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Evaluation of agonist and antagonist radioligands for somatostatin receptor imaging of breast cancer… Dude, Iulia; Zhang, Zhengxing; Rousseau, Julie; Hundal-Jabal, Navjit; Colpo, Nadine; Merkens, Helen; Lin, Kuo-Shyan; Bénard, François Apr 17, 2017

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RESEARCH ARTICEvaluation ofradioligandsimaging of bemission tomIulia Dude1, Zhengxing ZhangKuo-Shyan Lin1,2 and François* Correspondence:fbenard@bccrc.ca1Department of MolecularOncology, BC Cancer AgencyResearch Centre, 675 West 10thAve, Vancouver V5Z 1 L3, BC,Canada2Department of Radiology,University of British Columbia,Vancouver, BC, Canadato ensure high specific activity (137 – 281 MBq/nmol at the end of synthesis).g) andusclecomparedr uptakein ZR-75-1enouslyEJNMMI Radiopharmacy                and Chemistry Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 DOI 10.1186/s41181-017-0023-y(Continued on next page)expressing cell line, which may differ from previously published transfected modelsin the number of low-affinity, antagonist-specific binding sites. The relative merit ofuptake (18.4 ± 2.9%ID/g), followed by Ga-DOTATATE (15.2 ± 2.2%ID/68Ga-NODAGA-JR11 (12.2 ± 0.8%ID/g). Tumor-to-blood and tumor-to-mratios were also higher for the agonists (>40 and >150 respectively),to the antagonist (15.6 ± 2.2 and 45.2 ± 11.6 respectively).Conclusions: The antagonist 68Ga-NODAGA-JR11 had the lowest tumoand contrast compared to agonists 68Ga-DOTATOC and 68Ga-DOTATATExenografts.The main contributing factor to this result could be the use of an endogNOD scid gamma mice bearing ZR-75-1 tumors were injected intravenouslywith the labeled peptides and used for PET/CT imaging and biodistribution at1 h post-injection. We found that 68Ga-DOTATOC had the highest tumor68©LpiLE Open Accessagonist and antagonistfor somatostatin receptorreast cancer using positronography1, Julie Rousseau1, Navjit Hundal-Jabal1, Nadine Colpo1, Helen Merkens1,Bénard1,2*AbstractBackground: The somatostatin receptor subtype 2 (sstr2) is expressed on amajority of luminal breast cancers, however SPECT and scintigraphy imagingwith agonistic sstr2 probes has been sub-optimal. High affinity antagonists canaccess more binding sites on the cell surface, resulting in higher tumor uptakeand improved sensitivity. We compared the tumor uptake and biodistribution ofthe antagonist 68Ga-NODAGA-JR11 with two agonists 68Ga-DOTA-Tyr3-octreotide(68Ga-DOTATOC) and 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE), in the human,sstr2-positive, luminal breast cancer model: ZR-75-1.Results: Peptides were assayed for binding affinity using a filtration-basedcompetitive assay to sstr2. natGa-DOTATOC and natGa-DOTATATE had excellentaffinity (inhibition constant Ki: 0.9 ± 0.1 nM and 1.4 ± 0.3 nM respectively)compared to natGa-NODAGA-JR11 (25.9 ± 0.2 nM). The number of binding siteson ZR-75-1 cells was determined in vitro by saturation assays. Agonist 67/natGa-DOTATOC bound to 6.64 ± 0.39 × 104 sites/cells, which was 1.5-fold higher than67/natGa-NODAGA-JR11 and 2.3-fold higher than 67/natGa-DOTATATE. All three68Ga-labeled peptides were obtained in good decay-corrected radiochemicalyield (61-68%) and were purified by high performance liquid chromatographyThe Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Internationalicense (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,rovided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andndicate if changes were made.99mTc-depreotide, have been explored clinically in patients with sstr2-positive breast can-Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 2 of 16cer (Alberini et al. 2000; van Eijck et al. 1994; Wang et al. 2008). Sensitivity suffered inthese initial studies, partly due to the low resolution of conventional scintigraphy, as wellas the lower and more heterogeneous expression of sstr2 on breast carcinoma comparedto NETs (Alberini et al. 2000; van Eijck et al. 1994; Wang et al. 2008).More recently, a number of reports have identified several antagonist somatostatinanalogs that visualized sstr2-positive tumors better than conventional agonists in bothpreclinical and clinical cases. Ginj et al. first demonstrated this finding by comparing(Continued from previous page)agonists versus antagonists for sstr2 breast cancer imaging warrants furtherinvestigation, first in preclinical models with other sstr2-positive breast cancerxenografts, and ultimately in luminal breast cancer patients.Keywords: Somatostatin receptor, Breast cancer, Antagonists, Positron emissiontomography, Peptides, JR11, ZR-75-1BackgroundThe somatostatin family of G-protein coupled receptors is comprised of five differentsubtypes which are variably expressed on many cancer types, most notably neuroendo-crine tumors (NETs). Somatostatin receptor subtype 2 (sstr2) is the most commonlyoverexpressed subtype, and hence several high-affinity radiolabeled peptides (mostly ag-onists) have been developed for this target (Fani et al. 2012a; Krenning et al. 1993;Kwekkeboom et al. 2010; Maecke & Reubi 2011; Reubi et al. 2001; Reubi 2003). Suchtracers have been used for diagnosis, as is the case with 111In-DTPA-D-Phe-octreo-tide (111In-pentatreotide), 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE) and68Ga-DOTA-Tyr3-octreotide (68Ga-DOTATOC), or therapy with 90Y-DOTATOCand 177Lu-DOTATATE (Fani et al. 2012a; Krenning et al. 1993; Kwekkeboom