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Theranostics 2013; 3(11):816-830. doi:10.7150/thno.6989 This issue Cite

Research Paper

Integrin α_vβ₃-Targeted Radiotracer ^99mTc-3P-RGD₂ Useful for Noninvasive Monitoring of Breast Tumor Response to Antiangiogenic Linifanib Therapy but not Anti-Integrin α_vβ₃ RGD₂ Therapy

Shundong Ji¹, Yumin Zheng^1,2, Guoqiang Shao¹, Yang Zhou¹, Shuang Liu¹

1. School of Health Sciences, Purdue University, IN 47907, USA.
2. Department of Nuclear Medicine, China-Japan Friendship Hospital, Beijing, 100029, China.

✉ Corresponding author: Dr. Shuang Liu, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, Indiana 47907. Phone: 765-494-0236; Fax 765-496-1377; Email: liu100purdue.edu.

Abstract

Purpose: ^99mTc-3P-RGD₂ is a ^99mTc-labeled dimeric cyclic RGD peptide that binds to integrin α_vβ₃ with high affinity and specificity. The purpose of this study was to demonstrate the utility of ^99mTc-3P-RGD₂ SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-α_vβ₃ treatment.

Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 × 10⁶ MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 ± 90 mm³ on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal ^99mTc-3P-RGD₂ SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of ^99mTc-3P-RGD₂ was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)]₂ (RGD₂) was used as the anti-α_vβ₃ agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD₂ dose.

Results: The tumor uptake of ^99mTc-3P-RGD₂ from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin α_vβ₃ and CD31. Changes in tumor vasculature were further confirmed by pathological H&E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of ^99mTc-3P-RGD₂ decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin α_vβ₃ expression levels before linifanib therapy.

Conclusion: ^99mTc-3P-RGD₂ is an excellent radiotracer for monitoring integrin α_vβ₃ expression during and after linifanib therapy. ^99mTc-3P-RGD₂ SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-α_vβ₃ therapy; but it would be difficult to use ^99mTc-3P-RGD₂ for accurate and noninvasive monitoring of early tumor response to anti-α_vβ₃ therapy.

Keywords: Linifanib, monitoring antiangiogenic therapy, integrin α_vβ₃, and ^99mTc-3P-RGD_2.

Introduction

Solid tumor cells require higher levels of oxygen and metabolites to support their rapid uncontrolled growth than normal cells [1-5]. This is accomplished by increased angiogenesis at tumor site and is often seen as the rate limiting step in tumor progression [3, 5]. Targeting tumor angiogenesis with small molecular inhibitors represents a promising strategy for controlling and eradicating tumor growth [6-8]. Linifanib (ABT-869) is a multi-targeted receptor tyrosine kinase inhibitor, specifically targeting vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors [9-14]. In vitro, linifanib was shown to inhibit the phosphorylation of members of VEGF and PDGF receptor families [11]. Treatment with linifanib resulted in pronounced regression of tumors in vivo in a variety of pre-clinical tumor models [12-14]. Clinical studies showed promising results in breast cancer, metastatic non-small cell lung cancer (NSCLC), and liver cancer [15, 16]. As antiangiogenic therapies have become more common, finding suitable translational biomarkers for antiangiogenic modulation of the tumor vasculature has become critically important.

Microvessel density (MVD) has been proposed as a prognostic indicator of progression, overall survival, and disease-free survival in cancer patients [17, 18]. Evaluation of MVD is typically performed by immunostaining endothelial cells in tumor tissues and counting the number of vessels using a high power field microscope. This approach is not practical for routine monitoring of antiangiogenic therapy due to its invasive nature. DCE (dynamic contrast-enhanced) MRI (magnetic resonance imaging) has been used to measure tumor perfusion properties [19-22], but DCE-MRI is technically challenging and standardization is complicated. [¹⁸F]FDG PET was used to monitor linifanib therapy by determining reduced metabolic activity in the “treated” tumors [13], but glucose metabolism may not be an ideal biomarker for monitoring antiangiogenic therapy because most of metabolic activities take place in tumor cells rather than tumor vasculature.

Altered integrin α_vβ₃ expression has been detected in carcinomas of breast [23-25], prostate [26], ovary [27], lung [28], melanomas [29], and gliomas [30, 31]. The integrin α_vβ₃ expression has been correlated with an aggressive phenotype and metastatic dissemination [32-35]. It was reported that the integrin α_vβ₃ is overexpressed on both tumor cells and neovasculature in breast cancer xenografts [36], and is an interesting target to develop new imaging probes for early cancer detection [37-39]. It is well-established that the cross-talk between integrins and receptor tyrosine kinases (e.g. VEGFR and PDGFR) is crucial for many cellular functions [40-42]. Integrin α_vβ₃ and PDGFR-β co-precipitate and PDGFR activation increases endothelial cell migration and preliferation. The functional association between VEGFR2/PDGFR and integrin α_vβ₃ is of reciprocal nature since each is able to promote activation of its counterpart. This mutually beneficial relationship provides the conceptual basis to use radiolabeled cyclic RGD peptides as radiotracers for tumor imaging [43-52] and noninvasive monitoring of anti-α_vβ₃ and antiangiogenesis therapy [52, 53]. While they are appreciated for their role as noninvasive tools to monitor tumor response to antiangiogenesis therapy [43-46], it remains untested if integrin α_vβ₃-targeted radiotracers are useful for monitoring the tumor response to anti-α_vβ₃ treatment.

