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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Discov. Author manuscript; available in PMC Apr 1, 2013.
Published in final edited form as:
PMCID: PMC3351274
NIHMSID: NIHMS356665
Imaging androgen receptor signaling with a radiotracer targeting free prostate specific antigen
David Ulmert,1,2 Michael J. Evans,3 Jason P. Holland,4,5 Samuel L. Rice,4,5 John Wongvipat,3 Kim Pettersson,9 Per-Anders Abrahamsson,2 Peter T. Scardino,1,4 Steven M. Larson,4,5 Hans Lilja,1,6,8* Jason S. Lewis,4,5*# and Charles L. Sawyers3,7*#
1Urology Service of the Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
2Department of Urology, Lund University, Skåne University Hospital, Sweden.
3Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
4Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
5Department of Radiology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
6Departments of Laboratory Medicine and Medicine (Genitourinary Oncology Service), Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
7Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, U.S.A., NY 10065.
8Department of Laboratory Medicine, Lund University, Skåne University Hospital, Sweden.
9Department of Biotechnology, Tykistokatu 6A Biocity 6th floor, University of Turku, Finland.
*Corresponding Authors: Hans Lilja, M.D., Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, 1250 York Avenue, New York, NY10065, Tel: 1212-639-6982, Fax: 16464222379, liljah/at/mskcc.org
Jason S. Lewis, Ph.D., Radiochemistry Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY10065, Tel: 16468883038, Fax: 16464220408, lewisj2/at/mskcc.org
Charles L. Sawyers, M.D., Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY10065, Tel: 16468882594, Fax: 16468882595, sawyersc/at/mskcc.org
D.U. and M.J.E. contributed equally to this work
#J.S.L. and C.L.S. are senior co-corresponding authors
Despite intense efforts to develop radiotracers to detect cancers or monitor treatment response, few are widely used due to challenges with demonstrating clear clinical utility. We reasoned that a radiotracer targeting a validated clinical biomarker could more clearly assess the advantages of imaging cancer. The virtues and shortcomings of measuring secreted prostate specific antigen (PSA), an androgen receptor (AR) target gene, in prostate cancer (PCa) patients are well documented, making it a logical candidate for assessing whether a radiotracer can reveal new (and useful) information beyond that conferred by serum PSA. Therefore, we developed 89Zr-5A10, a novel radiotracer that targets “free” PSA. 89Zr-5A10 localizes in an AR-dependent manner in vivo to models of castration resistant prostate cancer, a disease state where serum PSA may not reflect clinical outcomes. Finally, we demonstrate that 89Zr-5A10 can detect osseous PCa lesions, a context where bone scans fail to discriminate malignant and non-malignant signals.
Keywords: PET, prostate cancer, prostate specific antigen, androgen receptor, biomarker
Abbreviations: PSA, PCa, AR, CRPC, 18F-FDG, fPSA, mAbs, PET, MRI, PSMA, ITLC
The serendipitous discovery that 18F-FDG uptake reflects aberrant c-KIT signaling (and predicts response to imatinib therapy) in gastrointestinal stromal tumors has greatly simplified patient management[1], and more generally, underscores the promise of molecular imaging in oncology. However, deliberately engineering radiotracers to achieve similar success in other tumors has proven challenging, resulting in the high attrition rate of novel radiotracers in the clinic.
The target of a radiotracer necessarily frames its potential context of use (i.e. detection, response indicator), and candidates are often selected on the basis of preclinical evidence pointing to an upregulation in cancer. In this regard, it can be challenging to appropriately evaluate novel radiotracers in patients without a thorough appreciation of the patho-biological mechanism of target upregulation. Therefore, we reasoned that a radiotracer targeting a well studied, tumor-specific clinical biomarker reflective of oncogenic pathway activation could more rapidly document the unique advantages of studying patient response with a cognate non-invasive imaging tool.
