Purpose:

We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [89Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models.

Experimental Design:

[89Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR, CD46+, prostate-specific membrane antigen–negative (PSMA)] or 22Rv1 (AR+, CD46+, PSMA+) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545.

Results:

[89Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [89Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [89Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [89Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection).

Conclusions:

[89Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.

This article is featured in Highlights of This Issue, p. 1215

Translational Relevance

Despite recent progress, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. We recently identified CD46 as a novel lineage-independent mCRPC cell surface antigen with high expression in aggressive, late-stage, and treatment-resistant prostate cancer. Herein, we developed [89Zr]DFO-YS5 as a CD46 immunoPET imaging probe, and demonstrated that [89Zr]DFO-YS5 imaged both CD46-positive tumor cell line xenografts and patient-derived xenografts with excellent contrast. With a CD46-targeted antibody–drug conjugate (FOR46), currently in phase I trials, this CD46-targeting immunoPET radiotracer could be translated to clinical studies rapidly, aiding patient selection and/or assessment of treatment response.

Molecular imaging and targeted theranostic agents are playing an increasingly important role in prostate cancer detection and therapy (1). By pairing a molecular imaging agent labeled with a positron-emitting radionuclide with a therapeutic agent, such as a beta or alpha radionuclide, molecular imaging may help select patients for appropriate therapy in the context of heterogeneous target expression. In particular, prostate-specific membrane antigen (PSMA) is a well-established biomarker for prostate cancer, making it the target of a number of imaging and therapeutic approaches (2, 3, 5–10). However, PSMA is not uniformly well expressed in prostate cancer, and in many cases, its expression is heterogeneous or absent in both localized and metastatic prostate cancer (11–14). Therefore, there is an unmet clinical need for new theranostic targets and agents in prostate cancer to facilitate cancer detection and treatment for men with absent or heterogeneous expression of PSMA.

In 2006, we selected phage antibody display libraries on prostate cancer tissues by laser capture microdissection and discovered a panel of human antibodies that target prostate cancer cells residing in their natural tissue microenvironment (15). Subsequently, we showed by single-photon emission CT (SPECT)/CT that one of the selected human antibody fragments efficiently targeted prostate cancer in vivo (16). In 2018, we identified CD46 as the target bound by the antibody, identified the epitope with tumor selectivity and a new panel of CD46-targeting human antibodies, and developed an antibody–drug conjugate (ADC) for prostate cancer therapy (17). In particular, the lead antibody found in that study, YS5, demonstrated high-affinity binding to prostate cancer cell and tissue, with little or no binding to normal tissues, except for prostate epithelium and placental trophoblasts. CD46 is known to be a negative regulator of the complement cascade in the innate immune system (18–20). In contrast with lineage markers, such as PSMA, CD46 showed uniformly intense cell surface expression in dedifferentiated castration-resistant prostate cancer phenotypes, including both adenocarcinoma and treatment-emergent neuroendocrine prostate cancer (17). YS5 is a fully human, full-length IgG1 and has been developed into an ADC for metastatic castration-resistant prostate cancer (mCRPC) treatment (17). Currently, the ADC (FOR46) is in a phase I clinical trial (NCT03575819). Taken together, these data support the development of a CD46-directed theranostic agent for metastatic prostate cancer.

ImmunoPET is a noninvasive molecular imaging modality, which combines the excellent targeting specificity of antibodies or antibody fragments with the superior sensitivity and resolution of PET (4, 21, 22). 89Zr (T1/2 = 78.41 hours) has been widely used for antibody radiolabeling because its long decay time matches the circulation half-time of full-length antibodies (23–26). We hypothesize that a CD46-targeted immunoPET radiotracer could enable a whole-body assessment of CD46 expression, evaluate prostate cancer disease burden, and aid patient selection and treatment monitoring for CD46-targeted therapies.

In this study, we report the production and preclinical evaluation of 89Zr-radiolabeled human antibody, YS5 ([89Zr]DFO-YS5), as the first immunoPET probe targeting CD46-positive prostate cancer.

Compounds and proteins

The fully human CD46-targeted antibody, YS5, was produced and purified as described previously (17). The radiolabeling chelator, p-Isothiocyanate-benzyl-DFO (catalog No. B-705), was purchased from Macrocyclics, Inc. 89Zr oxalate was purchased from 3D Imaging and the Cyclotron Laboratory at University of Wisconsin, Madison (Madison, WI). Recombinant human CD46 Fc chimera protein was purchased from Sino Biological, Inc. Native human IgG protein was purchased from Abcam. Other chemicals were purchased from Sigma-Aldrich, Inc.

Antibody conjugation and radiolabeling

Conjugation of p-Isothiocyanate-benzyl-DFO to YS5: the buffer of 5 mg YS5 was exchanged to 0.1 mol/L Na2CO3-NaHCO3 buffer, pH 9, using a 30K MW centrifugal filter. The final volume was adjusted to 1 mL by adding 0.1 mol/L Na2CO3-NaHCO3 buffer, pH 9. p-Isothiocyanate-benzyl-DFO (1.3 mg) was dissolved in 208-μL DMSO. p-Isothiocyanate-benzyl-DFO solution (20 μL; five equivalents to YS5) was added to the 1-mL solution containing 5 mg of YS5. The mixture was incubated at 37°C for 45 minutes. The mixture was purified with a PD10 gel filtration column, by eluting with 0.25 mol/L sodium acetate solution, pH 6.

Conjugation of p-isothiocyanate-benzyl-DFO to nonspecific IgG was performed, with the procedure similar to the conjugation for YS5.

Radiolabeling: 5-μL (3 mCi) 89Zr oxalate, 5-μL 1 mol/L Na2CO3, 200-μL 2 mol/L NH4OAc, and 200-μg DFO-YS5 were incubated at room temperature for 1 hour. The mixture was subject to instant thin-layer chromatography (iTLC) for labeling yield, and then purified with a PD10 column by eluting with 0.9% normal saline. The final product was also analyzed for purity by iTLC.

