fluorescent image guided surgery with an anti-prostate ... · biochemical speciﬁcity to prostate...

Personalized Medicine and Imaging

Fluorescent Image–Guided Surgery with anAnti-Prostate Stem Cell Antigen (PSCA) DiabodyEnables Targeted Resection of Mouse ProstateCancer Xenografts in Real TimeGeoffrey A. Sonn1, Andrew S. Behesnilian1, Ziyue Karen Jiang1, Kirstin A. Zettlitz2,3,Eric J. Lepin2,3, Laurent A. Bentolila4,5, Scott M. Knowles2,3, Daniel Lawrence2,Anna M.Wu2,3, and Robert E. Reiter1

Abstract

Purpose: The inability to visualize cancer during prostatectomycontributes to positive margins, cancer recurrence, and surgical sideeffects. A molecularly targeted fluorescent probe offers the poten-tial for real-time intraoperative imaging. The goal of this study wasto develop a probe for image-guided prostate cancer surgery.

Experimental Design: An antibody fragment (cys-diabody,cDb) against prostate stem cell antigen (PSCA) was conjugatedto a far-red fluorophore, Cy5. The integrity and binding of theprobe to PSCA was confirmed by gel electrophoresis, size exclu-sion, and flow cytometry, respectively. Subcutaneous models ofPSCA-expressing xenografts were used to assess the biodistribu-tion and in vivo kinetics, whereas an invasive intramuscularmodelwas utilized to explore the performance of Cy5-cDb–mediatedfluorescence guidance in representative surgical scenarios. Finally,a prospective, randomized study comparing surgical resectionwith and without fluorescent guidance was performed to deter-

mine whether this probe could reduce the incidence of positivemargins.

Results: Cy5-cDb demonstrated excellent purity, stability, andspecific binding to PSCA. In vivo imaging showedmaximal signal-to-background ratios at 6 hours. In mice carrying PSCAþ andnegative (�) dual xenografts, the mean fluorescence ratio ofPSCAþ/� tumors was 4.4:1. In surgical resection experiments,residual tumors <1 mm that were missed on white light surgerywere identified and resected using fluorescence guidance, whichreduced the incidence of positive surgical margins (0/8) com-pared with white light surgery alone (7/7).

Conclusions: Fluorescently labeled cDb enables real-timein vivo imaging of prostate cancer xenografts in mice, and facil-itates more complete tumor removal than conventional whitelight surgery alone. Clin Cancer Res; 22(6); 1403–12. �2015 AACR.

See related commentary by van Leeuwen and van der Poel, p. 1304

IntroductionRadical prostatectomy remains the most common definitive

treatment option for the >250,000 men newly diagnosed withlocalized prostate cancer each year (1), with up to 85% of allprostatectomies in theUnited States performed robotically (2–4).Early extracapsular extension of prostate cancer is rarely visibleduring prostatectomy, and proximity of the prostate to the rec-

tum, urinary sphincter, and erectile nerves precludes wide localexcision. As such, positive surgical margin rates range from 6.5%to32% in contemporary series (5), and are directly correlatedwithpoor cancer control (6, 7). The posterolateral prostate and pros-tatic apex remain the most common sites for positive surgicalmargins (8), which are intimately associated with the neurovas-cular bundle controlling erectile function and urinary sphincter,respectively. As urinary incontinence and sexual dysfunctionremain major issues postoperatively (9), surgeon reluctance toremove healthy tissue in an attempt to reduce these side effectslikely account for these higher rates. The ability to visualize smallfoci of extracapsular extension of prostate cancer at the time ofsurgery may reduce the incidence of positive surgical marginswhile reducing damage to critical adjacent structures.

Several research teams have endeavored to develop cancerspecific optical imaging agents that use fluorescence to highlightcancer in real time during surgery. Intraprostatic free indocyaningreen (ICG) has been utilized clinically in prostatectomy as alymphangiographic agent in the detection of sentinel lymphnodes and for delineation of prostate by limited diffusion(9, 10). However, use of free ICG is limited by the lack ofbiochemical specificity to prostate or prostate cancer, and suffersfrom dye spillage from handling or manipulating fluorescenttissue. Cancer-specific optical agents that overcome these limita-tions are currently being developed by various groups in the

1Department of Urology, University of California, Los Angeles, Cali-fornia. 2Molecular andMedical Pharmacology, University of California,Los Angeles, California. 3Crump Institute for Molecular Imaging, Uni-versityofCalifornia, LosAngeles,California. 4DeparmentofChemistryand Biochemistry, University of California, Los Angeles, California.5California NanoSystems Institute, University of California, LosAngeles, California.

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

G.A. Sonn, A.S. Behesnilian, and Z.K. Jiang contributed equally to this article.

Corresponding Author: Robert E. Reiter, University of California, Los Angeles,66-128A Center for the Health Sciences, 10833 LeConte Ave, Los Angeles, CA90095-1738. Phone: 310-794-7224; Fax: 310-206-5343; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-0503

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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preclinical stage. These include activatable cell-penetrating pep-tides (ACPP) that are specifically activated by metalloproteinasesexpressed in anumber of solid tumors (11, 12), aswell as prostate-specific membrane antigen (PSMA) targeted small molecules(13), ligands (14), and intact antibodies labeled with fluorescentdyes (15–17).

