68Ga-TRAP-(RGD)3 Hybrid Imaging for the In Vivo Monitoring of αvß3-Integrin Expression as Biomarker of Anti-Angiogenic Therapy Effects in Experimental Breast Cancer

Objectives To investigate 68Ga-TRAP-(RGD)3 hybrid imaging for the in vivo monitoring of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer. Materials and Methods Human breast cancer (MDA-MB-231) xenografts were implanted orthotopically into the mammary fat pads of n = 25 SCID mice. Transmission/emission scans (53 min to 90 min after i.v. injection of 20 MBq 68Ga-TRAP-(RGD)3) were performed on a dedicated small animal PET before (day 0, baseline) and after (day 7, follow-up) a 1-week therapy with the VEGF antibody bevacizumab or placebo (imaging cohort n = 13; therapy n = 7, control n = 6). The target-to-background ratio (TBR, VOImaxtumor/VOImeanmuscle) served as semiquantitative measure of tumor radiotracer uptake. Unenhanced CT data sets were subsequently acquired for anatomic coregistration and morphology-based tumor response assessments (CT volumetry). The imaging results were validated by multiparametric ex vivo immunohistochemistry (αvß3-integrin, microvascular density–CD31, proliferation–Ki-67, apoptosis–TUNEL) conducted in a dedicated immunohistochemistry cohort (n = 12). Results 68Ga-TRAP-(RGD)3 binding was significantly reduced under VEGF inhibition and decreased in all bevacizumab-treated animals (ΔTBRfollow-up/baseline: therapy -1.07±0.83, control +0.32±1.01, p = 0.022). No intergroup difference in tumor volume development between day 0 and day 7 was observed (Δvolumetherapy 134±77 μL, Δvolumecontrol 132±56 μL, p = 1.000). Immunohistochemistry revealed a significant reduction of αvß3-integrin expression (308±135 vs. 635±325, p = 0.03), microvascular density (CD31, 168±108 vs. 432±70, p = 0.002), proliferation (Ki-67, 5,195±1,002 vs. 7,574±418, p = 0.004) and significantly higher apoptosis (TUNEL, 14,432±1,974 vs. 3,776±1,378, p = 0.002) in the therapy compared to the control group. Conclusions 68Ga-TRAP-(RGD)3 hybrid imaging allows for the in vivo assessment of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer.


Introduction
Hybrid imaging has evolved into an essential diagnostic modality for state-of-the-art tumor staging in modern clinical oncology, allowing for a non-invasive tumor characterization on the morphological, functional, and molecular level [1]. While 18 F-fluorodeoxyglucose (FDG) remains the most widely applied tracer in positron emission tomography (PET), the arsenal of diagnostic radiotracers has been expanded significantly over the last decade [2][3][4][5]. The development of radiolabeled arginylglycylaspartic acid (RGD) tracers has enabled the specific targeting of α v ß 3 -integrin, an endothelial and tumor cell receptor with a significant role in neoangiogenesis [6]. Integrins are a family of transmembrane proteins interacting with a variety of ligands from the extracellular matrix [7]. Mediating cell adhesion and extracellular-tointracellular signaling pathways, they have been identified as key players in tumor progression and metastasis [7]. α v ß 3 -integrin is overexpressed by angiogenic endothelium and tumor cells and is involved in various angiogenic signaling cascades including the Vascular Endothelial Growth Factor (VEGF) pathway [8,9]. VEGF augments endothelial cell migration and adhesion by indirect, receptor-mediated α v ß 3 -integrin activation [9]. Vice versa, inhibition of VEGF was shown to significantly suppress tumor α v ß 3 -integrin expression in line with a significant reduction of microvascular density [10][11][12]. α v ß 3 -integrin is therefore proposed a marker of angiogenic activity and a target structure for the in vivo imaging of tumor neoangiogenesis [8]. Inhibition of the VEGF pathway using the VEGF antibody bevacizumab as either single or combination therapy has become an established clinical anti-angiogenic treatment regimen applied in various tumor entities, including non-small-cell lung, colorectal, and breast cancer [13]. In vivo, intact murine VEGF is not recognized by bevacizumab, while cleaved murine VEGF can be recognized by this antibody in Western Blots [14].
However, a recent study confirmed the anti-VEGF activity of bevacizumab in murine models [15]. Accordingly, Wang and colleagues recently demonstrated that bevacizumab exhibits anti-VEGF activity in human colorectal carcinoma xenografts in mice [16].
The aim of the present study was to investigate the applicability of 68 Ga-TRAP-(RGD) 3 -PET/CT for the quantitative and longitudinal in vivo imaging of a low α v ß 3 -integrin expressing human breast cancer model. We therefore hypothesized that 68 Ga-TRAP-(RGD) 3 -PET/CT allows for the in vivo monitoring of α v ß 3 -integrin expression as biomarker of anti-angiogenic therapy effects in orthotopic MDA-MB-231 breast cancer xenografts in mice treated with the VEGF antibody bevacizumab over the course of one week.