et al.2010; Maecke & Reubi 2011; Reubi 2003).Similar to NETs, breast tumors differentially express somatostatin receptors com-pared to non-malignant tissue (Fani et al. 2012b). Several studies have evaluated the ex-pression of sstr2 in different patient cohorts, showing that 15-66% of breast tumorswere sstr-positive by autoradiography (Dalm et al. 2015; Foekens et al. 1989; Bootsmaet al. 1993), 30-85% by immunohistochemistry (Ciocca et al. 1990; Schulz et al. 1998),and 97-100% by mRNA analysis (Kumar et al. 2005; Vikic-Topic et al. 1995; Evans et al.1997). The high variability observed between reports may be due to heterogeneousintra-tumor receptor density (Reubi et al. 1990) and lack of patient stratification. Sstr2expression strongly correlates with luminal A markers (estrogen and progesterone re-ceptor), and is typically not found in the other breast cancer subtypes (Dalm et al.2015; Frati et al. 2014; Reubi & Torhorst 1989). Imaging with sstr agents can thereforebe applicable to a large patient population, as luminal A cancers comprise 75% of allbreast cancer cases (Kwan et al. 2009).Compared to NETs, breast cancer sstr2 density is lower and more heterogeneous as de-termined using autoradiography (Reubi et al. 2002; Cescato et al. 2011) and PET/CT im-aging (Elgeti et al. 2008). Diagnostic SPECT and scintigraphy with 111In-pentatreotide andan sstr3 agonist and sstr3 antagonist of similar binding affinities in a tumor of humanembryotic kidney (HEK) cells transfected with sstr3. Scatchard analysis of theseDOTA-JR11 delivered a 1.7 - 10.6 fold higher tumor dose than the agonist 177Lu-DOTATATE in a small pilot study comprised of 4 patients with advanced NETsDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 3 of 16(Wild et al. 2014).Several breast cancer patients with recurrent or metastatic luminal breast cancereventually develop resistance to endocrine therapies (Milani et al. 2014). The subset ofpatients that express high levels of sstr2 might benefit from treatment by peptide recep-tor radionuclide therapy (PRRT) with somatostatin analogs if sufficient radiotracer ac-cumulation is achieved (Dalm et al. 2016). Because antagonists can bind to more sites,it is possible that tumors with lower sstr2 density, such as breast cancers, might still bevisualized and treated with radiolabeled peptides.68Ga-NODAGA-JR11 showed excellent tumor uptake and biodistribution comparedto 68Ga-DOTATATE in a preclinical setting (Fani et al. 2012b), and ~15% higher SUV-max uptake in NET patients compared to 68Ga-DOTATOC (Nicolas et al. 2015). Theaim of this study was to compare a potent antagonist, 68Ga-NODAGA-JR11, and twocommonly used agonists, 68Ga-DOTATATE and 68Ga-DOTATOC, for in vivo breastcancer imaging using a human xenograft model with endogenous sstr2 expression. Weused the sstr2-positive luminal A breast cancer cell line ZR-75-1 (Subik et al. 2010) anddetermined the transcriptional expression of the five sstr subtypes in those cells. Thebinding affinity of the peptides to human sstr2 was measured using identical assay con-ditions, and biodistribution of the radiolabeled peptides was compared.MethodsGeneral methodsAll reagents and solvents were purchased from commercial sources and used withoutfurther purification except otherwise specified. Peptides DOTATATE, DOTATOC, andNODAGA-JR11 and the cold standards of their natural gallium (natGa) complexes wereprepared by standard fmoc solid-phase peptide synthesis according to literature proce-dures (Fani et al. 2012b; Heppeler et al. 1999). C18 Sep-Pak cartridges (1 cm3, 50 mg)were obtained from Waters Corporation (Milford, MA) and pre-washed with ethanolfollowed by deionized (DI) water prior to use. High performance liquid chromatog-raphy (HPLC) purification and quality control were performed on a semi-preparativecolumn (C18, 5 μm, 250 × 10 mm), or an analytical column (C18, 5 μm, 250 × 4.6 mm)compounds revealed that the antagonist bound to more sites on the tumor cells, result-ing in an overall higher tumor uptake, despite marginally lower binding affinity and nointernalization capacity (Ginj et al. 2006). Since then, several sstr2 antagonists, labeledwith either diagnostic or therapeutic isotopes, have been explored (Fani et al. 2012b;Cescato et al. 2011; Fani et al. 2011; Wild et al. 2011; Wild et al. 2014). Cescato et al.performed in vitro autoradiography on several sstr2-positive primary tumor samples,including breast carcinomas, and suggested that the antagonist 177Lu-DOTA-BASSbound to more sites on the tumor samples than the agonist 177Lu-DOTATATE(Cescato et al. 2011). In clinical studies involving NET patients, the diagnostic antagon-ist 111In-DOTA-BASS showed improved contrast compared the conventional agonist111In-pentatreotide (Wild et al. 2011). Furthermore, the therapeutic antagonist 177Lu-respectively, both purchased from Phenomenex (Torrance, CA) and used on an Agilent1260 infinity platform (Santa Clara, CA). Triethylammonium phosphate buffer (TEAP),gic (Burlington, Canada) and also purified following the same procedures. The activitywere separated on HPLC. The peptide content was calculated by comparing the concen-Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 4 of 16tration of selected amino acids to known standards.Radiolabeling68GaCl3 in 0.5 mL DI water was added into an 8 mL glass vial preloaded with 25 μg(30 μg for NODAGA-JR11) peptide precursor and HEPES buffer solution (0.7 