Effect of Peptide Receptor Radionuclide Therapy in Combination with Temozolomide against Tumor Angiogenesis in a Glioblastoma Model

Simple Summary Glioblastoma multiforme (GBM) is an aggressive brain tumor characterized by intense angiogenesis. Thus, tumor angiogenesis-related receptors, such as the cell adhesion molecule integrin αvβ3, are potential biomarkers for cancer diagnosis and therapy. In this study, we aimed to investigate the therapeutic efficacy of peptide receptor radionuclide therapy (PRRT) with 188Re-IDA-D-[c(RGDfK)]2 (11.1 MBq). Our results revealed that PRRT combined with temozolomide markedly reduced the tumor volume compared with monotherapy. In summary, 188Re-IDA-D-[c(RGDfK)]2 might be an effective radiotherapeutic agent for the treatment of GBM. Abstract Cell adhesion receptor integrin αvβ3 is a promising biomarker for developing tumor-angiogenesis targeted theranostics. In this study, we aimed to examine the therapeutic potential of peptide receptor radionuclide therapy (PRRT) with 188Re-IDA-D-[c(RGDfK)]2 (11.1 MBq). The results showed that the tumor volume was significantly decreased by 81% compared with the vehicle-treated group in U87-MG xenografts. The quantitative in vivo anti-angiogenic responses of PRRT were obtained using 99mTc-IDA-D-[c(RGDfK)]2 SPECT and corresponded to the measured tumor volume. PRRT combined with temozolomide (TMZ) resulted in a 93% reduction in tumor volume, which was markedly greater than that of each agent used individually. In addition, histopathological characterization showed that PRRT combined with TMZ was superior to PRRT or TMZ alone, even when TMZ was used at half dose. Overall, our results indicated that integrin-targeted PRRT and TMZ combined therapy might be a new medical tool for the effective treatment of glioblastoma.


Introduction
Angiogenesis is essential for tumor growth and metastasis [1]. Tumor angiogenesisrelated receptors are promising biomarkers for cancer diagnosis and therapy [2]. The cell adhesion molecule integrin α v β 3 is a specific marker of tumor angiogenesis and plays a crucial role in the advancement and metastatic spread of cancer [3]. Therefore, antagonists against integrin α v β 3 were designed and evaluated either for tumor-specific anticancer therapy or combined with various therapeutic anticancer agents [4]. In addition, integrin α v β 3 can be used to assess expression status in vivo noninvasively. Thus, it may be valuable for evaluating the efficacy of anti-integrin treatment in reducing tumor growth and spread in order to improve therapy planning and monitoring of anti-angiogenic therapies [5,6]. Tripeptide Arg-Gly-Asp (RGD) sequence has been proven effective as a specific binding motif for integrin receptors, and since, numerous radionuclide-labeled RGD peptides targeting integrin α v β 3 have been developed and used in positron emission tomography (PET) and single-photon emission computed tomography (SPECT), which can longitudinally diagnose tumor angiogenesis in cancer [7][8][9][10][11][12][13][14][15][16][17]. Theranostics combines diagnostic imaging and therapy into a single platform and is considered the next generation of personalized medicine [18]. Nuclear medicine imaging, along with radiotherapeutic agents, is effective in planning and monitoring biology-driven personalized radiotherapy. For instance, 99m Tc/ 188 Re is one of the most promising pairs owing to their favorable nuclear properties for diagnostic imaging (t 1/2 = 6 h, gamma energy of 141 keV) and tumor radiotherapy (t 1/2 = 17 h, maximum beta energy of 2.12 MeV), respectively. Moreover, 99m Tc and 188 Re can be easily obtained by periodic aseptic elution of 99 Mo/ 99m Tc-and 188 W/ 188 Re-generator, respectively, and are thus suitable for routine clinical use.
Glioblastoma multiforme (GBM) is a highly vascularized cancer [19]. Glioma cells produce proangiogenic factors, including vascular endothelial growth factor (VEGF); additionally, high levels of these factors are correlated with high-grade malignancy and poor prognosis [20,21]. Temozolomide (TMZ), which is spontaneously cleaved in vivo and generates the reactive DNA alkylating agent monomethyl triazenoimidazole carboxamide that promotes apoptosis, is used as the current standard chemotherapeutic agent for newly diagnosed GBM. It is typically used for the treatment of GBM in conjunction with radiation therapy.
In this study, we evaluated the possible use of peptide receptor radionuclide therapy (PRRT) of 188 Re-IDA-D-[c(RGDfK)] 2 with 99m Tc-IDA-D-[c(RGDfK)] 2 as a promising theranostic strategy in U87-MG human glioblastoma xenografts. Furthermore, as the main aim of this study, we examine the possible synergistic effect of PRRT and TMZ and whether this combination is more effective than individual compounds.

