Theranostic Agents for Photodynamic Therapy ofProstate Cancer by Targeting Prostate-SpecificMembrane AntigenXinning Wang1, Brian Tsui2, Gopolakrishnan Ramamurthy1, Ping Zhang3, Joseph Meyers1,Malcolm E. Kenney3, Jonathan Kiechle4, Lee Ponsky4, and James P. Basilion1,5
Abstract
Prostatectomy has been the mainstay treatment for men withlocalized prostate cancer. Surgery, however, often can result inmajor side effects, which are caused from damage and removalof nerves and muscles surrounding the prostate. A technologythat can help surgeons more precisely identify and removeprostate cancer resulting in a more complete prostatectomy isneeded. Prostate-specific membrane antigen (PSMA), a type IImembrane antigen highly expressed in prostate cancer, hasbeen an attractive target for imaging and therapy. The objectiveof this study is to develop low molecular weight PSMA-targetedphotodynamic therapy (PDT) agents, which would provideimage guidance for prostate tumor resection and allow forsubsequent PDT to eliminate unresectable or remaining cancercells. On the basis of our highly negatively charged, urea-based
PSMA ligand PSMA-1, we synthesized two PSMA-targeting PDTconjugates named PSMA-1-Pc413 and PSMA-1-IR700. Inin vitro cellular uptake experiments and in vivo animal imagingexperiments, the two conjugates demonstrated selective andspecific uptake in PSMA-positive PC3pip cells/tumors, butnot in PSMA-negative PC3flu cells/tumors. Further in vivophotodynamic treatment proved that the two PSMA-1–PDTconjugates can effectively inhibit PC3pip tumor progression.The two PSMA-1–PDT conjugates reported here may have thepotential to aid in the detection and resection of prostatecancers. It may also allow for the identification of unresectablecancer tissue and PDT ablation of such tissue after surgicalresection with potentially less damage to surrounding tissues.Mol Cancer Ther; 15(8); 1834–44. �2016 AACR.
IntroductionPSA testing has allowed significantlymoremen tobediagnosed
and treated for prostate cancer. Approximately 220,800 newdiagnoses and 27,540 deaths from the disease are projected in2015 in the United States (1). Over 90% of men have localizedtumors at initial screenings and are candidates for radical pros-tatectomies (2). However, at surgery, cancer has been shown toextend outside the prostate (pathologic stage C) in 20% to 42%ofpatients (3), surgery fails to halt the disease in approximately 20%of the patients who undergo radical prostatectomy, and recur-rence rate of this disease is more than 60% (4–6).
During radical prostatectomy, surgeons have difficulty acces-sing prostate cancer invasion; therefore, manymalignant nodulesescape detection, leading to disease recurrence. A retrospective
multivariate analysis by Wright and colleagues of incompleteresection of prostate cancer in more than 65,000 patients whounderwent radical prostatectomies (7) found that positive surgi-cal margins were associated with a 2.6-fold increased unadjustedrisk of prostate cancer–specific mortality and are an independentpredictor of mortality. This study also underscored the need forsurgeons to optimize surgical techniques to achieve negativesurgical margins to increase sound oncological outcomes. How-ever, surgical achievement of this without side effects is challeng-ingbecause theprostate is surroundedbymanynerves andmusclefibers controlling different excretory and erectile functions that aredifficult but necessary to avoid. In 1983, Walsh defined nervelocations around the prostate and inspired a number of "nerve-sparing" surgical approaches, including robotic-assisted laparo-scopic prostatectomy (8). Unfortunately, the success of theseapproaches to mitigate side effects is mixed, (9, 10) and surgicalapproaches are still associated with significant morbidity, forexample, incontinence (3%–74%) and impotence (30%–90%;refs. 11–16). There remains an unmet clinical need to improvesurgical techniques for identifying and removing cancerous tissuewithout damaging surrounding tissues during prostatectomy.Recently, Neuman and colleagues showed that the near-infrared(NIR) fluorescence probe YC-27 can improve the surgical treat-ment of prostate cancer and reduce the rate of positive surgicalmargins in real-time laparoscopic extirpative surgery (17).
Photodynamic therapy (PDT) is a minimally invasive therapyused clinically in the treatment of cancers and other diseases(18–20). PDT uses photosensitizers which are pharmacologicallyinactive until exposed to light in the presence of oxygen. The activedrug forms reactive oxygen species such as singlet oxygen to kill
1Department of Radiology and NFCR Center for Molecular Imaging,Case Western Reserve University, Cleveland, Ohio. 2School of Medi-cine, Case Western Reserve University, Cleveland, Ohio. 3DepartmentofChemistry,CaseWesternReserveUniversity,Cleveland,Ohio. 4Urol-ogy Institute, University Hospitals Case Medical Center, Cleveland,Ohio. 5Department of Biomedical Engineering, CaseWestern ReserveUniversity, Cleveland, Ohio.
Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: James P. Basilion, Case Western Reserve University,11100 Euclid Ave, Wearn Building B-44, Cleveland, OH 44106-5056. Phone: 216-983-3264; Fax: 216-844-4986; E-mail: [email protected]
cells. Various agents, including porphyrins and phthalocyanines,have been examined as photosensitizers (21–23). Most photo-sensitizers are fluorescent and some can emit NIR light that can beused for in vivo imaging to identify the location of cancer cells andprovide image-guided PDT treatment, potentially leading toimproved therapeutic accuracy and outcome (21). The mainchallenge for PDT treatment is off-target tissue accumulation andactivation of photosensitizer leading to cell death in normal tissue(24). Development of a highly selective delivery method forphotosensitizers will minimize side effects and generate bettertherapeutic outcomes.