mL,pH 5). The vial was sealed with a screw cap and heated in a microwave oven (catalognumber: DMW7700WDB, Danby Appliance, Findlay, Ohio) as described previously(Lin et al. 2015). Heating time was 60 s, and the microwave power level was set to “2.”Reaction temperature was not determined. The reaction mixture was cooled, and dir-ectly injected into the HPLC semi-preparative column (4.5 mL/min) for purification.was measured using a Capintec (Ramsey, NJ) CRC®-25R/W dose calibrator.Binding affinityThe binding affinity of natGa-labeled compounds, natGa-DOTATOC, natGa-DOTATATEand natGa-NODAGA-JR11 to sstr2 was determined using a membrane-based competitionbinding assay. Somatostatin-28 (SRIF-28) purchased from Bachem (Torrance, CA) wasused as a known positive control for affinity determination. Purified Chinese hamsterovary-K1 (CHO-K1) membranes overexpressing human sstr2 (Perkin Elmer, Waltham,MA) were incubated with 125I-[Tyr11]-somatostatin-14 (125I-[Tyr11]-SRIF14, PerkinElmer) and competing non-radioactive ligand in a 96-well, 1.2 μm glass fibre filter plate(EMD Millipore, Darmstadt, Germany). Prior to assay, the plate filters were pre-soaked in0.1% polythylenimine (Sigma, St. Louis, MO) for 1 h at ambient temperature. Followingpre-incubation, membranes (25 μg/well), 125I-[Tyr11]-SRIF14 (0.05 nM) and various con-centrations of competing peptides (10 μM to 1 pM) were diluted in assay buffer (25 mMHEPES, pH 7.4, 10 mM MgCl2, 1 mM CaCl2, 0.5% BSA) and incubated for 1 h at 27 °Cwith moderate shaking. Once complete, the incubation mixture was aspirated through thefilters, followed by 6 washes with 200 μL ice-cold wash buffer (50 mM Tris–HCl pH 7.4,0.2% BSA). Each filter was removed and counted on a PerkinElmer WIZARD 2480gamma counter. The inhibition constant (Ki) was calculated by fitting the data to a one-site Fit-Ki curve in GraphPad Prism v7.02. To ensure that the concentration of our pep-tides, and hence our determination of Ki, was accurate, the peptide concentration was de-termined by amino acid analysis at the SPARC BioCentre (Toronto Hospital for SickChildren, Toronto, Canada), where peptides were hydrolyzed and comprising amino acidsphosphate buffered saline (PBS) and acetonitile (MeCN) were used for HPLC elution.TEAP buffer (pH 7.3) was prepared by titrating triethylamine (8 mL) with o-phosphoric acid in DI water (1 L), and PBS (pH 7.4) was prepared by dissolving PBSpowder or tablets in DI water. The pH was monitored using a Denver InstrumentUltraBasic Benchtop pH meter (Bohemia, NY), and solvents were filtered using 0.2 μmfilters (Whatman, GE Healthcare or Durapore, Merck Milliproe) prior to use.68Ga waseluted from a 50 mCi 68Ge/68Ga generator (iThemba LABS, South Africa) and purifiedaccording to reported methods (Lin et al. 2015). 67Ga-citrate was purchased from Isolo-HPLC conditions, buffers and retention times are described in Table 1. The 68Ga-la-beled peptide was collected and diluted with 50 mL 0.05 M ammonium formateity control was done using the analytical column (2 mL/min) and the same conditionssupplemented with 10% FBS from VWR Life Science Seradigm (Radnor, PA). Jurkat cellsDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 5 of 16were grown in the same base media, and contained 10% heat-inactivated FBS and 1 mMsodium pyruvate. HeLa cells were grown in DMEM+GlutaMAXTM (Life Technologies)with 10% FBS. HEK-sstr5 cells were grown in DMEM+GlutaMAXTM with 10% FBS, andcontained 0.5 mg/mL G418 to maintain sstr5 expression. All cell cultures were exposedto 100 I.U./mL penicillin/streptomycin (Life Technologies) and grown in a humidifiedatmosphere at 37 °C with 5% CO2.Quantitative-PCRThe transcriptional expression of sstr1, sstr2, sstr3, sstr4 and sstr5 in ZR-75-1 cells wasdetermined relative to reference gene hypoxanthine phosphoribosyltransferase 1(HPRT1) using qPCR. Total RNA from ZR-75-1 cells was purified using the GenE-described in Table 1. Specific activity was calculated via dividing the radioactivityinjected into HPLC (analytical column) by the mass of the tracer. The mass of the ra-diotracer was calculated based on UV absorbance from a standard curve, constructedusing serial dilutions of corresponding natGa cold-standard. Radiolabeling with 67Ga forsaturation binding assays was performed according to the same procedures for thepreparation of their 68Ga analogs.Cell cultureAll imaging and biodistribution studies were performed using the human breast carcin-oma, ER-positive cell model ZR-75-1 purchased from ATCC (Manassas, VA). In addition,HeLa cells, Jurkat cells (ATCC) and sstr5-transfected HEK-293 cells (HEK-sstr5, giftedfrom Dr. Stefan Schultz, Universitaetsklinikum, Jena, Germany) were used forquantitative-PCR (qPCR) standard curve construction. ZR-75-1 cells were cultured inRPMI 1640 +GlutaMAXTM media purchased from Life Technologies (Carlsbad, CA) andsolution and passed through a C18 light Sep-Pak cartridge. The product was elutedwith 90% ethanol in saline and formulated in saline for animal studies. The Sep-Pakpurification was performed to remove HPLC solvents (especially MeCN), concentratethe product, and formulate the tracer in solution suitable for injection into mice. Qual-Table 1 HPLC conditions and retention times (tR)Tracer HPLC conditions tR on semi-preparative column tR on analytical column68Ga-NODAGA-JR11 77% / 23% PBS/ MeCN 14.3 min 5.3 min68Ga-DOTATOC 79% / 21% PBS / MeCN 19.2 min 6.8 min68Ga-DOTATATE 81% / 19% TEAP / MeCN 20.4 min 7.4 minluteTM Mammalian total RNA miniprep kit (Sigma), treated with amplification gradeDNase I (Sigma), and measured using a NanoDropTM spectrophotometer. 