Materials and Methods
The study was approved by the Government of Upper Bavaria Committee of Animal Research (Gz.: 55.2-1-54-2532-82-2014). It was conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All efforts were taken to minimize animal suffering. The mice were kept in individually-ventilated cages (n = 4 per cage; constant temperature 26˚C, relative air humidity 65%, 18 room air changes per hour, light-dark-cycle 12 hours). Animals were nourished ad libitum with water and dedicated small animal nutrition. In order to assure environmental enrichment, nest boxes and nestlets were provided. After tumor cell inoculation, animals were monitored once a day and tumor growth was determined using a caliper. Weight and tumor growth were noted on a daily basis. Abnormal inactivity was considered to indicate pain and was treated by analgesia (buprenorphin 0.5 mg/kg body weight s.c.). Imaging was performed under isoflurane anesthesia (2.5% in 1.0 L 100% O 2 / min for induction and 1.5% in 1.0 L 100% O 2 /min for maintenance, respectively). The animals were euthanized if one of the following conditions were present: no tumor growth, tumor size >1.5 cm, ulceration of tumors, weight loss >20%, apathy, remarkable defense reaction when tumors were palpated, remarkable breathing difficulties, lameness, and a non-physiologic body posture. Animals were euthanized under inhalation anesthesia (5.0% isoflurane in pure oxygen) by intracardial injection of a saturated potassium chloride solution.