Prostate-specific membrane antigen (PSMA) is a uniquemembrane-bound glycoprotein originally discovered in theandrogen-dependent LNCaP human prostate adenocarcinomacell line (25). PSMA is overexpressed in prostate cancer. Expres-sion of PSMA correlates with the stage of disease and Gleasonscore (26). PSMA expression is also higher in prostate cancercells from hormone-refractory patients, (26, 27) and increasedPSMA expression has been shown to be an independent markerof disease recurrence (27–29). In addition to being overex-pressed in prostate cancer, PSMA is also expressed in theneovasculature of many solid tumors (30), making PSMA apromising target for both imaging (31–33) and treatment ofprostate cancer (34–36). The objective of this study is todevelop PSMA-targeted PDT agents that could be used forsurgical guidance and allow for subsequent PDT to eliminateunresectable or "missed" cancer cells. Previously, we havedeveloped a peptide-based, highly negatively charged PSMAligand (PSMA-1; Supplementary Fig. S1) for PSMA-targetedimaging of prostate cancer (37, 38). In this study, we havedesigned two PSMA-1–based PDT conjugates (PSMA-1–PDT),PSMA-1-Pc413 (Fig. 1A) and PSMA-1-IR700 (Fig. 1C). Pc413 isan analogue of the second-generation phthalocyanine PDTdrug Pc4 which is currently in clinical trials (39), and IR700is a commercially available NIR phthalocyanine dye which hasPDT activity (40–42). The two conjugates reported here werefound to be effective as theranostic conjugates, allowing bothtargeted-bioimaging and targeted-PDT of prostate cancer.
Materials and MethodsGeneral
PSMA targeting peptide Glu-CO-Glu'-Amc-Ahx-Glu-Glu-Glu-Lys-NH2 (PSMA-1) was synthesized by Fmoc chemistry asreported previously (38). (S)-2-(3-((S)-5-amino-1-carboxypen-tyl)ureido)pentanedioic acid (Cys-CO-Glu; Supplementary Fig.S1) was custom-made by Bachem Bioscience Inc. Pc413 wasprovided by M.E. Kenney's laboratory. All the other chemicalswere purchased from Sigma-Aldrich Inc.
Synthesis of PSMA-1-SMCCCoupling of PSMA-1 to sulfosuccinimidyl-4-(N-maleimido-
methyl)cyclohexane-1-carboxylate (sulfo-SMCC; Thermo FisherScientific) was performed in 100 mmol/L phosphate buffer, pH7.0. PSMA-1 (200 nmol) was dissolved in 100 mL of phosphatebuffer; then 400 nmol of sulfo-SMCC in 500 mL of phosphatebuffer was added (Supplementary Fig. S2A). The reactionmixturewas left at room temperature overnight. The crude product waspurified by preparative high-pressure liquid chromatography(HPLC) using gradient A (Supplementary Methods). Yield:78%. Retention time: 24.8 minutes. MALDI-MS: C58H87N11O23,
Synthesis of PSMA-1-Pc413Pc413 was first dissolved in 1mL of DMF, then PSMA-1-SMCC
in 100 mL of 100 mmol/L phosphate buffer, pH 8.0 was added(Supplementary Fig. S3A). The reaction mixture was stirred atroom temperature for 2 hours and purified by preparative HPLCusing gradient B (Supplementary Methods) to get purified PSMA-1-Pc413. Yield: 63%. Retention time: 15.4 minutes. MALDI-MS:C102H134N20O25SSi3, 1996.9 (found, M-C7H19NOSi); 1996.3(calculated; Supplementary Fig. S3B and S3C).
Synthesis of PSMA-1-IR700Coupling of PSMA-1 to IRDye700DX NHS ester (LI-COR
Biosciences) was performed in 100 mmol/L phosphate buffer,pH 7.0. PSMA-1 (1mg) in 200 mL of phosphate buffer was addedto 0.5 mg of IRDye700DX NHS ester in 200 mL of phosphatebuffer (Supplementary Fig. S4A). The reaction was performed atroom temperature overnight. The crude product was purified bypreparative HPLC using gradient C (Supplementary Methods).Yield: 43%. Retention time: 28.7 minutes. MALDI-MS: C116
H125N21Na4O44S6Si3, 1840.9 (found,M-2C14H30NNa2O10S3Si);1841.0 (calculated; Supplementary Fig. S4B and S4C).
Cell cultureRetrovirally transfected PSMA-positive PC3pip cells and
transfection control PC3flu cells were obtained from Dr. MichelSadelain in 2000 (Laboratory of Gene Transfer and GeneExpression, Gene Transfer and Somatic Cell Engineering Facil-ity, Memorial-Sloan Kettering Cancer Center, New York, NY).The cells were last checked by Western blot analysis in 2014; nogenetic authentication was performed. Cells were maintainedin RPMI1640 medium (Invitrogen Life Technology) with2 mmol/L L-glutamine and 10% FBS at 37�C and 5% CO2
under a humidified atmosphere.
Competitive binding assayThe assay was carried out as reported previously (38) by
incubation PC3pip cells with different concentrations ofPSMA-1–PDT in the presence of 12 nmol/L N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[3H]-methyl-L-cysteine(3H-ZJ24;Supplementary Fig. S1; GE Healthcare Life Sciences). Radioac-tivity of cell pellet was counted by scintillation counter. Theconcentration required to inhibit 50% of binding was deter-mined (IC50) by GraphPad Prism 3.0.
In vitro cellular uptake studiesPC3pip and PC3flu cells were plated on coverslips at about
70% confluency. Twenty-four hours later, cells were incubatedwith 1 mmol/L of PSMA-1-Pc413 or PSMA-1-IR700 for varioustimes. After incubation, cells were washed three times with PBS,fixed with 4% paraformaldehyde, counterstained with 2-(4-ami-dinophenyl)-6-indolecarbamidine dihydrochloride (DAPI),mounted with Fluor-Mount aqueous mounting solution, andobserved under Leica DM4000B fluorescence microscopy (LeicaMicrosystem Inc). Competition experiments were performed bycoincubation of PC3pip and PC3flu cells with 1 mmol/L of PSMA-1-Pc413 or PSMA-1-IR700 and 10 mmol/L of Cys-CO-Glu (Sup-plementary Fig. S1; ref. 38) for 4 hours.
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In vitro phototoxicity assayCell viability in the dark and under light exposure was evalu-
ated by CellTiter 96 Aqueous Cell Proliferation Assay (PromegaCorporation). Cells (3,000/well) were seeded in 96-well cultureplates the day before treatment. On treatment day, cells wereincubated with 1 mmol/L of PSMA-1-Pc413 or PSMA-1-IR700 for1 or 4 hours. After incubation, cells were washed three times with200 mL of cold RPMI media and irradiated under light (>500 nm;Apollo Horizon Projector, Acco Brands; Supplementary Fig. S5Afor the spectrum of the light and Supplementary Method) withirradiance at 8.3 mW/cm2 and radiant exposure at 0.5 J/cm2.Twenty-four hours later, CellTiter 96 Aqueous reagent was addedto each well. After a 3-hour incubation at 37�C, the absorbance at490 nm was measured.
