targeted brain tumor radiotherapy using an auger emittera novel approach for treating gbm is...

CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING

Targeted Brain Tumor Radiotherapy Using an AugerEmitter A C

Giacomo Pirovano1, Stephen A. Jannetti1,2,3, Lukas M. Carter1, Ahmad Sadique1, Susanne Kossatz1,Navjot Guru1, Paula Dem�etrio De Souza Franca1, Masatomo Maeda1, Brian M. Zeglis1,4,5,6,Jason S. Lewis1,5,7,8, John L. Humm9, and Thomas Reiner1,5,10

ABSTRACT◥

Purpose:Glioblastomamultiforme is a highly aggressive form ofbrain cancer whose location, tendency to infiltrate healthy sur-rounding tissue, and heterogeneity significantly limit survival, withscant progress having been made in recent decades.

Experimental Design: 123I-MAPi (Iodine-123 Meitner-AugerPARP1 inhibitor) is a precise therapeutic tool composed of a PARP1inhibitor radiolabeled with an Auger- and gamma-emitting iodineisotope. Here, the PARP inhibitor, which binds to the DNA repairenzyme PARP1, specifically targets cancer cells, sparing healthytissue, and carries a radioactive payload within reach of the cancercells’ DNA.

Results:The high relative biological efficacy of Auger electronswithin their short range of action is leveraged to inflict DNAdamage and cell death with high precision. The gamma ray

emission of 123I-MAPi allows for the imaging of tumor progres-sion and therapy response, and for patient dosimetry calculation.Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model.In vitro and in vivo studies demonstrate high tumor uptake anda prolonged survival in mice treated with 123I-MAPi whencompared with vehicle controls. Different methods of drugdelivery were investigated to develop this technology for clinicalapplications, including convection enhanced delivery and intra-thecal injection.

Conclusions: Taken together, these results represent the firstfull characterization of an Auger-emitting PARP inhibitor whichdemonstrate a survival benefit in mouse models of GBM andconfirm the high potential of 123I-MAPi for clinical translation.

IntroductionGlioblastoma multiforme (GBM) is one of the deadliest forms of

solid tumors, with a 5-year survival rate as low as 5% (1). Clinicalintervention typically consists of maximal safe surgical resectionfollowed by adjuvant chemoradiotherapy. This therapeutic regimen,

unfortunately, imparts only limited improvements to survival (2). Inaddition, the GBM molecular heterogeneity represents a robust chal-lenge in need of better imaging tools that would allow for themonitoring of therapy response and lead to better and more person-alized therapeutic plans. Furthermore, the presence of the blood–brainbarrier (BBB) biochemically limits the pharmacokinetics of manyGBM drug candidates.

A novel approach for treating GBM is therefore urgently needed,one that would address both pharmacodynamic as well as pharma-cokinetic hurdles (3, 4). Promising delivery strategies to overcomethese obstacles in the clinics include aligning novel targeting schemeswith improved drug delivery approaches including convection-enhanced delivery (CED; refs. 5, 6) and intrathecal injection (7, 8).

A known molecular biomarker for most tumors, including GBM,is PARP1 (9–15). PARP1 is recruited to the nucleus of cancer cellsand binds DNA as a single-strand break repair enzyme (16). Thiscentral role has been successfully leveraged for the development ofvarious PARP inhibitors, both as a monotherapy and in combina-tion with other therapeutics (17, 18). Modified PARP inhibitorshave also been widely used for tumor detection and imaging due totheir cancer specificity and their high target-to-backgroundcontrast (14, 15, 19–25); more recently, they have found theranosticapplications (26).

Here, we focus on PARP1-based radiotherapy with a more spo-radically utilized type of radioactive emission: Auger radiation. Augeremitters are an extremely potent radioactive source for targetedradiotherapy, characterized by their greater linear energy transfer,incredibly short range, and ability to cause complex, lethal DNAdamage as compared with traditional x-rays or b-particles (27–32).Previous attempts to use Auger emitters as cancer therapies have notbeen successful, due to the limited range of the radiation emitted andthe difficulty of reliably delivering the lethal electrons close enough tothe DNA target (<100 Å; refs. 31, 33–35).

1Department of Radiology, Memorial Sloan Kettering Cancer Center, New York,New York. 2Department of Biochemistry, Hunter College, The City University ofNew York (CUNY), New York, New York. 3PhD Program in Biochemistry, TheGraduate Center, The City University of NewYork (CUNY), NewYork, NewYork.4Department of Chemistry, Hunter College, The City University of New York(CUNY), New York, New York. 5Department of Radiology, Weill Cornell MedicalCollege, NewYork, NewYork. 6PhD Program in Chemistry, TheGraduate Center,The City University of New York (CUNY), New York, New York. 7MolecularPharmacology Program, Memorial Sloan Kettering Cancer Center, New York,New York. 8Department of Pharmacology, Weill Cornell Medical College, NewYork, New York. 9Department of Medical Physics, Memorial Sloan KetteringCancer Center, New York, New York. 10Chemical Biology Program, MemorialSloan Kettering Cancer Center, New York, New York.

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

G. Pirovano and S.A. Jannetti contributed equally to this article.

Current address for P. Dem�etrio De Souza Franca: Department of Otorhino-layrngology and Head and Neck Surgery, Federal University of Sao Paulo, SP,Brazil; and current address for S. Kossatz: Department of Nuclear Medicine,University Hospital Klinikum Rechts der Isar, Technical University Munich,Munich, Germany.

Corresponding Author: Thomas Reiner, Memorial Sloan Kettering CancerCenter, 1275YorkAvenue, NewYork, NY 10044. Phone: 646-888-3461; Fax: 646-422-0408; E-mail: [email protected]

Clin Cancer Res 2020;26:2871–81

doi: 10.1158/1078-0432.CCR-19-2440

�2020 American Association for Cancer Research.

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In this study, we developed and characterized Iodine-123 Meitner-Auger PARP1 inhibitor (123I-MAPi), the first Auger-based theranosticPARP inhibitor able to directly deliver its lethal payload within a 50 Ådistance of the DNA of GBM cancer cells (Fig. 1A). This distance iswithin the Auger radius of action, resulting in an effective preclinicalcancer treatment drug and leading to improved survival in a preclinicalGBM model. We used the 159 keV gamma ray to image tumorprogression using single-photon emission CT (SPECT) imaging andcalculate dosimetry and treatment efficacy using SPECT imaging.