Although the exquisite specificity of antibodies offers greatpromise in the targeted delivery of fluorescent dyes to cancer cellsin vivo, the large size of intact antibodies (�155 kDa) results inslow tumor penetration and long circulating half-life. The use ofantibody fragments enables preservation of cancer specificity withfaster tumor targeting kinetics and potentially greater intratu-moral diffusion (18, 19). Indeed, the rapid penetration of thefragments may enable imaging within 2 hours of injection (20).Recombinant humanized antibody fragments (minibodies anddiabodies) have been developed that retain their specificity toprostate cancer and have been used successfully to image softtissue and bone metastases using positron emission tomography(PET) in both transgenic and xenograft models (21, 22).

In the current study, we employed diabodies that target theprostate stem cell antigen (PSCA), a glycosyl phosphatidylino-sitol-anchored glycoprotein that is expressed on the cell surfaceof virtually all prostate cancers with minimal background innormal prostate or surrounding tissues (23). Higher levels ofPSCA have been correlated with poor prognosis and metastaticdisease (24, 25). PSCA is also upregulated in a substantialpercentage of bladder and pancreatic cancers (26, 27). It is botha promising therapeutic and imaging target (21, 28). Thera-peutic monoclonal antibodies against PSCA have been evalu-ated in phase I and II clinical trials. A clinical trial using anengineered humanized anti-PSCA minibody has recently begunpatient enrollment (R.E. Reiter; unpublished data).

With a goal of offering molecular imaging–guided surgery tomen undergoing prostatectomy, we employed a humanized anti-PSCA cys-diabody fragment, A2, and site specifically labeled itwith a far-red fluorescent dye, Cy5, via maleimide chemistry. Thepurity and specificity of the probe was evaluated in vitro. Optimalin vivo imaging parameters were determined by imaging humanprostate cancer xenograft–bearing mice. We performed real-timefluorescently guided surgery to remove invasivemouse xenograftsand elucidate the potential clinical utility of this probe in detect-ing small foci of residual prostate cancer. We also performed a

prospective randomized study to assess the ability offluorescentlyguided surgery to reduce positive surgical margins using anintramuscular model that yields difficult to resect tumors.

Materials and MethodsReagents

The 2B3 A2 cys-diabody, (cDb, 50 kDa) was developed andvalidated for preclinical in vivo targeting of PSCA at UCLA (LosAngeles, CA; ref. 29). It was derived by yeast affinity maturationof a humanized monoclonal anti-PSCA antibody, 2B3, andengineered to contain a C-terminal–free cysteine that forms aninter-chain disulfide bond stabilizing dimerization. Upon mildreduction this disulfide bond can be broken and free thiols areavailable for site-specific labeling away from the antigen-bind-ing site using, for example, maleimide chemistry. A2 cDb waspurified from mammalian cell culture supernatant usingimmobilized metal affinity chromatography. Protein concen-trations were determined photometrically and purity was ana-lyzed by SDS-PAGE. Detailed biodistribution data for the A2cDb was previously determined (21). Nonspecific binding wasnot seen. Fluorescent signals were present in liver, kidney, andbladder due to the metabolism and urinary excretion of theprobe. Cy5 Maleimide (649 nm absorbance, 670 nm emission)was purchased from GE Healthcare.

Synthesis of Cy5-cDb probeTo achieve optimal conjugation efficiencies, the diabody was

first concentrated using an Amicon Ultra-0.5 mL (10 K) Cen-trifugal Filter Device (Millipore) to a concentration greater than2.8 mg/mL. Then, 50 mmol/L diabody was reduced in 40-foldmolar excess of TCEP for 2 hours at room temperature. A 20-fold molar excess of Cy5 maleimide dissolved in dimethylfor-mamide was then added to the reduced diabody and themixture was incubated for 2 hours at room temperature. Afterincubation, excess dye was removed using a 2 mL Zeba DesaltSpin Column (Thermo Scientific). Cy5 and diabody concentra-tions were then measured using a spectrophotometer at 650and 280 nm, respectively. The ratio of Cy5 to diabody wascalculated to confirm the number of fluorophore moleculesconjugated to each diabody molecule.

Size exclusionSize exclusion chromatography (SEC) was performed using a

Superdex 75 HR 10/30 column (GE Healthcare Life Sciences) onan AKTAPurifier and PBS asmobile phase at aflow rate of 0.5mL/minute. Both A280 for protein detection and A650 for fluorophoredetection were monitored during elution. Retention time wascompared to following standard proteins: bovine serum albumin(66 kDa), carbonic anhydrase (29 kDa), and cytochromeC (12.4 kDa; Sigma-Aldrich, Saint Louis).