Preparation of Tumor-Bearing Mice
A suspension of human glioblastoma U87-MG cells was prepared (5 × 10 6 cellsmL −1 ). A total of 0.1 mL of cell suspension was injected subcutaneously into the right flank of a 6-7-week-old male BALB/c nu/nu nude mice (20-25 g, Orient Bio Inc., Seongnam, Korea). In the case of pharmacokinetic studies of 99m Tc-IDA-D-[c(RGDfK)] 2 , we used tumor xenograft mice whose tumor cells were inoculated in the right shoulder and performed the SPECT imaging study when tumor volume reached 68.7 ± 18.8 mm 3 at 10 days after injection of U87-MG cells. Evaluations of integrin-targeted blocking and radiotherapy treatment on the growth of U87-MG xenografts were carried out when tumor volume reached 59.4 ± 14.9 mm 3 (For authentic "cold" Re-peptide (

Immunohistochemistry
Tumor tissues were harvested on day 14 after the treatment and immediately fixed in 10% formalin solution. Frozen tissue sections (5 µm) were placed onto glass slides. The tumor sections were stained with anti-integrin α v β 3 rabbit antibody (1:1000, Millipore) at 4 • C for 16 h. The sections were then incubated with anti-rabbit secondary antibody (biotinylated) at room temperature for 1 h. The detection system (Vector Laboratories, Burlingame, CA, USA) was applied according to the manufacturer's instructions. The nuclei were counter-stained with hematoxylin (Invitrogen, Carlsbad, CA, USA). For assessment of tumor vascularization, mouse anti-human CD31 monoclonal antibody (diluted 1:40, Dako) staining was performed on acetone-fixed cryosections using a blood vessel staining kit (ECM590, Millipore Ireland, Cork, Ireland). For another assessment of integrin α v β 3 receptors in dissected tumor tissues, anti-integrin α v β 3 (SC-7312, Santa Cruz Biotech, Santa Cruz, CA, USA) was used. Primary antibody against γH2AX (diluted 1:50, Abcam) was applied to acetone-fixed cryosections and incubated overnight. Secondary donkey anti-goat antibody (FITC conjugated, Invitrogen, Carlsbad, CA, USA) was applied and incubated for 1 h. Immunohistochemical staining was performed at 30 days after excision of tumor due to the half-life of radioisotope (Re-188). Images were acquired using Axioscope A1 fluorescent microscope (Carl Zeiss, Jena, Germany) or AxioCam MRc5 (Carl Zeiss, Jena, Germany) and analyzed with Axiovision software (version 4.4, Carl Zeiss Meditec, Jena, Germany). The nuclei were counter-stained with hematoxylin (Invitrogen, Carlsbad, CA, USA).

Confocal Microscopy
Fluorescence images of Q-dot 605-D-[c(RGDfK)] 2 were collected using a Zeiss LSM510 META Confocal Imaging System with a Chameleon laser system (Carl Zeiss, Jena, Germany). All images were taken with an EC-Plan Neo-Fluar 40× (NA 1.3) oil immersion lens. The Q-dot 605 was excited at 543 nm, and emission was monitored from 590 to 620 nm. Images were analyzed using Zeiss LSM software (Carl Zeiss, Jena, Germany).