In this study, we evaluated ^99mTc-3P-RGD₂, a ^99mTc-labeled cyclic RGD peptide dimer [54, 55], for its capability to monitor integrin α_vβ₃ expression during breast tumor growth and after treatment with linifanib and E[c(RGDfK)]₂ (RGD₂) in the xenografted MDA-MB-435 human breast tumor model. ^99mTc-3P-RGD₂ is of our particular interest due to the linear relationship between its tumor uptake and integrin α_vβ₃ expression [56]. ^99mTc-3P-RGD₂ SPECT/CT has been successfully used for quantification of radioactivity accumulation, delineation of tumor necrosis [57], and monitoring progression of breast cancer lung metastases [58]. RGD₂ was used as anti-α_vβ₃ agent due to its higher integrin α_vβ₃ binding affinity than that of c(RGDfK) because of dimerization [54]. MDA-MB-435 cell line was of interest due to its high potential for metastasis. Despite the controversy associated with its identity [59-61], more recent results confirmed that MDA-MB-435 is indeed a poorly differentiated and aggressive breast tumor cell line [62]. The main objective of this study was to demonstrate the utility of ^99mTc-3P-RGD₂ SPECT/CT as a molecular imaging tool for monitoring early tumor response to antiangiogenesis therapy in small animals, and to illustrate its limitations in monitoring the efficacy of anti-α_vβ₃ treatment.

MATERIALS AND METHODS

Chemicals and Analytical Method. Linifanib (ABT-869) was from AbbVie Inc. (North Chicago, IL). Trisodium triphenylphosphine-3,3',3''-trisulfonate (TPPTS) and tricine were purchased from Sigma-Aldrich (St. Louis, Missouri), and were used without further purification. Cyclic RGD peptides, E[c(RGDfK)]₂ (RGD₂) and PEG₄-E[PEG₄-c(RGDfK)]₂ (3P-RGD₂: PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), were made by Peptides International Inc. (Louisville, KY). HYNIC-3P-RGD₂ (HYNIC = 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl) was prepared using the method described in our previous communication [55]. Na^99mTcO₄ was obtained from Cardinal HealthCare (Chicago, IL). The radio-HPLC method used a LabAlliance system equipped with a β-ram IN-US detector and Zorbax C₁₈ column (4.6 mm x 250 mm, 300 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 1 mL/min. The mobile phase was isocratic with 90% solvent A (25 mM NH₄OAc buffer, pH = 5.0) and 10% solvent B (acetonitrile) at 0 - 5 min, followed by a gradient mobile phase from 10% B at 5 min to 40% B at 20 min.

Radiolabeling and Dose Preparation. ^99mTc-3P-RGD₂ was prepared according to the literature method [55] using lyophilized kit formulation containing 20 μg HYNIC-3P-RGD₂, 5 mg TPPTS, 6.5 mg tricine, 40 mg mannitol, 38.5 mg disodium succinate hexahydrate and 12.7 mg succinic acid. ^99mTc-labeling was accomplished by adding ~ 1.5 mL of Na^99mTcO₄ solution (1,110 - 1,850 MBq). The reconstituted vial was heated in a 100 ^oC water bath for 20 - 30 min. Radiochemical purity determined by radio-HPLC must be >95% before being used for both imaging and biodistribution studies. Doses for biodistribution were prepared by dissolving the radiotracer in saline to a concentration of ~1.1 MBq/mL. For SPECT/CT imaging, doses were prepared by dissolving the radiotracer in saline to a concentration of 370 - 740 MBq /mL. The dose solution was filtered with a 0.20 μm Millex-LG filter before being injected into animals. Each animal was injected with 0.1 - 0.2 mL of the dose solution.

Cell Culture. MDA-MB-435 cell line was obtained from ATCC (American Type Culture Collection, Manassas, VA). Tumor cells were grown at 37 ºC in Minimal Essential Medium (Alpha Modification) supplemented with 10% fetal bovine serum (FBS, ATCC) and 1% penicillin and streptomycin solution (GIBCO Industries Inc., Langley, OK), and at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were grown as monolayers and were harvested or split when they reached 90% confluence to maintain exponential growth.

Animal Model. Biodistribution and imaging studies were performed using athymic nude mice bearing MDA-MB-435 breast cancer xenografts in compliance NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86-23, revised 1985). The protocol for animal studies has been approved by the Purdue University Animal Care and Use Committee (PACUC). Female athymic nu/nu mice were purchased from Harlan (Indianapolis, IN) at 4 - 5 weeks of age. Each animal implanted with 5 × 10⁶ MDA-MB-435 cells into the fat pad. The tumor volume was measured every two days for the first two weeks, every day for next 3 weeks, and calculated by the formula: (length × width²)/2. Animals were subjected to biodistribution and imaging at specified times.

Linifanib Treatment Protocol. Groups (n = 5) were size-matched with an average tumor volume of 180 ± 90 mm³ one day before baseline SPECT/CT imaging. Vehicle (0.15% hydroxypropylmethyl cellulose, 2% ethanol, 5% Tween 80, 20% PEG400, 73% saline) or linifanib treatment was initiated at a dose of 12.5 mg/kg on day 0 after baseline imaging. Linifanib was dosed orally twice daily, with a time interval of >8 h between consecutive doses.

