Abstract
Abraxane (nanoparticle albumin-bound paclitaxel) is an anticancer drug approved by the Food and Drug Administration. However, the mechanism of action of Abraxane is complex, and no established biomarker is available to accurately monitor its treatment outcomes. The aim of this study was to investigate whether the integrin-specific PET tracer 18F-FPPRGD2 (investigational new drug 104150) can be used to monitor early response of tumors to Abraxane therapy. Methods: Orthotopic MDA-MB-435 breast cancer mice were treated with Abraxane (25 mg/kg every other day, 3 doses) or phosphate-buffered saline. Tumor volume was monitored by caliper measurement. PET scans were obtained before and at different times after the start of treatment (days 0, 3, 7, 14, and 21) using 18F-FPPRGD2 and 18F-FDG. The tumoricidal effect was also assessed ex vivo by immunohistochemistry. Results: Abraxane treatment inhibited the tumor growth, and a significant difference in tumor volume could be seen at day 5 after the initiation of treatment. The tumor uptake of 18F-FPPRGD2 in the Abraxane-treated group was significantly lower on days 3 and 7 than at baseline but returned to the baseline level at days 14 and 21, indicative of relapse of the tumors after the treatment was halted. Immunohistologic staining confirmed that the change of 18F-FPPRGD2 uptake correlated with the variation of integrin level in the tumor vasculature induced by Abraxane treatment. No significant change of tumor (rather than vascular) integrin expression was observed throughout the study. No significant decrease of 18F-FDG uptake was found between the treated and the control tumors on days 3, 14, and 21, although an increase in 18F-FDG tumor uptake of treated mice, as compared with the control mice, was found on day 7. The increase of 18F-FDG on day 7 was related to the inflammatory response during therapy. Conclusion: Abraxane-mediated downregulation of integrin αvβ3 expression on tumor endothelial cells can be quantitatively visualized by PET. The change of integrin expression precedes that of tumor size. Consequently, 18F-FPPRGD2 PET is superior to 18F-FDG PET in monitoring early response to treatment, favoring its potential clinical translation.
Paclitaxel was originally derived from the bark of the Pacific Yew tree in the early 1960s. It can disrupt microtubule reorganization and lead to G2/M cell cycle arrest (1). As a potent cytotoxic agent, paclitaxel is widely used against various refractory and metastatic malignancies (2,3). However, it is also well known for its ability to produce hypersensitivity reactions and its limited aqueous solubility (4,5). The clinical use of paclitaxel has been improved significantly by formulating this drug in polyethoxylated castor oil–free, albumin-bound, 130-nm particles named Abraxane (nanoparticle albumin-bound paclitaxel; Abraxis BioScience).
Abraxane is an important therapeutic breakthrough, because it addresses the toxicities associated with solvents in taxane-based chemotherapy and minimizes the occurrence of severe anaphylactic reactions (6,7). In addition, compared with paclitaxel, Abraxane can improve drug delivery into the tumor (8,9). The antiangiogenesis activity of paclitaxel has been observed even at low concentrations, which did not affect cellular microtubule assembly (10,11). Paclitaxel also showed an inhibitory effect on the growth of transplanted human oral squamous cell carcinoma and reduced the expression of vascular endothelial growth factor (VEGF) and CD31 (12). However, contradictory reports also exist that Abraxane triggers reactionary angiogenesis by upregulating VEGF-A expression in a breast cancer model (2).
Integrins are composed of a family of heterodimeric glycoproteins responsible for the regulation of cellular activation, migration, proliferation, survival, and differentiation (13). One of the most important members of this receptor class is integrin αvβ3, which is preferentially expressed on several types of cancer cells, including melanoma, glioma, and ovarian and breast cancers (14). Integrin αvβ3 is also expressed on proliferating endothelial cells associated with neovascularization in both malignant tumors and normal tissue but not in quiescent blood vessels (15,16).
Because peptides containing arginine-glycine-aspartic acid (RGD) can bind strongly to integrin αvβ3, many RGD peptide probes have been developed for multimodality imaging of integrin expression (17–21). Targeting the αvβ3 integrin receptor could provide us with a tool to visualize and quantify integrin αvβ3 expression levels; early cellular and molecular effects of therapies (especially on vascular modulation), without having to rely on invasive histologic staining; consequential changes in blood perfusion and permeability; and terminal morphologic changes, such as substantial tumor mass or volume reduction (14).