Tumor model and study setup
In a previous study, we confirmed the low tumor cell α v ß 3 -integrin expression of the investigated MDA-MB-231 (ATCC 1 HTB-26™, Manassas, VA) cell line by flow cytometry experiments [10]. 3 x 10 6 MDA-MB-231 cells were resuspended in 0.05 mL of 1:1 Matrigel™ (BD Biosciences, San Jose, CA) / phosphate-buffered saline (PBS) solution and injected into the mammary fat pads of n = 25 Severe Combined Immunodeficiency (SCID) mice (age: 7 to 8 weeks; Harlan Laboratories Inc., Indianapolis, IN). After reaching a tumor size of 0.5 cm, the animals were assigned to either the imaging (n = 13) or the immunohistochemistry cohort (n = 12).
Imaging cohort (n = 13): The animals were randomly assigned to either the therapy (n = 7) or the control group (n = 6). After the baseline scan (day 0), animals were treated daily with either bevacizumab (therapy group; Avastin 1 , Hoffmann-La Roche AG, Basel, Switzerland; 5 mg/kg body weight) or a volume-equivalent placebo solution (control group; 0.9% sodium chloride) for one week (days 1 to 6). The therapeutic agents were injected into the peritoneal cavity using a 27-gauge needle. On day 7, the follow-up imaging was performed.
Immunohistochemistry cohort (n = 12): In order to exclude competitive blocking effects between the primary anti-α v ß 3 -integrin antibody used for the immunohistochemical staining and receptor-bound 68 Ga-TRAP-(RGD) 3 , the ex vivo immunohistochemical validation was conducted in a separate animal cohort. Analogously to the imaging cohort, mice were randomly assigned to either the therapy (n = 6) or the control group (n = 6), and the abovedescribed one-week therapy regimen was carried out accordingly. On day 7, the animals were sacrificed and tumors were explanted and cut in half in order to undergo the immunohistochemical workup. One half was cryopreserved in liquid nitrogen at -196˚C for 1 min and then stored at -80˚C, while the other half was fixed in formalin.  Ga-TRAP-(RGD) 3 was synthesized as described previously [20][21][22] with slight modifications. Briefly, 25 μg of the precursor (AVEBETRIN, SCINTOMICS GmbH, Fürstenfeldbruck, Germany) dissolved in 288 μL 1 M Na-acetate were labelled with 300-400 MBq 68 GaCl 3 eluted in 2 mL 0.1 N HCl from a 68 Ge/ 68 Ga-generator (IGG100-50M, Eckert & Ziegler Radiopharma GmbH, Berlin, Germany). After heating for 10 min at 90˚C followed by purification via C18 cartridge the final injection solution was prepared by adjusting the pH with phosphate buffer to 7.2. Radiochemical purity of each preparation was >98% determined by radio-high performance liquid chromatography. The in vivo properties of 68 Ga-TRAP-(RGD) 3 in mice were characterized previously, including blocking, metabolite, and biodistribution studies [20].

Imaging protocol
Imaging was performed under isoflurane anesthesia (2.5% in 1.0 L 100% O 2 /min for induction and 1.5% in 1.0 L 100% O 2 /min for maintenance, respectively). 68 Ga-TRAP-(RGD) 3 (20 MBq in a total volume of 100 μL) was injected via the lateral tail vein. The animals were then placed within the aperture of a dedicated small animal PET (Inveon Dedicated PET, Preclinical Solutions, Siemens Healthcare Molecular Imaging, Knoxville, TN) and a 7-minute transmission scan employing a rotating 57 Co source was started 53 min after injection. Transmission scans were acquired for scatter and attenuation correction. Subsequently, an emission scan (threedimensional listmode acquisition) was initiated from 60 to 90 min after radiotracer injection.  3 -PET/CT baseline scan (day 0), animals of the imaging cohort were treated daily with either bevacizumab (therapy group) or a volume-equivalent placebo solution (control group) for 6 days. 68 Ga-TRAP-(RGD) 3 -PET/CT followup scan was performed on day 7. Animals of the immunohistochemistry cohort were randomized to a therapy and a control group and treated analogously to the imaging cohort with either bevacizumab (therapy group) or placebo (control group) for 6 days. On day 7, the animals of the immunohistochemistry cohort were sacrificed and the tumors were explanted in order to undergo immunohistochemical workup with regard to α v ß 3 -integrin expression, microvascular density (CD31), proliferation (Ki-67), and apoptosis (TUNEL). An unenhanced CT (Somatom Force, Siemens Healthcare, Erlangen, Germany; 35 mAs, 100 kV, slice thickness 0.6 mm; reconstructed in axial, coronal, and sagittal planes) data set in identical animal position relative to the PET scan was acquired, serving for anatomic coregistration, tumor localization, and morphology-based assessments of tumor response (CT volumetry at day 0 and day 7).