Singlet oxygen detectionPhoto-induced singlet oxygen generation was detected by the
chemicalmethodusing1,3-diphenylisobenzofuran (DPBF) as thechemical quencher (43, 44). The decay ofDPBFwasmonitored bythe absorption at 411 nm. Briefly, DPBF (50 mmol/L) in ethanolwas added to PSMA-1–PDT conjugate solutions in PBS or inRPMI1640 media. The irradiation wavelength used to excitePSMA-1-Pc413 and PSMA-1-IR700 was 672 nm and 690 nm,respectively. The absorbance at 411 nm was measured on anInfinite M200 Plate Reader (Tecan Group Ltd.).
In vivo NIR imaging studiesAnimal experiments were performed according to guidelines of
the animal care and use committee at Case Western Reserve
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Figure 1.
A, chemical structure of PSMA-1-Pc413. B, in vitro cellular uptake results of PSMA-1-Pc413. PSMA-positive PC3pip cells and PSMA-negative PC3flu cells on coverslipswere incubatedwith noprobe (0minute) or 1mmol/L of PSMA-1-Pc413 for 15minutes, 30minutes, 1 hour, and4hours. Nucleiwere stained usingDAPI (false color blue)and uptake of PSMA-1-Pc413 was assessed by fluorescence microscopy (false color red). Specificity of PSMA-1-Pc413 for PSMA binding was evaluated byincubation of PC3pip and PC3flu cells with 1 mmol/L of PSMA-1-Pc413 and 10 mmol/L of Cys-CO-Glu, last column. Signal in PC3pip cells was significantly competed byCys-CO-Glu, suggesting the binding of PSMA-1-Pc413 to PSMA is specific. Images are taken at 40�. Representative images are shown from three independentexperiments. C, chemical structure of PSMA-1-IR700. D, in vitro cellular uptake results of PSMA-1-IR700. PSMA-positive PC3pip cells and PSMA-negative PC3flucells on coverslips were incubated with no probe (0 minute) or 1 mmol/L of PSMA-1-IR700 for 15 minutes, 30minutes, 1 hour, and 4 hours. The nuclei were stained byDAPI (false color blue) and uptake of PSMA-1-IR700 was assessed by fluorescence microscopy (false color red). Specificity of PSMA-1-IR700 to PSMA wasevaluated by incubation of PC3pip and PC3flu cells with 1 mmol/L of PSMA-1-IR700 and 10 mmol/L of Cys-CO-Glu, last column. Signal in PC3pip cells was significantlycompeted by Cys-CO-Glu, suggesting the binding of PSMA-1-IR700 to PSMA is specific. Images are taken at 40�. Representative images are shownfrom three independent experiments.
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Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1836
University (IACUC#120024). Six- to eight-week-old male athy-mic nude mice were implanted subcutaneously with 1 � 106 ofPSMA-negative PC3flu and PSMA-positive PC3pip on the left andright dorsum, respectively. Animals were used when tumorlengths reached 10mm. PSMA-1–PDTwas injected intravenouslyvia the tail vein. Fluorescence imaging was performed using theMaestro In Vivo Imaging System (Perkin-Elmer). During imaging,mice were anesthetized with isoflurane. After imaging, the micewere euthanized, and tissues were harvested for ex vivo imaging.For in vivo competition experiments, mice were coinjected with 1nmol of PSMA-1-Pc413 and 1,000 nmol of ZJ-MCC-Ahx-YYYG(Supplementary Fig. S1), an analogue of PSMA-1 with similarbinding affinity (37), or 1 nmol of PSMA-1-IR700 conjugate and1,000 nmol of PSMA-1. Different competitors were used forPSMA-1-Pc413 and PSMA-1-IR700 due to their different phar-macokinetic behaviors. Multispectral images were unmixed intotheir component spectra (PSMA probes, autofluorescence, andbackground). Component images were used to quantitate theaverage fluorescence intensity associated with the tumors bycreating regions of interest around the tumors.
In vivo PDT treatment of subcutaneous PC3pip tumorsSix- to eight-week-oldmale athymic nudemicewere implanted
subcutaneously with 1 � 106 PC3pip cells. Tumor volume wasmeasured in vivo using calipers and calculated by the followingformula: Tumor volume¼ length�width� depth (45). Tumorsreaching approximately 50 mm3 in volume were selected for thestudy. To test the PDT activity of PSMA-1-Pc413, selected micewere divided into 4 groups with each group having 5 mice.Groups: (i) no treatment; (ii) mice were injected at a dosage of0.1 mg/kg; (iii) mice were injected at a dosage of 0.25 mg/kg; or(iv) mice were injected at a dosage of 0.5 mg/kg. The mice wereimaged before injection, immediately following injection, and24 hours postinjection. At 24 hours postinjection, the tumorswere subjected to PDT treatment using 672-nm light from a diodelaser (AppliedOptronics Corp; see Supplementary Fig. S5B for thespectrumof the laser light). The 672-nm laser diodewas chosen asit was previously used with success in prior studies (46) due to itsproximity to the peak of Pc413 absorbance (676 nm) and becauseof the availability of laser diodes. Light was delivered through aGRIN-lens-terminate multimode fiber (OZ Optics) and tumorswere illuminated with irradiance of 33.3 mW/cm2, radiant expo-sure of 150 J/cm2 at 672 nm (Supplementary Methods). Themice were imaged posttreatment and allowed to recover. Formice receiving PSMA-1-IR700, selected mice were divided intothree groups with each group having 5 mice. Groups: (i) notreatment; (ii) mice were injected at a dosage of 0.25 mg/kg ondays 0, 4, and 8 and treated with 50 J/cm2 of laser light(Supplementary Methods) at 1 hour postinjection on these 3days; (iii) mice were injected at a dosage of 0.5 mg/kg on day 0,4, and 8 and treated with light at 1 hour postinjection on these3 days. A diode LED light source (L690-66-60, MarubeniAmerica Co.; see Supplementary Fig. S5C for the spectrum ofthe diode LED) was used to irradiate PSMA-1-IR700 at 690 nmdue to its proximity to the peak of IR700 absorbance (690 nm)as reported by others (41–43). Mice were treated with irradi-ance of 31.8 mW/cm2 and radiant exposure of 50 J/cm2. Noradiant exposure higher than 50 J/cm2 was tried, otherwise theLED light unit would be too hot. Mice were monitored daily,and tumor size was measured every other day until animalswere euthanized.
