Antisecretory Factor Mediated Inhibition of Cell Volume ... · mg/kg/day, Selleck Chemicals, 5days...

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Cell Death and Survival Antisecretory FactorMediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma Shirin Ilkhanizadeh 1,2 , Hanna Sabelstr om 1,3 , Yekaterina A. Miroshnikova 4 , Aaron Frantz 1,3 , Wen Zhu 5 , Aurora Idilli 6,7 , Jon N. Lakins 4 , Christin Schmidt 1,3 , David A. Quigley 2,8 , Trenten Fenster 1,3 , Edith Yuan 1,3 , Jacqueline R. Trzeciak 1,3 , Supna Saxena 1,3 , Olle R. Lindberg 2,9,10 , Janna K. Mouw 4 , Jason A. Burdick 11 , Sergey Magnitsky 12 , Mitchel S. Berger 2,9 , Joanna J. Phillips 2,9,10 , Daniele Arosio 6,7 , Dandan Sun 5 , Valerie M. Weaver 4,2 , William A. Weiss 1,9,2 , and Anders I. Persson 1,9,3,2 Abstract Interstitial uid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how uid dynamics impacts tumor progression and can be targeted ther- apeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temo- zolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-medi- ated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 77790. Ó2018 AACR. Introduction Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 to 15 months (1). Growing in the connes of the skull, GBMs in patients produce elevated intracranial pressures (10100 mm Hg) com- pared with matched controls (05 mm Hg; ref. 2). In GBM, tumor cells displace nonneoplastic astrocytes, which, together with vascular abnormalities, lead to failure in barrier properties, induc- ing vessel permeability that allows plasma and uid to leak into the tumor tissue, inducing cerebral edema and increasing inter- stitial uid pressure (IFP) (3). Fluid accumulation compresses the tumor and surrounding normal tissue and reduces cellular uptake in solid tumors (3). Although antiangiogenic therapies effectively reduce IFP, they may also contribute to invasion and treatment resistance (3, 4). Changes in cell volume caused by water and ion transport across the cell membrane are necessary for proliferation and migration of GBM cells (5). Invading GBM cells maximally decrease their cell volume by 30% to 35%, a value that represents all free unbound cytoplasmic water (6). Any volume reduction beyond this value is expected to compromise the survival of GBM cells (7). Therefore, we hypothesized that IFP-reducing strategies that prevent osmotic adaptation of tumor cells should reduce tumor progression without driving invasiveness. In the brain, transport of water and ions across the plasma membrane by aquaporins and the sodium-potassium-chloride cotransporter 1 (NKCC1) is necessary for glial cells to adapt to osmotic challenges (8, 9). Although knockdown of aquaporins reduced migration and survival of GBM cells, therapeutic targeting of these integral membrane proteins remains a major challenge (10). Expression of NKCC1 is lower in the brain and increases 1 Department of Neurology, University of California, San Francisco, San Francisco, California. 2 Brain Tumor Research Center (BTRC) at the Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. 3 Sandler Neurosciences Center, University of California, San Francisco, San Francisco, California. 4 Department of Surgery, University of California, San Francisco, San Francisco, California. 5 Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania. 6 Institute of Biophysics, CNR and FBK, Trento, Italy. 7 CIBIO, University of Trento, Trento, Italy. 8 Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California. 9 Department of Neurological Surgery, University of California, San Francisco, San Francisco, California. 10 Department of Pathology, University of California, San Francisco, San Francisco, California. 11 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania. 12 Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). S. Ilkhanizadeh and H. Sabelstrom contributed equally to this article. Corresponding Author: Anders I. Persson, Department of Neurology, University of California, San Francisco, 675 Nelson Rising Lane, NS-270B, San Francisco, 94158 CA. Phone: 415-606-3604; Fax: 415-476-0133; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-17-0413 Ó2018 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org 777 on April 3, 2020. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst February 5, 2018; DOI: 10.1158/1541-7786.MCR-17-0413

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Cell Death and Survival

Antisecretory Factor–Mediated Inhibition of CellVolume Dynamics Produces Antitumor Activity inGlioblastomaShirin Ilkhanizadeh1,2, Hanna Sabelstr€om1,3, Yekaterina A. Miroshnikova4,Aaron Frantz1,3,Wen Zhu5, Aurora Idilli6,7, Jon N. Lakins4, Christin Schmidt1,3,David A. Quigley2,8, Trenten Fenster1,3, Edith Yuan1,3, Jacqueline R. Trzeciak1,3,Supna Saxena1,3, Olle R. Lindberg2,9,10, Janna K. Mouw4, Jason A. Burdick11,Sergey Magnitsky12, Mitchel S. Berger2,9, Joanna J. Phillips2,9,10, Daniele Arosio6,7,Dandan Sun5, Valerie M.Weaver4,2,William A.Weiss1,9,2, and Anders I. Persson1,9,3,2

Abstract

Interstitial fluid pressure (IFP) presents a barrier to druguptake in solid tumors, including the aggressive primary braintumor glioblastoma (GBM). It remains unclear how fluiddynamics impacts tumor progression and can be targeted ther-apeutically. To address this issue, a novel telemetry-basedapproach was developed to measure changes in IFP duringprogression of GBM xenografts. Antisecretory factor (AF) is anendogenous protein that displays antisecretory effects in animalsand patients. Here, endogenous induction of AF protein orexogenous administration of AF peptide reduced IFP andincreased drug uptake in GBM xenografts. AF inhibited cellvolume regulation of GBM cells, an effect that was phenocopied

in vitro by the sodium-potassium-chloride cotransporter 1(SLC12A2/NKCC1) inhibitor bumetanide. As a result, AFinduced apoptosis and increased survival in GBM models.In vitro, the ability of AF to reduce GBM cell proliferation wasphenocopied by bumetanide and NKCC1 knockdown. Next,AF's ability to sensitize GBM cells to the alkylating agent temo-zolomide, standard of care in GBM patients, was evaluated.Importantly, combination of AF induction and temozolomidetreatment blocked regrowth in GBM xenografts. Thus, AF-medi-ated inhibition of cell volume regulation represents a novelstrategy to increase drug uptake and improve outcome in GBM.Mol Cancer Res; 16(5); 777–90. �2018 AACR.

IntroductionGlioblastoma (GBM) is the most common and aggressive

primary brain tumor with a median survival of 12 to 15 months

(1). Growing in the confines of the skull, GBMs in patientsproduce elevated intracranial pressures (10–100 mm Hg) com-paredwithmatched controls (0–5mmHg; ref. 2). In GBM, tumorcells displace nonneoplastic astrocytes, which, together withvascular abnormalities, lead to failure in barrier properties, induc-ing vessel permeability that allows plasma and fluid to leak intothe tumor tissue, inducing cerebral edema and increasing inter-stitial fluid pressure (IFP) (3). Fluid accumulation compresses thetumor and surrounding normal tissue and reduces cellular uptakein solid tumors (3).

Although antiangiogenic therapies effectively reduce IFP, theymay also contribute to invasion and treatment resistance (3, 4).Changes in cell volume caused by water and ion transport acrossthe cellmembrane arenecessary for proliferation andmigrationofGBM cells (5). Invading GBM cells maximally decrease their cellvolume by 30% to 35%, a value that represents all free unboundcytoplasmic water (6). Any volume reduction beyond this value isexpected to compromise the survival of GBM cells (7). Therefore,we hypothesized that IFP-reducing strategies that prevent osmoticadaptation of tumor cells should reduce tumor progressionwithout driving invasiveness.

