Radium-223 Inhibits Osseous Prostate Cancer Growth by Dual ...clincancerres.aacrjournals.org/content/clincanres/early/2017/06/12/... · Cancer Therapy: Preclinical Radium-223 Inhibits

Cancer Therapy: Preclinical

Radium-223 Inhibits Osseous Prostate CancerGrowth by Dual Targeting of Cancer Cells andBone Microenvironment in Mouse ModelsMari I. Suominen1, Katja M. Fagerlund1, Jukka P. Rissanen1, Yvonne M. Konkol1,Jukka P. Morko1, ZhiQi Peng1, Esa J. Alhoniemi2, Salla K. Laine3, Eva Corey4,Dominik Mumberg5, Karl Ziegelbauer5, Sanna-Maria K€ak€onen3,6, Jussi M. Halleen1,Robert L. Vessella4, and Arne Scholz5

Abstract

Purpose: Radium-223 dichloride (radium-223, Xofigo), a tar-geted alpha therapy, is currently used for the treatment of patientswith castration-resistant prostate cancer (CRPC)with bonemetas-tases. This study examines the mode-of-action and antitumorefficacy of radium-223 in two prostate cancer xenograft models.

Experimental Design: Mice bearing intratibial LNCaP orLuCaP 58 tumors were randomized into groups (n ¼ 12–17)based on lesion grade and/or serum PSA level and administeredradium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals.X-rays and serum samples were obtained biweekly. Soft tissuetumors were observed macroscopically at sacrifice. Tibiae wereanalyzed by gamma counter, micro-CT, autoradiography andhistology.

Results: Radium-223 inhibited tumor-induced osteoblasticbone growth and protected normal bone architecture, leading toreduced bone volume in LNCaP and abiraterone-resistant LuCaP

58models. Furthermore, radium-223 resulted in lower PSA valuesand reduced total tissue and tumor areas, indicating that treat-ment constrains prostate cancer growth in bone. In addition,radium-223 suppressed abnormal bone metabolic activity asevidenced by decreased number of osteoblasts and osteoclastsand reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in theintratumoral bone matrix. DNA double-strand breaks wereinduced in cancer cells within 24 hours after radium-223 treat-ment, and PSA levels were significantly lower 72 hours aftertreatment, providing further evidence of the antitumor effects.

Conclusions: Taken together, radium-223 therapy exhibits adual targeting mode-of-action that induces tumor cell deathand suppresses tumor-induced pathologic bone formationin tumor microenvironment of osseous CRPC growth in mice.Clin Cancer Res; 1–12. �2017 AACR.

IntroductionProstate cancer is the secondmost frequently diagnosed cancer

worldwide and the third leading cause of cancer-relatedmortalityin men in developed countries (1). Although surgery and radio-therapy offer effective treatment against localized prostate cancer,curative options for metastatic prostate cancer remain elusive.Hormone ablation therapy represents the most common thera-peutic option for locally advanced or widespread prostate cancer.However, most patients eventually develop resistance to andro-

gen deprivation therapy and progress towards the terminal stageof the disease, castration-resistant prostate cancer (CRPC). Inaddition to the treatment resistance, the risk of developing met-astatic bone disease secondary to prostate cancer, and increasedbone loss due to androgen deprivation therapy represent signif-icant clinical obstacles in the treatment of patients with advancedprostate cancer (2).

Bonemetastases accompanyingprostate cancer feature a stateofaccelerated bone turnover with abnormal activation of bothosteoclasts and osteoblasts, and consequent abnormal bone for-mation, resulting in fragile, disorganized woven bone (2). Thisleads to increasedriskof skeletal fractures, immobility, severebonepain, significantdecrease inqualityof life, increasedmortality and,consequently, remarkable economic burden (3, 4). Therefore, theelimination of cancer in bone and the restoration and/or preser-vation of healthy bone morphology are important goals in themanagement and treatment of prostate cancer.

Abiraterone [cytochrome P (CYP17) inhibitor] and enzaluta-mide (androgen receptor inhibitor) have been approved by theFDA and the EuropeanMedicines Agency (EMA) for the treatmentof patients with CRPC. Although these secondary androgensuppression agents show high efficacy in Phase III trials (5–8),their impact on bone microenvironment remains largelyunknown. One report has suggested that CYP17 inhibition withabiraterone has direct effects on bone anabolic and antiresorptiveactivity (9).

1Pharmatest Services Ltd., Turku, Finland. 2Avoltus Oy, Turku, Finland. 3AurexelLife Sciences Ltd., Askainen, Finland. 4Department of Urology, University ofWashington, Seattle, Washington. 5Bayer AG, Pharmaceuticals Division, DrugDiscovery, Therapeutic Research Groups Oncology, Berlin, Germany. 6Depart-ment of Cell Biology and Anatomy, University of Turku, Turku, Finland.

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

Current address for J.P. Rissanen: PreclinApps Ltd., Raisio, Finland.

Current address for Y.M. Konkol: Department of Cell Biology and Anatomy,University of Turku, Turku, Finland.

Corresponding Author: Arne Scholz, Bayer AG, M€ullerstr. 178, 13353, Berlin,Germany. Phone: 49-30-4681-6369; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-2955

�2017 American Association for Cancer Research.

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Radium-223 dichloride (radium-223, Xofigo) is the firsttargeted alpha therapy approved by FDA and EMA for thetreatment of patients with CRPC with symptomatic bonemetastases and no known visceral metastatic disease.Radium-223 is an alpha particle (a)-emitting calcium-mimeticthat selectively binds to hydroxyapatite in the bone, targetsareas of increased bone turnover such as osteoblastic bonemetastases and improves the median overall survival in a largephase III trial in patients with CRPC and bone metastases (10).In addition, radium-223 has also been shown to significantlydelay the manifestation of the first clinically identified symp-tomatic skeletal events (SSE; ref. 11).