Cell culture

Cell lines were obtained from the ATCC. 22Rv1 cells or DU145 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2. MC38 cells were grown in DMEM with high glucose, supplemented with 10% FBS, 100 U penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2. Cells were removed from flasks for passage or for transfer to assay plates by incubating them with 0.25% trypsin. Cells were subcultured every 3–4 days.

In vitro Kd measurement

Kd value of [89Zr]DFO-YS5 against CD46-expressing cell lines, DU145 or 22Rv1, was determined by a saturation binding assay. For the Kd measurement of [89Zr]DFO-YS5 against DU145, DU145 cells were plated in 48-well plates (250 μL/well) 48 hours before testing (triplet) in RPMI1640 medium supplemented with 10% FBS. The cell number was about 2.5 × 105 cells per well when the assay was performed. The growth medium was removed and washed with PBS three times. PBS with 1% nonfat milk was added to each well and incubated for 1 hour. The buffer was removed and various concentrations (200 μL/well, 0.0005–5 nmol/L) of [89Zr]DFO-YS5 in saline were added to cells. The cells were incubated in this buffer for 1 hour at room temperature. Then, the radioactive medium was removed by pipet, cells were washed with PBS twice, and 250 μL of 5 N NaOH was added to lyse the cells. The lysate was transferred to 2-mL vials, and the bound radioactivity was counted using a Hidex Gamma Counter. Kd value was determined by nonlinear regression one site–specific binding using GraphPad Prism Software (GraphPad Software). For the Kd measurement of [89Zr]DFO-YS5 against 22Rv1, the same procedure was followed, except for using 22Rv1 cells instead of DU145.

For the Kd measurement of [89Zr]DFO-YS5 against CD46 recombinant protein, 1 μg/mL CD46 in PBS was placed in a 96-well Nunc MaxiSorp Plate (100 μL/well, Invitrogen) and kept at 4°C for 24 hours before testing (triplet). This enabled binding of the CD46 protein directly to the plate. The buffer was removed and the wells were washed with PBS three times. PBS with 1% nonfat milk was added to each well and incubated for 1 hour to minimize subsequent nonspecific protein binding. The buffer was removed and various concentrations (100 μL/well, 0.0005–5 nmol/L) of [89Zr]DFO-YS5 in saline were added, and incubated for 1 hour at room temperature. Then, the radioactive solution was removed by pipet. The plate was washed with PBS twice, and 250 μL of 5 N NaOH was added to denature the protein and remove the protein and any bound radioactivity from the plate. The solubilized protein-bound activity was transferred to small vials, and the bound radioactivity was counted using a Hidex Gamma Counter. Kd value was determined by nonlinear regression one site–specific binding using GraphPad Prism Software (GraphPad Software).

CD46 magnetic beads target binding fraction assay

HisPur Ni-NTA Magnetic Beads (catalog No. 88831) were purchased from Thermo Fisher Scientific. The DynaMag-2 Magnet (catalog No. 12321D) was purchased from Thermo Fisher Scientific. To 40-mL PBS, 20-μL tween20 was added to make the PBST solution. Vials were divided as testing group A, blocking group B, and control group C (triplicate in each group). HisPur Ni-NTA magnetic beads (20 μL) and 380-μL PBST were added to the vials in groups A, B, and C. Samples were vortexed, beads were trapped by the DynaMag-2 magnet, and the supernatant was removed. PBST (360 μL) and 40 μL of 25 μg/mL CD46 were added to groups A, B, and C, the supernatant was removed, and the beads were washed with PBST once. [89Zr]DFO-YS5 (2 ng) in 1% milk PBS was added to groups A and B, and 2 ng [89Zr]DFO-IgG was added to group C. YS5 antibody (10 μg) was added to the blocking group B. Samples were diluted to 400 μL per vial using 1% milk PBS, incubated for 30 minutes, and washed with 1% milk PBS twice. The activity of beads, 2 ng [89Zr]DFO-YS5 and 2 ng [89Zr]DFO-IgG, was read using Hidex Gamma Counter. Binding percentage was calculated by beads activity/2 ng [89Zr]DFO-YS5 activity for groups A and B or beads activity/2 ng [89Zr]DFO-IgG activity of group C.

Xenograft models

All animal studies were conducted according to Institutional Animal Care and Use Committee–approved protocols at University of California, San Francisco (San Francisco, CA) and University of Virginia (Charlottesville, VA). Male 5- to 6-week-old athymic mice (nu/nu, homozygous; purchased from The Jackson Laboratory or Charles River Laboratories) were housed under aseptic conditions, and received subcutaneous tumor cell inoculation. In brief, 3–5 × 106 cells in a 200-μL 1:1 mixture of complete medium and Matrigel (Thermo Fisher Scientific) were injected in the thigh or shoulder of the animals. All mice were subjected to undergo PET imaging, as well as biodistribution analysis, when the tumor reached a size of 300–500 mm3.

The LTL-331, LTL-331R, and LTL-545 patient-derived xenografts (PDXs) were obtained from the Living Tumor Laboratory (Vancouver, British Columbia, Canada; ref. 27). In brief, PDX tissue (∼5 mm × 5 mm) was passaged in intact NOD/SCID gamma (NSG) mice subcutaneously. Mice were subjected to PET imaging, as well as biodistribution analysis, when the tumor reached a size of 300–500 mm3.

In vivo [89Zr]DFO-YS5 and [68Ga]Ga-PSMA-11 PET imaging studies

Approximately 3–5 weeks after tumor implantation, animals with tumors reaching 300–500 mm3 were anesthetized by isoflurane inhalation. For [68Ga]Ga-PSMA-11 PET/CT imaging, methods were identical to those reported previously (28). For [89Zr]DFO-YS5 PET imaging, 3.70–5.55 MBq (100–150 μCi, 10 μg/mouse) of [89Zr]DFO-YS5 in saline was administered through tail vein. The animals were imaged at various timepoints with a 20-minute acquisition time by using microPET/CT (Inveon, Siemens Medical Solutions) or Albira Trimodal PET/SPECT/CT Scanner (Bruker Corporation). PET imaging data were acquired in list mode and reconstructed using an iterative 2D OSEM reconstruction algorithm (for Inveon data) or Albira Software Suite (for Albira data) provided by the manufacturer. The resulting image data were then normalized to the administered activity to parameterize images in terms of %ID/mL. Imaging data were viewed and processed using an open source Amide software. CT images were acquired following PET, and the CT data were used for attenuation correction for PET reconstruction, and anatomic reference.