18F-FPPRGD2 is an 18F-labeled dimeric RGD peptide recently developed in our laboratory that has higher receptor binding affinity than monomeric RGD peptide counterparts and enhanced tumor uptake (22). An exploratory investigative new drug application (IND 104150) for 18F-FPPRGD2 was recently approved by the Food and Drug Administration for the first tests in humans. In this study, we performed longitudinal PET studies to compare the abilities of 18F-FPPRGD2 and 18F-FDG to evaluate the treatment efficacy of Abraxane in an MDA-MB-435 human breast cancer xenograft model.
MATERIALS AND METHODS
Radiochemistry
18F-FPPRGD2 was synthesized as previously reported (22). In brief, PEG3-E[c(RGDyK)]2 (denoted as PRGD2) was added to dried 4-nitrophenyl 2-18F-fluoropropionate, followed by the addition of N,N-diisopropylethylamine. The labeled peptide was purified by reversed-phase high-performance liquid chromatography on a semipreparative C-18 column. After trapping with a C-18 cartridge preactivated with 5 mL of ethanol and 10 mL of water, the product was washed with 2 mL of water and eluted with 2 mL of ethanol. After the ethanol solution was blown dry with a slow stream of N2 at 60°C, the 18F-labeled peptide was redissolved in phosphate-buffered saline (PBS) solution and passed through a 0.22-μm Millipore filter into a sterile multidose vial for in vivo experiments. Radiochemical yield was approximately 95%. The specific activity was approximately 37 TBq/mmol.
Preparation of MDA-MB-435 Breast Cancer Model
The MDA-MB-435 human breast carcinoma tumor model known to express medium levels of integrin αvβ3 (23) was used for the in vivo imaging studies. The MDA-MB-435 cell line was purchased from American Type Culture Collection and grown in Leibovitz's L-15 medium supplemented with 10% (v/v) fetal bovine serum under a 100% air atmosphere at 37°C. The MDA-MB-435 tumor model was generated by orthotopic injection of 5 × 106 cells in the left mammary fat pad of female athymic nude mice (Harlan Laboratories). The mice were used for studies when the tumor volume reached about 250 mm3 (∼10–14 d after implantation). Tumor growth was followed by caliper measurements of perpendicular diameters of the tumor. The tumor volume was estimated by the formula tumor volume = a × (b2)/2, where a and b were the tumor length and width, respectively, in millimeters. All animal experiments were performed in compliance with the guidelines for the care and use of research animals established by Stanford University's Animal Studies Committee.
Longitudinal Small-Animal PET Scans
PET scans and image analysis were performed using an Inveon microPET scanner (Siemens Medical Solutions). Each MDA-MB-435 tumor–bearing mouse was injected via the tail vein with 3.7 MBq (100 μCi) of 18F-FPPRGD2 or 7.4 MBq (200 μCi) of 18F-FDG under isoflurane anesthesia. Five-minute static scans were acquired at 1 h after injection. For the 18F-FDG scan, mice were maintained under isoflurane anesthesia during the injection, accumulation, and scanning periods and were kept fasting for 4 h before tracer injection. For the 18F-FPPRGD2 scan, mice were not anesthetized during the tracer accumulation period and were not kept fasting before tracer injection. The images were reconstructed and quantified using procedures described in the supplemental materials (supplemental materials are available online only at http://jnm.snmjournals.org).
As shown in Table 1, 40 mice underwent baseline PET with 18F-FPPRGD2 (n = 20) or 18F-FDG (n = 20) (day 0) when the tumors reached a size of around 250 mm3. Then the tumor-bearing mice were randomly divided into the following 4 groups (n = 10/group): 18F-FPPRGD2 control, 18F-FPPRGD2 treatment, 18F-FDG control, and 18F-FDG treatment. Mice in the treatment groups received 3 doses of Abraxane diluted in PBS, and control mice received PBS only. Doses of Abraxane (25 mg/kg) or PBS were administered on days 0, 2, and 4. PET scans were repeated on days 3, 7, 14, and 21. At each time point, 1 mouse from each group was sacrificed, and the tumors were excised for histopathology.