Imaging Protocol for SPECT/CT. Longitudinal SPECT/CT imaging was performed at -1, 1, 4 and 11 days after initiation of linifanib treatment using a u-SPECT-II/CT scanner (Milabs, Utrecht, The Netherlands) equipped with 0.6 mm multi-pinhole collimators. Linifanib was dosed two hours prior to SPECT/CT. One hour before SPECT/CT imaging, ^99mTc-3P-RGD₂ (37 - 55.5 MBq) was administered via the lateral tail vein. Anesthesia was induced using an air flow rate of 350 mL/min and approximately 3.0% isoflurane. After induction of anesthesia, the air flow rate was reduced to 250 mL/min with approximately 2.0% isoflurane. SPECT images were acquired using 75 projections over 30 minutes. After SPECT acquisition, CT imaging was performed using 'normal' acquisition settings (2 degree intervals) at 45 kV and 500 μA. SPECT reconstruction, data processing and quantification of organ uptake were performed according to literature methods [56, 57]. Reconstructed images were visualized as both orthogonal slices and maximum intensity projections. The tumor uptake of ^99mTc-3P-RGD₂ was reported as the percentage of injected dose (%ID) and percentage of injected dose per unit volume (%ID/cm³), and was compared with that obtained from biodistribution.

RGD₂ Treatment Protocol. Each mouse was implanted subcutaneously with 5 × 10⁶ MDA-MB-435 cells into the fat pads. Seven days after inoculation of MDA-MB-435 cells, the tumor-bearing mice were randomly divided into 2 groups: the vehicle-treated group (n = 5) and RGD₂-treated group (n = 5) administered intravenously by tail-vein injection of RGD₂ at a dose of 50 mg/kg. The RGD₂-treatment was continued for 10 days (a dose every two days with a total of 5 doses). Animals in the vehicle-treated group were administered with equal volume of saline. SPECT/CT imaging was performed at week 1 or week 3 after the final dose of RGD₂ treatment. Quantification of organ uptake and tumor-to-background ratios were performed according to the methods described in our previous reports [56, 57]. Muscle was used as background. The region fo interest (ROI ~ 0.01cm³) was drawn over the contralateral forelimb. Tumor/muscle (T/M) ratios were calculated on the basis of %ID/cm³ uptake values of ^99mTc-3P-RGD₂ in tumor and muscle of the same animal, and were reported as the means ± the standard deviation.

Biodistribution Protocol. The tumor-bearing mice (tumor sizes = 0.05 - 1.5 g) were used for biodistribution studies. Each animal (20 - 25 g) was administered with ~110 kBq of ^99mTc-3P-RGD₂ by tail vein injection. Animals were sacrificed by sodium pentobarbital overdose (~200 mg/kg) at 60 min post-injection (p.i.). Blood was withdrawn from the heart of tumor-bearing mice. Tumors and normal organs (brain, eyes, heart, spleen, lungs, liver, kidneys, muscle and intestine) were harvested, washed with saline, dried with absorbent tissue, weighed, and counted on a Perkin Elmer Wizard - 1480 γ-counter (Shelton, CT). The organ uptake was calculated as the percentage of injected dose (%ID) and percentage of injected dose per gram of tissue (%ID/g).

Tumor Immunohistochemistry. Tumor tissues were harvested from animals, and were snap-frozen in the OCT (optical cutting temperature) solution (Sakara, Torrance, CA). Tumor tissues were then cut into slices (5 μm). After drying at room temperature, slices were fixed with ice-cold acetone for 10 min, and dried in air for 20 min at room temperature. Sections were blocked with 10% goat serum for 30 min at room temperature, and then were incubated with the hamster anti-integrin β₃ antibody (1:100, BD Biosciences, San Jose, CA) and rat anti-CD31 antibody (1:100, V:V; BD Biosciences) for 1 h. After incubating with Cy3-conjugated goat anti-hamster and FITC-conjugated goat anti-rat secondary antibodies (1:100, Jackson ImmunoResearch Inc., West Grove, PA) and washing with PBS, the fluorescence was visualized with a Nikon fluorescence microscope. All pictures were taken under 200x magnification with the same exposure time.

Statistical Analysis. All data were expressed as means plus/minus standard deviation based on results from five animals. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. The level of significance was set at p < 0.05. GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA) was used for linear and nonlinear regression analysis.

RESULTS

Monitoring of Breast Tumor Growth. Figure 1A shows 3D and transverse views of SPECT/CT images of a tumor-bearing mouse at 5, 7, 14, 21, 28 and 35 days after inoculation of MDA-MB-435 breast tumor cells. Small tumors (Figure 1A: D5) was readily detected with excellent contrast. When the tumor size was 0.15 - 0.40 cm³, the radioactivity distribution was relatively uniform. After week 3, the tumors were >0.50 cm³, and the radioactivity distribution heterogeneity was significant due to tumor necrosis (Figure 1A: D28). By week 5, there was extensive necrosis in MDA-MB-435 breast tumors. Necrotic regions had little radioactivity accumulation (Figure 1A: D28 - D35) due to lack of integrin α_vβ₃ expression (Figure 1B).