Tumor Growth Measurement
For caliper measurements, tumor length (longitudinal diameter) and tumor width (transverse diameter) were measured, and the tumor volume was calculated according to the following formula: tumor volume = (length × width 2 )/2.
Animal CT imaging was performed using NanoSPECT/CT (Bioscan Inc., Washington, DC, USA) consisting of a low-energy X-ray tube and a precision-motion translation stage. A total of 180 projections were acquired with the X-ray source set at 45 kVp and 177 mA. Two-dimensional slices were reconstructed using an Exact Cone Beam Filter Back Projection algorithm with a Shepp-Logan filter. CT images were reconstructed on a voxel/pixel size of 0.20:0.192 mm, providing image sizes (x, y, z) of 176 × 176 × 136 with an image resolution of 48 mm.

SPECT Image Analysis
Animal SPECT/CT imaging was acquired using a NanoSPECT/CT using low-energy and high-resolution pyramid collimator. Mice were placed in a prone position on the bed and kept under anesthesia with 2% isoflurane. SPECT images were obtained at 0 to 180 min after intravenous injection of 99m Tc-IDA-D-[c(RGDfK)] 2 (18.5 MBq, n = 4). After SPECT imaging, whole-body CT images were obtained in 24 projections over a 10 min period using a 4-head scanner with 4 × 9 (1.4 mm) pinhole collimators in helical scanning mode. Image reconstruction and quantification of micro-SPECT and CT images was performed using the software programs HiSPECT (version 1.0, Bioscan Inc. Washington, DC, USA) and InVivoScope software (version 1.43, Bioscan Inc. Washington, DC, USA), respectively. The percentage of the injected dose per gram of tissue (%IDg −1 ) was determined from the radionuclide uptake in the region of interest (ROI) on the tumor after intravenous injection of 99m Tc-IDA-D-[c(RGDfK)] 2 .

Statistical Analysis
Statistical software SPSS version 10.1 (SPSS Inc., Chicago, IL, USA) was used for analyzing results. Multiple group comparisons were made using one-way ANOVA followed by post hoc test (Bonferroni correction). Differences with a p-value less than 0.05 were considered as significant.

Pharmacokinetic Studies of 99m Tc-IDA-D-[c(RGDfK)] 2 in U87-MG Xenografts
As shown in Figure 1A, 99m Tc-IDA-D-[c(RGDfK)] 2 had prominent tumor accumulation and retention potential with rapid general clearance mainly through the kidneys and to a lesser extent through the liver. The quantified tumor uptake of 99m Tc-IDA-D-[c(RGDfK)] 2 was measured from the ROI of SPECT images and expressed as a percentage of the injected dose per gram tissue (%IDg −1 ) ( Figure 1B).

Statistical Analysis
Statistical software SPSS version 10.1 (SPSS Inc., Chicago, IL, USA) was used for analyzing results. Multiple group comparisons were made using one-way ANOVA followed by post hoc test (Bonferroni correction). Differences with a p-value less than 0.05 were considered as significant.