Taken together, these results illustrate the tremendous potential of123I-MAPi as an Auger-emitting PARP inhibitor and a theranosticagent for GBM treatment.

Materials and MethodsGeneral

Sodium [123I]iodide in 0.1 NNaOHwith a specific activity of 7.14�107 GBq/g was purchased from Nordion. 4-(4-fluoro-3-(4-(3-iodobenzoyl)piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one

Figure 1.123I-MAPi Synthesis, binding, and efficacy in vitro.A, 123I-MAPi binding toPARP1 candeliver lethal Auger radiationwithin approximately 100Å, enough to affect DNA incancer cells when bound. B, RP-HPLC coelution of 123I-MAPi with nonradioactive I-127 analog. C, Synthetic scheme of 123I-MAPi, (a) 0.1 mol/L NaOH, Chloramine T,AcOH, MeOH 20m, RT; (b) 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, HBTU, DMAP, 2,6-Lutidine, DMSO, ACN, 2 h, 65�C. D, Cellular uptakeof 123I-MAPi in U251 GBM cell line. Blocking performed with 100-fold incubation of olaparib prior to treatment. Sodium Iodine-123 represented by red line. Michaelis–Menten curve fitting. E, Alamar Blue assay comparing 123I-MAPi efficacy with olaparib at equal molar concentrations. 123I-MAPi EC50¼ 69 nmol/L. Nonlinear fit fourparameters variable slope. F, DNA damage characterization in vitro showing immunofluorescence of g-H2AX (red), DAPI nuclear staining (blue), PARP1 expression(green), and merged images.G,Quantification of the number of g-H2AX foci within the nucleus of cells after treatment with 123I-MAPi (n¼ 85), 127I-PARPi (n¼ 125),and vehicle control (n ¼ 140). �� , P < 0.001, Kruskal–Wallis test. DSB, double-strand breaks.

Translational Relevance

Glioblastoma is one of the deadliest forms of solid tumors. Itsheterogeneity, invasiveness, and location make it difficult totreat without significant side effects. We developed an Auger-emitting theranostic PARP inhibitor, Iodine-123 Meitner-AugerPARP1 inhibitor (123I-MAPi), which is able to deliver a lethaldose of radiation specifically to tumor cells. The combinedbiophysical properties of Iodine-123 and the small-moleculePARP inhibitor convey a lethal payload in close proximity tocancer cells’ DNA with limited damage to healthy surroundingtissue. The emitted gamma rays of Iodine-123 make 123I-MAPisuitable for single-photon emission CT imaging. We presentdifferent delivery methods to overcome the blood–brain barrierand reach tumor cells within the brain. This study couldrepresent an essential milestone in the clinical development ofPARP-targeted Auger therapies.

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Clin Cancer Res; 26(12) June 15, 2020 CLINICAL CANCER RESEARCH2872




was synthesized as described previously (36). Olaparib (AZD2281)waspurchased from LC Laboratories. PARPi-FL was synthesized asdescribed previously (14, 37). Water (>18.2 MWcm�1 at 25�C) wasobtained from an Alpha-Q Ultrapure Water System and acetonitrile(AcN) as well as ethanol were of high-performance liquid chroma-tography (HPLC) grade purity. Sterile 0.9% saline solution was usedfor all in vivo injections. HPLC purification and analysis was per-formed on a Shimadzu UFLC HPLC system equipped with a DGU-20A degasser, an SPD-M20A UV detector, a LC-20AB pump system,and a CBM-20A Communication BUS module. A LabLogic Scan-RAM radio-TLC/HPLC-detector was used to detect activity. HPLCsolvents (buffer A: water, buffer B: AcN) were filtered before use.Purification of 2,5-dioxopyrrolidin-1-yl 3-(iodo-123I)benzoate wasperformed with method 1 (flow rate: 1 mL/minute; gradient: 15 min-utes 5%–95% B; 17 minutes 100% B; 20 minutes 100%–5% B); QCanalysis was performed with Method 1. Method 1 was performed on areversed-phaseC18WatersAtlantis T3Column (C18-RP, 5mm,6mm,250 mm). Purification of the final product was performed on a C6Waters Spherisorb Column (C6, 5 mm, 4.6 mm � 250 mm) withmethod 2 (flow rate: 1.5 mL/minute; isocratic: 0–30 minutes 35% B.

Cell cultureThe human glioblastoma cell line U251 was kindly provided by the

laboratory of Dr. Blasberg (MSKCC,NewYork, NY). Cells were grownin Eagle minimal essential medium (MEM), 10% (vol/vol) heat-inactivated FBS, 100 IU2 penicillin, and 100 mg/mL streptomycin,purchased from the culture media preparation facility at MSKCC(New York, NY). TS543 cells are a patient-derived glioblastoma stemline kindly provided by the laboratory of Dr. Mellinghoff (MSKCC,New York, NY). These cells were grown in suspension in Neuro-CultTM NS-A Proliferation Kit with proliferation supplement(StemCell Technologies, catalog no. 05751), 20 ng/mL RecombinantHuman EGF (StemCell Technologies, catalog no. 02633), 10 ng/mLRecombinant Human Basic Fibroblast Growth Factor (StemCellTechnologies, catalog no. 02634), 2 mg/mL heparin (StemCellTechnologies, catalog no. 07980), 1� antibiotic–antimicotic (LifeTechnologies Gibco, catalog no. 15240-062), and 2.5 mg/mL Plas-mocin (InvivoGen, Cat ant-mpp). All cells were tested for Myco-plasma contamination.

ImmunofluorescenceCells were plated on a coverslip slide on the bottom of a 6-well plate

and incubated overnight. 123I-MAPi, 127I-PARPi, or vehicle controlswere added to the media and cells were returned to the incubatorovernight. Cells were then fixed in 4% paraformaldehyde/PBS, per-meabilized, and stained with anti-g-H2AX antibody (Millipore Sigma,05-636) and anti-PARP1 antibody (Invitrogen, PA5-16452). DAPIwas used to localize nuclei. Coverslips were mounted on slides formicroscopy imaging.

ImmunoblottingCells were lysed in RIPA (Thermo Fisher Scientific, catalog no.