In a mouse model of osteolytic breast cancer bone metasta-sis, radium-223 reduced development of osteolytic lesions andimproved survival by inducing DNA double-strand breaks intumor cells and decreasing the number of osteoclasts (12).Whether radium-223 impact on CRPC bone metastasis occursvia a similar mechanism has not been investigated yet. Here, weexploited two mouse models of osteoblastic reaction resultingfrom prostate cancer growth in the bone environment mim-icking the devastating bone disease observed in prostate cancerpatients to investigate the efficacy and mode-of-action ofradium-223.

Materials and MethodsCompounds

Radium-223 was synthesized at Institute for Energy and Tech-nology (Norway) as a solution containing 60 kBq/mL of radium-223 dichloride. Abiraterone acetate (abiraterone, Zytiga) wasobtained from Janssen Biotech.

CellsLNCaP cells were purchased from the ATCC (November 2013)

andmaintained in standard cell culture conditions as indicated byprovider for three weeks until the experiment was started. Cellswere authenticated in June 2014 using short tandem repeat

analysis (GenePrint10 system, Promega) at the Institute forMolecular Medicine Finland (FIMM, Helsinki, Finland). LuCaP58 patient-derived xenograft (PDX) was licensed from the Uni-versity of Washington (13) and propagated subcutaneously inintact male SCID mice (CB17/lcr-Prkdcscid/lcrCrl, Charles RiverLaboratories) until the experimentwas started (July 2013). LuCaP58 tumors were demonstrated asMycoplasma free in January 2013using the IDEXX PCR test (IDEXX). For intratibial inoculation,tumors were harvested and processed to single-cell suspension asdescribed before (13). Briefly, the cells were mechanically disso-ciated and placed in Hank's balanced salt solution (HBSS). Redblood cells were lysed with a solution containing 0.15 mol/LNH4Cl, 0.01 mol/L NaHCO3, and 0.09 mmol/L EDTA using 5:1(vol:vol) ratio at room temperature, and the reaction was stoppedwith 3-fold volume of HBSS. The cell suspension was washed atleast two times with PBS. The viability of the cell suspension wasapproximately 20%.

In vivo modelsAll experiments were approved by the Animal Experiment

Board of Finland and performed according to the guidelines ofthe European Union directive 2010/63/EU. Mice were keptunder pathogen-free and controlled conditions and fed 2916Teklad Global diet (Harlan Laboratories, B.V., Horst, the Neth-erlands). The effects of radium-223 were studied in cell line–based LNCaP and patient-derived LuCaP 58 prostate cancerxenograft models in mice. With LuCaP 58 mice, three individ-ual studies were carried out to assess the efficacy and mode-of-action of radium-223 as well as sensitivity for abiraterone. Theadministration dose of 300 kBq/kg was selected based on aprevious dose escalation study in mouse model of breast cancerbone metastasis, representing 12% of the severely toxic doseof radium-223 to 10% of the mice (STD10) after single admin-istration (12). For the cell line–based model, LNCaP cells(2 � 106 cells in 20 mL of PBS) were inoculated into the rightproximal tibia of 7-week-old male NOD SCID mice (NOD.CB17-Prkdcscid/NcrCrl, Charles River, Germany). The mice wereanesthetized via isoflurane inhalation (IsoFlo vet, Abbot Lab-oratories). Analgesia was provided with buprenorphine(0.3 mg/mL Temgesic, RB Pharmaceuticals Ltd.) once beforethe intratibial inoculation (0.1 mg/kg, s.c.), and for 2 days afterthe intratibial inoculation and for the last 5 days of theexperiment (0.02 mg/mL in drinking water). After 6 weeks,mice with tumor-induced osteoblastic, mixed and osteolyticchanges as observed by radiography were randomized into twogroups (n ¼ 13–14 per group) based on lesion score (1–3) andserum prostate-specific antigen (PSA) value before treatment.Radium-223 (300 kBq/kg) and vehicle (28 mmol/L sodiumcitrate, Institute for Energy Technology, Norway) were admin-istered intravenously (i.v.) one day after randomization, and at4 weeks after first dose, followed by sacrifice 6 weeks after thefirst dose.

Blood samples were collected from the saphenous vein 6weeksafter LNCaP tumor cell inoculation and biweekly thereafter andadditionally by cardiac puncture at sacrifice. Body weight wasmeasured twice weekly. Tumors in soft tissues, including liver,adrenal glands, heart, lungs and pancreas, were observed macro-scopically at sacrifice. The tumor-bearing and contralateralhealthy tibiae were collected, weighed and measured for radi-um-223 activity using a gamma counter (1260 Multigamma II,LKB/Wallac). Serum PSA levels were analyzed using the

Translational Relevance

Radium-223 dichloride (Xofigo) is a targeted alpha therapydemonstrating improved overall survival in castration-resis-tant prostate cancer patients with bone metastases. All themechanisms-of-action of radium-223 in prostate cancer bonemetastasis remain incompletely understood. This study indi-cates that radium-223 therapy possesses a dual mode-of-action that inhibits tumor growth and suppresses tumor-induced abnormal bone formation, both essential players inthe destructive vicious cycle of osteoblastic bone metastasis inprostate cancer. Our findings define themechanisms bywhichradium-223 confers its observed clinical benefits and demon-strate the antitumor efficacy of radium-223 in an abiraterone-resistant patient-derived prostate cancer model with osseoustumor growth. These and our previously published data in abreast cancer bone metastasis model provide biological basisfor developing new combinations and sequential treatmentstrategies for patients with prostate and potentially other solidcancers with bone metastases. Clinical studies on these addi-tional cancer types are ongoing.

