1, more recent studies have identified small molecules that can trigger this form of death in a manner that is independent from Ras or Rac1. Specifically, the indolyl chalcone 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), which was used in the present study, can induce intense macropinocytosis, leading to methuosis in cultured GBM cells at low micromolar concentrations [28]. Similar results were obtained in TMZ-resistant glioma cells [29].

Thus, this study aims to (i) evaluate in a systematic manner whether genes implicated in macropinocytosis are deregulated in gliomas, employing analyses of mRNA expression levels in clinical sample datasets and (ii) evaluate whether the induction of severe vacuolization and macropinocytosis with small molecules may represent an interesting strategy to enhance the uptake and/or efficiency of chemotherapeutic agents by gliomas as detailed in the Discussion section.

## **2. Results**

#### *2.1. Evaluation of The Deregulation of Macropinocytosis in Human Gliomas*

To evaluate whether macropinocytosis is deregulated in gliomas, we made use of mRNA expression databases of human samples. Table A1 (Appendix A) summarizes the 38 selected main proteins participating in either the initiation/regulation or the formation and recycling of macropinosomes, with their detailed function in that process. The GDS4290 Henry Ford database regroups the mRNA expression array data of 180 patients: 23 non-tumoral samples (from epilepsy patients) and 157 glioma samples. We first performed a systematic, statistical comparison of the mRNA expression of the 38 targets between normal brain samples (*n* = 23), grade II (*n* = 45), grade III (*n* = 31) and grade IV (*n* = 81) glioma samples through non-parametric Kruskall–Wallis comparison and two-tailed tests. Detailed results per probe, provided in Table A1, highlighted that 14/38 targets were overexpressed in glioma while 9/38 were downregulated. This means that more than 60% (23/38) of genes playing key roles in macropinocytosis are deregulated in glial tumors.

As expected, *EGFR* and *PDGFR* were upregulated according to the malignancy grade of the tumor (Figure 1a,b), but other key players in macropinocytosis and methuosis were also upregulated according to the grade of the tumor, including actin dynamic-related gene products (*SWAP70*, several *ARPC* involved in the Arp2/3 complex), their upstream activator adenosine diphosphate ribosylation factors 6 (*ARF6*), and actin-rich membrane ruffles associated protein Rab34 (Figure 1d,f,h). In contrast, the expression level of GIT1 that mediates the deactivation of Arf6 is decreased (Figure 1c), along with the other genes involved in the closure of the macropinosomes, such as *PAK1*, *CTBP1* and *PDL1* (Table A1, Figure 2). *RAB20* and *SNX5*, two markers of initial macropinosome maturation, are both upregulated (Table A1; Figure 1e,g; Figure 2). Thus, the expression of genes encoding numerous proteins associated with macropinocytosis is deregulated in glioma in comparison to normal brain tissues; these changes are additionally correlated with the grade of the tumor (Figure 1). Figure 2 schematically illustrates the macropinocytosis process, noting the proteins analyzed and the changes observed in their expression in glioma tumors. According to the high number of targets whose mRNA levels were found deregulated, we proceeded with an unsupervised hierarchical clustering of the 180 patient samples on the basis of those 38 gene expression signatures, and we observed a clear tendency of grouping according to the grade of the sample (Figure 3a). While normal brain tissue samples grouped on the left, the GBM samples were distinctly clustered on the right of the Euclidian-based tree (Figure 3a). Note that while grade II samples also appeared grouped between non-tumoral brain samples and grade IV samples, grade III samples were spread among grade II or grade IV samples (Figure 3a).

**Figure 1.** mRNA expression level of selected targets involved in macropinocytosis in patient brain samples. Data are expressed as mean ± SEM of mRNA intensities of each probe expressed in log 2 ratio. NB: normal brain; grade II, III and IV: glioma samples according to the histopathological grade of the tumor. (**a**) epidermal growth factor receptor (*EGFR*), (**b**) platelet-derived growth factor receptor A (*PDGFRA*) and *PDGFRB*, (**c**) *GIT1*, (**d**) *ARF6*, (**e**) *RAB20*, (**f**) *RAB34*, (**g**) *SWAP70* and (**h**) *SNX5*. Note that only probes with the highest intensities are presented for clarity of the figure with respect to *EGFR* and *SNX5*. Statistical comparisons with the non-tumoral brain samples are represented by (\*), statistical comparisons between grade II and IV by ( ), between grade III and IV by (#) and between grade II and III by (§). All statistics are based on two-tailed tests according to conventional thresholds: *p* < 0.05 (\*, , # or §), *p* < 0.01 (\*\*, , ## or §§) and *p* < 0.001 (\*\*\*, , ### or §§§).

**Figure 2.** Macropinocytosis process and its deregulation in glioma. Upregulated genes are framed in red, downregulated ones in green, and those genes for which no change was observed are framed in grey. The stimulation of EGFR and PDGFR induces the activation of K-Ras and H-Ras. These Ras GTPases activate both Arf6 and Pak1 to initiate the actin polymerization. Arf6 recruits ARNO (Arf guanine nucleotide exchange factor) for the activation of Arf1 to enable the WAVE regulatory complex (WRC), composed by Wasf1 and the WAVE complex, which regroups Nckap1, Brk1, Cyfip and Abi1. WRC activates Arp2/3, which induces actin branch formation and actin polymerization (1) for the formation of macropinosomes with the help of Rab34, Swap70 and PI(3,4,5)P3. Both Ctbp1 (activated by Pak1) and PLD1 (activated by Arf6 and Ctbp1) are essential for the closure of the macropinocytic cup and the final fission from the plasma membrane (2). Rab5 is a marker of early endosome as well as Rab21, which remains on the intermediate endosome (3). SNX1 and SNX5 play a role in macropinosome maturation (3). Markers of intermediate endosomes are Rab21, Rab20 and Rab7 (4). Rab7 is also a marker of late endosome as well as Lamp1 (5). Rab11 is a marker of endosome recirculating to the plasma membrane (6).

In order to evaluate whether this deregulation has any impact on the survival of the patient, we carried out the same analysis on the data from the GDS53733 database of 70 samples of grade IV GBM patients (16 short term survival patients (<12 months); 23 long term survival patients (>36 months); and 31 intermediate survival patients). We found only very limited statistically significant changes, i.e., with respect to probes for *SWAP70* and *CYFIP1* and, to a lesser extent, *PDGFRA*, *EGFR* and *ABI1* (Table S1). Nevertheless, the unsupervised heatmap clustering revealed a tendency of grouping of the samples from long surviving GBM patients into a subcluster (Figure 3b). In addition, we noticed that many targets overexpressed in GBM samples of the 180 series were downregulated in long surviving

patients, including *RAB20*, *RAB34*, *SWAP70*, *CYFIP1*, *ARPC1B*, *ARPC2* and *ARPC4*. The dataset is certainly limited in size and further investigation integrating all clinical aspects should be conducted to determine whether deregulated macropinocytosis could be a predictive marker for clinical outcome of the patient.

**Figure 3.** Heatmaps of 38 macropinocytosis gene signatures. (**a**) Dataset GSE4290 regrouping 180 samples (columns) of non-tumoral tissues (N/A) or glial tumors of various histological origin and grade according to the legend. For each gene, the data of the different probes have been averaged to be presented as a single line. Clustering has been made on the basis of the Euclidian distance. (**b**) Similar heatmap made on the dataset from GSE53733 regrouping 70 samples of GBM patients with different overall survival according to the legend. Overexpressed genes are in red, and downregulated genes are in blue (see scale next to the figures).

*2.2. Morphological Comparison of Glioma Cells Treated by Honokiol, 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP) and Vacquinol-1*

Next, we examined the effects of three compounds that alter vesicle trafficking or autophagy in glioma cells. Two are known for their propensity to induce macropinocytosis—i.e., vacquinol-1 [27] and MOMIPP [29]—while the last one, honokiol, is a natural bioactive polyphenol extracted from several parts of Magnolia genus tree [30] (Figure 4). The latter displays various well-known pharmacological properties, such as anti-oxidant [31], neuro-protective [32] and anti-inflammatory effects in both microglia and astrocytes [33]. These properties explain the traditional use of honokiol for the treatment of thrombotic stroke, gastrointestinal complaints, anxiety and nervous disturbance [34]. Honokiol has also been shown to display interesting anti-tumoral properties against glioma cells, where it may trigger p53-mediated cell cycle arrest and apoptosis [35] or alternative autophagic cell death [36].

**Figure 4.** Chemical structure of the three compounds used in this study.

Surprisingly, we observed striking cytoplasmic vacuolization in cells exposed to honokiol at concentrations slightly lower than its IC50 in Hs683 (40 μM) and U373 (35 μM) glioma cells (Figure 5 with respect to the U373 cell line; Figure S1 for Hs683 cells). Although the vacuolization appeared delayed with honokiol in comparison to MOMIPP and vacquinol-1, the progressive increase in the number and size of highly refringent vacuoles and their apparent homotypic fusion resembled the morphological features observed with the macropinocytosis inducers at equipotent concentrations (close to their own IC50 as determined by means of MTT colorimetric assay, i.e., 3 μM for MOMIPP and 5 μM for vacquinol-1; see Figure S2 for the MTT curves).

### *2.3. Characterization of the Vacuoles Induced by Honokiol, Vacquinol-1 and MOMIPP*

To obtain more insight regarding these vacuolization processes, we utilized different inhibitors of vacuolization, i.e., bafilomycin A1, ethyl-isopropyl-amiloride (EIPA), EHT1864, PP2 and filipin.

Bafilomycin A1 is an inhibitor of the vacuolar H+-ATPase that plays crucial roles in maintaining low pHs of late endosomes and lysosomes. Bafilomycin A1 was accordingly shown to block the endosomal and endosome-lysosomal fusion during macropinocytosis [37]. Additionally, bafilomycin A1 has been suggested to also inhibit nascent macropinosome formation, similarly to the Na+/H+ exchanger inhibitor, by disrupting the fine tuning of submembranous pH needed for the recruitment and activation of Rac1 and Cdc42 to membrane ruffles [38]. As illustrated in Figure 6 with respect to the U373 cell line (Figure S3 for HS683 cells), bafilomycin A1 almost completely inhibited the vacuolization induced by each of the three compounds. Consistently, similar results were obtained with respect to the Na+/H+ exchanger inhibitor EIPA [9] when its concentration adapted to the cell line and the compound under investigation in accordance with the timing of the vacuolization that they induced (Figure 6 and Figure S3).