89900) buffer containing protease inhibitor at 4�C. Lysateswere run onan SDS-Page gel (Bio-Rad). Bound antibodies were detected bydeveloping film from nitrocellulose membranes exposed to chemilu-minescence reagent (Thermo Fisher Scientific, catalog no. 34077).PARP1 primary antibody (Santa Cruz Biotechnology, catalog no. sc-7150, 0.2 mg/mL) and goat anti-rabbit IgG-HRP secondary antibody(Santa Cruz Biotechnology, catalog no. sc-2004, 1:10,000 dilution). Ananti-b-actin antibody (Sigma, catalog no. A3854, 1:1,000) was used asloading control.

Synthesis of 123I-MAPi123I-MAPi was obtained similar to synthetic procedures reported

before (26). First, 2,5-dioxopyrrolidin-1-yl 3-(iodo-123I)benzoatewas obtained by adding N-succinimidyl-3-(tributylstannyl) benzo-ate (250 mg, 0.5 mmol) in 10 mL of AcN to a solution containingmethanol (40 mL), chloramine T (9 mg, 32 nmol), acetic acid (3 mL),and 123I-NaI in NaOH 0.1 mol/L (2.5 mCi). After the reactionsolution was driven for 20 minutes at room temperature, thereaction was purified by HPLC (method 1), and 2,5-dioxopyrroli-din-1-yl 3-(iodo-123I)benzoate at 15.1 minutes. The collected puri-fied fraction was concentrated to dryness in vacuum, reconstitutedin a solution of 80 mL can, and added to a solution of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (0.3 mg, 0.9mmol) in 20 mL DMSO, N,N,N0,N0-Tetramethyl-O-(1H-benzotria-zol-1-yl)uronium hexafluorophosphate (HBTU; 0.3 mg, 0.8 mmol) in20mLDMSO, 4-Dimethylaminopyridine (DMAP; 0.1mg, 0.8mmol) in20 mL DMSO and 10 mL triethylamine. The reactionmixture stirred inan Eppendorf ThermoMixer for 2 hours at 65�C (500 rpm). Afterward,the reaction mixture was injected and purified by HPLC (method 2)and 123I-MAPi was collected at RT ¼ 25.5 minutes (RY > 70%; RP >99%) and concentrated to dryness under vacuum. 123I-MAPi wasformulatedwith 30%PEG300/70% saline (0.9%NaCl) for both in vitroand in vivo assays. Coelutionwith nonradioactive 127I-PARPi referencecompound confirmed the identity of the radiotherapeutic. 123I-MAPiwas synthesized with molar activity of 3.93 � 0.10 GBq/mmol.

Internalization assayUptake of 123I-MAPi was tested in vitro (three replicates). 5 � 105

U251 cells were plated 24 hours prior to the experiment (n¼ 3).Mediawas changed and 1 hour later 3.7 kBq of 123I-MAPi were added to thecells. For blocking, cells were incubated with a 100-foldmolar excess ofolaparib 1 hour before adding 123I-MAPi. Media was removed, andcells werewashedwith PBS and lysed (1mol/LNaOH) at different timepoints. The lysate was collected, and uptake was determined byradioactivity on a gamma counter.

Viability assayU251 GBM cells were incubated with 0–296 kBq of 123I-MAPi or

equivalent dose of olaparib overnight and then washed and incubatedfor 4 days in normal media (three replicates, n¼ 3 each). Viability wasdetermined by AlamarBlue assay as indicated by the manufacturer.

Apoptosis assayU251 GBM cells were incubated with 370 kBq of 123I-MAPi and

apoptosis was detected at 1 and 24 hours posttreatment using an in situDirect DNA Fragmentation (TUNEL) Assay Kit (Abcam, ab66110)and following manufacturer's instructions.

Colony formation assayColony formation assay (CFA) was performed using U251 cells.

Cells were plated as single-cell suspensions and left to attach for8 hours prior to irradiation or treatment (n ¼ 3). Colony formationwasmeasured at 2weeks post 123I-MAPi treatment and comparedwithEBIR. Cells were treated adding 0–23 kBq of compound in each well.Colony count was normalized on plating efficiency at 0 kBq for eachtreatment. External beam irradiation (EBIR) was performed using aShepperd Irradiator Cs-137 at Memorial Sloan Kettering CancerCenter (New York, NY). Radiobiological parameters of the linear-quadratic model were determined via nonlinear regression withinGraphPad software, and relative biological effectiveness was deter-mined by interpolation, using 37% survival as the endpoint.

PARP1-targeted Auger Radiotheranostic in Glioblastoma

AACRJournals.org Clin Cancer Res; 26(12) June 15, 2020 2873




Animal workAll in vivo experiments were performed with female athymic nude

CrTac:NCr-Fo mice purchased from Taconic Laboratories at age 6–8weeks. During subcutaneous injections,micewere anesthetized using2% isoflurane gas in 2 L/minute medical air. 1� 106 TS543 cells wereinjected in the right shoulder subcutaneously in 150 mL volume of 50%media/matrigel (BD Biosciences) and allowed to grow for approxi-mately 2 weeks until the tumors reached about 8 mm in diameter(100 � 8 mm3).

For intravenous injections, the lateral tail vein was used. Mice werewarmed with a heat lamp, placed in a restrainer, and the tail wassterilized with alcohol pads before injection.

For orthotopic injections, TS543 cells (5 � 105 cells in 2 mLgrowth media) were injected 2 mm lateral and 1 mm posterior to thebregma using a Stoelting Digital New Standard Stereotactic Deviceand a 5 mL Hamilton syringe and allowed to grow for 3 weeks beforetreatment. All mouse experiments were performed in accordancewith protocols approved by the Institutional Animal Care and UseCommittee of MSKCC and followed NIH guidelines for animalwelfare.

SPECT/CT imagingSPECT/CT scans were performed using a small-animal NanoS-

PECT/CT from Mediso Medical Imaging Systems. For subcutane-ous TS543 xenografts, 123I-MAPi (2.3 � 0.5 MBq in 20 mL 30%PEG300 in 0.9% sterile saline) was administered intratumorally assingle injection. At chosen time points postinjection, the mice wereanesthetized with 1.5%–2.0% isoflurane (Baxter Healthcare) at 2mL/minute in oxygen and SPECT/CT data were acquired for 60minutes.

