Am J Cancer Res 2019;9(9):2037-2046 www.ajcr.us /ISSN:2156-6976/ajcr0100033

Original Article Immuno-PET imaging of VEGFR-2 expression in with 89Zr-labeled

Miao Li1,2, Dawei Jiang2, Todd E Barnhart2, Tianye Cao2, Jonathan W Engle2, Weiyu Chen2, Weibo Cai2

1Department of Radiology, The First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, Shaanxi, People’s Republic of China; 2Departments of Radiology and Medical Physics, University of Wisconsin-Madison, 1111 Highland Avenue, Madison 53705, Wisconsin, United States Received July 24, 2019; Accepted August 6, 2019; Epub September 1, 2019; Published September 15, 2019

Abstract: The detection and monitoring of prostate cancer (PrCa) malignancies using most of the conventional strategies is challenging. As an over-expressed biomarker of PrCa, the vascular endothelial receptor 2 (VEGFR-2) can be delineated by non-invasive imaging to address such issue. Herein, we report the positron emission tomography (PET) of VEGFR-2 expression in a PrCa mice models by composing a novel tracer, [89Zr]zirconium-labeled clinical VEGFR-2 antibody (Ramucirumab), i.e. 89Zr-Df-R. The VEGFR-2 expression levels among three different PrCa cell lines (PC-3, LNCAP and LAPC-4) were confirmed by flow cytometry. The immuno-PET imaging and bio-distribution (Bio-D) study were conducted in subcutaneous PrCa mice models via the 89Zr-Df-R. The regions of interest (ROI) data showed that the uptake of 89Zr-Df-R in the positive PC-3 (9.5±3 %ID/g) tumors are obviously higher than those ones in the negative LNCAP (6.0±1.7 %ID/g) or LAPC-4 (4.3±0.7 %ID/g) tumors at 120 hours post-injection, while the ac- cumulation of 89Zr-Df-R in PC-3 tumors (4.3±1.2 %ID/g)) could be significantly reduced by the blockade of unlabeled Ramucirumab. These quantitative data coincide with the Bio-D data and proves the specificity. Additionally, the im- muno-fluorescent staining results confirmed the expression pattern of VEGFR-2 among various PrCa tumors. Finally, the flow cytometry of PC-3 tumor tissue further proved that the binding of 89Zr-Df-R to VEGFR-2 primarily occurs on the PC-3 tumor cells. In summary, the description of the VEGFR-2 expression in PrCa by in-vivo PET with 89Zr-Df-R is feasible and it may shed light on the early detection of foci and dynamic monitoring of anti-VEGFR-2 therapy in PrCa.

Keywords: Positron emission tomography (PET), vascular endothelial 2 (VEGFR-2), prostate cancer, ramucirumab, zirconium-89

Introduction stage of PrCa [4]. Therefore, a targeted, non- invasive, comprehensive and dynamic imaging Prostate cancer (PrCa) is estimated as the modality is highly demanded in the clinical prac- second-leading cause for all the death induced tice of PrCa theranostics. by cancer in the United States (US), and is also the major cancer type around the world in male The vascular endothelial growth factor receptor [1, 2]. The conventional methods ranging from 2 (VEGFR-2/Flk-1/KDR) is an over-expressed the determination of serum prostate-specific biomarker in both tumor neo-vasculature and antigen (PSA), digital rectal examination, trans- PrCa malignancies [5]. As a trans-membrane rectal ultrasound (TRUS)-guided biopsy, mag- kinase receptor expressed on the vascular and netic resonance imaging (MRI) to the MRI-gui- lymphatic endothelium, the VEGFR-2 is critical ded biopsy have been used for detecting and for the proliferation/migration of vascular en- monitoring PrCa [3]. However, it is still challen- dothelial cell via the VEGF signaling. The high ging to achieve a good tissue-specificity with expression of VEGFR-2 was found in both of fine patient-compliance and acceptable repre- PrCa tumor cells and cultured cell lines [6-10]. sentativeness of sampling simultaneously by It has been well confirmed that VEGFR-2 is these ways. Besides, it is also difficult to obtain associated with PrCa progression [11-13], and a pathologic specimen for early diagnosis and the differentiation stage of PrCa tumor is also intervention during the onset or asymptomatic related to the expression level of VEGFR-2 [6, ImmunoPET imaging of VEGFR-2 in prostate cancer

14]. So, VEGFR-2 is becoming a promising tar- bought from the Macrocyclics, Inc. (Dallas, TX). get for the theranostics of PrCa [4, 10, 15, 16]. AlexaFluor488-laebled goat anti-rabbit anti- body was purchased from the Molecular Pro- Ramucirumab (i.e. IMC-1121B) is a recombi- bes, Inc. (Eugene, OR). AlexaFluor488-labe- nant humanized IgG1 led goat anti-human antibody was obtained which can target VEGFR-2 and further inhibit its from the Life Technologies of Invitrogen, Inc. activation by VEGF. It exhibited convincing (Eugene, OR). AlexaFluor647 labeled rat anti- results in the phase II clinical trial for the the- mouse CD31 antibody was bought from Bio- rapy of PrCa [17]. [64Cu]copper-labeled Ramu- Legend, Inc. (San Diego, CA). Pharmingen rat cirumab (64Cu-R) has been successfully em- anti-mouse CD31 antibody was purchased ployed as a positron emission tomography from BD Bioscience, Inc. (San Diego, CA). Cy3- (PET) tracer that dynamically evaluated the labeled donkey anti-rat antibody was obtained tumor angiogenesis in a murine model of non- from Jackson ImmunoResearch Laboratories, small cell lung cancer (NSCLC) [18, 19]. More Inc. (West Grove, PA). PD-10 desalting cartridge importantly, the uptake of 64Cu-R within the was bought from GE Healthcare (Piscataway, tumor kept increasing even at the last time- NJ). All other buffers and reagents were bought point of imaging, i.e. 72 h post-injection (p.i.) from Thermo Fisher Scientific (Fair Lawn, NJ). [18]. It implied that the contrast between the tumor and background tissue could be im- Deferoxamine-modification and 89Zr-labeling of proved by an extended scanning timeframe. ramucirumab Alternatively, nuclides with longer decay half- The p-SCN-Df dissolved in 10 μL of dimethyl life (t1/2) may show a great potential in enhanc- ing the quality of immuno-PET imaging mediat- sulfoxide (DMSO) and the Ramucirumab dis- ed by the Ramucirumab. solved in the mixture (300 μL) of phosphate buffered saline (PBS 1 ×; 10 mM, pH 7.4)/