To establish this concept, we selected PSA based on the large body of research highlighting the virtues and shortcomings of measuring the secreted forms of this protein in PCa[24]. Originally, it was hoped that measuring total concentrations of PSA in serum might revolutionize PCa management by allowing early detection of subclinical disease and precise monitoring of residual disease after therapy. This hope was based on the fact that PSA is expressed almost exclusively by prostate epithelia and, as an AR target gene, its expression reflects AR pathway activity. Years of work to clinically validate this biomarker have revealed certain limitations. For example, despite strong associations between metastasis or death from prostate cancer and PSA levels in blood[5], the inability to distinguish PSA produced by normal versus malignant prostate tissue limits its general utility in primary screening. Moreover, apart from a few contexts in which a dramatic reduction confirms successful therapeutic intervention (e.g. post radical prostatectomy), interpreting changes in serum PSA levels in response to therapy has been problematic. At first glance, this may seem surprising since PSA expression is tightly coupled to AR signaling. However, the ability to detect PSA in serum not only requires expression but also secretion and leakage into the circulation—two processes that are very poorly understood[2]. Also, it is well documented that only a very small percentage of intratumoral PSA is secreted into perivascular space[6], and the rate determining step to serum circulation is undefined. These considerations raise the possibility that a non-invasive tool measuring tumor-associated PSA expression could more clearly reflect AR-driven changes in PSA expression.
PSA is initially produced as a catalytically active serine protease (“free” PSA [fPSA]), and subsequent to its release into the perivascular space, it is rapidly and irreversibly converted to non-catalytic forms (“complexed” PSA)[7, 8]. We therefore reasoned that 5A10, a monoclonal antibody that specifically recognizes an epitope adjacent to the catalytic cleft of PSA[911] (and therefore selectively binds fPSA), could in principle target tumor-associated PSA. In considering radiolabeling strategies, we noted that our recent studies with 89Zr-labeled mAbs yielded high contrast images of tumors, with low radiotracer uptake in normal tissues[1214]. Based on these observations, we prepared 89Zr-labeled 5A10.
We conjugated 89Zr to 5A10 with the chelator desferrioxamine B via a previously established synthetic route (see Methods and Supplementary Fig. S1)[12, 14]. To determine the affinity of 89Zr-5A10 for fPSA, competition binding assays were conducted, and the bioconjugation of 5A10 resulted in virtually no loss of affinity for purified fPSA (Supplementary Fig. S2).
We began in vivo studies by administering 89Zr-5A10 to intact male mice inoculated with subcutaneous xenografts of LNCaP-AR (a PSA positive PCa model derived from parental LNCaP overexpressing wild type AR[15]). This model was chosen because the magnitude of PSA production and circulation in mouse serum closely approximates that observed in patients with advanced disease (Supplementary Table S1). Tissues were harvested at multiple time points post injection to determine the kinetics of radiotracer biodistribution (Fig. 1a). Peak tumor-associated activity was observed 24 hours post injection (19.59 ± 4.9 % ID/g, tumor-to-muscle ratio: 22.80 ± 18.6), and with few exceptions, little 89Zr-5A10 accumulation was observed in host tissues (Fig. 1a, Supplementary Fig. S3, and Supplementary Tables S2 and S3). PET studies showed a region of contrast at the tumor, supportive of the biodistribution data (Fig. 1b). Using 100-fold excess of unlabeled 5A10 to compete 89Zr-5A10 uptake, we confirmed the specificity of the biological interaction between 89Zr-5A10 and LNCaP-AR (Fig. 1c, Supplementary Fig. S4, and Supplementary Tables S4 and S5). Moreover, there was little incorporation of the non-specific radiotracer 89Zr-labelled mouse IgG1 in LNCaP-AR at 24 hours post injection (Fig. 1c, Supplementary Fig. S5, and Supplementary Tables S6 and S7). As expected, PC3 xenografts, an AR- and PSA-negative model of human PCa, showed little avidity for 89Zr-5A10 at 24 hours post injection (Fig. 1c, Supplementary Fig. S6 and Supplementary Table S8). Finally, subcutaneous CWR22Rv1 xenografts were avid for 89Zr-5A10, another AR- and PSA-positive PCa model (Fig. 1c, Supplementary Fig. S6 and Supplementary Table S9).