For orthotopic TS543 tumor-bearingmice, 123I-MAPi (614.2 kBq in5 mL 30% PEG300 in 0.9% sterile saline) was injected intracraniallyusing the same coordinates as for tumor cell injection (2 mm lateraland 1 mm posterior to the bregma using a Stoelting Digital NewStandard Stereotactic Device and a 5 mL Hamilton syringe). At chosentime points postinjection, the mice were anesthetized with 1.5%–2.0%isoflurane (Baxter Healthcare) at 2 mL/minute in oxygen. SPECT/CTdata were collected for 60 minutes.

Ex vivo biodistribution for intratumoral administration routeBiodistribution studies were performed in subcutaneous TS543

xenograft-bearing mice. Mice were randomized and divided in twogroups (blocked and unblocked, ntotal ¼ 6) and 123I-MAPi wasadministered intratumorally (average injected activity 1.702 �0.629 MBq in 20 mL, 30% PEG300 in 0.9% sterile saline). The blockedgroup was preinjected (1 mg/mouse in 100 mL 30% PEG300 in 0.9%sterile saline) 60minutes prior to treatment with olaparib (100mmol/Lstock inDMSO).Mice were sacrificed by CO2 asphyxiation at 18 hourspostinjection and counted in a WIZARD2 automatic g-counter (Per-kinElmer). Uptake was expressed as a percentage of injected dose pergram (%ID/g) using the following formula: [(activity in the targetorgan/grams of tissue)/injected dose].

Survival studiesMice were inoculated orthotopically at week 0 with TS543 cells (5

� 105 cells in 2 mL growth media). Cells were injected 2 mm lateraland 1 mm posterior to the bregma using a Stoelting Digital NewStandard Stereotactic Device and a 5 mL Hamilton syringeand allowed to grow for 3 weeks before treatment. At week 3, micewere randomly grouped into cohorts, and intratumorally injectedwith 123I-MAPi or 30% PEG300 in 0.9% sterile saline vehicle using

the same stereotactic coordinates as for tumor implantation. Micewere monitored daily thereafter. Study endpoint was determined onthe basis of animals’ sign of discomfort, pain, or significant weightloss.

Orthotopic CED modelALZET Osmotic Pumps were implanted subcutaneously into the

mice to slowly deliver an infusion into the brain using the samecoordinates as for the tumor cell injection as described previously (26).Control mice received an ALZET Osmotic Pump Model 1003D withBrain Infusion Kit 3 containing 30% polyethylene glycol (PEG)/PBSvehicle. Treatment mice received 123I-MAPi (average pump activity481� 111 kBq in 100 mL, 30% PEG300 in 0.9% sterile saline). Deliveryflowwas 1mL/hour, over 5 days. Pumpswere surgically removed 5 dayspostimplantation and mice monitored daily thereafter.

Intrathecal injectionBrain tumor–bearing mice were anesthetized and injected in the

intrathecal space (injection site from L3/5 or L4/5 to prevent spinalcord injury) with 2.0MBq of 123I-MAPi in 50 mL 30% PEG300 in 0.9%sterile saline. 123I-MAPi were injected and the mice imaged 1 hourpostinjection.

Hematoxylin–eosin stainingBrains were collected from mice at the time of death. The collected

brains were embedded in Tissue-Tek O.C.T. compound (SakuraFinetek), flash-frozen in liquid nitrogen, and cut into 10-mm sectionsusing a Vibratome UltraPro 5000 Cryostat (Vibratome). Sections weresubsequently subjected to hematoxylin–eosin (H&E) staining formorphologic evaluation of tissue pathology.

In vivo g-H2AX analysisMice were injected intracranially at 4 weeks posttumor implanta-

tion with either 123I-MAPi or vehicle for control (n¼ 3/cohort). Micewere perfused 1 hour postinjection and brains were fixed in 4%paraformaldehyde. Brains were then sliced and stained for g-H2AXas a marker for double-strand breaks (DSB).

Liver enzymes analysisAnimals (n ¼ 5/cohort) were injected systemically with either

123I-MAPi or Vehicle control. After 24 hours, blood was collected viaretro-orbital bleeding. Enzymes levels were determined by the Anti-tumor Assessment Core Facility at Memorial Sloan Kettering CancerCenter (New York, NY).

Organ-level and cell-level dosimetryDosimetry calculations are described in detail in the Supplementary

Materials.

Data and material availabilityAll data necessary for interpreting the manuscript have been

included in the main manuscript or in the Supplementary Materials.Additional information may be requested from the authors.

ResultsSynthesis of 123I-MAPi and in vitro validation

We previously showed that it is possible to successfully conjugatea PARP inhibitor with radioiodine without altering the high affinityto the target—maintaining an IC50 in the nanomolar range (11 � 3nmol/L) and a logPCHI of 2.3 (38). 123I-MAPi is a novel, previously

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unreported isotopolog, and was synthesized with a final molaractivity of 3.93 � 0.10 GBq/mmol. Radiochemical purity was 99.1� 0.9% for all prepared compounds (Fig. 1B).

Pharmacologic properties determined with 127I-PARPi suggest that123I-MAPi (Fig. 1C; Supplementary Fig. S1A), retains the sameproperties as 131I-PARPi, which have been shown to be similar to theFDA-approved PARP inhibitor olaparib (26, 38).

In vitro internalization was tested on U251 cells expressing PARP1(Supplementary Fig. S1B). 123I-MAPi (37 kBq/well) was added toadherent cells in monolayer and uptake was calculated by measuringgamma radiation in cell lysates at different time points. We confirmedrapid cellular internalization, with 50% of the final total uptake beingreached after 5 to 10 minutes postincubation and with an uptakeplateau reached at 1 hour posttreatment (Fig. 1D). The final totaluptake was approximately 8% of added activity, Vmax ¼ 8.2 � 0.2 %compared with blocked uptake, Vmax ¼ 3.8 � 0.1 % (Michaelis-Menten fit, R2 ¼ 0.974 and R2 ¼ 0.879, respectively). Uptake wasblocked with a 100-fold excess dose of olaparib to show targetspecificity. Na-123I was used as a control and showed significantlylower uptake, Vmax ¼ 2.5 � 0.1. The two dominant targets for 127I-PARPi, the stable-isotope labeled form of themolecule, are PARP1 andPARP2 (26), similar to what has been previously reported for olapariband other modified PARP inhibitors (15).