2.0 μg oftotal ZR-75-1 RNA was reverse transcribed in a 20 μL reaction using SuperScript®VILOTM cDNA synthesis kit from Invitrogen (Carlsbad, CA). qPCR was set up in 384-well plates, in a total volume of 10 μL; each reaction containing 1 μL template cDNA,500 μM forward and reverse primers, 250 μM probe, and 1X SsoAdvancedTM universalprobes supermix from Bio Rad (Hercules, CA). Each reaction was performed in tripli-cates and repeated 3 times. Predefined primers (forward and reverse) and probes for allDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 6 of 16confluence in 12-well plates, and growth media was replaced with reaction media(RPMI, 1% BSA, 100 I.U./mL penicillin/streptomycin) 1 h before the assay. Cells weretreated in duplicates with 67/natGa-DOTATOC, 67/natGa-DOTATATE or 67/natGa-NODAGA-JR11 in 500 μL reaction media and incubated for 1 h at 25 °C. Excess cold-standard (1.2 μM) was used to block receptors and determine non-specific binding.After the incubation period, the reaction media was aspirated, and the cells werewashed 3 times with cold PBS. Cells were lysed and collected with 1 M NaOH andcounted in a PerkinElmer WIZARD 2480 gamma counter. The number of binding sitesper cell was calculated and fitted to a one-site binding model in GraphPad Prism 7.02to determine the dissociation constant (Kd) and number of binding sites (Bmax).Estrogen pellet implant and tumor inoculationAll animal studies were done in compliance with the Canadian Council on Animal Careguidelines and were approved by the Animal Care Committee of University of BritishColumbia (Vancouver, Canada). Immunodeficient female NOD.Cg-PrkdcsciIl2rgtm1Wjl/SzJ mice (NOD scid gamma) were obtained from an in-house breeding colony at theAnimal Resource Centre of the BC Cancer Agency Research Centre and also fromsix genes were purchased from Integrated DNA Technologies (Coralville, Iowa); seeAdditional file 1 for assay names. The QuantstudioTM 6 K Flex Real-Time PCR systemfrom Thermo Fisher (Carlsbad, CA) was used for amplification and detection. The con-centration of each target was determined by interpolating the Ct value from respectivestandard curves of known concentrations. To construct the standard curves, RNA fromcell lines with known expression of sstr subtypes was purified and reverse-transcribedas described above. Target sstr genes were PCR amplified using Q5® high-fidelity DNApolymerase (New England BioLabs, Ipswich, MA) as per the manufacturer’s instruc-tions, using the same primers as the qPCR reactions (without the fluorogenic probe).PCR products were separated on a 2% agarose gel, and target bands were extracted andpurified using the Monarch® DNA gel extraction kit (New England BioLabs). Theamount of DNA was quantified using Qubit® dsDNA HS assay kit (Thermo Fisher) andthe number of copies/μL was calculated using the following formula:copies=μL ¼ DNA Concentration g=μLð Þamplicon length bpð Þ  650 g=mol 6:022 n1023copies=molStandard curves were constructed from 10-fold serial dilutions (105 copies/μL to 1copy/μL) and assayed by qPCR in triplicates. All standard curves were repeated 2–3times. Sstr1 transcripts were amplified from HeLa cells, sstr2 from ZR-75-1 cells, sstr3from Jurkat cells, sstr4 from the ChantestTM human sstr4 receptor cell line (irradiatedcells) from Charles River Laboratories (Wilmington, MA), and both sstr5 and HPRT1from HEK-sstr5 cells. Representative standard curves, primer and probe information, andcycling conditions for both PCR and qPCR can be found in the Additional file 1.Saturation binding assaysSaturation binding assays were performed in vitro on ZR-75-1 cells using variable con-centrations (0.1 – 100 nM) of 67/natGa-labeled tracers. Cells were grown to near-Jackson Laboratory. To sustain the growth of the ER-positive ZR-75-1 cell model, ani-mals were administered a 1.7 mg, 60-day slow-release estrogen pellet from Innovativeorgans were harvested, rinsed in PBS, patted dry and weighed. Organ uptake was mea-Tumor-bearing mice were injected i.v. with 8–9 MBq of 68Ga-labeled peptide. Static PETStatistical analysis was performed using GraphPad Prism v7.02 software. Transcrip-trol had a Ki of 3.7 ± 1.7 nM (n = 5) in our assays. Representative inhibition curves areDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 7 of 16shown in Fig. 1. Multiple batches of 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11 were prepared in good radiochemical yield (61 ± 5, 62 ± 8 and 68 ±13% respectively, n = 3), purity (>98%) and high specific activity (251.6 ± 33.9, 197.3 ±85.2 and 138.8 ± 2.6 MBq/nmol respectively, n = 3). The particular tracer preparationsused for animal studies corresponded to radiochemical yields of 62, 66 and 58% andtional sstr expression results were tested using a one-way ANOVA. Binding affinity,Bmax values, Kd values and in vivo organ uptakes were compared between the threegroups using a one-way ANOVA. The difference was considered statistically significantif the p value was < 0.05. Non-statistically significant findings were indicated as “ns.”ResultsBinding affinity and radiolabelingnatGa-DOTATOC and natGa-DOTATATE had an inhibition constant (Ki) in the lownanomolar range (0.9 ± 0.1 nM, n = 4 and 1.4 ± 0.3 nM, n = 3 respectively), while the Kiof