In this study, the feasibility of characterizing NaHCO3-Na2CO3 buffer (0.1 M, pH 9.2) = 1:1 VEGFR-2 expression in PrCa by using [89Zr]zirco- (v/v) were mixed in the ratio of 20:1 (mol/mol). nium-labeled Ramucirumab (89Zr-Df-R) was ver- The mixture was oscillated at room tempera- ified in the nude mouse models bearing subcu- ture (RT) for 2 h, then purified by PD-10 car- taneous xenograft of different PrCa cell lines tridge with PBS 1 × as the mobile phase. The (PC-3, LNCAP and LAPC-4) [6-10]. For this pur- fractions of resulting Df-R product were col- pose, 1) the expression of VEGFR-2 in PrCa cell lected. lines and tumor tissue cells was measured by flow cytometry; 2) Ramucirumab was radio- Nuclide 89Zr was produced by an onsite PE- labeled with 89Zr via chelation chemistry and Ttrace cyclotron (GE Healthcare, Milwaukee, evaluated in-vivo; 3) the uptake of radio-activity WI) using a reaction of 89Y(p,n)89Zr. The [89Y] quantified from the regions of interest (ROI) in yttrium foil (250 μm, 99.9% in purity) was bom- PET images and the bio-distribution (Bio-D) of barded by 16.4 MeV proton with the current of 89Zr-Df-R within major organs were investigat- 5 mA for 2 h. Then the foil was dissolved in con- ed; 4) ex-vivo immuno-fluorescent staining of centrated HCl (Ultrex grade; Mallinckrodt; Du- PrCa tumor sections was conducted to depict blin, Ireland) and the solution was loaded onto the expressing profile of VEGFR-2. a hydroxamate-functionalized resin column. After a washing by 6 N HCl, the product 89Zr was Materials and methods eluted by 1 M oxalic acid. The 89Zr oxalate prod- uct was collected. Chemicals Approximately 61 MBq (1.65 mCi) of 89Zr oxa- Ramucirumab (Cyramza®) was obtained from late was added into 500 μL of HEPES buffer the Eli Lilly, Inc. (Indianapolis, IN). The 55B11 (0.5 M, pH 7.0). The pH value was adjusted to rabbit anti-human VEGFR-2 antibody was pur- 7.0-7.5 with 1 M Na2CO3 solution. Then 200 μg chased from the Cell Signaling Technology, Inc. of Df-R (200 μg/mCi) was added into the mix- (Danvers, MA). 1-(4-isothiocyanatophenyl)-3-[6, ture. The reaction was carried out in a constant 17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylh- temperature shaker at 37°C with oscillation ydroxylamino)-6,11,17,22-tetraazaheptaeico- (700 rpm) for 1 h. The reaction mixture was sine]thiourea (p-SCN-deferoxamine or Df) was purified via the PD-10 cartridge. Finally, the

2038 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer fractions of 89Zr-Df-R product were collected Prism software (ver. 7.0; GraphPad, Inc.; San and passed through 0.22 μm filter for in-vivo Diego, CA). injection [20-22]. All the procedures of animal study were in com- Cell culture pliance with the regulations by the Institutional Animal Care and Use Committee (IACUC), Three Human PrCa cell lines (PC-3, LNCAP and University of Wisconsin-Madison (UW-Madison). LAPC-4) were from the American Type Culture The xenograft tumor block was collected right Collection (ATCC; Manassas, VA). The PC-3 and after the mouse bearing PC-3 tumor was sacri- LNCAP cells were grown in Roswell Park Me- ficed. Then the tumor tissue was immersed in morial Institute (RPMI)-1640 medium (Gibco by cold PBS 1 ×, cut into tiny pieces, homogenized Life Technologies, Inc.; Grand Island, NY) with and filtered by Felcon cell strainer (25 μm in high glucose and supplemented with 10% fetal pore size; Corning; Corning, NY) for collection. bovine serum (FBS; Gemini Bio-products, Inc.; The cells are washed with cold 2% bovine West Sacramento, CA). The LAPC-4 cells were serum albumin (in PBS 1 ×) and re-suspended grown in the same RPMI-1640 medium (high in cold PBS 1 ×. The following steps are similar glucose), 20% FBS, 1% sodium pyruvate (NaPyr) to the staining of cell lines. AlexaFluor640 anti- and 0.25 mmol/L beta-mercaptoethanol mouse CD31 antibody was used for vascula- (β-MeOH). All the cells were incubated at 37°C ture staining. Ramucirumab and AlexaFluor488 in a humidified constant temperature incubator anti-human antibody was used as the primary/ with 5% CO and were harvested for flow cytom- 2 secondary antibody for the staining of VEGFR-2, etry or tumor implantation at the confluence of respectively. ~80%. PET imaging and bio-distribution Flow cytometry