Comparison of Breast Tumor Uptake Values from SPECT/CT and Biodistribution. To validate u-SPECT-II/CT as a tool for quantification of the tumor uptake of ^99mTc-3P-RGD₂, we performed a biodistribution study using animals with tumor size being 0.01 - 1.2 g, and compared the tumor uptake values to those from SPECT/CT quantification. Figure 2 exhibits the relationship between the tumor size (cm³) and the tumor uptake (%/ID for total radioactivity and %/ID/g or %/ID/cm³ for radioactivity concentration) determined by SPECT/CT (A and C) and biodistribution (B and D). Apparently, the tumor uptake of ^99mTc-3P-RGD₂ from SPECT/CT was almost identical to that from biodistribution. Similar results were also seen in the U87MG model [64]. u-SPECT-II/CT is excellent imaging platform for accurate quantification of tumor uptake of ^99mTc-3P-RGD₂ in small animals.

Tumor Uptake Changes during Tumor Growth. When the tumors were very small (<0.1 cm³), their uptake of ^99mTc-3P-RGD₂ were low due to lack of integrin α_vβ₃ contribution from neovasculature. Between weeks 2 and 3, the tumor size was in the range of 0.15 - 0.30 cm³, and the %ID/g tumor uptake of ^99mTc-3P-RGD₂ peaked (Figure 2: B and D). Between weeks 4 and 5, the tumor size was >0.5 cm³. During this period, the % ID tumor uptake of ^99mTc-3P-RGD₂ was enhanced steadily with increased tumor size (Figure 2: A and C), but its % ID/g uptake decreased slowly due to necrosis (Figure 1B), maturation of blood vessels, and larger interstitial space [63]. The overall relationship between tumor volume and % ID uptake of ^99mTc-3P-RGD₂ was linear (Figure 2: A: R² = 0.923; and C: R² = 0.859). The relationship between tumor size and %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ was best described as quadratic polynomial (Figure 2: B and D).

 Fig 1 

A: The 3D (upper panel) and transverse (lower panel) views of SPECT/CT images of a mouse with bearing MDA-MB-435 breast cancer xenografts at 5, 7, 14, 21, 28 and 35 days after inoculation of MDA-MB-435 human breast cancer cells. The animal was administered with 37 - 55.5 MBq of ^99mTc-3P-RGD₂ via the lateral tail vein. B: Microscopic images (Original magnification: ×200) of tumor slice selected from the necrotic and viable regions after immunohistochemical staining for integrin β₃ and CD31. The tumor tissue was harvested on day 28 days after inoculation of MDA-MB-435 cells. CD31 was used as a biomarker for endothelial cells on blood vessels (both mature vasculature and neovasculture). Integrin β₃ was visualized with Cy3 (red) and CD31 was visualized with FITC (green). The yellow color in overlay images indicates the presence of integrin β₃ on neovasculature.

 Fig 2 

Left: Linear relationship between the tumor size (cm³) and the tumor uptake (%/ID for total radioactivity accumulation) determined by SPECT/CT quantification (A) and biodistribution (C). Right: Relationship between the tumor size (g) and tumor uptake (%/ID/g or %/ID/cm³ for radioactivity concentration) obtained from SPECT/CT quantification (B) and biodistribution (D: n = 26).

Integrin α_vβ₃ Expression Changes during Tumor Growth. Figure 3 displays selected microscopic images of the slices obtained from the xenografted MDA-MB-435 tumors (0.05 g, 0.13 g, 0.28 g, 0.55 g and 0.88 g) after immunohistochemical staining for integrin β₃ and CD31. As expected, small tumors (<0.05g) had few microvessels with very little integrin α_vβ₃ expression. When tumors became larger (0.2 - 0.5 g), the blood vessel density was increased, as illustrated by the enhanced yellow and orange colors on overlay images. When tumors were > 0.5 g, the tumor blood vessel density decreased while the total integrin α_vβ₃ expression level was increased due to more contributions from MDA-MB-435 tumor cells, which was completely consistent with the %ID tumor uptake values of ^99mTc-3P-RGD₂ (Figure 2: B and D).

 Fig 3 

Microscopic images of frozen tumor slices from the xenografted MDA-MB-435 tumors (0.05 g, 0.13 g, 0.28 g, 0.55 g and 0.88 g) after immunohistochemical staining for integrin β₃ and CD31. CD31 was used as a biomarker for tumor endothelial cells. Integrin β₃ was visualized with Cy3 (red), and CD31 with FITC (green). Yellow color indicates the integrin β₃ on new blood vessels.

Monitoring Tumor Response to Linifanib Therapy. Figure 4A compares tumor volume changes in both vehicle (n = 5) and linifanib (n = 5) treated groups. A significant difference in tumor volume (Figure 4A: p < 0.05) was seen between vehicle and linifanib-treated groups after 4 days of linifanib treatment. This effect is further illustrated by SPECT/CT images (Figure 4B) obtained at -1, 1, 4 and 11 days after initiation of linifanib therapy. Figure 5 shows SPECT/CT quantification data to illustrate early breast tumor response to linifanib antiangiogenesis therapy. In the linifanib-treated group, there was a dramatic reduction in both %ID and %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ as early as 24 h. It was clear that ^99mTc-3P-RGD₂ SPECT can detect much earlier biological changes (decrease in integrin α_vβ₃ expression level) as a result of linifanib therapy. The %ID tumor uptake of ^99mTc-3P-RGD₂ increased steadily in the vehicle-treated group (Figure 5A), while its %ID/cm³ tumor uptake decreased slowly over 11 day study period (Figure 5B).

