Comparing Gly11/dAla11-Replacement vs. the in-Situ Neprilysin-Inhibition Approach on the Tumor-targeting Efficacy of the 111In-SB3/111In-SB4 Radiotracer Pair

Background: The GRPR-antagonist 68Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from 68Ga to 111In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized 111In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of 111In-SB4: 111In-[dAla11]SB3. Methods: The biological responses of 111In-SB3/SB4 were compared in PC-3 cells and animal models. Results: Gly11/dAla11-replacement deteriorated GRPR-affinity (SB4 IC50: 10.7 ± 0.9 nM vs. SB3 IC50: 4.6 ± 0.3 nM) and uptake in PC-3 cells (111In-SB4: 1.3 ± 0.4% vs. 111In-SB3 16.2 ± 0.8% at 1 h). 111In-SB4 was more stable than 111In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified 111In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. 111In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with 111In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for 111In-SB4). Conclusions: Replacement of Gly11 by dAla11 improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of 111In-SB4 vs. unmodified 111In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of 111In-SB3, and potentially other peptide radiotracers.

In the present work we have compared the two stabilization strategies, namely the structural DAla 11 /Gly 11 -substitution in the peptide sequence vs. the in situ NEP-inhibition approach [23], for their efficacy to improve tumor uptake and overall pharmacokinetics of resulting 111 In-radiotracers in mice. For this purpose, we first developed SB4, [DAla 11 ]SB3, and directly compared the in vitro and in vivo behavior of 111 In-SB4 vs. 111 In-SB3 in the same experimental models. We next studied the impact of NEP-inhibition after PA-coinjection on the in vivo stability and the tumor targeting capabilities of the two 111 In-radiotracers.

Peptides and Radioligands
The new SB4 peptide conjugate was generated by single Gly 11 /DAla 11 -replacement in the SB3 peptide chain (Figure 1a). Both SB3 and SB4 carrying the universal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at their N-terminus could be labeled with 111 In at molecular activities of 3.7-7.4 MBq 111 In/nmol peptide. Quality control by radioanalytical HPLC demonstrated in all cases labeling yields >98% in a >96% radiochemical purity. Hence, the resultant 111 In-SB3 and 111 In-SB4 radioligands were used without further purification in all subsequent experiments. A representative radiochromatogram of analysis of labeling reaction product 111 In-SB4 is included in Figure 1b Figure 2b). In both cases, the bulk of radioactivity remained bound to the cell-membrane with only a small portion internalizing into the cells, as consistent with a radioantagonist profile [11]. However, 111 In-SB4 showed much lower overall uptake in PC-3 cells (1.9 ± 0.5% of total added activity) vs. 111 In-SB3 (16.2 ± 0.8% of total added activity; p < 0.0001), further demonstrating the negative impact of the Gly 11 /DAla 11 -substitution on the interaction ability of the forming radiotracer with the GRPR. The two 111 In-SB3 and 111 In-SB4 radiotracers exhibited different metabolic stability in peripheral mouse blood. As revealed by HPLC analysis of blood samples collected from mice at 5 min after radioligand injection, the DAla 11 -substituted 111 In-SB4 was markedly more stable (80 ± 3% intact, n = 3) than non-modified 111 In-SB3 (56 ± 2% intact, n = 3; P<0.001), revealing the positive influence of the adopted structural intervention on metabolic stability; representative radiochromatograms for 111 In-SB3 and 111 In-SB4 are shown in Figure 3a, b, respectively. Radiochromatograms of HPLC analysis of mouse blood samples collected 5 min pi, of (a) 111 In-SB3 (55% intact radiotracer) or (b) 111 In-SB4 (77% intact radiotracer) without PA-coinjection; the respective radiochromatograms of (c) 111 In-SB3 (98.9% intact radiotracer), or (d) 111 In-SB4 (99.7% intact radiotracer) with PA-coinjection are also included; the tR of parent radiopeptide was determined by coinjection with the respective radioligand sample in the column (HPLC system 2) and is indicated here by the arrow.

