MRI with Magnetic Nanoparticles Monitors ... - Springer Link

B Academy of Molecular Imaging and Society for Molecular Imaging, 2010 Published Online: 18 June 2010

Mol Imaging Biol (2011) 13:314Y320 DOI: 10.1007/s11307-010-0357-2

RESEARCH ARTICLE

MRI with Magnetic Nanoparticles Monitors Downstream Anti-Angiogenic Effects of mTOR Inhibition Alexander R. Guimaraes,1,2,4 Robert Ross,3 Jose L. Figuereido,1,2 Peter Waterman,1,2 Ralph Weissleder1,2 1

Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA 3 Lank Center for Genitourinary Oncology, Dana Farber Cancer Institute, Boston, MA, USA 4 Division of Abdominal Imaging, Department of Radiology, Massachusetts General Hospital, Boston, MA, 02114, USA 2

Abstract Purpose: To study the effect of mammalian target of rapamycin (mTOR) inhibition on angiogenesis with magnetic resonance imaging (MRI) using magnetic iron oxide nanoparticles (MNP). Procedures: One million CAK-1 renal cell carcinoma cells were subcutaneously implanted into each of 20 nude mice. When tumors reached ∼750 μl, four daily treatment arms began and continued for 4 weeks: rapamycin (mTOR inhibitor) 10 mg/kg/day; sorafenib (VEGF inhibitor) high dose (80 mg/kg/day) and low dose (30 mg/kg/day); and saline control. Weekly MRI (4.7 T Bruker Pharmascan) was performed before and after IV MION-48, a prototype MNP similar to MNP in clinical trials. Vascular volume fraction (VVF) was quantified as ΔR2 (from multi-contrast T2 sequences) and normalized to assumed muscle VVF of 3%. Linear regression compared VVF to microvascular density (MVD) as determined by histology. Results: VVF correlated with MVD (R2 =0.95). VVF in all treatment arms differed from control (pG0.05) and declined weekly with treatment. VVF changes with rapamycin were similar to high-dose sorafenib. Conclusion: This study demonstrates noninvasive, in vivo anti-angiogenic monitoring using MRI of mTOR inhibition. Key words: Magnetic resonance imaging, MRI, Magnetic nanoparticle imaging, Ultrasmall superparamagnetic iron oxide nanoparticle, Renal cell cancer, mTOR, Angiogenesis

Introduction

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enal cell carcinoma (RCC) is the third most common genitourinary tumor with an estimated number of cases

Alexander R. Guimaraes and Robert Ross contributed equally as first authors to this article. Funding Funding provided by the Renal Spore-Dana Farber Cancer Institute & MGH-AstraZeneca Strategic Alliance Brief Article Significance—showing the application of steady state MRI with magnetic nanoparticles to monitor the anti-angiogenic effect of rapamycin on xenograft model in vivo. Correspondence to: Alexander Guimaraes; e-mail: [email protected]

numbering greater than 50,000 worldwide in 2007. Although surgery offers cure for a majority of patients with localized tumors (960%), 30-40% of patients will present with metastatic disease, offering a dismal prognosis. The cloning of the von Hippel-Lindau tumor suppressor gene has uncovered a role in upregulating multiple growth factors (e.g., hypoxia inducble factor (HIF)-1), and mammalian target of rapamycin (mTOR), some of which may have effects on angiogenesis through upregulation of vascular endothelial growth factor (VEGF) or platelet-derived growth factor. Drugs that inhibit the VEGF pathway, including sunitinib (Sutent®, Pfizer) and sorafenib (Nexavar®, Bayer/ Onyx), have been approved for renal cell cancer for their