Figure 1
Figure 1
89Zr-5A10 specifically localizes to multiple preclinical models of AR- and fPSA-positive PCa
We next asked whether 89Zr-5A10 could detect androgen-regulated elevations in fPSA expression. Castrate male mice were inoculated with LNCaP-AR, and after tumor formation, animals received no manipulation or a surgically implanted subcutaneous testosterone pellet. 89Zr-5A10 was administered 7 days post manipulation, and biodistribution studies were conducted 24 hours post injection 89Zr-5A10 localization was significantly higher in LNCaP-AR xenografts exposed to testosterone compared to control (Fig. 1d, Supplementary Fig. S7 and Supplementary Table S10). Intratumoral PSA levels also increased, as expected (Supplementary Table S11). Similar results were observed with CWR22Rv1 xenografts (Supplementary Fig. S8, and Supplementary Tables S12 and S13). Collectively, these results show that 89Zr-5A10 can faithfully reflect intratumoral AR signaling.
We next tested if pharmacological inhibition of AR can be quantified in vivo with 89Zr-5A10 PET using the antiandrogen MDV3100, whose clinical activity is correlated with responses in the LNCaP-AR model[16, 17]. Castrate male mice were inoculated with subcutaneous LNCaP-AR xenografts, and tumor-bearing mice were randomized into groups receiving a daily oral gavage of vehicle, or MDV3100 at 10, 40, or 80 mg/kg. Seven days post initiation of treatment, 89Zr-5A10 was administered, and biodistribution studies were conducted 24 hours post injection (Fig. 2a, Supplementary Fig. S9 and Supplementary Table S14). As expected, this course of therapy inhibited tumor growth and fPSA protein in the tumor (Supplementary Fig. S10 and Supplementary Table S15). Accordingly, tumor-associated 89Zr-5A10 was significantly decreased by a 40 and 80 mg/kg dose of MDV3100 (Fig. 2a). Statistically significant changes in tumor-associated 89Zr-5A10 were observed by PET between groups of animals receiving a daily oral gavage of vehicle or 80 mg/kg MDV3100 (Figs. 2b and 2c, Supplementary Table S16). In addition, a significant increase in tumor-associated 89Zr-5A10 was observed in a separate treatment arm of mice receiving subcutaneous testosterone pellets. Notably, the fold change in tumor-associated 89Zr-5A10 closely mirrored the fold change in expression of intratumoral fPSA. For example, the testosterone pellet resulted in ~2.5 fold increase in tumor-associated 89Zr-5A10 (Supplementary Table S10), very close to the ~2.6 fold increase in fPSA expression in the LNCaP-AR tumors (Supplementary Table S11). Collectively, these results highlight the ability of 89Zr-5A10 to measure pharmacologically triggered changes in intratumoral AR signaling.
Figure 2
Figure 2
89Zr-5A10 detects pharmacological inhibition of AR in vivo
One of the challenges in evaluating experimental therapies like MDV3100 is assessing therapeutic effects on individual metastatic lesions in patients with diffuse disease[18]. Although bone is the most common site of metastatic spread in PCa[19], they remain particularly challenging to characterize, because the most widely used nuclear medicine technologies (e.g. 99mTc-MDP, 18F-NaF) do not directly image the tumor but, rather, target nearby normal bone repair[20]. Therefore, these scans cannot distinguish between malignant and non-malignant disease (e.g. injury or degenerative joint disease). Furthermore, response to antiandrogen therapy cannot be efficiently assessed since resolution of tumor-induced bone repair can lag clinical response by months or years.