The Expression of VEGFR-2 on tumor cell sur- PET imaging was conducted on an Inveon Mi- face along with the binding affinity of Df-R was cro-PET/CT scanner (Siemens Medical Solu- verified among three different PrCa cell lines tions USA, Inc.). Nude mice bearing PrCa tumors (PC-3, LNCAP and LAPC-4) by flow cytometry. were injected with 5-10 MBq (0.14-0.27 mCi) of 89 Cells were harvested at 80% confluence and Zr-Df-R via lateral tail vein. For the blockade suspended in the cold flow buffer (~1 × 7 10 group of nude mice bearing PC-3 tumors, 1.5 cells/mL). The cell suspension split into ~1.5 × mg of unlabeled (cold) Ramucirumab was in- 106 cells/tube was blocked and incubated with jected to each mouse at 24 h before the injec- 89 cold PBS 1 × (as blank cell control), the goat tion of Zr-Df-R. The original imaging was con- anti-rabbit/human antibody (as the controls of ducted by 5-15 min of static acquisition at pre- secondary antibody only; 5 μg/mL), 55B11 [23] defined time-points p.i. without attenuation or and Df-R (both as the primary antibodies; 10 scatter correction, respectively. The PET imag- μg/mL) for 1 h in ice bath, respectively. The es were reconstructed by three-dimensional or- cells engaged with 55B11 and Df-R were dered subset expectation maximization (OSE- washed with cold PBS 1 × and then incubated M3D) algorithm. The outlining of region of in- with the Alexa488 anti-rabbit/human antibod- terest (ROI) and quantification of uptake in ma- ies (5 μg/mL) for 1 h in ice bath and darkness, jor organ was performed on Inveon Research respectively. All the samples engaged with sec- Workshop (IRW) software (Siemens, Inc.). The ondary antibodies were washed with cold PBS percentage of injected dose per gram (%ID/g) 1 ×. Finally, cells in each sample were re-sus- was calculated by dividing the tissue activity in pended in 300 μL of cold PBS 1 ×. The 5-Laser MBq/g, which was converted from the uptake LSR Fortessa cytometer (Becton-Dickinson, in ROI volume, with the total radioactivity of Inc.; San Jose, CA) was employed to test the injection. cells. On FlowJo software (ver. X.0.7; Tree Star, Inc.; Ashland, OR), shift values of the top 1% of Right after the PET scanning at the last time the positive counts in fluorescent intensity were point of 120 h p.i., all the mice were euthanized gated on the histograms of LAPC-4 blank cells and dissected immediately. The blood, major as the baseline control. This gate was applied organs and tumors were collected and weighed. to all the histograms of other samples and the The radioactivity of all the blood and tissue resulting data were analyzed in the GraphPad samples were determined using a Wizard 2480

2039 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

ondary antibody (5 μg/mL) for 1 h at RT. Coverslips were mounted to the sections using Vectashield medium with 4’,6- diamidino-2-phenylindole (DA- PI; Vector Laboratories, Inc.). All the fluorescent imaging was carried out on an A1R confocal microscope (Nikon, Inc.; Melvi- lle, NY).

Statistical analysis

Quantitative data were pre- sented as mean ± standard deviation (SD). Means of up- take were compared using the Student’s t-test. Means of cell count percentage in flow cy- tometry were compared using the one-way ANOVA. For both, P < 0.05 were considered as statistically significant.

Figure 1. The VEGFR-2 expression in prostate cancer (PrCa) cell lines mea- Results and discussion sured by flow cytometry. The percentage of cell counts are from the same gate of top positive cells in fluorescent intensity in the three PrCa cell lines. Sample groups: Anti-r 2nd Ab only, the controls engaging with Alexa488 VEGFR-2 expression among anti-rabbit secondary antibody only; Anti-h 2nd Ab only, the controls engag- PrCa cell lines and tumor tis- ing with Alexa488 anti-human secondary antibody only; 55B11 as 1st Ab, sue the samples engaging with rabbit anti-human 55B11 as the primary anti- body; Df-R as 1st Ab, the samples engaging with deferoxamine-conjugated The VEGFR-2 expression, the Ramucirumab as the primary antibody (for PC-3 cell line, n = 4; for LAPC-4 in-vitro binding profile of de- and LNCAP cell lines, n = 3). feroxamine-conjugated Ramu- cirumab (Df-R) in PrCa cell automatic γ-counter (PerkinElmer, Inc.; Wal- lines and the in-vivo VEGFR-2 expression in tham, MA) and recorded as %ID/g. PrCa tumor tissue were all delineated by flow cytometry (Figures 1 and S1). As shown in Immuno-histology Figure 1, the PC-3 cells after immune-staining (with 55B11 and Df-R respectively) showed sig- Tumor tissue was frozen immediately after the nificant shifts compared to those of LAPC-4, dissection for Bio-D study and cut into slices LNCAP and control samples, demonstrating the of 5 μm in thickness by the Experimental Pa- highest expression of VEGF-2 in PC-3 cells. thology Laboratory in the Carbone Cancer Notably, the binding affinity of the Ramucirumab Center, UW-Madison. Tumor sections were fixed was not affected by the Df-conjugation. As in cold acetone for 10 min and dried in air for 3 shown in the Figure S2, the signal of CD31 fr- min at RT. Then the sections were blocked with om the PC-3 tumor tissue cells is much weak- 5% donkey serum for 30 min at RT. The blocked er than that of VEGFR-2. It indicates that the sections were incubated with 55B11 as the pri- VEGFR-2 is mostly expressed in the PC-3 tumor mary antibody (5 μg/mL) for overnight at 4°C tissue rather than vasculature. According to the flow cytometry results of PC-3 tumor tissue, and then incubated with AlexaFluor488 anti- VEGFR-2 is dominantly expressed on the tumor rabbit antibody as the secondary antibody for 1 cells. h at RT. The adjacent sections of the same tumors were also stained with rat anti-mouse The product of synthesis and radio-labeling CD31 (vascular endothelium biomarker) as the primary antibody (10 μg/mL) overnight at 4°C, The p-SCN-Df was successfully conjugated to followed by Cy3 anti-rat antibody as the sec- Ramucirumab at a ratio of 20:1 (mol/mol), sh-