R. Ross, et al.: MRI Molecular Imaging of mTOR Inhibition

anti-angiogenic effects. Inhibitors of the mammalian target of mTOR have also been approved for the treatment of metastatic RCC and have only recently been shown to have histologic anti-angiogenic effect. All three of these drugs modify the natural history of metastatic RCC and temsirolimus, in particular, improves overall survival in those with highly aggressive disease [1–3]. The mechanism of action of sorafenib is based on its selective inhibition of the VEGF receptor and as a result sorafenib has potent anti-angiogenic qualities [1, 2, 4–7]. The mTOR pathway is also implicated in angiogenesis, which may be either through its direct impact on the phosphatidylinositol 3-kinase pathway or indirectly through inhibition of HIF. Both of these mechanisms impact VEGF production, in addition to facilitating proliferation of endothelial cells through serine/threonine kinase Akt activation and anti-apoptotic mechanisms [8, 9]. Accumulating evidence also suggests mTOR to be a crucial component in angiogenesis [1, 2, 4, 8, 10–15]. Studies of non-invasive imaging based biomarkers of treatment efficacy can help to better understand the mechanism of action of anti-angiogenic agents both in models and patients with renal cell cancer. Recently dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been utilized to assess the vascular leak associated with anti-angiogenic therapies [12, 16–18]. DCE-MRI has been used to assess mTOR inhibition in animal models and has not demonstrated any significant changes in leak or changes correlating with histologic changes (e.g., microvessel density). MRI approaches using magnetic nanoparticles (MNP) have recently been validated for the examination of tumor vascularity in vivo [19, 20]. The MNP have profound T2* shortening properties. The MNP has a long-lived intravascular state with slow leakage and eventual phagocytosis by macrophage harboring in resident lymph nodes and the reticuloendothelial system. The long-lived intravascular properties of the MNP can be exploited for steady-state imaging of angiogenesis. The advantages of this imaging technique include the use of MRI with its higher soft-tissue resolution and the fact this technique employs steady-state vascularity measurements, obviating the need for complicated and unreliable input function calculations. Moreover, nanoparticles soon should be clinically available, making this technique readily translatable. This approach has been employed in preclinical models to monitor the anti-angiogenic effects of a vascular targeting agent in mouse models of cancer (e.g., pancreatic, fibrosarcoma) [20, 21]. The aim of this study was to further verify MRI using MNP as a robust, imaging biomarker of angiogenesis by evaluating the in vivo effects of mTOR inhibition on the vascularity of a mouse model of RCC, comparing these effects to an established anti-angiogenic agent, sorafenib. These findings may lend further support to utilizing this approach to uncover the subtle anti-angiogenic effects of novel chemotherapeutic strategies.

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Procedures Animal Model All studies were approved by the small animal institutional animal care and use committee at the Massachusetts General Hospital. One million CAK-1 (American Type Culture Collection) cells in 100 ul Matrigel (BD Biosciences; Bedford, MA, USA) were implanted into the right flanks of n=20 athymic nu/nu mice (MGH Cox 7). Tumors were allowed to reach the size of 500 mm3 prior to starting any treatment protocol. Mice were divided into four treatment arms (n=5/ arm) for the comparison of mTOR inhibition as compared to conventional anti-angiogenic therapies. All therapies were given by oral gavage and included the following: (a) highdose sorafenib (80 mg/kg/day), (b) low-dose sorafenib (30 mg/ kg/day), (c) rapamycin (10 mg/kg/day), and (d) control saline vehicle. Rapamycin was commercially available as an oral solution (1 mg/ml rapamycin) with inactive ingredients including the following: phosphatidylcholine, propylene glycol, mono- and di-glycerides, ethanol, soy fatty acids, ascorbyl palmitate, and polysorbate 80. Sorafenib (200 mg tablets) were crushed, weighed, and then dissolved in a solution of 0.5% methylcellulose and 0.4% polysorbate-80 to produce a suspension with a final concentration of 6 mg/ml. Animals were treated daily for 28 days and then euthanized using carbon monoxide.

Contrast Agent The MNP utilized was a dextran coated iron oxide nanoparticle measuring 26 nm with an R1 32.4 s−1 mM−1 and R2 130.5 s−1 mM−1 with a concentration of iron 13.55 mg/mL.