Pharmacokinetic Studies of 99m Tc-IDA-D-[c(RGDfK)]2 in U87-MG Xenografts
As shown in Figure 1A, 99m Tc-IDA-D-[c(RGDfK)]2 had prominent tumor accumulation and retention potential with rapid general clearance mainly through the kidneys and to a lesser extent through the liver. The quantified tumor uptake of 99m Tc-IDA-D-[c(RGDfK)]2 was measured from the ROI of SPECT images and expressed as a percentage of the injected dose per gram tissue (%IDg −1 ) ( Figure 1B).  To anticipate the therapeutic efficacy and side effects of 188 Re-IDA-D-[c(RGDfK)] 2 , we assessed the pharmacokinetic (PK) parameters of 99m Tc-IDA-D-[c(RGDfK)] 2 in three tumorbearing nude mice. PK parameters were derived using nonlinear regression curve fitting. The area under the curve (AUC 0-∞ ) that was obtained by plotting concentration versus time for 99m Tc-IDA-D-[c(RGDfK)] 2 in the liver and muscle was proportionally 10-and 200-fold lower than that in the tumor, respectively. The AUC 0 -∞ of tumor was slightly higher than that of the kidneys. The maximum concentrations (C max ) of 99m Tc-IDA-D-[c(RGDfK)] 2 in the tumor and kidneys were 13.3 and 24.0% IDg −1 , respectively.
The elimination half-life (T 1/2 elim ) of 99m Tc-IDA-D-[c(RGDfK)] 2 in the liver was 83.39 min, which was 1.10-fold higher than that in the tumor. The clearance rate (Cl) and T 1/2 elim of 99m Tc-IDA-D-[c(RGDfK)] 2 in the kidneys were 0.053 mLmin −1 and 28.54 min, respectively. These results partially showed that the kidneys eliminated the radionuclide uptake more rapidly than the tumor. However, these PK parameters from SPECT imaging indicated that the repeated dosing and renal toxicity of 188 Re-IDA-D-[c(RGDfK)] 2 need to be considered before pharmacological evaluation. crovessels was decreased to 31% at 5 mgkg −1 and 26% at 10 mgkg −1 in the 185/187 Re-IDA-D-[c(RGDfK)]2-treated group. In contrast, the 188 Re-IDA-D-[c(RGDfK)]2-treated group showed markedly increased destruction of tumor tissue microvessels in a linear radioactivity dose-dependent manner. Moreover, immunohistochemical staining with anti-integrin v3 antibody revealed the suppression of tumor growth in groups treated with 11.1 and 18.5 MBq 188 Re-IDA-D-[c(RGDfK)]2 accompanied by an 82% and 92% decrease in integrin expression levels, respectively, compared with the control.  Immunohistochemical analysis of the dissected tumor tissues with the anti-human CD31 monoclonal antibody and anti-integrin α v β 3 antibody showed reduced microvessel density and anti-angiogenic effects in tumor tissues ( Figure 2D,E). These results indicated that integrin-targeted PRRT was superior to integrin-targeted treatment inhibition. CD31 immunostaining data supported the significant anti-angiogenic effect of 188 Re-IDA-D-[c(RGDfK)] 2 with increasing radiotherapy doses [27]. The % area of CD31 positive microvessels was decreased to 31% at 5 mgkg −1 and 26% at 10 mgkg −1 in the 185/187 Re-IDA-D-[c(RGDfK)] 2 -treated group. In contrast, the 188 Re-IDA-D-[c(RGDfK)] 2 -treated group showed markedly increased destruction of tumor tissue microvessels in a linear radioactivity dose-dependent manner. Moreover, immunohistochemical staining with anti-integrin α v β 3 antibody revealed the suppression of tumor growth in groups treated with 11.1 and 18.5 MBq 188 Re-IDA-D-[c(RGDfK)] 2 accompanied by an 82% and 92% decrease in integrin expression levels, respectively, compared with the control.

Selectivity of Radiotherapy
After determining the optimal effective radiotherapeutic dose at 11.1 MBq, we investigated whether 188 Re-IDA-D-[c(RGDfK)] 2 has a selective anti-angiogenic effect on U87-MG tumors compared with the negative control peptide 188 Re-IDA-D-[c(RADfK)] 2 , which does not bind integrin α v β 3 owing to the addition of a single methyl group, changing glycine to alanine [28,29]. We analyzed the ability of 188