In the brain, transport of water and ions across the plasmamembrane by aquaporins and the sodium-potassium-chloridecotransporter 1 (NKCC1) is necessary for glial cells to adapt toosmotic challenges (8, 9). Although knockdown of aquaporinsreducedmigration and survival ofGBMcells, therapeutic targetingof these integral membrane proteins remains a major challenge(10). Expression of NKCC1 is lower in the brain and increases

1Department ofNeurology,University of California, SanFrancisco, San Francisco,California. 2Brain Tumor Research Center (BTRC) at the Helen Diller FamilyComprehensive Cancer Center, University of California, San Francisco, SanFrancisco, California. 3Sandler Neurosciences Center, University of California,San Francisco, San Francisco, California. 4Department of Surgery, University ofCalifornia, San Francisco, San Francisco, California. 5Department of Neurology,University of Pittsburgh, Pittsburgh, Pennsylvania. 6Institute of Biophysics, CNRand FBK, Trento, Italy. 7CIBIO, University of Trento, Trento, Italy. 8Department ofEpidemiology and Biostatistics, University of California, San Francisco, SanFrancisco, California. 9Department of Neurological Surgery, University ofCalifornia, San Francisco, San Francisco, California. 10Department of Pathology,University of California, San Francisco, San Francisco, California. 11Department ofBioengineering, University of Pennsylvania, Philadelphia, Pennsylvania.12Department of Radiology and Biomedical Imaging, University of California,San Francisco, San Francisco, California.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

S. Ilkhanizadeh and H. Sabelstr€om contributed equally to this article.

Corresponding Author:Anders I. Persson, Department of Neurology, Universityof California, San Francisco, 675 Nelson Rising Lane, NS-270B, San Francisco,94158 CA. Phone: 415-606-3604; Fax: 415-476-0133; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-17-0413

�2018 American Association for Cancer Research.

MolecularCancerResearch

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with glioma grade (11), shown to regulate tumor cell invasion(12). We recently demonstrated that the FDA-approved NKCC1inhibitor bumetanide abrogated regulatory volume increase andpotentiated temozolomide-induced cytotoxicity of human GBMcells (13). However, poor brain penetrance and side effects fromthe highly potent diuretic bumetanide limit its chronic use (14,15). Is there an alternative approach to regulate tumor cell volumethat reduces IFP and produces antitumor activity in GBM?

Efforts to identify agents that prevent cholera toxin-inducedhypersecretion from intestine lead to the identification of aprotein in intestinal organs that demonstrated antisecretoryproperties (16). Purification of this antisecretory factor (AF)protein identified an eight-amino acid N-terminal sequencethat displayed antisecretory properties in higher vertebrates andhumans (17–20). Endogenous AF protein can be induced inanimals and humans within days following treatment with aspecially processed cereal (SPC; refs. 21, 22). Exogenousadministration of AF protein through oral route (Salovum) ornasal uptake of AF peptide produced more immediate effects(23, 24). Endogenous and exogenous induction of AF proteinwas well tolerated in animals and patients (21, 25). More recentstudies show that AF can reverse fluid dysregulation, includingintracranial pressure, in the brain and other organs (22–24, 26–28). The mechanism of action has been linked to targets knownto regulate fluid homeostasis, including chloride permeabilityacross cell membranes (29), and lipid content, including phys-ical interaction of AF with the lipid raft protein flottilin-1 (30,31). AF's ability to reduce intracranial pressure in the injuredbrain, lower IFP in subcutaneous tumors, and increase druguptake in solid subcutaneous tumors suggests that it might alsobe effective in GBM (22, 23).

Here, we used a telemetry-based approach to measure IFPduring GBM progression in patient-derived xenografts (PDX). Incontrast to previousmethodology thatmeasured IFP in tumors ofanaesthetized animals for a few hours (32, 33), this non-invasiveapproach enabled us to remotely and continuously measure IFPduring tumor progression. Oral or nasal AF therapy reduced IFPlevels and increased drug uptake in this PDXmodel. Oral admin-istration of SPC diet (endogenous AF induction) inhibited tumorgrowth and extended survival in GBM xenografts. Upon closerexamination, approximately 40% of tumor cells displayedreduced tumor cell volume along with increased apoptotic indexfollowing administration of SPC diet versus control diet. AFinduction reduced NKCC1 expression in GBM xenografts andtreatment with AF peptide, more potently than bumetanide,inhibited regulatory volume increase and chloride permeabilityin GBM tumorsphere cultures. Using a compression force biore-actor, we show that AF peptide, bumetanide, and NKCC1 knock-down blocked compression-induced proliferation of GBMtumorspheres. Importantly, a combination of SPC diet andtemozolomide completely prevented GBM progression andregrowth of PDXs. Thus, AF therapy represents a novel IFP-reducing strategy that produces antitumor activity in GBM.

Materials and MethodsMice

All mouse experiments were approved by and performedaccording to the guidelines of the UCSF Institutional AnimalCare and Use Committee. For PDX experiments, we purchased4- to 6-week-old athymic nu/nu mice (Simonsen Laboratories).

Intracranial injectionsWe stereotactically injected 5 � 104 GBM43 cells, 3 �

105GBM43 cells, or 3 � 105 GBM6 cells expressing the fireflyluciferase gene (LUC) into the frontal cortex of adult athymicmice. We also injected 2 � 105 mouse GBM cells expressinghuman EGFRvIII (pEGFRlow), mCherry, and LUC (34) into adultFVBnmice (Charles River Laboratories). Injections were done at adepth of 3mm, and the coordinates were 2-mm anterior and 1.5-mm lateral of the right hemisphere relative to Bregma. Wemeasured body weight and bioluminescence every second dayduring progression. Bioluminescencewasmeasuredusing aXeno-gen IVIS imager, and survival was plotted using Kaplan–Meiercurves. Symptomatic mice were sacrificed according to ethicalguidelines approved by the UCSF Institutional Animal Care andUse Committee.

Treatment studiesFor endogenous AF protein induction, mice were administered

(as indicated in figures) SPC diet (10% SPC from POA Pharmacold-pressed with 90% Global Protein Rodent Diet, Envigo).Control mice were treated with 100% Global Protein RodentDiet. For exogenous AF administration, nasal AF was injectedacutely preceding a single tail vein injection of doxorubicin asstated for uptake studies below. For long-term exogenous AFadministration, mice were given 4% Salovum (approximately0.8 g/kg/day, egg yolk powder, POA Pharma) dissolved in 3%glucose-containing water. Mice were administered erlotinib (100mg/kg/day, Selleck Chemicals, 5 days on, 2 days off for 3 weeks).Temozolomide hydrochloride (50 mg/kg/day, Selleck Chemi-cals) was administered through oral gavage on days 10 to 11.

Implantation of IFP recording probesBefore transplantation, each pressure transducer (PA-C10,Data

Sciences International) was calibrated and sterilized according toDSI instruction manual. An offset reading was recorded for eachprobe and was accounted for in pressure readings throughout theexperiment. An opening (approximately 1 mm diameter) wasdrilled into the right side of the parietal skull 2-mm anterior and1.5-mm lateral of the right hemisphere relative to Bregma. Ster-ilized, custom pan slot vented pure titanium screws (UnitedTitanium Inc) were screwed in the drilled hole. Injections of GBMcells were done at a depth of 3 mm. Subsequently, the tip of thesensor was inserted into the same hole to a depth of 3 to 4 mm.Surgical glue was applied around and on the top of the screw tofixate the probe. The transducer device was then placed under theskinon theback of themice. Themousewas put back in its cage forrecovery.