2040 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

kinetics of tumors and major organs are coincident with the typical style of antibody trace- rs (Figure 2). The accumulati- on of 89Zr-Df-R within the PC-3 tumors kept increasing and reached the peak at 96 h p.i. (9.6±2.3 %ID/g), from 3.5±1.4 (at injection time-point) to 9.5±3.0 %ID/g (120 h p.i.) (n = 3). The LNCAP and LAPC-4 tu- mors appear much weaker up- takes, with signals ranging fr- om 4.1±1.4 to 6.0±2.0 %ID/g (LNCAP) and 2.1±0.5 to 4.3± 0.7 %ID/g (LAPC-4), respective- ly (n = 4; P < 0.05). Moreover, the radioactivity accumulated in the blood pool and liver have an analogous kinetic charac- teristic among three PrCa mod- Figure 2. The uptake kinetics of the region of interest (ROI) in the posi- els, indicating the non-specific tron emission tomography (PET) images of the three prostate cancer (PrCa) accumulation of 89Zr-Df-R were models (for the models bearing PC-3 tumors without/with pre-blockade, n similar (Table S1). The whole- = 3; for the models bearing LAPC-4 and LNCAP tumors, n = 4). P < 0.05. body maximum-intensity pro- jections (MIP) of mice are com- owing a high radio-chemical yield (> 80%) for Zr pared in Figure 3. As it can be illustrated, the labeling. After the PD-10 purification, the spe- 89Zr-Df-R uptake was pronounced within PC-3 cific activity of final 89Zr-Df-R tracer is 11.7±3.1 tumors, while the uptake in LNCAP and LAPC-4 mCi/mg (432.9±114.7 MBq/mg). tumors were modest.

In-vivo PET imaging and ex-vivo bio-distribution In the blocking study, with the administration of using the 89Zr-Df-R 1.5 mg unlabeled (cold) Ramucirumab 24 h in advance, the uptake of 89Zr-Df-R in PC-3 tumors Cells suspended in cold PBS 1 × were mixed (2.8±0.9, 4.7±1.3, 4.8±1.2, 4.6±1.2, 4.2±1.2, with Matrigel (Corning by Discovery Labware, 4.3±1.2 %ID/g at 4, 24, 48, 72, 96, 120 h p.i., Inc.; Bedford, MA) in the ratio of 1:1 (v/v). The respectively; n = 3) declined significantly P( < mixture was inoculated with an insulin syrin- 0.05). However, the radioactivity in blood pool ge pre-cooled on ice, ~5 × 106 cells in 100 μL (5.0±1.5-11.4±1.2 %ID/g) and liver (5.5±1.1- of mixture for each male athymic nude mouse 6.7±1.7 %ID/g) were still comparable to those (4-5 weeks in age; Envigo; Cambridge shire, among the un-blocking PC-3 groups (blood UK). The inoculation site was right under skin pool: 4.1±1.0-13.0±1.0 ID%/g; liver: 5.2±1.2- layer and ~10 mm from the injection point on 7.6±0.6 ID%/g) (Figure 2; Table S1). The appar- the right hind limb of mouse. The soft bubble ent variation of the 89Zr-Df-R uptake between under skin form by the inoculated mixture was the two PC-3 groups (without/with pre-block- adsorbed by mice slowly in 1-2 days. Tumors ade, Figure 3) and the significant difference of emerged at ~10 days post-inoculation. The uptake kinetics in the major organs (Figure 2) growth of tumor was monitered by palpation further confirmed the specificity of 89Zr-Df-R from then on. At ~4 weeks post-inoculation, towards VEGFR-2. tumors reached 5-10 mm in diameter and were utilized for in-vivo studies. In order to monitor The radioactive Bio-D data acquired validates the potential growth of 89Zr-Df-R uptake at the the quantitative uptake withdrawn from the later stage, the final imaging time-point was PET ROI (Figure 4). The radio-activity with- extended to 120 h p.i.. The quantitative uptake in PC-3 tumors without blockade (10.0±3.3

2041 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

12.7 h) would quickly decay and an enhanced tracer uptake might be achieved after that, 89 the Zr (t1/2 78.4 h), a positron

emitter with a longer decay t1/2 was employed in current stu- dy. In this work, the absolute uptake value of 89Zr-Df-R in the PrCa PC-3 tumors is compara- ble to that of 64Cu-R in the NSCLC HCC4006 tumors at 48 h p.i.. However, the uptake plot of 89Zr-Df-R at the later time- points successfully provided a clearer PET imaging by an en- hanced contrast of target/ non-target tissues. Additionally, there is a baseline accretion in the VEGFR-2-negative LAPC-4 tumor and pre-blocked VEGFR- 2-positive PC-3 tumors, which may be contributed by an en- hanced permeability and reten- tion (EPR) effect. This is a com- mon phenomenon in the PET imaging with antibody tracers [24].