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Clin Cancer Res; 2017 Clinical Cancer ResearchOF2




Quantikine Human Kallikrein 3/PSA Enzyme-Linked Immuno-sorbent Assay (ELISA) Kit (R&D Systems) and N-terminal pro-peptide of type I procollagen (PINP) concentration was deter-mined using the Rat/Mouse PINP enzymeimmunoassay (EIA) Kit(IDS Ltd.). Themice were sacrificed with CO2 followed by cervicaldislocation.

For the PDXmodel, LuCaP 58 cells as single-cell suspension of1� 106 cells in 20 mL of PBS harvested from subcutaneous tumorswere inoculated into the bone marrow cavity of tibiae of SCIDmice (Charles River Laboratories). Mice with apparent bonechanges at 7 or 9 weeks after tumor inoculation were selectedfor the study. Blood samples were collected at 3 weeks after cellinoculation, and biweekly thereafter as described above. To assessthe efficacy, animals were randomized into two groups (n ¼ 15–17/group) based on serum PSA levels; the first mice reached theinclusion criteria (PSA > 1 ng/mL) at 7 weeks and the rest at9 weeks after tumor inoculation. Radium-223 (300 kBq/kg) or28 mmol/L sodium citrate (vehicle) were administered intrave-nously on the first day after randomization and 4 weeks later,followed by sacrifice 6weeks after the first dose, or earlier if weightloss exceeded 20%.

In the mode-of-action study, LuCaP 58 mice were randomizedinto groups (n¼ 12 per group) based on PSA level at 7 weeks aftertumor inoculation. Radium-223 (300 kBq/kg) was administeredas a single dose, followed by sacrifice at 24, 48, or 72 hours. Micein the vehicle group were sacrificed at 72 hours.

For comparison of the efficacy between radium-223 and abir-aterone in LuCaP 58 PDXmodel, mice were randomized (n¼ 17/group) based on serum PSA levels and administered with vehicle(28 mmol/L sodium citrate for radium-223 and 5% benzylbenzoate in 95% peanut oil for abiraterone), radium-223(300 kBq/kg, i.v., on the first day after randomization and 4weekslater) or abiraterone (daily, 200mg/kg, p.o.), followed by sacrifice6 weeks after the first dose.

Histology, histomorphometry, and autoradiographyTibiae were fixed in 4% PFA (paraformaldehyde) for 1–2 days,

decalcified in 14% to 10%EDTA for 2weeks, processed to paraffinblocks and then cut into 4-mm sections. Tumor and bone areaswere measured from mid-sagittal sections of each tibia stainedwith hematoxylin and eosin (H&E;Harris hematoxylin, CellPath)or Masson–Goldner Trichrome (MGT), using MetaMorph imageanalysis software (Molecular Devices, LLC).

Multinucleated (>1 nuclei) osteoclasts in the tumor-boneinterface were counted in TRACP (tartrate-resistant acid phospha-tase)-stained sections and normalized to the length of the tumor-bone interface. The whole section was analyzed. Osteoblasts werecounted from MGT-stained sections based on morphology andlocation. Two microscope fields at �10 magnification wereanalyzed.

The effect of radium-223 on inducing DNA double-strandbreaks was evaluated by immunohistochemical staining ofg-H2AX molecules (JBW301, Millipore) as previously described(14). Four microscope fields (when possible) were counted forg-H2AX–positive tumor cells using �40 objective. g-H2AX pos-itive osteoblasts and multinucleated osteoclasts on bone werecounted in TRACP and g-H2AX double-stained sections. Samples(n ¼ 6/group) were obtained from mice sacrificed at 24, 48, or72 hours after radium-223 administration, and analyzed in onemid-sagittal section per sample using Leica DM4000 B ResearchMicroscope (Leica Microsystems).

For autoradiography, undecalcified tibiae (n ¼ 6/group)from radium-223-treated mice sacrificed at 24, 48, or 72 hourspost dosing were embedded in methyl methacrylate, cut into4-mm-thick sections and after plastic removal dipped in IlfordK5 emulsion (Polysciences Inc.). The sections were then held ina light-protected box for 3 days at room temperature, processedaccording to manufacturer's instructions and counterstainedwith MGT staining.

Radiographic and micro-CT imagingRadiography was performed at two weeks and at sacrifice (days

22–25) and micro-CT imaging at sacrifice. For radiography, theanimals were anesthetized by inhalation of isoflurane and x-rayedin an anteroposterior position using the Faxitron SpecimenRadiographic SystemMX- 20 D12 (Faxitron Corp.) with FaxitronDicom 3.0 software. At least one radiograph (both hind limbs)per animal were taken at each analysis point (34 kV, 7 seconds,2-fold magnification). The formation of tumor-induced osteo-blastic, mixed and osteolytic changes in bone (lesion area) wasdetermined from the images with the MetaMorph software.Micro-CTmeasurements on the fixed tibiae were performed usingSkyScan 1072 scanner (Bruker) using voxel size of 7 mm. Themeasurement regionwas6-mmwide, starting from0.2mmbelowthe growth plate and the lower threshold for total bone intensitywas 80. Trabecular and cortical bone could not be analyzedseparately due to the massive destruction of normal bone archi-tecture in the control group. Thus, only total bone was analyzedwith lower intensity threshold of 80.