Data post-processing and analysis
The PET data sets were analyzed using dedicated post-processing software (Inveon Acquisition Workplace, Siemens Medical Solutions, Knoxville, TN). Data sets were reconstructed as static images using OSEM 3D and MAP 3D algorithms with 4 and 32 iterations, respectively. PET image parameters were as follows: 256 x 256 matrix, 159 slices, slice thickness 0.796 mm, zoom factor 100%, spatial resolution 1.5 mm. PET data sets were normalized and corrected for attenuation and scatter as well as for randoms, dead time, and decay. CT Volumes-of-Interest (VOI) were superimposed on PET images to determine corresponding volumes for quantification of the PET signal. Fig 2 provides details on VOI selection. The target-to-background ratio (TBR, Volume-of-Interest (VOImax tumor /VOImean muscle ) was determined as semiquantitative measure of tumor radiotracer accumulation before (day 0) and after treatment (day 7). As the radiotracer uptake is normalized to a reference tissue (muscle), TBR is, compared to the standardized uptake value, less sensitive to inter-and intraindividual variations in mouse weight, radiotracer blood clearance, and radiotracer dose. Tumor regions with spillover from the urinary bladder were excluded from the analysis. Tumor volumes [μL] at baseline and follow-up were measured by CT volumetry using the above-mentioned post-processing software. PET and CT data sets were analyzed coregistered as side-by-side examinations. unenhanced CT with tumor (green, asterisk) and muscle (violet, arrow) VOIs; C: fused PET and CT data sets; D: fused PET and CT data sets with tumor (pink, asterisk) and muscle (violet, arrow) VOIs. Tumor and muscle VOIs selected on the unenhanced CT (B) were superimposed on the PET data sets (D) to allow for a quantification of the PET signal. Note that tumor regions with significant signal spillover from the urinary bladder were excluded from the quantitative analysis (D).

Validation of 68 Ga-TRAP-(RGD) 3 binding specificity in MDA-MB-231 xenografts using autoradiography and immunofluorescence imaging
Target specificity of 68 Ga-TRAP-(RGD) 3 was validated in two steps. First, we conducted autoradiography experiments of radiotracer-incubated tumor sections with and without blocking of the α v β 3 -integrin receptor using a specific antibody (step 1). Second, the specific α v β 3 -integrin receptor blocking in the autoradiography experiments was confirmed by secondary immunofluorescence stainings (step 2).
Step 1: For autoradiography, tumor cryosections (6 μm) mounted on glass slides were unfrozen and dried at room temperature for 2 h. Half number of the slide-mounted tumor sections were washed with binding buffer (Tris-HCl 50 mM, pH 7.4) and air-dried for 1.5 h. For the blocking experiments, the remaining half of the slide-mounted tumor sections were incubated with the primary anti-α v β 3 -integrin antibody (LM609: 1:20, Merck Millipore, Darmstadt, Germany) for 1 h, then washed with binding buffer (Tris-HCl 50 mM, pH 7.4) and air-dried for 1.5 h. Subsequently, all slides (without and with α v β 3 -integrin receptor blocking) were incubated with 68 Ga-TRAP-(RGD) 3 (5 MBq in 50 mL Tris-HCl 50 mM), washed with binding buffer, and air-dried for 1 h. The slides were then placed on autoradiography imaging plates (BAS cassette2 2025 imaging plates, Fujifilm, Tokyo, Japan) for 2.5 h, and imaging plates were subsequently scanned by autoradiography (CR-35-BIO, 25 μm resolution, Elysia-Raytest GmbH, Straubenhardt, Germany). Image analysis was performed using dedicated post-processing software (AIDA image analyzing software V4.50, Elysia-Raytest GmbH, Straubenhardt, Germany). Signal intensity (intensity per area; background subtraction) was extracted in adjacent blocked and unblocked tumor sections. The ratio between unblocked and blocked tumor sections was calculated.

Statistical analysis
The statistical analysis was performed using SPSS 23 for Windows (IBM Corp., Armonk, NY). Descriptive statistical data were expressed as arithmetic means with standard deviations at 95% confidence intervals. Wilcoxon/Mann-Whitney U tests were applied for intra-and intergroup comparisons of the quantitative parameters. Linear correlations between non-normally distributed variables were assessed by Spearman's test. Autoradiographic signal intensities in unblocked and adjacent blocked tumor sections were compared using a paired student's t test. Statistical significance was assumed for p-values <0.05.