Doxorubicin and erlotinib uptake studiesAs bioluminescence levels reached 108 p/s/cm2/sr, we admin-

istered 9 mg/kg doxorubicin hydrochloride (in 0.9% NaCl;Sigma-Aldrich) by tail vein injection to SPC-treatedmice followedbyharvesting of tumor tissue 15minutes later (35).Weperformedintranasal injections of AF-16 (9 mg in PBS/mouse, PhoenixPharmaceuticals Inc.) peptide 20 minutes prior to doxorubicininjections in a separate cohort of xenografted mice. Mice wereperfused and brains were harvested and sectioned as 15 mmsections on glass slides. The slides were counterstained with DAPIandmountedwithProLongGold antifade reagent (ThermoFisherScientific). Images were acquired using the Zeiss M1 AxioImagerand the Zen software. For quantification of doxorubicin, tumor

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regionswere identified, and themeanfluorescence intensity of thered channel (emission at 590 nm) was determined using theimage analysis tool in Zen. The background was removed on allimages to the same level until only nuclear doxorubicin could befound. Threemice in each group (control, AF, SPC)were analyzedwith three brain sections for each mouse and four regions in eachbrain section. Results were normalized to a sample with nodoxorubicin administration.

Erlotinib was dissolved in OraPlus and administered (100mg/kg) through oral gavage to mice followed by dissection of tumortissue onehour later.We appliedprevious protocols using LC/MS-MS to quantify erlotinib (Selleck Chemicals) in tumor isolates(36). Erlotinib-D6 hydrochloride (Toronto Research Chemicals)was used as an internal standard.

Measurement of IFPEach cage (containing one mouse with an implanted device)

was placed on top of a receiver (RPC-1, Data Sciences Interna-tional), which was connected to a computer equipped withPonemah Software System (Data Sciences International). Con-tinuous IFP could then be measured in awake and freely movingmice. The devices could be turned on and off when being heldclose to amagnet (provided byData Sciences International). Eachmousewas recorded for at least 2 hours each day at the same time.The collected data were analyzed using the Ponemah SoftwareSystem following protocol provided by DSI.

In vivo MRIMagnetic resonance imaging (MRI) was performed with a 7T

preclinical horizontal boremagnet interfacedwithAgilent imagingconsole. Animals were anaesthetized by inhalation of 2% isoflur-ane with 98% oxygen and placed into a costume made linearpolarized birdcage coil with 21 mm ID. The body temperatureof the animals was kept at 35�C during imaging experiments.Monitoring of the heartbeat, breathing, and temperature of theanimals was done withMRI-compatible small-animal life supportsystem (SA Instruments Inc,). To identify tumor location in themouse brain, T2-weighted spin echo MRI was performed with thefollowing parameters: repetition time (TR)¼ 3 seconds, echo time(TE) ¼ 42 ms, 10 slices with 1 mm thickness and approximately120 mm in plane resolution, 2 repetitions.

Because diffusion is inversely proportional to water pressure,wehypothesized that high intratumoral pressure canbe correlatedwith apparent diffusion coefficient (ADC) of water molecules inthe tissue. Measurements of ADC were performed with diffusion-weighted spin echo pulse sequence and the following parameters:TR ¼ 3 seconds, TE ¼ 42 ms, b ¼ 0, 1,027 s/mm2, diffusiongradients were applied along readout direction, 10 slices with 1mm thickness and approximately 120 mm in plane resolution. Tominimize motion artifacts, cardiac and breathing gating wasemployed. At the end of the imaging experiment, ADC map wasreconstructed and average ADC values were calculated for thenormal and tumor brain tissues. Color ADC maps were createdusing ImageJ software (NIH image). A conversion of gray scaleinto color images was performed with "spectrum" color map.

High intratumor pressure can potentially prevent extravasationof a contrast agent into the tumor tissue. To test this hypothesis,we performed dynamic contrast enhancement experiments. Weadministered 100 mL 83mmol/LGd-DTPA (Magnevist, Bayer) viaa catheter in the tail vein. Images were obtained from 4 centralslices of tumor tissue with temporal resolution of approximately

14.9 seconds. Gradient echo pulse sequence was utilized with thesame resolution as diffusion images (�120 mm). Parameters ofthe sequence were TR¼ 29ms, TE¼ 3.6ms, flip angle¼ 500, andtotal acquisition time, approximately 25 minutes.

MicroscopyImages were acquired using Zeiss M1 AxioImager or Zeiss 780

confocalmicroscopes andZen software. For quantification, tumorregions were identified and region of interest was determined,avoiding the edge of tumor/section and necrotic regions. Themean fluorescence intensity was determined using the imageanalysis tool in Zen.

Tissue preparationBrains were fixed for 24 hours in 10% formalin buffered

solution, paraffin-embedded, sectioned at 5 mm, and dehydrated.Frozen tissue sections (20 mm) were collected onto glass slidesusing a cryostat (Leica). Mice were perfused transcardially usingPBS followed by 4% paraformaldehyde (PFA). Brains were har-vested and incubated overnight in 4% PFA at 4�C and transferredto 30% sucrose at 4�C. Brains were frozen embedded in OCTcompound (Tissue-Tek) and sectioned using a cryostat (Leica) as15 or 30 mm sections on glass slides (SuperFrost Plus, ThermoFisher Scientific).

IHCParaffin-embedded sections were stained for GFAP (Agilent)

and Iba1 (Wako) using the Ventana system at the UCSF Neuro-surgery Tissue Core. Glass slides with PFA-fixed frozen sectionswere washed 3 times for 10 minutes in Tris-buffered saline (TBS,pH 7.4). The sections were then incubated in blocking solution(5% donkey serum, 1% BSA, 0.3% Triton X-100) for 1 hour atroom temperature. Primary antibodies were diluted in blockingsolution and incubated on sections overnight at 4�C. Antibodiesused: cleaved caspase-3 (1:500, Cell Signaling Technology),NKCC1, (1:200, Santa Cruz Biotechnology), pNKCC1 (1:500,MRC PPU Reagents), CD31 (1:250, BD Biosciences), CD45(1:500, BD Biosciences), GFAP (1:1000; Thermo Fisher Scientif-ic), Ki67 (1:100, Novus), AF (1:200, Phoenix PharmaceuticalsInc.), and Phalloidin (1:100, Thermo Fisher Scientific). Sectionswere washed in TBS and incubated with the appropriate Alexa488(1:500), 555 (1:500), 647 (1:500) secondary antibodies (ThermoFisher Scientific) in blocking solution for 2 hours at room tem-perature. The sections were washed in TBS 3 times for 10 minutes(DAPI was included in the second wash), followed by mountingwith ProLong Gold antifade reagent (Thermo Fisher Scientific).