Statistical methodsStatistical analysis was performed with statistical software R

(ref. 15; version 3.1.0 or newer). All statistical analyses wereperformed as two-sided tests. For end-point analyses, data nor-mality andhomogeneity of variancewere evaluated before furtheranalyses. In the case of violating these assumptions, either log orother appropriate transformation (e.g., square root, reciprocal)was applied. If the assumptions were fulfilled as such or after atransformation, the group comparisons were carried out usingone-way ANOVA followed by Tukey's HSD post hoc test (in thecase of statistically significant ANOVA; P < 0.05). If the assump-tions were not fulfilled, Kruskal–Wallis test followed by theMann–Whitney U test for pairwise comparisons (in the case ofsignificant Kruskal–Wallis test; P < 0.05) were used. If an analysiscontained only one comparison, either the Student t test (nor-mally distributed data) or theMann–WhitneyU test (non-normaldata) was applied. For the analysis of the proportion of animalswith visceral metastases, Fisher's exact test was used. The para-meters assessed in multiple time points as well as total activitiesbetween control and tumor-bearing tibiae were analyzed usingeither fixed or mixed models (estimated using R package nlme(16). The hypotheses (e.g., comparisons between two groups atspecific time points) were tested usingmodel contrasts. In the caseofmultiple comparisons, the P values were adjusted to avoid falsepositives. Contrasts and the corresponding P values were com-puted using R package multcomp (17).

ResultsRadium-223 inhibits tumor growth in prostate cancermouse models

The effects of radium-223 on tumor growth were investigatedin LNCaP cell line and LuCaP 58 PDX models in mice

Radium-223 Mode-of-Action in Prostate Cancer

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Figure 1.

Radium-223 suppresses LNCaP and LuCaP 58 prostate cancer growth in bone in mouse models. A, Serum PSA levels in mice bearing LNCaP tumors, measuredbiweekly during dosing (mean � SD, n ¼ 10–14, P ¼ 0.02771). B, Serum PSA levels in mice bearing LuCaP 58 PDX tumors, measured biweekly during dosing(mean � SD, n ¼ 16–18, P ¼ 0.00191). C, Tumor area in mice bearing LNCaP tumors (n ¼ 12–13, P ¼ 0.00928). D, Tumor area in mice bearing LuCaP 58 tumors(n ¼ 11, P ¼ 0.09817). E, Total tissue area in mice bearing LNCaP tumors (n ¼ 7–10, P ¼ 0.91945). F, Total tissue area in mice bearing LuCaP 58 tumors(n ¼ 11, P ¼ 0.0697). G, The length of tumor–bone interface in mice bearing LNCaP tumors (n ¼ 6–10, P ¼ 0.00122). H, The length of tumor–bone interface in micebearing LuCaP 58 tumors (n¼ 8–11, P¼ 0.00014). In box plots, horizontal lines show 5th, 25th, 50th, 75th, and 95th centiles and crosses indicate mean values; ns¼ notsignificant; � , P < 0.05; ��, P < 0.01; ��, P < 0.001.

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Figure 2.

Radium-223 inhibits tumor-inducedosteoblastic reaction resulting fromLNCaP and LuCaP58prostate cancer growth in bone inmice.A andB,Representativemicro-CT reconstructions of healthy and tumor-bearing tibiae in (A) LNCaP and (B) LuCaP 58 models imaged from the medial side (measurement area starting0.5 mm below the growth plate). Respective sagittal sections are shown on the right. Bone volume of tibiae in mice bearing (C) LNCaP (n ¼ 12–13, P ¼ 0.00450)or (D) LuCaP 58 (n¼ 8–10, P <0.001) tumors. In box plots, horizontal lines show5th, 25th, 50th, 75th and 95th centiles and crosses indicatemean values. Representativeradiographs of healthy and tumor-bearing tibiae in (E) LNCaP and (F) LuCaP 58 models. Red line delineates the osteoblastic/osteolytic/mixed lesion area.Osteoblastic/mixed lesion area measured biweekly during dosing in mice bearing (G) LNCaP tumors (mean � SD, n ¼ 13, P ¼ 0.00366) and (H) LuCaP 58 tumors(mean � SD, n ¼ 11–17, P ¼ 0.01702). I, PINP levels in mice bearing LNCaP tumors were measured biweekly (mean � SD, n ¼ 13, P < 0.001). J, PINP levels in micebearing LuCaP 58 tumors were measured biweekly (mean � SD, n ¼ 11–15, P ¼ 0.00548); � , P < 0.05; ��, P < 0.01; ��, P < 0.001.






(Supplementary Table S1). Serum PSA levels and lesion areas(osteoblastic, mixed, and osteolytic changes in bone determinedusing radiography) were measured biweekly and tumor growthwas evaluated histologically. Mice treated with radium-223(300 kBq/kg, twice with 4-week interval) exhibited lower serum

PSA levels in comparison with vehicle control (LNCaP, P ¼0.02771; LuCaP 58, P ¼ 0.00191; Fig. 1A and B). Despite theclear reduction compared with control, PSA level remained stableor increased during time course in radium-223-treated LNCaPand LuCaP 58 xenografts, respectively, similar to clinical

Figure 3.