natGa-NODAGA-JR11 was higher (25.9 ± 0.2 nM, n =3, p < 0.001). The SRIF-28 con-images were acquired 55 min post-injection for 10 min using an Inveon microPET/CTscanner (Siemens, Erlangen, Germany) as described previously (Lin et al. 2015). A base-line CT scan was used for localization and attenuation correction. Mice were promptlyeuthanized after imaging, and biodistribution studies were undertaken as described above.Statisticssured either in a WIZARD 2480 gamma counter (Perkin Elmer) or Cobra II auto-gamma counter (Packard), both calibrated with standards of known 68Ga activity. Up-take was normalized to the injected dose and to the respective weight of the organ, andexpressed as percent injected dose per gram of tissue (%ID/g).PET/CT imagingResearch of America (Sarasota, FL) subcutaneously in the dorsal space of the neck. 3–5days post pellet-implant, 10 million ZR-75-1 cells were re-suspended in a mixture of1:1 PBS and Matrigel (Corning Inc., Corning, NY) and inoculated subcutaneously onthe right shoulder. Tumors were grown for 5–6 weeks, until they reached a size of 7–11 mm in diameter.Biodistribution studiesMice were sedated using 2 mL/min of O2 with 2% isoflurane and injected intravenously(i.v.) with 1–2 MBq of 68Ga-labeled peptide. Mice were allowed to roam freely for60 min prior to euthanasia by 2 mL/min of O2 with 4% isoflurane followed by CO2 as-phyxiation. Blood was promptly collected by cardiac puncture and weighted. Internalspecific activities of 281.2, 218.3 and 136.9 MBq/nmol for 68Ga-DOTATOC, 68Ga-DOTATATE and 68Ga-NODAGA-JR11 respectively.Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 8 of 16Saturation binding assays67Ga-labeled peptides used for saturation binding assays were prepared using the sameFig. 1 Representative inhibition curves for natGa-DOTATOC, natGa-DOTATATE, natGa-NODAGA-JR11 and SRIF-28 against the binding of 125I-[Tyr11]-SRIF14 to sstr2-overexpressing CHO-K1 membranesprocedures as the 68Ga-analogs. 67Ga-NODAGA-JR11, 67Ga-DOTATOC and 67Ga-DOTATATE were labeled in 4, 57, 25% decay-corrected radiochemical yield, > 99%radiochemical purity and 11.5, 392, 444 MBq/nmol specific activity respectively. Invitro saturation binding assays revealed that agonist 67/natGa-DOTATOC bound tomore sites on ZR-75-1 cells (6.64 ± 0.39 × 104 sites/cell) compared to both 67/natGa-DOTATATE (2.85 ± 0.02 × 104 sites/cell, p < 0.001) and 67/natGa-NODAGA-JR11(4.39 ± 0.32 × 104 sites/cell, p < 0.001). The dissociation constant (Kd) in these assayswas lowest for 67/natGa-DOTATATE (0.55 ± 0.015 nM), followed by 67/natGa-NODAGA-JR11 (1.19 ± 0.06 nM, p < 0.001) and finally 67/natGa-DOTATOC (2.70 ± 0.13nM, p < 0.001). See Table 2 for Bmax and Kd values, and Fig. 2 for representative saturationbinding curves.Transcriptional Sstr expressionWe calculated the target gene/HPRT1 copy number ratio for all five somatostatin sub-types and found predominant expression of sstr2. The normalized expression of sstr2to HPRT1 was 0.055 ± 0.0083 (n = 3), and < 0.00005 for all other subtypes (n = 3 each,Table 2 Saturation binding results for respective radiotracers with ZR-75-1 cells67/natGa-NODAGA-JR11 (n = 3) 67/natGa-DOTATOC(n = 3) 67/natGa-DOTATATE (n = 3)Bmax(× 104 sites/cell)4.39 ± 0.32 6.64 ± 0.39 2.85 ± 0.02Kd (nM) 1.19 ± 0.06 2.70 ± 0.13 0.55 ± 0.02transcript in our cDNA preparation. In comparison, we identified 12,915 ± 2218 copies/μL (n = 4) HPRT1 transcripts, and 681 ± 148 (n = 3) copies/μL sstr2 transcripts.Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 9 of 16Tumor and organ uptakep < 0.001) (Fig. 3). For all subtypes except sstr2, we calculated < 10 copies/μL of targetFig. 2 Representative saturation binding curves for 67/natGa-NODAGA-JR11, 67/natGa-DOTATOC and 67/natGa-DOTATATE to ZR-75-1 cells in vitro. The non-specific binding, determined by blocking with excess cold-standard, was subtracted and only specific binding is shownA full overview of tracer biodistribution is presented in Table 3. Among the three testedtracers, we found that the antagonist 68Ga-NODAGA-JR11 had the lowest tumor uptake(12.2 ± 0.8%ID/g) compared to agonists 68Ga-DOTATOC (18.4 ± 2.9%ID/g, p < 0.001) and68Ga-DOTATATE (15.2 ± 2.2%ID/g, ns) (Fig. 4). 68Ga-NODAGA-JR11 had tumor-to-blood and tumor-to-muscle ratios of 15.6 ± 2.2 and 45.2 ± 11.6 respectively compared to68Ga-DOTATOC (41.1 ± 5.7, p < 0.001 and 171.5 ± 55.3, p < 0.01 respectively) and 68Ga-DOTATATE (44.7 ± 11.7, p < 0.001 and 152.0 ± 60.8, p < 0.01 respectively). 68Ga-Fig. 3 Relative transcriptional expression of sstr subtypes normalized to housekeeping gene HPRT1(n = 3 for each)Table 3 Biodistibution of 68Ga-NODAGA-JR11, 68Ga-DOTATOC and 68Ga-DOTATATE in NOD scidgamma ZR-75-1 tumor-bearing mice68Ga-NODAGA-JR11n = 568Ga-DOTATOCn = 668Ga-DOTATATEn = 6Mean SD Mean SD Mean SDTumor 12.21 0.78 18.44 2.87 *** 15.22 2.20Blood 0.80 0.10 0.45 0.09 *** 0.35 0.06 ***Fat 0.23 0.11 0.18 0.09 0.28 0.15Ovaries 0.67 0.22 0.51 0.07 0.89 0.35Uterus 0.86 0.22 0.54 0.11 0.84 0.10Intestines 0.90 0.20 2.39 0.30 *** 7.35 0.44 ***Stomach 1.36 0.78 2.75 1.25 8.29 3.06 ***Pancreas 9.29 2.03 11.01 1.32 51.56 5.47 ***Spleen 0.39 0.05 0.46 0.11 0.84 0.38Kidneys 14.12 1.65 9.27 1.73 *** 8.45 1.73 ***Adrenals 2.00 0.65 9.52 3.78 ** 15.20 7.26 **Liver 0.99 0.12 0.71 0.17 1.95 0.52 ***Lungs 4.72 0.71 22.93 6.12 *** 28.66 2.94 ***Heart 0.46 0.06 0.30 0.04 * 0.67 0.13 **Muscle 0.28 0.07 0.11 0.02 *** 0.11 0.04 ***Bone 0.26 0.04 0.20 0.03 0.25 0.03Brain 0.05 0.02 0.03 0.01 0.03 0.01Tumor to Background RatiosBlood 15.56 2.20 41.13 5.68 *** 44.65 11.74 ***Muscle 45.15 11.56 171.51 55.33 ** 151.95 60.75 **Kidneys 0.87 0.12 2.01 0.24 *** 1.88 0.48 ***Organ uptake is expressed as mean ± standard deviation (SD) in units of percent injected dose per gram of tissue (%ID/g).The tumor uptake is highlighted in bold. Means were statistically compared to the respective organ uptake of 68Ga-NODAGA-JR11 and p values < 0.05, < 0.01, and < 0.001 were expressed as *, ** and *** respectivelyFig. 