With these considerations in mind, we asked if 89Zr-5A10 can more clearly distinguish a skeletal PCa lesion. Osseous tumors were established in intact male mice via injection of LNCaP-AR in the tibia of the left hindlimb, and tumor development was confirmed by MRI after 7 weeks (Supplementary Figs. S11 and S12). PET/CT studies showed high contrast in the tumor-bearing hindlimb, compared to the contralateral limb (Fig. 3a). Consistent with this observation, co-registered PET/MRI images showed an alignment of the PET and MRI contrast in the tibia (Fig. 3b and Supplementary Fig. S13). Post mortem, autoradiography of a surgically excised tibia showed that positron emissions from 89Zr-5A10 co-aligned with the topography of the LNCaP-AR lesion, defined by histology (Supplementary Fig. S14). To further confirm that 89Zr-5A10 does not cross-react with bone remodeling, bone fractures were induced surgically in a separate cohort of mice by puncturing the tibia in the right hindlimb. Both 18F-NaF and 99mTc-MDP readily localized to the site of repair, while 89Zr-5A10 did not (Fig. 3c). Collectively, these results highlight the unique specificity of 89Zr-5A10 for PCa tumors in the bone.
Figure 3
Figure 3
89Zr-5A10 specifically targets prostate cancer in the bone microenvironment in vivo
In this report, we show that changes in the expression of PSA, an AR regulated, prostate-specific gene, can be measured non-invasively with the novel radiotracer 89Zr-5A10. In support of its suitability for quantifying AR signaling in CRPC, 89Zr-5A10 readily localized to multiple AR- and PSA-positive PCa models, and quantitatively measured declines in fPSA synthesis induced by antiandrogen therapy in a clinically validated xenograft model of CRPC. Since 89Zr-5A10 specifically targets PCa cells rather than the non-malignant skeletal pathologies that phenocopy the changes induced by cancers on bone scans, this radiotracer offers the opportunity for more accurate staging and better treatment selection. Owing to the abundant expression of PSA also in benign pathologies of the prostate[2], unambiguously detecting PCa lesions within the prostate gland with 89Zr-5A10 may be challenging. In this respect, the most exciting immediate clinical application for 89Zr-5A10 is likely the opportunity to study AR-driven tumor activity in individual lesions in a heterogeneous disease to enhance the clinical assessment of advanced disease.
We previously reported that changes in prostate-specific membrane antigen (PSMA), a cell surface protein whose expression is suppressed by AR, can also serve as a noninvasive marker for imaging of AR signaling[21]. Although PSMA imaging with PET is attractive because several human-ready targeting agents already exist[22, 23], PSMA expression is not prostate-specific and the clinical impact of AR-directed therapy on PSMA expression is not known[24]. Also, a formal comparison in LNCaP-AR xenografts showed that 89Zr-5A10 PET resulted in a more compelling change in tumor localization post therapy than 89Zr-J591, a radiotracer derived from a humanized monoclonal antibody to PSMA (Supplementary Fig. S15).
The precise mechanism by which 89Zr-5A10 allows visualization of AR- and fPSA-positive PCa models is unclear. Our data and published work suggest the following model. Prior to secretion, fPSA exists as a proteolytically active protein,[25] then is transiently present in the pericellular space prior to sequestration by extracellular binding proteins that preclude recognition by 5A10. We propose that visualization of PCa cells by 89Zr-5A10 is dependent on this ability of 5A10 to recognize fPSA in this unique context. Future studies are required to refine our understanding of the biological basis of localization of 89Zr-5A10 to tumor in situ, which may also further enhance our understanding of the patho-biological mechanism(s) of fPSA secretion and processing. Regardless of the mechanism, the molecular imaging tool presented here could have near term clinical impact, particularly since the dosimetry of other 89Zr-labeled mAbs has been determined to be favorable for humans and will soon be examined in clinical trials[26].