In Vivo Comparison of 111
In-SB3 and 111 In-SB4

Stability of 111 In-SB3 and 111 In-SB4 in Healthy Mice
The two 111 In-SB3 and 111 In-SB4 radiotracers exhibited different metabolic stability in peripheral mouse blood. As revealed by HPLC analysis of blood samples collected from mice at 5 min after radioligand injection, the DAla 11 -substituted 111 In-SB4 was markedly more stable (80 ± 3% intact, n = 3) than non-modified 111 In-SB3 (56 ± 2% intact, n = 3; p < 0.001), revealing the positive influence of the adopted structural intervention on metabolic stability; representative radiochromatograms for 111 In-SB3 and 111 In-SB4 are shown in Figure 3a,b, respectively. The two 111 In-SB3 and 111 In-SB4 radiotracers exhibited different metabolic stability in peripheral mouse blood. As revealed by HPLC analysis of blood samples collected from mice at 5 min after radioligand injection, the DAla 11 -substituted 111 In-SB4 was markedly more stable (80 ± 3% intact, n = 3) than non-modified 111 In-SB3 (56 ± 2% intact, n = 3; P<0.001), revealing the positive influence of the adopted structural intervention on metabolic stability; representative radiochromatograms for 111 In-SB3 and 111 In-SB4 are shown in Figure 3a, b, respectively. Radiochromatograms of HPLC analysis of mouse blood samples collected 5 min pi, of (a) 111 In-SB3 (55% intact radiotracer) or (b) 111 In-SB4 (77% intact radiotracer) without PA-coinjection; the respective radiochromatograms of (c) 111 In-SB3 (98.9% intact radiotracer), or (d) 111 In-SB4 (99.7% intact radiotracer) with PA-coinjection are also included; the tR of parent radiopeptide was determined by coinjection with the respective radioligand sample in the column (HPLC system 2) and is indicated here by the arrow.  Radiochromatograms of HPLC analysis of mouse blood samples collected 5 min pi, of (a) 111 In-SB3 (55% intact radiotracer) or (b) 111 In-SB4 (77% intact radiotracer) without PA-coinjection; the respective radiochromatograms of (c) 111 In-SB3 (98.9% intact radiotracer), or (d) 111 In-SB4 (99.7% intact radiotracer) with PA-coinjection are also included; the t R of parent radiopeptide was determined by coinjection with the respective radioligand sample in the column (HPLC system 2) and is indicated here by the arrow.
It is interesting to note that coinjection of the NEP-inhibitor PA stabilized both 111 In-SB3 and 111 In-SB4 in mouse circulation (Figure 3c,d, respectively; p < 0.001), revealing NEP as the major degrading protease in vivo. Hence, the in-situ NEP-inhibition strategy turned out to be more efficacious in metabolically stabilizing the radioligand than the structural modification approach, pursued herein via Gly 11 /DAla 11 -replacement. It is interesting to note that coinjection of the NEP-inhibitor PA stabilized both 111 In-SB3 and 111 In-SB4 in mouse circulation (Figure 3c, d, respectively; P<0.001), revealing NEP as the major degrading protease in vivo. Hence, the in-situ NEP-inhibition strategy turned out to be more efficacious in metabolically stabilizing the radioligand than the structural modification approach, pursued herein via Gly 11 /DAla 11 -replacement.  Both tracers showed rapid blood clearance with quite similar distribution patterns in all non-GRPR-expressing tissues at all time points. The radioactivity cleared from the body of mice via the kidneys and the urinary tract. However, significant differences in the uptake of the two tracers were evident in the PC-3 tumors and the tissues physiologically expressing the GRPR, such as the pancreas. . Data is expressed as average ± sd %ID/g, n = 4 and plots were drawn in the same scale of uptake for easy comparison; statistically significant differences are indicated by ** or ++ between controls and block or PA groups, respectively, at 4 h pi, as estimated by one-way ANOVA with Tukey's post-hoc analysis. Bl = blood, Li = liver, He = heart, Ki = kidneys, St = stomach, In = intestine, Sp = spleen, Mu = muscle, Lu = lungs, Fe = femur, Pa = pancreas and Tu = PC-3 tumor.
Both tracers showed rapid blood clearance with quite similar distribution patterns in all non-GRPR-expressing tissues at all time points. The radioactivity cleared from the body of mice via the kidneys and the urinary tract. However, significant differences in the uptake of the two tracers were evident in the PC-3 tumors and the tissues physiologically expressing the GRPR, such as the pancreas. Thus, tumor uptake of non-modified 111 In-SB3 were 8.8 ± 3.0%ID/g at 4 h pi declining to 4.4 ± 0.3%ID/g at 24 h pi, whereas the respective values for 111 In-SB4 were 3.0 ± 0.8%ID/g (p < 0.01) and 0.9 ± 0.1%ID/g (p < 0.0001). Likewise, 111 In-SB3 exhibited higher pancreatic uptake compared to 111 In-SB4 both at 4 h pi (3.5 ± 0.4%ID/g and 1.6 ± 0.8%ID/g, respectively; p < 0.01) and at 24 h (0.6 ± 0.1%ID/g and 0.1 ± 0.0%ID/g, respectively; p < 0.001). It should be noted that tumor and pancreas values achieved by 111 In-SB4 at 4 h pi were significantly reduced after coinjection of excess [Tyr 4 ]BBN, suggesting a GRPR-mediated uptake (p < 0.001). For 111 In-SB3 however, no significant change was found adopting the one way ANOVA test with Tukey's post-hoc analysis due to the wide variance of values and the unpaired two tailed Student's t test was additionally conducted revealing highly significant differences (p < 0.001).
The effect of metabolic stabilization of both radiotracers during NEP-inhibition was studied in separate animal groups co-injected with a 300 µg dose of PA. As a result, the tumor uptake was markedly improved for both radioligands, but was most affected in the case of unmodified 111 In-SB3 compared to the more in vivo robust 111 In-SB4. Thus, in the case of 111 In-SB3 a 4.3-fold enhancement of tumor uptake was accomplished at 4 h pi by PA coinjection (38.3 ± 7.9%ID/g -PA -vs. 8.8 ± 3.0%ID/g -control; p < 0.001) with only 2.4-fold improvement achieved for 111 In-SB4 (7.3 ± 0.3%ID/g -PA -vs. 3.0 ± 0.8%ID/g -control; p < 0.001). Likewise, higher enhancement was observed in the uptake of 111 In-SB3 compared to 111 In-SB4 after PA-coinjection in all tissues with physiological GRPR-expression, especially in the mouse pancreas (33.1 ± 6.2%ID/g vs. 4.9 ± 0.7%ID/g, respectively; p < 0.001).