Histology studies with H&E stainingPC3pip tumors were harvested 1 day after single PDT treatment
with 0.5mg/kg of PSMA-1–PDT conjugates. Tumors were fixed in4% paraformaldehyde and embedded into paraffin blocks. Serial10-mm slice sections were fixed on slide glasses and hematoxylinand eosin (H&E) stained. Sections were photographed using the10� objective. Control tumors were harvested frommice receivedsame dose of drugs, but without light illumination.
Statistical analysisStatistical analyses were carried out using Microsoft Excel.
Student t test was used to compare the treatment effects withcontrols. P < 0.05 was used to determine statistical significance.
ResultsSynthesis
The synthesis and characterization of PSMA-1-Pc413 andPSMA-1-IR700 is shown in Supplementary Figs. S2–S4. PSMA-1 and Pc413 were coupled through the heterobifunctional linkersulfo-SMCC (Supplementary Figs. S2A and S3A). PSMA-1-IR700was synthesized by reaction of PSMA-1 with commercially avail-able IRDye700DX NHS ester in PBS (Supplementary Fig. S4A).PSMA-1-Pc413 has maximum absorbance (lmax) at 676 nm andmaximum emission (lEm) at 678 nm (Supplementary Fig. S6A)and PSMA-1-IR700 has lmax at 690 nm and lEm at 698 nm(Supplementary Fig. S6B), both concurring those of Pc4 (47)and IR700 (40). Conjugation of PSMA-1 and Pc413 resulted in amolecule with better solubility characteristics than the parentPc413, likely due to the presence of three negatively chargedglutamate residues in PSMA-1. As a result, PSMA-1-Pc413 (logPvalue ¼ �1.35 � 0.14) is water soluble. The conjugate PSMA-1-IR700, logP ¼ �2.46 � 0.22, also has good water solubility. Thewater solubility of our conjugates eliminates the need for acomplicated drug formulation for systemic delivery.
In vitro competition binding resultsCompetition binding assay showed that both PSMA-1-Pc413
(IC50¼2.1�0.22nmol/L) andPSMA-1-IR700 (IC50¼2.2�0.15nmol/L) had binding affinities greater than 4.6-fold comparedwith the related ligand Cys-CO-Glu (Supplementary Fig. S1;ref. 38; IC50 ¼ 10.2 � 0.31 nmol/L; Table 1) and an affinitysimilar to that measured in previous studies using PSMA-1-Cy5.5or PSMA-1-IR800 (38).
In vitro uptake resultsTo examine the uptake of PSMA-targeted PDT conjugates,
in vitro cellular uptake of PSMA-1-PC413 and PSMA-1-IR700 inPSMA-positive PC3pip cells and PSMA-negative PC3-flu cellswere performed and visualized by fluorescence microscopy. No
Table 1. In vitro competition binding results of PSMA-1-Pc413 and PSMA-1-IR700
Cys-CO-Glu PSMA-1-Pc413 PSMA-1-IR700
IC50 (nmol/L) 10.2 � 0.31 2.1 � 0.22 2.2 � 0.15
NOTE: The assay was carried out by incubating PC3pip cells (5 � 105) withdifferent concentrations of PSMA-1–PDT conjugates in the presence of 12 nmol/LN-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[3H]-methyl-L-cysteine(3H-ZJ24).The concentration required to inhibit 50% of binding is determined (IC50) byGraphPad Prism 3.0. Values represent mean � SD of three independent experi-ments. Both PSMA-1-Pc413 and PSMA-1-IR700 showed better binding affinitythan the related ligand Cys-CO-Glu.
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detectable amount of fluorescence uptake was observed in PSMA-negative PC3flu cells for either PSMA-1 conjugates (Fig. 1). Incontrast, for both targeted PDT agents, fluorescence intensity inPSMA-positive PC3pip cells increased with prolonged incubationtime (Fig. 1B andD). When an excess amount of Cys-CO-Glu wasincluded in the incubation, no fluorescence signal was observed,confirming that cellular uptake of fluorescence was attributed tothe specific binding of PSMA-1–PDT conjugates to PSMA. Onceinternalized into PC3pip cells, the PSMA-1–PDT conjugates werelocated in the perinuclear position, concurring with our previousresults with PSMA-NIR conjugates (38).
In vitro cytotoxicity of PDTTo determine the in vitro phototoxicity of PSMA-1-Pc413,
PC3pip, and PC3flu cells were incubated with PSMA-1-Pc413 at37�C for 1hour and then exposed to lightwith radiant exposure of0.5 J/cm2. At 1 mmol/L of PSMA-1-Pc413, 65.0� 1.8% of PC3pipcells were killed, whereas only 22.5 � 3.0% PC3flu cells werekilled at the same condition (P¼ 0.0012; Fig. 2A). In the presenceof 10 mmol/L of Cys-CO-Glu, the PDT activity of PSMA-1-Pc413
(1 mmol/L) to PC3pip cells was reduced approximately 6-fold(Supplementary Fig. S7), suggesting dependence onPSMAexpres-sion. In contrast, no changeswere observed in its activity to PC3flutumors. The presence of excess amount of Cys-CO-Glu also hadno effect on the PDTactivity of unconjugated PC413. These resultsindicated that PSMA-1-Pc413 can preferentially kill PSMA-posi-tive PC3pip cells.
When cells were treated with PSMA-1-IR700, however, nophototoxocitywasobserved evenwithprolonged incubation time(4 hours) and higher radiant exposure (up to 2.0 J/cm2; Fig. 2B).We also utilized a LED diode as the light source and obtainedsimilar results. To begin to understand the difference in PDTefficacy between the agents, we measured singlet oxygen gener-ation of the conjugates.