 Fig 4 

A: Comparison of tumor volumes in vehicle and linifanib-treated in the MDA-MB-435 models. Tumor volume was determined by caliper measurements. *: p < 0.05, significantly different from the vehicle-treated group. #: p < 0.01, significantly different from the vehicle-treated group from that specific date until the end of study. B: Transverse views of selected SPECT/CT images from athymic nude mice bearing MDA-MB-435 breast tumors in the vehicle (upper panel) and linifanib-treated groups (lower panel). SPECT/CT images were obtained at -1, 1, 4 and 11 days after initiation of linifanib therapy to illustrate the early tumor response in terms of tumor uptake and integrin a_vβ₃ expression levels. Arrows indicate the presence of tumors.

 Fig 5 

Direct comparison of the %ID tumor uptake (top) and %ID/cm³ tumor uptake (bottom) of ^99mTc-3P-RGD₂ in the vehicle (n = 5) and linifanib-treated (n = 5) groups. The tumor uptake values were calculated from quantification of SPECT/CT images. *: p < 0.05, significantly different from the vehicle-treated group. #: p < 0.01, significantly different from the vehicle-treated group.

Comparison of Tumor Response in Different Animal Models. Figure 6 compares the changes in %ID/cm³ tumor uptake and T/M ratios of ^99mTc-3P-RGD₂ after linifanib treatment in three different xenografted tumor-bearing animal models (U87MG with high integrin α_vβ₃ expression on tumor cells and tumor neovasculature; MDA-MB-435 with moderate integrin α_vβ₃ expression on tumor cells and neovasculature; and PC-3 with low integrin α_vβ₃ expression on tumor cells and neovasculature). The %ID/cm³ tumor uptake and T/M ratios of ^99mTc-3P-RGD₂ in U87MG glioma and PC-3 prostate models were obtained from our previous studies [64]. The degree of tumor response (Figure 6A: defined by the %ID/cm³ tumor uptake change of ^99mTc-3P-RGD₂) to linifanib therapy was highest for U87MG gliomas, moderate for MDA-MB-435 breast tumors, and lowest for PC-3 prostate tumors. T/M ratio changes in the xenografted U87MG and MDA-MB-435 tumors were more significant than those in PC-3 tumors. There was no significant difference (Figure 6B) in T/M ratio changes between the U87MG and MDA-MB-435 models within experiemtnal errors.

 Fig 6 

Comparison of changes in %ID/cm³ tumor uptake (A) and T/M ratios (B) of ^99mTc-3P-RGD₂ after linifanib treatment in three different tumor-bearing animal models (U87MG: high integrin α_vβ₃ expression on tumor cells and neovasculature; MDA-MB-435: moderate integrin α_vβ₃ expression on tumor cells and neovasculature; PC-3: low integrin α_vβ₃ expression on tumor cells and neovasculature). Tumor uptake values were calculated from quantification of SPECT/CT images. The %ID/cm³ tumor uptake and T/M ratios of ^99mTc-3P-RGD₂ in the U87MG and PC-3 models were obtained from our previous report [64]. The changes in %ID/cm³ tumor uptake and T/M ratios were calculated by subtracting the value on a specific date (+1, +4 and +11) from the value on day -1. *: p < 0.05, significantly different from those in the U87MG and PC-3 models. ^#: p < 0.01, significantly different from those in the U87MG and MDA-MB-435 models.

Effects of Linifanib on Tumor Vasculature and Integrin α_vβ₃ Expression. Immunohistochemical staining was performed to study the impact of linifanib treatment on integrin α_vβ₃ and CD31 expression in MDA-MB-435 tumors. Figure 7 (top) shows overlay images of the tumor slices stained with hamster anti-integrin β₃ (red color) or rat anti-CD31 (green color) antibody in both vehicle and linifanib-treated groups. The yellow color in overlay images indicates the presence of integrin β₃ on new blood vessels. It was quite clear that the effect of linifanib therapy was on both tumor cells and tumor neovasculature as indicated by the disappearance of red (breast tumor cells) and yellow/orange (neovasculature) colors for integrin α_vβ₃ in the overlay images. After 24 h of linifanib therapy, the integrin α_vβ₃ on neovasculature disappeared almost completely while the tumor cell integrin α_vβ₃ expression level decreased steadily during the 11-day period (Figure 7: top). This was further supported by the pathological H&E staining data of tumor slices in vehicle and linifanib-treated groups (Figure 7: bottom) at -1 and 1 days. Within 24 h after initiation of linifanib therapy, the blood vessel density was dramatically reduced. These data clearly show that linifanib treatment had a strong impact on both breast tumor cells and neovasculature.

Relationship between Tumor Response and Integrin α_vβ₃/CD31expression. Figure 8 shows plots of the %ID/cm³ tumor uptake changes after linifanib treatment against integrin α_vβ₃ (left) and CD31 (right) expression levels in the U87MG, MDA-MB-435 and PC-3 models at the time of diagnosis. The tumor uptake change was calculated by deducting the %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ on days 1, 4 and 11 from its %ID/cm³ tumor uptake on -1 day for the same animal. Integrin α_vβ₃ and CD31 expression levels were obstained from the tumors (0.1 - 0.3 g) without significant necrosis. Obviously, there was an excellent linear relationship between %ID/cm³ tumor uptake changes (the tumor response or therapeutic effect) and the total integrin α_vβ₃ expression levels (integrin α_vβ₃ density) at the time of diagnosis (-1 day). The same linear relationship was also seen between the tumor response and the CD31 expression levels (blood vessel density).