Cell cultureCWR22Rv1 cells that express minimal levels of endogenous

PSCA were obtained from ATCC and cultured in RPMI1640medium containing 10% FBS, 1� sodium pyruvate and 1%penicillin–streptomycin–glutamine (PSG). A PSCA-expressinglentivirus was used to transduce these cells to generate a22Rv1-PSCAþ line, as previously described (30). Quantitativeflow cytometry with the murine 1G8 anti-PSCA antibody previ-ously showed little or no expression of PSCA on 22Rv1 cells, with

Translational Relevance

The ability to visualize cancer in real time usingmolecularlytargeted fluorescent probes has the potential to transform themodern practice of cancer surgery. In the case of prostatecancer, the inability to differentiate cancer from normal sur-rounding structures contributes both to incomplete cancerremoval and surgical side effects. Technology for fluorescenceimaging in humans during robotic surgery is commerciallyavailable, but its application is limited by the lack of prostate-specific optical probes. We report the development and val-idation of a novel targeted optical imaging probe using anantibody fragment against a cell surface marker of prostatecancer. Such probes will be useful for maximizing resection ofprimary and metastatic cancers while minimizing damage tocritical adjacent tissues.

Sonn et al.

Clin Cancer Res; 22(6) March 15, 2016 Clinical Cancer Research1404




2.2� 106 PSCA antigens on 22Rv1-PSCAþ cells (30). LAPC-9 cellsthat endogenously express PSCA were passaged in vivo andexplanted tumors were processed into single-cell suspensionbefore injection into nude mice as described previously (28).

Flow cytometry analysisBoth parental and PSCAþ 22Rv1 cells were dissociated with

glucose- EDTA and stained with the Cy5-cDb probe (1 mg per 1�106 cells) on ice for 1 hour followed by 3�washes with PBSþ 2%FBS. Amurine anti-human PSCA antibody, 1G8 (28), was used aspositive control followedbyAlexa Fluor 647-goat-anti-mouse IgGas secondary antibody (Invitrogen). Fluorescence signal wasacquired and analyzed using a FACSCalibur flow cytometer(BD Biosciences).

Measurement of affinity using an Attana Cell 200 C-Fast systemThe antigen, human PSCA-mouseFc fusion protein, was

immobilized on an LNB-carboxyl sensor chip by amine cou-pling at a concentration of 50 mg/mL according to the manu-facturer's protocol, resulting in a frequency shift of 200 Hz.Binding experiments were performed with HBS-T (10 mmol/LHEPES, 150 mmol/L NaCl, 0.005% Tween20, pH 7.4) asrunning buffer and the temperature was controlled at 22�C.Varying concentrations of the antibody fragments (300–3nmol/L, quadruplicates) were analyzed for binding and thechip surface was regenerated using 10 mmol/L glycine pH 2.5between each sample. Buffer injections prior to each samplewere used as internal reference and an activated/deactivated(no antigen) surface chip was used for external reference. Datawere collected and analyzed using the Attana Attach�e softwareand the binding curves were fit using a mass transport limitedbinding model.

Xenograft modelsAll procedures were approved by the UCLA Animal Research

Committee. Five- to 8-week-old male athymic nude mice wereused for all experiments.Micewere fed irradiated, alfalfa-free food(Harlan Laboratories) to reduce nonspecific fluorescent signal.For subcutaneous models, 1.5 � 106 22Rv1 or LAPC-9 cells weremixed with Cultrex (1:1, v/v, Trevigen, Inc) and injected into thesubcutaneous space overlying the shoulder on the dorsal surfaceof the mice. Particularly, in 22Rv1 experiments, the parental andthe PSCA-overexpressing lines were implanted in the same mice,with the parental tumor on the left shoulder and the PSCAþ tumoron the right. When combined tumor size approached 15 mm, invivo imaging was performed (typically 10–14 days after injectionof 22Rv1, 21 days after injection of LAPC-9) and animals eutha-nized. For the intramuscular model, cells were injected into eitherthe bilateral flank muscles (in the preliminary surgical resectionexperiments) or into the right thigh muscles (in the prospectiverandomized study).

In vivo imagingImaging was performed using the IVIS 200 In Vivo Imaging

System (Xenogen). Cy5 was excited using a 615 to 665 nmbandpass filter and emission was measured at 695 to 770 nm.These settings are optimized for the dye Cy5.5, which has peakabsorbance and emission at wavelengths approximately 25 nmlonger than Cy5. Exposure time was 1 second and binning was setat medium. Prior to imaging, the probe was prepared by mixingthe Cy5-cDb, 4 mL of 10% human serum albumin, and sterilenormal saline to bring the injected volume to 100 mL. Twenty-five

micrograms or indicated doses of diabody were used in imagingexperiments. The probe was intravenously injected via the lateraltail vein. Animals were anesthetized with 2% isoflurane duringimaging sessions. Imaging was performed at 6 hours or indicatedtime points after probe administration. When multiple time-points were used, animals were allowed to awaken after imaging,and then reanesthetized for each time point. Following the finaltime point for each mouse, mice were sacrificed by isofluraneoverdose and cervical dislocation. The skinwas then removed andthe mice were again imaged with the whole-body mouse imagingsystem. Regions of interest (ROI) were delineated over the tumorsites with reference to white light. Maximal fluorescence intensityin the ROIs was analyzed using Living Image software. In micewith 2 tumors (22Rv1 experiments), the fluorescence intensity ofthe PSCAþ tumor was compared with the PSCA� tumor. In micewith 1 tumor (LAPC-9 experiment), the fluorescence intensity ofthe tumor was compared with the body (background).