For the serial PET imaging study of DU145 tumor detection, PET images were acquired at 24, 48, 72, 96, 120, 144, and 168 hours in tumor-bearing mice postinjection of [89Zr]DFO-YS5 [3.7–7.4 MBq (100–200 μCi)].

To validate the specificity of tumor targeting, PET imaging was also performed at 24, 48, 72, and 96 hours postinjection of [89Zr]DFO-YS5 [5.92–7.03 MBq (160–190 μCi)], with groups of mice bearing both DU145 (target positive) and MC38 (target negative) tumors. As another blocking control, a group of mice with DU145 received 300 μg of unlabeled YS5 at 48 hours before the injection of [89Zr]DFO-YS5 [5.92–7.03 MBq (160–190 μCi)], followed by PET imaging 48 hours later. As a comparison of the tumor targeting, a control mAb IgG (nonbinding control) was also radiolabeled with 89Zr ([89Zr]DFO-IgG) in the same process and the PET imaging of DU145 tumor–bearing mice was recorded at 48 hours followed by terminal biodistribution study.

Biodistribution studies

The tumor-bearing mice were sacrificed at various timepoints postinjection of [89Zr]DFO-YS5. Blood was collected by cardiac puncture. Major organs (liver, heart, kidney, lung, spleen, stomach, small intestine, large intestine, pancreas, muscle, subcutaneous tumor, and bone) were harvested, weighed, and counted in an automated Gamma Counter (Hidex). The percent injected dose per gram of tissue (%ID/g) was calculated by comparing with standards of known radioactivity.

Autoradiography

Four days postinjection of [89Zr]DFO-YS5, mice were sacrificed and tissues were immediately collected and flash frozen in optimal cutting temperature compound on dry ice. Tissues were sectioned on a microtome at a thickness of 20 μm and immediately mounted on glass slides. The slides were then exposed on a GE phosphor storage screen for 1 hour, and the screen was developed on an Amersham Typhoon 9400 phosphor imager. The autoradiography images were processed using ImageJ software.

Flow cytometry

Cell surface CD46 expression was analyzed by flow cytometry using methods described previously (17). Briefly, the YS5 IgG1 was conjugated to Alexa Fluor 647 using an Alexa Fluor 647 Monoclonal Antibody Labeling Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Alexa Fluor 647–labeled YS5 IgG1 was incubated with monodispersed cells isolated from PDXs (LTL-331, LTL-331R, and LTL-545) at room temperature for 1 hour, and washed three times with PBS to remove unbound antibody. Binding was analyzed by Flow Cytometry (BD Accuri C6, BD Biosciences) with median fluorescence intensity (MFI) recorded for each sample.

Statistical analysis

Data were analyzed using the unpaired, two-tailed Student t test and one-way ANOVA. Differences at the 95% confidence level (P < 0.05) were considered to be statistically significant.

Synthesis and in vitro analysis of [89Zr]DFO-YS5

The radiolabeling of 89Zr oxalate to DFO-YS5 was accomplished in a typical, two-step procedure by first conjugating with deferoxamine (DFO), and then subsequent chelation of the isotope (Fig. 1A). Using five equivalents of p-SCN-Bn-DFO, an average of 1.33 chelators was added to the antibody, as determined by matrix-assisted laser desorption/ionization–mass spectrometry (Supplementary Fig. S1). The intermediate DFO-YS5 could be stored at −20°C for 12 months without detectable loss of binding activity after radiolabeling with 89Zr. [89Zr]DFO-YS5 was isolated in 74% ± 11% (n = 6) yield based on starting 89Zr oxalate with molar activities ranging from 274.2 to 351.1 MBq/mg (39.7–50.8 GBq/μmol, 7.42–9.49 mCi/mg, and 1.07–1.37 Ci/μmol). Radiopharmaceutical purity was greater than 95% in all cases (Fig. 1B). Size-exclusion chromatography demonstrated no evidence of aggregation (Supplementary Fig. S2).

Figure 1.

Synthesis and in vitro analysis of [89Zr]DFO-YS5. A, Synthesis scheme of [89Zr]DFO-YS5. Reaction conditions: (1) 0.1 mol/L Na2CO3-NaHCO3 buffer, pH 9.0, 37°C, 45 minutes. (2) NH4OAc (2 mol/L), room temperature, 1 hour. B, iTLC analysis of [89Zr]DFO-YS5, demonstrating greater than 98% purity. C, Kd measurement of [89Zr]DFO-YS5 on the DU145 cell line, determined by a saturation binding assay (Kd = 6.7 ± 0.3 nmol/L). D, Kd measurement of [89Zr]DFO-YS5 on the 22Rv1 cell line, determined by a saturation binding assay (Kd = 7.2 ± 0.9 nmol/L). E, Kd measurement of [89Zr]DFO-YS5 on CD46 recombinant protein, determined by a saturation binding assay (Kd = 6.0 ± 0.6 nmol/L). F, Competition radioligand binding assay using [89Zr]DFO-YS5, demonstrating similar IC50 values for YS5 versus DFO-YS5 (23.1 ± 1.8 and 37.8 ± 2.5 nmol/L, respectively). G, Magnetic bead–based radioligand binding assay, demonstrating target binding fraction of 79.5% ± 2% for [89Zr]DFO-YS5.

Figure 1.