Singlet oxygen (1O2) is believed to play a key role in theefficacy of PDT. We used DPBF to quantify the production ofsinglet oxygen by each of our PDT agents by following thechanges in the absorbance at 411 nm (43, 44). As shown in Fig.2C, the absorbance at 411 nm decreased quickly after PSMA-1-Pc413 or PSMA-1-IR700 in PBS was added to DPBF solution
*
In vitro phototoxicity of PSMA-1-IR700
Generation of singlet oxygen by PSMA-1-PDTconjugates in PBS
C
A
D
B
Generation of singlet oxygen by PSMA-1-PDT conjugates in RPMI1640 media
A, phototoxicity of PSMA-1-Pc413. PC3pip and PC3flu cellswere incubatedwith different concentrations of PSMA-1-Pc413 or Pc413 at 37�C for 1 hour in RPMImedium,cellswere then treatedby lightwith radiant exposure at 0.5 J/cm2. PSMA-1-Pc413 showed selectivePDT activity in PSMA-positive PC3pip cells (� ,P<0.05). Values aremean � SD of eight replicates. B, phototoxicity of PSMA-1-IR700. PC3pip and PC3flu cells were incubated with 1 mmol/L of PSMA-1-IR700 or IR700 at37�C for 4 hours in RPMImedium, the cellswere then kept in dark or exposed to lightwith different radiant exposure. However, no phototoxicitywas observed. Valuesare mean � SD of eight replicates. C, generation of singlet oxygen by PSMA-1-Pc413 and PSMA-1-IR700 in PBS. Generation of singlet oxygen was detected by theindirect DPBF method. The absorbance at 411 nm (OD) decreased significantly when PSMA-1-Pc413 or PSMA-1-IR700 in PBS was added to DPBF solution and lightirradiation was applied, suggesting a high efficacy in generation of reactive 1O2. Values are mean � SD of three replicates. D, generation of singlet oxygen byPSMA-1-Pc413 and PSMA-1-IR700 in RPMI1640media. PSMA-1-Pc413 can effectively generate singlet oxygen in RPMI media as shown by the reduced absorbance at411 nm (OD); however, PSMA-1-IR700 was not as good in generating single oxygen in RMPI1640 media as in PBS. Values are mean � SD of three replicates.
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Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1838
and light irradiation was applied, suggesting high efficacy ingeneration of reactive 1O2. When the test was performed inRPMI1640 media containing 10% FBS (Fig. 2D), the decay ofDPBF resembled that in PBS for PSMA-1-Pc413; however, thedecrease of absorbance at 411 nm was significantly less in RPMImedia than in PBS for PSMA-1-IR700, indicating that PSMA-1-IR700 was not effective in generating singlet oxygen in RPMImedia, potentially explaining the poorer in vitro cytotoxicity ofPSMA-1-IR700.
In vivo imagingAnimals bearing both PSMA-positive PC3pip and PSMA-
negative PC3flu tumors were used to demonstrate noninvasiveimaging and examine the biodistribution of PSMA-1–PDT con-jugates in vivo. Selective uptake was observed in PSMA-positivePC3pip tumors. As shown in Fig. 3A, the fluorescence intensityof PSMA-1-Pc413 in PC3pip tumors increased gradually, peak-ing at 24 hours postinjection, and then slowly declined. Thefluorescence was also observed in PC3flu tumors, but was 1.5-fold less than that in PC3pip tumors after 2 hours postinjection(P ¼ 0.029). High fluorescence was observed in the upper backof the animals at early time points, butwas cleared after 24 hourspostinjection; this is likely due to nonspecific accumulation in
the fatty region of the neck (46). To confirm that binding ofPSMA-1-Pc413 is specific to PSMA, in vivo competition experi-ments were performed. Previous in vivo competition experi-ments have shown that ZJ-MCC-Ahx-YYYG (SupplementaryFig. S1), an analogue of PSMA-1 with similar binding affinity(37), can effectively compete with PSMA-1-IR800 and PSMA-1-Cy5.5 in vivo (38); therefore, we used it again to compete withPSMA-1-Pc413 in vivo. When the mice were coinjected with 1nmol of PSMA-1-Pc413 and 1,000 nmol of ZJ-MCC-Ahx-YYYG,the fluorescent intensity in PC3pip tumors decreased (Fig. 3Cand D). At 24 hours postinjection, fluorescence in PC3piptumors decreased about 30% (P ¼ 0.0067), whereas no changein fluorescence intensity was observed in PC3flu tumors (P ¼0.345). In all cases, inclusion of the unlabeled competitor ligandreduced binding in PSMA-positive PC3pip tumors to uptakelevels measured in the receptor-negative PC3flu tumors. Fivedays postinjection, mice were euthanized and tissues such asskin, liver, stomach, heart, lung, spleen, kidneys, PC3pip tumor,PC3flu tumor, and bladder were taken for ex vivo imaging.PC3pip tumor showed bright fluorescent signal, whereas othertissues had minimal amount of fluorescence signal (Fig. 3B).
PSMA-1-IR700 showed different pharmacokinetics com-pared with PSMA-1-Pc413. PSMA-1-IR700 reached highest
PSMA-1-Pc413+ 1000X ZJ-MCC-Ahx-YYYG, in vivo competition Maestro quantification of PSMA-1-Pc413
Figure 3.
Imaging of PSMA-1-Pc413 in mice bearing flank PC3pip and PC3flu tumors. A, in vivoMaestro imaging of a typical mouse treated with PSMA-1-Pc413. Mice received 1nmol of PSMA-1-Pc413 via tail vein injection and then were imaged at the designated times. Representative images are shown of n ¼ 5. B, ex vivo imagingof mice organs at 5 days postinjection of PSMA-1-Pc413. The fluorescent signal in PC3pip tumor was significantly higher than in other organs. C, in vivo Maestroimaging of mice injected with 1 nmol of PSMA-1-Pc413 and 1,000 nmol of a selective PSMA receptor binding molecule, ZJ-MCC-Ahx-YYYG. Images are on the samescale as in Fig. 3A. Blockade of fluorescent uptake in PC3pip tumors was observed. D, quantification of fluorescent signal intensity in PC3pip and PC3flutumors from the mice used in Fig. 3A and C. Values represent mean � SD of 5 animals.