SurgeryFor all surgery experiments, 25 mg of Cy5-cDb was injected

intravenously on the day of operation. Mice were imaged in vivoon the IVIS at 5 hours to confirm fluorescence detection in thetumor. Surgery was performed at 6 hours. Animals were sacrificedimmediately before surgery in accordance with our AnimalResearch Committee protocol to reduce unnecessary duress.Tumors were resected using a fluorescence dissecting microscope(LeicaM205 FA, LeicaMicrosystems) at 10�magnification underwhite light or with excitation and emission parameters for Cy5(excitation 620/60 nm and emission 700/75 nm). Images of thefluorescence signal were captured with a Leica DFC360 FXmono-chrome camera and displayed on an adjacent computer monitor.After surgery, any residual fluorescent tissue and/or tumor mar-gins were surgically collected, fixed in formalin, and sent to theUCLA pathology core facility for hematoxylin and eosin (H&E)staining and histologic analysis. In the feasibility experiment, amargin of remaining nonfluorescent tissue was also removed andprocessed as control.

ResultsDevelopment and characterization of Cy5-cys-Db

The engineered anti-PSCA cDb contains a C-terminal cysteineresidue that canbe reduced to enable site specific labeling (Fig. 1A;ref. 31). After reduction, the dimeric diabody is held togethersolely by noncovalent interchain interactions. Therefore,completely reduced diabody migrates on SDS-PAGE as a 25 kDamonomer band, whereas the nonreduced diabody migrates as a50 kDa band (Supplementary Fig. S1A). Following reduction, thecysteine moiety is available for binding to the Cy5 dye viamaleimide chemistry (Fig. 1A). We investigated a variety ofdifferent concentrations of dye to maximize site-specific conju-gation. Optimal conjugation was obtained using a 15-fold molarexcess of Cy5 relative to the cys-diabody (SupplementaryFig. S1B). To assess reproducibility of the diabody–Cy5 conjuga-tion, the dye:protein ratio was checked (n ¼ 8). The mean dye:protein ratio was 1.31 (SD ¼ 0.04). To evaluate purity andintegrity of the conjugate, a size exclusion experiment was per-formed. This demonstrated good purity (major peak representingaCy5-cDbmolecule)withno signof protein aggregation andonlya small amount of unbound dye (late elution peaks; Fig. 1B). Boththe unconjugated diabody and the Cy5-cDb eluted with similarretention times (22.4 and 22.5 minutes, respectively) indicating

Fluorescent Image–Guided Surgery with an Anti-PSCA Diabody

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that reduction of the cys-diabody and conjugation of the Cy5 didnot interfere with dimer maintenance.

To determine whether the Cy5–cDb conjugate maintains itsbinding to PSCA in vitro, a series of flow cytometry experimentswere performed using PSCA overexpressing 22Rv1 cells (Fig. 1C).Specific binding of Cy5-cDb to PSCAwas demonstrated, althougha low level of binding to 22Rv1-PSCA� cells was also observeddue to low endogenous levels of PSCA expression. We furtherconfirmed these results in an endogenous PSCA-expressing pan-creatic cell line, Capan-1 (data not shown). Furthermore, to

investigate whether Cy5 labeling had affected the binding affinityquantitatively, quartz crystal microbalance (QCM) measure-ments were conducted using an Attana Cell 200 C-Fast systemto assess the interaction of Cy5-cDb to recombinant PSCA pro-tein, which was immobilized on an LNB carboxyl sensor chip,in comparison with the mock-treated cDb control. As shownin Fig. 1D, both Cy5-cDb (0.42 nmol/L) and mock-labeled cDb(1.96 nmol/L) had apparent affinity in the low nanomolar rangeconfirming that reducing the C-terminal disulfide bridge and site-specific conjugation of maleimide-Cy5 did not impair binding of

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Figure 1.Biochemical and functionalcharacterization of Cy5-conjugatedanti-PSCA cys-diabody. A, schematicof the conjugation reaction. Anti-PSCA cys-Diabody was first reducedby TCEP to open up the interchaindisulfide bond, revealing cysteineresidues that can then be conjugatedto Cy5 via maleimide chemistry. B,size exclusion chromatography ofpurified A2_cDb and Cy5-labeledA2_cDb. Retention time of standardproteins is indicated. C, flowcytometry analysis comparingbinding of Cy5-cDb to 22Rv1 cells thatover express PSCA (left) and theparental line (right) that expressesvery low level of PSCA. Strongbinding of Cy5-cDb to PSCA (orangeline) was evident compared with thepositive control (red line). Right, asmall amount of binding to thePSCA- 22Rv1 cells (orange line)was also present. D, quartz crystalmicrobalance (QCM) measurementsof the interaction between mocklabeled cDb or Cy5-cDb andrecombinant PSCA. The tablesummarizes the on and off rates andBmax for indicated samples.Mock-treated cDb had an apparentaffinity of 1.96 nmol/L and Cy5-cDb0.42 nmol/L, both in the lownanomolar range.