Experimental Design for Longitudinal 18F-FDG and 18F-FPPRGD2 Imaging of Abraxane Treatment Efficacy and Ex Vivo Histopathology
Fluorescence Staining
Frozen tumor tissue slices (8 μm) were fixed with cold acetone for 20 min and dried in the air for 30 min at room temperature. After blocking with 1% bovine serum albumin for 30 min, the sections were incubated with humanized antihuman integrin αvβ3 antibody Abegrin (24) (20 μg/mL) for 1 h at room temperature and then visualized with cy3-conjugated donkey antihuman secondary antibodies (1:300; Jackson ImmunoResearch Laboratories). For the overlay staining of CD31 and CD61, slices were incubated with rat antimouse CD31 antibody (1:200; BD Biosciences) and hamster anti-β3 CD61 antibody (1:200; BD Biosciences) and then visualized with cy3-conjugated goat antirat and fluorescein isothiocyanate–conjugated goat antihamster secondary antibody (1:300; Jackson ImmunoResearch Laboratories). For the overlay staining of Ki67 and F4/80, slices were incubated with rabbit antimouse Ki76 antibody (1:200; BD Biosciences) and rat antimouse F4/80 antibody (1:200; Abcam, Inc.) and then visualized with cy3-conjugated goat antirabbit and fluorescein isothiocyanate–conjugated goat antirat secondary antibody (1:300; Jackson ImmunoResearch Laboratories). After being washed 5 times with PBS, the slices were mounted with 4′-6-diamidino-2-phenylindole (DAPI)-containing mounting medium and observed under an epifluorescence microscope (X81; Olympus). Each experiment was performed in pairs, and the pairs were then repeated twice.
Statistical Analysis
Quantitative data were expressed as means ± SD. Means were compared using 1-way ANOVA and a Student t test. P values less than 0.05 were considered statistically significant. Unpaired Student t tests were used to evaluate differences between the 2 treatment groups, and paired Student t tests were performed for differences between time points on the activity curve within a treatment condition.
RESULTS
Abraxane Treatment–Inhibited MDA-MB-435 Tumor Growth
Abraxane is effective in delaying MDA-MB-435 tumor growth. Intravenous administration of 3 doses of Abraxane (25 mg/kg; days 0, 2, and 4) resulted in a reduction in tumor volume. As shown in Figure 1A, a time-related increase in tumor volume was observed in the PBS control group, in which the tumors showed an average fractional tumor volume (V/V0) of 1.27, 1.42, 1.51, 1.78, and 2.63 on days 3, 5, 7, 14, and 21, respectively. Abraxane treatment resulted in a V/V0 of 0.99, 0.78, 0.67, 0.51, and 0.65 on days 3, 5, 7, 14, and 21, respectively. The average tumor size of the control group became significantly smaller than that of the treatment group starting from day 5 (P < 0.05). In the treated mice, an increase of fractional tumor volume from day 14 (V/V0 = 0.51) to day 21 (V/V0 = 0.65) was noted. This pattern of initial response during and immediately after the treatment, and then regrowth after the treatment was stopped, is common in many other experimental cancer therapy studies and clinical cancer trials (25–27). Mouse body weight was monitored as an indicator of the toxicity of Abraxane. As evidenced in Figure 1B, it is clear that Abraxane had no observable side effects at the low dosage used in this study.
Antitumor activity of Abraxane in established MDA-MB-435 xenografts. (A) 3 doses of Abraxane (intravenous injection, 25 mg/kg/dose on days 0, 2, and 4) led to substantial tumor regression but also regrowth after day 14. (B) Body weight of animals treated with PBS or Abraxane.
PET with 18F-FPPRGD2 and 18F-FDG
Static PET images at 1 h after injection of 18F-FDG (Fig. 2) or 18F-FPPRGD2 (Fig. 3) were acquired at day 0 (baseline, before Abraxane treatment) and at days 3, 7, 14, and 21 after the Abraxane treatment was initiated. The tumor region-of-interest analysis is summarized in Figure 4. As shown in Figures 2 and 4A, there was little fluctuation of 18F-FDG uptake in the control mice at different days after tumor inoculation. Abraxane treatment led to some increase of 18F-FDG uptake at days 3 and 7, which returned to the baseline level at days 14 and 21. Figures 3 and 4B describe the tumor uptake pattern of 18F-FPPRGD2 with and without Abraxane treatment. After Abraxane treatment, the MDA-MB-435 tumor uptake of 18F-FPPRGD2 significantly decreased from 1.22 ± 0.08 percentage injected dose per gram (%ID/g) (day 0) to 1.08 ± 0.10 %ID/g (day 3, P < 0.05) and 0.63 ± 0.06 %ID/g (day 7, P < 0.01), which represents a decrease of up to 48%. At days 14 and 21, the 18F-FPPRGD2 in the treated mice increased to the level in the control mice, with tumor uptake of 1.42 ± 0.14 and 1.23 ± 0.19 %ID/g, respectively.
Representative decay-corrected whole-body coronal images of female athymic nude mice bearing orthotopic MDA-MB-435 tumors at 1 h after intravenous injection of 18F-FDG (7.4 MBq/mouse) on days 0, 3, 7, 14, and 21 after treatment was initiated. Increased uptake of 18F-FDG was observed on days 3 and 7, decreasing to baseline level on days 14 and 21. (A) PBS control. (B) Abraxane treatment.