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accumulation in PC3pip tumor within 30 minutes postinjec-tion, then dropped rapidly (Fig. 4A). Compared with PSMA-1-Pc413, PSMA-1-IR700 demonstrated better selectivity. At4 hours postinjection, the signal in PC3pip tumor (23.3 �3.8 counts/s) was more than 3.6-fold higher than in PC3flutumor (6.4 � 3.3 counts/s; P ¼ 0.0018). For in vivo competitionexperiments, we first tried to use excess amount of ZJ-MCC-Ahx-YYYG to compete with PSMA-1-IR700 as we did withPSMA-1-Pc413; however, ZJ-MCC-Ahx-YYYG failed to blockthe binding of PSMA-1-IR700. PSMA-1-IR700 had differentpharmacokinetics from PSMA-1-Pc413, clearing very quicklyfrom the body. To better compete for binding, we testedunconjugated PSMA-1(Supplementary Fig. S1; ref. 38). Whenmice were coinjected with excess amount of PSMA-1, thesignals in PC3pip tumor reduced significantly (Fig. 4C). At4 hours after coinjection of 1 nmol of PSMA-1-IR700 and 1,000nmol of PSMA-1, the fluorescent signal in PC3pip tumor was86% (P ¼ 0.0002) lower compared with animals receivedPSMA-1-IR700 only (Fig. 4D). In contrast, no significantchange was observed in the signals in PC3flu tumors (P ¼0.065). Ex vivo tissue images showed that PSMA-1-IR700 wasmainly accumulated in PC3pip tumors (Fig. 4B). Some signalwas observed in the kidneys but was weaker than that inPC3pip tumors.
In vivo photodynamic treatment of PC3pip tumorsTo test the photodynamic efficacy of PSMA-1-Pc413, mice-
bearing PC3pip tumors were irradiated with 150 J/cm2 of lightat 672 nm at 24 hours postinjection to take advantage of themaximum peak of PSMA-1-Pc413 tumor accumulation. Ex vivoimages of tissues at 24 hours postinjection of 0.5mg/kg of PSMA-1-Pc413 showed that PSMA-1-Pc413 was mainly located insidePC3pip tumor (Supplementary Fig. S8A and S8B), showing goodselectivity of the conjugate. Mice receiving no drug and no lightwere used as controls. Control groups in which mice receive lightonly or drug only were not included in this study as light alone ordrug alone results in no PDT effect (42, 48). Maestro imagesshowed that fluorescence signal in PC3pip tumor increased whenmore drug was administered (Fig. 5A). Treated tumors showedimmediate loss of fluorescence indicating photobleaching fromthe activation of Pc413 (Fig. 5B). The loss of fluorescence was alsoobserved whenmice were treated with 50 J/cm2 of light. Swellingaround the treated site was observedwithin hours after treatment.Tumor volume was significantly reduced starting on day 5 post-injection formice receiving 0.25mg/kg (P¼ 0.001) or 0.50mg/kg(P ¼ 0.0004) of PSMA-1-Pc413 when compared with untreatedcontrols (Fig. 5C). As the drug dose increased, improved treat-ment efficacy was observed. The increased drug accumulation inthe tumor, therefore, likely led to better treatment results. At the
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Imaging of PSMA-1-IR700 in mice bearing flank PC3pip and PC3flu tumors. A, in vivo Maestro imaging of mice treated with PSMA-1-IR700. Mice received 1 nmol ofPSMA-1-IR700 through tail vein injection and then were imaged at the indicated time points. Representative images of n ¼ 5 mice are shown. Selectiveuptake in PC3pip tumors was observed. B, ex vivo imaging of mice organs at 48 hours postinjection of PSMA-1-IR700. The fluorescent signal in PC3pip tumor wassignificantly higher than in other organs. C, in vivoMaestro imaging ofmice injected with 1 nmol of PSMA-1-IR700 and 1,000 nmol of PSMA-1. Images are on the samescale as in Fig. 4A. Blockade of fluorescent uptake in PC3pip tumors was observed. D, fluorescent signal quantification of PSMA-1-IR700 in PC3pip and PC3flutumors from mice used in Fig. 4A and C. Values represent mean � SD of 5 animals.
Wang et al.
Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1840
dose of 0.25 and 0.5 mg/kg, tissue damage was observed in someanimals due to tumor damage/necrosis; no other adverse effectswere observed.
For mice treated with PSMA-1-IR700, we first tried to treatthe animals at 2 hours and 4 hours postinjection to allowthe conjugate to distribute in the tumor and clear from the body;however, no PDT effect was observed. We then moved thetreatment time to 1 hour postinjection to maximize tumor-asso-ciated drug at the time of irradiation. Ex vivo tissue images taken at1 hour postinjection of 0.5 mg/kg of PSMA-1-IR700 showedfluorescent signal was mainly observed in the kidneys, followedby the PC3pip tumor, but not in other organs (SupplementaryFig. S8C and S8D), indicating that 1 hour postinjection can offeracceptable selectivity. To improve the treatment outcome, micereceived PSMA-1-IR700 on days 0, 4, and 8 and were treated withlight at 1 hour postinjection. Similar to PSMA-1-Pc413, fluores-cence signal in PC3pip tumors was dose dependent (Fig. 5D).Fluorescence signal in PC3pip tumors disappeared after irradia-
tion to light (Fig. 5E). The loss offluorescencewas also observed ata lower dose of 30 J/cm2 light. Significant tumor growth inhibi-tion was observed starting on day 7 after PDT treatment. Com-paredwith untreated controlmice, tumor sizes inmice exposed toPDT treatments were significantly reduced. The PDT effect ofPSMA-1-IR700 was dose dependent (Fig. 5F). Treatment did notaffect physical appearance and activity of the mice; no overttoxicity was observed.
Histologic studiesFor histologic analysis, tumors were extracted 24 hours after
single PDT treatment with PSMA-1–PDT. Tissues were processedusing H&E staining. Pathologic analysis showed dramatic differ-ences between the treated and untreated tumors (Fig. 6). Nucleiin cancer cells treated with PSMA-1–PDT conjugates weremuch smaller compared with untreated tumors, indicating thatthe cells were damaged; in contrast the untreated tumor was notdamaged and the cells were intact (49).