We tested the cancer-specific efficacy of 123I-MAPi in vitro bytreating U251 GBM cells with 123I-MAPi. Comparing 123I-MAPi withthe previously published b-emitting 131I-PARPi, we observed a 16-foldincrease in EC50 potency (EC50 of

131I-PARPi being 1148� 1 nmol/L;Supplementary Fig. S1C). 123I-MAPi treatment proved to reduceviability at safe concentrations of 123I-NaI (Supplementary Fig. S1Dand S1E). Critically, in the molar concentration range used in thesestudies, olaparib showed negligible effect in terms of cell viability,suggesting that clonogenic inactivation arises exclusively from I-123radiotoxicity as opposed to PARP1 inhibition (Fig. 1E). 123I-MAPiproved to be capable of killing cancer cells with a submicromolar EC50

(EC50 ¼ 68.9 � 1.1 nmol/L, R2 ¼ 0.999). At the same concentrations,olaparib did not show any effect in terms of cell viability, suggestingthat the observed effect is due to the PARP1 inhibitor–mediated closeproximity to the target DNA.

To characterize the induction of DNA damage in cancer cellsafter treatment with 123I-MAPi, we performed immunofluorescenceanalysis of the levels of g-H2AX foci, a known marker for DSBs (39).We treated U251 GBM cells with 740 kBq 123I-MAPi in 20 mL of30% PEG/PBS added to the media in each well (2 mL) andcompared it with the equivalent dose of nonradioactive 127I-PARPi and vehicle control (Fig. 1F; Supplementary Fig. S2A).123I-MAPi–treated cancer cells showed a significantly higher num-ber of g-H2AX foci in the cell nucleus (P < 0.001, Kruskal–Wallistest; Fig. 1G). We performed a TUNEL assay to detect apoptosisafter treatment with 123I-MAPi in vitro and detected increasedlevels of apoptotic cells at 24 hours posttreatment as comparedwith control (Supplementary Fig. S2B; P < 0.05).

Efficacy of 123I-MAPi in a CFAWe tested 123I-MAPi in comparison with externally applied photon

radiation, assessing clonogenic survival. CFAs were performed in6-well plates to compare efficacy of 123I-MAPi in cell killing whencompared with standard EBIR. U251 GBM cells were plated andtreated with Cs-137 g-rays (662 keV) or by adding 123I-MAPi to thewells (Supplementary Fig. S3A).

As expected with high linear energy transfer (LET) radiationtreatment, cells treated with the Auger-emitting small molecule

showed a steep decline in survival, confirming the high efficacy intumor cell killing when compared with EBIR (SupplementaryFig. S3B). We derived first-order estimates of relative biologicaleffectiveness of on-target 123I-MAPi through cell dosimetry calcula-tions (Supplementary Information), which suggest an increase intherapeutic potency when 123I-MAPi is bound to PARP1 (i.e., whenDNA is within reach of its Auger emissions). Radiobiological para-meters were calculated after linear-quadratic model fit of the data: 123I-MAPi a ¼ 19.8 Gy�1, b ¼ N/A. EBIR a ¼ 0.269 Gy�1, b ¼ 0.0588Gy�1. 123I-MAPi D37 ¼ 0.05 Gy, EBIR D37 ¼ 2.41 Gy. Relativebiological effectiveness (RBE) was calculated for 123I-MAPi: RBE atD37 ¼ 48.4, RBE (aI-123/aEBIR) ¼ 73.4 (Supplementary Table S1).

Biodistribution and toxicity of 123I-MAPi in vivoTS543 patient-derived glioblastoma stem cells were used to grow

tumors in athymic nude mice to investigate the biodistribution of 123I-MAPi. Tumors were grown subcutaneously on the animals’ rightshoulder. Mice were then randomized and divided into two cohorts(n ¼ 3/group) one of which was used for blocking. Blocking wasperformed systemically with an intravenous injection of olaparib1 hour prior to 123I-MAPi treatment. 123I-MAPi was injected intra-tumorally for this model. SPECT/CT images showed that 123I-MAPitumor uptake was retained at 1, 6, and 18 hours postinjection innonblocked animals, but was strongly reduced at 6 and 18 hours inblocked animals (Fig. 2A; Supplementary Fig. S4A). Biodistribution at18 hours was also examined and showed higher tumor uptake (33.4�28.0 %ID/g) compared with the tumor of blocked animals (0.4 � 0.1%ID/g; Fig. 2B). Tumor-to-muscle ratios in 123I-MAPi–treated miceversus blocked mice were more than 500 and 5, respectively, with lessthan 1%ID/g in all clearing organs.

Auger particles are highly cytotoxic when they can directly interactwith the DNA and cause complex damage. However, they are signif-icantly less so in the cellular cytosol, where they are beyond the reach oftheir DNA target (27). As liver clearance is the main route of excretionof 123I-MAPi that could cause dose-limiting problems in the clinic, weinvestigated potential clinical liver failure by observing specific livertoxicity in our preclinical study. We compared tumor and liveraccumulation of PARPi-FL, a thoroughly characterized fluorescentanalogue of the same PARP1 inhibitor with comparable biodistribu-tion and tumor uptake after intravenous injection (14, 15, 20, 38, 40).PARPi-FL was injected intravenously (50 mg/mouse) 2 hours beforecollecting the animals’ tumor and liver (Fig. 2C; SupplementaryFig. S5A). Organs were then sectioned, and tissue sections weredigitalized. Hoechst (150mL/mouse of 10mg/mL)was used for nuclearcounterstaining. PARPi-FL accumulated in the nucleus of GBM cellsas confirmed by colocalization with Hoechst signal. Livers werecollected and imaged with an inverted confocal microscope.PARPi-FL accumulation was observed in the cytoplasm of liver cells(Fig. 2D), suggesting that liver cells are protected fromAuger toxicity.To confirm this, we performed an enzyme analysis assay in the blood ofmice injected with and without prior injection of 123I-MAPi. 1.09 �0.04 MBq were injected in n ¼ 5 mice for the treated cohort, whereasthe vehicle control cohort (n¼ 5) received 150mLof 30%PEG/PBS.Nosignificant variations were observed in treated mice compared withcontrol (Supplementary Fig. S5B), suggesting a limited systemictoxicity of the injected molecule, likely due to the large distance ofthe Auger emitter from the DNA (Supplementary Fig. S5C).