Synthesis and in vitro analysis of [89Zr]DFO-YS5. A, Synthesis scheme of [89Zr]DFO-YS5. Reaction conditions: (1) 0.1 mol/L Na2CO3-NaHCO3 buffer, pH 9.0, 37°C, 45 minutes. (2) NH4OAc (2 mol/L), room temperature, 1 hour. B, iTLC analysis of [89Zr]DFO-YS5, demonstrating greater than 98% purity. C, Kd measurement of [89Zr]DFO-YS5 on the DU145 cell line, determined by a saturation binding assay (Kd = 6.7 ± 0.3 nmol/L). D, Kd measurement of [89Zr]DFO-YS5 on the 22Rv1 cell line, determined by a saturation binding assay (Kd = 7.2 ± 0.9 nmol/L). E, Kd measurement of [89Zr]DFO-YS5 on CD46 recombinant protein, determined by a saturation binding assay (Kd = 6.0 ± 0.6 nmol/L). F, Competition radioligand binding assay using [89Zr]DFO-YS5, demonstrating similar IC50 values for YS5 versus DFO-YS5 (23.1 ± 1.8 and 37.8 ± 2.5 nmol/L, respectively). G, Magnetic bead–based radioligand binding assay, demonstrating target binding fraction of 79.5% ± 2% for [89Zr]DFO-YS5.

Close modal

The binding affinity of [89Zr]DFO-YS5 was measured in a saturation binding assay by incubating CD46-expressing cell lines or recombinant CD46 with increasing concentration of [89Zr]DFO-YS5. The dissociation constant value, Kd, was 6.7 ± 0.3 nmol/L for the DU145 cell line, 7.2 ± 0.9 nmol/L for the 22Rv1 cell line, and 6 ± 0.6 nmol/L for a recombinant CD46 protein (Fig. 1CE). A competition radioligand binding assay was developed using [89Zr]DFO-YS5, in the presence of varying concentrations of competing YS5 or DFO-YS5. In this assay, the IC50 value for YS5 was 23.1 ± 1.8 nmol/L, and for DFO-YS5 was 37.8 ± 2.5 nmol/L (Fig. 1F). We adopted a recently described magnetic bead–based radioligand binding assay to determine the target binding fraction of the labeled [89Zr]DFO-YS5 (Fig. 1G). In this assay, the binding of [89Zr]DFO-YS5 was 79.5% ± 2%, while marked reductions to 18.4% ± 1.8% were seen in the presence of 10-fold excess of cold YS5. Nonspecific IgG demonstrated minimal binding of 5.2% ± 3.6%. Taken together, these data demonstrate that [89Zr]DFO-YS5 can be synthesized in an efficient, reproducible manner, with minimal loss of binding affinity.

Longitudinal PET imaging and biodistribution analysis of [89Zr]DFO-YS5 in DU145 tumor model

ImmunoPET images of [89Zr]DFO-YS5 recorded in DU145 tumor–bearing mice between 24 and 168 hours are presented in Fig. 2, Supplementary Fig. S3; Supplementary Table S1. The in vivo tumor targeting of [89Zr]DFO-YS5 was also quantified by conducting biodistribution studies in DU145 tumor–bearing mice at 24, 48, 72, 96, and 168 hours after intravenous administration. The data revealed that high DU145 tumor uptake was observed at 24 hours (11.4 ± 2.6 %ID/g), with a steady increase through 48 (14.1 ± 1.8 %ID/g) and 72 hours (14.8 ± 4.6 %ID/g), and reaching 18.2 ± 10.9 %ID/g at 168 hours (Fig. 2B). This gradually increasing accumulation of [89Zr]DFO-YS5 in tumor was along with the extraction of the activity from the blood (24 hours, 10.9 ± 1.4 %ID/g; 48 hours, 5.7 ± 0.9 %ID/g; 72 hours, 3 ± 0.4 %ID/g; 96 hours, 2 ± 0.3 %ID/g, and 168 hours, 3 ± 0.3 %ID/g). These increasing tumor uptake corresponded with high tumor-to-muscle ratios of 15.6 ± 7.5, 26.3 ± 4.7, 19.4 ± 2.2, 59 ± 16.5, and 53.6 ± 42.3 for the times at 24, 48, 72, 96, and 168 hours, respectively (Fig. 2C).

Figure 2.

[89Zr]DFO-YS5 detects tumors in male nu/nu mice with subcutaneous DU145 xenografts. A, Maximum-intensity projections of [89Zr]DFO-YS5 from 24 to 168 hours. B, Biodistribution of [89Zr]DFO-YS5 in various tissues at timepoints from 24 to 168 hours. C, Tumor-to-nontarget organ ratio of [89Zr]DFO-YS5 from 24 to 168 hours.

Figure 2.

[89Zr]DFO-YS5 detects tumors in male nu/nu mice with subcutaneous DU145 xenografts. A, Maximum-intensity projections of [89Zr]DFO-YS5 from 24 to 168 hours. B, Biodistribution of [89Zr]DFO-YS5 in various tissues at timepoints from 24 to 168 hours. C, Tumor-to-nontarget organ ratio of [89Zr]DFO-YS5 from 24 to 168 hours.

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Comparison of [89Zr]DFO-YS5 and [68Ga]PSMA-11 PET/CT in the CD46- and PSMA-positive 22Rv1 xenograft model

To further evaluate the imaging ability of [89Zr]DFO-YS5, and compare it with the PSMA-based prostate cancer imaging agent, [68Ga]PSMA-11, [89Zr]DFO-YS5 or [68Ga]PSMA-11 was administered to athymic mice xenografted with 22Rv1 cells subcutaneously. Mice administered with [89Zr]DFO-YS5 were imaged 4 days after intravenous injection and then sacrificed for a biodistribution study. As shown in Fig. 3A, the probe specifically localized at the tumor site and had a low accumulation in other organs. The biodistribution study showed that the tumor uptake was 14.5 ± 3.2 %ID/g at 4 days postinjection, whereas the uptake in all other organs was below 5% (Fig. 3B; Supplementary Table S2). Mice administered with [68Ga]PSMA-11 were imaged 1 hour after intravenous injection and then sacrificed for a biodistribution study. As shown in Fig. 3C, and as expected on the basis of our own previous study (28), the [68Ga]PSMA-11 demonstrated high tumor, spleen, and, especially, kidney uptake. The biodistribution study showed 3.74 ± 0.76 %ID/g uptake at tumor site and more than 120 %ID/g uptake in kidney because of its high expression level of PSMA (Fig. 3D; Supplementary Table S2). These results demonstrate that [89Zr]DFO-YS5 can specifically image CD46-positive prostate cancer with favorable imaging characteristics compared with [68Ga]PSMA-11 in the 22Rv1 model.