Tumor volume
Contrary to the changes in α v ß 3 -integrin expression detected by 68 Ga-TRAP-(RGD) 3 -PET, there was no significant intergroup difference in tumor volume development over the course of the experiment (Δvolume therapy 134±77 μL, Δvolume control 132±56 μL; p = 1.000). There was no significant difference in baseline tumor volumes between therapy and control group (volume therapy-baseline 289±162 μL, volume control-baseline 248±89 μL; p = 0.628). No significant correlation between tumor volumes and TBR values at baseline and follow-up was observed (Spearman's ρ -0.23; p = 0.261). Table 1 displays individual tumor volumes before and after treatment.

Discussion
In this experimental study, 68 Ga-TRAP-(RGD) 3 -PET/CT allowed for the non-invasive monitoring of anti-angiogenic effects in a low α v ß 3 -integrin expression orthotopic human breast cancer model in mice, generating complementary and additional information to morphologybased tumor response assessments. 68 Ga-TRAP-(RGD) 3 binding was significantly reduced following VEGF inhibition, analogously to the significant suppression of α v ß 3 -integrin expression observed in the immunohistochemistry cohort. In untreated animals, 68 Ga-TRAP-(RGD) 3 uptake increased over the course of the experiment and α v ß 3 -integrin expression was found to be significantly higher.
Our observations are in accordance with a previous study investigating a 68 Ga-labeled pegylated RGD dimer probe for monitoring the early anti-angiogenic effects of an endostatin therapy in heterotopic human lung cancer xenografts in mice [23]. Validated by ex vivo immunohistochemistry, the authors found the RGD radiotracer uptake to be significantly reduced following anti-angiogenic therapy, reflecting tumor response significantly earlier than  [12]. Despite a reduced α v ß 3 -integrin expression under VEGF inhibition, they observed an increased binding of 68 Ga-NODAGA-RGD in the investigated A-431 xenografts. Rylova et al. therefore concluded that RGD radiotracer uptake might not necessarily reflect the changes in α v ß 3 -integrin expression on the molecular level. They hypothesized that bevacizumab may activate α v ß 3 -integrin, causing a higher affinity to 68 Ga-NODAGA-RGD and consequently increased radiotracer uptake in vivo. Assuming a highaffinity state of α v ß 3 -integrin however, one would expect an increased binding of the primary anti-α v ß 3 -integrin antibody used in the immunohistochemical stainings and consequently false-high α v ß 3 -integrin levels. However, in line with our results, the authors found a reduced α v ß 3 -integrin expression in the therapy group. In addition, VEGF is known to indirectly activate α v ß 3 -integrin [9]. It therefore becomes unlikely that VEGF inhibition also causes α v ß 3integrin activation. The validation experiments in the present study revealed that binding of the primary anti-α v ß 3 -integrin antibody used in the immunohistochemical analysis is effectively blocked by receptor-bound radiotracer. Our results suggest that intraindividual immunohistochemical validation, i.e., α v ß 3 -integrin staining of tumors previously exposed to RGD radiotracers, may only be of limited value. We therefore conclude that the assessment of the α v ß 3 -integrin receptor status parallel to RGD PET imaging requires a dedicated immunohistochemistry animal cohort. Competitive blocking effects between receptor-bound radiotracer and the α v ß 3 -integrin-specific antibody may also be a possible explanation for the contrary observations reported by Rylova et al. [12]. Bevacizumab is the humanized form of mouse VEGF Mab A4.6.1 and therefore shows no cross-reactivity with circulating murine VEGF [24]. Nevertheless, ex vivo immunohistochemistry revealed significant anti-angiogenic effects of bevacizumab in the investigated MDA-MB-231 human breast cancer model in mice, as indicated by the suppression of endothelial marker CD31 and α v ß 3 -integrin expression under therapy. In line with these findings, significant antiangiogenic effects of bevacizumab have also been observed in various other in vivo models of human cancer in murines [16,25,26]. Although not targeting murine VEGF, bevacizumab binds to tumor-derived human VEGF when applied in vivo [24]. As only human tumor cells and no stromal/endothelial cells were injected into the mammary fat pads of the nude mice in our study, the vasculature of the xenografts must be of murine origin [27]. However, as shown by Millauer et al. [28], the murine VEGF receptor 2 (= flk-1) shows a high affinity for human VEGF, resulting in upregulation of angiogenesis. Consequently, in vivo blocking of human VEGF using a human VEGF-specific monoclonal antibody (2C3) showed significant antiangiogenic effects in MDA-MB-231 human breast cancer xenograft-bearing mice [29]. Therefore, bevacizumab exhibits significant anti-angiogenic effects in human tumor xenograft models in which angiogenesis is also driven by human VEGF [24]. Accordingly, Liang et al. found that the response of human cancer xenografts to bevacizumab is highly dependent on the grade of host stroma invasion and the presence of stroma-derived VEGF [30]. The authors demonstrated that human tumor xenografts with low murine stroma invasion show a better response to bevacizumab than those with high murine stroma invasion, as the angiogenic response predominantly relies on tumor-derived, human VEGF [30]. Bevacizumab thus blocked the tumor-derived but not the host-derived VEGF in the MDA-MB-231 human breast Table 2. Immunohistochemistry cohort: individual values of immunohistochemical parameters for therapy and control group. Subsequent to follow-up imaging on day 7, tumors were explanted to undergo multiparametric immunohistochemistry with regard to α v ß 3 -integrin expression, microvascular density (CD31), proliferation (Ki-67), and apoptosis (TUNEL). Results are expressed as mean number of positively-stained microvessels (α v ß 3 -integrin and CD31) or cells (Ki-67 and TUNEL) per ten high-power fields at 200x magnification.