Preparation of Zirconium-89
Zirconium-89 was produced via the 89Y(p,n)89Zr transmutation reaction on an EBCO TR19/9 variable beam energy cyclotron (Ebco Industries Inc., Richmond, British Columbia, Canada) in accordance with previously reported methods[27]. 89Zr-oxalate was isolated in high radionuclidic and radiochemical purity >99.9%, with an effective specific-activity of 195–497 MBq/µg, (5.27–13.31 mCi/µg).
Preparation of radiolabeled constructs
Desferrioxamine B (DFO) was conjugated to antibodies using the following protocol. Antibody (in PBS) was added to a centrifuge vial and the pH adjusted to 9.5 – 10.0 with Na2CO3(aq.). Four equivalents of [Fe(N-succDFO-TFP)] were added, and the reaction was conducted at room temperature for 1 hour. The pH of the reaction was then adjusted to 3.9 – 4.2 by the slow addition of 0.25 M H2SO4(aq.), and a 10-fold excess of ethylenediaminetetraacetic acid disodium salt was added. The reaction was incubated in a water bath at 38 °C for 1 hour. The DFO-conjugated antibody was purified by size-exclusion chromatography (Sephadex G-25 M, PD-10 column, GE Healthcare).
Typical radiolabeling reactions were conducted according to previously reported methods[12] used for labeling monoclonal antibodies with 89Zr. Briefly, 89Zr-oxalate (429 MBq, [11.6 mCi]) in 1.0 M oxalic acid (250 µL) was adjusted to pH7.1–7.7 with 1.0 M Na2CO3(aq.). DFO-conjugated 5A10 (500 µL, 2.0 mg/mL [1.0 mg of protein], in sterile saline) was added and the reaction was mixed gently. The reaction was incubated at room temperature for between 1–2 hour and progress was monitored with ITLC (DTPA, 50 mM, pH7). After 2 hour, crude radiolabeling yields and RCP were typically >80–90%. 89Zr-5A10 was purified by using spin-column centrifugation (4 mL total volume, >30 kDa particle retention, Amicon Ultra-4, Millipore, Billerica, MA; washed with 4×3 mL sterile saline). The radiochemical purity of the final 89Zr-5A10 (formulation: pH5.5–6.0; <500 µL; sterile saline) was measured by ITLC and size-exclusion chromatography. In the ITLC experiment the 89Zr-5A10, 89Zr-IgG and 89Zr-DFO remain at the baseline (Rf = 0.0), whereas 89Zr4+(aq.) ions and the complex 89Zr-DTPA elute with the solvent front (Rf = 1.0). The final radiochemical yield of the purified 89Zr-5A10 was typically >70% and the product was formulated in sterile saline with a radiochemical purity >99% (n = 5) and a specific-activity of 195.0±8.0 MBq/mg (5.27±0.2 mCi/mg) of protein. Supplementary Figure S1 shows a typical radio-ITLC chromatogram of the crude and purified (formulated) 89Zr-5A10.
Cell lines
PC3 and CWR22Rv1 were purchased from ATCC (Manassas, VA) and AR and PSA expression was annotated by immunoblot and ELISA. The cell lines were cultured according to the manufacturer’s instructions. LNCaP-AR, parental LNCaP stably overexpressing wild type AR, was previously developed and reported by the Sawyers lab[15]. The cell line was authenticated for AR overexpression and PSA expression by immunoblot.
Animal studies
All animal experiments were conducted in compliance with institutional guidelines at MSKCC. Male CB-17 SCID mice (6–8 weeks old) were obtained from Taconic Farms Inc. (Hudson, NY), LNCaP-AR, 22Rv1, and PC3 tumors were inoculated in the right flank by subcutaneous injection of 1.0×106 cells in a 200 µL cell suspension of a 1:1 v/v mixture of media with Matrigel (Collaborative Biomedical Products Inc., Bedford, MA). Tumors developed after 3–7 weeks. MDV3100 was dissolved in DMSO so that the final DMSO concentration when administered to animals would be 5%. The formulation of the vehicle is 1% carboxymethyl cellulose, 0.1% Tween-80 and 5% DMSO. MDV3100 or vehicle was administered daily via gavage. Tumor volume (V / mm3) was estimated with caliper measurements.