In contrast, the Rac1 inhibitor, EHT1864 [26], has only slight effects on honokiol and vacquinol-1-induced vacuolizations and no effect on MOMIPP–induced vacuolization (Figure 6 and Figure S3). Alternatively to oncogenic RAS stimulation, Src was also demonstrated to participate in macropinosome membrane ruffling via PI3K [15,16]. We therefore made use of the Src inhibitor PP2 [39]. This later inhibited vacuolization induced by honokiol at 25 μM (Figure S4). We tried higher concentrations to possibly inhibit MOMIPP and vacquinol-1 vacuoles but encountered solubility issues making us impossible to conclude at this stage whether Src may or not be involved in their vacuolization processes.

The last inhibitor, filipin, is a cholesterol binding agent [26] known to inhibit clathrin-independent endocytosis. Consistently, macropinocytosis occurs in cholesterol-rich membrane domains [40]. When used at nontoxic concentrations, i.e., 1 μg/mL, filipin had no effect on the vacuole formation induced by each of the three compounds (Figure S5). Previous studies demonstrated that filipin effectively impaired MOMIPP-induced vacuolization in a different cell line, but it was at higher concentrations, i.e., 12 μg/mL. However, in our cellular models, such high concentrations proved to be too toxic.

**Figure 5.** Morphological illustration of the vacuoles formed after treatment of the U373 cell line with honokiol (35 μM), 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP) (3 μM), or vacquinol-1 (5 μM) over time. The accumulation of vacuoles is only visible after 24 h of treatment with honokiol, but those induced by treatment with vacquinol-1 occurs after 3 h and after only 1 h of treatment with MOMIPP. The experiment has been conducted two times in triplicate.

**Figure 6.** Effects of various inhibitors on vacuole formation following honokiol, vacquinol-1, and MOMIPP treatment in U373 cells. Representative brightfield illustrations of U373 cells treated with or without the inhibitors are as follows: (**a**) pre-treated 1 h with bafilomycin A1 (100 nM) before honokiol (35 μM), MOMIPP (3 μM) or vacquinol-1 (5 μM) treatment, co-treatment with EHT1864 (25 μM) and the compound. Bafilomycin A1 inhibits the vacuoles induced by each compound but not ER-derived dark dense vacuoles induced by honokiol (white arrow). EHT1864 slightly inhibits vacuolization induced by honokiol and vacquinol-1 but not for vacuolization induced by MOMIPP. (**b**) Cells were co-treated with EIPA (10, 30 and 75 μM) and each compound. EIPA inhibits the vacuoles induced by each compound except the ER-derived dark dense vacuoles induced by honokiol (white arrow; similar than bafilomycin A1 effects). Each experiment has been conducted at least twice in triplicate.

Finally, we utilized fluorescent probes to further decipher the origin of vacuoles. Lucifer yellow is a fluid-phase tracer that is internalized intracellularly by endocytic processes including macropinocytosis [12]. We observed, as expected, that both MOMIPP and vacquinol-1 increased the number of positive lucifer-yellow vacuoles—a feature that was also observed with honokiol, but to a lesser extent, with several vacuoles remaining negative (Figure 7a and Figure S6a).

**Figure 7.** Lucifer yellow and fluorescent 10 kDa dextran uptake by U373 cells treated with honokiol, MOMIPP, or vacquinol-1. (**a**) Illustration of Lucifer yellow staining (100 μg/mL during the whole treatment period). Exposure time is the same for all conditions. The experiment has been conducted three times in duplicate. (**b**) Illustration of fluorescent 10 kDa dextran uptake. Again, cells were treated with each compound in presence of 10 kDa Texas-Red labeled dextran (125 μg/mL) for 24 h. Some vacuoles are positive with MOMIPP and vacquinol-1 but none with honokiol. Exposure time has been adjusted for each condition (550 ms for the control, 490 ms for honokiol, 650 ms for MOMIPP and 160 ms for vacquinol-1). The experiment has been conducted twice in duplicate. (**c**) Quantitative dextran 10 kDa uptake by U373 and HS683 determined by flow cytometry after 24 h and 30 h, respectively. Data are expressed as box plots: line, median; boxes, percentiles 25–75; and whiskers, non-outlier ranges of six experiments. Statistical comparisons with untreated cells are based on Mann–Whitney tests according to conventional thresholds: *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*).

As macropinocytosis differs from other endocytic processes by its capacity to internalize large extracellular volumes and high molecular weight molecules, we evaluated this feature by means of 10 kDa and 70 kDa fluorescent dextran staining. Figure 7b and Figure S6b show that each of the three compounds seemed to trigger the uptake of 10 kDa dextrans that was observed in small vacuoles, but rarely in the most enlarged ones. MOMIPP and vacquinol-1 seemed to uptake 70 kDa dextrans, contrary to honokiol, which had no positive vacuole (Figure S7). A quantitative assay was conducted with 10 kDa Texas Red-labeled dextran. Significant increased uptake under treatment with each of the three compounds was confirmed, except in the case of U373 cells treated 24 h with honokiol (Figure 7c), and the most marked increase was obtained with vacquinol-1. By contrast, acridine orange labelled most of the enlarged vacuoles induced by honokiol in red, suggesting acidic content (Figure 8 and Figure S8). Interestingly, while only few of MOMIPP-induced vacuoles were also stained red (Figure 8 and Figure S8), vacquinol-1-induced vacuoles were positive for both red and green fluorescence after acridine orange staining [41]. When looking for lysosomal staining, these organelles were readily observed in cells treated either with honokiol or MOMIPP (Figure S9). Thus, the principal difference between these was that most of the large vacuoles remained unstained by acridine orange with MOMIPP, whereas most of them appeared acidic in honokiol-treated cells while being lyso-Tracker negative (acidic; Figure S9). This could suggest that the large vacuoles induced by honokiol might result from fusion of endosomes with lysosomes or autophagolysosomes at some point during their biogenesis, while MOMIPP completely blocks vacuole fusion with lysosomes (Figure 8 and Figures S8 and S9). Finally, although vacquinol-1 appears to trigger the strongest increase in macropinocytosis tracers (Lucifer Yellow and high molecular weight dextrans), few enlarged vacuoles have been observed, suggesting different effects on macropinosome maturation and recycling.

**Figure 8.** Illustration of the fluorescent characterization of the vacuoles using different fluorescent cell compartment trackers in U373 cells. Two kinds of vacuoles can be observed after treatment with honokiol: red arrows show acridine orange positive vacuoles possibly derived from macropinocytosis, and white arrows show ER-positive vacuoles. Some vacuoles induced by MOMIPP are red-positive with acridine orange (red arrow) but none to the ER-tracker. Vacquinol-1-induced vacuoles are red-positive (red arrow), but also green-positive stained by acridine orange. This experiment has been conducted three times in duplicate.

We also used probes for mitochondria and ER to assess the contributions made by these compartments to the vacuoles induced by honokiol. We observed some ER-positive vacuoles only in the honokiol-treated cells (Figure 8 and Figure S8). Both mitochondria and ER are thought to be a source of vacuolization during paraptosis [42]. Thus, honokiol differed from both MOMIPP and vacquinol-1 and could induce paraptosis features in these glioma cell lines, similar to what was previously described in leukemia cells [43]. ER swelling does not play any role in macropinocytosis. Accordingly, ER-positive vacuoles were still present after treatment with the inhibitors of macropinocytosis (Bafilomycin A1, EIPA and PP2; Figure S4). Note that none of the treatments triggered mitochondrial swelling (Figure S9). This supports the hypothesis that honokiol may induce paraptosis in addition to macropinocytosis according to the other organelle markers detailed above (see Discussion section).

## *2.4. Evaluation of the Effects of Honokiol, Vacquinol-1 and MOMIPP on Intracellular Temozolomide (TMZ) Concentration*

To highlight the possibility of using these inducers of vacuolization to enhance the penetration of chemotherapeutic agents in cancer cells, we pre-treated glioma cells with the three compounds of interest before the addition of the treatment with TMZ. TMZ was chosen as the current first-line chemotherapeutic agent against newly diagnosed GBM [44,45] and for its pro-autophagic effects in glioma cells [46,47]. Honokiol was used 22 h, vacquinol-1 15 h and MOMIPP 3 h before the addition of TMZ. These timepoints were selected according to the time required to visualize the beginning of the vacuolization with each compound. The quantification of intracellular TMZ in Hs683 and U373 cells was achieved through HPLC-MS after two hours of subsequent exposure to TMZ at 200 μM with or without the macropinocytosis inducers. Figure 9 shows that all three compounds significantly increased the intracellular TMZ concentration in both Hs683 and U373 cells, except in the case of MOMIPP-pretreated Hs683 cells. Note that we pre-treated cells only for 3 h with MOMIPP, and longer incubation periods may be required in these cells.

**Figure 9.** Temozolomide (TMZ) concentrations in U373 and Hs683 cell lysates after two hours of treatment with TMZ (200 μM). Cells were pre-treated with honokiol for 22 h (HONO + TMZ), vacquinol-1 for 15 h (VAC + TMZ) or MOMIPP for 3 h (MOMIPP + TMZ). Data are expressed as fold-change TMZ concentrations compared to its own control (TMZ alone) by box plots: line, median fold change; boxes, percentiles 25–75; whiskers, non-outlier ranges of 12 replicates. TMZ concentration of the cells treated with TMZ alone was normalized to 1. Statistical comparison has been made by the Mann–Whitney test. *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*).

Despite apparent differences in their effect, certain considerations, including the concentration of the compound itself, the duration of the pretreatment required, or their ability to cross the blood brain barrier (BBB), are all crucial to be taken into account for future in vitro and in vivo combinations. Furthermore, this experiment only highlighted that the use of those compounds could help to increase the intracellular concentration of TMZ. Whether these effects actually related to macropinocytosis induction and/or other mechanisms should be evaluated in more depth. Such investigations would help decipher whether this strategy could be used with other kinds of drugs and pharmaceuticals. Other aspects and advantages of combinations (pro-methuotic or pro-paraptotic effects, additive or synergisctic effects) should also be considered, as discussed below.

#### **3. Discussion**

In this study, we showed for the first time that genes associated with macropinocytosis were deregulated in human glioma brain tumors. This conclusion is based on a systematic statistical analysis of the mRNA dataset from Henry Ford Hospital by regrouping non-tumoral human samples in comparison to gliomas samples of grades II, III and IV. Figure 2 summarizes the genes of the macropinocytosis process whose expression levels were evaluated in this study and whether they were up- or downregulated. Hereafter, we discuss these results briefly in relation to the scientific knowledge currently available regarding the main deregulated targets in glioma biology.