Imaging of an orthotopic GBM model using 123I-MAPiTo test 123I-MAPi in a more realistic model of GBM, we orthoto-

pically implanted TS543 cells into the right brain hemisphere. This






GBMmodel proved to be very consistent in growth and take rate, as wemonitored by MRI imaging of the head. 50,000 cells were injected atweek 0, which resulted in rapid disease progression leading to animaldeath at week 7 (Fig. 3A). On the basis of this data, we decided todeliver 123I-MAPi treatment at week 3. 123I-MAPi was injected intra-tumorally using the same stereotactic coordinates as for tumorimplantation. Animals were then imaged at 1 and 18 hours posttreat-ment. Full-body SPECT/CT images were acquired, showing retentionof 123I-MAPi in the tumor at 18 hours postinjection (Fig. 3B; Sup-plementary Fig. S6A).

Therapeutic efficacy of 123I-MAPi in a GBM mouse modelWe monitored the clinical potential utility of 123I-MAPi in treating

GBM in the above described orthotopic mouse model. 123I-MAPi–treated mice were then monitored daily, using day 98 (the end of Week14), measured from the day of xenografting, as the study endpoint.Weinjected an intratumoral single dose (0.37–1.11MBq) of 123I-MAPi forthe treated cohort (n ¼ 10) and the same volume of vehicle for thecontrol cohort (n¼ 12). Survival data confirmed an improved survivalfor the 123I-MAPi treatment cohort, with a median survival of 58 days

as opposed to 40 days observed for the control cohort. Log-rank curvecomparison showed a significant difference, with P ¼ 0.009(Fig. 3C; Table 1). Animals brains were imaged ex vivo with H&Estaining and in vivo with MRI showing a diffuse presence of cancercells in vehicle-treated mice as opposed to the treatment (Fig. 3D).Mice brains (n ¼ 3/cohort) were also stained for the presence ofg-H2AX foci. We found a significantly increased number of DSB at1 hour postintratumoral injection of 123I-MAPi when comparedwith vehicle (P < 0.0001, Kruskal–Wallis test; SupplementaryFig. S6B and S6C).

Improved delivery of 123I-MAPi for clinical translationIn clinics, to improve biodistribution toward a more effective

targeted therapy, the radiopharmaceutical can be administered direct-ly into the tumor compartment. For brain tumors, this is achievedthrough intratumoral injection, intrathecal injection, or CED, anapproach that elevates the injection pressure so as to impel the agentacross the BBB. To allow for clinical translation of 123I-MAPi, we firstbuilt a preclinical model of CED. We implanted an ALZET osmoticdelivery pump with a subcutaneous catheter in tumor-bearing mice.

Figure 2.

Imaging and biodistribution of 123I-MAPi in subcutaneous TS543 mouse model. A, Specific tumor uptake of 123I-MAPi at 18 hours after local injection. Blocking wasperformed intravenously 1 hour before injection. T, tumor; Th, thyroid. B, Ex vivo biodistribution of 123I-MAPi at 18 hours after local injection. C, Uptake of PARPi-FL intumor and liver of TS543 tumor-bearing mice show nuclear uptake for tumor and cytoplasmic uptake for liver tissue. D, Quantification of PARPi-FL uptake in thenucleus and cytoplasm of tumor and liver tissue. Tumor control was performed by intravenous injection of blocking olaparib dose. Liver control was performed byinjection of vehicle. Nuclear versus cytoplasmic uptakewas automatically detected using an ImageJ script to detect colocalization of Hoechst (intravenously injected5 minutes before extracting the organs) and PARPi-FL (Student t test, �� , P < 0.01; �� , P < 0.001).

Pirovano et al.





This delivered the content of the subcutaneous reservoir at a flow rateof 1 mL/h over the course of approximately 100 hours through acannula connected to the mouse brain. Subcutaneous reservoirs werefilled with 100 mL of 123I-MAPi for the treatment cohort (n ¼ 8) andwith 100 mL of vehicle for the control cohort (n ¼ 8). Five dayspostimplantation, we surgically removed the pumps, as per manu-facturer's instruction, and monitored mice survival. Kaplan–Meiersurvival plots confirmed the therapeutic efficacy of 123I-MAPi–treatedmice presenting a median survival of 72 days as opposed 48 days inthe control cohort, a statistically significant difference of 50% (log-rankP ¼ 0.0361; Fig. 4A; Table 2). Dosimetry for the CED experiments

(Fig. 4B; Supplementary Fig. S6D) confirmed accumulation in thetumor with minimal absorbed dose to healthy organs not in directproximity to the implanted pump (Fig. 4C). Importantly, while notfeasible in a mouse model, normal organ doses would be significantlyreduced in a corresponding clinical scenario where the radionuclidereservoir would be external, shielded, and the administration per-formed over a shorter timescale.

Intrathecal injection of 123I-MAPi for a further simplified clinicaltranslation

We then investigated a more broadly used and technically feasiblebrain drug delivery method for delivering 123I-MAPi across the BBB.Tumor-bearing mice were injected with 50 mL of 123I-MAPi intrathe-cally at 3 weeks after orthotopic tumor implantation. SPECT/CTimaging was performed at 1 hour postinjection to visualize 123I-MAPi accumulation in the brain (Supplementary Fig. S7A). Imagingconfirmed favorable pharmacokinetics and specific tumor uptake,suggesting intrathecal delivery as a feasible technique for our smallmolecule. Unfortunately, this technique leads to surgery-related highlevels of stress in the mice model preventing us from being able tomonitor survival.

Figure 3.

TS543glioblastoma stem-cellmousemodel and 123I-MAPi efficacy.A,MRImonitoring of TS543 xenograft disease progression.B,SPECT/CT imagingofGBMwith 123I-MAPi single injection. Images were taken at 18 hours after local injection. Cl, clearing organs; T, tumor. C, Kaplan–Meier curve of mice injected with 123I-MAPi (localsingle injection 370 kBq–1.11 MBq, n¼ 10) comparedwith vehicle injection (n¼ 12). Log-rank (Mantel–Cox) test, �� , P <0.01.D,Cytology staining of untreatedmice atweeks 3 and 7 after tumor implantation and cytology staining and MRI imaging of treated mouse brain at 14 weeks postimplantation.

Table 1. Survival of single-dose treatment with 123I-MAPi.

nMedian survival(days) Pa

IT injection vehicle 12 50IT injection 123I-MAPi 10 58 0.0094

��

aLog-rank (Mantel–Cox) test; ��, P < 0.01.