Selectivity of Radiotherapy
After determining the optimal effective radiotherapeutic dose at 11.1 MBq, we investigated whether 188 Re-IDA-D-[c(RGDfK)]2 has a selective anti-angiogenic effect on U87-MG tumors compared with the negative control peptide 188 Re-IDA-D-[c(RADfK)]2, which does not bind integrin v3 owing to the addition of a single methyl group, changing glycine to alanine [28,29]. We analyzed the ability of 188    Based on macroscopic observations, the dissected tumors in the 188 Re-IDA-D-[c(RGDfK)] 2treated group appeared less vascularized than those in the vehicle-and negative control peptide-treated groups ( Figure 3C). Histological examination also revealed significant differences among the groups. The 188 Re-IDA-D-[c(RGDfK)] 2 -treated tumor tissues showed a decrease in microvessel density of approximately 60% as assessed by CD31 immunostaining ( Figure 3D) and a significant decrease in positive integrin α v β 3 staining. Therefore, the anti-angiogenic effects of 188 Re-IDA-D-[c(RGDfK)] 2 might be partly responsible for the delay in tumor growth. We also found that beta radiation from 188

Theranostics for Tumor Angiogenesis
As an integrin-targeted theranostic strategy, 99m Tc-IDA-D-[c(RGDfK)] 2 SPECT was performed in all tested experimental groups (Figure 3) to assess the extent of damage in integrin receptor due to noninvasive beta irradiation of PRRT (Figure 4). Changes in the radionuclide uptake of 99m Tc-IDA-D-[c(RGDfK)] 2 in the tumor region were measured every 7 d. Vehicle-and negative control peptide-treated groups showed a rapid increase in the integrin-mediated uptake levels at 7 d of treatment (13.5 ± 1.8 and 13.1 ± 1.5% IDg −1 , respectively) and then a slight decrease at 14 d of treatment owing to the rapid enlargement of tumor size. In contrast, SPECT/CT analysis revealed that treatment with 188 Re-IDA-D-[c(RGDfK)] 2 significantly suppressed integrin α v β 3 expression and reduced tumor growth in vivo. The tumor volume of the 188 Re-IDA-D-[c(RGDfK)] 2 -treated group at 14 d of treatment was similar to that of the vehicle-treated group at 7 d of treatment ( Figure 4A); however, the percentage IDg −1 in the tumor region displayed differences between the two groups (13.5 ± 1.8 and 9.02 ± 0.6% IDg −1 , respectively; p < 0.05).

PRRT Combined with TMZ
To evaluate the combined antitumoral activity of internal radiation and chemotherapy in gliomas, we selected the anticancer drug TMZ (Figure 5), which represents the standard therapy for glioblastoma, although its dose-dependent side effects often limit its use.
TMZ treatment significantly reduced tumor size compared with saline treatment but without any significant dose-dependent differences. Although no dose-dependent responses were observed until day 14 of treatment, histological results showed that high doses of TMZ (5 mgkg −1 ) reduced integrin expression in tumor tissues ( Figure 5C,D). The combined use of 188 Re-IDA-D-[c(RGDfK)] 2 and TMZ (2 mgkg −1 ) resulted in the best anti-tumor results as confirmed by histological examination that demonstrated a significant reduction in microvessel density (97.9% reduction of positive % area for CD31 expression) and integrin receptors (98.5% reduction of positive % area for integrin α v β 3 expression) ( Figure 5C). These results revealed that combined therapy with 188 Re-IDA-D-[c(RGDfK)] 2 and TMZ could deplete the angiogenic process and retard glioblastoma progression.