Consistent with existing literature, we found *EGFR* and *PDGFR* to be upregulated in gliomas [19,20,48]. These are potent inducers of macropinocytosis [18]. *H-RAS*, by contrast, was found to be downregulated, but only the mRNA level was investigated in the present study. Indeed, increased active wild-type Ras activity has been reported in high-grade glioma and may contribute to increased macropinocytosis as well [17]. Interestingly, Src plays key roles in EGF-triggered macropinocytosis associated with enhanced migration and further fusion of macropinosomes with lysosomes [16]. In this study we observed that the Src inhibitor PP2 impaired acid vacuolization induced by honokiol (see below).

We also found that several other key actors of macropinocytosis, such as Rab34, Arp2/3 complex and SWAP70, were significantly overexpressed according to the grade of the tumor (Figure 1, Table A1). These are all implicated in the organization of the actin cytoskeleton and ruffling of the plasma membrane during macropinocytosis [49–51]. Our results are consistent with previous data suggesting that overexpression of these proteins contributes to glioma cell invasion [52]. In this context, expression levels of *ARP2/3* and *RAB34* correlate with the grade of the tumor and/or the survival of the patient, with both being upregulated from low-grade to high-grade gliomas [53,54]. *RAB34* was additionally associated with poor patient survival [54]. Regarding other Rab proteins, only few studies have been published regarding their roles and/or deregulation in glial tumors. The increased expression of *RAB20* found in the present study could suggest a role in macropinocytosis in glioblastoma. Downregulation of *RAB21* by siRNA significantly inhibited cell proliferation and remarkably induced cell apoptosis [55], but its overexpression in glioblastoma was not reported, and no link to enhanced macropinocytosis was suggested in those tumors. In addition, mRNA coding for Arf6 was also found to be overexpressed in GBM samples (Figure 1d). During macropinocytosis, active Arf6 recruits the Arf nucleotide binding site opener (ARNO) that activates Arf1 via its guanine exchanger activity and allows the recruitment of the WAVE protein regulatory complex (WRC). This heteropentameric complex of WASP family proteins composed of WAVE, Cyfip1, Nap1 (*NCKAP1*), Abi1 and Brk1, in turn activates Arp2/3 to initiate actin polymerization [51,56]. Recently, a potential role for Arf1 in glioblastoma progression was suggested [48], and over-expression of Arf6 was shown to enhance glioma cell migration both in vitro and in vivo [48,57,58].

While we did not observe significant changes in *LAMP1* expression, Jensen et al. [59] found that *LAMP1* is more highly expressed in glioblastoma than in diffuse and anaplastic astrocytomas, even though its expression does not correlate with the overall survival of the patient [59,60]. *CTBP1/BARS* and its downstream target *PLD1*, whose activation triggers the closure and the final fission of macropinosomes from the plasma membrane [9,61,62], were found downregulated in high-grade tumors at the mRNA level herein. Those results are not consistent with previous data indicating positive correlation of Ctbp protein antigen expression with the histopathologic grade of the glioma [63] and worse survival of the patient [64]. The discrepancies could be related to evaluation at the mRNA versus protein expression level. Further, the downregulation of *PAK1* mRNA is not easy to integrate when considering that both protein phosphorylation and localization (cytoplasmic versus nuclear) appeared essential for its function and prognosis value in glioblastoma [65].

Nevertheless, as highlighted by Figure 2, the macropinocytosis process appeared obviously deregulated in glioma, and particularly in GBM (Figure 1). Accordingly, we showed that the mRNA expression signature of these 38 genes taken together, and covering most of the macropinosome formation, maturation and turn-over processes, enabled discriminating GBM from non-tumoral samples and lower grade glioma on the basis of unsupervised analysis (Figure 3). Macropinocytosis could, thus, participate in GBM aggressiveness, notably when considering its contribution for nutrient uptake and exosome GBM crosstalk [21,22]. Even if a systematic comparison of each target alone, according to the survival of GBM patient, did not reveal any significant changes (Table S1), long surviving GBM patients displayed a trend of grouping together on the basis of these 38 genes' signatures. A study at the protein level, and with full patient characterizations and follow-ups, should be conducted to further confirm an upregulation of macropinocytosis in GBM, and whether it might be of prognosis value for the patient.

Recent findings have suggested that stimulation of macropinocytosis in cancer cells can lead to increased chemotherapeutic efficiency [22–25,66]. Thus, we herein propose taking advantage of this possible Achilles' heel by the use of macropinocytosis-stimulating agents to combat GBM. Hyperstimulation of macropinocytosis with small molecules may ultimately induce methuotic cell death in glioma cells, as previously shown [26]. However, when used for short periods of time or at sub-lethal concentrations, methuosis-inducing compounds may be useful for increasing the intracellular concentrations of chemotherapeutic agents.

Therefore, we compared the effects of three potential compounds of interest—honokiol, vacquinol-1 and MOMIPP. We observed high morphological similarities between the vacuolization processes induced by those three compounds in terms of refringency, increase in number followed by apparent fusion, and cytoplasmic accumulation (Figure 5 and Figure S1). Although they are all inhibited by both bafilomycin A1 and EIPA, one unique inhibitor of macropinocytosis, our results indicate that the vacuoles they induce may differ in terms of origin and capacity to fuse with lysosomes. Vaquinol-1 actually appeared to stimulate macropinosome formation according to the marked increase in the uptake of Lucifer Yellow and dextrans (Figure 7 and Figure S6). The fact that it induces vacuoles of intermediate pH, as revealed by acridine orange staining (positive in both red and green fluorescences; Figure 8 and Figure S8) and the absence of increase in lysosomal content, are two features that differentiate vacquinol-1 from MOMIPP- and honokiol-induced effects. Indeed, these two latter display slight increases in lysosomal content assessed by Lyso-tracker. However, the enlarged vacuoles are not stained with that tracker. (Figure S9). Such enlarged vacuoles were not obtained with vacquinol-1 (Figure 5 and Figure S1). Important differences were also obtained with respect to uptake and acidity evaluated by acridine orange staining. In the case of honokiol, most of those large vacuoles appeared acidic as revealed by their red staining with acridine orange, while MOMIPP-induced enlarged vacuoles remained negative. We conclude that the three compounds affect macropinocytosis process and/or endosomal traffic in high-grade glioma cells, but that the mechanisms underlying those effects markedly differ from one compound to the other. In particular, the fusion of the vacuoles with lysosomal compartments seems to still occur with respect to honokiol, and partly to vacquinol-1 treatments, but not with MOMIPP. In addition, honokiol is the only compound in the current study that also induces ER-derived vacuolization (Figure 6 and Figure S8). Note that ER stress has been shown previously to inhibit endocytic pathways, including macropinocytosis [67,68]. This effect may also be linked to the ability of honokiol to induce paraptosis-like cell death, as previously reported in leukemia cells [43], and autophagy in glioblastoma [36,69]. Interestingly, vacquinol-1 was also recently shown

to trigger mitophagy in GBM cells [70]. Although both macropinocytosis and autophagy represent two "opposite" major mechanisms to provide nutrient supply and recycling from extracellular versus intracellular sources respectively, they seem to share upstream and downstream regulation whose links remain to be fully deciphered [71]. In contrast, methuosis results from the accumulation of unprocessed macropinosomes that fuse together, rather than fusing with lysosomes or recycling to the plasma membrane [27], as described previously with respect to the MOMIPP chalcone.

Obviously, further studies are required to clarify if and how each of these compounds affect membrane ruffling, macropinosome formation, trafficking, fusion, and recycling independently to other effects such as ER swelling or autophagy.

Nevertheless, we observed that all three compounds were able to significantly increase intracellular TMZ concentration, even if TMZ uptake was excellent [47]. This study provides a proof of concept, and it encourages further investigations with drugs whose intracellular and/or BBB penetrations are more problematic. This could be the case with respect to antibody-based therapies, including depatuxizumab mafodotin, a new antibody-drug conjugate with promising clinical results notably in EGFR-amplified GBM cases [72,73]. Typically, patients with EGFR amplification may benefit from macropinocytosis-interfering drugs, such as those studied herein.

Honokiol and MOMIPP have been proven to cross the BBB [35,74,75]. Although vacquinol-1 also displays an adequate BBB penetration, its systemic toxicity requires a lowering dose or local delivery [76]. At a tolerable dose, vacquinol-1 alone allowed a reduction in tumor size, but it did not increase the overall survival of the GBM preclinical model [76]. Thus, toxicological issues have to be taken into account for future development. Honokiol is widely used and freely available as a phytotherapeutic complement in several countries for medicinal properties against anxiety, for facilitation of sleep, support of cognitive functions, and antioxidant effects. Further, possible additional and/or synergistic effects between macropinocytosis inducers and chemotherapeutic agents may be expected. Accordingly, honokiol has already been shown to increase TMZ cytotoxic effects in vitro [77].  Recent studies pointed JNK Kinase as an important signal for both honokiol-induced effects on stem cells [78] and cytotoxic effects of indoylchalcones such as MOMIPP [75].

Numerous perspectives remain to be addressed, including the following: (1) evaluation of whether macropinocytosis deregulation may be linked to patient survival and response to chemotherapy, (2) deciphering how each compound affects macropinocytosis and other endocytosis processes, and (3) testing the efficiency of combined treatments for the proposed molecules investigated herein with chemotherapeutic agents, with a particular attention to new therapeutic drugs characterized by limited intracerebral brain pharmacokinetics.

#### **4. Materials and Methods**

#### *4.1. Cell Lines and Compounds*

The Hs683 human oligodendroglioma cell line was obtained from the American Type Culture Collection (ATCC, code HTB-138) and the human glioblastoma U373 cell line from the European Collection of Cell Culture (ECACC, code 08061901). Cells were cultivated in RPMI 1640 (Gibco, Thermofisher, Dilbeek, Belgium) culture medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 0.6 mg/mL L-glutamine (Gibco), 200 IU/mL penicillin–streptomycin (Gibco), and 0.1 mg/mL gentamicin (Gibco) at 37 ◦C with 5% CO2. Cultures were checked twice a month for mycoplasma.

Honokiol was purchased from Sigma-Aldrich (St. Louis, MO, USA) as well as EIPA and PP2, Vaquinol-1 and EHT1864 from Selleckchem (Houston, TX, USA), Bafilomycin A1 from Cayman chemicals (Selleckchem), and filipin complex from AG Scientific (San Diego, CA, USA). MOMIPP was synthesized and characterized as previously described [29].