Figure 3.

[89Zr]DFO-YS5 and [68Ga]PSMA-11 imaging and biodistribution in the 22rV1 xenograft model reveals favorable imaging characteristics for [89Zr]DFO-YS5. A, Maximum-intensity projection (MIP) PET/CT, coronal CT, and microPET/CT fusion images obtained 7 days following administration of [89Zr]DFO-YS5 reveal high tumor uptake with low background tissue retention. B, Biodistribution analysis of [89Zr]DFO-YS5 in 22Rv1 xenografts, obtained 7 days following administration of [89Zr]DFO-YS5, demonstrate high tumor retention. C, Maximum-intensity projection PET/CT, coronal CT, and μPET/CT fusion images obtained 60 minutes following administration of [68Ga]PSMA-11 reveal high tumor uptake with expected high abdominal radiotracer accumulation. D, Biodistribution analysis obtained 60 minutes following administration of [68Ga]PSMA-11 reveals high tumor, spleen, and kidney uptake.

Figure 3.

[89Zr]DFO-YS5 and [68Ga]PSMA-11 imaging and biodistribution in the 22rV1 xenograft model reveals favorable imaging characteristics for [89Zr]DFO-YS5. A, Maximum-intensity projection (MIP) PET/CT, coronal CT, and microPET/CT fusion images obtained 7 days following administration of [89Zr]DFO-YS5 reveal high tumor uptake with low background tissue retention. B, Biodistribution analysis of [89Zr]DFO-YS5 in 22Rv1 xenografts, obtained 7 days following administration of [89Zr]DFO-YS5, demonstrate high tumor retention. C, Maximum-intensity projection PET/CT, coronal CT, and μPET/CT fusion images obtained 60 minutes following administration of [68Ga]PSMA-11 reveal high tumor uptake with expected high abdominal radiotracer accumulation. D, Biodistribution analysis obtained 60 minutes following administration of [68Ga]PSMA-11 reveals high tumor, spleen, and kidney uptake.

Close modal

Specificity of CD46 tumor targeting in vivo: PET imaging and biodistribution in CD46-positive and CD46-negative tumors, together with blocking and control antibody studies

To verify the specificity of CD46 targeting of [89Zr]DFO-YS5, a series of control experiments were performed, including imaging in CD46-negative tumors, a blocking study, and comparison with a control, nonbinding antibody. Biodistribution studies using a nonbinding, control antibody IgG group and a blocking group were compared against [89Zr]DFO-YS5 alone in the DU145 model (Fig. 4A and B; Supplementary Table S3). As expected, high targeting of [89Zr]DFO-YS5 was observed with tumor uptake at 14.1 ± 1.8%ID/g at 48-hour postinjection. In contrast, much lower tumor uptake using nonbinding 8[89Zr]DFO-IgG or [89Zr]DFO-YS5 with blocking YS5 group was observed at 3.3 ± 1 %ID/g (P < 0.01) and 7.5 ± 1.2 %ID/g (P < 0.01), respectively (Fig. 4; Supplementary Fig. S4A). The data of tumor-to-nontarget ratios in DU145 tumor targeting with [89Zr]DFO-YS5 also showed the significant difference compared with [89Zr]DFO-IgG control group (tumor/liver, P < 0.001; tumor/kidney, P = 0.004; tumor/spleen, P < 0.001; and tumor/muscle, P < 0.001) and blocking group (tumor/liver, P = 0.021; tumor/kidney, P = 0.002; tumor/spleen, P < 0.001; and tumor/muscle, P < 0.001).

Figure 4.

[89Zr]DFO-YS5 targeting specificity in subcutaneous tumor models. A, Maximum-intensity projections (MIP) and μPET/CT of [89Zr]DFO-YS5, [89Zr]DFO-IgG, and blocking YS5+[89Zr]DFO-YS5 in DU145 subcutaneous xenograft tumor mice at 48 hours. B, Biodistribution of [89Zr]DFO-YS5, [89Zr]DFO-IgG, and YS5+[89Zr]DFO-YS5 in DU145 subcutaneous xenograft tumor mice at 48 hours. C, Maximum-intensity projections and μPET/CT of [89Zr]DFO-YS5 in male nu/nu mice with subcutaneous DU145 and MC38 xenografts at 96 hours. D, Biodistribution of [89Zr]DFO-YS5 in male nu/nu mice with subcutaneous DU145 and MC38 xenografts at 96 hours.

Figure 4.

[89Zr]DFO-YS5 targeting specificity in subcutaneous tumor models. A, Maximum-intensity projections (MIP) and μPET/CT of [89Zr]DFO-YS5, [89Zr]DFO-IgG, and blocking YS5+[89Zr]DFO-YS5 in DU145 subcutaneous xenograft tumor mice at 48 hours. B, Biodistribution of [89Zr]DFO-YS5, [89Zr]DFO-IgG, and YS5+[89Zr]DFO-YS5 in DU145 subcutaneous xenograft tumor mice at 48 hours. C, Maximum-intensity projections and μPET/CT of [89Zr]DFO-YS5 in male nu/nu mice with subcutaneous DU145 and MC38 xenografts at 96 hours. D, Biodistribution of [89Zr]DFO-YS5 in male nu/nu mice with subcutaneous DU145 and MC38 xenografts at 96 hours.

Close modal

In contrast to the high absolute tumor uptake observed in the DU145 model, much lower accumulation of [89Zr]DFO-YS5 in MC38 (CD46 negative) tumors was observed at 96 hours (Fig. 4C and D; Supplementary Fig. S4B; Supplementary Table S4). Specifically, [89Zr]DFO-YS5 uptake in the MC38 tumors at 96 hours (MC38 vs. DU145: 4.7 ± 2.3 vs. 14.8 ± 6.4 %ID/gram; P = 0.026) showed a statistically significant reduction in tracer accumulation, compared with uptake in CD46-positive DU145 tumors.