The expressing profile of Figure 3. The typical positron emission tomography (PET) maximum-intensi- VEGFR-2 in tumor tissue con- ty projections (MIP) of the PC-3 (unblocked/pre-blocked), LNCAP and LAPC- firmed by immuno-histology 4 prostate cancer (PrCa) models. Arrows point to the xenograft tumor of each group at 96 h p.i. The immuno-fluorescent stain- ing and confocal microscopy %ID/g, n = 3) is notably higher than that in were conducted for further confirming the LNCAP/LAPC-4 tumors (5.4±1.6, 5.2±0.6 VEGFR-2 expressions among three tumors. As %ID/g, respectively; n = 4), while the uptake shown in Figure 5, the fluorescence from VE- was greatly prohibited by the Ramucirumab GFR-2 (in green) overlays with PC-3 and LNCAP blockage in PC-3 tumors (4.3±1.0 %ID/g, n = 3). tumor cells (nucleus, in blue) and the tumor These Bio-D data well matches the uptake in vasculature (CD31, in red), but the VEGFR-2 sig- ROI quantification, indicating the novel specific- nal in LNCAP tumor is much weaker than that in ity towards VEGFR-2 for 89Zr-Df-R as well. These PC-3 tumor. In LAPC-4 tumor, the VEGFR-2 sig- data well demonstrate that the PET uptake val- nal is minimal. No overlay between the VEGFR- ues can reliably represent the in-vivo distribu- 2 and the nucleus of tumor cells (in blue) or tion of the tracer. CD31 (in red) was observed. These immuno- histological results confirmed the expressing The replacement of 64Cu with 89Zr profile of VEGFR-2 in the tumor tissues are con- sistent with the results of previous PET/Bio-D 64 To date, the Cu-R is the only antibody-based studies. PET tracer that has been utilized for imaging VEGFR-2 [18]. The authors claimed that the The signal of CD31 from mouse vasculature is increasing trend of 64Cu-R uptake in tumor was much weaker than that of the VEGFR-2 on still observed at the last time-point (72 h p.i.) of tumor cells. It coincides with the results from 64 scanning. In consideration that the Cu (t1/2 the flow cytometry of PC-3 tumor tissue. In con-

2042 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

is not only a rational thera- peutic target (e.g. for Cabo- zantinib), but also a diagnostic biomarker for PrCa.

Advantages exhibited by immuno-PET in the imaging of VEGFR-2

Compared with classical path- ological approaches such as the biopsy and histo-chemical staining, the non-invasive mo- lecular imaging shows great advantages in mapping in-vivo biomarkers, including: 1) dyna- mic and real-timely surveil- Figure 4. The radioactive ex-vivo bio-distribution in the tumors and major lance; 2) direct and selecti- organs of the three prostate cancer (PrCa) models (for the models bearing ve collection of information; 3) PC-3 tumors without/with blockade, n = 3; for the models bearing LAPC-4 comprehensiveness through- and LNCAP tumors, n = 4). P < 0.05. out the whole-body; 4) favor- able patient-compliance. Since sideration that the Ramucirumab is human- now, several imaging modalities including the specific, we believe the deposit of 89Zr-Df-R ultrasonic imaging (via the BR55 micro-bubble) tracer within PC-3 tumor tissue is primarily con- and fluorescent imaging (quantum dot) have tributed by the ones binding to the surface been applied to the characterization of VEG- VEGFR-2 on the PC-3 tumor cells, which is con- FR-2 expression in PrCa [3, 28, 29]. How- sistent with our previous study [18]. This expla- ever, either of them can be parallel with the nation corresponds to the reported studies in nuclear medicine imaging in terms of the sensi- terms of the targeting potency and human-spe- tivity and depth of tissue penetration. In other cific binding ability of Ramucirumab [25, 26]. words, the selectivity is not nice enough. Among all the existed modalities of molecular imaging, Discussion PET is an optimally quantitative and the most sensitive technique. Traditional PET tracers for VEGFR-2 as an ideal target for PrCa PrCa are mostly based on small molecules or ligands, such as choline or VEGF. Those tracers The VEGFR-2 pathway is widely involved in vari- usually suffer from the sub-optimal accuracy, ous biological processes of PrCa ranging from low affinity or unsatisfying selectivity in the the angiogenesis, tumor cell proliferation to detection of primary tumor and metastasis. bone metastasis [10-13, 15, 16]. Thus, the Previously, the 64Cu-labeled peptoid was report- monitoring of the VEGFR-2 expression level ed for the imaging of VEGFR-2, but its relatively within the solid tumors is valuable for the per- weak affinity and the fast excretion from blood sonalized management of PrCa: 1) the early circulation result in a low uptake within the detection of primary lesions would greatly im- tumor [30, 31]. In sharp comparison, antibody prove the outcome, with a nearly 100% 5-year (or its fragment)-based tracers are able to offer survival rate after a successful early diagnosis; better specificity and pharmaco-kinetic perfor- 2) the localization of bone metastasis, which is mance simultaneously during the immuno-PET the primary cause of mortality in PrCa, may imaging [27]. help the optimization of the clinical decision- making; 3) it facilitates the stratification of pa- The merits of ramucirumab tracer tients who can benefit from a particular anti- VEGFR-2 therapy; 4) it promotes the evaluation Notably, with the approval by the Food and of VEGFR-2 profile during intervention; 5) it can Drug Administration of the US, the Ramucirumab serve as a screening tool of ideal in employed here is fully safe and translatable pre-clinical phase [4, 27]. Undeniably, VEGFR-2 [32]. Meanwhile, the 89Zr-labeled Ramucirumab