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the cys-diabody to PSCA. Small differences in Kd might beattributed to minor inaccuracy in protein concentration due tothe presence of BSA in the samples, whichwas required during thepurification procedure to stabilize the diabody.

In vivo tumor imagingTo determine whether the Cy5-cDb probe binds to PSCA-

expressing tumors in vivo, we first performed imaging experimentswith subcutaneous xenografts. Four mice, each bearing PSCAþ

and PSCA� 22RV1 xenografts, were imaged on the IVIS 200(excitation 615–665 nm, emission 695–770 nm) at 6 hoursafter intravenous injection of 25 mg of the probe. This revealedfluorescence in the PSCAþ tumor and lack of fluorescence in the

non–PSCA-expressing tumor (Fig. 2A and B). Strong autofluor-escence was detected from the skin and intestines at this wave-length, and fluorescence was also identified in the kidneys due torenal excretionof the probe.We repeated the experiment using theendogenously PSCA-expressing LAPC-9 xenograft to rule out anymodel-specific effects. We consistently observed positive fluores-cent signal only in PSCAþ tumors, with a mean tumor-to-muscleratio of 4.48 (SD 0.52; Supplementary Fig. S2). Furthermore, toconfirm that the positive signal observed in PSCAþ tumors wasattributable to Cy5-cDb and not preferential uptake of free Cy5dye, we imaged mice bearing both PSCAþ and PSCA� 22Rv1xenografts with 5 mg of free Cy5. At 4 hours after injection, PSCA�

tumors (n ¼ 4) exhibited greater fluorescence than their PSCAþ

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Figure 2.Determination of the optimal imaging parameters for in vivo detection of PSCA-expressing tumors using Cy5-cDb. Fluorescent signal intensity increases from darkred to yellow. A and B, a mouse bearing 22rv1 PSCAþ (right shoulder) and PSCA� (left shoulder) tumors was photographed (A) and fluorescent imaged (B)6 hours post intravenous administration of the Cy5-cDb probe. The skin was removed to avoid interference from autofluorescence. PSCA-specific fluorescentsignalsweredetectedon the right shoulder. Background signal is present in the skin and kidneys (probe excretion). C, dose determination. 2.4, 12, or 25mgof Cy5-cDbwas intravenously administrated to PSCA (þ) and (�) 22rv1 tumor bearing-mice (shown are 5 animals in each group). Fluorescent imagingwas performed at 6 hoursafter skin removal. Red arrow: PSCA (þ) tumor; green arrow: PSCA (�) tumor. Mean ratios of PSCA � tumors in each dose were also indicated. D–F, timingdetermination. Five PSCA (þ) and (�) 22rv1 tumor-bearing mice received 25 mg of Cy5-cDb intravenously and were then imaged at indicated time points.D, serial images of a representative animal over time. Red arrow: PSCA (þ) tumor; green arrow: PSCA (�) tumor; yellow arrow: background signal from stomach.E, maximum fluorescence intensity over time determined bymeasurement of respective PSCA (þ) and (�) tumor regions of interest. F, PSCA (þ) to PSCA (�) ratiosover time calculated on the basis of E. As the skin was not removed for (E) and (F), the levels and ratios shown will underestimate the true � ratio, thoughprovides the optimal time point for imaging.






counterparts.MeanPSCAþ to PSCA� ratiowas0.55 (SD0.18; datanot shown).

Dose determination. To determine the optimal dose for in vivoimaging, mice bearing PSCAþ and PSCA� 22Rv1 xenografts wereinjected with 2.4 mg, 12 mg, and 25 mg (n ¼ 5) of the probe andimaged at 2, 4, and 6 hours using the IVIS 200. PSCAþ tumorsfluoresced more than PSCA� tumors at each dose. Post mortemPSCAþ to PSCA� ratios at 6 hours were 1.58 (SD 0.52), 2.43(SD 0.58), and 4.47 (SD 1.63) for the 2.4 mg dose, 12 mg dose, and25 mg dose, respectively (Fig. 2C). Based upon this result, subse-quent imaging experiments were performed using the 25 mg dose.

Optimal time for imaging. To evaluate the optimal interval fromprobe injection to in vivo imaging, mice with PSCAþ and PSCA�

22Rv1 xenografts were administered 25 mg of Cy5-cDb andserially imaged at 1, 2, 4, 6, 8, and 24 hours (n ¼ 5). Maximumfluorescence intensity and ratios from PSCAþ and PSCA� tumorswere determined at all timepoints (Fig. 2D–F).Maximal PSCAþ tonegative ratio and fluorescence intensity were reached at 6 hourspostinjection (Fig. 2E and F). To remove interference from skinautofluorescence, the experiment was repeated at the 2, 6, and 24

hour timepoints without skin overlying the tumor. Again, peakPSCAþ to PSCA�

fluorescence intensity ratio was achieved at 6hours. The post mortem ratios were 1.94 (SD 0.66) at 2 hours,4.04 (SD 1.52) at 6 hours, and 3.53 (SD 1.58) at 24 hours (P ¼0.06; Supplementary Fig. S3).