PET imaging and biodistribution of [89Zr]DFO-YS5 and [68Ga]PSMA-11 in DU145/22Rv1 dual tumor model

Next, we explored [89Zr]DFO-YS5 and [68Ga]PSMA-11 imaging in mice bearing DU145 and 22Rv1 xenografts, to determine the ability of the CD46-targeted agent to image PSMA-negative prostate cancer. [89Zr]DFO-IgG imaging was performed as a control agent to evaluate the enhanced permeability and retention (EPR) effect. As expected, on the basis of the single-tumor experiments outlined above (Fig. 3A and B), both DU145 and 22Rv1 showed high uptake of [89Zr]DFO-YS5 (13.3 ± 3.9 and 11.2 ± 2.5 %ID/g at 4 days postinjection; Fig. 5A and D; Supplementary Table S5). The tumor-to-muscle ratio was 28.3 ± 9.3 and 23.1 ± 2.8, respectively. As a comparison, [89Zr]DFO-IgG showed a moderate uptake in the DU145 and 22Rv1 xenografts, and the tumor-to-muscle ratio was much lower than the [89Zr]DFO-YS5 group (Fig. 5CE; Supplementary Table S5). In [68Ga]PSMA-11 imaging (Fig. 5F and G), the PSMA-negative cell line, DU145, showed low uptake (0.28 ± 0.06 %ID/g) compared with the PSMA-positive cell line, 22Rv1 (4.04 ± 1.31 %ID/g). An autoradiography study performed after tumor was dissected demonstrated good uptake of [89Zr]DFO-YS5 (Fig. 5B; Supplementary Fig. S5). The distribution was not homogenous, probably because of the vasculature of the tumor, central necrosis, and/or the large size of the antibody. Taken together, these data demonstrate the feasibility of imaging CD46-positive, PMSA-negative tumors with [89Zr]DFO-YS5.

Figure 5.

Comparison of [89Zr]DFO-YS5 and [68Ga]PSMA-11 PET in a dual 22Rv1 and DU145 prostate cancer tumor model demonstrates feasibility for imaging PSMA-negative tumors with μPET/CT. A, Maximum-intensity projection (MIP) μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-YS5 reveal high tumor uptake. B, Autoradiography of 22Rv1 and DU145 tumor sections. C, Maximum-intensity projection μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-IgG reveal low tumor uptake. D, Biodistribution of [89Zr]DFO-YS5 and [89Zr]DFO-IgG. E, Tissue-to-organ ratio of [89Zr]DFO-YS5 and [89Zr]DFO-IgG biodistribution. F, Maximum-intensity projection μPET/CT, coronal CT, and coronal μPET/CT slices obtained 60 minutes after administration of [68Ga]PSMA-11 reveal high tumoral uptake in 22Rv1, but low in DU145. G, Biodistribution data matching imaging data in F.

Figure 5.

Comparison of [89Zr]DFO-YS5 and [68Ga]PSMA-11 PET in a dual 22Rv1 and DU145 prostate cancer tumor model demonstrates feasibility for imaging PSMA-negative tumors with μPET/CT. A, Maximum-intensity projection (MIP) μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-YS5 reveal high tumor uptake. B, Autoradiography of 22Rv1 and DU145 tumor sections. C, Maximum-intensity projection μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-IgG reveal low tumor uptake. D, Biodistribution of [89Zr]DFO-YS5 and [89Zr]DFO-IgG. E, Tissue-to-organ ratio of [89Zr]DFO-YS5 and [89Zr]DFO-IgG biodistribution. F, Maximum-intensity projection μPET/CT, coronal CT, and coronal μPET/CT slices obtained 60 minutes after administration of [68Ga]PSMA-11 reveal high tumoral uptake in 22Rv1, but low in DU145. G, Biodistribution data matching imaging data in F.

Close modal

PET imaging and biodistribution of [89Zr]DFO-YS5 in clinically relevant patient-derived tumor models

We also tested the imaging ability of [89Zr]DFO-YS5 in the LTL-331 patient-derived adenocarcinoma xenograft model, and the LTL-331R and LTL545 neuroendocrine prostate cancer models (27). In contrast with other xenograft models, these PDX tumors were grown in the more severely immunosuppressed NSG mouse model. Fc blocking was performed with excess cold IgG, reported to reduce Fc-mediated splenic retention of antibody (29). Despite Fc blocking, splenic retention of antibody was still greater than in the nu/nu model. Tumor retention of the antibody was high in the LTL-331 model, measuring 18.8 ± 5.3 %ID/g (Fig. 6A and D; Supplementary Table S6). Similar findings were seen in a bone metastasis model using the LTL-331 xenograft model, where tumor was introduced directly into the tibia by injection (30). Similar to the subcutaneous xenograft model, a high degree of tumor uptake was observed (Supplementary Fig. S6). The neuroendocrine prostate model, LTL-331R, demonstrated a high degree of [89Zr]DFO-YS5 uptake, slightly less than the LTL-331 model, measuring 12.5 ± 1.8 %ID/g (Fig. 6B and D; Supplementary Table S6). The LTL-545 neuroendocrine prostate cancer demonstrated very high [89Zr]DFO-YS5 uptake, measuring 32 ± 7.8 %ID/g (Fig. 6C and D; Supplementary Table S6). We performed flow cytometry study to determine CD46 cell surface expression level, and found that all three PDXs expressed CD46, and LTL-545 expressed the highest level, with lower expression in LTL-331 and LTL-331R (Fig. 6F), consistent with the result from PET imaging and biodistribution studies. These data demonstrate the utility of CD46-targeted imaging in more clinically relevant PDX models, suggesting feasibility for subsequent clinical translation. Moreover, they support the use of CD46-directed imaging and therapy in advanced, neuroendocrine prostate cancer phenotypes.

Figure 6.