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In vivo photodynamic treatment of PSMA-positive PC3pip tumors. Values represent mean� SD of 5 animals. A, quantification of PSMA-1-Pc413 fluorescent signal inPC3pip tumors. Signal in PC3pip tumors increased when mice received increased dose of PSMA-1-Pc413. B, loss of fluorescent signal in PC3pip tumors afterPSMA-1-Pc413 PDT treatment. Mice received 0.5 mg/kg PSMA-1-Pc413 and were treated by laser light (672 nm) with radiant exposure of 150 J/cm2 at 24 hourspostinjection. Tumor is indicated by the red circle. This loss of fluorescence after PDT indicated the activation of PSMA-1-Pc413 by light.C, tumor growth inhibition byPSMA-1-Pc413 PDT treatment in PC3pip tumors. Tumors were irradiated with 150 J/cm2 light (672 nm) at 24 hours postinjection (red arrow). Significant tumorregression was observed and the response was dose dependent. Values represent mean � SD of 5 tumors. P values are obtained by comparison withcontrol group (� , P < 0.05).D, quantification of PSMA-1-IR700 fluorescent signal in PC3pip tumors. Signal in PC3pip tumors increased when mice received increaseddose of PSMA-1-IR700.E, loss offluorescent signal in PC3pip tumors after PSMA-1-IR700PDT treatment.Mice received0.5mg/kgPSMA-1-IR700andwere treatedbylight (690 nm) with radiant exposure at 50 J/cm2 at 1 hour postinjection. Tumor is indicated by the red circle. This loss of fluorescence after PDT indicatedthe activation of PSMA-1-IR700 by light. F, tumor growth inhibition by PSMA-1-IR700 PDT treatment in PC3pip tumors. Mice received PSMA-1-IR700 on days0, 4, and 8 (red arrows). PDT treatment was performed at 1 hour postinjection. Significant tumor regression was observed and the response was dose dependent.Values represent mean � SD of 5 tumors. P values are obtained by comparison with control group (� , P < 0.05).
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DiscussionEffective PSMA-targeted photoimmunotherapy has been dem-
onstrated by Watanabe and colleagues (43) both in vitro and invivo. Compared with antibody, low molecular weight moleculesare easy to be synthesized and more cost-effective. To date, theonly PSMA-targeted PDT agent is a PSMA inhibitor conjugate ofpyropheophorbide, as reported in 2009 (50). Selective cell killingwas observed in PSMA-positive LNCaP cells, but no in vivo resultshave been reported since then. In this article, we report thesynthesis and in vitro and in vivo studies of two PSMA-1–PDTconjugates, PSMA-1-Pc413 and PSMA-1-IR700. Both conjugateshad improved binding affinity compared with the related ligandCys-CO-Glu (38), and their binding affinities were similar to theunconjugated PSMA-1 (ref. 38; Table 1). In vitro cellular uptakeexperiments demonstrated that the two PSMA-1–PDT conjugatesare selectively and specifically taken up by PSMA-positive PC3pipcells but not by PSMA-negative PC3flu cells (Fig. 1). In vivoimaging studies showed that both PSMA-1-Pc413 and PSMA-1-IR700 selectively accumulate in PSMA-positive PC3pip tumor,although PSMA-1-IR700 had better selectivity than PSMA-1-Pc413 (Figs. 3 and 4). The measurable nonspecific uptake ofPSMA-1-Pc413observed inPSMA-negative PC3flu tumorsmayberelated to the in vivo stability of the molecule. The nonspecificbinding to tumors was also observed when unconjugated Pc413was administered to mice. As it is similar in level to free Pc413,theremay be a stability issue with the agent andwe are working toinvestigate this potential issue. If this is the case, proposed
synthesis to correct this is to conjugate PSMA-1 to Pc413 throughthe tetraazatetrabenzoporphyrin group instead of through theaxial siloxy group similar to the synthesis of PSMA-1-IR700.
In vivo imaging studies found that PSMA-1-Pc413 and PSMA-1-IR700 showed dramatically different pharmacokinetics (Figs. 3and 4), with PSMA-1-IR700 accumulation and clearance from thetumor and the body occurring much more rapidly. This might beattributed to differences in the hydrophobicity of the two mole-cules; the more hydrophobic PSMA-1-Pc413 (LogP ¼ �1.35 �0.14)may adhere to blood proteins, remaining in the body longerthan the less hydrophobic PSMA-1-IR700 (LogP¼�2.46�0.22).This agrees with previous evidence showing that fluorophores canaffect the pharmacokinetic behavior of the conjugate (38). Furtherthis explains why it is critical to match the pharmacokinetics ofcompetitive ligands to that of the PSMA-1–PDT molecules foreffective competition studies. In ex vivo organ imaging, fluores-cence was mainly observed in PSMA-positive PC3pip tumors,indicating the potential for a low toxicity to other organs duringPDT treatment, due to selective irradiation of the lesion and lowlevels of PDT agents in nontargeted tissues. Furthermore, theproposed use of these ligands to guide surgery and then selectivelyirradiate remaining inoperable tumor tissue will further reduceoff-target toxicity by specifically localizing light to target tissues.
In vitro phototoxicity experiments (Fig. 2A) showed that PSMA-1-Pc413 was more potent against PSMA-positive PC3pip cellsthan for PSMA-negative PC3flu cells. This result was consistentwith the in vitro cellular uptake results (Fig. 1B), in which selec-tively higher cellular uptake of PSMA-1-Pc413 was observed forPSMA-positive PC3pip cells than for PSMA-negative PC3flu cells.No phototoxicity was observed for either cell typewhen cells weretreated with PSMA-1-IR700 in RPMI media with 10% FBS (Fig.2B). Further singlet oxygen generation studies demonstrated thatboth PSMA-1-Pc413 and PSMA-1-IR700 are able to producesinglet oxygen effectively when activated by light in PBS. Theconfluence of the absorption of the PDT agents and the emissionwavelengths along with measurement of similar absorption byeach agent (Figs. S5 and S6) suggests that these are not majorcontributors to the difference in efficacy. PSMA-1-IR700 was,however, less efficient in generating singlet oxygen than PSMA-1-Pc413 when activated by light in RPMI media with 10% FBS(Fig. 2D), explaining lack of phototoxicity of PSMA-1-IR700 invitro.Others have reportedphotoimmunotherapy in tissue culturecells when IR700 was conjugated to antibody (40, 42); however,this is not the case with our low molecular weight conjugate. Atthis time, it is unclear what causes this difference in results but itmight be related to molecules in RPMI medium with 10% FBSinteracting with the "unprotected" IR700 bound to PSMA-1attenuating free-radical generation, compared with the "pro-tected" IR700 already in close proximity to a large antibody(40, 42), perhaps eliminating medium-dependent inhibition offree-radical generation.