Surgical resection of tumors under fluorescence guidanceTo assess the feasibility and utility of optical imaging to aid

complete tumor resection, we established intramuscular xeno-grafts of PSCAþ 22RV1 (n ¼ 7; Fig. 3 and Supplementary Fig. S4)and LAPC-9 (n ¼ 3; Supplementary Fig. S5), in which tumors areinvasive and therefore difficult to resect under white light alone.Upon tumor establishment, 25 mg of Cy5-cDb were administeredintravenously and surgery was performed 6 hours later. We firstmade a skin incision and exposed the surface of thigh muscle(Fig. 3A). We attempted to resect the tumor completely underwhite light alone (Fig. 3B, left). Next, the fluorescent mode wasturned on to evaluate for residual signal (Fig. 3B, right). Areaswithresidualfluorescencewere surgically resected,fixed, and examinedhistologically to confirm that the fluorescence was indeed comingfrom cancer cells (Fig. 3C). A final fluorescent image was takenafter the secondary surgery (Fig. 3D) and tissues from the tumor

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Figure 3.Cy5-cDb enabled fluorescence-guided surgical resection of PSCA-expressing tumors. A, white light andfluorescent image of 22Rv1 PSCA (þ)intramuscular tumor (arrow) prior toresection. B, fluorescent image afterwhite light surgical resection showsresidual cancer that is not clearly seenon white light. C, H&E of residualfluorescent tissue from Bdemonstrating tumor (�) and normaladjacent muscle. D, fluorescent imageshowing absence of signal followingfluorescence-guided surgicalresection. H&E staining and systematicsectioning of tissue from this areaconfirmed absence of tumor.

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bed were collected and examined histologically to confirm theabsence of residual tumor. As demonstrated in Fig. 3A, thesetumors were often indistinct compared with muscle, and theextent and margins difficult to readily identify under white light.Of the 10 tumors, 8 had postresection sites (six 22Rv1 and twoLAPC-9) that haddetectable residualfluorescence followingwhitelight surgery, some areas as small as <1 to 2 mm. In some cases,there were multiple sites of residual fluorescence adjacent to asingle tumor. Histology of residual fluorescent foci was positivefor cancer in all 8 cases (exemplified by Fig. 3B and C).

Prospective comparison of surgical margins followingresection with or without the aid of fluorescence

To demonstrate the ability of fluorescent surgery to reducepositive surgical margin rates in a controlled, objective manner,we next performed a prospective randomized study comparingwhite light versus white light and fluorescent surgery. As shownin Fig. 4A, 17 nude mice received intramuscular implantation ofPSCAþ 22Rv1 xenografts on their thighs 21 to 22 days before theintravenous injection of Cy5-cDb. Mice were then randomizedinto two cohorts to receive either just a white light surgery or anadditional fluorescence-guided surgery. The surgeon was blindedto the groupings while performing the first-round white lightsurgery to insure that all 17 mice received the same unbiasedoperation, with the goal of removing as much tumor as possiblewhile preserving adjacent normal tissues (akin to radical prosta-tectomy). As expected, the xenografts were invasive into the thighmusculature and demonstrated strong fluorescence and contrastwith adjacent nerves and normal tissue (Supplementary Fig. S6).Followingfirst stagewhite light resection, thefluorescent lightwasturned on to assess margin status; all mice (n ¼ 17) had residualfluorescent signals (Fig. 4B). At this point,mice randomized to thefluorescence cohort underwent secondary surgery to removeresidual fluorescing tissue, which was then subjected to histologicstaining and analysis. Consistent with the previous pilot study, allremaining fluorescent tissues collected from the 9 animals in thiscohort contained residual cancer (Fig. 4C). Finally, to determinefinal margin status, remaining thigh musculature was harvestedand examined for thepresence of prostate cancerwith the aid of anexpert uropathologist. Positive surgical margins were found in 8of 8mice in thewhite light only cohort but 0 of 9mice assigned tofluorescent image–guided surgery (Fig. 4D and SupplementaryFig. S7).

DiscussionThe inability to visualize cancer during surgery leads to incom-

plete cancer removal and surgical side effects. This is especiallytrue in prostatectomy, where small foci of extracapsular extensionof prostate cancer can easily be overlooked, leading to positivesurgical margins and compromised onocologic control. Thisencourages surgeons to resect healthy tissue in an attempt atgood oncologic control, leading to substantial urinary and sexualside effects. The ideal imaging agent is one that is specific to cancer(or the cancer-bearing organ where the entire organ is to beextirpated), safe, sensitive enough to detect small amounts ofresidual tumor, and has a half-life to allow visualization through-out the critical span of the operation. To date, no such opticalprobe is clinically available.