4 PET maximum intensity projection images of a. 68Ga-DOTATOC, b. 68Ga-DOTATATE and c. 68Ga-NODAGA-JR11 in ZR-75-1 tumor bearing miceDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 10 of 16Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 11 of 16DOTATATE had the highest uptake in non-tumor sstr2-positive organs such as intestines,stomach, pancreas, adrenal glands and lungs, followed by 68Ga-DOTATOC and lastly by68Ga-NODAGA-JR11. The excretion profile of all three tracers was predominantly renal,with high uptake in the kidneys and bladder, and low uptake in the liver. The agonist68Ga-DOTATATE had the lowest kidney uptake (8.5 ± 1.7%ID/g), compared to 68Ga-DOTATOC (9.3 ± 1.7%ID/g) and 68Ga-NODAGA-JR11 (14.1 ± 1.7%ID/g).DiscussionIn this study, we used a human breast cancer cell model with endogenous sstr2 expres-sion (ZR-75-1 cells) to compare tumor uptake of the antagonist 68Ga-NODAGA-JR11with two routinely used agonists 68Ga-DOTATOC and 68Ga-DOTATATE. Most stud-ies evaluating sstr tracers in vivo typically used a rat pancreatic cell model, i.e. AR42J(Froidevaux et al. 2002), or HEK cells transfected with somatostatin receptors (Fani et al.2012b; Ginj et al. 2006; Fani et al. 2011), which may not adequately represent a breast can-cer phenotype. We chose to use the chelator NODAGA instead of DOTA for the antag-onist, as 68Ga-NODAGA-JR11 showed better binding affinity and higher tumor uptakecompared to 68Ga-DOTA-JR11 in a preclinical model (Fani et al. 2012b). 68Ga-NODAGA-JR11 is a more potent antagonist, and thus a better candidate for comparisonwith the current gold-standard agonists 68Ga-DOTATOC and 68Ga-DOTATATE.We looked at the transcriptional expression of all five sstr subtypes in ZR-75-1 cellsin vitro and found predominant expression of sstr2, with negligible expression of theother four subtypes. The HPRT1-normalized expression level was 0.055 ± 0.0083 forsstr2, and < 0.00005 (p < 0.001) for sstr1, sstr3, sstr4 and sstr5. Previous studies have re-ported a strong correlation between sstr mRNA and protein expression, suggesting thattranscriptional studies are adequate for profiling this receptor family (Kumar et al.2005; Wang et al. 2008; Schaer et al. 1997). Breast carcinoma samples typically showvaried expression of all five subtypes, often with 2 or 3 subtypes co-expressed on thesame sample (Kumar et al. 2005; Schaer et al. 1997). Although sstr1, sstr3 and sstr5were identified at high levels in a number of cases, sstr2 was the most commonlyexpressed (Reubi et al. 2001; Kumar et al. 2005; Vikic-Topic et al. 1995; Evans et al.1997; Schaer et al. 1997).We tested the binding affinities of the peptides natGa-DOTATOC, natGa-DOTATATEand natGa-NODAGA-JR11 to human sstr2 in a filtration-based, competition bindingassay. For the agonists natGa-DOTATOC, natGa-DOTATATE and SRIF-28, our inhib-ition constants (Ki) were 0.9 ± 0.1, 1.4 ± 0.3 and 3.7 ± 1.7 nM respectively. Our Ki valuesare comparable to the IC50 values reported by Reubi et al. (Ki values were not re-ported), which were 2.5 ± 0.50, 0.2 ± 0.04 and 2.7 ± 0.30 nM respectively (Reubi et al.2000). As IC50 values are dependent on the concentration of substrates used in a spe-cific assay, they are not reproducible between laboratories, and it is recommended thatKi values are calculated. AlthoughnatGa-DOTATATE was reported to have very highaffinity to sstr2 (12 fold higher than natGa-DOTATOC) (Reubi et al. 2000), we did notobserve this in our experiments. This may explain, in part, why the diagnostic perform-ance of both peptides is similar in clinical studies, with perhaps a slight advantage for68Ga-DOTATOC (Poeppel et al. 2011).We observed a significantly lower binding affinity for the antagonist natGa-NODAGA-JR11 (Ki = 25.9 ± 0.2 nM) compared to the two agonists, and also comparedDude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 12 of 16to the IC50 reported by Fani et al. (IC50 = 1.2 ± 0.2 nM) (Fani et al. 2012b). These differ-ences could be partially attributed to differences in methodology and assay conditions.We used a filtration-based binding assay with an SRIF-14 based competing hot ligand(125I-[Tyr11]-SRIF14), whereas other reports used an autoradiography approach and theSRIF-28 analog: 125I-[Leu8, DTrp22, Tyr25]-somatostatin-28 (Fani et al. 2012b). A stan-dardized assay system and a direct comparison between classical protein binding assaysand autoradiography methods would be valuable to improve our understanding of thestructure-activity relationship of these ligands. When assessing binding affinity basedon Kd values, we observed a different relationship, more closely resembling that re-ported in the literature. 