Western blottingFor tissue specimen, samples were frozen in dry ice and stored

at �80�C until processed. Samples were homogenized and son-icated in cell lysis buffer (Cell Signaling Technology). For cells,samples were washed in ice-cold PBS before lysis with cell lysisbuffer (Cell Signaling Technology). All samples were cleared bycentrifugation at 16,000 rpm for 10minutes at 4�Candquantifiedusing the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).Equal amounts of total protein lysates were loaded and resolvedon a 4% to 12% Bis-Tris Gel with MOPS running buffer andtransferred to PVDF membranes. Membranes were blotted withantibodies againstNKCC1 (SantaCruzBiotechnology), p-histoneH3, pAKT, and AKT (Cell Signaling Technology), b-actin (BDBiosciences), and GAPDH (Millipore), all used at 1 mg/mL

GBM Aggressiveness Is Reduced by Antisecretory Factor

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dilution. Antibodies were detected with horseradish peroxidase–linked antibody against mouse (Calbiochem), rabbit (Calbio-chem), or goat (Santa Cruz Biotechnology) immunoglobulin G,followed by ECL (Amersham).

Cell volume measurementsTomeasure approximate values of total cell volume and nucleus

volume in GBM xenografts, we used DAPI and Phalloidin488-stained sections and performed z-stacks using a Zeiss 780 confocalmicroscope. Cell and nuclei volumes were measured using a scriptin the Imaris 8.0.1 software (Bitplane Inc.). In brief, parameterswereset to "DetectNucleus andCell." DAPIwas set as the source channeland "Nucleus Diameter" was set to 5.00 mmol/L to distinguishindividual cell nuclei. "Cell Detection Type" was set to "Cell BodyDetection," and the source channel was set to the Phalloidin488channel. Cell membrane width was set to 0.700 mmol/L. Mitoticcells and apoptotic cell bodies were excluded from analysis.

Proliferation assayTo assess proliferation of humanGBMtumorspheres, we plated

0.2 � 104 GBM43 cells per well in NBE media on polyornithine/laminin–coated 96-well plates and analyzed theDNAcontent 0 to8 days using the Cyquant NF proliferation assay (Thermo FisherScientific; ref. 37). In brief, cells were lysed and simultaneouslyincubated at 37�C with a fluorescent probe to label nonfragmen-ted DNA as an indirect measure of the number of cells. Thefluorescence intensity wasmeasured at Ex/Em: 485/530 nmusinga Tecan microplate reader. A standard curve was generated byplotting number of plated cells (1,000–40,000) against corre-spondingfluorescent values, resulting in the equation y¼ xþ150,R2 ¼ 1. The number of cells in each sample was calculated usingthis equation. Experiments were done in triplicates.

3D hyaluronic acid gel cultureCells were plated on nonadherent plates for 4 days to allow

formation of tumorspheres. Tumorspheres were then collected,spun down, and resuspended in 2% solution of 30% methacry-lated 65 kDa hyaluronic acid (HA; generous gift from Dr. JasonBurdick,UniversityofPennsylvania, Philadelphia, PA) in completeneurobasal media [-A, Invitrogen; supplemented with 1% v/v B27supplement (Gibco), 0.5% v/v N2 supplement (Gibco), 20 ng/mLFGF-2 (PeproTech), 20 ng/mL EGF (Sigma-Aldrich), 2 mmol/LL-glutamine, and penicillin/streptavidin)]. Gels were photo-crosslinked with 0.05% Irgacure 2959 (Sigma-Aldrich) with365 nm UV light (10 minutes at a distance of 1.5 inches awayfrom the top of the constructs).

Compression bioreactorCustom-built compression bioreactor (Tissue Growth Technol-

ogies) with 12 individual compression wells operating underclosed loop position/force feedback with computer control andcustom software was utilized for compression studies. For allexperiments, trapezoid input was sent to the servo controller withforce ramping from 0% to 30% at a rate of 0.0005N/s, held at 30%for 48 hours, then ramped down to 0% at a rate of 0.005N/s; at thispoint, the constructs were extracted from the bioreactor and imme-diately subjected to fixation or RNA/protein sample collection.

RNA/protein isolation from 3D HA gelsImmediately upon completion of compression studies, HA gels

were removed from the bioreactor assembly and the triplicates

combined in single Eppendorf tubes with either RNA (700 mL)or protein (300 mL) lysis buffers (Cell Signaling Technology).Gels were then crushed mechanically, sonicated briefly, andRNA/proteins were allowed to diffuse out into the supernatanton ice for 2 hours. Subsequently, HA gel–RNA/protein solutionwas sonicated at maximum speed at 4�C for 10 minutes;supernatant was then aliquoted into new tubes. Protein sam-ples were treated identically to standard Western blot protocol;RNA was isolated using RNeasy Mini Kit (Qiagen) and furtherpurified to remove any remaining glycosaminoglycans (ZymoResearch); all samples were then flash frozen on dry ice andstored in �80�C.

Microarray and bioinformaticsHuman GBM tumorspheres (GBM5, GBM14, GBM34, and

GBM43) embedded in HA gels were subjected to no compressionor compression (48 hours, 30%) using a compression forcebioreactor (Tissue Growth Technologies) with/without incuba-tion with 1 ng/mL AF peptide. We isolated GBM cells and totalRNA was prepared as mentioned in qRT-PCR section (Qiagen).The concentration and quality of RNA was determined usingNanoDrop, followed by use of a RNA 6000 Nano Kit (Agilent)and bioanalyzer chips (Applied Biosystems) to determine RINvalues. We then sent 300 ng total RNA (50 ng/mL) to the UCSFInstitute for Human Genetics for further processing. In brief, 150ng was used in the first- and second-strand cDNA synthesis andIVT cRNA synthesis. We then performed cRNA purification, andsamples were normalized to 13.33 mg cRNA in 24 mL volume.Samples then go through a second cycle of cDNA synthesis andRNase H hydrolysis. The cDNA was purified and normalized to5.5 mg cDNA in 31.2 mL volume. We then performed cDNAfragmentation, labeling, and hybridized 90 mL of the sample inthe Affymetrix GeneTitan Hybridization Oven instrument for 16hours at 48�C. The arrays were processed with the AffymetrixGeneTitan Fluidics Station instrument, using the HuGene-2_1-stfluidics script and GeneTitan Hybridization, Wash, and Stain Kitfor WT Array Plates. Then, the arrays were scanned in the Affyme-trix GeneTitan Multi-Channel (MC) instrument with the AGCCGeneTitan Instrument Control. Quality control of samples wasperformedusing theAffymetrix Expression console, anddatawereinitially analyzed using Affymetrix Transcriptome Analysis Con-sole. CEL files were normalized by RMA using the oligo packagein R (38). Differential expression was assessed by paired t testusing the samr package with a 5% FDR threshold (39), andGene Ontology enrichment analysis was assessed using BiNGO(40). Raw microarray data for this study has been deposited atthe Gene Expression Omnibus (GEO) repository under acces-sion GSE81473.

Volume measurements of tumorspheresWe first diluted 2� concentrated 70 mm HA with neurobasal

media, added igracure, and mixed 1:1 with 20 to 30 humanGBM tumorspheres (100–200 mm diameter spheres 4 days afterplating) preceding polymerization for 7 minutes. Whole cell–embedded HA gels were stained with DAPI or Alexa Fluor 488Phalloidin (Thermo Fisher Scientific) and imaged (50 mmconfocal z stacks with 0.2 mm z-steps) on a Nikon Eclipse Ti-Ebase microscope equipped with the Yokogawa CSU-X1 confo-cal scanner and Andor's iXon3 EMCCD camera. Cell volumewas subsequently quantified using 3D Suite of Image J Software(NIH, Bethesda, MD).