PRRT Combined with TMZ
To evaluate the combined antitumoral activity of internal radiation and chemotherapy in gliomas, we selected the anticancer drug TMZ (Figure 5), which represents the standard therapy for glioblastoma, although its dose-dependent side effects often limit its use. TMZ treatment significantly reduced tumor size compared with saline treatment but without any significant dose-dependent differences. Although no dose-dependent responses were observed until day 14 of treatment, histological results showed that high doses of TMZ (5 mgkg −1 ) reduced integrin expression in tumor tissues ( Figure 5C,D). The combined use of 188 Re-IDA-D-[c(RGDfK)]2 and TMZ (2 mgkg −1 ) resulted in the best antitumor results as confirmed by histological examination that demonstrated a significant reduction in microvessel density (97.9% reduction of positive % area for CD31 expression)

Discussion
In this study, we demonstrated that 99m Tc-IDA-D-[c(RGDfK)] 2 SPECT could perform the role of diagnostics and assess integrin α v β 3 target density changes during treatment with 188 Re-IDA-D-[c(RGDfK)] 2 in U87-MG-bearing mice. 99m Tc-IDA-D-[c(RGDfK)] 2 SPECT might be helpful not only for selecting individuals for RGD peptide-based treatment but also for evaluating any changes in integrin α v β 3 levels and presumably in tumor size after anti-angiogenic treatment. Our preclinical and clinical results suggest that 99m Tc-IDA-D-[c(RGDfK)] 2 could be a novel radiopharmaceutical medical tool.
Based on our previous findings on 188 Re-IDA-D-[c(RGDfK)] 2 accumulation in tumor (12.3 ± 1.7% IDg −1 at 30 min post-injection) [18], we performed an integrin-targeted radionuclide therapy using 188 Re-IDA-D-[c(RGDfK)] 2 in the U87-MG xenograft model. Several previous studies have performed radiotherapy in a subcutaneous mouse xenograft using 177 Lu, 90 Yo, or 67 Cu-labeled RGD peptide and reported their low therapeutic efficacy and requirement in multiple injections of even high doses (37 MBq) [16,31,32]. However, our current findings indicated that 188 Re-IDA-D-[c(RGDfK)] 2 effectively suppressed tumor growth, presumably by destroying integrin with Re-188 beta emission. Furthermore, the radioactivity (11.1 MBq) used in the present study was relatively low (0.018 MBqg −1 ) and could be appropriate for clinical application. Nonetheless, multiple injections of 188 Re-IDA-D-[c(RGDfK)] 2 were required every 4 d, and thus, further optimization of radiotherapy is needed along with a detailed toxicity test. Moreover, although the antitumor efficacy results found using the subcutaneous tumor model are better than those obtained by [16,31,32], a further criticality of this work could lie in not having used an orthotopic model to be able to explore the BBB crossing ability of our system. However, this aspect will be explored in the future.
Although angiogenesis plays a critical role in tumor growth, it is uncertain whether the PRRT of 188 Re-IDA-D-[c(RGDfK)] 2 could be considered a direct cancer therapy. TMZ is an FDA-approved DNA alkylation agent that typically improves survival rates in glioblastoma patients. Since 188 Re-IDA-D-[c(RGDfK)] 2 and TMZ have different mechanisms of actions and stoichiometric ratios, we hypothesized that their combination might have a synergistic effect in the treatment of glioma cancer. Reported combination studies show that relatively low doses of the TMZ regime (<7 mgkg −1 , once a week) were effective in the reduction in tumor growth of U87-MG xenografts [33,34]. In our study, we selected the 2 and 5 mgkg −1 doses for the TMZ single treatment by considering the incidence of adverse effects related to TMZ. Moreover, we performed PRRT combined with the lower one of these two TMZ doses for the purpose of suggesting that a relatively low dose of TMZ could show an inhibition effect of tumor growth in combination therapy.