## *4.2. MTT Colorimetric Assay*

Cell viability was determined using a colorimetric assay as described previously [79]. Cells were seeded in 96 well plates (Sarstedt AG & CO, Nümbrecht, Germany) and were grown for 24 h. They were then treated with honokiol, vacquinol-1, or MOMIPP at concentrations ranging from 0.01 to 100 μM or left untreated for 72 h. The viability was estimated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) mitochondrial reduction into formazan measured at 570 nm with a spectrophotometer (680XR, Bio-Rad Laboratories, Berkeley, CA, USA; reference wavelength 610 nm). The experiment was realized two times in sextuplicate for each cell line and each compound.

#### *4.3. Characterization of Vacuoles*

#### 4.3.1. Phase Contrast Microscopy for Morphological Observations

In order to observe the morphological changes induced by treatments, pictures of living cells were taken with the Imager M2 fluorescence microscope (Carl Zeiss, Zaventem, Belgium) coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss). Cells were seeded on glass coverslips in six-well plates (Sarstedt AG & CO) and allowed to attach and start growing for 24 h. Afterwards, cells were either left untreated or treated with each compound as follows: 35 μM and 40 μM of honokiol for U373 and Hs683 cell lines, respectively; and 5 μM of vacquinol-1 and 3 μM of MOMIPP for both cell lines.

For the characterization of the vacuolization by using inhibitors, cells were either pre-treated 1 h with bafilomycin A1 (100 nM), filipin (1 μg/mL), or co-treated with EHT1864 (25 μM), EIPA (10, 30 or 75 μM) or PP2 (25 μM). At different time points, coverslips were washed twice in PBS, transferred onto microscope slides, and four pictures were taken per slide. Each experimental condition was tested twice in triplicate.

#### 4.3.2. Fluorescent Microscopy Assays

Fluorescent probes that stained different cellular compartments were used to characterize the origin of the vacuoles. The dapoxyl ER-tracker blue-white dye, the Lyso-tracker red dye, the Mito-tracker green dye, and 10 kDa and 70 kDa Texas-Red labeled dextran were all obtained from Molecular Probes (Life Technologies, Merelbeke, Belgium). We also used Lucifer yellow (Lucifer Yellow CH, lithium salt) from Biotium (Fremont, CA, USA) and acridine orange from Sigma-Aldrich. Briefly, the cell seeding and treatment procedures were similar to the ones used for the phase contrast microscopy (Section 4.3.1). The dye solutions were simply added to the culture medium 1 h before the end of the treatment periods, excepted for the Lucifer yellow and both dextrans 10 kDa and 70 kDa, which were added for the whole duration of the treatment. The concentrations of the dyes were as follows: ER tracker, 0.5 μM; Lyso tracker, 75 nM; Mito tracker, 200 μM; Lucifer yellow, 100 μg/mL; acridine orange, 1 μg/mL; and dextrans 10 kDa and 70 kDa, 125 μg/mL; At the end of the treatment period, the procedure was similar to that of phase-contrast microscopy to take pictures of living cells with the Imager M2 fluorescence microscope (Carl Zeiss) coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss). The experiment was realized at least two times in duplicate.

### 4.3.3. Flow Cytometry

In order to quantify the internalization of Texas-red labeled dextran 10 kDa (Molecular Probes), cells were treated with honokiol (35 μM for U373 and 40 μM for Hs683 cells), MOMIPP (3 μM), or vacquinol-1 (5 μM), or left untreated in the presence of Texas-red labeled dextran 10 kDa (125 μg/mL). After 24 h for U373 and 30 h for Hs683, cells were washed twice with PBS, detached with trypsin-EDTA (Gibco) and centrifuged. The supernatant was removed and cells were resuspended in 250 μL of PBS for flow cytometry analysis with the Beckmann Gallios apparatus (Beckmann Coulter, Analis, Suarlee, Belgium). Each sample recorded 10,000 events, and the experiment was conducted once in sextuplicate.

## *4.4. Quantification of Intracellular TMZ*

## 4.4.1. Sample Preparation

For the experiment, cells were seeded in T75 flasks (Sarstedt AG & CO). When the confluence of the flasks was around 75%, cells were pre-treated with honokiol (35 μM for U373 and 40 μM for Hs683), MOMIPP (3 μM), vacquinol-1 (5 μM), or left untreated for different periods of time (3, 15 and 22 h) before the addition of TMZ (200 μM) for 2 h. After the treatment, the culture medium was removed, the cells were washed twice with cold PBS (Gibco)), scrapped in 200 μL of ice-cold methanol (VWR International, Oud-Heverlee, Belgium), and sonicated for 30 s. As TMZ is stable at pH < 4 [80], 50 μL of cell lysate was diluted in 50 μL of an acid internal standard solution (2 μM theophyline and 0.1% formic acid in methanol).

### 4.4.2. LC-MS Process, Data Acquisition and Analysis

Ten microliters from the prepared sample were injected into the liquid chromatography (LC) system. Analyses were performed with a rapid resolution LC (RRLC) 1200 series from Agilent Technologies (Santa Clara, CA, USA). Separation was performed on a Zorbax Eclipse XDB-C18 Rapid Resolution HT column (4.6 × 50 mm, 1.8 μm particle size) from Agilent Technologies, preceded by a Zorbax Eclipse XDB-C18 pre-column (4.6 × 5 mm, 1.8 μm particle size) using water supplemented with a 0.1% formic acid/acetonitrile gradient. A 6520 series electrospray ion source (ESI)–quadrupole time-of-flight (QTOF) from Agilent Technologies was used for the MS analyses. Initial ESI–Q-TOF parameters were as follows: positive mode; capillary voltage of 4500 V; dynamic high resolution acquisition mode (2 Hz); gas temperature of 350 ◦C; drying gas flow of 9 L/min; nebulizer pressure of 50 psig; fragmentor voltage of 130; and skimmer voltage of 65 V. Data was acquired using the Mass Hunter Acquisition software (Agilent Technologies, version B.04 SP3) and analyzed by Mass Hunter Quantitative Analysis software (Agilent Technologies, version B.07 SP1). TMZ was monitored at an m/z value of 195.0625 and theophyline at an m/z value of 181.0720. A quantitative curve was performed over the range 0.1 to 10 μM of TMZ.

#### *4.5. Statistical Analyses*

The mRNA expression analyses were conducted on two datasets of human samples publicly available on the NCBI GEO repository. The GSE4290 dataset, from the Henry Ford Hospital, was published in 2006 [81]. The GSE53733 dataset regroups the mRNA expression data sets of 70 human glioblastoma (grade IV) primary tumors from the German Glioma Network and was published in 2014 [73]. Both expression data were generated using the same microarray platform (Affymetrix Human Genome U133 Plus 2.0 Array).

The microarray analysis was based on the robust multi-array average (RMA) expression values, which were obtained with affy package v1.56 of Bioconductor/R packages (http://www. bioconductor.org). The heatmaps were made using the pheatmap R package v1.0.10. The samples were grouped by annotation or clustered using hierarchical clustering using the average linkage on the Euclidian distance.

In both databases, we collected the data corresponding to the probes of 38 target genes involved in macropinocytosis (see list in Table A1). Comparison of the expression level for each probe was conducted using the Kruskal–Wallis test (a nonparametric, one-way analysis of variance), followed by two-tailed tests using Statistica (Statsoft, Tulsa, OK, USA).

For in vitro biological assay comparisons (flow cytometry and TMZ quantification), we conducted non-parametric Mann–Whitney tests using Statistica.

#### **5. Conclusions**

In conclusion, we found that more than 60% of the 38 macropinocytosis-related genes studied herein are overexpressed or down-regulated in GBM patient samples. These 38 genes may constitute a signature discriminating GBM from non-tumoral samples and lower grade gliomas. Those results suggest that macropinocytosis may play important roles in GBM aggressiveness.

We then proposed to make use of compounds that interfere with this endocytotic process to increase anti-cancer drug uptake. The three compounds selected for this purpose, i.e., honokiol, vacquinol-1 and MOMIPP indeed allowed significant increase in intracellular TMZ concentration in vitro. This study provides with a first proof of concept and paves the way to use macropinocytosis deregulators in combination with chemotherapeutic agents. Considering that EGFR is an activator of macropinocytosis, patients with amplified or mutated EGFR may represent better candidate for this approach.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/11/3/411/s1, Table S1: Statistical comparison of mRNA expression data of macropinocytosis gene probes in the GSE53733 dataset, Figure S1: Morphological illustration of the vacuoles formed after treatment of Hs683 cell line with honokiol (40 μM), MOMIPP (3 μM) or vacquinol-1 (5 μM) over time, Figure S2: MTT curves of the three products used in this study, Figure S3: Effects of various inhibitors on vacuoles formation following honokiol, vacquinol-1 and MOMIPP treatment in Hs683 cells, Figure S4: Illustration of the ER-Tracker staining of U373 (a) and Hs683 (b) in presence of honokiol with or without the various vacuolization inhibitors, Figure S5: Effects of filipin on vacuoles formation following honokiol, vacquinol-1 and MOMIPP treatment in U373 and Hs683 cells, Figure S6: Illustration of lucifer yellow (a) and fluorescent 10 kDa dextran (b) uptake in Hs683 cells treated with honokiol, MOMIPP or vacquinol-1, Figure S7: Illustration of fluorescent 70 kDa dextran internalization in U373 (a) and Hs683 (b) cells treated with honokiol, MOMIPP, vacquinol-1, Figure S8: Illustrations of the fluorescent characterization of the vacuoles using different fluorescent cell compartment trackers in Hs683 cells, Figure S9: Illustration of the fluorescent marking of the vacuoles using mitochondrial and lysosomal cell compartment trackers in U373 (a) and Hs683 (b) cells.

**Author Contributions:** Conceptualization, V.M.; Cellular experiments, M.C., G.R. and A.-S.L.; mRNA dataset management and analysis: R.J.; Analytical HPLC-MS methodology and results analyses, C.D. and P.V.A.; Materials supply, W.A.M. (MOMIPP); Data interpretation, M.C., V.M. and W.A.M.; Writing—Original Draft Preparation, M.C. and V.M..; Critical Review & Editing, W.A.M.

**Funding:** This research was supported by the Belgian Brain Tumor Support (BBTS) and the United States National Institutes of Health (R01 CA 115495).