Magnetic Resonance Imaging MRI was performed at 4.7 T on a Bruker imaging system (Pharmascan, Karlsruhe, Germany). Animals were imaged at baseline and weekly until the termination of therapy. Animals were anesthetized during imaging with 1-1.5% inhaled isofluorane and monitored during imaging with respiratory monitoring. Imaging protocols included a Triplane and axial RARE localizer. Multi-slice multiecho T2weighted imaging was performed prior to and following intravenous injection of magnetic MNP (10 mg/kg Fe). The following parameters were utilized: flip angle=90°; matrix size (128×64); TR=2500 ms; TE=16 equally spaced echoes at 8.6 ms intervals ranging from 8.6 ms to 137 ms; field of view ðFOVÞ ¼ 4:24 2:12cm, slice thickness=1 mm. T1weighted imaging was performed following the administration of intravenous Gd-DTPA utilizing the following parameters: flip angle=90; matrix size (256 × 256); TR= 700 ms; TE=14 ms; field of view ðFOVÞ ¼ 4:24 2:12cm, slice thickness=1 mm. Vascular volume fractions (VVF) measures were calculated from the pre- and post-contrast MNP-enhanced images as described in detail elsewhere [19, 22, 23]. A fundamental

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assumption is that the change in the transverse relaxation rate (ðR2 ¼ 1=T2Þ and ðR2 ¼ 1=T2Þ) relative to the pre-injection baseline is proportional to the perfused local blood volume per unit tumor volume (V) multiplied by a function (f) of the plasma concentration of the agent (P). R2 ¼ k f ðPÞ V Assuming a steady state for MNP distribution, the R2 was fit by using a mono-exponential fitting algorithm for the multi-TE data (Osirix). A region of interest incorporating the center three to four slices of the tumor as well as nearby muscle within the same slices were analyzed prior to and following MNP administration. ΔR2 was then converted to absolute tumoral VVF by scaling measurements to muscle with a known VVF of 3%. In addition, analysis was performed on a pixel-by-pixel basis to further assess geographic distribution of VVF. Data are reported as VVF±standard error of the mean. Statistical analysis, using a two-tailed paired t test, compared different time points for each treatment paradigm (GraphPad Prism 4.0, La Jolla, CA, USA).

Histology and Immunohistochemistry After 28 days of therapy, following MRI, animals were euthanized and tumors were excised, frozen in Optimal Cutting Temperature compound (Sakura Finetek, Tokyo, Japan) and sectioned in 5 μm slices. Adjacent sections were then incubated with 0.3% hydrogen peroxide to inhibit endogenous peroxidase activity and then incubated with primary rat anti-mouse CD31 (PECAM-1; BD Pharmingen), rat anti-mouse VEGF (Abcam), or rabbit polyclonal phospho-p70 S6 kinase (Thr421/ Ser424; cell signaling) antibodies. After washing with phosphate-buffered saline, species-appropriate biotinylated secondary antibodies were applied, followed by avidin-peroxidase complex (Vectastain ABC kit; Vector Laboratories). The reaction was visualized with 3-amino-9-ethyl carbazole substrate (Sigma Chemical Co). Sections were counterstained with Mayer’s hematoxylin solution (Sigma) and mounted. Images were captured with a digital camera (Nikon DXM1200-F, Nikon Inc, Melville, NY, USA) using imaging software ACT-1 (version 2.63). To determine microvessel density (MVD), three representative sections per tumor were analyzed. Results are reported as mean vessel number±SD per 20× high power field. Linear regression analysis compared MRI VVF measurements to MVD. Statistical analysis using a two-tailed unpaired t test compared each treatment paradigm (GraphPad Prism 4.0, La Jolla, CA, USA).