2043 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

Figure 5. The confocal imaging of tumor tissues from the three prostate cancer (PrCa) models after immuno-fluo- rescent staining. Sample groups: DAPI, the nucleus stained by DAPI; VEGFR-2, the expression of VEGFR-2 stained by 55B11 rabbit anti-human antibody as the primary antibody; CD31, the expression of vascular endothelium bio- marker CD31. well conservers the affinity and selectivity of Conclusion Ramucirumab towards the VEGFR subtypes Herein, the 89Zr-Df-R has been well proved to be (VEGFR-1/-2) [18]. Moreover, the biological t1/2 utilized as a novel tracer for the in-vivo monitor- of intact antibody aligns to the longer decay t1/2 of 89Zr better in circulation, leading to the high- ing of VEGFR-2 expression in PrCa via immuno- er contrast at tumor site and alleviated interfer- PET. Prominent binding and specific uptake ence from the background (e.g. in blood poor) in the VEGFR-2-positive malignancy can be ac- in the extended period (Table S1) [33]. Finally, hieved in the PC-3 cell line and its xenograft the 89Zr-Df-R facilitates the imaging of primary model, which are validated by the results of in- PrCa foci, since the molecular size of intact vitro and ex-vivo studies. The profile of VEGFR-2 antibody (typically ~150 kDa) exceeds the thre- expression in PrCa presented by the immuno- 89 shold of renal clearance (typically 40~50 kDa). PET with Zr-Df-R is feasible and superior to It provides a minimal background radio-activity the reported imaging modalities. The potential application of this tracer may shed light on the in urinary system (Table S1) [27]. Consequently, early focal detection, patient stratification, the hepatic clearance becomes the dominant route monitoring of anti-VEGFR-2 therapy and the for the elimination of 89Zr-Df-R from circulation medication screening in PrCa. and accounts for the non-specific accumulation in liver (Table S1). All these results demonstrate Acknowledgements that the 89Zr-labeled Ramucirumab shows great potential as a novel PET tracer for the further This work was supported, in part, by the Uni- study and translation. versity of Wisconsin-Madison, the National In-

2044 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer stitutes of Health (P30CA014520) and the cells generated after prolonged treatment with National Natural Science Foundation of China 6. Eur J Cancer 2004; 40: 1066- (Program for Young Scholars, Grant No. 817- 1072. 03468). The VEGFR-2 expression in PrCa cell [9] Darrington E, Zhong M, Vo BH and Khan SA. lines and tumor tissue cells, the uptake values Vascular endothelial growth factor A, secreted in response to transforming growth factor-be- of 89Zr-Df-R in in-vivo PET images and ex-vivo ta1 under hypoxic conditions, induces auto- bio-distribution. crine effects on migration of prostate cancer cells. Asian J Androl 2012; 14: 745-751. Disclosure of conflict of interest [10] de Brot S, Ntekim A, Cardenas R, James V, Al- legrucci C, Heery DM, Bates DO, Odum N, Pers- None. son JL and Mongan NP. Regulation of vascular endothelial growth factor in prostate cancer. Address correspondence to: Drs. Weiyu Chen and Endocr Relat Cancer 2015; 22: R107-123. Weibo Cai, Departments of Radiology and Medical [11] Lee RJ and Smith MR. Targeting MET and vas- Physics, University of Wisconsin-Madison, Wisconsin cular endothelial growth factor receptor signal- 53705, USA. Tel: 608-471-9849; E-mail: chenwy@ ing in castration-resistant prostate cancer. stanford.edu (WYC); Tel: 608-262-1749; E-mail: Cancer J 2013; 19: 90-98. [email protected] (WBC) [12] Roberts E, Cossigny DA and Quan GM. The role of vascular endothelial growth factor in meta- References static prostate cancer to the skeleton. Prostate Cancer 2013; 2013: 418340. [1] Pernar CH, Ebot EM, Wilson KM and Mucci LA. [13] Migliozzi M, Hida Y, Seth M, Brown G, Kwan J, The epidemiology of prostate cancer. Cold Coma S, Panigrahy D, Adam RM, Banyard J, Spring Harb Perspect Med 2018; 8. Shimizu A and Bielenberg DR. VEGF/VEGFR2 [2] Siegel RL, Miller KD and Jemal A. Cancer sta- autocrine signaling stimulates metastasis in tistics, 2019. CA Cancer J Clin 2019; 69: 7-34. prostate cancer cells. Current Angiogenesis [3] Smeenge M, Tranquart F, Mannaerts CK, de 2014; 3: 231-244. Reijke TM, van de Vijver MJ, Laguna MP, Po- [14] Ferrer FA, Miller LJ, Lindquist R, Kowalczyk P, chon S, de la Rosette J and Wijkstra H. First-in- Laudone VP, Albertsen PC and Kreutzer DL. Ex- human ultrasound molecular imaging with a pression of vascular endothelial growth factor VEGFR2-specific ultrasound molecular con- receptors in human prostate cancer. Urology trast agent (BR55) in prostate cancer: a safety 1999; 54: 567-572. and feasibility pilot study. Invest Radiol 2017; [15] Lee RJ and Smith MR. and pros- 52: 419-427. tate cancer: inhibiting seed and disrupting [4] Huss WJ, Hanrahan CF, Barrios RJ, Simons JW soil? Clin Cancer Res 2014; 20: 525-527. and Greenberg NM. Angiogenesis and prostate [16] Dai J, Zhang H, Karatsinides A, Keller JM, Kozl- cancer: identification of a molecular progres- off KM, Aftab DT, Schimmoller F and Keller ET. sion switch. Cancer Res 2001; 61: 2736-2743. Cabozantinib inhibits prostate cancer growth [5] Kollermann J and Helpap B. Expression of vas- and prevents tumor-induced bone lesions. Clin cular endothelial growth factor (VEGF) and Cancer Res 2014; 20: 617-630. VEGF receptor Flk-1 in benign, premalignant, [17] Hussain M, Rathkopf D, Liu G, Armstrong A, and malignant prostate tissue. Am J Clin Pathol Kelly WK, Ferrari A, Hainsworth J, Joshi A, Ho- 2001; 116: 115-121. zak RR, Yang L, Schwartz JD and Higano CS. A [6] Jackson MW, Roberts JS, Heckford SE, Ric- randomised non-comparative phase II trial of ciardelli C, Stahl J, Choong C, Horsfall DJ and cixutumumab (IMC-A12) or ramucirumab (IMC- Tilley WD. A potential autocrine role for vascu- 1121B) plus mitoxantrone and prednisone in lar endothelial growth factor in prostate can- men with metastatic docetaxel-pretreated cas- cer. Cancer Res 2002; 62: 854-859. tration-resistant prostate cancer. Eur J Cancer [7] Stadler WM, Cao D, Vogelzang NJ, Ryan CW, 2015; 51: 1714-1724. Hoving K, Wright R, Karrison T and Vokes EE. A [18] Luo H, England CG, Graves SA, Sun H, Liu G, randomized Phase II trial of the antiangiogenic Nickles RJ and Cai W. PET imaging of VEGFR-2 agent SU5416 in hormone-refractory prostate expression in lung cancer with 64Cu-labeled cancer. Clin Cancer Res 2004; 10: 3365- ramucirumab. J Nucl Med 2016; 57: 285-290. 3370. [19] Laffon E and Marthan R. A three-time-point [8] Steiner H, Berger AP, Godoy-Tundidor S, Bjar- method for assessing kinetic parameters of tell A, Lilja H, Bartsch G, Hobisch A and Culig Z. (64)Cu-labeled ramucirumab trapping in VEG- An autocrine loop for vascular endothelial FR-2 positive lung tumors. Phys Med 2017; 43: growth factor is established in prostate cancer 1-5.