PET imaging of [89Zr]DFO-YS5 detects tumors in PDX models, including in neuroendocrine prostate cancer. Maximum-intensity projection (MIP) μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-YS5 in the LTL-331 (A), LTL-331R (B), and LTL-545 (C) tumor models. D, MicroPET/CT fusion images on the same scale, demonstrating greater uptake in the LTL-545 model when compared against LTL-331 or 331-R. E, Biodistribution analysis obtained 4 days after administration of [89Zr]DFO-YS5 reveal high tumor uptake in the xenograft models, particularly for the LTL-545 neuroendocrine prostate cancer. F, Flow cytometry analysis of CD46 cell surface expression in PDXs. MFI values for LTL-331 (adenocarcinoma), LTL-331R (neuroendocrine), and LTL-545 (neuroendocrine) are 40,804, 40,473, and 286,645, respectively. Ctrl, an isotype-matched nonbinding antibody control.

Figure 6.

PET imaging of [89Zr]DFO-YS5 detects tumors in PDX models, including in neuroendocrine prostate cancer. Maximum-intensity projection (MIP) μPET/CT, coronal CT, and coronal μPET/CT slices obtained 4 days after administration of [89Zr]DFO-YS5 in the LTL-331 (A), LTL-331R (B), and LTL-545 (C) tumor models. D, MicroPET/CT fusion images on the same scale, demonstrating greater uptake in the LTL-545 model when compared against LTL-331 or 331-R. E, Biodistribution analysis obtained 4 days after administration of [89Zr]DFO-YS5 reveal high tumor uptake in the xenograft models, particularly for the LTL-545 neuroendocrine prostate cancer. F, Flow cytometry analysis of CD46 cell surface expression in PDXs. MFI values for LTL-331 (adenocarcinoma), LTL-331R (neuroendocrine), and LTL-545 (neuroendocrine) are 40,804, 40,473, and 286,645, respectively. Ctrl, an isotype-matched nonbinding antibody control.

Close modal

In this study, we report a novel CD46-targeted immunoPET probe, [89Zr]DFO-YS5, and its prostate cancer imaging abilities in multiple prostate cancer models, including mCRPC cell line xenografts (DU145 and 22Rv1) and PDX models with both adenocarcinoma (LTL-331) and neuroendocrine subtypes (LTL-331R and LTL-545). The radiopharmaceutical could be synthesized in high yield, and in vitro study demonstrated that [89Zr]DFO-YS5 has a high binding affinity for CD46. The in vivo study showed that this probe localized specifically at CD46-positive tumor with an excellent contrast to off-target organs. The specific binding of [89Zr]DFO-YS5 to CD46-positive tumors was verified by appropriate control experiments, including blocking, isotype control, and CD46-negative tumor imaging. The uptake of [89Zr]DFO-YS5 in the CD46-negative MC38 xenograft and the uptake of a nonbinding probe, [89Zr]DFO-IgG, in the CD46-positive xenografts (DU145 and 22Rv1) were all low and close to 5 %ID/g. This moderate degree of uptake in these control groups compared with muscle or blood is attributed to the EPR effect of antibody in tumor (31). To show key differentiating features of CD46 from lineage markers, such as PSMA, we compared [89Zr]DFO-YS5 with a PSMA-based imaging probe, [68Ga]PSMA-11. Importantly, [89Zr]DFO-YS5 could detect PSMA-negative/CD46-positive DU145 tumors. Finally, we demonstrated the ability to detect more clinically relevant human prostate cancer models, including adenocarcinoma and neuroendocrine prostate cancer PDXs. In these models, we found that the expression of CD46 measured with flow cytometry correlated with uptake of [89Zr]DFO-YS5, with the highest expression in LTL-545, and lower expression in LTL-331 and LTL-331R. One important finding was that [89Zr]DFO-YS5 had high uptake in neuroendocrine prostate cancer models, LTL-331R and LTL-545, suggesting feasibility of detecting this aggressive prostate cancer phenotype. Taken together, these results demonstrate that [89Zr]DFO-YS5 is an excellent imaging probe for detecting prostate cancer in preclinical models, spanning a wide variety of phenotypes, including adenocarcinoma, neuroendocrine prostate cancer, and PSMA-negative tumors.

Our study differentiates from prior research performed with CD46-directed molecular imaging agents. One interesting feature of CD46 is that it is required for measles virus infection. Thus, viruses have been used to both image and treat CD46-positive prostate cancer using an oncolytic virus (32). In the reporter gene strategy, a measles virus is genetically modified to induce overexpression of the sodium iodide symporter in the target cell (33). The cancer cells could then be imaged with 123I and treated with 131I. While this strategy indicates feasibility of CD46-directed imaging and therapy in prostate cancer, clinical translation of this method would be challenging due to the requirement of the use of a virus in the imaging protocol. We previously labeled a single-chain antibody fragment against CD46 with 99mTc, and imaged its biodistribution in mouse models using SPECT imaging (16). The study showed high tumor uptake and tumor-to-blood (12:1) and tumor-to-muscle (70:1) ratios. However, when compared against the PET/CT method detailed herein, this prior study demonstrated inferior spatial resolution and imaging characteristics of SPECT imaging (16). Moreover, a very high degree of renal uptake was observed. To overcome these challenges, we have labeled YS5, a novel internalizing full-length antibody, with the positron-emitting isotope, 89Zr. An additional advantage of this method is feasibility for subsequent clinical translation, given that an ADC using the YS5 platform is already in clinical trials. However, one important potential caveat is that the YS5 antibody does not cross-react with murine CD46. Therefore, the mouse models presented herein may underestimate background uptake in human tissues. In addition, PET imaging with full-length antibodies also has important limitations. Notably, imaging is typically performed at least 3 days and up to 1 week after radiopharmaceutical injection, which may present a practical challenge for patients. Moreover, the use of long-lived isotopes, such as 89Zr, also imparts greater radiation dose to patients. Nevertheless, 89Zr antibody imaging is a promising overall method for tumor detection, with several agents now translated into the clinic (34–40).