 Fig 7 

A: Overlay images of MDA-MB-435 tumor tisues after immunohistochemical staining for integrin β₃ and CD31 in vehicle (upper panel) and linifanib-treated groups (lower panel) to illustrate the changes in integrin β₃ expression level and blood vessel density during linifanib therapy. Tumor tissues were harvested at -1, 1, 4 and 11 days after initiation of linifanib therapy. CD31 was the marker for tumor blood vessels (both mature and newly formed), which was visualized with FITC (green color). Integrin β₃ was visualized with Cy3 (red color). The yellow color in overlay images indicates the presence of integrin β₃ on new blood vessels. B: Images of selected histological slice (H&E stained) of tumor tissues from animals in the vehicle (upper panels) and linifanib-treated (lower panels) groups to illustrate vascular density changes after only one day of linifanib therapy. Red color indicates the presence of blood vessels.

 Fig 8 

Linear relationship between the %ID/cm³ tumor uptake change at days 1 (top), 4 (middle) and 11 (bottom) after linifanib therapy and the expression levels of integrin α_vβ₃ (left) and CD31 (right) in three animal models. The %ID/cm³ tumor uptake values of ^99mTc-3P-RGD₂ were calculated from SPECT/CT quantification, and reported as the mean plus/minus standard error of the mean based on results from five animals (n = 5). The %ID/cm³ tumor uptake values and integrin α_vβ₃/CD31 expression levels in the U87MG and PC-3 tumors were obtained from our previous report [64]. The %ID/cm³ tumor uptake change was calculated by deducting the %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ on days 1, 4 and 11 from its original value on -1 day (before linifanib therapy) in the same animal. The average %ID/cm³ tumor uptake change is used as an indicator of tumor response to linifanib antiangiogenesis treatment.

Effects of RGD₂ Therapy on Tumor Volume. Figure 9A shows the tumor growth curves during 42 days of study period for both vehicle (n = 5) and RGD₂-treated (n = 5) groups. No significant difference in tumor volumes was found over first 3 days after initiation of RGD₂ therapy. After two doses of RGD₂ (4 days after initiation of RGD₂ therapy), there was significant tumor volume reduction in RGD₂-treated group. By the time of the final dose (10 days after initiation of RGD₂ therapy), the tumor volume in the vehicle-treated group was almost 3x larger than that in RGD₂-treated group. Obviously, there was a significant therapeutic effect from RGD₂ treatment on the basis of tumor volume change. However, RGD₂ treatment had to be stopped after five doses of RGD₂ because of toxicity, as indicated by significant weight loss (Figure 9B).

 Fig 9 

Tumor growth curves (A) and body weight changes (B) for vehicle (n = 5) and RGD₂-treated (n = 5) groups in the xenografted MDA-MB-435 breast tumor model. Tumor size was calculated on the basis of caliper measurements according the formula (length × width²)/2. Body weight was measured by balance. RGD₂ treatment had to be stopped after five doses of RGD₂ because of toxicity, as indicated by significant weight loss. The body weight recovered after termination of RGD₂ treatment.

Effects of RGD₂ Therapy on Tumor Uptake. SPECT/CT images (Figure 10: top) were obtained before RGD₂ treatment, and at 21 and 35 days after inoculation of MDA-MB-435 cells. Tumor volumes (A), tumor uptake (B: %ID and C: %ID/cm³) and T/M ratios (D) were calculated from SPECT/CT quantification. Since the tumor retention time of RGD₂ was ~15 h [65], a 7-day interval between the final RGD₂ dosing and the first SPECT/CT imaging study was used to make sure that there was no significant blockage of ^99mTc-3P-RGD₂ binding to integrin α_vβ₃. As expected, there was significant reduction in tumor volume (Figure 10A) and %ID tumor uptake (Figure 10B) of ^99mTc-3P-RGD₂ between the vehicle and RGD₂-treated animals. However, its %ID/cm³ tumor uptake was comparable in vehicle and RGD₂-treated groups within experimental errors (Figure 10C: p > 0.05) at 7 days post RGD₂ treatment. By 21 days after RGD₂ therapy, the %ID/cm³ uptake in RGD₂-treated group was significantly higher than that in vehicle group (Figure 10C: p < 0.05) while T/M ratios were almost identical at both time points (Figure 10D). RGD₂ therapy resulted in significant reduction in both the tumor volume and %ID tumor uptake (the tumor radioactivity) of ^99mTc-3P-RGD₂ (Figure 10: A and B); but it also led to the enhanced %ID/cm³ tumor uptake (the radioactivity density) of ^99mTc-3P-RGD₂ (Figure 10C).

 Fig 10 

Top: The 3D and transverse views of SPECT/CT images of tumor-bearing mice before RGD₂ treatment, as well as on day 21 and 35 after inoculation of MDA-MB-435 cells (or on day 7 and 21 after final dose of RGD₂). SPECT image at day 7 was used only for comparison purposes. Bottom: SPECT/CT quantification data, including tumor volumes (A), %ID tumor uptake (B), %ID/cm³ tumor uptake (C), and T/M ratios (D) in vehicle (n = 5) and RGD₂-treated (n = 5) groups. *p < 0.05 and # p < 0.01: significantly different from the vehicle-treated group.