No. Group (T/C) a α v ß 3 -integrin CD31
Ki-67 TUNEL cancer xenografts investigated in the present study. This is in support of the defined study purpose, which was not to understand the host-specific VEGF response but to investigate a novel, α v ß 3 -integrin-targeted radiotracer for monitoring anti-angiogenic effects in a small animal model of human breast cancer. Our findings indicate that human, tumor-derived VEGF substantially triggers neovascularization in the investigated MDA-MB-231 human breast cancer model in mice and that a one-week bevacizumab monotherapy can be successfully applied to inhibit angiogenesis in this particular tumor model. However, it has to be acknowledged that residual, pro-angiogenic effects caused by uninhibited murine VEGF may still be present.
Our results confirm the applicability of 68 Ga-TRAP-(RGD) 3 in the investigated, low α v ß 3integrin expression tumor model. In a rodent model of myocardial infarction however, 68 Ga-TRAP-(RGD) 3 allowed for the assessment of α v ß 3 -integrin expression as imaging biomarker of myocardial repair, but, despite the higher target affinity in vitro [20], did not show a higher uptake than 68 Ga-NODAGA-RGD or 18 F-Galacto-RGD [31]. It can thus be postulated that the individual performance of different RGD radiotracers may vary depending on the investigated microstructures and tumor models. In the investigated MDA-MB-231 xenograft model, 68 Ga-TRAP-(RGD) 3 allowed for a robust and reproducible dual time-point in vivo imaging and generated moderate-to-good TBR.
α v ß 3 -integrin has recently gained attention as potential imaging target for the non-invasive in vivo characterization of the tumor microenvironment, and first clinical studies investigating radiolabeled RGD compounds in humans have been published [17,32]. In particular, the inhuman use of RGD-based radiotracers proved to be safe with tumor-specific uptake profiles   [6,33]. A recent clinical study even demonstrated the potential of combined functional parameters of 68 Ga-NOTA-RGD-and 18 F-FDG-PET/CT for the non-invasive molecular phenotyping of human breast cancer [34]. The combination of both radiotracers allowed for the distinction of breast cancer subtypes with regard to the receptor status (i.e., expression of the estrogen receptor, progesterone receptor, and Human Epidermal Growth Factor Receptor 2), although the underlying mechanisms still need to be identified. Therefore, the clinical translation and routine use of RGD-based hybrid imaging are likely to increase within the next years. The present study may provide a preclinical basis for future clinical studies investigating RGDbased hybrid imaging for monitoring therapy response to molecular cancer therapies. The expression of the α v ß 3 -integrin receptor and its distribution within the tumor (endothelial vs. cellular) should be assessed prior to clinical RGD-based hybrid imaging. This will help clinicians to interpret the imaging results as PET imaging is limited in its ability to distinguish between signal derived from tumor cells and signal derived from the tumor microenvironment. In the future, it will be of particular interest to investigate RGD PET-derived imaging biomarkers in correlation to clinical end points such as progression-free and overall patient survival. RGD-based hybrid imaging may allow for a non-invasive real-time molecular phenotyping of breast cancer under anti-angiogenic treatment, adding complementary molecular imaging biomarkers of therapy response to morphology-based and functional tumor response assessments. As part of multimodal imaging protocols, RGD-based hybrid imaging may help to identify patients who potentially benefit from targeted treatment regimens and therefore contribute to a tailored, personalized cancer therapy.