Representative decay-corrected whole-body coronal images of female athymic nude mice bearing orthotopic MDA-MB-435 tumors at 1 h after intravenous injection of 18F-FPPRGD2 (3.7 MBq/mouse) on days 0, 3, 7, 14, and 21 after treatment was initiated. Decreased tumor uptake of 18F-FPPRGD2 was observed on days 3 and 7 but was restored to baseline level on days 14 and 21. (A) PBS control. (B) Abraxane treatment.
Quantitative small-animal PET region-of-interest analysis of tumor uptake for 18F-FDG (A) and 18F-FPPRGD2 (B).
Abraxane-Induced Inflammation and Inhibition of Proliferation
To evaluate the therapeutic effect of Abraxane on MDA-MB-435 tumors, we performed Ki67 immunofluorescence staining on tumor sections. The percentage of Ki67-positive cells (Ki67 staining index) in untreated tumors was around 72% ± 17%, independent of the tumor size (Fig. 5). Consistent with the tumor growth inhibition, significantly delayed cell proliferation was observed in the Abraxane-treated mice at days 3 and 7 with a Ki67 SI of 17% ± 7% and 22% ± 5%, respectively (P < 0.01) (Supplemental Fig. 1). The Ki67 SI was restored to the baseline level at days 14 and 21.
Ex vivo overlay staining of tumor sections from PBS- and Abraxane-treated MDA-MB-435 tumors on days 3, 7, and 14. Green = Ki67; red = F4/80; blue = DAPI.
The tumor sections were also stained against macrophage-specific marker F4/80 to evaluate Abraxane-induced inflammation. Compared with control tumors, a more pronounced inflammatory reaction was observed at days 3 and 7 after treatment. At day 14, F4/80-positive macrophages decreased significantly (P < 0.01) (Supplemental Fig. 2).
Effect of Abraxane on Tumor Integrin Expression
To further investigate the mechanism of altered 18F-FPPRGD2 uptake during and after Abraxane treatment, integrin expression was evaluated by immunofluorescence staining. Because integrin on tumor vascular endothelial cells was of murine origin and that on tumor cells was of human origin, we stained tumor sections with both antimurine integrin β3 and antihuman integrin αvβ3 antibodies. As shown in Figure 6A, MDA-MB-435 tumor cells had positive human integrin αvβ3 staining, which was unaffected by Abraxane treatment.
(A) Ex vivo human integrin staining of tumor sections from PBS- and Abraxane-treated MDA-MB-435 tumors on days 3, 7, and 14. (B) Overlay staining of CD31 (red), murine integrin β3 (green), and DAPI (blue) of tumor sections from PBS- and Abraxane-treated MDA-MB-435 tumors on days 3, 7, and 14.
Murine β3 was colocalized with murine CD31, indicating that the expression of murine integrin β3 was mainly on the tumor vascular endothelial cells. A significant morphologic change of tumor vasculature, including the collapse of microvessel cavities, was observed in Abraxane-treated tumors at different times. In addition, after Image J (National Institutes of Health) analysis, we found that the mean murine β3 fluorescence intensity decreased significantly at days 3 (P < 0.05) and 7 (P < 0.01) (Supplemental Fig. 3) after Abraxane treatment; the treated tumors and control tumors had similar vascular integrin levels at days 14 and 21, correlating with the 18F-FPPRGD2 imaging studies.
DISCUSSION
The ability to visualize and quantify integrin αvβ3 expression levels noninvasively in vivo provides new opportunities to document integrin expression, greatly assist the understanding of mechanisms of angiogenesis and antiangiogenic treatment, and monitor treatment efficacy. We have previously reported a series of radiolabeled RGD monomers and multimers for imaging integrin αvβ3 expression (18,22,28–32). Among them, 18F-FPPRGD2, with relatively high tumor integrin-specific accumulation and favorable in vivo kinetics, has made its way into the clinic for cancer diagnosis and for treatment response monitoring (22).
There have been several studies using suitably labeled monomeric RGD peptide tracers to monitor chemotherapy or antiangiogenic treatment efficacy. For example, paclitaxel therapy in Lewis lung carcinoma tumor–bearing mice significantly retarded tumor growth and was accompanied by a corresponding reduction of tumor glucosamino 99mTc-D-c(RGDfK) uptake, as determined by γ-camera imaging, correlating well with tumor αv integrin expression levels (33). Similarly, paclitaxel therapy reduced the microvessel density in Lewis lung carcinoma tumor–bearing mice and resulted in significantly reduced tumor uptake of 18F-AH111585. ZD4190 (a small-molecular-weight VEGF receptor-2 tyrosine kinase inhibitor) therapy resulted in a significant decrease in 18F-AH111585 uptake in Calu-6 tumors, compared with vehicle control-treated Calu-6 tumors (34). Dasatinib, an Src-family kinase inhibitor, reduced 64Cu-DOTA-c(RGDfK) uptake in a U87MG xenograft model. In contrast, tumor 18F-FDG uptake showed no significant reduction with dasatinib therapy (35). All these studies supported the feasibility of noninvasive imaging using RGD-based probes to monitor antiangiogenic therapy response.