Radium-223 suppresses pathologicbone formation in LNCaP and LuCaP 58models of prostate cancer growth inbone.A, Representative MGT staining ofbone architecture and tumor area inLNCaP and LuCaP 58 tumor-bearingmice treated with vehicle or radium-223(300 kBq/kg, i.v.). Turquoise indicatesbone, pale pink tumor, and dark pinkbone marrow; scale bar, 1 mm; �25magnification.B,Total bone area inmicebearing LNCaP tumors (n ¼ 12–13, P ¼0.00059). C, Total bone area in micebearing LuCaP 58 tumors (n ¼ 11, P ¼0.05883). D, Trabecular bone arearelative to bone marrow area in micebearing LNCaP tumors (n ¼ 12–13, P ¼0.43710). E, Trabecular bone arearelative to bone marrow area in micebearing LuCaP 58 tumors. (n ¼ 11, P ¼0.0212). F, The number of osteoblastsrelative to bone surface in mice bearingLNCaP tumors (n¼ 12–13, P¼ 0.00127).G, The number of osteoblasts relative tobone surface in mice bearing LuCaP 58tumors (n ¼ 11, P ¼ 0.0014). H, Thenumber of osteoclasts relative totumor–bone interface (TBI) in micebearing LNCaP tumors (n ¼ 6–10, P ¼0.01207). I, The number of osteoclastsrelative to TBI in mice bearing LuCaP 58tumors (P¼ 8–11, 0.19644). In box plots,horizontal lines show 5th, 25th, 50th, 75th,and 95th centiles and crosses indicatemean values. ns, not significant; � , P <0.05; �� , P < 0.01; �� , P < 0.001.

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observations (18, 19). Histological evaluation of tumor-bearingtibiae at sacrifice revealed reduced tumor area (LNCaP,P ¼ 0.00928, 68% reduction; LuCaP 58, P ¼ 0.09817, 25%reduction), total tissue area (consisting of bone, bone marrowand tumor; LNCaP, P ¼ 0.00076, 16% reduction; LuCaP 58,P¼ 0.00655, 26% reduction), and tumor-bone interface (LNCaP,P¼ 0.00122, 82% reduction; LuCaP 58, P¼ 0.00014) in radium-223–treated mice as compared with vehicle control (Fig. 1C–H).In LuCaP58mice, the reduction in tumor areawas not statisticallysignificant. This difference may be due to the fact that the LuCaP58 tumor was growing more aggressively than LNCaP, as indi-cated by a 5-fold higher average tumor area in LuCaP 58 micecompared with LNCaP model. Furthermore, the proportion ofbone marrow occupied by tumor cells (not including area oftumor-induced bone) was decreased in radium-223–treatedLNCaP xenografts, whereas in LuCaP 58 xenografts no differencewas observed (LNCaP, P ¼ 0.02486, 57% decrease; LuCaP 58,P ¼ 0.72855, 2% decrease) presumably due to more aggressivetumor growth. In addition, the weight of tumor-bearing tibiae atsacrifice was decreased in LuCaP 58 xenografts in response toradium-223 treatment (38% decrease; P ¼ 0.02697). Takentogether, these data indicate that radium-223 inhibits tumorgrowth in both LNCaP and LuCaP 58 xenograft models.

Radium-223 inhibits tumor-induced bone formation in bothLNCaP and LuCaP 58 xenograft models

SREs associated with prostate cancer growth in bone are causedby pathologic and structurally weak bone resulting from abnor-mal activation of both osteoclasts and osteoblasts by prostatecancer. To better understand the role of radium-223 in preventingthe SREs, the effects of radium-223 on bone were investigated inLNCaP and LuCaP 58 xenograft models. In both models, miceadministered with radium-223 at 300 kBq/kg, twice with 4-weekinterval, exhibited significantly reduced bone volume (LNCaP,P ¼ 0.00450, 17% reduction; LuCaP 58, P < 0.001, 28% reduc-tion), bone surface area (LNCaP,P<0.001, 22%reduction; LuCaP58, P < 0.001, 45% reduction), and bone surface density (LNCaP,P<0.001, 21% reduction; LuCaP 58, P<0.001, 30% reduction) intumor-bearing tibiae in comparison with vehicle control, asmeasured by micro-CT (Fig. 2A–D). The lesion area consistingof mainly osteoblastic, but also mixed and osteolytic tumor-induced pathologic changes as observed by radiography wasdecreased in both LNCaP (P ¼ 0.00366) and LuCaP 58(P ¼ 0.01702) tumor-bearing tibiae (Fig. 2E–H). In addition,radium-223 treatment resulted in lower levels of bone formationbiomarker PINP in LNCaP (P < 0.001) and LuCaP 58(P ¼ 0.00548) models in comparison with untreated animals(Fig. 2I and J). These results indicate that radium-223 suppressesthe abnormal bone formation in tumor microenvironment.

The beneficial effect of radium-223 on tumor-induced patho-logical bone effects was further supported by histomorphometryof LNCaP and LuCaP 58 tumor-bearing tibiae showing markedlyreduced total bone area (LNCaP, P < 0.00059, 16% reduction;LuCaP58,P¼0.059, 30% reduction) and relative trabecular bonearea (LNCaP, P¼ 0.00564, 12% reduction; LuCaP 58, P¼ 0.0212,56% reduction) in LNCaP and LuCaP 58 models in comparisonwith vehicle control (Fig. 3A–E). Similarly to tumor area, thereduction in total bone area was not significant in LuCaP 58model. Histological analyses revealed that tibiae were filled withLuCaP 58 tumor and additional ectopic tumor growth wasobserved (Fig. 3A and B), resulting in higher total tissue area