Discussion
Radiotracers based on GRPR-antagonists have been lately attracting much attention in nuclear medicine compared to agonists, largely because of their higher inherent biosafety [11,12]. First radioligands developed for nuclear medicine purposes were based on linear native BBN-/GRP-sequences, which, following systemic administration and subsequent GRPR-binding, internalized into target cells while at the same time activating the receptor and eliciting adverse effects [2][3][4]. In contrast, radiolabeled GRPR-antagonists, that neither internalize nor activate the GRPR upon binding, turned out to be more appropriate for human use [20]. Moreover, recent studies have shown that radiolabeled GRPR-antagonists unexpectedly achieved superior tumor targeting and better pharmacokinetics compared to their agonist counterparts [12]. It should be noted that GRPR-antagonists are actually synthetic compounds designed to inhibit the action of agonists in the body, preferably for long periods of time. Accordingly, they are tailored to better resist in vivo degradation through strategic chemical changes of their structure, as for example by introduction of unnatural amino acids, reduction or methylation of amide bonds and other means [20]. It is therefore, reasonable to assume that radiolabeled GRPR-antagonists for nuclear medicine use would also be more stable than agonists.
Following this rationale, we have been exploring the impact of in vivo metabolic stability on the biological profile of radiolabeled GRPR-antagonists developed by our group. In one set of compounds, suitable chelators were coupled at the N-terminal of the potent GRPR-antagonist [DPhe 6 ,Leu 13 -NHEt]BBN(6-13) via different linkers allowing for labeling with 99m Tc, 68 Ga, 111 In and 177 Lu [15,16,22]. As expected, the resultant radiotracer uptake in PC-3 xenografts in mice was found to depend on in vivo stability. The latter was influenced by structural changes of the radiotracer not only on the peptide chain, but also the linker or the radiometal chelate. For example, in a small library of 99m Tc-DB1 mimics [22], the DAla 11 -substituted analog was the most in vivo robust member, in agreement with the higher stability reported for similarly substituted DAla 11 /Gly 11 -BBN-like or DAla 24 /Gly 24 -GRP-like analogs [21]. In another example, it was the radiometal/radiometal-chelate affecting in vivo stability and tumor uptake. Thus, by switching radiometal from 68 Ga to 111 In (or 177 Lu) in SB3 we observed a drastic drop of metabolic stability translating into impaired targeting of 111 In-SB3 PC-3 tumors in mice compared to 68 Ga-SB3 [17,18].
In an effort to identify the major protease(s) involved in the fast in vivo degradation of radiolabeled [DPhe 6 ,Leu 13 -NHEt]BBN(6-13) analogs, we have co-injected specific protease-inhibitors along with the radiopeptide and applied HPLC analysis to monitor potential changes in radiometabolite patterns induced in mouse blood [17,23]. Of great significance is the finding that coinjection of the NEP-inhibitor PA stabilized most GRPR-radioantagonists in vivo, leading to remarkable enhancement of radiolabel uptake in the implanted PC-3 tumors. The above results established NEP as a leading cause of in vivo catabolism of studied radioligands. Furthermore, the innovative concept of enhancing tumor targeting via in-situ altering the immediate milieu of the radiopeptide on its way to the target instead of modifying its structure warrants further investigation.
In the present study we first introduced SB4, a single DAla 11 /Gly 11 -substution derivative of SB3. This modification was shown to enhance radiotracer stability without greatly impairing other biological features (e.g. receptor affinity or in vivo tumor targeting) in a series of 99m Tc-DB1 mimics [22]. In the present study however, we observed a ≈2.5-fold drop of affinity to the GRPR by comparing SB3 (IC 50 = 4.6 ± 0.3 nM, n=3) to SB4 (IC 50 = 11.2 ± 1.1 nM, n = 3; p < 0.001). We have not compared the receptor affinities of the In-metalated species, given that the type and position of the forming metal-chelate was identical in the two analogs, which differed only at position 11 of the peptide chain. Furthermore, the overall specific uptake of the respective 111 In-radioltracers in PC-3 cells after 1 h incubation at 37 o C significantly dropped from 16.2 ± 0.8% ( 111 In-SB3) to 1.9 ± 0.5% ( 111 