**Conflicts of Interest:** MOMIPP is included in a patent licensed to Systems Oncology, Inc. As a co-inventor, W.A.M. receives a portion of licensing fees and royalties. The other authors declare no conflict of interest.

## **Table A1.** *Cont.*

**Table A1.** *Cont.*

oncogene family

## **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article*

## **Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma**

**Monserrat Llaguno-Munive 1,2, Mario Romero-Piña 1, Janeth Serrano-Bello 3, Luis A. Medina 4 , Norma Uribe-Uribe 5, Ana Maria Salazar 6, Mauricio Rodríguez-Dorantes 7 and Patricia Garcia-Lopez 1,\***

1 Laboratorio de Farmacología, Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Ciudad de México 14080, Mexico<EMAIL_ADDRESS>(M.L.-M<EMAIL_ADDRESS>(M.R.-P.)

Received: 10 November 2018; Accepted: 20 December 2018; Published: 22 December 2018

**Abstract:** The standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemo/radiotherapy. A major limitation on patient improvement is the high resistance of tumors to drug treatment, likely responsible for their subsequent recurrence and rapid progression. Therefore, alternatives to the standard therapy are necessary. The aim of the present study was to evaluate whether mifepristone, an antihormonal agent, has a synergistic effect with temozolomide (used in standard therapy for gliomas). Whereas the mechanism of temozolomide involves damage to tumor DNA leading to apoptosis, tumor resistance is associated with DNA damage repair through the O6-methylguanine-DNA-methyltransferase (MGMT) enzyme. Temozolomide/mifepristone treatment, herein examined in Wistar rats after orthotopically implanting C6 glioma cells, markedly reduced proliferation. This was evidenced by a decreased level of the following parameters: a proliferation marker (Ki-67), a tumor growth marker (18F-fluorothymidine uptake, determined by PET/CT images), and the MGMT enzyme. Increased apoptosis was detected by the relative expression of related proteins, (e.g. Bcl-2 (B-cell lymphoma 2), Bax (bcl-2-like protein 4) and caspase-3). Thus, greater apoptosis of tumor cells caused by their diminished capacity to repair DNA probably contributed significantly to the enhanced activity of temozolomide. The results suggest that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide during chemotherapy for GBM.

**Keywords:** glioblastoma; temozolomide; mifepristone; MGMT; drug resistance; apoptosis

## **1.  Agents for GBM therapy encounter obstacles nonexistent in the treatment of non-neurological cancers. For instance, the blood-brain barrier restricts the passage of many chemotherapeutic agents due to certain well-known characteristics, such as the presence of tight junctions between cells and the lack of fenestrae [2,3].

The current treatment for GBM consists of surgery followed by radiotherapy and chemotherapy with temozolomide. Unfortunately, the great tumor resistance to this drug limits its effectiveness and often leads to recurrence and rapid tumor progression [4]. Subsequent to the oral administration of temozolomide, its spontaneous breakdown at physiological pH produces monomethyltriazene 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC), which in turn reacts with water to liberate 5-aminoimidazole-4-carboxamide (AIC) and the highly reactive methyldiazonium cation. The latter methylates DNA purine residues, preferentially O-6 guanine (O6-MeG, 6%) in guanine-rich regions [5], but also N-7 guanine (N7-MeG, 70%) and N-3 adenine (N3-MeA, 9%). Alkylation of the O6 site on guanine promotes the insertion of a thymine instead of a cytosine during DNA replication, which can result in cell death [5].

Tumor cells often show chemoresistance to temozolomide, continuing to proliferate after treatment. Drug resistance is due in part to the overexpression of MGMT (O6-methylguanine-DNA-methyltransferase), an enzyme involved in the repair of temozolomide-induced DNA damage. There is an inverse relationship between the levels of MGMT expression and the cellular response to temozolomide [6]. Hence, MGMT expression is a relevant prognostic factor for the response to temozolomide therapy, with an elevated expression linked to drug resistance [7].

Clinical and experimental data have established the resistance of GBM to apoptosis. Defects in apoptotic mechanisms foster tumorigenesis and contribute to the resistance to temozolomide, since its cytotoxic activity is exerted in part through the triggering of apoptosis [8]. In recent years, new molecules have been developed to attempt to overcome the problem of tumor resistance to temozolomide, but with little impact on the prognosis and survival of GBM patients [9–11]. One strategy for improving GBM treatment is to seek new chemo-sensitizing agents that enhance the effectiveness of temozolomide by increasing the apoptosis of tumor cells through a decrease in the activity of the DNA repair enzyme.

One possibility is mifepristone, which blocks the capacity of progesterone to stimulate the growth, migration and invasion of human astrocytoma cells lines (such as U373, U87 and D54) [12,13]. This synthetic steroid is used as an abortifacient drug because of its anti-progestational and anti-glucocorticoid action. Our group previously demonstrated an important role of mifepristone in chemo-radio-sensitization, describing its synergistic effect with cisplatin and radiotherapy to act against proliferation of cervical cancer cell lines and cervix xenografts [14]. We also studied the addition of mifepristone to temozolomide-based chemotherapy (accompanied by radiation), observing improved antiproliferative activity on a glioma xenograft in the flank of nude mice. The tumor growth rate was slower than that found with radiation alone or temozolomide alone, indicating a chemo- and radio-sensitizing effect [15].

However, the flank tumor model has drawbacks, especially the absence of the environment provided by the normal brain parenchyma and blood brain barrier. Since these factors are known to influence drug delivery to the tumor [16], orthotopic models are preferred. The aim of the present study was to evaluate, with an immunocompetent orthotopic model based on the intracranial implantation of a glioma cell line in rats, whether mifepristone is able to modulate the growth of temozolomide-treated tumors. The possible mechanism of action of mifepristone as a chemo-sensitizing agent was explored by determining the levels of MGMT (a DNA repair enzyme) as well as the expression of proteins as Bcl-2 (B-cell lymphoma 2), Bax (bcl-2-like protein 4) and caspase that participate in the apoptotic pathway.

## **2. Results**

## *2.1. Tumor Growth Assessed by Molecular Imaging*

Rats were subjected to four treatments: the vehicle only, temozolomide only, mifepristone only, and mifepristone/temozolomide. PET/CT scans were performed at baseline (day 0 of drug treatment, 2 weeks post-implantation) and at the end of the experiment (day 21). In the resulting images (Figure 1a), the presence of red reflects the uptake of 18F-fluorothymidine (18F-FLT), a tumor cell growth marker. It is measured as total lesion proliferation (TLP).

**Figure 1.** Antitumor activity in the orthotopic rat model of a glioma. (**a**) PET/CT images showing 18F-FLT (18F-fluorothymidine) tumor uptake for the distinct groups. The images on the left represent the beginning of drug treatment (baseline, two weeks after tumor cell implantation, considered as day 0) and those on the right depict the end of the third week of treatment (day 21). Red reflects the uptake of (18F-FLT). (**b**) The proliferative activity of tumors, measured as total lesion proliferation (TLP). Data are expressed as the mean ± SEM of three animals. \* A significant difference (*p* < 0.05) between the Mifeprostone/Temozolomide (Mif/Tz) group and the other groups with implantation of glioma cells (the vehicle only, Tz only and Mif only).

After 21 days of treatment, there was an increase in TLP and consequently in the tumor size for the control, temozolomide and mifepristone groups, and a decrease in this parameter for the mifepristone/temozolomide group (Figure 1b). At day 21, both the increase for the vehicle only (control) animals and the decrease for the mifepristone/temozolomide group were significant compared to baseline. Moreover, a significantly lower rate of proliferation was found for the combination treatment than for each of the other three groups (the control, temozolomide only and mifepristone only).

#### *2.2. Determination of Body Weight and Overall Survival*

Tumor growth was also assessed by means of weight loss (Figure 2a). All animals continued to gain weight during the first two weeks post-implantation of tumor cells. Subsequently, the rats treated with the vehicle or temozolomide only rapidly lost weight, evidencing accelerated tumor proliferation.

**Figure 2.** The effect of tumor growth in the orthotopic rat model of a glioma, evaluated by weight loss and overall survival. (**a**) Relative weight of rats in the five groups: sham surgery (without implanting glioma cells) ( ), and the implantation of glioma cells followed by each of the treatments: vehicle only (ȣ), temozolomide only (ȣ), mifepristone only (ȣ), and the mifepristone/temozolomide combination (ȣ). Each point represents the mean ± SEM of six animals. \* A significant difference (*p* < 0.05) between Mif/Tz and the other groups (Sham, Control, Tz only and Mif only) on day 24. # A significant difference (*p* < 0.05) between Mif/Tz and Mif only on day 31. † A significant difference (*p* < 0.05) between the Mif/Tz and the Sham group on day 31. (**b**) Survival analysis of the same groups for up to 50 days after implantation (day 0 = day of surgery).

The rats in the group given mifepristone only displayed a less drastic weight loss. All animals in these three groups survived between 17 and 32 days (Figure 2b). In contrast, the rats receiving mifepristone/temozolomide maintained their weight throughout the study (Figure 2a), and those having no tumor cell implantation (sham operation) gained weight. The latter two groups were still alive when all the rats of the other three groups had died, and most survived to the end of the study (50 days post-implantation) (Figure 2b).

#### *2.3. Histological and Immunohistochemical Analysis*

At the end of the treatments, the brain tissue was processed for histological examination, applying hematoxylin and eosin (H&E) stain to the tissue sections (Figure 3a–e). The tissue of the animals treated with the vehicle only, temozolomide only or mifepristone only showed hypercellularity, mitosis, pleomorphism and necrosis (Figure 3b–d). These characteristics were exhibited to a lesser

extent in the rats administered mifepristone/temozolomide (Figure 3e), but with a hypo- versus hypercellular lesion.

**Figure 3.** Haematoxylin and eosin (H&E) staining and immunohistochemical analysis of glioma tissue. Sections of tumor tissue were stained with haematoxylin and eosin (**a**–**e**): Mitosis (black arrows), nuclear pleomorphism (red arrows) and necrosis (yellow arrows). Immunostaining of Ki-67 (**f**–**j**), showing few cell nuclei positive to this protein in the tumors of the Mif/Tz group compared to those of the individual treatments (Tz or Mif). The images are representative of three animals per treatment. Magnification 40×.