in these mice compared with LNCaP xenografts (Fig. 1E and F).With regard to bone cells, radium-223 reduced the number ofosteoblasts relative to bone surface in histologic sections of bonemetastases in both models (Fig. 3F and G; LNCaP, P ¼ 0.00127,90% reduction; LuCaP 58,P¼ 0.0014, 77% reduction) and a totaleradication of osteoclasts at tumor-bone interfacewas observed inLNCaP model (P¼ 0.01207) in response to radium-223 therapy.In contrast, in LuCaP 58 xenografts no difference was observed(P¼0.19644) in thenumber of osteoclasts relative to tumor-boneinterface in response to radium-223 therapy (Fig. 3H and I),although the tumor-bone interface length and thus also theabsolute number of osteoclasts at tumor-bone interface wasdecreased (P ¼ 0.00106, 61% reduction). Taken together, thesedata demonstrate that radium-223 decreases the tumor-inducedpathologic bone formation by suppressing the accelerated boneturnover in tumor-bearing tibiae.

Interestingly, unlike radium-223, abiraterone treatment wasnot able to reduce tumor growth or pathologic bone changesin intratibial LuCaP 58 PDX model (Supplementary Fig. S1,Supplementary Table S2), suggesting that radium-223 couldbe a potential option for patients with acquired abirateroneresistance.

Visceral metastases in radium-223–treated miceTo evaluate the effect of radium-223 in the development of

metastases in soft tissue, mice bearing LuCaP 58 cells weremacroscopically evaluated for visceral metastases. The numberof mice affected by visceral spread was lower in the radium-223treatment group; however, the differences were not statisticallysignificant (Fig. 4). In mice bearing LNCaP cells, tumor growthwas not observed in other sites than in the inoculated tibiae.

Radium-223 is deposited in the intratumoral bone matrix,allowing local targeting of a particles

The localization of radium-223 dichloride particles withinosteoblastic tumors was studied by autoradiography in micesacrificed at 24, 48, and 72 hours after single intravenous admin-istration of radium-223. In mice intratibially inoculated withLuCaP 58 cells, a vast majority of radium-223 deposits wasobserved within the bone matrix and especially in the vicinityof activated osteoblasts. In addition, a few radium-223 depositsco-localized with prostate cancer cells (Fig. 5A). The mean valuefor total activity (gamma counts perminute) was higher in tumor-bearing tibiae compared with control non-tumored tibiae at24 and 48 hours (Fig. 5B; 24 hours, P ¼ 0.00590; 48 hours,P ¼ 0.00332).

A single radium-223 injection induces immediate effectson tumor cells, osteoblasts, and osteoclasts in prostatecancer growing in bone

The immediate effects on tumors were studied after a singledose of radium-223 in LuCaP 58 tumor-bearing mice. Interest-ingly, radium-223 decreased serum PSA level in comparison withvehicle control (P¼ 0.04926) at 72 hours post injection (Fig. 5C).Furthermore, there was a trend for increased relative necrotictumor area in tumor-bearing tibiae over time, although signifi-cance was not reached (Fig. 5D). The effect of radium-223 oninducing DNA double-strand breaks was evaluated by immuno-histochemical staining of g-H2AX molecules in LuCaP 58 tumor-bearing tibiae (14). DNA double-strand breaks were increased intumor cells at all time points (P¼ 0.00297, 0.03665, and 0.00714






for 24, 48, and 72 hours, respectively) and in osteoblasts andosteoclasts at 72 hours after dosing (P ¼ 0.03148 and 0.00071,respectively), compared with vehicle-treated mice at 72 hours(Fig. 5E–G).

DiscussionMetastasis to bone has detrimental effects on bone, quality of

life, and survival (20, 21). Although currently used palliativetherapies for bone metastases in CRPC, such as zoledronic acidand the RANK-L inhibitor denosumab, are capable of reducingSREs and provide pain palliation (22–25), the targeted alphatherapy radium-223 is the first drug with proven beneficialeffect on overall survival (10). However, it has not been yetinvestigated in detail whether the beneficial clinical effects inprostate cancer patients are solely due to radium-223 affectingosteoblasts and/or osteoclasts, or if the treatment also has adirect effect on prostate cancer cells. Our results from preclin-ical studies with two different prostate cancer xenograft models,LNCaP representing osteoblastic/mixed lesions and LuCaP 58PDX representing osteoblastic lesions, demonstrate markedtumor growth suppression, and inhibition of tumor-inducedbone alteration in radium-223-treated animals. We have pre-viously reported similar findings with radium-223 treatment ina mouse model of osteolytic breast cancer bone metastasis (12),indicating that radium-223 is effective regardless of the primarytumor origin and the type of tumor-induced pathologicaleffects on bone. In addition to the distinct tumor-inducedreactions in bone, the observed differences in certain para-meters may derive from the use of different mouse strains anddistinct properties of the tumor cells. LNCaP is a cell line–derived model whereas LuCaP 58 model represents a PDXmodel. Furthermore, LuCaP 58 tumors grow very aggressively

in comparison with LNCaP tumors, resulting in extra-osseoustumor growth and consecutively increased total tissue area.

Radium-223 uptake was recently characterized in healthymice as well as in osteoblastic (LNCaP) and osteolytic (PC3)prostate cancer–bearing animals (26). This study showed thatbone, specifically the areas of active bone remodeling andvascular supply, is the main target site for radium-223 accu-mulation. In mice with prostate cancer growing in bone,radium-223 accumulated on the bone surface next to themalignant site (26). Here, we assessed radium-223 depositionpattern in LuCaP 58 model representing a very strong osteo-blastic component and demonstrated the efficient depositionof radium-223 into the intratumoral bone matrix, with themost substantial occurrence in the proximity of activatedosteoblasts. A portion of radium-223 deposits was found inthe midst of prostate cancer cells. The deposition pattern was inagreement with the increased uptake measured using gammacounter. However, the extent and nature of co-localization withtumor cells remains to be elucidated. The localization ofradium-223 to the bone microenvironment adjacent to tumor,combined with the short track length of a-ray (2–10 celldiameters, < 100 mm), effectively limits the damage only toadjacent cells. This is further substantiated by high tolerabilityin mice and a low rate of hematological side effects in clinicaltrials with radium-223 (27–30).