In this study, we investigated the potential of 18F-FPPRGD2 to accurately assess therapeutic response of human mammary carcinomas to Abraxane treatment and to be translated into the clinic for integrin αvβ3 imaging. In vivo small-animal PET showed that there was a significant reduction in the tumor uptake of 18F-FFPPRGD2 as early as day 3 after the initiation of Abraxane treatment. Moreover, this reduction in tumor accumulation of 18F-FPPRGD2 was observed before a tumor size decrease could be discerned by caliper measurement, suggesting that 18F-FPPRGD2 can detect early responses preceding clinical regression. We also performed a comparison study with 18F-FDG as an imaging tracer. 18F-FDG showed increased tumor uptake at days 3 and 7, despite positive tumor response to Abraxane treatment.
It is worth mentioning that our longitudinal scans showed an initial decrease and subsequent restoration of 18F-FPPRGD2 after Abraxane treatment, as is consistent with the pattern of initial growth inhibition and tumor relapse after treatment is stopped. However, the change of 18F-FPPRGD2 uptake accurately predicted the tumor response a few days before the anatomic tumor size change could be accurately measured. Immunohistochemical staining also confirmed that the change of 18F-FPPRGD2 uptake reflected tumor vascular integrin level, rather than tumor cell integrin expression (which was unaffected by Abraxane treatment). The exact mechanism by which Abraxane affects the vascular integrin but not tumor cell integrin expression is not well understood and will be a subject of future studies.
Despite the cytoxicity of Abraxane, 18F-FDG uptake was somewhat increased at the early stage of treatment, possibly because of the combined effect of reduced tumor cell proliferation (Ki67 index) and increased macrophage infiltration (F4/80 staining) in regressing tumors. It has been well documented that 18F-FDG can show high uptake when inflammatory cells are present (36,37)—which is the potential limitation of 18F-FDG when used to monitor therapeutic response. 18F-FPPRGD2 is superior to 18F-FDG, because the uptake of 18F-FPPRGD2 seems not to be influenced by tumor-associated macrophages after therapy. Thus, it can identify MDA-MB-435 tumor response to Abraxane more accurately.
The promising imaging results elucidated in this longitudinal study suggest the usefulness of quantitative 18F-FPPRGD2 PET to evaluate treatments that produce an antiangiogenic effect and a change in vascular integrin level. However, we have to bear in mind that tumor uptake and accumulation of a given radiotracer are not solely dependent on receptor expression. Several other factors, including vascular density and volume, vascular permeability, and interstitial fluid pressure, also affect the distribution (38,39). To explore the usefulness of 18F-FPPRGD2 in monitoring treatment response, studies in various tumor models with different integrin αvβ3 expression patterns and different sensitivities to certain therapeutics should be conducted. In certain cases, receptor binding potential derived from dynamic scans, instead of tumor uptake or tumor-to-background ratio, might be needed to eliminate the influence of tumor microenvironment on the pharmacokinetics of 18F-FPPRGD2. 18F-FPPRGD2 alone or in combination with additional functional imaging modalities can enhance our mechanistic understanding of how novel molecular therapeutic strategies affect tumors and allow early prediction of treatment efficacy and potential relapse, leading to better individualization of chemotherapy.
CONCLUSION
Our data illustrate that 18F-FPPRGD2 is a promising PET tracer that allows noninvasive evaluation of response to treatments that affect integrin αvβ3 level, before size changes can be found. It also has the potential to provide earlier clinical opportunities to adjust anticancer drug doses and intervals to maintain sustained antitumor effect and avoid relapse. 18F-FPPRGD2 is superior to 18F-FDG in monitoring anticancer treatment, because 18F-FPPRGD2 imaging is not significantly affected by the presence of infiltrating macrophages in regressing tumors.
Acknowledgments
We thank Dr. Henry S. Eden for proofreading this manuscript. This project was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH); the Radiology and Imaging Sciences Department, NIH Clinical Center; and the Intramural Research Program, NIBIB, NIH.
- © 2011 by Society of Nuclear Medicine
REFERENCES
- Received for publication June 25, 2010.
- Accepted for publication September 17, 2010.
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