Immunohistochemical staining of brain tumor tissue was employed to detect the presence of Ki-67 (Figure 3f–j), a protein expressed by proliferating cells. Compared to the groups receiving the vehicle only, temozolomide alone or mifepristone alone, rats given mifepristone/temozolomide displayed a lower proportion of Ki-67 positive cells in tumors. Indeed, the stained sections from the latter group were similar to those from the sham animals, indicating few cells undergoing proliferation. For animals administered mifepristone only, there was slightly diminished proliferation compared to those in the control and temozolomide only groups. In the mifepristone/temozolomide group, a close correlation can be observed between a lower level of Ki-67 and the significant reduction in tumor growth illustrated in the PET/CT images.

## *2.4. Expression of Apoptotic Proteins*

The combination treatment also affected the expression of apoptotic proteins in tumor cells (Figure 4). Compared to the control group, animals with temozolomide only or mifepristone only displayed a slight increase in the Bax/Bcl-2 ratio, but no change in the expression of cleaved caspase 3. In animals given mifepristone/temozolomide, contrarily, a significant increase was found in the Bax/Bcl-2 ratio and the expression of cleaved caspase 3, revealing a synergy between these two drugs.

**Figure 4.** Expression of apoptotic proteins. Representative western blot results and densitometric analysis of Bcl-2, Bax and Cl-Caspase 3. Data are expressed as the mean ± SEM from three independent experiments. \* Indicates a significant difference (*p* < 0.05) between Mif/Tz and the other groups with tumor cell implantation (given Tz, Mif or the vehicle).

## *2.5. Expression of MGMT*

MGMT, an enzyme involved in DNA repair, plays a significant role in the resistance of glioma tumors to temozolomide. Western blot data and band intensity showed the protein expression of MGMT to be strikingly downregulated in the mifepristone/temozolomide group and slightly decreased in the mifepristone only group compared to the animals administered temozolomide only (Figure 5).

**Figure 5.** Expression of MGMT (O6-methylguanine-DNA-methyltransferase). Representative western blot results and densitometric analysis of the MGMT protein. Data are expressed as the mean ± SEM of three independent experiments. \* A significant difference (*p* < 0.05) between Mif/Tz and the other groups with tumor cell implantation (given Tz, Mif or the vehicle).

#### **3. Discussion**

One of the major problems in the treatment of GBM is the drug resistance of tumors and the repopulation carried out by cells that escape chemotherapy. The standard treatment for this disease, consisting of surgery followed by radiation therapy plus chemotherapy with temozolomide, yields a median survival of only 1–2 years. Therefore, it is essential to find new strategies for treating GBM in order to lengthen patient survival and avoid recurrence.

The current study evaluated whether the combination of mifepristone and temozolomide could give a better outcome than temozolomide only applied to glioma tumors in rats. Mifepristone is known to act as an antagonist of progestins, glucocorticoids and androgens by blocking progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR), respectively [17,18]. However, the role of this antihormonal agent as a chemo-sensitizer in GBM has scarcely been explored. Although we previously suggested the potential of mifepristone as a chemo-radio-sensitizer for the standard treatment of a glioma tumor based on a subcutaneous xenograft model of GBM [15], the present intracerebral model more closely resembles the native niche of such a tumor in the central nervous system.

A significant difference in tumor growth was observed between the animals administered mifepristone/temozolomide and those given the vehicle only, temozolomide only or mifepristone only. Compared to the latter three groups, the animals submitted to the combination treatment exhibited significantly reduced tumor growth and greater survival time.

The level of Ki-67 was herein scrutinized due to its expression by proliferating cells in all phases of the active cell cycle and its absence in resting cells (G0). Ki-67 was abundant in the tumor cells of the groups given temozolomide only, mifepristone only or the vehicle only, but was scarcely detected in tumor cells of the animals receiving mifepristone/temozolomide. Hence, the mifepristone/ temozolomide combination blocked the proliferation of glioma cells.

The PET/CT scans, which measured 18F-FLT uptakes, were in agreement with the Ki-67 results, revealing a lack of tumor cell growth with the combined treatment. 18F-FLT displays minimal uptake in normal brain tissue because the expression level of thymidine kinase-1 is very limited in neurons and glia. Thus, 18F-FLT/PET may be advantageous for examining tumor recurrence [19,20].

Mifepristone is known to diminish the proliferation of cancer cells of reproductive and non-reproductive origin [21] and to inhibit the in vitro growth of cancer cells derived from the nervous system, breast, prostate, and ovary. It also gives rise to G1-S blockage of the cell cycle through inhibition of cdk2 activity in human ovarian cancer cells [22].

Whereas progesterone is known to generate the proliferation of tumor cells, the progesterone/ mifepristone combination has proven to block the proliferative effect. This combination also significantly decreases the tumor area compared to progesterone only administered to rats [23]. Experimental studies have indicated that progesterone is capable of stimulating the infiltration and migration of astrocytes to the rat cortex, exerting its effects through two mechanisms. The first implies an interaction with nuclear PR (nPR), while the second requires the participation of membrane receptors (mPR) [24]. PR expression exists in several types of brain tumors, including meningiomas and gliomas, and increases with histological malignancy [25]. Moreover, mPRs are expressed in GBM cell lines such as U251 and U87 [26]. Upon evaluating the expression of PRs in C6 glioma cells, Su et al. observed no expression of classical nuclear PRs but identified significant levels of mPRs [27].

Although mifepristone plus temozolomide herein diminished tumor growth, its mechanism of action as a chemo-sensitizing agent is unknown. The promotion of apoptosis by mifepristone has been documented in distinct tumor types. Li et al. demonstrated that this drug produces an antiproliferative effect on human SGC-79901 gastric adenocarcinoma cells by downregulating Bcl-XL expression and upregulating caspase 3 activity [28]. Moreover, mifepristone inhibits cell growth by arresting cell cycle progression at the S phase, triggers apoptosis by activating caspase-3, and modulates the genes involved in apoptosis (including BCL2/BAX and FAS/FASLG) in Ishikawa cells [29]. According to Gonzalez-Agüero et al., progesterone significantly increases the growth of U373 and D54 human astrocytoma cells, an outcome blocked by mifepristone in both cases. Additionally, mifepristone administered without progesterone reduced the growth of these two cell lines [12], evidencing a possible antiproliferative effect on gliomas.

Mifepristone/temozolomide treatment presently exerted a strong downregulation of the expression of the antiapoptotic protein Bcl-2 and an upregulation of the expression of two apoptotic proteins (Bax and cl-caspase-3) (Figure 4). This suggests that mifepristone/temozolomide induces apoptosis in glioblastoma tumors in vivo, which could be relevant for improving the effectiveness of standard therapy for GBM.

On the other hand, the epigenetic silencing of MGMT was shown to enhance the response of GBM patients to chemotherapy based on alkylating agents, leading to longer mean survival. The median survival of patients treated with temozolomide is greater when the promoter of MGMT is hypermethylated [30,31]. Whereas most patients are resistant to temozolomide, epigenetic inactivation of MGMT, occurring in approximately 40% of patients, is associated with a better response. [32]. Therefore, the depletion of MGMT is important for increasing tumor sensitivity to temozolomide.

It is still unknown whether mifepristone epigenetically inhibits MGMT. However, some previously described mechanisms could partially account for the current results. Different nuclear transcription factors, including SP1 (protein 1-binding), AP-1 (activator protein), NF-κB (nuclear factor for the polypeptide gene enhancer in B cells) and HIF-1α (hypoxia inducible factor-1α), can also activate the transcription of the MGMT gene [33]. Some of the aforementioned transcription factors may participate in MGMT gene regulation by mifepristone. It has recently been documented that MGMT expression is associated with SP1 expression in glioma cell lines. Since knockdown of SP1 strongly reduced MGMT protein expression, SP1 is one of the main factors regulating MGMT [34]. MGMT expression also depends on p53, a transcription factor that sequesters SP1 and prevents its binding to MGMT [34]. Additionally, there is evidence of mifepristone increasing apoptosis due to p53 activation in several cancer cell lines [35,36]. Hence, the present findings could be partially explained by some of these mechanisms. Further studies are needed to clearly define the mechanisms responsible for the inhibition of MGMT by mifepristone.

Glucocorticoids are frequently used in glioblastoma therapy to address edema. According to some authors, they are involved in eliciting the expression of the MGMT gene, meaning that they could contribute to an elevated MGMT protein level. Biswas et al. and Ueda et al. detected an upregulation of MGMT in glioblastoma cell lines during glucocorticoid treatment [37,38]. Furthermore, Horiguchi et al. found that dexamethasone acts as a positive regulator of hepatic MGMT expression, an effect reversed by concomitant administration of its antagonist mifepristone [39]. The present results are the first, to our knowledge, in relation to the influence of mifepristone on MGMT expression when administered alone. The current mifepristone only treatment was able to decrease the levels of MGMT compared to the group given the vehicle only and temozolomide only. This could account for the enhancement of sensitivity of tumor cells to temozolomide, observed in the group receiving the mifepristone/temozolomide combination (Figure 5).

A correlation existed between a higher MGMT expression and greater resistance of the tumor to treatment (the latter reflected in the lower survival of the animals). Whereas the rats administered temozolomide only did not survive more than 24 days, those with the mifepristone/temozolomide combination showed a remarkable increase in mean survival. About 75% of the animals receiving the latter treatment survived to the end of the experiment (50 days).

A possible mechanism of mifepristone for improving the efficacy of temozolomide is proposed (Figure 6). Drug resistance to temozolomide is associated with DNA damage repair, which impedes apoptosis. Among the numerous mechanisms utilized by glioma cells to resist temozolomide-induced DNA damage, the avoidance of apoptosis is probably one of the most frequently studied. It has been reported that glioma tumors have elevated levels of the anti-apoptotic proteins Bcl-2 and reduced levels of the apoptotic protein Bax, giving rise to a predisposed anti-apoptotic state correlated with resistance to chemotherapy.

**Figure 6.** Schematic portrayal of the possible mechanisms of treatment with mifepristone/ temozolomide. This combination decreased the level of anti-apoptotic protein Bcl-2 and increased the levels of pro-apoptotic proteins Bax and cl-caspase-3, leading to greater cellular apoptosis. As described elsewhere, temozolomide releases a compound, O6-guanine, that causes DNA damage and cell death. However, there are different mechanisms of repair or avoidance employed by tumor cells. For example, resistance to temozolomide treatment is reportedly related to the presence of the MGMT enzyme, which removes the methyl group from O6-guanine and thus restores the cellular replication of tumor cells. A lower expression of MGMT was herein detected when administering mifepristone/temozolomide versus temozolomide alone. The combination treatment perhaps allows methylation to occur in the purine bases of DNA at the O6 position of guanine without removing the methyl groups, and consequently contributes to an increase in the effect of temozolomide.