DiscussionIn this article, we show the results for the first preclinical charac-

terization of an Auger-emitting theranostic PARP inhibitor. Wepresent a functioning workflow for the synthesis of a stable 123I-MAPi compound which proved to be effective in vitro at reducing theviability of GBM cell lines. The lethality of 123I-MAPi suggests that theDNA is in range of the Auger electrons when it is complexed withPARP1. It was further established that GBM cell death is inducedthrough radiogenic damage rather than PARP inhibition at thesetracer levels.

In vivo studies with human tumor xenograft models also show 123I-MAPi drug treatment efficacy. The 159 keV gamma emission of I-123allowed for quantification of drug uptake in tumor-bearing mice bySPECT/CT imaging. The isotope is ideal for a proposed theranosticPARPi clinical agent because of its widespread use in nuclear medicinefor thyroid disorders (Na123I) and pediatric tumors (123I-MIBG).

Animal studies employing the nonradioactive drug olaparib showprolongation of blood pool clearance and blocked tumor uptake,proving 123I-MAPi–binding specificity.

To illustrate the potential advantages of clinical translation of 123I-MAPi, and to better illustrate the impact of Auger therapeutics, welooked at the agent's potential subcellular biodistribution. In vitrog-H2AX analysis showed a significantly increased number of DSBs incells treated with 123I-MAPi when compared with 127I-PARPi orvehicle control (P < 0.001, Kruskal–Wallis test; SupplementaryFig. S2D). Furthermore, by looking at the foci morphology, it ispossible to observe larger and more clustered foci in the 123I-MAPi–treated samples as compared with spontaneously induced andbackground foci (Supplementary Fig. S2A–S2C). These observationswere also confirmed in vivo, showing elevated levels of g-H2AX focipost 123I-MAPi treatment in GBM cells when compared with control(Supplementary Fig. S6C). In this study, we did not notice anyvariation in the levels of PARP1 (green staining) as expected due toa positive feedback loop of self-amplification of PARP1 expres-sion (21). For this reason, we decided to not include a potentialPARP1 amplification effect in the kinetic modeling for dosimetrystudies. We performed a CFA to test efficacy of 123I-MAPi in U251cells. The obtained data show a steep drop in survival, which wasexpected due to the high-LET nature of Auger electrons. Comparisonwith EBIR allowed us to calculate the RBE, which corroborated thepotential of Auger-emitting molecules for targeted radiotherapy, afast-growing and promising field (41, 42). This proved to be asignificant improvement compared with a previously published

Figure 4.

Improved delivery of 123I-MAPi with an in vivo CED model. A, Kaplan–Meier survival study of pump-implanted mice shows an improvement of survival of 123I-MAPi–treated mice (n¼ 8) when compared with control (n¼ 8). Treatment mice osmotic pumps were loaded with 481� 111 kBq. Log-rank (Mantel–Cox) test, � , P < 0.05.Organ-level (B) and3Ddosimetry (C) estimates calculated byMonteCarlo simulation for subcutaneouspumpadministration. Equivalent doses assume themeasureddeterministic RBE of 48.4 for 123I-MAPi tumor tissue and assume RBE of 1.0 elsewhere. P, pump; T, tumor.

Table 2. Survival of CED dose treatment with 123I-MAPi.

nMedian survival(days) Pa

IT injection vehicle 8 48IT injection 123I-MAPi 8 72 0.0361�

aLog-rank (Mantel–Cox) test; � , P < 0.05.

Pirovano et al.





version of the molecule, 131I-PARPi, where the RBE of the emittedelectron is significantly lower. There has been a prior phase I/II clinicaltrial in which the therapeutic effects of two antibodies targeting coloncancer were studied [131I-A33 (43) and 125I-A33 (29)]. I-125 is anAuger electron–emitting radionuclide with comparable properties toI-123 (44). This study showed that the maximum tolerated activity for131I-A33 was 2.78 GBq/m2 (75 mCi/m2) in heavily pretreated patients,whereas for 125I-A33, bone marrow toxicity was not seen after admin-istered activities as high as 12.95 GBq/m2 (350 mCi/m2). In this studyas in the A33 trial, it is expected that the short-range emissions from I-123 will result in far lower normal tissue toxicity (including dose-limiting organs). In vivo, olaparib-based PARP radiotherapeutics arelikely going to be cleared via the hepatobiliary pathway (15) and theliver is therefore a potential organ for Auger-specific radiotoxicity. Inpreparation for clinical translation of 123I-MAPi, we performed studiesto investigate liver toxicity. We found near-exclusive extracellular orperinuclear localization of 123I-MAPi in liver cells (i.e., the DNA of theliver parenchymal cells is out of range ofmost of the emitted low-rangeAuger electrons) as shownwith the fluorescent analog PARPi-FL. Thisis in stark contrast to the observed nuclear accumulation of 123I-MAPiin GBM cells (Fig. 2C). The cytoplasmic liver accumulation is alsocorroborated by a liver enzyme analysis we performed on injectedmice. We did not find significant changes of enzyme levels whencompared with control mice (Supplementary Fig. S5B). The radiobi-ological effectiveness of Auger-emitting compounds is criticallydependent on the proximity of the electron emitter to the cellularDNA (45, 46). This distance-dependent relationship for DSB produc-tion has been shown for I-125 (47). Cell-level dosimetry analysisshowed a noteworthy increase in cell sterilization as a function ofabsorbed dose with 123I-MAPi in comparison with external photonirradiation, resulting in high estimates of relative biological effective-ness and suggesting that intranuclearly delivered radiation doses fromPARP1-bound 123I-MAPi are nearly 50 times as potent as that fromexternally directed photons. This estimation, though dependent on theparameters of the Monte Carlo simulation geometry, is in agreementwith the expected equivalent dose for Auger electron emitters, which iscomparable with that of intracellularly incorporated 5.3 MeV a-par-ticles (35, 48). The dose was calculated to the tumor cells usingPARaDIM v1.0, a Monte Carlo method, using as input the time-integrated activity coefficients frommeasured data (49). These cellulardose estimates were confirmed against standard cellularmethods fromMIRDcell (50). We do, however, caution that these cell dose and RBEestimates strongly depend on the measured cell size, end point,reference radiation, and model assumptions. A limitation of thisstudy is that the RBE is calculated starting from in vitro observations.This is because the orthotopic mouse model made it challenging todose-paint the tumor with external beam irradiation without simul-taneously irradiating the whole brain. On the basis of the limitedliterature on the potential use of I-123–labeled pharmaceuticals fortherapy (51, 52)—although this could be different in using I-123–labeled PARP agents)—projected in-human dose could start from animaging dose of 8 mCi and collect biodistribution data for dosimetrycalculations, guiding a phase I study in patients.