Preparation of osseous tumor grafts and bone fracture model
Prior to surgery, castrate male SCID mice were anesthetized with ketamine, and an incision was be made in the left hind limb. The tibia was punctured using a bone drill, and 1 × 105 cells (22Rv1 or LNCaP-AR) injected into the cavity. The puncture was closed with bone wax, the incision sutured, and animals received a palliative dose of carprofen (5 mg/kg) once daily for three days post surgery. Tumor development was followed with bioluminescence imaging, and confirmed with MRI. The bone fracture model was prepared similar to the osseous tumor model, excluding injection of cells and application of bone wax.
Biodistribution studies
Biodistribution studies were conducted to evaluate the uptake of 89Zr-5A10 in human prostate cancer xenograft models. Mice received 89Zr-5A10 (1.11–1.85 MBq, [30–50 µCi], 5.7–9.5 µg of protein, in 200 µL sterile saline for injection) via intravenous tail-vein injection (t = 0 h). Animals (n = 4–5, per group) were euthanized by CO2 asphyxiation at 1, 4, 12, 24, 48, 72, 96, and 120 hours post-injection and blood was immediately harvested by cardiac puncture. Sixteen tissues (including the tumor) were removed, rinsed in water, dried in air for 5 minutes, weighed and counted on a gamma-counter for accumulation of 89Zr-radioactivity. The tumor tissues were partitioned for biodistribution or PSA ELISA. The mass of 89Zr-5A10 formulation injected into each animal was measured and used to determine the total number of counts per minute by comparison to a standard syringe of known activity and mass. Count data were background- and decay-corrected and the tissue uptake measured in units of percentage injected dose per gram (% ID/g) for each sample was calculated by normalization to the total amount of activity injected.
Small-animal PET imaging
PET imaging experiments were conducted on a microPET Focus 120 scanner (Concorde Microsystems). In repeated studies (n=4) mice were administered formulations of 89Zr-5A10 (10.4–12.6 MBq, [280–340 µCi], 53.1–64.5 µg of protein, in 200 µL sterile saline for injection) via i.v. tail-vein injection. Approximately 5 minutes prior to recording PET images, mice were anesthetized by inhalation of 1–2% isoflurane (Baxter Healthcare, Deerfield, IL)/oxygen gas mixture and placed on the scanner bed. PET images were recorded at various time-points between 1 – 120 hours post-injection. List-mode data were acquired using a γ-ray energy window of 350–750 keV, and a coincidence timing window of 6 nanoseconds. For all static images, scan time was adjusted to ensure a minimum of 20 million coincident events were recorded. Data were sorted into 2-dimensional histograms by Fourier re-binning, and transverse images were reconstructed by filtered back-projection into a 128×128×63 (0.72×0.72×1.3 mm) matrix. The reconstructed spatial resolution for 89Zr was 1.9 mm full-width half maximum at the center of the field-of-view. The image data were normalized to correct for non-uniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection but no attenuation, scatter, or partial-volume averaging correction was applied. An empirically determined system calibration factor (in units of [mCi/mL]/[cps/voxel]) for mice was used to convert voxel count rates to activity concentrations. The resulting image data were then normalized to the administered activity to parameterize images in terms of %ID/g. Manually drawn 2-dimensional regions-of-interest were used to determined the maximum and mean %ID/g (decay corrected to the time of injection) in various tissues. Images were analyzed by using ASIPro VM™ software (Concorde Microsystems).