67/natGa-DOTATATE bound sstr2 receptors on ZR-75-1 cellswith the lowest Kd (0.55 ± 0.02 nM) compared to67/natGa-DOTATOC (2.70 ± 0.13 nM)and 67/natGa-NODAGA-JR11 (1.19 ± 0.06 nM). It appears that 67/natGa-NODAGA-JR11binds receptors on ZR-75-1 cells with high affinity, but is not as potent when compet-ing with reference ligand 125I-[Tyr11]-SRIF14. These findings are interesting and unex-pected, and indicate several factors that must be considered when choosing assayconditions to measure affinities.When tested in vivo, the agonist 68Ga-DOTATOC had the highest tumor uptake(18.4 ± 2.9%ID/g) compared to 68Ga-DOTATATE (15.2 ± 2.2%ID/g, ns) and 68Ga-NODAGA-JR11 (12.2 ± 0.8%ID/g, p < 0.001). 67/natGa-DOTATOC also had the highestBmax value in saturation binding assays (6.64 ± 0.39 × 104 sites/cell), even higher than thatobserved for antagonist 67/natGa-NODAGA-JR11 (4.39 ± 0.32 × 104 sites/cell, p < 0.001).These results contrast recently published reports stating that antagonists can achievehigher tumor uptake by binding more receptor sites (Fani et al. 2012b; Ginj et al. 2006). Itis possible that in a cell model where the target G-protein coupled receptor isoverexpressed without concomitant overexpression of the associated G-protein,there would be a greater number of receptors in a low-affinity state (unbound byG-protein), and therefore significantly higher Bmax values observed for antagonistscompared to agonists (Kenakin 1997). Overexpression of, not only the receptors, butalso the associated G-protein, would be required to have a more representative model.When Ishihara et al. also overexpressed the G-protein in COS cells transfected with secre-tin receptor, the number of agonists binding sites increased from 1.8% (of the total seenby the antagonist) to 15% (Ishihara et al. 1991). In an endogenous model, such as the oneused herein, where most receptors are found in a high affinity state (bound by the G-protein), imaging with antagonists might not be more advantageous. Similar to our stud-ies, Wadas et al. did not observe higher tumor uptake with antagonist 64Cu-CB-TE2A-sst2-ANT compared to agonist 64Cu-CB-TE2A-Y3-TATE when using the endogenouslyexpressing sstr2 model, AR42J (Wadas et al. 2008). It is also interesting that the agonists67/natGa-DOTATOC and 67/natGa-DOTATATE showed differing number of binding sites(6.64 ± 0.39 and 2.85 ± 0.021× 104 sites/cell respectively) on the ZR-75-1 cells used in thisstudy. This is an unexpected finding, which could explain why both these compoundshave comparable tumor uptake clinically, despite differences in binding affinity.We observed that other sstr2-positive organs such as pancreas, adrenal glands, intes-tine and stomach had very high uptake with agonist 68Ga-DOTATATE compared tothe other two radiotracers. This finding can be explained by injected peptide amount,which was lowest for 68Ga-DOTATATE (15.6 ± 4.4 pmol/mouse) compared to 68Ga-DOTATOC (33.0 ± 33.5 pmol/mouse) and 68Ga-NODAGA-JR11 (40.3 ± 21.5 pmol/Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 13 of 16mouse). A biphasic relationship between uptake in receptor-positive organs andinjected peptide mass has been reported, with maximum tumor uptake reached be-tween 10 – 100 pmol/mouse (Bernhardt et al. 2003; de Jong et al. 1999; Notni et al.2016). In other sstr2-positive organs, maximum uptake is achieved at lower peptidedoses, presumably due to lower absolute receptor quantities (more regionally concen-trated) and therefore lower saturation limits in these organs (Bernhardt et al. 2003; deJong et al. 1999; Notni et al. 2016). In our studies, low capacity organs (i.e. pancreas,adrenals, intestine and stomach), but not high capacity organs (i.e. tumor), showed ele-vated uptake with 68Ga-DOTATATE, indicating this to be a peptide mass effect.Although tumor uptake is also influenced by peptide dose, we believe the massdifferences in our studies were not significant enough to cause major differencesbetween the three groups. De Jong et al. showed optimal CA20948 uptake when111In-DOTATOC was injected in 0.5 μg/rat (~30 pmol/mouse), with uptake > 80%of the maximum in the 0.25 – 1 μg/rat range (~15 – 60 pmol/mouse) (de Jonget al. 1999). Similarly Bernhardt et al. showed > 70% of maximum tumor uptakewith 111In-pentetreotide (GOT1 cell model) when the tracer was injected between6.4 – 664 pmol/mouse (Bernhardt et al. 2003). Indeed, normalization of peptide con-tent would remove some variability, and enable a more accurate comparison.Tumor-to-blood and tumor-to-muscle ratios were lower for 68Ga-NODAGA-JR11(15.6 ± 2.2 and 45.2 ± 11.6 respectively) compared to 68Ga-DOTATOC (41.1 ± 5.7 and171.5 ± 55.3 respectively) and 68Ga-DOTA-TATE (44.7 ± 11.7 and 152.0 ± 60.8 respect-ively). 68Ga-NODAGA-JR11 had a ~2 fold higher uptake in the blood and muscle com-pared to the other two agonists, accounting for the lower tumor contrast. These resultsdiffer significantly from recently reported clinical data in subjects with neuroendocrinetumors (Nicolas et al. 2016). The improved contrast reported by Nicolas et al. might becaused by a combination of lower specific activity, combined with a lower binding affin-ity of 68Ga-NODAGA-JR11 (68Ga-OPS202) to sstr2, which may decrease tracer accu-mulation in low capacity binding sites (Nicolas et al. 2016). Alternatively, differentpharmacokinetic properties between mice and humans may also contribute to theseconflicting results.All three tested peptides had predominant renal clearance. Exogenous estrogen pel-lets are known to cause hydronephrosis and urine retention (Gakhar et al. 2009; Ing-berg et al. 2012), thus we expected higher than normal kidney uptake due to theindirect