Consequently, the inhibition of Bcl-2 through mifepristone treatment, fostering higher levels of Bax and Cl-Caspase, may be an effective strategy for overcoming the resistance of tumors to apoptosis when administering temozolomide. Although new small molecules have been investigated for the inhibition of the Bcl-2 family of proteins in numerous cancer types, their application to GBM has been scarcely explored.

Temozolomide generates numerous DNA adducts (e.g., O6-methylguanine) that are regarded as very cytotoxic. Contrarily, MGMT removes alkylating adducts from the O6 position of guanine and thus protects glioma cells from this cytotoxicity. A decrease in the levels of MGMT by mifepristone would impede the repair of temozolomide-induced damage, leading to an eventual accumulation of adducts, a reduction of replication and the triggering of cell death.

Mifepristone has been widely studied as an abortifacient and contraceptive drug. According to recent evidence and the current findings, this drug seems to have great potential for cancer treatment. It offers palliative benefits for a wide variety of human cancer types, including patients with glioblastoma (demonstrating that it successfully crosses the blood-brain barrier) [40,41]. Mifepristone has only a mild adverse effect, even when taken daily for up to 13 years [42]. Therefore, long-term administration of mifepristone may be feasible and clinically well-tolerated in combination with temozolomide to treat patients suffering from a glioma. We propose a Phase I clinical trial to test this combination in the near future.

## **4. Materials and Methods**

### *4.1. Drugs and Reagents*

Mifepristone and temozolomide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), ethylenediaminetetracetic acid (EDTA) and SDS were purchased from Gibco, BRL (Grand Island, NY, USA). High-quality water for the solutions was processed with a Milli-Q Reagent Water System (Continental Water Systems; El Paso, TX, USA). Temozolomide was prepared in DMSO at a final concentration of 4% and mifepristone was reconstituted in polyethylenglycol and saline solution. All standard solutions were stored at −20 ◦C until use.

#### *4.2. Animals*

Male Wistar rats, (200–230 g) were supplied by the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran (INCMNSZ), Mexico City, Mexico. All procedures for the care and handling of the animals were approved by the Ethics Committee of the "Instituto Nacional de Cancerología" (INCan, Mexico City, Mexico), and were in accordance with Mexican Federal Regulations for Animal Experimentation and Care (NOM-062-ZOO-1999, Ministry of Agriculture, Mexico).

#### *4.3. Cell Culture*

The glioma C6 cell line was acquired from American Type Culture Collection (Rockville, MD, USA). It was routinely maintained as a monolayer in DMEM, supplemented with 5% fetal bovine serum and incubated at 37 ◦C in 5% CO2 atmosphere at high humidity. Cells were harvested with 1mM EDTA.

### *4.4. Tumor Cell Implantation*

Posterior to anaesthetization with a combination of tiletamine hydrochloride (10 mg/kg) and acepromazine maleate (0.4 mg/kg) administered s.c., each animal was placed in a stereotactic device for surgery. After fastening the head in the frame, a midline incision was made and bregma was identified. The skull was then drilled at the coordinates of 2.0 mm right from bregma and 6 mm deep (hippocampus), in accordance with the Paxinos and Watson atlas [43].

C6 cells were harvested, washed three times and diluted in DMEM to a concentration of 7.5 × 105 in a volume of 3 μL. Employing an infusion pump and a 27-gauge needle, these cells were slowly implanted at a depth of 6 mm from the dura mater. The injection was made over a 6-min period. Upon closing the scalp, the rat was returned to the animal facility. The rats of the sham group were submitted only to the surgical procedure, without the implantation of tumor cells. Animals were subsequently weighed 3 times/week during the study.

## *4.5. Treatments*

At two weeks post-implantation of tumor cells, the animals were divided into four groups (*n* = 6–8): (A) temozolomide only (5 mg/kg/day i.p), (B) mifepristone only (10 mg/kg/day s.c.), (C) mifepristone plus temozolomide (the same doses), and (D) control animals (vehicle only). A fifth group consisted of sham animals (inoculated only with DMEM, in the absence of glioma cells). The drug treatments were administered in three cycles of five consecutive days over a period of three weeks. The antitumor activity of temozolomide is schedule-dependent, with multiple administrations being more effective than a single treatment. In clinical practice, the recommended dose of temozolomide is 75–200 mg/m2 given orally for 5 consecutive days every 28-day cycle (5/28 d) [44,45].

#### *4.6. Tumor Growth Assessed by Molecular Imaging*

Brain tumor growth was measured with a microPET/CT scanner (Albira ARS, Oncovision Valencia, Spain). PET/CT images were acquired at 2 weeks post-implantation (day 0 of drug treatment) and subsequently on a weekly basis up to the time of euthanization. 300 μCi of 18 F-FLT was injected into the caudal vein of rats under O2/isoflurane anesthesia (1–3% isoflurane in 100% oxygen). The images were acquired at 40–60 min post-injection.

## *4.7. Determination of Body Weight and Overall Survival*

Tumor growth was also evaluated by monitoring weight loss during treatments and recording the global survival of animals throughout the study, which lasted 50 days.

## *4.8. Histology and Immunohistochemistry*

At the end of the experiment, all rats were euthanized and perfused with saline solution followed by 4% paraformaldehyde. Brains were removed and immersed in 4% paraformaldehyde for 2 weeks. The brain tissue was embedded in paraffin and sliced into sections (2 mm thick) on the coronal plane. The tissue slices were stained using the H&E method, and cell proliferation examined according to the level of the Ki-67 protein. A section of rat spleen was employed as a positive control.

## *4.9. Western Blot Analysis*

Tissue samples were homogenized with a lysis buffer containing protease inhibitors. Proteins were obtained by centrifugation at 10,000 g and 4 ◦C, separated on 10% polyacrylamide gel and quantified by bicinchoninic acid assay (BCA). Colored markers (Bio-Rad, Hercules, CA, USA) were included to establish size. Proteins were then electrophoretically transferred from the gel onto PVDF membranes (Amersham, Buckinghamshire, UK), which were blocked with 5% non-fat dry milk at room temperature for 2 h. Membranes were incubated overnight at 4 ◦C with antibodies against MGMT (sc-166528, 1:500), Bcl-2 (sc-7382, 1:1000), Bax (sc-20067, 1:1000), actin (sc-69879, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and cleaved caspase 3 (9661, 1:1000; Cell Signaling Technology, MAB230, R&D Systems, Minneapolis, MN, USA). After washing, the membranes were incubated with anti-mouse or anti-rabbit secondary antibodies, IgG-HRP (1:1500 and 1:3000, respectively; Santa Cruz Biotechnology) for 1 h.

Blots were developed with a chemiluminescent substrate (ECL Prime). Chemiluminescent bands were developed on Kodak autoradiography film in a darkroom and their densities were measured on Image Studio software (version, 5.2 Li-cor, Lincoln, NE, USA). In each figure, representative blot images were selected from the same gel.

## *4.10. Statistical Analysis*

Values are expressed as the mean ± SEM (standard error of the mean). Statistical analysis was performed with one-way analysis of variance (ANOVA) on SPSS Base 20.0 software (SPSS Inc., Chicago, IL, USA). Differences were scrutinized with multiple comparisons between groups. When necessary, the comparison of means was Bonferroni adjusted. In all cases, a value of *p* < 0.05 was considered significant.

## **5. Conclusions**

The present results indicate that mifepristone was capable of enhancing the inhibitory effect of temozolomide on the proliferation of glioma tumors in an orthotopic rat model of glioblastoma. Hence, this antihormonal drug could possibly be beneficial as a sensitizer for temozolomide in standard therapy for GBM. Since standard GBM treatment is of limited effectiveness, evidenced by low patient survival, the current findings are encouraging and suggest the importance of a Phase I clinical study to further explore the combination of mifepristone and temozolomide.

**Author Contributions:** M.L.-M. participated in the experimental procedures for tumor cell implantation, in data processing and the analysis of the results. J.S.-B. took part in experimental procedures, while M.R.-P. and L.A.M. carried out the evaluation of tumor growth by molecular imaging. N.U.-U. contributed to histology and immunohistochemistry. A.M.S. and M.R.-D. designed the Western blot experiments. P.G.-L. planned and supervised the study, coordinated all activities, and wrote the manuscript. All authors read and approved the final version of the manuscript.

**Funding:** This work was partially financed by CONACYT (México) through grant CB-258823.

**Acknowledgments:** Monserrat Llaguno Munive, a doctoral student of the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), is grateful for having received a fellowship 330137 from CONACYT. We thank Bruce Allan Larsen for proofreading the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article*

## **Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma**

**Seçkin Akgül 1,2,† , Ann-Marie Patch 3,†, Rochelle C.J. D'Souza 1, Pamela Mukhopadhyay 3 , Katia Nones 3, Sarah Kempe 3, Stephen H. Kazakoff 3, Rosalind L. Jeffree 4 , Brett W. Stringer 1 , John V. Pearson 3, Nicola Waddell 3,‡ and Bryan W. Day 1,5,\*,‡**

Received: 24 December 2018; Accepted: 2 February 2019; Published: 6 February 2019

**Abstract:** Glioblastomas are the most common and lethal neoplasms of the central nervous system. Neighbouring glioma cells maintain extreme degrees of genetic and phenotypic variation that form intratumoural heterogeneity. This genetic diversity allows the most adaptive tumour clones to develop treatment resistance, ultimately leading to disease recurrence. We aimed to model this phenomenon and test the effectiveness of several targeted therapeutic interventions to overcome therapy resistance. Heterogeneous tumour masses were first deconstructed into single tumour cells, which were expanded independently as single-cell clones. Single nucleotide polymorphism arrays, whole-genome and RNA sequencing, and CpG methylation analysis validated the unique molecular profile of each tumour clone, which displayed distinct pathologic features, including cell morphology, growth rate, and resistance to temozolomide and ionizing radiation. We also identified variable sensitivities to AURK, CDK, and EGFR inhibitors which were consistent with the heterogeneous molecular alterations that each clone harboured. These targeted therapies effectively eliminated the temozolomide- and/or irradiation-resistant clones and also parental polyclonal cells. Our findings indicate that polyclonal tumours create a dynamic environment that consists of diverse tumour elements and treatment responses. Designing targeted therapies based on a range of molecular profiles can be a more effective strategy to eradicate treatment resistance, recurrence, and metastasis.