Taken together, the data presented herein demonstrate that [89Zr]DFO-YS5 is an effective radiopharmaceutical for imaging CD46-positive prostate cancer. CD46 is highly expressed in prostate cancer, including metastatic prostate cancer and treatment-emergent small-cell/neuroendocrine prostate cancer (17). Theranostic targeting with PSMA is highly promising for both imaging and therapy for prostate cancer. However, PSMA expression is not uniform in prostate cancer, and some cases demonstrate either heterogeneous or absent PSMA expression (11–13). Some prostate cancer cells express low level of PSMA, which could not be detected through PSMA-based imaging. Our imaging study in the dual DU145 and 22Rv1 model (Fig. 5) mimics the situation sometimes seen in the clinic, with heterogeneous target expression (14). Thus, CD46-targeted imaging and therapy could be a more effective or complementary option for PSMA-negative prostate cancer.

Overall, the high reproducibility of labeling, high tumor expression, and high tumor uptake of [89Zr]DFO-YS5 suggest a strong potential for translation of this imaging method to the clinic. A direct application of this probe could be a companion diagnostic to CD46-targeted therapy. With the CD46 ADC (FOR46, NCT03575819) now ongoing in multi-center phase I trials for mCRPC, a need may develop for appropriate selection of CD46-positive patients who will likely respond to therapy. CD46-targeted immunoPET imaging could be used to confirm CD46 expression in mCRPC in a quantitative manner, aid patient selection, and improve therapeutic outcomes in the ongoing or future trials. Similarly, these data also suggest feasibility for future CD46-directed radioligand therapy development.

Y. Su reports a patent for US20170362330A1 issued, licensed, and with royalties paid. R. Dreicer reports personal fees from Tavanta Therapeutics, Bayer, Eisai, Propella, Merck, EMD Serono, Astellas, Novartis, Exelixis, Pfizer, and Seagen outside the submitted work. F. Feng reports personal fees from Celgene, Blue Earth Diagnostics, Astellas, Janssen, Roivant, Myovant, Genentech, Bayer, PFS Genomics, and Serimmune outside the submitted work. R. Aggarwal reports grants from Janssen, AstraZeneca, Amgen, Novartis, Xynomic Pharmaceuticals, and Merck and personal fees from Dendreon, Clovis Oncology, and Advanced Accelerator Applications outside the submitted work. M.J. Evans reports other from ORIC Pharmaceuticals and grants from Soteria Biotherapeutics outside the submitted work. Y. Seo reports other from Fortis Therapeutics, Inc. outside the submitted work. B. Liu reports other from Fortis Therapeutics, Inc outside the submitted work, as well as patents for US956742B2 issued, licensed, and with royalties paid from Fortis Therapeutics, US20170362330A1 issued, licensed, and with royalties paid from Fortis Therapeutics, and US8865873B2 issued, licensed, and with royalties paid from Fortis Therapeutics, and is a board member of Fortis Therapeutics, Inc, and a consultant of Merck Sharpe & Dohme Corp. R.R. Flavell reports grants from Department of Defense, Prostate Cancer Foundation, and UCSF Precision Imaging of Cancer and Therapy Award during the conduct of the study, and grants from Fukushima SiC outside the submitted work. J. He reports grants from NIH during the conduct of the study and holds equity shares in Molecular Imaging and Therapeutics Inc., which were converted to equity shares in Fortis Therapeutics Inc., which licensed intellectual properties from the University of California and is taking the anti-CD46 antibody–drug conjugate to clinical trials. No disclosures were reported by the other authors.

S. Wang: Conceptualization, resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. J. Li: Conceptualization, resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. J. Hua: Conceptualization, resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. Y. Su: Conceptualization, resources, data curation, formal analysis, validation, investigation, methodology, writing-review and editing. D. Beckford-Vera: Conceptualization, resources, data curation, formal analysis, supervision, validation, investigation, visualization, methodology, project administration, writing-review and editing. W. Zhao: Data curation, validation, investigation, methodology, writing-review and editing. M. Jayaraman: Data curation, validation, investigation, methodology, writing-review and editing. T.L. Huynh: Data curation, validation, investigation, methodology, writing-review and editing. N. Zhao: Data curation, formal analysis, validation, investigation, methodology, writing-review and editing. Y.-H. Wang: Data curation, investigation, methodology, writing-review and editing. Y. Huang: Data curation, investigation, methodology, writing-review and editing. F. Qin: Data curation, formal analysis, validation, investigation, methodology, writing-original draft, writing-review and editing. S. Shen: Data curation, validation, writing-review and editing. D. Gioeli: Data curation, formal analysis, validation, writing-review and editing. R. Dreicer: Data curation, project administration, writing-review and editing. R. Sriram: Resources, investigation, methodology, writing-review and editing. E.A. Egusa: Resources, investigation, methodology, writing-review and editing. J. Chou: Resources, supervision, validation, investigation, methodology, writing-review and editing. F. Feng: Resources, data curation, supervision, funding acquisition, validation, investigation, methodology, writing-review and editing. R. Aggarwal: Formal analysis, investigation, writing-review and editing. M.J. Evans: Resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-review and editing. Y. Seo: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, project administration, writing-review and editing. B. Liu: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. R.R. Flavell: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. J. He: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing.

This work was supported by grants from the NIH/NCI R01CA223767 (to B. Liu and J. He) and CCSG P30 CA44579 (University of Virginia Cancer Center). Part of the PET imaging data was acquired through the University of Virginia Molecular Imaging Core Laboratory, with NIH S10OD021672 funding for the Albira Si trimodal scanner. R.R. Flavell was supported by a David Blitzer Prostate Cancer Foundation Young Investigator Award, R37 CA251980, a UCSF Research Allocation Program Precision Imaging of Cancer and Therapy Award (funded by UCSF Cancer Center Support grant No. P30CA082103), and the Prostate Cancer Research Program of the Congressionally Directed Medical Research Programs (DOD CDMRP) PC190656. MALDI-MS data were provided by the Mass Spectrometry Facility, Department of Chemistry, University of Alberta (Edmonton, Alberta, Canada).

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

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Supplementary data