effects of estrogen.Beyond diagnosis, sstr2-targeting tracers can also be used therapeutically. Diag-nostic somatostatin radiotracers, such as the ones evaluated in this study, can iden-tify breast cancer lesions and monitor response to therapy by PRRT. Treatment ofsstr2-positive breast cancer with therapeutic agents such as 177Lu-DOTATATE,177Lu-DOTATOC and 177Lu-DOTA-JR11 could be particularly valuable to patientsthat develop resistance to conventional endocrine therapy. The efficacy and safetyof 177Lu-DOTATATE was demonstrated in an international, multi-centric phase IIIclinical trial, and showed a potent tumor response and very favourable toxicity inpatients with metastatic midgut NETs (NCT01578239) (Strosberg et al. 2016). Simi-larly, the safety and tolerability of 177Lu-DOTA-JR11, also known as 177Lu-OPS201,is currently being tested in phase I/II clinical trials for NET patients(NCT02592707).Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 14 of 16ConclusionWe compared the tumor uptake and biodistribution of two well-known agonists andone antagonist in vivo using ZR-75-1 tumors, a human breast cancer xenograft withendogenous sstr2 expression. In this model, the antagonist 68Ga-NODAGA-JR11 hadthe lowest tumor uptake and contrast among the three tracers; a finding that differssignificantly from recently published reports. This result may be explained by the useof an endogenously expressing sstr2 cell model, which would have fewer low-affinitybinding sites compared to transfected models. More studies are needed to determine ifantagonists are better radiotracers for sstr2 breast cancer imaging than agonists, par-ticularly in other breast cancer xenografts, and ultimately in luminal breast cancer patients.Additional fileAdditional file 1: Supplemental information. Figure S1. Representative standard curve for absolute quantificationqPCR experiments. Table S1. Standard curve parameters. Table S2. PCR Cycling conditions. Table S3. qPCRCycling conditions. (DOCX 38 kb)Abbreviations%ID/g: Percent injected dose per gram; 111In-Pentatrotide: 111In-DTPA-D-Phe-octreotide; 125I-[Tyr11]-SRIF14: 125I-Tyr11-somatostatin 14; ANOVA: Analysis of variance; BSA: Bovine serum albumin; cDNA: Coding DNA; Ct: Cycle threshold;CT: Computed tomography; DI: Deionized; DMEM: Dulbecco Modified Eagle Medium; DNA: Deoxyribonucleic acid;DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTATATE: DOTA-Tyr3-octrotate; DOTATOC: DOTA-Tyr3-octrotide; ER: Estrogen receptor; FBS: Fetal bovine serum; HEK: Human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC: High performance liquid chromatography; HPRT1: Hypoxanthinephosphoribosyltransferase 1; i.v.: Intravenous; IC50: Half maximal inhibitory concentration; Ki: Inhibition constant;MeCN: Acetonitrile; mRNA: Messenger RNA; natGa: Natural gallium; NET: Neuroendocrine tumor; NOD scidgamma: NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; NODAGA: 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid; ns: Notsignificant; PBS: Phosphate buffered saline; PCR: Polymerase chain reaction; PET: Positron emission tomography;PR: Progesterone receptor; PRRT: Peptide receptor radionuclide therapy; qPCR: Quantitative polymerase chain reaction;RNA: Ribonucleic acid; RPMI: Rosewell Park Memorial Institute; SD: Standard deviation; SPECT: Single photon emissioncomputed tomography; SRIF-28: Somatostatin-28; Sstr1-5: Somatostatin receptor subtype 1–5; SUV: Standard uptakevalue; TEAP: Triethylammonium phosphate; tR: Retention time; Tris: 2-Amino-2-hydroxymethyl-propane-1,3-diolAcknowledgementsThe authors would like to acknowledge Dr. Chengcheng Zhang, Dr. Joseph Lau and Gemma Dias for meaningfuldiscussion and input at all steps of this study. We would also like to thank the Animal Resource Centre (ARC) at the BCCancer Agency Research Center for animal monitoring and care, and Dr. Jinhe Pan for help eluting the 68Ge/68Gagenerator.FundingThis study was funded by the Canadian Breast Cancer Foundation.Authors’ contributionsFB and KSL conceived and designed the complete study. Binding affinity assays, qPCR experiments, saturation bindingassays, and biodistribution studies were performed by ID. Animal procedures and monitoring were primarily done byID with some assistance and critical input from HM. ZZ was responsible for chemical synthesis and quality control ofcold standards and radiotracers. NC, NHJ and JR were responsible for PET/CT image acquisition. The manuscript wasdrafted by ID with critical revisions from FB, KSL and ZZ. All authors read and approved the final manuscript.Competing interestsThe authors of this manuscript have no competing interests.Consent for publicationNot applicable.Ethics approvalAll animal studies were done in compliance with the Canadian Council on Animal Care guidelines and were approvedby the Animal Care Committee of University of British Columbia (Vancouver, BC, Canada).Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Dude et al. EJNMMI Radiopharmacy and Chemistry  (2017) 2:4 Page 15 of 16prospective cohort studies of breast cancer survivors. Breast Cancer Res. 2009;11:R31.Kwekkeboom DJ, Kam BL, van Essen M, Teunissen JJ, van Eijck CH, Valkema R, et al. Somatostatin-receptor-basedimaging and therapy of gastroenteropancreatic neuroendocrine tumors. Endocr Relat Cancer. 2010;17:R53–73.Lin KS, Pan J, Amouroux G, Turashvili G, Mesak F, Hundal-Jabal N, et al. 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