To force amore favorable biodistribution toward tumor targeting inclinical targeted radionuclide studies directed at brain tumors, inves-tigators have proposed intratumor injection (53), intrathecal injec-tion (54), andmore recently CED to administer the antibody across theBBB and into tumor tissue—for example, to treat diffuse intrinsicpontine glioma (55). The above approaches have shown the ability tosignificantly improve the ratio of the radiation dose delivered to tumorrelative to normal dose-limiting tissues. Many clinics do have the

capability to perform intratumoral and intrathecal injection with orwithout a CED system (56–58). This delivery method, albeit notsimple, is becoming more widely used as more patients present withtumors growing in the central nervous system, a consequence of theimproved efficacy of chemotherapy for the treatment of vascular-accessible disease. To emulate these studies in a preclinical setting, weadopted an orthotopic GBM model in mice and performed a localinjection of 123I-MAPi using an intratumoral osmotic pump deliverysystem. This system allowed for constant and prolonged delivery of thedrug directly to the tumor and its microenvironment. Dosimetryestimates based on imaging, tissue specimen counting, and treatmentresponse shown by Kaplan–Meier survival data are suggestive of thehigh potential clinical impact of this approach.

CEDs and Ommaya reservoirs are emerging as effective chemo-therapy and targeted radionuclide delivery methods for patients withpreviously inaccessible metastatic disease. We have developed a meth-od to perform CED injection of novel radiopharmaceuticals in pre-clinical models of cancer. We also performed intrathecal injectionwhich would allow tumor imaging and therapy in clinical settings.This technique is routinely performed in most hospitals forchemotherapy drug delivery and allows clinicians to access thebrain without opening the patient's skull. SPECT/CT imaging canvisualize and quantify radiolabeled drug uptake in tumor and itsdispersion throughout the tissues of the body. We have demon-strated this capability with a newly developed theranostic agent,123I-MAPi. We have shown that it can access the brain wheninjected intrathecally and using the intratumoral osmotic pumpdelivery system. While the favorable pharmacokinetics wasaccompanied by stressful side effects in some mice (on accountof the comparatively small volumes of the murine skull), this hasnot been, nor is it expected to be, a limiting factor for humanstudies.

To conclude, we characterized the first Auger-emitting PARPinhibitor in vivo which presented promising therapeutic results in apreclinical glioma model. The physical properties of Auger emission,paired with the biological distribution of a PARP inhibitor, make itpossible to speculate that a dose escalation in patients could achievehigh tumoricidal doseswith limitednormal tissue toxicity.The159keVgamma ray emitted is at the sweet spot for gamma camera imaging inpatients and will allow for monitoring of treatment delivery across theBBB, redistribution, and precise dose evaluation, leading to trulypersonalized treatment plans (Supplementary Fig. S7B and S7C).123I-MAPi has the potential to be a new and potent clinical agent forthe treatment of brain tumors when used in conjunction with newintrathecal/CED administration methods.

Disclosure of Potential Conflicts of InterestS. Kossatz holds ownership interest (including patents) in and is an unpaid

consultant/advisory board member for Summit Biomedical Imaging. J.S. Lewis holdsownership interest (including patents) in Summit Biomedical Imaging. T. Reiner is apaid consultant for and reports receiving commercial research grants from Ther-agnostics; reports receiving other commercial research support from Pfizer andSummit Biomedical Imaging; and holds ownership interest (including patents) inand is an unpaid consultant/advisory boardmember for Summit Biomedical Imaging.No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design:G. Pirovano, S.A. Jannetti, A. Sadique, J.S. Lewis, J.L. Humm,T. ReinerDevelopment of methodology: G. Pirovano, S.A. Jannetti, S. Kossatz, T. ReinerAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): G. Pirovano, S.A. Jannetti, A. Sadique, S. Kossatz, N. Guru,P. Dem�etrio De Souza Franca, M. Maeda






Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Pirovano, S.A. Jannetti, L.M. Carter, J.S. Lewis,J.L. Humm, T. ReinerWriting, review, and/or revision of the manuscript: G. Pirovano, S.A. Jannetti,L.M. Carter, S. Kossatz, N. Guru, P. Dem�etrio De Souza Franca, B.M. Zeglis, J.S. Lewis,T. ReinerAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): G. Pirovano, S.A. Jannetti, A. Sadique, T. ReinerStudy supervision: S.A. Jannetti, B.M. Zeglis, T. Reiner

AcknowledgmentsWe thank Dr. Cameron Brennan and the Brain Tumor Center for the GBM cell

line model. We thank Dr. Ingo Mellinghoff and Dr. Beatriz Salinas for theirhelpful discussions and for providing their expertise. We thank Dr. Pat Zanzonicoand Valerie Longo for technical support and help with animal imaging. We thankDr. Elisa de Stanchina and Dr. Vanessa R. Thompson from the Antitumor FacilityCore. We thank the Molecular Cytology Core Facility, the Animal Imaging Core

Facility, and the Radiochemistry & Molecular Imaging Probes Core Facility atMemorial Sloan Kettering Cancer Center. We thank Dr Ronald A. Ghossein, MDfor consultation on pathology slides. We also thank Garon Scott for editing themanuscript. This work was supported by NIH under grants R01 CA204441, P30CA008748, R43 CA228815, R35 CA232130, and K99 CA218875. The authorsthank the Tow Foundation and MSK's Center for Molecular Imaging & Nano-technology and Imaging and Radiation Sciences Program. L.M. Carter acknowl-edges support from the Ruth L. Kirschstein Postdoctoral Fellowship (NIH F32-EB025050).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 26, 2019; revised October 7, 2019; accepted February 12, 2020;published first February 17, 2020.

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2020;26:2871-2881. Published OnlineFirst February 17, 2020.Clin Cancer Res Giacomo Pirovano, Stephen A. Jannetti, Lukas M. Carter, et al. Targeted Brain Tumor Radiotherapy Using an Auger Emitter

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