In-SB4) of total added activity (p < 0.0001). This drop followed the same trend of receptor affinity deterioration observed in the unlabeled species and further corroborated the negative effect of DAla 11 /Gly 11 -substution on the in vitro interaction of SB4/ 111 In-SB4 with the GRPR (Figure 2). However, the in vivo stability of 111 In-SB4 in peripheral mouse blood (80 ± 3% intact, n = 3) was found significantly increased compared to 111 In-SB3 (56 ± 2% intact, n = 3; p < 0.001) (Figure 3). It is interesting to note that the pursued DAla 11 /Gly 11 -replacement, albeit improving in vivo stability, negatively influenced the interaction with the GRPR, eventually translating into poorer uptake of 111 In-SB4 in PC-3 tumors in mice (Figure 4). At both the 4 h and the 24 h pi time intervals unmodified 111 In-SB3 (8.8 ± 3.0%ID/g and 4.4 ± 0.3%ID/g, respectively) showed superior uptake compared to 111 In-SB4 (3.0 ± 0.8%ID/g and 0.9 ± 0.1%ID/g, respectively; p < 0.01), revealing the negative impact of this modification on the overall performance of resulting radiotracer. This unfavorable result was found to be surprisingly pronounced in the 111 In-SB3/ 111 In-SB4 pair compared to what was expected from previous observations on other analogs [21] and highlights the fact that extrapolation of structural changes across molecules may lead to different or even opposite effects.
Aiming at assessing the impact of the alternative NEP-inhibition approach on the in vivo performance of 111 In-SB3 and 111 In-SB4, we have co-injected PA and studied the changes induced on the in vivo stability and tumor uptake of both radioligands at 4 h pi. Firstly, we observed an impressive full stabilization of both radiotracers in mice blood ( Figure 3). Most importantly, this stabilization translated into remarkably enhanced tumor values for both non-modified 111 In-SB3 (38.3 ± 7.9%ID/g; p < 0.001) and the DAla 11 /Gly 11 -modified 111 In-SB4 (7.3 ± 0.3%ID/g; p < 0.001). It is clearly evident that in-situ NEP-inhibition is a direct and more effective strategy than structural intervention to improve tumor targeting in this pair of GRPR-radioantagonists.
However, several issues need to be addressed first before proposing this exciting approach for clinical application of GRPR-radioantagonists or of a broader spectrum of peptide analogs. Thus, protease-inhibitor administration as such or in combination with the radiopeptide in question has to be proven safe for patients. Next, the increased costs of extended toxicology tests and production of GMP-grade compounds for clinical testing should be taken into account. Eventually, it is expected that approval by ethical committees will become more challenging when injection of combinations of substances is planned. On the other hand, the in situ protease-inhibition concept, once clinically established for a certain radiopeptide, may be more easily extrapolated thereafter to a broader spectrum of radiopeptide candidates for clinical translation. Undoubtedly, this methodology provides a powerful preclinical tool to: (i) identify enzymes truly involved in the degradation of biodegradable peptide radioligands, (ii) reveal cleavage sites and (iii) indicate the range of feasible improvements for a particular tumor-target model induced by radiopeptide stabilization.
Radioiodination of [Tyr 4 ]BBN was performed using 125 I ([ 125 I]NaI in 0.1 N NaOH (pH 12-14) provided by Perkin Elmer) according to the chloramine-T methodology, as previously described [22]. Reversed-phase HPLC was performed on a Waters Chromatograph based on a 600E multisolvent delivery system coupled to a Waters 2998 photodiode array detector and a Gabi gamma-detector (Raytest, RSM Analytische Instrumente GmbH). Data processing and chromatography were controlled by the Empower Software (Waters, Vienna, Austria). For quality control 2 µL aliquots of the radiolabeling solution were quenched with 28 µL of an acetate buffered solution of DTPA (1 mM, pH 4.6) and a Symmetry Shield RP18 cartridge column (5 µm, 3.9 mm × 20 mm, Waters) was used for analyses. Solutes were eluted with 0.1% TFA/MeCN applying a linear gradient starting from 0% MeCN and a 2% increase per min at 1 mL/min flow rate (system 1). The radiochemical labeling yield exceeded 98% and the radiochemical purity was >99%. Samples of 111 In-SB3 and 111 In-SB4 were analyzed before and after completion of all biological experiments.