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Calcein-AM–based cell volume measurementsCell volume change was determined using calcein as a

marker of intracellular water volume, which was establishedpreviously (41). Briefly, cells on coverslips either coated withpoly-L-lysine (50 mg/mL; GC22) or polyornithine (50 mg/mL)and laminin (5 mg/ml; GBM43) were incubated with 0.5 mmol/Lcalcein-AM for 30 minutes at 37�C. The cells were placed in aheated (37�C) imaging chamber (Warner Instruments) on aNikon Ti Eclipse inverted epifluorescence microscope equippedwith perfect focus, a 40� Super Fluor oil immersion objectivelens, and a Princeton Instruments MicroMax CCD camera.Calcein fluorescence was monitored using a FITC filter set(excitation 480 nm, emission 535 nm, Chroma Technology).Images were collected every 60 seconds with MetaFluor image-acquisition software (Molecular Devices). Regions of interest(0.5 mm in diameter) were selected in the cytoplasm of 5 to 15cells. Baseline drift resulting from photobleaching and dyeleakage was corrected as described before. The fluorescencechange was plotted as a function of the reciprocal of the relativeosmotic pressure, and the resulting calibration curve applied toall subsequent experiments as described previously (41). TheHEPES-buffered isotonic solution contained (in mmol/L, pH7.4): 100 NaCl, 5.4 KCl, 1.3 CaCl2, 0.8 MgSO4, 20 HEPES, 5.5glucose, 0.4 NaHCO3, and 70 sucrose with 310 mOsm deter-mined using an osmometer (Advanced Instruments). Anisos-motic solutions (262 and 432 mOsm) were prepared byremoval or addition of sucrose.

Use of ClopHensor for chloride imagingClopHensor is based on a chloride-sensitive GFP and can be

combined with real-time optical detection of chloride fluxes inliving cells. ClopHensor can uniquely compensate for pH changesthat are known to influence chloride levels (42). Electroporationof the ClopHensor plasmid was used to induce ClopHensorexpression into cultured GBM cells. Cells were maintained in ahumidified atmosphere at 37�C containing 5% CO2 duringmicroscopy analysis. Wide-field fluorescence images wereacquired using a customized iMIC (Till Photonics) microscopeequipped with a 0.65 NA plan-N 40� air objective (Olympus), ahigh-stability xenon lamp, and a 14-bit CCD camera (StingrayF145B-30fps, Allied Vision Technologies). The excitation filters458/10 (Chroma), 482/18 (BrightLine HC), and 563/9 (Sem-rock) weremounted on the lamp fast filter switching device, and adual channel filter set (dichroic: HC-Beamsplitter BS R488/561,emitter: Brightline HC 523/610, Semrock) was used in the filtercube. Thus, cyan, green, and red channels were respectivelyacquired with excitation (emission) wavelengths at 458 � 10(525 � 25), 482 � 18 (525� 25), and 563 � 9 (610 � 30) nm.Intracellular chloride concentration was measured in time-lapsefrom cells exposed to hypertonic stress in the following sequence:5-minute isotonic HEPES-MEM (317 mOsm), 20-minute hyper-tonicHEPES-MEM(432mOsm), and 10-minute isotonicHEPES-MEM. Cells were preincubated for 10 minutes in isotonic condi-tion before time-lapse acquisition. Bumetanide (10 mmol/L) andAF peptide (5 mg/mL) were preincubated and present throughoutthe experiment. Osmolarity was measured using a cryoscopicosmometer (Osmomat 030, Gonotec GmbH). Three-channelimages were acquired every 60 seconds, and ratio analysis wasperformed as described previously (42), where chloride concen-tration and pH were calculated from the cyan-over-red (chloride)and the green-over-cyan (pH) ratios.

Statistical analysisStatistical tests were performed using GraphPad Prism v6.0

software. Statistical analyses for experiments with two groupswere performed using Student t test. Statistical analyses for experi-ments withmore than two groups were performed using one-wayANOVA followed by post hoc tests (for in vitro proliferation andtherapy studies). Kaplan–Meier survival analysis was conducted,and the P value of the comparison between survival curves wasdetermined to be significant by the log-rank (Mantel–Cox) test.

ResultsAF reduces IFP and increases MRI-based ADC levels in GBMxenografts

To assess how AF treatment impacted fluid dynamics, weadministered adiet containing 10%SPC (to induceAF) beginning6 days following orthotopic engraftment of patient-derivedGBM43 cells into athymic mice, and then analyzed effects byMRI. Treatment with SPC diet significantly increased ADC levelsin tumors, a measure of fluid movement on MRI (Fig. 1A and B;Supplementary Fig. S1A and B). To confirm that increased ADClevels following SPC treatment were mediated by AF, we nextattempted nasal delivery of AF peptide, a preclinical method fordelivery of therapeutics (43).Wenext repurposed telemetry-basedmethodology (44) to measure IFP during tumor progression byinjecting LUC-expressing GBM43 cells into the frontal cortex,followed by immediate implantation of a pressure sensor (Fig.1C). A radiofrequency signal emitted from the pressure sensortransmitter placed within intracranial human GBM43 xenograftsenabled parallel measurements of IFP and LUC during tumorprogression (Fig. 1D and E). We observed no changes in thenumbers of tumor-associated macrophages or reactive astrocytesfollowing insertion of the pressure sensor (Supplementary Fig.S2A–S2D). Immunostainings using an antibody recognizing theantisecretory region of the AF protein showed that SPC dietincreased expression AF protein levels in tumor cells but not inthe surrounding brain (Fig. 1F). We observed no induction of AFprotein in CD31þ tumor vasculature and GFAPþ reactive astro-cytes, and low levels in CD45þ tumor-associated immune cells(Fig. 1F). Administration of SPC diet suppressed IFP levels com-paredwith animals administered control diet (Fig. 1G). Similarly,IFP levels were normalized for up to 30 minutes after nasaladministration of AF peptide (Fig. 1H). Thus, AF therapy repre-sents a novel IFP-reducing strategy.

AF increases drug uptake in GBMAdministration of SPC diet increased uptake of the contrast

agent gadolinium and reduced clearance in tumor tissue ofGBM43 xenografts compared with controls (Supplementary Fig.S3A and S3B), suggesting that reduced IFP levels promote druguptake. The uptake of doxorubicin, a topoisomerase II inhibitor,can be assayed on the basis of its fluorescence (35). Doxorubicin(intravenous injection) demonstrated increased (7-fold) uptakein nuclei of tumor cells following administration of SPC or nasalinjections of AF peptide in GBM43 xenografts (Fig. 2A–E), result-ing in increased survival of mice (Fig. 2F). Moreover, we observedincreased uptake of the EGFR inhibitor erlotinib along withextended survival following SPC treatment in GBM43 tumors(Supplementary Fig. S3C–S3E). As a consequence ofAF's ability tolower IFP levels, AF represents a novel therapeutic to promotedrug uptake in GBM.

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AF treatment inhibits GBM tumor growthGBM xenografts typically showed rapid elevation of IFP during

late stages of tumor development (Fig. 3A). In contrast, miceadministered SPC diet delayed increases in bioluminescence andmaintained low IFP in GBM43 xenografts (Fig. 3A). As tumorgrowth and IFP eventually increased in SPC-treated mice, weobserved a characteristic drop in IFP at terminal stages of tumordevelopment. Tumor tissue from SPC-treated mice showedreduced proliferation (Ki67) and induction of apoptosis (cleaved

caspase-3) comparedwith control tumors (Fig. 3B andC). Endog-enous AF protein induction through SPC treatment extendedoverall survival in orthotopic GBM43 xenografts (Fig. 3D). Exog-enous administration of AF protein was achieved by dissolvingSalovum (500-fold enrichment of AF protein in sterile egg yolkpowder) in the drinking water. Similarly to SPC diet, Salovumtreatment increased survival in GBM43 xenografts (Fig. 3D). SPCdiet and Salovum treatment had no effect on body weight (Sup-plementary Fig. S4A and S4B). In contrast to mesenchymal

Figure 1.