Discussion

In this study, we found that ^99mTc-3P-RGD₂ is an excellent radiotracer for imaging MDA-MB-435 tumors (Figure 1A). As shown in Figure 2, the uptake values (%ID and %ID/cm³) of ^99mTc-3P-RGD₂ from SPECT/CT is almost identical to those from biodistribution. There was also a linear relationship between tumor size and %ID uptake of ^99mTc-3P-RGD₂ (Figure 2: A and C). These results strongly suggested that the tumor uptake obtained from SPECT/CT images can accurately reflect the total tumor integrin α_vβ₃ expression. ^99mTc-3P-RGD₂ is useful for monitoring integrin α_vβ₃ expression changes during tumor growth (Figure 1A).

[¹⁸F]AH111585 has been used to monitor sunitinib therapy response [43-45], and its tumor uptake was significantly reduced 2 days after sunitinib therapy. It was believed that [¹⁸F]AH111585 was better than [¹⁸F]FDG for imaging angiogenesis [45]. ⁶⁴Cu-DOTA-RGD was used to monitor dasatinib therapy [46]. It was also found that dasatinib was able to significantly reduce the uptake of ⁶⁴Cu-DOTA-RGD in the “treated” tumors. A significant challenge for [¹⁸F]AH111585 and many ¹⁸F-based PET radiotracers to assume a wide-spread clinical utility is their poor clinical availability. In contrast, ^99mTc-3P-RGD₂ is readily prepared with very high specific activity using a kit formulation [54, 55]. From availability point of view, ^99mTc-3P-RGD₂ has significant advantages of the corresponding ¹⁸F PET radiotracers.

Inhibition of VEGF and PDGF receptors with linifanib normalizes tumor vasculature [9-11, 13, 14]. Since growth factors (e.g. VEGF) increase integrin α_vβ₃ expression [40-42], inhibition of VEGF and PDGF receptors could lead to a decrease of integrin α_vβ₃ expression. This makes integrin α_vβ₃ a mechanistically relevant biomarker to assess the tumor response to linfanib therapy. In this study, we found significant decreases in the tumor uptake (%ID and %ID/cm³) of ^99mTc-3P-RGD₂ prior to tumor volume changes after linifanib treatment (Figures 4). Changes in tumor vasculature were confirmed by IHC staining data (Figure 7A) and pathological H&E analysis (Figure 7B).

It is interesting to note that the therapeutic effect of linifanib is not limited to tumor blood vessels. It is well-established that linifanib is a multi-targeted receptor tyrosine kinase inhibitor [9-14]. Since VEGF and PDGF receptors are overexpressed on tumor cells and neovasculature [41, 42], it is not surprising that linifanib has inhibitory effects on both tumor cells and tumor vasculature. There are two phases for the tumor response to linifanib therapy (Figure 5): a dramatic decrease in the tumor uptake (%ID and %ID/g) of ^99mTc-3P-RGD₂ within the first 24 h after initiation of linifanib therapy, and a steady tumor uptake reduction over the next 10 days of study period. The IHC and H&E staining data (Figure 7) suggest that the rapid decrease in tumor uptake of ^99mTc-3P-RGD₂ is most likely induced by antiangiogenic effect of linifanib on tumor neovasculature while the slow tumor uptake reduction is caused by either its direct inhibitory effect on tumor cells or slow tumor cell apoptosis as a result of normalization of tumor vasculature [9-11, 13, 14]. This explanation is supported by the sharp decrease of CD31 signals (Figure 7A) and tumor microvessel density (Figure 7B) over the first 24 h after initiation of linifanib treatment, followed by slow disappearance of fluorescence intensity from integrin β₃ on tumor cells over the next 10 days of study period (Figure 7A).

In our previous study [64], we found that there was a significant decrease in tumor uptake (%ID and %ID/cm³) and T/M ratios of ^99mTc-3P-RGD₂ in the xenografted U87MG model while no significant changes in tumor volume or tumor uptake of ^99mTc-3P-RGD₂ were seen in the xenografted PC-3 model after linifanib treatment. In this study, we found that the changes in tumor volume (Figure 4) and tumor uptake of ^99mTc-3P-RGD₂ (Figure 6A) in the MDA-MB-435 model were between those observed in the U87MG and PC-3 models. This is consistent with the relative integrin α_vβ₃ expression levels on three xenografted tumors [56, 66], and reciprocal relationship between integrin α_vβ₃ and VEGFR2 [40-42]. We also found that there is a linear relationsip between the integrin α_vβ₃/CD31 expression levels and the tumor response, as indicated by changes in %ID/cm³ tumor uptake and T/M ratios of ^99mTc-3P-RGD₂ (Figure 8). Highly vascularized tumors (e.g. U87MG) with higher expression level of integrin α_vβ₃ and CD31 have better tumor response to linifanib therapy than poorly vascularized tumors (e.g. PC-3) with low expression levels of integrin α_vβ₃ and CD31. ^99mTc-3P-RGD₂ has the potential as a pre-treatment screening tool to select appropriate patients who will benefits most from the antiangiogenic linifanib treatment. If the tumor in cancer patient has a high expression level of integrin α_vβ₃, as indicated by the high tumor uptake of ^99mTc-3P-RGD₂ in SPECT/CT images at the time of diagnosis, antiangiogenic linifanib therapy would more likely be effective. If the tumor has very little integrin α_vβ₃ expression (low tumor uptake of ^99mTc-3P-RGD₂), the antiangiogenic therapy would not be effective regardless the amount of linifanib administered into each cancer patient.