Cell Lines and Culture
Human androgen-independent prostate adenocarcinoma PC-3 cells endogenously expressing the human GRPR (LGC Promochem, Teddington, UK) were used in the present study [24]. Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin, and kept in a controlled humidified atmosphere containing 5% CO 2 at 37 • C. Passages were performed weekly using a trypsin/EDTA (0.05%/0.02% w/v) solution. All culture media were purchased from Gibco BRL, Life Technologies and supplements were provided by Biochrom KG Seromed.

Competition Binding in PC-3 Cell-Membranes
Competition binding experiments against [ 125 I-Tyr 4 ]BBN were performed with SB3, SB4, or [Tyr 4 ]-BBN (reference) in PC-3 cell membranes, prepared as previously reported [25]. For the assay, triplicates per concentration point (concentration range: 10 −13 -10 −6 M) of each test peptide were incubated together with the radioligand (~40,000 cpm per assay tube, at a 50 pM concentration) in PC-3 cell-membrane homogenates in a total volume of 300 µL binding buffer (BB, 50 mM HEPES pH 7.4, 1% BSA, 5.5 mM MgCl 2 , 35 µM bacitracin) for 1 h at 22 • C in an Incubator-Orbital Shaker (MPM Instr. SrI, Italy). Binding was interrupted by ice-cold washing buffer (WB, 10 mM HEPES pH 7.4, 150 mM NaCl) and rapid filtration (Whatman GF/B filters presoaked in BB) on a Brandel Cell Harvester (Adi Hassel Ing. Büro, Munich, Germany). Filters were washed with ice-cold WB and counted in an automatic well-type gamma counter (NaI(Tl) 3´´-crystal, Cobra Packard Auto-Gamma 5000 series instrument). The half maximal inhibitory concentration (IC 50 ) values were calculated using nonlinear regression according to a one-site model applying the PRISM 2 program (Graph Pad Software, San Diego, CA) and represent the mean±sd from three independent experiments performed in triplicate.