AF increases MRI-based ADC levels and lowers IFP in GBM xenografts. A and B, MRI-based measurements of ADC levels following acute nasal injections ofAF peptide or chronic induction of AF protein (SPC diet) showed increased ADC levels in GBM43 xenografts versus control. C, Setup for telemetry-based recordingsof IFP in GBM xenografts. D–E, Parallel measurements of tumor growth (bioluminescence) and IFP (mmHg) in GBM43 xenografts. F, Immunoreactivity forAF protein 10 days following administration of SPC diet to GBM43 xenografts showing high expression in tumor cells (dotted line; tumor border) but not in CD31þ

endothelial cells, GFAPþ reactive astrocytes, or CD45þ immune cells. Scale bar: top, 50 mm; bottom, 10 mm. G, IFP measurements in GBM43 xenografts. SPC dietlowered IFP levels versus control. H, Nasal administration of AF peptide effectively reduced IFP levels for 10 to 30 minutes. � , P < 0.05; �� , P < 0.01.

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GBM43 tumors that display NF1 mutations, proliferative GBM6tumors express EGFRvIII. SPC diet and Salovum treatment sig-nificantly increased survival in GBM6 xenografts (Fig. 3D). To testwhether AF therapy shows antitumor activity in an immunocom-petent GBMmodel, we administered SPC diet to mice allograftedwith Ink4a/Arf–null neural stem cells expressing humanEGFRvIII,mCherry, and LUC (Fig. 3E; ref. 34). Treatment with SPC dietinduced AF protein in mCherryþ tumors and resulted in massiveapoptosis (Fig. 3F and G). Complete tumor regression wasobserved in 5 of 6 allografted mice following treatment with SPCdiet (Fig 3H; Supplementary Fig. S4C). Thus, AF treatmentreduced proliferation and induced apoptosis of GBM cells, ulti-mately leading to reduced tumor growth and extended survival.

AF inhibits NKCC1 activity and osmotic adaption of GBM cellsWe have previously demonstrated that the NKCC1 inhibitor

bumetanide prevents restoration of cell volume by reducingtransport of sodium, potassium, and chloride ions across theplasma membrane in GBM cultures (13). As a regulator ofchloride permeability across neuronal membranes, AF proteinor its derived octapeptide regulated permeability of chloride ionsacross neuronal membranes (45, 46). To test whether AF induc-tion similar to bumetanide regulates cell volume inGBMcells (13,47), we analyzed tumor cell volume following SPC treatment inGBM43 xenografts. Actin stainings showed that AF induction intumor cells reduced cell growth (size) at terminal stages comparedwith control tumors (Fig. 4A and B). Measurements of actin(Phalloidin488) stainings from z-stacks with Imaris softwareprovided an approximation for cell volumes (Fig. 4C and D).Tumor cells in SPC-treatedmice displayed reduced relative valuesof total cell and nuclear volumes compared with controls (Fig. 4E

and F). Using an antibody that recognizes residues 198-217 ofhuman NKCC1, including the Thr203, Thr207, and Thr212phosphorylation sites, we found reduced immunoreactivity intumor cells of SPC-treated GBM43 xenografts (Fig. 4G andH). Tofurther evaluate the effects of AFon cell volume regulationofGBMcells in vitro, we loaded cultured GBM43 cells with cell-permeablecalcein dye (calcein-AM), an established marker of intracellularcell volume (41). Treatment with AF peptide (5 mg/mL) orbumetanide (10 mmol/L) inhibited regulatory volume increaseof GBM43 cells grown under hypertonic conditions (Fig. 4I). Incontrast to bumetanide-treated cells, tumor cells incubated withAFpeptide failed to surpass thebaselinewhen returned to isotonicconditions (Fig. 4J and K). AF peptide and bumetanide alsoprevented restoration of cell volume in GC22 (GBM) cultures(Supplementary Fig. S5A–S5C). We have shown that bumetanideregulates chloride permeability in GBM cells (13). Electropo-ration of GBM43 cells with the ratiometric chloride sensorClopHensor (42) demonstrated that AF increased the chlorideconcentration when tumor cells were transferred to hypertonicconditions (Fig. 4L and M). Thus, AF's ability to reduce NKCC1activation in vivo and its paralleled effects with bumetanide onosmotic adaptation in vitro provide a rationale for reduced cellsurvival in AF-treated GBM xenografts.

Compression-induced GBM cell proliferation is blocked by AFin a NKCC1-dependent manner

Total and phosphorylated NKCC1 were expressed on patient-derived GBM cells (Fig. 5A; Supplementary Fig. S6A). Immuno-blotting confirmed that NKCC1 protein was expressed in humanGBM tumorsphere cultures (Fig. 5B). Incubation of adherenthuman GBM cells with AF peptide had no effect on cell

Figure 2.

AF treatment increases doxorubicin uptake in GBM xenografts. A, Mice xenografted with GBM43 cells were given a combination of doxorubicin (Dox) andSPC diet or nasal AF.B–D,Doxorubicin (red) and DAPI (blue) in tumors 20minutes after tail vein injection of doxorubicin. Scale bar, 40 mm. E and F,Quantification offluorescence (red) shows increased uptake of doxorubicin in tumor tissues of mice treated with SPC diet or nasal AF (n ¼ 3; �� , P < 0.01, one-way ANOVA).Kaplan–Meier (n¼ 6 for control, n¼ 5 for AF, and n¼ 9 for SPC; �� , P < 0.01; ��� , P < 0.001; one-way ANOVA for control vs. AF and SPC, respectively) curve showingsurvival benefit following combination treatments with doxorubicin and SPC diet or nasal AF.

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proliferation (Fig. 5C; Supplementary Fig. S6B). To mimic con-ditions during progression, human primary GBM cells wereexpanded as tumorspheres for 4 days and mixed in a HA-based3D matrix followed by compression (30%) for 48 hours using a

force bioreactor (Fig. 5D). Compression significantly expandedthe average tumorsphere size across GBMs (GBM5, GBM14,GBM43; Fig. 5E). Smaller spheres showed even further expansionin response to compression (Supplementary Fig. S7A–S7D).

Figure 3.