Co-registered PET/CT
Computed tomography images were acquired on a small-animal Siemens/CTI microCAT II (Siemens Medical Solutions, Malvern, PA) scanner with an 8.5 cm axial by 5.0 cm transaxial FOV. Co-registered PET/CT images were recorded and mapped to a matrix in accordance with previously reported methods[28].
Magnetic resonance imaging
Mouse prostate MR images were acquired on a Bruker 4.7T Biospec scanner operating at 200 MHz and equipped with a 400 mT/m ID 12 cm gradient coil (Bruker Biospin MRI GmbH, Ettlingen, Germany). A custom-built quadrature birdcage resonator with ID of 32 mm was used for RF excitation and acquisition (Stark Contrast MRI Coils Research Inc., Erlangen, Germany). Mice were anaesthetized with oxygen and 1% isoflurane gas. Animal breathing was monitored by using a small animal physiological monitoring system (SA Instruments, Inc., Stony Brook, New York). T2 weighted scout images along three orthogonal orientations were first acquired for animal positioning. The T2-weighted fast spin-echo RARE sequence (Rapid Acquisition with Relaxation Enhancement) was used to acquire axial mouse pelvic images with a slice thickness of 0.8 mm, FOV 30 mm × 34 mm with a spatial resolution of 117 × 133 µm. The following acquisition parameters, TR = 4.5 s, TE= 40 ms, RARE factor 8, and an acquisition time of 20 minutes were used.
PSA detection in serum and tumor tissues
Free PSA and total PSA were measured with a dual-label immunofluorometric assay (DELFIA Prostatus™ PSA Free/Total PSA; Perkin-Elmer Life Sciences) according to manufacturer’s recommendations. This assay measures free PSA and complexed PSA in an equimolar fashion[29], and the cross-reactivity of PSA-ACT for free PSA is <0.2% [10]. The lower limits of detection are 0.05 µg/L for total PSA (CV = 5.0% at 2.32 µg/L) and 0.04 µg/L for free PSA (CV = 5.9% at 0.25 µg/L). For detection, the 1235 automatic immunoassay system from Perkin-Elmer Life Sciences was used. Complexed PSA concentrations were calculated by subtracting free PSA from total PSA.
Affinity tests of 89Zr-labeled 5A10
The capture mAb H117 was immobilized onto microtiter plates through physical adsorption by using low-fluorescence Maxisorp strips (Nunc, Roskilde, Denmark). The wells were coated with 1 µg of the mAb H117 in 100 L of buffer containing 0.2 mol/L NaH2PO4 buffer overnight at 35 °C. Coated wells were washed twice with DELFIA wash solution, and were then saturated for 3 hours at room temperature with 300 µL of a solution containing diethylenetriaminepentaacetic acid (DTPA)-treated BSA (1 g/L), sorbitol (60 g/L), Germall II (1 g/L), and 50 mmol of NaH2PO4. After saturation, the wells were aspirated, dried, and stored at 4 °C sealed plastic bags with desiccant until use.
Using a fresh sample of 89Zr-5A10, affinity assays were conducted by adding a solution of fPSA (25 µL, 47.7 ng/mL) in 100 µL of DELFIA assay buffer to each well from the previously prepared plates. After incubation for 1 hour at room temperature, the solution was aspirated, and the wells were washed twice with assay buffer. The binding assay was initiated with the addition of 200 µL aliquots of assay buffer containing 20 µg of 89Zr-5A10, and 0.0, 0.002, 0.02, 0.2, 0.5, 1, 2, 4, 6, 8, or 10 µg of unlabeled 5A10. All reactions were conducted in duplicate. The reactions were incubated, with slow shaking, for 2 hours at which time the wells were aspirated and rinsed four times with DELFIA wash solution. The bound activity was determined in a NaI (Tl) - well counter (Perkin Elmer 2480 Automatic Gamma Counter).