It is important to note that there are no accurate techniques to quantify absolute integrin α_vβ₃ expression levels. Western blotting was used to determine the total integrin α_vβ₃ protein concentration in tumor tissues [44, 45, 56, 67, 68]; but the percentage of integrin α_vβ₃ recovery from the tumor tissue and its activation state remained unknown. We have been using IHC staining with anti-integrin β₃ antibodies to determine the relative expression levels of integrin α_vβ₃ in the tumor tissues [56, 66]. This technique is a better fit to reflect the activation state of integrin α_vβ₃ in acetone-fixed tumor tissues since only the activated integrin α_vβ₃ is able to bind the fluorescence-labeled anti-integrin β₃ monoclonal antibody. We also found that the primary antibody is very important for IHC staining of tumor tissues. In this study, we use hamster anti-human integrin β₃ antibody for staining of integrin α_vβ₃ on both tumor cells and neovasculature mainly because of the murine nature of xenografted tumor tissues. It must be noted that quantification of absolute fluorescent intensity is operator-dependent and subjected to individual interpretation. Tumors of different sizes might have different integrin α_vβ₃ expression patterns because of tumor necrosis. The integrin α_vβ₃ expression pattern is heterogeneous even in the same tumor tissue. Thus, the relative expression levels of integrin α_vβ₃ and CD31 only represent the status under specific experimental conditions, such as the primary integrin α_vβ₃ monoclonal antibody, animal species, inoculation location of tumor cells, tumor types, and tumor size.

As illustrated in this study, there is significant challenge in using ^99mTc-3P-RGD₂ to monitor early tumor response to RGD₂ therapy. Since ^99mTc-3P-RGD₂ and RGD₂ target the same receptor, there must be a waiting period to allow RGD₂ to dissociate completely from integrin α_vβ₃ sites before using ^99mTc-3P-RGD₂ to determine integrin α_vβ₃ level changes after RGD₂ therapy. Otherwise, it would be difficult to determine if changes in tumor uptake are actually caused by its blocking effect or therapeutic effect. It is also important to note that the waiting period also depends on tumor retention time of the anti-α_vβ₃ agent. If its tumor retention time is short (2 - 3 h), it is possible to image tumors with ^99mTc-3P-RGD₂ 24 h (~10 half-lives) after anti-α_vβ₃ therapy. Since its tumor retention time is ~15 h [65], it would take 6 - 7 days for RGD₂ to dissociate completely from integrin α_vβ₃ sites. During this period, the tumor might undergo significant biological changes. Therefore, it is difficult to use the integrin α_vβ₃-targeted radiotracers (e.g. ^99mTc-3P-RGD₂) to accurately monitor early tumor response to anti-α_vβ₃ therapy.

There is no doubt that RGD₂ can significantly reduce the tumor size (Figure 10A) and %ID tumor uptake (Figure 10B) of ^99mTc-3P-RGD₂; but its %ID/cm³ tumor uptake in RGD₂-treated group was higher (p < 0.05) than that in vehicle-treated group (Figure 10C). It is possible that RGD₂ was able to block the integrin α_vβ₃ sites in RGD₂-treated group, resulting in tumor growth inhibition, as indicated by tumor volume reduction. Once RGD₂ therapy stopped, RGD₂ slowly dissociated from tumor cells and neovasculature. As a result, integrin α_vβ₃ is activated again. Recently it was reported that low concentrations of integrin α_vβ₃ and α_vβ₅ inhibitors (e.g. Cilengitide) can stimulate tumor growth and angiogenesis by altering α_vβ₃ integrin and VEGFR-2 trafficking, thereby promoting endothelial cell migration to VEGF [69]. That might explain why the %ID/g tumor uptake of ^99mTc-3P-RGD₂ in RGD₂-treated group was higher than that in vehicle-treated group 3 weeks after RGD₂ therapy (Figure 10C). More studies are needed to understand the integrin α_vβ₃ expression or activation state changes after anti-α_vβ₃ RGD₂ therapy, as well as their relationship with the %ID/g tumor uptake of ^99mTc-3P-RGD₂.

CONCLUSION

In this study, we clearly show that ^99mTc-3P-RGD₂ SPECT/CT is a useful molecular imaging tool for monitoring the integrin α_vβ₃ expression changes during breast tumor growth or during/after linifanib antiangiogenic therapy. ^99mTc-3P-RGD₂ SPECT/CT is also a useful pre-treatment screening tool to identify the cancer patient who will benefits best from antiangiogenic therapy with linifanib or related antiangiogenic drugs of the same or similar mechanism. However, it is difficult to use the integrin α_vβ₃-targeted radiotracers, such as ^99mTc-3P-RGD₂, for monitoring the early tumor response to anti-α_vβ₃ therapy with Cilengitide or RGD₂. This conclusion may also apply to other radiotracers and anti-cancer drugs that target the same receptor.

Acknowledgements

Authors thank AbbVie Inc. (North Chicago, IL) for the use of Linifanib. This work was supported by Purdue University, the Challenge Research Award from Purdue Cancer Center, the Indiana Clinical and Translational Sciences Institute funded in part by grant Number TR000006 (Clinical and Translational Award) from the National Institutes of Health, National Center for advancing Translational Science, and R01 CA115883 (S.L.) from the National Cancer Institute.

COMPETING INTERESTS

The authors have declared that no competing interest exists.

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Author contact

Corresponding author: Dr. Shuang Liu, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, Indiana 47907. Phone: 765-494-0236; Fax 765-496-1377; Email: liu100purdue.edu.

Received 2013-6-24
Accepted 2013-8-6
Published 2013-10-12

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