AF treatment reduces tumor growth in GBM models. A, Parallel IFP and bioluminescence measurements showed that treatment with SPC diet reduced IFPlevels and tumor growth comparedwith control GBM43 xenografts.B andC, SPC treatment reduced proliferation (Ki67) and induced apoptosis (cleaved caspase-3)in GBM43 xenografts. Scale bar, 30 mm. (n ¼ 3; � , P < 0.05, Student t test; D) Kaplan–Meier curves showing increased survival in GBM43 xenografts treatedwith SPC diet (n¼ 7 for control and n¼ 12 for SPC; �� , P < 0.01; log-rank Mantel–Cox test) or Salovum (n¼ 7 for control and n¼ 5 for Salovum; � , P < 0.05; log-rankMantel–Cox test) compared with control. SPC diet and Salovum also increased survival in EGFRvIII-expressing GBM6 xenografts (n ¼ 8 for control, n ¼ 13 forSPC, and n¼ 10 for Salovum; �,P <0.05; �� ,P <0.01; one-wayANOVA).E, Immunocompetent GBMmodel based on established neurospheres from the subventricularzone of Ink4a/Arf–null FVBn mice that were lentivirally transduced to express human EGFRvIII, mCherry, and LUC. Mouse GBM cells were allografted intoadult FVBn mice and administered SPC diet on day 6. F–H, SPC treatment resulted in AF induction in tumor cells (dotted line; tumor border) and induction ofapoptosis (cleaved caspase-3). Scale bar: top, 50 mm; bottom, 20 mm (n ¼ 3; �� , P < 0.01, Student t test). Kaplan–Meier (n ¼ 5 for control and n ¼ 5 for SPC;�� , P < 0.01; log-rank Mantel–Cox test) curve showing increased survival following SPC treatment in GBM43 xenografts.

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Figure 4.

AF prevents restoration of cell volume and chloride permeability under hyperosmolar conditions. A–F, Phalloidin (F-actin, green) and DAPI (nuclei, blue)stainings show significantly reduced cellular and nuclear volumes after SPC treatment of mice xenografted with GBM43 cells. C, Orthogonal view show thatPhalloidin (arrows) is expressed around DAPIþ nuclei. D, Imaris software was used to demonstrate a correlation between approximate values for cell (Phalloidin,F-actin) and nuclear (DAPI) volumes in GBM cells. Scale bar, 20 mm (n ¼ 3; �, P < 0.05, Student t test). G and H, SPC treatment reduced immunoreactivity ofphosphorylated NKCC1 (pNKCC1) in GBM43 tumors. I, AF peptide (5 mg/mL) and bumetanide (BMT, 10 mmol/L) did not affect loading of calcein-AM dye (baseline)or retention (calibration) in GBM43 cells. Scale bar, 10 mm. J, Cell shrinkage was induced with hypertonic exposure in both bumetanide and AF peptide–treated cells with no regulatory volume increase response recorded within 20 minutes of hypertonic exposure. K, Quantification of regulatory volume increasedata (n ¼ 3; � , P < 0.05, one-way ANOVA). L, Time-lapse recordings of intracellular chloride concentrations in GBM14 cells in the presence of bumetanideor AF peptide. M, Quantifications following adjustment for pH changes showing changes in [Cl-] following incubation with bumetanide or AF peptide versuscontrol (median values of 30, 39, and 39 time-lapse experiments for bumetanide, AF, and control, respectively).

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Figure 5.

NKCC1 is necessary for AF-mediated compression-induced proliferation in GBM tumorspheres. A, NKCC1 is expressed in human GMB43 cells at the cellmembrane and in the cytoplasm. Scale bar, 10 mm. B, NKCC1 protein is expressed in four human GMB tumorsphere cultures. C, AF treatment has no proliferativeeffects on GBM43 cells grown under atmospheric pressure. D, Human GBM tumorspheres were grown under static compression (30%) for 48 hours in HAgels. E, Tumorsphere size wasmeasured following 48-hour compression (30%) for human GBM tumorspheres grown in 3D HA gels (n¼ 3; �� , P < 0.01; ��� , P < 0.001,Student t test). F, AF and NKCC1 knockdown reversed compression-induced expansion of tumorsphere size. Scale bar, 100 mm (inset 7 mm). G, Knockdownof NKCC1 by shRNA in GBM43 cells.H, shRNA-mediated knockdown of NKCC1 and the NKCC1 inhibitor bumetanide (BMT) restored compression-induced expansionof GBM tumorspheres. No further effects of AF or bumetanide were found in shNKCC1 cells. I, AF peptide reverses the protein levels of pH3 and pAKT in cellssubjected to static pressure (30%) for 48 hours (n ¼ 3; ��� , P < 0.001, one-way ANOVA).

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However, AF peptide blocked compression-induced expansion ofGBM tumorspheres following static compression (30%) for 48hours (Fig. 5F).

Similarly, shRNA-mediated knockdown of NKCC1 or the FDA-approved NKCC1 inhibitor bumetanide restored compression-induced expansion of GBM tumorspheres (Fig. 5F–H). The aver-age tumorsphere size was not further reduced when shRNANKCC1 cells were incubated with AF peptide or bumetanide,

consistent with NKCC1 as a target of both agents (Fig. 5H). Thenormalizing effect of AF peptide on compression-induced tumor-sphere size was attributed to restored cell proliferation as levels ofphosphorylatedmitotic proteins histoneH3 (pH3) and AKTwerenormalized (Fig. 5I). Transcriptional profiling of patient-derivedGBMs (GBM5, GBM14, GBM43, GBM34) showed that compres-sion significantly altered the expression profile of >3,000 genes(Supplementary Fig. S7E and S7F). Expression profiles of

Figure 6.

Combined AF and temozolomide treatment blocks regrowth in GBM xenografts. A, Bioluminescence data of mice treated with control diet, SPC, temozolomide(TMZ; 5 mg/kg, 2 consecutive days) alone, or in combination. B, Temozolomide alone or in combination with SPC diet effectively reduced tumor growth at earlystages of tumor development. C and D, Bioluminescence showing regrowth of tumors following temozolomide treatment, but not after a combination oftemozolomide and SPC diet. E, Kaplan–Meier (n ¼ 9 for control, n ¼ 8 for SPC, n ¼ 9 for temozolomide, and n ¼ 7 for SPC þ temozolomide; �� , P < 0.01; log-rankMantel–Cox test) curve displays regrowth after approximately 50 days after temozolomide treatment. Tumors in mice treated with a combination ofSPC and temozolomide never displayed regrowth.

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compressed cells incubated with AF peptide were not distinguish-able from cells under atmospheric conditions. Compressionpreferentially regulated genes involved in translational control(SNORA60, SNORA75, MIR21), stress response (ATG5, FOSB,GADD45B), and members of the solute carrier family (SLCA5P2,SLC1A4, SLC9A6; Supplementary Table S1). Thus, our data showthat NKCC1 function is necessary for compression-induced pro-liferation and transcriptional changes in GBM cells, a phenotypethat was completely reversed by AF peptide.

A combination of AF and temozolomide treatment blocksprogression and regrowth in GBM xenografts

Temozolomide is an integral part of standard care in GBMpatients (1). We have previously demonstrated that bumetanide-mediated NKCC1 inhibition accelerated temozolomide-inducedapoptosis and reduced temozolomide-induced migration inhuman GBM cultures (13, 47). Temozolomide alone or in com-binationwith SPC diet effectively reduced bioluminescence levelsin GBM43 xenografts (Fig. 6A and B). However, temozolomide-treated GBM43 xenografts underwent regrowth and 70% suc-cumbed to the disease displayed 30 to120days postinjection (Fig.6C–E). In contrast, mice administered temozolomide in combi-nation with SPC showed cessation of tumor growth and 100%overall survival at 120 days, compared with either monotherapy.These data demonstrate that AF therapy, similar to NKCC1inhibition in vitro, accelerates temozolomide-induced apoptosisof GBM cells, preventing tumor regrowth in xenografted mice.