Using an antibody fragment labeled with a fluorescent dye, wedeveloped andvalidated anoptical imagingprobe (Cy5-cDb) that

specifically recognizes prostate cancer xenografts expressing thecell surface glycoprotein PSCA. The far-red fluorescent dye Cy5was chosen because of the stability and ease of conjugation ofcyanine dyes, the feasibility of performing site-specific labeling ofour cys-diabody, the lack of affinity change with Db-Cy5 conju-gation, and the availability of equipment for whole-body andmicroscopic imaging during surgery. The probe enabled fluores-cent imaging in real time to guide surgical resection of tumors inmice. The small size of the diabody (50 kDa, below the thresholdfor renal clearance) enabled administration and imaging on thesame day, and persisted throughout the course of surgery.

In vitro and in vivo characterization of the Cy5-cDb probedemonstrated high affinity to recombinant PSCA protein as wellas excellent specificity for PSCA-expressing cells. When injectedintravenously into mice, optimal imaging was achieved within 6hours, and inmany cases, strong signal to backgroundwas presentwithin 2 hours of administration. This compares favorably to astudy by Nakajima and colleagues in which prostate cancerxenografts were imaged using an anti-prostate–specific mem-brane antigen (PSMA) intact antibody conjugated to ICG, wheretime from administration to imaging was 2 days (16).

When using the probe to guide surgical resection of tumors inmice, the signal was sufficiently strong to be seen easily and usedto guide surgery at a video-quality frame rate. Furthermore, usingfluorescence, residual tumor fragments <1 mm in size were oftenidentified after standard surgical resection with white light.

We demonstrated the ability of real-time fluorescent-imageguided surgery to significantly reduce positive surgical marginrates when compared with white light surgery alone in an intra-muscular model of prostate cancer. An intramuscular model waschosen to provide a scenario inwhich complete removal of cancerwould present a challenge for the surgeon under white light,examining the hypothesis that our diabody probe can aid inidentifying and completely resecting small foci of residual cancerfollowing white light surgery. Although no small animal modelcaptures the complexities of radical prostatectomy, we believe ourfindings highlight the potential utility in identifying small foci ofextracapsular extension of cancer during prostatectomy thatwould otherwise be missed. We envision this can play a role inthe posterolateral prostate during nerve sparing, and may aid inthe apical dissection, which lacks distinct capsule and surgicalplanes. Presence of fluorescence may prompt intraoperative fro-zen sectionswhen the exact extent of cancer is unclear in vital areassuch as the neurovascular bundle (32, 33).

Several other research groups have investigated similar strate-gies of targeted fluorescent molecular imaging. Jiang and collea-gues imaged tumor xenografts that overexpress matrix metallo-proteinases 2 and 9 using activatable cell penetrating peptides(ACPP) that are cleaved by the MMPs, thereby activating fluores-cence (11). The group injected their ACPPs intravenously intomice bearing isografts two days prior to imaging, then resected thetumors and demonstrated improved survival in mice whosetumors were removed using fluorescence guidance (12). Theability of the ACPP to image prostate cancer has yet to beinvestigated. Nakajima and colleagues developed an activatableantibody–fluorophore conjugate made of a humanized anti-PSMA antibody (J591) linked to ICG. They used this to performin vivo imaging of prostate cancer xenografts expressing PSMA(16). As with the study using ACPPs, thismethod is limited by the�2 days required from administration to imaging. The use of thisprobe to guide surgical resection has not been published. The






T

T Fully grown tumor

White light surgery

T

T Positive margin

T

N = 9

Secondary fluorescent

surgery

T Negative margin

N = 17 Margin evaluation by

histology

TN = 9

N = 8

A

CB

*

*

**

D

E

*

Figure 4.Cy5-cDb–directed fluorescence-guided surgery enabled complete removal of infiltrative intramuscular prostate cancer. A, schematics of the study design. B and C,representative white light (B) and fluorescent (C) images of a tumor bed after white light only surgery revealing tumor that would otherwise have been missed.D, the residual fluorescent tissue from (B and C) proved to be cancer (�) by H&E staining (10� magnification). E, representative H&E staining of tumormargins from the white light surgery alone [top; with residual tumors (�)] and the fluorescence-guided surgery cohorts (bottom; no residual tumors).

Sonn et al.





strategy of using an antibody fragment to shorten the intervalfrom administration to imaging was employed by Oliveira andcolleagues They used an anti-EGFR antibody fragment (7D12nanobody) labeled with the NIR dye IRDye800CW. This enabledtumor visualization as early as 30minutes postinjection (20). Useof this probe in surgical resection has not been reported. Morerecently, Neuman and colleagues reported the use of a lowmolecular weight NIR fluorescent agent (YC-27) that targetsPSMA in preclinical models of prostate cancer (13, 34). Survivalsurgery in a mouse subcutaneous xenograft model comparingwhite light to real-time fluorescent was performed 20 hourspostinjection, with no recurrence in the fluorescent group(0/8). Investigators noted adequate tumor contrast with YC-27within 6hours of administration. Although todate clinical studiesof fluorescently labeled prostate probes have not been reported,Maurer and colleagues recently described the use of a hand-heldgamma camera to detect signal from a gallium-labeled PSMA PETprobe in metastatic lymph nodes intra-operatively (35). Onewould predict that use of an optical label would improve thesensitivity of detection. Consistent with this hypothesis, we aredeveloping dual PET/optical tracers to enable PET imaging fol-lowed by fluorescently guided surgery.