2045 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

[20] England CG, Jiang D, Ehlerding EB, Rekoske [26] Clarke JM and Hurwitz HI. Targeted inhibition BT, Ellison PA, Hernandez R, Barnhart TE, Mc- of VEGF receptor 2: an update on ramucirum- Neel DG, Huang P and Cai W. (89)Zr-labeled ab. Expert Opin Biol Ther 2013; 13: 1187- for imaging of T-cell infiltration in a 1196. humanized murine model of lung cancer. Eur J [27] Hong H, Zhang Y, Sun J and Cai W. Positron Nucl Med Mol Imaging 2018; 45: 110-120. emission tomography imaging of prostate can- [21] Kang L, Jiang D, England CG, Barnhart TE, Yu cer. Amino Acids 2010; 39: 11-27. B, Rosenkrans ZT, Wang R, Engle JW, Xu X, [28] Tardy I, Pochon S, Theraulaz M, Emmel P, Pas- Huang P and Cai W. ImmunoPET imaging of santino L, Tranquart F and Schneider M. Ultra- CD38 in murine lymphoma models using (89) sound molecular imaging of VEGFR2 in a rat Zr-labeled . Eur J Nucl Med Mol prostate tumor model using BR55. Invest Ra- Imaging 2018; 45: 1372-1381. diol 2010; 45: 573-578. [22] Kang L, Jiang D, Ehlerding EB, Barnhart TE, Ni [29] Kwon H, Lee J, Song R, Hwang SI, Lee J, Kim D, Engle JW, Wang R, Huang P, Xu X and Cai W. YH and Lee HJ. In vitro and in vivo imaging of Noninvasive trafficking of prostate cancer angiogenesis using anti-vas- and PET imaging of CD30 in lung cancer mu- cular endothelial growth factor receptor 2 anti- rine models. Mol Pharm 2018; 15: 1627- body-conjugated quantum dot. Korean J Radi- 1634. ol 2013; 14: 30-37. [23] Smith NR, Baker D, James NH, Ratcliffe K, Jen- [30] Hao G, Hajibeigi A, Leon-Rodriguez LM, Oz OK kins M, Ashton SE, Sproat G, Swann R, Gray N, and Sun X. Peptoid-based PET imaging of vas- Ryan A, Jurgensmeier JM and Womack C. Vas- cular endothelial growth factor receptor (VEG- cular endothelial growth factor receptors VEG- FR) expression. Am J Nucl Med Mol Imaging FR-2 and VEGFR-3 are localized primarily to 2011; 1: 65-75. the vasculature in human primary solid can- [31] Ceci F, Castellucci P, Cerci JJ and Fanti S. New cers. Clin Cancer Res 2010; 16: 3548-3561. aspects of molecular imaging in prostate can- [24] Heskamp S, van Laarhoven HW, van der Graaf cer. Methods 2017; 130: 36-41. WT, Oyen WJ and Boerman OC. Radionuclide [32] Javle M, Smyth EC and Chau I. Ramucirumab: imaging of drug delivery for patient selection in successfully targeting angiogenesis in gastric targeted therapy. Expert Opin Drug Deliv 2014; cancer. Clin Cancer Res 2014; 20: 5875-5881. 11: 175-185. [33] Tsai WK and Wu AM. Aligning physics and phys- [25] Aprile G, Bonotto M, Ongaro E, Pozzo C and Gi- iology: engineering antibodies for radionuclide uliani F. Critical appraisal of ramucirumab delivery. J Labelled Comp Radiopharm 2018; (IMC-1121B) for cancer treatment: from ben- 61: 693-714. chside to clinical use. Drugs 2013; 73: 2003- 2015.