Results VVF Correlates with Histologic MVD Measurements T1-weighted MRI axial images of mice status post xenograft implantation of RCC (CAK-1) xenograft in the right flank

(Fig. 1a) and a three-dimensional volume rendered image of the mouse abdomen and pelvis and entire tumor (Fig. 1b). Superimposed over the tumor is a pseudocolorized VVF map whose voxel color, and color bar on the far left is representative of the VVF. Note the heterogeneous vascularity throughout the tumor revealed by MNP-enhanced MRI with correlative CD31-stained histlogic slice from the corresponding center slice of the tumor (Fig. 1c). Quantitative analysis using mean VVF was compared to histologically derived CD-31-stained MVD. Least squares linear regression analyses was performed comparing VVF to MVD demonstrating excellent correlation R2 =0.95 (pG0.05; Fig. 1d).

MRI Using VVF Demonstrates Downstream Anti-Angiogenic Effects of Mtor Inhibition Figure 2 demonstrates T1-weighted MRI of mice with pseudocolorized VVF maps superimposed over the center of the tumor within the right flank for each treatment arm (rapamycin (top row), sorafenib (middle row), and control (bottom row)) of the trial at baseline, and at the end of therapy. Note the heterogeneity of signal intensity and color spread throughout all cohorts at baseline, with marked decrease in vascularity in the rapamycin (top row)- and sorafenib (high dose—middle row)-treated animals as compared to control (bottom row). Of note, only three mice survived all 3 weeks of therapy within the rapamycin-treated arm, with two mice perishing from respiratory failure of unknown etiology. VVF was quantified each week in each cohort and graphed (mean±SEM; Fig. 2d). Note the parallel, rapid decrease in VVF in both the sorafenib- and rapamycintreated cohorts as compared to control with a statistically significant decrease in VVF (pG0.05) at week3 relative in both cohorts relative to differences noted in the control cohort. Although there was a decrease in mean VVF in control mice, there was no statistically significant difference in VVF comparing week3 to baseline measurements in control mice. MRI VVF measurements also demonstrated dose response differences between low and high-dose sorafenib most notable and exceptional at week2, but these differences did not demonstrate statistical significance (Fig. 1e). Histologic analysis (Fig. 3) demonstrates decreased anti CD-31 (first row) and anti-VEGF (second row) staining within the rapamycin- (second column) and sorafenib (third column)-treated mice demonstrating expected downstream anti-VEGF effect of sorafenib, and concordant, anti-VEGF downstream effect of rapamycin, with no such effect noted in the untreated, control arm (first column). Furthermore, when phospho-p70 antibody was stained (third row), there was diffuse, patchy staining throughout the sorafenib- and control-treated cohorts, with expected marked, decreased staining noted in the rapamycin cohort, which confirmed the mTOR inhibitory effect within this cohort. There were statistically significant differences in p70 staining and VEGF staining comparing rapamycin treated to control mice values (pG0.0001, labeled *). As well, there were statistically


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Fig. 1. T1-weighted MRI axial images of mice status post xenograft implantation of RCC (CAK-1) xenograft in the right flank (a) and a three-dimensional volume rendered image of the mouse abdomen and pelvis and entire tumor (b). Superimposed over the tumor is a pseudocolorized VVF map whose voxel color, and color bar on the far left is representative of the VVF. Note the heterogeneous vascularity throughout the tumor revealed by MNP enhanced MRI with correlative CD-31 stained histlogic slice from the corresponding center slice of the tumor (c). Quantitative analysis using mean VVF was compared to histologically derived CD-31 stained MVD. Least squares linear regression analyses was performed comparing VVF to MVD demonstrating excellent correlation R2 =0.95 (pG0.05) (d).

significant differences in p70 and VEGF staining comparing sorafenib treated-mice to rapamycin-treated mice (pG0.0001, labeled +). Interestingly, however, there was a statistically significant difference in p70 staining comparing sorafenibtreated mice to control mice (pG0.001, labeled *).