DiscussionHigh IFP drives compression and osmotic swelling in solid

tumors. In patient-derived GBM xenografts, we used telemetry-basedmethodology to show that IFP levels parallel tumor growthduring progression. Here, we show that AF therapy reduced IFPand increased drug uptake in GBM xenografts. Collectively, AF'seffects on osmotic adaptation in vitro, its ability to reduce NKCC1activation in vivo, and its inability to block compression-inducedproliferation in NKCC1 knockdown cells suggest that intactNKCC1 function is necessary for AF-mediated antitumor activityin GBM. These results suggest that AF therapy represents an IFP-reducing approach that should be tested in clinical trials for GBMpatients.

Our data suggest that AF regulates osmotic adaptation byinhibiting NKCC1 function, resulting in reduced survival andincreased chemosensitivity of GBM cells. The activity of NKCC1 isstimulated by low chloride ion levels during swelling in astrocytes(48). NKCC1 promotes proliferation via decreasing intracellularchloride concentrations, driving efflux through chloride ion chan-nels and triggering membrane depolarization and voltage-sensi-tive calcium influx (49). The neurotransmitter GABA also regu-lates osmotic swelling in neural stem cells through type A receptorGABAAR signaling (50). NKCC1 knockdown or bumetanidetreatment inhibits GABAAR-induced depolarization and reducedproliferation of neural progenitors (51). Interestingly, low levelsof AF peptide reduced GABA diffusion and chloride ion conduc-tance across cell membranes (29, 45). It is therefore plausible thatAF and bumetanide treatment regulate osmotic adaptation andproliferation by converging on GABAARs in human GBMs.

Antiangiogenic therapies effectively reduce IFP levels in solidtumors but can also contribute to invasive recurrent tumors. Wefound that SPC treatment induced endogenous AF protein in

tumor cells but not in the surrounding tumor vasculature (Fig.1F). Previous studies showed that NKCC1 knockdown or bume-tanide treatment disrupted migration of GBM cells (11, 47).Interactions between activated NKCC1 and ezrin, acting as alinker between membrane proteins and the actin cytoskeleton,were critical topromote invasionofGBMcells (11). Thus, osmoticregulators that inhibit NKCC1 function on tumor cells rather thantumor vasculature may achieve the benefits of antiangiogenictherapies without also contributing invasive resistance.

Activated by growth factor signaling pathways geneticallyaltered in a majority of GBMs and transforming when overex-pressed (52), NKCC1 is emerging as a contributor to GBMaggressiveness. The expression of NKCC1 along with kinasesincluding upstream with-no-K (Lysine) kinase family members,oxidative stress response kinase (OSR1), and ste-20-related, pro-line-alanine-rich kinase (SPAK) all increase with glioma grade(11, 12). Administration of oral SPC takes advantage of anendogenous response, leading to induction of AF protein inregions of active fluid secretion and osmotic imbalance. On thebasis of our findings and previous work, blood–brain barrierpermeable small-molecule inhibitors that target NKCC1 or itsupstream kinases represent a promising strategy to block osmoticadaptation of tumor cells, to improve outcome in GBM patients.

The interplay between extracellular matrix mechanics and fluidpressure in solid tumors remains unclear. Here, we found that AF-mediated reduction in IFP was paralleled by reduced swelling ofGBM cells. More work is needed to determine to what extentosmotic pressure in tumor cells contributes to elevated IFP andpoor drug uptake in solid cancers. Although our studies suggestthat endogenous AF induction and exogenous AF administrationinhibit NKCC1 activation, the systemic action on NKCC1 func-tion and other potential genes need further clarification. Preced-ing clinical trials, additional studies are needed to determine theoptimal treatment regimen to achieve AF-mediated NKCC1 inhi-bition in GBMs.

Here, we report that AF-mediated inhibition of osmotic adap-tation of GBM cells reduced IFP levels and tumor growth. Thesefindings imply that osmotic adaptation is critical for survival ofGBM cells. As a result, strategies that regulate osmotic adaptationemerge as tractable approaches to improve outcome in cancerpatients.

Disclosure of Potential Conflicts of InterestW.A. Weiss has ownership interest (including patents) in Stem Synergy

Therapeutics. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: S. Ilkhanizadeh, H. Sabelstr€om, D. Sun, V.M. Weaver,W.A. Weiss, A.I. PerssonDevelopment of methodology: S. Ilkhanizadeh, Y.A. Miroshnikova,J.N. Lakins, J.A. Burdick, S. Magnitsky, D. Arosio, V.M. Weaver, A.I. PerssonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Ilkhanizadeh, H. Sabelstr€om, Y.A. Miroshnikova,A. Frantz, W. Zhu, A. Idilli, C. Schmidt, T. Fenster, E. Yuan, J.R. Trzeciak,O.R. Lindberg, S. Magnitsky, J.J. Phillips, D. Sun, V.M. Weaver, A.I. PerssonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ilkhanizadeh, H. Sabelstr€om, Y.A. Miroshnikova,W. Zhu, A. Idilli, D.A. Quigley, T. Fenster, E. Yuan, S. Saxena, S. Magnitsky,J.J. Phillips, D. Arosio, D. Sun, V.M. Weaver, W.A. Weiss, A.I. PerssonWriting, review, and/or revision of the manuscript: S. Ilkhanizadeh,H. Sabelstr€om, D.A. Quigley, E. Yuan, J.K. Mouw, J.A. Burdick, S. Magnitsky,D. Arosio, D. Sun, V.M. Weaver, W.A. Weiss, A.I. Persson

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Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Ilkhanizadeh, E. Yuan, S. Saxena, A.I. PerssonStudy supervision: M.S. Berger, D. Sun, V.M. Weaver, W.A. Weiss, A.I. Persson

AcknowledgmentsThis work was supported byU54CA163155 (to A.I. Persson,W.A.Weiss, and

V.M. Weaver), R21NS088114 (to A.I. Persson), R01NS091620 (to W.A. Weiss),1U01CA217864 (to W.A. Weiss), R01NS081117 (to J.J. Phillips),R01NS075995 (to D. Sun), Children's Tumor Foundation (to W.A. Weiss),the SamuelWaxmanCancer Research Foundation (toW.A.Weiss), theNationalBrain Tumor Society (to A.I. Persson), the Loglio Collaborative (to M.S. Berger,A.I. Persson, and W.A. Weiss), the TDC Foundation (to A.I. Persson), and theGuggenheimEndowment Fund (toA.I. Persson), the Swedish Brain Foundation(to S. Ilkhanizadeh), the Swedish Childhood Cancer Foundation (to S. Ilkha-nizadeh and O.R. Lindberg) and the Foundation BLANCEFLOR Boncompagni

Ludovisi (to S. Ilkhanizadeh andH. Sabelstr€om), the Swedish Research Council(to H. Sabelstr€om), the European Molecular Biology Organization(H. Sabelstr€om), and the American Brain Tumor Association Basic ResearchFellowship in memory of Joel A. Gingras Jr (to H. Sabelstr€om).

The authors thank patients for giving their consent and the staff at the UCSFNeurosurgery Tissue Core for providing biopsies. We also thank Stefan Lange atSahlgrenskaUniversityHospital in Sweden for providing information regardingAF function.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 31, 2017; revised December 13, 2017; accepted January 24,2018; published first February 5, 2018.

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2018;16:777-790. Published OnlineFirst February 5, 2018.Mol Cancer Res   Shirin Ilkhanizadeh, Hanna Sabelström, Yekaterina A. Miroshnikova, et al.   Produces Antitumor Activity in Glioblastoma

Mediated Inhibition of Cell Volume Dynamics−Antisecretory Factor

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