In in vivo PDT experiments (Fig. 5C), PSMA-1-Pc413 demon-strated strong inhibition on the growth of PC3pip tumor. Afteronly a single dose, a significant difference in tumor growth wasmeasured between treated and untreated tumors. PSMA-1-IR700required three doses to inhibit PC3pip tumor growth (Fig. 5F).This lower efficacy of PSMA-1-IR700 may be due to lower radiantexposure (50 J/cm2) compared with PSMA-1-Pc413 treatment(150 J/cm2). Posttreatment toxicity was not observed for eitheragent based on physical appearance and activity of the mice,consistent with the ex vivo organ images.
Histologic analysis of PC3pip tumors. Cell damage was observed in PDT-treatedPC3pip tumors as compared with nontreated controls. A, control PC3pip tumorreceiving PSMA-1-PC413. Mice received 0.5 mg/kg of PSMA-1-PC413 and tumorswere taken out at 48 hours postinjection without light irradiation.B, PC3pip tumors receiving PSMA-1-Pc413 and treated with light. Mice received0.5 mg/kg of PSMA-1-PC413, irradiated with light with radiant exposure at150 J/cm2 light at 24 hours postinjection. Tumors were taken out at 24 hoursafter PDT treatment. C, control PC3pip tumor receiving PSMA-1-IR700. Micereceived 0.5 mg/kg of PSMA-1-IR700 and tumors were taken out at 24 hourspostinjection without light irradiation. D, PC3pip tumors receiving PSMA-1-IR700 and treated with light. Mice received 0.5 mg/kg of PSMA-1-IR700, andtreated by light with radiant exposure at 50 J/cm2 light at 1 hour postinjection.Tumors were taken out at 24 hours after PDT treatment.
Wang et al.
Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1842
There is an unmet need to visualize and completely resectprostate cancerswhichhavemovedoutside of the prostate capsuleduring surgical resection of prostate cancer. We have generatedtwo PSMA-targeting PDT conjugates, PSMA-1-Pc413 and PSMA-1-IR700. Our results demonstrated that the synthesized conju-gates have the following advantages as imaging agents: (i) theconjugates bind avidly to cancer cells with quantitative clearancefrommost healthy tissues, allowing rapid tumor visualization; (ii)the conjugates can enter into cancer cells, providing stable tumorcontrast; (iii) the conjugates bind specifically to the tumormarkerPSMA, which help create highly defined boundaries of the tumor.These features will allow discrimination between diseased, nor-mal, and neural tissues, help surgeons identify extracapsulardisease, which is currently invisible during prostatectomy, anddecide the aggressiveness of the surgical approach during theprocedure. Furthermore, in addition to tracking tumor location,our conjugates have PDT activity. Selective targeting of PDT agentsto cancer cells with PSMA-1 and selective irradiation with light totargeted lesions may result in selective destruction of cancer cellsas low expression levels of PSMA inhealthy tissueswould not leadto enough PSMA-1–PDT agent accumulation for a pharmacologiceffect. This has been demonstrated using antibody targeted IR700in mixed culture studies to demonstrate PDT killing of cellsoverexpressing the targeted cell surface receptor only (42). It ispossible to envision that the described PSMA-1–PDT conjugatescan be potentially used to ablate cancer contained within thegland, that is, partial prostatectomy. They could also be used todestroy unresectable tissues and/or missed cancer cells, leading toimproved quality and success of radical prostatectomies. Thesmall amount of tissue remaining after the surgery will reducethe burden for elimination of cancer by PDT and improve thetherapeutic window for PDT by reducing collateral damage. Thisapproach will reduce the need for subsequent medical treatment.Furthermore, PSMA is also expressed on the neovasculature ofmany different solid tumors, so this approach could be used forPDT delivery and ablation for these diseases as well.
ConclusionOur rationally designed dual-functional PSMA-1–PDT conju-
gates have potential to serve as anticancer agents and to ourknowledge represent the first PSMA-targeted PDT agents. Throughin vitro and in vivo studies, we have demonstrated that there aredifferences in the PDT efficacy of each of the two targeted agents
that are likely related to the PDT moiety and not the targetingligand, as the affinity remains unchanged after conjugation toeither of the PDT agents. The effectiveness and features of ourPSMA-1–Pc413 conjugate suggest that it has potential clinicalutility andmay represent a next-generation theranostic PDTagent.The dual use of the developed PSMA-1–Pc413 conjugate mayoffer surgeons photoablation as an adjunct to surgical resection tospare proximate nerves and muscles and eliminate stray cancertissue and cells, potentially reducing the frequency of unresectedtumor and cancer recurrence.
Disclosure of Potential Conflicts of InterestJ.P. Basilion is on the SAB at Akrotome Imaging and Vergent Biosciences,
received a commercial research support fromAkrotome Imaging, has ownershipinterest (including patents) in Akrotome, and is a consultant/advisory boardmember for Akrotome Imaging and Vergent Biosciences. No potential conflictsof interest were disclosed by the other authors.
Authors' ContributionsConception and design: X. Wang, J. Kiechle, L. Ponsky, J.P. BasilionDevelopment of methodology: X. Wang, P. Zhang, J. Meyers, J.P. BasilionAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X.Wang, B. Tsui, G. Ramamurthy, J. Meyers, J. Kiechle,L. Ponsky, J.P. BasilionAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Wang, B. Tsui, J. Meyers, J. Kiechle, J.P. BasilionWriting, review, and/or revisionof themanuscript:X.Wang, B. Tsui, J. Kiechle,L. Ponsky, J.P. BasilionAdministrative, technical, or material support (i.e., reporting or organizingdata, constructingdatabases):G.Ramamurthy, P. Zhang, J.Meyers, J.P. BasilionStudy supervision: M.E. Kenney, L. Ponsky, J.P. Basilion
AcknowledgmentsThe authors acknowledge the laboratory of Dr. John Crabb at the Cole Eye
Institute, Cleveland Clinic for assistance with mass spectrometry.
Grant SupportThis work was supported by National Foundation of Cancer Research (to
J. P. Basilion) and Alpha Omega Alpha Carolyn L. Kuckein Student ResearchFellowship (to B. Tsui).
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 August 31, 2015; revised April 18, 2016; accepted May 25, 2016;published OnlineFirst June 13, 2016.
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Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics1844
2016;15:1834-1844. Published OnlineFirst June 13, 2016.Mol Cancer Ther Xinning Wang, Brian Tsui, Gopolakrishnan Ramamurthy, et al. by Targeting Prostate-Specific Membrane AntigenTheranostic Agents for Photodynamic Therapy of Prostate Cancer
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