Internalization Assay in PC-3 Cells
The overall cell association -internalization of 111 In-SB3 and 111 In-SB4 was assessed in PC-3 cells. Briefly, PC-3 cells were seeded in six-well plates (~1 × 10 6 cells per well) 24 h before the experiment. Approximately 50,000 cpm of either 111 In-SB3 or 111 In-SB4 (corresponding to 250 fmol total peptide in 150 µL of 0.5% BSA/PBS) was added alone (total) or in the presence of 1 µM [Tyr 4 ]BBN (non-specific). Cells were incubated at 37 • C for 1 h and incubation was interrupted by placing the plates on ice, removing the supernatants and rapid rinsing with ice-cold 0.5% BSA/PBS. Cells were then treated 2 × 5 min with acid wash buffer (2 × 0.6 mL, 50 mM glycine buffer pH 2.8, 0.1 M NaCl) at room temperature and supernatants were collected (membrane-bound fraction). After rinsing with 1 mL chilled 0.5% BSA/PBS, cells were lyzed by treatment with 1 N NaOH (2 × 0.6 mL) and lysates were collected (internalized fraction). Sample radioactivity was measured in the γ-counter and total cell-associated (internalized+membrane bound) radioactivity was determined vs. total added activity. Results represent the average values ± sd of four experiments performed in triplicate.

In Vivo Stability Tests
For stability experiments (approved protocol # 1609, Prefecture of Attica), healthy male Swiss albino mice (30 ± 5 g, NCSR "Demokritos" Animal House Facility) were used. The radioligand, 111 In-SB3 or 111 In-SB4, was injected as a 100 µL bolus (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) MBq, 3 nmol total peptide) in the tail vein together with injection solution (100 µL; control) or with a phosphoramidon (PA)-solution (100 µL injection solution containing 300 µg PA). Animals were euthanized and blood (0.5-1 mL) was directly withdrawn from the heart and transferred in a pre-chilled EDTA-containing Eppendorf tube on ice. Blood samples were centrifuged for 10 min at 2000 g/4 • C and plasma was collected. After addition of an equal volume of ice-cold MeCN the mixture was centrifuged for 10 min at 15,000 g/4 • C. The supernatant was concentrated under a N 2 -flux at 40 • C to 0.05-0.1 mL, diluted with saline (0.4 mL), filtered through a 0.22 µm Millex GV filter (Millipore, Milford, USA) and analyzed by RP-HPLC. The Symmetry Shield RP18 (5 µm, 3.9 mm × 20 mm) column was eluted at a flow rate of 1.0 mL/min with the following linear gradient (system 2): 0% B at 0 min to 10% B in 10 min and then in 40 min to 30% B; A = 20 mM ammonium acetate and B = MeCN. The t R of the intact radiopeptide was determined by coinjection with the 111 In-SB3 or 111 In-SB4 reference in the HPLC.

Induction of PC-3 Xenografts in SCID Mice
A suspension containing freshly harvested human PC-3 cells (≈150 µL of a ≈1.2 × 10 7 cells) was subcutaneously injected in the flanks of female SCID mice (15 ± 3 g, six weeks of age at the day of arrival, NCSR "Demokritos" Animal House Facility). The animals were kept under aseptic conditions and 4 weeks later developed well-palpable tumors (80-200 mg) at the inoculation sites (approved protocol # 1610, Prefecture of Attica).