Autoradiography and tissue histology
After mice were sacrificed, the tibia, including the tumor, was surgically excised and embedded in OCT (Miles Inc., Elkhart, IN) and snap frozen on dry ice in a cryo-mold. Sets of ten contiguous 5 µm thick tissue sections were cut using a Microm HM500 cryostat microtome (Microm International, Walldorf, Germany) and arrayed onto poly-L-lysine coated glass microscope slides. Tissue sections were fixed in 10% phosphate-buffered formalin for 5 minutes, washed twice, air dried and stained with hematoxylin and eosin (H&E). Stained tissue sections were placed in a film cassette against a Fuji film BAS-MS2325 imaging plate (Fuji Photo Film Co, Tokyo, Japan) to acquire digital autoradiograms. The slides were exposed for 48 hours, ~168 hours after injection of 89ZrCl or 89Zr-5A10. Exposed phosphor plates were read by a Fujifilm BAS-1800II bio-imaging analyzer (Fuji Photo Film Co, Tokyo, Japan) generating digital images with 50 µm pixel dimensions. Digital images were obtained with an Olympus BX60 System Microscope (Olympus America Inc, Melville, NY) equipped with a motorized stage (Prior Scientific Inc, Rockland, MA). Subsequently, H&E images were acquired to the same resolution as the DAR data. DAR images were manually aligned to the H&E images using rigid planar transforms.
Statistical analyses
Data were analyzed by using the unpaired, two-tailed Student’s t-test. Differences at the 95% confidence level (P<0.05) were considered to be statistically significant.
Significance
This report establishes that AR-dependent changes in PSA expression levels can be quantitatively measured at tumor lesions using a radiotracer that can be rapidly translated for human application, and advances a new paradigm for radiotracer development that may more clearly highlight the unique virtues of an imaging biomarker.
Supplementary Material
Acknowledgments
We thank Drs. Howard Scher, Naga Vara Kishore Pillarsetty, Brett Carver, and Pat Zanzonico for comments, Valerie Longo, Thomas Ku, Michael Doran, Dr. Sara Cheal, Dr. Katharina Braun, Dr. Sean Carlin, Dr. Carl Le, Dov Winkleman, William Golden, and Brad Beattie for technical assistance.
Financial Support: D.U., M.J.E., C.L.S. and J.S.L. were supported in part by the Geoffrey Beene Cancer Research Center of MSKCC. D.U. was supported in part by the Tegger Foundation. M.J.E. was supported in part by the Brain Tumor Center at MSKCC. J.S.L., S.L.R. and J.P.H. were supported by the Office of Science (BER), U.S. Department of Energy (DE-SC0002456). M.J.E. and S.L.R. were supported in part by a training grant in molecular imaging from the National Institutes of Health (R25-CA096945). S.M.L. was supported by the Ludwig Center for Cancer Immunotherapy at MSKCC and the National Cancer Institute (P50-CA86483). C.L.S. was supported in part by the Howard Hughes Medical Institute. H.L. was supported in part by National Cancer Institute (R33 CA127768-02), the Swedish Cancer Society (3455), the Swedish Research Council (Medicine-20095), and the Sidney Kimmel Center for Prostate and Urologic Cancers. P.T.S. and H.L. were supported in part by the Prostate Cancer SPORE through the National Cancer Institute (P50-CA92629) and the David H. Koch Fund through the Prostate Cancer Foundation. Technical services provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by the National Institutes of Health (R24-CA83084, P30-CA08748).
Footnotes
Conflicts of Interest: C.L.S. is a co-inventor of MDV3100 and owns stock in the company (Medivation) that is developing the drug for PCa treatment. H.L. is a co-inventor of free PSA-assays for in vitro diagnostics in blood. This article does not make any claims about the efficacy of MDV3100 or the diagnostic value of free PSA measurements in the blood; it merely uses MDV3100 and free PSA measurements in serum as tools to evaluate the 5A10 antibody-based radiotracer described herein.
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