2046 Am J Cancer Res 2019;9(9):2037-2046 ImmunoPET imaging of VEGFR-2 in prostate cancer

Figure S1. The histograms of VEGFR-2 expression in prostate cancer (PrCa) cell lines measured by flow cytometry. The expression in the three PrCa cell lines. Sample groups: Anti-r 2nd Ab only, the controls engaging with fluores- cent anti-rabbit secondary antibody only; Anti-h 2nd Ab only, the controls engaging with fluorescent anti-human secondary antibody only; 55B11 as 1st Ab, the samples engaging with 55B11 rabbit anti-human antibody as the primary antibody; Df-R as 1st Ab, the samples engaging with Df-Ramuci- rumab as the primary antibody.

Figure S2. The VEGFR-2 expression in PC-3 tumor tissue cells measured by flow cytometry. Panels: (A) blank cells; (B) the control engaging with Al- exa488 anti-human secondary antibody only; (C) the sample engaging with Alexa647 anti-mouse CD31 antibody only; (D) the sample engaging with both Alexa647 anti-mouse CD31 antibody and Ramucirumab (as the pri- mary antibody). Vertical axis, the emission signal from the Alexa488 fluoro- phore; Horizontal axis, the emission signal from the Alexa 647 fluorophore.

1 ImmunoPET imaging of VEGFR-2 in prostate cancer

Table S1. The quantitative uptake values of 89Zr-Df-Ramucirumab from the region of interest (ROI) in positron emission tomography (PET) images of the tumors and major organs in the three prostate cancer (PrCa) models. The Values are reported as mean± SD Mean value of uptake at the time-point post-injection (%ID/g) Group Tissue 4 h 24 h 48 h 72 h 96 h 120 h PC-3 Tumor 3.5±1.4 6.6±1.7 8.4±2.1 8.9±1.9 9.6±2.3 9.5±3.0 (positive, n=3) Heart 13.0±1.0 8.4±0.8 7.3±1.1 6.0±0.9 4.8±1.0 4.1±1.0 Liver 7.6±0.6 6.0±0.7 5.4±0.6 5.4±0.6 5.5±0.7 5.2±1.2 Spleen 5.2±1.0 4.4±0.9 3.7±1.0 4.0±0.9 4.4±0.9 3.8±0.7 Kidney 5.5±0.5 3.3±0.6 2.7±0.2 2.3±0.2 2.5±0.7 1.8±0.2 Muscle 1.1±0.2 1.4±0.3 1.3±0.1 1.1±0.2 1.0±0.2 1.0±0.1 LNCAP Tumor 4.1±1.4 5.0±1.7 6.0±2.0 5.6±1.6 6.0±1.7 5.8±1.9 (negative, n=4) Heart 14.2±3.5 8.9±3.3 7.1±3.4 6.3±3.5 5.4±3.4 5.0±3.1 Liver 8.5±2.6 5.9±2.0 5.2±2.0 4.8±1.3 5.2±2.0 5.0±1.6 Spleen 4.5±0.8 4.1±0.7 3.7±0.7 4.2±1.0 4.0±1.6 3.8±1.3 Kidney 4.8±0.6 3.5±0.9 2.6±1.1 2.6±1.1 2.2±0.9 2.2±0.8 Muscle 1.2±0.6 1.3±0.6 1.4±0.6 1.3±0.4 1.1±0.3 1.1±0.3 LAPC-4 Tumor 2.1±0.5 3.5±0.6 4.0±0.7 4.1±0.8 4.1±0.9 4.3±0.7 (negative, n=4) Heart 13.0±0.5 8.4±2.8 6.2±2.6 5.1±2.4 4.4±2.3 3.8±2.3 Liver 8.2±0.9 6.0±1.0 5.4±1.2 5.0±1.4 5.2±1.5 5.1±1.6 Spleen 4.2±1.8 3.5±0.5 3.1±1.0 3.2±1.1 3.0±0.4 4.0±1.1 Kidney 5.1±0.6 3.6±0.6 2.7±0.4 2.6±0.6 2.1±0.6 2.2±0.8 Muscle 1.1±0.2 1.1±0.2 1.1±0.4 1.0±0.4 0.8±0.2 0.9±0.3 PC-3 Tumor 2.8±0.9 4.7±1.3 4.8±1.2 4.6±1.2 4.2±1.2 4.3±1.2 (blocked, n=3) Heart 11.4±1.2 8.6±1.7 7.1±1.5 6.1±1.7 5.7±1.7 5.0±1.5 Liver 6.7±1.7 5.9±1.1 5.8±1.0 5.5±1.1 5.6±0.9 5.6±1.0 Spleen 5.8±2.2 5.2±2.2 5.2±1.4 3.8±0.2 5.2±1.2 4.9±1.2 Kidney 5.6±0.6 4.1±1.0 3.5±0.9 3.0±0.8 2.7±0.7 2.7±0.7 Muscle 1.4±0.2 1.5±0.3 1.2±0.4 1.2±0.2 0.9±0.2 0.9±0.2

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