Radium-223 inducedDNAdouble-strand breaks in tumor cells24, 48, and 72 hours after dosing in mice bearing LuCaP 58tumors. In addition, serum PSA level compared with vehiclecontrol was decreased already at 72 hours post treatment, indi-cating that the induction of DNA double-strand breaks is asso-ciated with significant tumor cell death. Furthermore, clear tumornecrosis was observed in tumor-bearing tibiae treated with radi-um-223. The observed radium-223 deposition pattern, togetherwith its early effects on tumor cells, osteoblasts and osteoclasts,suggests direct and potent radiation effects on both tumor andbone cells in osseous tumor growth.

In addition to bone metastases, prostate cancer also causesvisceralmetastases to soft tissue in approximately 20%of patientsparticipating in first-line studies for CRPC (31–33). Additionalpreclinical studies are necessary to investigate the possible rolesand mode-of-action of radium-223 in inhibiting overall meta-static spread. The ability of radium-223 to trigger tumor cell deathand to prevent both osteolytic and osteoblastic metastasis pro-gression in the bone as well as secondarymetastases to soft tissuesconcurrently would render this agent a very powerful therapy inthe treatment of diverse solid tumors.

The observed decreases in both overall tumor burden asmeasured by PSA and in pathologic bone changes as deter-mined using the bone formation marker PINP in response toradium-223 treatment are in line with changes in PSA andalkaline phosphatase (ALP) observed in clinical setting(18, 19). Our preclinical data also suggest a dependencybetween decreased osteoblastic activity and decreased tumorburden in radium-223–treated mice as measured by variousparameters reflecting pathologic bone formation and tumorgrowth, respectively. Future studies with larger treatmentgroups are needed to determine if there is an associationbetween ALP and response to radium-223.

There are currently multiple therapies with very distinctmechanisms of actions and unique toxicities available for thetreatment of CRPC (5, 10, 31–38). Regrettably, clinical

Figure 4.

The effect of radium-223 treatment on visceral metastases in LuCaP 58 prostatecancer PDXmodel in mice. The number of mice with visceral metastases amongmice treated with 28 mmol/L sodium citrate as vehicle or with radium-223(300 kBq/kg, i.v.). The bars represent all mice and mice with smaller (PSA < 5ng/mL) and larger (PSA > 5 ng/mL) amounts of tumor growth. The numbersabove the bars represent the number of mice with visceral metastasis per thenumber of mice in a respective group.

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application has revealed that a large number of patients acquireresistance to most therapies after a short period of treatmentand moreover, multiple patients exhibit de novo resistance. Ofnote, our data, similarly to clinical situation show that radium-223 is active in abiraterone-resistant prostate cancer, excludingthe unlikely possibility that abiraterone resistance impairs theefficacy of radium-223. Resistance has not been described in

association with alpha therapy. However, the impact of geneticalterations, such as mutations in or copy-number variation ofDNA repair mechanism genes, on the antitumor efficacy ofradium-223 treatment in preclinical and clinical studies needsto be elucidated. For example, publicly available genomic datain LNCaP cells reveal several defects in DNA repair genes, suchas ATM and BRCA2 (39).

Figure 6.

Radium-223 therapy exhibits a dualtargetingmode-of-action that destroystumor cells and inhibits tumor-inducedpathologic bone reaction. A, Tumor-induced dysregulation of boneremodeling is a key driver in prostatecancer–derived bone metastases.Prostate cancer bone metastases arecommonly associated with abnormalosteoblastic bone growth. Prostatecancer cells release paracrine factorsthat affect both osteoblastic andosteoclastic activity and stimulateabnormal bone remodeling.Osteoblasts, in turn, secrete factorsthat promote progression of theprostate cancer metastases andstimulate osteoclast activity. Thegrowth factors released from bone byactivated osteoclasts further stimulatetumor growth. This complex viciouscycle of prostate cancer bonemetastasis results in increased tumorgrowth and altered bone structurestability, resulting eventually in bonedestruction. RANKL, receptor activatorof nuclear factor kappa-B ligand. B,Radium-223 is a targeted alpha therapywith a dual targeting mode-of-action.Radium-223 inhibits the vicious cycle ofprostate cancer bone metastases byinducing tightly localized cytotoxiceffects through double-stranded DNAbreaks in osteoblasts, osteoclasts, andprostate cancer cells.