**Keywords:** glioblastoma; intratumoural heterogeneity; tumour resistance; personalised therapy; targeted therapy; combination therapy; drug screens

#### **1. Introduction**

Glioblastoma remains an aggressive disease despite decades of comprehensive research. The overall and progression-free survival has not changed since the introduction of concomitant and adjuvant radiotherapy and chemotherapy in the form of temozolomide (TMZ) in 2005 [1,2]. This poor outcome is partially linked to its original name, "glioblastoma multiforme", as glioblastoma displays extreme degrees of genetic and phenotypic variation. This intertumoural heterogeneity has been evidenced by the characterisation of numerous tumour samples that showed distinct and unique molecular profiles, including gene mutations, chromosomal alterations, copy number variations, epigenetic alterations, and protein or RNA expression signatures [3–7]. Thus, the classification of glioblastoma into different subgroups based on their molecular profiles has been suggested to simplify the complexity of the disease, and also facilitate personalised treatment strategies [8]. However, recent studies have identified another phenomenon: intratumoural heterogeneity, which refers to individual cells/clones within a single tumour mass that are associated with different molecular characteristics [9]. Intratumoural heterogeneity poses additional challenges, as current pathological and molecular diagnostics are based only on a small region of the tumour, which may not be representative of the tumour mass as a whole [9].

Initial attempts to better understand glioblastoma intratumoural heterogeneity was undertaken on spatially distinct regions of a single tumour, and analysing these distinct tumour fragments revealed both unique and common copy number alterations in different fragments [10]. Subsequent studies extended these findings and reported a wide spectrum of transcriptional programs, cell states (differentiation vs. stemness), and proliferation capacity between individual tumour cells/clones isolated from the same tumour mass [11,12]. Clonal diversity was further demonstrated by the discovery of a mosaic and mutually exclusive amplification of three receptor tyrosine kinase (RTK) genes in intermingled subpopulations of glioblastoma cells. The heterogeneous but co-existing distribution of these *EGFR*, *MET*, and *PDGFRA* gene amplifications suggests that glioblastoma may undergo a dynamic evolution during tumour progression that creates diversity within a single mass [13]. Importantly, the involvement of multiple kinases in tumour development raises the question of whether therapies or a combination of therapies targeting multiple oncogenic signals are needed to eradicate the whole tumour mass.

Intratumoural heterogeneity arises by the continuous acquisition of molecular alterations during tumour progression. As tumour growth proceeds, individual cells and clones persistently compete for nutrient, oxygen and space within the tumour microenvironment. In this selective environment, clones evolve and acquire alterations that enable them to survive and proliferate, essentially becoming dominant subclones while others either perish or remain quiescent [14]. Current treatment, including chemotherapy and radiotherapy, also provides strong selective pressures which trigger clonal evolution responses. Although treatment induces death in a significant proportion of the tumour, surviving cells acquire new alterations, becoming resistant to therapy and enabling tumour recurrence [14,15]. In support of this notion, it has been found that the mutation rate (mutation per megabase) in low-grade gliomas increased from (0.2–4.5) to (31.9–90.9) when they relapse as secondary glioblastomas. Importantly, >98.7% of these alterations have been associated with TMZ treatment and did not exist in the pre-treatment primary tumours. Thousands of de novo mutations and novel oncogenic signatures observed in these TMZ-resistant clones suggest that tumours branch out into new molecular profiles and evolve into even more malignant states after treatment [16].

It is therefore imperative to capture and recapitulate the ever-fluctuating intratumoural heterogeneity in order to fully comprehend the complex biology of glioblastoma. Furthermore, designing and testing rationalised therapeutic interventions in consideration of this phenomenon has important clinical implications. Here, we show that individual tumour clones have a wide range of genetic and biological features which ultimately determine their response to several clinically relevant compounds. Our results shed further light on the complexity and heterogeneity present within glioblastoma tumours and highlight that, despite this diversity, both treatment-resistant and sensitive

clones can be effectively targeted. These findings may help to inform future clinical trial development to overcome tumour heterogeneity to improve clinical outcomes for glioblastoma sufferers.

## **2. Results**

## *2.1. Single-Cell Clonal Model Development to Assess Intratumoural Heterogeneity in Glioblastoma*

To model intratumoural heterogeneity, we developed a three-step approach (Figure 1A). Firstly, we prepared a polyclonal primary cell line from patient-derived tumour tissue. We then deconstructed this polyclonal cell line into individual cells and established single-cell clones grown under serum-free conditions. The passage number of the clones was kept to a minimum to reduce culture induced alterations. Next, we undertook a number of genomics analyses, including Single nucleotide polymorphism (SNP) arrays, RNA sequencing, and whole genome sequencing (WGS), allowing us to profile each clone in great detail. Secondly, we analysed the biological response of the clones to the clinical standards of care by treatment with TMZ and ionizing radiation (IR). This allowed us to identify a number of treatment sensitive and resistant clones. Lastly, we used our detailed knowledge of the clones to guide our treatment decisions to rationally target and eliminate resistant tumour cell populations. These three steps were achieved in a structured workflow (Figure 1B).

**Figure 1.** Modelling tumour heterogeneity. (**A**) Schematic representation of the three main steps used to investigate intratumoural heterogeneity in glioblastoma. Step 1, deconstructing a polyclonal tumour into single-cells that were expanded clonally prior to comprehensive genomics analyses. Step 2, assessment of individual clones' responses to the current standards of care, including temozolomide (TMZ) and IR. Step 3, drug screen development and rationale target validation. (**B**) A schema of the workflow used to undertake this study.

#### *2.2. Single-Cell Clones Exhibit Unique Molecular Relationships with a Spectrum of Growth Rates*

The copy number events in each sample, which were assessed by SNP array, were used to elucidate the clonal relationships between samples. Genotype analysis confirmed a high degree of similarity between all samples, with a concordance of ≥0.954 from pairwise comparisons of >400K polymorphic

positions. The most variable cross sample LogR ratio values (the total normalised signal intensity of the two alleles), which indicate the copy number status of each array probe, were analysed using unsupervised hierarchical clustering to generate a relationship diagram (Figure 2A). The clustering indicated two major groups with all bulk tumour and matched normal samples in the first and cell lines in the second that contained three further identifiable subgroups of tumour clones (Figure 2A, yellow boxes).

The proliferation rate of each tumour clone was assessed for 12 single-cell clones and the polyclonal line to determine whether the observed molecular clonal SNP array variances correlated with mitogenic potential (Figure 2B). Interestingly, we observed a wide spectrum of growth rates among the tumour clones. For instance, while clone-F reached confluency in approximately 5 days, clone-B did not reach 50% confluency even after 8 days when the assay was ended. Other clones fell between these two extremes, which led to three main proliferation categories: high, medium, and low (Figure 2B). Using this relatively straight-forward two-parameter approach, using the SNP array and proliferative potential, we were able to separate tumour clones from each other.

**Figure 2.** Single-cell clones exhibit unique molecular relationships and growth rates. (**A**) single nucleotide polymorphism (SNP) array-based dendrogram of 12 single-cell clones (A–L), parental polyclonal line (Poly), normal cortex tissue (Cortex), peripheral blood (Blood), aspirated tumour tissue (Aspirate), and surgically resected tumour tissue (Tumour). Three subgroups were found among the tumour clones, which are indicated with yellow boxes. (**B**) Growth rates of the 12 single-cell clones (A–L) and polyclonal cell line were recorded every 3 h for a period of 8 days using an IncuCyte instrument. The cell growth was categorised as high, medium or low. Growth curves for each single-cell clone are labelled with a different colour, except for the grey curves, which represent the clones that were not subjected to further comprehensive genomics analyses.

## *2.3. Comprehensive Genomic Analyses Identify Relationships between Single-Cell Clones*

Based on the SNP array clustering and growth characteristics, we selected six clones (clones A–F) to further model intratumoural heterogeneity. These six clones were distributed within the three main arms of the SNP array cluster and covered the spectrum of observed growth rates (Figure 2A,B). We performed comprehensive genomic analyses for these selected clones, including whole genome sequencing, RNA sequencing, and CpG methylation profiling (Figure 3).

Genomic analysis focusing on the somatic differences between the clones revealed heterogeneity across all data types. Somatic DNA changes were catalogued for each sample, including copy number, structural variants (SVs), single nucleotide variants (SNVs) and small insertions/deletions (indels). Comparative analysis of the SNVs indicated that 3490 out of 8696 (40.14%, Supplementary Table S1 and Figure S1A–C) were shared by all tumour and clonal samples. These shared variants are likely to have occurred early on in the development of this cancer and are present in most cells as trunk variants. Genomic heterogeneity between the cell line clones was apparent as a further 3492 of 8696 (40.16%) SNVs were identified to be unique to one sample, with clone-A containing the most (1345) unique SNVs (Supplementary Table S1).

**Figure 3.** Comprehensive genomic analyses of single-cell clones. (**A**) Genomic proportion of copy number alteration. For clonal samples B–F, >84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. (**B**) Number of somatic structural variants identified per sample with the type indicated by colours. (**C**) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). (**D**) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. (**E**) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.

The copy number data indicated that five of six clonal samples (B–F) had undergone whole genome duplication (WGD). For these samples, >84% of their genome was copy number 3 and 4 (range 84.82% to 85.9%) as compared to ≤3% for the other samples (Figure 3A and Supplementary Table S2). The WGD event increased copies of both alleles, and therefore it is difficult to assign a functional

consequence at a gene level to such a global event. Therefore, a WGD correction was applied for these samples that revealed that all samples were relatively copy-number quiet, with <20% of the genome judged to be copy-number aberrant (i.e., not diploid or polyploidy heterozygous, Figure 3A).

Large structural rearrangements (SVs) were detectable in all samples at an average of 20.11 SVs (range 14 to 35 SVs) (Figure 3B, Supplementary Table S3 and Figure S1D–F). Of the 83 SVs detected, just 10 SVs (12.05%) were identified as early trunk events and were shared by all the samples. The majority 65 of 83 SVs (75.31%) were unique to individual samples. Clone-C harboured the highest number of somatic structural variants (35 SVs) with more than double the number of events in the bulk tumour sample (16 SVs). The predicted consequences for all rearrangements indicated that 43 genes were likely to have been disrupted, resulting in loss of function for those genes, none of which affected known cancer driver genes. There were three inter-gene fusion events identified, but none included known cancer driver genes or targets for current therapies (Supplementary Table S3).