Discussion MRI provides high spatial resolution, non-invasive imaging of anatomy with high soft tissue contrast. We have shown in various xenograft, murine models, that MRI enhanced with intravenously administered, long-circulating MNP provides a non-invasive, sensitive assessment of VVF, which is a surrogate marker of MVD, and angiogenesis [19, 20, 24]. We have further validated this technology with high correlation to histologic gold standards (MVD; R2 =0.95) in a RCC (CAK-1) xenograft model, and have shown in vivo evidence and temporal, non-invasive monitoring of downstream anti-angiogenic effects of mTOR inhibition through rapamycin treatment within this model, which were as efficacious as known anti-angiogenic therapies. This technology, further, was highly sensitive and demonstrated dose response with statistically significant differences in VVF comparing high dose to low-dose anti-angiogenic, specifi-

cally sorafenib, therapies. This difference in VVF did not last through the entire therapeutic time of treatment and a possible explanation may include that the therapeutic response maximized earlier in the high-dose cohort than in the low-dose cohort. Histologic analyses demonstrated expected anti-angiogenic effect of sorafenib treatment. As well, there was similar antiangiogenic effect from rapamycin treatment. Quantitative analysis confirmed decreases in MVD in both the high-dose sorafenib and rapamycin-treated mice, which approached, but did not reach statistical significance (pG0.10). The finding that VVF decreases were statistically significant in these two cohorts (high-dose sorafenib and rapamycin) as compared to control animals, suggests that this technique may be an improved, non-invasive surrogate biomarker of anti-angiogenic therapy. Quantitative, histologic analysis of phospho-p70 antibody staining demonstrate statistically significant differences comparing rapamycin-treated animals as compared to both control and sorafenib-treated animals. The finding of decreased phospho-p70 staining in the sorafenib-treated cohort was not statistically significant when compared to control. Furthermore, VEGF antibody staining demonstrated statistically significant decreases in the rapamycin-treated

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Fig. 2. T1-weighted MRI of mice with pseudocolorized VVF maps superimposed the center of the tumor within the right flank for each treatment arm (rapamycin (top row), sorafenib (middle row), and control (bottom row)) of the trial at baseline, and at the end of therapy. Note the heterogeneity of signal intensity and color spread throughout all cohorts at baseline, with marked decrease in vascularity in the rapamycin (top row)- and sorafenib (high dose—middle row)-treated animals as compared to control (bottom row). Of note, only three mice survived all 3 weeks of therapy within the rapamycin-treated arm. VVF was quantified each week in each cohort and graphed (mean±SEM; Fig. 2d). Note the parallel, rapid decrease in VVF in both the sorafenib and rapamycin-treated cohorts as compared to control with a statistically significant decrease in VVF (pG0.001) at week3 relative in both cohorts relative to control. Although there was a decrease in mean VVF in control mice, there was no statistically significant difference in VVF comparing week3 to baseline measurements in control mice. MRI VVF measurements also demonstrated dose response differences between low and high-dose sorafenib most notable and exceptional at week2 (pG0.05; Fig. 2e).

cohort as compared to the control, while not demonstrating any significant difference between the control and the sorafenib-treated cohort. There was, however, a statistically significant difference between the rapamycin-treated cohort and the sorafenib-treated cohort, which supports the upregulation of VEGF when comparing these two different treatment arms. In summary, our results demonstrate that MRI measures of VVF quantify changes following targeted therapies. MRI VVF correlates highly to histopathologic indices of MVD and may serve as a surrogate marker of angiogenesis confirming previous results. Furthermore, the sensitivity of this technique may provide physiological insight into potential downstream anti-angiogenic effects associated with other chemotherapeutic strategies, may serve as a preclinical outcome measure of dose scheduling given the accuracy in discriminating between low and high-dose dosing schedules, and, lastly, may allow for pretreatment determination of potential therapeutic efficacy with those tumors

demonstrating low vascular volume fraction, possibly precluding anti-angiogenic treatement strategies [19, 20, 24].