Although the results using our probe to resect tumors in miceare promising, several limitations are acknowledged. First, imag-ing and surgery were performed in xenograft tumor models usinghuman prostate cancer cell lines. The humanized diabody doesnot recognize mouse PSCA. Therefore, evaluating the signal tobackground that will be achieved for cancer surgery in humanswill require use of genetically engineered PSCA knock-in mousemodels or an antibody fragment capable of recognizing bothhuman and murine PSCA. Second, while this proof-of-conceptstudy demonstrates the ability to find and resect small foci ofresidual cancer following white light surgery and significantlyreduce positive surgical margins, no small animal model existsthat recapitulates the complexities of prostatectomy, and as suchthe extent to which this improves surgical outcomes will have tobe determined clinically (12). Third, the use of afluorescent probeto improve detection and resection of lymphnodemetastasis is anexciting potential application. We have yet to investigate thefeasibility of this application by evaluating lymph node imagingwith our probe. Fourth, autofluorescence at the emission wave-lengthofCy5 is greater than that for near-infrared (NIR) dyes (36).This is particularly apparent in skin and the gastrointestinal tract,although it can be reduced by dietary changes (37). We acknowl-edge that the current trend for imaging applications is towardsNIRdyes. Cy5was chosen given the ease of conjugationof cyaninedyes and the feasibility of performing site-specific binding to ourcys-diabody. In addition, Cy5 is compatible with commonlyavailable light sources and CCD cameras at our facility withoutsignificant alterations. We are currently developing ICG and IR-800–labeled diabodies. Fifth, the differential fluorescencebetween benign human prostate and prostate cancer has yet tobe evaluated.

Notwithstanding these limitations, the development of noveltargeted fluorescent probes has tremendous translational poten-tial. Optical probes are relatively easy to produce and imaging

technology is relatively inexpensive (20). In fact, a fluorescentcamera is already available for the Da Vinci robot (IntuitiveSurgical), the surgical system used to perform the overwhelmingmajority of robotic prostatectomies in the world. To make use ofthis technology, amethod to selectively deliver thefluorescent dyeto cancer cells is required. The Cy5-cDb probe developed hereinenables targeted detection and resection of cancers expressingPSCA using an antibody fragment that enables same day admin-istration and imaging. Efforts to translate this probe into the clinicare ongoing.

ConclusionWe successfully synthesized amonoclonal antibody fragment–

fluorophore conjugate consisting of a humanized anti-PSCA dia-body linked to the fluorescent dye Cy5 that enables same dayadministration and in vivo imaging. This probe was used success-fully to resect residual cancer fragments in mice under real-timefluorescent guidance, and demonstrated a significant reduction inpositive surgical margins compared with white-light surgeryalone.

Disclosure of Potential Conflicts of InterestE.J. Lepin is an employee of ImaginAb. A.M. Wu has ownership interest

(including patents) in and is a consultant/advisory board member for Imagi-nAb. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G.A. Sonn, A.S. Behesnilian, Z.K. Jiang, K.A. Zettlitz,S.M. Knowles, A.M. Wu, R.E. ReiterDevelopment of methodology: G.A. Sonn, A.S. Behesnilian, Z.K. Jiang,K.A. Zettlitz, E.J. Lepin, L. Bentolila, S.M. Knowles, R.E. ReiterAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G.A. Sonn, A.S. Behesnilian, Z.K. Jiang, K.A. Zettlitz,E.J. Lepin, L. Bentolila, D.J.P. Lawrence, R.E. ReiterAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G.A. Sonn, A.S. Behesnilian, Z.K. Jiang, K.A. Zettlitz,R.E. ReiterWriting, review, and/or revision of the manuscript: G.A. Sonn, A.S. Behesni-lian, Z.K. Jiang, K.A. Zettlitz, L. Bentolila, D.J.P. Lawrence, A.M. Wu, R.E. ReiterAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.S. BehesnilianStudy supervision: Z.K. Jiang, E.J. Lepin, L. Bentolila

AcknowledgmentsThe authors thank VadimGoldshteyn for his expert technical assistance with

the study. In vivo fluorescence imaging was performed at the California Nano-Systems Institute Advanced Light Microscopy/Spectroscopy and the Macro-Scale Imaging Shared Facilities at UCLA.

Grant SupportThisworkwas supported by theNCI P50CA092131UCLA SPORE in Prostate

Cancer, and R01CA174294Multifunctional ImmunoPET Tracers for Pancreaticand Prostate Cancer.

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 March 2, 2015; revised July 20, 2015; accepted October 6, 2015;published OnlineFirst October 21, 2015.

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Sonn et al.




2016;22:1403-1412. Published OnlineFirst October 21, 2015.Clin Cancer Res Geoffrey A. Sonn, Andrew S. Behesnilian, Ziyue Karen Jiang, et al. Prostate Cancer Xenografts in Real TimeAntigen (PSCA) Diabody Enables Targeted Resection of Mouse

Guided Surgery with an Anti-Prostate Stem Cell−Fluorescent Image

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