Glioblastoma growth is closely associated with the formation of new vessels. In the early stages of glioma development, there is no apparent disruption of the BBB; tumor own vasculature has not yet been formed, and the tumor mass is sustained by normal brain vessels. As glioma progresses and aggravates, endothelial cells derived from normal vessels are roughly separated from the vessel main structure and form new angiogenic spots associated with the tumor site. In this context, the importance of our findings about the combined effect of 2 mg TMZ with the radionuclide emerges, since giving the best therapeutic output in the treatment of angiogenic depletion with glioblastoma progress retardation enables keeping the blood-brain barrier intact and thus avoiding progression of the disease [35].
Compared with the relatively inadequate blocking response of cold Re-RGD peptide in integrin receptors (Figure 2), treatment with 188 Re-IDA-D-[c(RGDfK)] 2 had a very significant effect on tumor growth in the U87-MG xenograft model, even at relatively small doses (3.7 MBq). Beta irradiation from RGD peptide inhibited tumor growth, presumably by suppressing angiogenesis and destroying integrin α v β 3 in the tumor. In addition, 188 Re-IDA-D-[c(RGDfK)] 2 bound to integrin α v β 3 had a crossfire effect that led to DNA damage with subsequent tumor cell death ( Figure 3E). Consequently, our current findings indicated that 188 Re-IDA-D-[c(RGDfK)] 2 was an effective integrin-targeted radiotherapy.
The most important issue in targeted radiotherapy is the selection of agents that will provide optimal treatment efficacy with minimum undesired side effects due to ineffective exposure. While agent selection is nominally based on tissue type or tumor selectivity, it is impossible to predict its effectiveness in cases of multiple lesions or new tumors at different sites. Therefore, it is desirable to combine similar radiotherapeutic agents and evaluate their behavior for specific localization of a particular tumor target. In the present study, we demonstrated an integrated radiodiagnostic and radiotherapeutic agent set. 99m Tc-IDA-D-[c(RGDfK)] 2 was used to evaluate the possible extent of tumor localization through binding to a tumor-specific integrin α v β 3 target and further assess the change in integrin α v β 3 target density during treatment with 188 Re-IDA-D-[c(RGDfK)] 2 ( Figure 4A). Our results showed that 99m Tc-IDA-D-[c(RGDfK)] 2 SPECT might be useful not only for selecting individuals for RGD peptide-based treatment but also for evaluating changes in integrin α v β 3 levels and presumably tumor size after radiotherapeutic treatment. While 188 Re-IDA-D-[c(RGDfK)] 2 provides the possibility of using integrin-targeted radiotherapy in tumors, there are several limitations to our study. First, we used multiple injections of 188 Re-IDA-D-[c(RGDfK)] 2 every 4 d because of the in vivo rapid clearance of RGD peptide. Second, our in vivo efficacy studies did not include any toxicity test, and the safety of radiotherapy was only estimated by the mouse body weight. Although 188 Re-IDA-D-[c(RGDfK)] 2 showed a potential PRRT for tumor angiogenesis, special care such as with the kidneys and liver function test panel assay is required to prevent renal and hepatic toxicity. Third, we used only U87-MG glioblastoma cells over a 14 d treatment period. Future studies should be performed in complementary glioma models for a longer period.
The present study suggested that the combination of PRRT and TMZ might be an effective and synergistic glioblastoma treatment. Internal radiotherapy of tumor angiogenesis combined with chemotherapy and angiogenesis imaging could help to successfully develop new anti-angiogenesis drugs. Further studies examining the efficacy of combined therapy in other glioma models are needed to confirm whether 188 Re-IDA-D-[c(RGDfK)] 2 can improve the treatment of GBM.

Conclusions
In conclusion, the results of biodistribution, pharmacokinetics, in vivo SPECT imaging, and anti-angiogenic radiotherapy efficacy studies suggest that 99m Tc-and 188 Re-IDA-D-[c(RGDfK)] 2 are promising theranostic tools in the field of tumor-induced angiogenesis. Combined therapy with PRRT and TMZ showed more cytotoxic effects than monotherapy. Overall, 188 Re-IDA-D-[c(RGDfK)] 2 might be a valuable and innovative radiotherapeutic agent for the treatment of GBM.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cancers13195029/s1, Figure S1: Comparison of methods for measuring of tumor volume and radionuclide uptake, Figure S2