Summary Statement In summary, our results demonstrate that MRI measures of VVF using magnetic nanoparticles quantify changes following targeted chemotherapeutic strategies with excellent correlation to histopathologic indices of MVD and may serve as a surrogate marker of angiogenesis. Acknowledgements. The authors would like to acknowledge grant support from the Dana Farber Renal Spore where Dr. Ross, received funding to support this work from a career development award. In addition, the authors would like to acknowledge funding support from the AstraZeneca Pharmaceuticals, Inc. where Dr. Guimaraes received funding to support part of this work. Lastly, the authors would like to acknowledge Claire Kaufman and Carlos Rangel for technological support in imaging and data analysis


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Fig. 3. Histologic analysis demonstrates expected and concordant decreased anti CD-31 (first row) and anti-VEGF (second row) staining within the rapamycin (second column)- and sorafenib (third column)-treated mice demonstrating expected downstream anti-VEGF effect of sorafenib, and concordant, anti-VEGF downstream effect of rapamycin, with no such effect noted in the untreated, control arm (first column). Furthermore, when phospho-p70 antibody was stained (third row), there was diffuse, patchy staining throughout the sorafenib and control-treated cohorts, which confirmed the lack of mTOR inhibition from these two strategies. In converse, there was expected, marked, decreased staining noted in the rapamycin cohort, which confirmed the mTOR inhibitory effect. There were statistically significant differences in p70 staining and VEGF staining comparing rapamycin treated to control mice values (pG0.0001, labeled *). As well, there were statistically significant differences in p70 and VEGF staining comparing sorafenib-treated mice to rapamycin-treated mice (pG0.0001, labeled +). Interestingly, however, there was a statistically significant difference in p70 staining comparing sorafenib-treated mice to Control mice (pG0.001, labeled *).

Appendix The theory behind the use of superparamagnetic contrast agents with long vascular half lives to determine blood volumes by MRI, which is supported by detailed numerical simulations, is that the change in gradient-echo transverse relaxation rate (ΔR2*) relative to the pre-injection baseline is proportional to the local blood volume times some function of the plasma concentration of the agent or R2 ¼ k f ðPÞ V . If a steady state concentration of the contrast agent is assumed in the blood plasma then there is a simple linear relationship between ΔR2* and blood volume at any time (t) and the formula reduces to R2ðtÞ ¼ K V ðtÞ. Stated another way, V ðtÞ ¼ ½R2ðtÞ=K where V(t) is the blood volume, ΔR2* (t) is the change in the transverse relaxation rate of the region, and the constant K includes the contrast agent blood pool concentration and is therefore dose dependent. While this technique allows for easy measurement of total blood volume in a given voxel, tissue slice, or entire organs, additional methods more sensitive to microvessels have also been developed.

These methods are based on the property that compartmentalization of these magnetic nanoparticles also induces long-range magnetic field perturbations that extend over many microns and increase both transverse relaxation rates, R2 and R2*, of the tissues. The enhancement of R2 and R2* caused by these agents can be expressed as follows: R2 ¼ 1 T 2post 1 T 2pre 1=TE ln Spost Spre a n d R2 ¼ 1 T2post 1 T2pre 1=TE ln Spost Spre where S is the signal intensity, TE the echo time, and T2 is the transverse relaxation time. It has previously been shown that there exists a unique relationship between ΔR2 (spin echo), ΔR2* (gradient echo) as a function of vessel diameter and contrast agent concentration. The ΔR2 peaks for vessels 1-2 μm in diameter whereas ΔR2* is fairly independent of vessel size beyond 3-4 μm. These studies thus provide the rationale for spin-echo imaging being more sensitive to microvasculature, while gradient-echo imaging is more sensitive to total vasculature. Ultimately, gradient echo/spin ratio imaging could theoretically be utilized to measure vessel size noninvasively, as the ΔR2*/ΔR2 ratio increases nearly linearly with vessel size. Sequestered magnetic nanoparticles, as occurs with uptake by

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