Figure 5.Radium-223 is deposited in the intratumoral bone matrix and induces DNA double-strand breaks in tumor cells, osteoblasts, and osteoclasts in LuCaP 58 mousemodel of prostate cancer growth in bone. A, Autoradiography analysis of undecalcified tissue sections was used to define the localization of radium-223 particles(black dots) in osteoblastic/mixed bone metastases. Analysis was done at 24 hours after single intravenous administration of radium-223 (300 kBq/kg).Green arrows indicate radium-223 deposition in the proximity of osteoblasts, and blue arrows point to radium-223 deposits within the tumor bed; scale bar, 100 mm;�200 magnification. B, Total activity of healthy and contralateral tumor-bearing tibiae of mice treated with single dose of radium-223 measured with agammacounter (cpm, counts perminute; n¼ 11–12).C,Relative serumPSA72hours after radium-223 dosing (%of value onday0;mean�SD, n¼ 11–12,P¼0.04926).D, Relative necrotic tumor area in animals sacrificed 24, 48, or 72 hours after a single dose of radium-223 and 72 hours after vehicle dosing (n ¼ 4–5, P ¼ 0.27134).E, g-H2AX–positive tumor cells (n/microscope field). The animals were sacrificed 24, 48, or 72 hours after a single radium-223 dose and 72 hours after vehicle dosing(n¼ 4–5; P¼0.00297, 0.03665, and 0.00714 for 24, 48, and 72 hours, respectively. F, g-H2AX positive osteoblasts (% of all osteoblasts). The animalswere sacrificed24, 48, or 72 hours after a single radium-223 dose and 72 hours after vehicle dosing (n ¼ 3–5; P ¼ 0.03148 for 72 hours). G, g-H2AX positive osteoclasts(% of all osteoclasts). The animals were sacrificed 24, 48, or 72 hours after a single radium-223 dose and 72 hours after vehicle dosing (n ¼ 4–5; P ¼ 0.00714for 72 hours). In box plots, horizontal lines show 5th, 25th, 50th, 75th, and 95th centiles and crosses indicate mean values. ns ¼ not significant; � , P < 0.05; ��,P < 0.01; �� , P < 0.001.

Suominen et al.





Radium-223 was recently shown to induce T-cell–mediatedlysis in human prostate, breast, and lung carcinoma cells (40).In our study with preclinical CRPC models established inimmunocompromized mice, the host immune response andpotential immunotherapeutic role of radium-223 in prostatecancer could not be addressed. Additional preclinical studiesusing immunocompetent mice will be useful in evaluating theimmunotherapeutic effects of radium-223 in prostate cancerand the efficacy of radium-223 in combination with immunecheckpoint inhibitors, such as PD-(L)1 inhibitors. Clinicalevaluation of this is currently ongoing in a Phase I studyevaluating the safety and tolerability of radium-223 in combi-nation with atezolizumab (NCT02814669).

Taken together, our results indicate that radium-223 therapyexhibits a dual targeting mode-of-action that inhibits diseaseprogression via tightly localized cytotoxic effects on tumor cellsand stabilization of the bone microenvironment in bone metas-tases (Fig. 6).

On the basis of clinical findings, our previously publishedresults (12) and the data presented here, the potential of radi-um-223 in delaying time to SREs and bonemetastases in patientswith earlier phase of prostate cancer and also inpatientswithbonemetastases from other solid tumors need to be evaluated. Thefocus of investigation is now on developing optimal combina-tions of thenew therapeutic agents to effectively target the primarytumor as well as preventmetastasis to bone, resulting in increasedsurvival and lower patient morbidity. Preliminary data from apost-hoc study exhibited improved overall survival in patientstreated with radium-223 and concomitant abiraterone, enzalu-tamide, or denosumab, raising positive expectations for furtherresearch (30).

Disclosure of Potential Conflicts of InterestM.I. Suominen, K.M. Fagerlund, J.P. Rissanen, and J.M. Halleen hold own-

ership interest (including patents) in Pharmatest Services Ltd. D. Mumberg and

A. Scholz hold ownership interest (including patents) in Bayer AG.No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.I. Suominen, J.P. Rissanen, D. Mumberg,K. Ziegelbauer, S.-M. K€ak€onen, A. ScholzDevelopment of methodology: M.I. Suominen, K.M. Fagerlund, E. Corey,A. ScholzAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.I. Suominen, Y.M. Konkol, J.P. Morko, Z. PengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.I. Suominen, E.J. Alhoniemi, S.K. Laine,S.-M. K€ak€onen, A. ScholzWriting, review, and/or revision of the manuscript: M.I. Suominen,K.M. Fagerlund, J.P. Rissanen, Y.M. Konkol, J.P. Morko, E.J. Alhoniemi,S.K. Laine, E. Corey, D. Mumberg, K. Ziegelbauer, S.-M. K€ak€onen, J.M. Halleen,R.L. Vessella, A. ScholzAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.M. Fagerlund, J.P. Rissanen, Y.M. Konkol,S.K. Laine, E. Corey, S.-M. K€ak€onenStudy supervision: S.-M. K€ak€onen, J.M. Halleen, A. Scholz

AcknowledgmentsThe authors thank Natalia Habilainen-Kirillov, Riikka Kyt€omaa, Anniina

Luostarinen, Johanna €Orling, and Jani Sepp€anen for their skillful technicalassistance. Aurexel Life Sciences Ltd. (www.aurexel.com) is acknowledged forthe editorial support funded by Bayer AG.

Grant SupportThe establishment of the LuCaP 58 PDX was supported by grants from the

National Institutes of Health (to E. Corey, National Institutes of Health,NIHPO1 CA085859 and NIHPO1 CA163227). Bayer AG has paid PharmatestServices Ltd. for the execution of the experiments. Editorial support was fundedby Bayer AG.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 28, 2016; revised January 5, 2017; accepted March 29,2017; published OnlineFirst March 31, 2017.

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Published OnlineFirst March 31, 2017.Clin Cancer Res Mari I. Suominen, Katja M. Fagerlund, Jukka P. Rissanen, et al. in Mouse ModelsDual Targeting of Cancer Cells and Bone Microenvironment Radium-223 Inhibits Osseous Prostate Cancer Growth by

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