Limitations
We acknowledge several limitations of the study. First, 68 Ga-TRAP-(RGD) 3 showed a renal clearance with impaired visualization of lesions adjacent to the urinary system, which is a known limitation of RGD radiotracers [6]. Accordingly, tumor regions with spillover from radiotracer in the urinary bladder were not included for the analysis. However, this is a common difficulty with many radiolabeled peptides, especially when labeled with short half-life radionuclides. Second, RGD radiotracers are small molecules with potential extravasation and may therefore also bind to α v ß 3 -integrin expressed by tumor cells. It cannot be quantified to what degree the RGD radiotracer is bound by the endothelium and to what degree the RGD radiotracer is bound by the tumor cells. However, we were able to show that α v ß 3 -integrin expression is predominantly reserved to the endothelium in the investigated tumor model and that the MDA-MB-231 tumor cells only exhibit marginal α v ß 3 -integrin expression [10]. Thus, our experiments confirmed that there is no relevant α v ß 3 -integrin expression in the extravascular compartment. Nevertheless, subtle enhanced permeability and retention-mediated effects as well as marginal radiotracer binding to the tumor cells cannot be completely excluded. However, these would be present to the same degree in both therapy and control group with no significant impact on whole-tumor TBR. Third, although overexpressed by angiogenic endothelium, α v ß 3 -integrins are expressed ubiquitously, e.g., in the gut and the liver, hence compromising the assessment of tumor TBR by elevated background signal and spillover [6]. Therefore, the respective tumor VOI needs to be selected accordingly. Fourth, the investigation of additional α v ß 3 -integrin-targeted radiotracers would allow for a side-byside comparison of binding specificity and tumor signal in the examined MDA-MB-231 model of breast cancer.

68
Ga-TRAP-(RGD) 3 proved to be applicable for the longitudinal in vivo monitoring of α v ß 3integrin expression as surrogate of neoangiogenesis in the investigated MDA-MB-231 xenografts in which α v ß 3 -integrin expression remains predominantly reserved to the endothelium. 68 Ga-TRAP-(RGD) 3 allowed for a robust and reproducible dual time-point in vivo imaging and, despite a low overall α v ß 3 -integrin expression, generated moderate-to-good TBR. It can be concluded that 68 Ga-TRAP-(RGD) 3 allows for the in vivo imaging of tumor models with low overall α v ß 3 -integrin expression.