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JMA74.pdf
Dynamic Fluorescence Imaging For The Detection of Vascular Changes in Anti-Angiogenic Drug Therapy Jonghwan Lee1, Thomas Pöschinger4, Sonia L. Hernandez2, Jianzhong Huang3, Tessa Johung3, Jessica Kandel3, Darrell J. Yamashiro2, Andreas H. Hielscher1,5 1Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, 500 West 120th St., New York, NY 10027 2Department of Pediatrics, Columbia University, 630 West 168th Street, New York, NY 10032 3Department of Surgery, Columbia University, 177 Fort Washington Ave., New York, NY 10032 4 Institut für Medizinische Physik ,Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestr. 91, 91052 Erlangen, Germany 4Department of Radiology, Columbia University, 630 West 168th St., New York, NY 10032 E-mail:
[email protected]
Abstract : We show that dynamic fluorescence imaging with indocyanine green can be used to detect changes in the vasculature of a small animal Ewing sarcoma model in response to antiangiogenic drug treatments. !2009 Optical Society of America OCIS codes!"(170.3880) Medical and Biological Imaging;
1. Introduction The inhibition of tumor angiogenesis has been emerging as an efficient therapeutic strategy for eliminating neoplastic tumor vasculature [1-4]. Four anti-angiogenic drugs have been approved by FDA since 2004. These agents, especially bevacizumab (Avastin®) target mostly vascular endothelial growth factor (VEGF), which is ubiquitously expressed in almost all human tumors. Although, there have been promising clinical results, diverse responses of different tumor types to VEGF blockade have appeared. To obtain a robust quantification of angiogenesis or anti-angiogenesis responses, a reliable monitoring method for angiogenic effects is highly desirable [3,5]. While many candidate biomarkers were tested in several studies, there are no validated biomarkers of antiangiogenesis for routine preclinical and clinical use and most of the early detection studies have used invasive methods including the tumor excision [5-7]. In this study, we hypothesize that dynamic fluorescence imaging with Indocyanine Green (ICG) is sufficiently sensitive to detect changes in tumor vasculature in response to anti-angiogenic therapy. ICG is a watersoluble tricarbocyanine dye that is well characterized and clinically approved for use in humans. It has an intense emission in the near infrared wavelengths range, where light experiences lower scattering and higher penetration in the blood and other tissues [8,9]. 2. Method We intrarenally implanted human Ewing sarcoma cells engineered to express luciferase (SK-NEP1-luc) in 8 NCR nude mice. After anesthetizing mice with IP injection of the mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg), the left flank prepared in a sterile manner was incised to expose the left kidney and an inoculum of 106 SKNEP1-luc tumor cells in 0.1 ml of PBS was injected with a 25g needle. The flank muscles and the skin were closed with a suture and staples respectively. The tumors were allowed to grow until they reached approximately 1g as assessed by biweekly bioluminescence measurement with a Xenogen IVIS apparatus. The treatment schedule consisted of!20 mg/kg bevacizumab every 3 days intravenous injections. Bioluminescence, autofluorescence and dynamic fluorescence imaging were performed with a Maestro 2 In Vivo imaging system (CRI, Inc., Woburn, MA). For the bioluminescence measurements we used 3 minutes exposure time and a 560 nm (± 10 nm) emission filter set. For the dynamic fluorescence imaging, we used 65 ms exposure time, a 704 nm ((± 20 nm) ) excitation filter set, a 820 nm (± 10 nm) emission filter set and a 5 frames/second imaging frame rate. All imaging data sets of autofluorescence and dynamic fluorescence were calibrated based on ICG degradation measurement and summed up respectively. The autofluorescence signal was subtracted from the dynamic fluorescence signal. Figure 1. The experimental set up in the Figure 1 shows a photo of the experimental set-up. Two 45-degree side system chamber. mirrors allow one to view three sides of the animal and two directional
!
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JMA74.pdf tumor views were used to evaluate the fluorescence signal. The mice were anesthetized by the gas mixture of 1.8% isofluorene and oxygen and the breathing rate was maintained around 60 bits/minutes. For the injection of luciferin, ICG and bevacizumab, the tail vein was catheterized using a 30g needle with long tubing. Before the first treatment with bevacizumab, we acquired bioluminescence and fluorescence baseline data. Bioluminescence images were obtained 2 minutes after the injection of 70 !L luciferin through the IV catheter. 30 Minutes after the bioluminescence imaging, without changing the mice position, an autofluorescene image of the animals were obtained and this was immediately followed by an ICG injection and the acquisition of the dynamic fluorescence images.. The dynamic fluorescence image data was acquired for 3 minutes, starting from the intravenous injection time of the 60 !L, 200 !M ICG in saline. A total of 900 frames of data (5 frames/second) were acquired. In addition to these baseline measurements, both bioluminescence and fluorescence images were taken at 1 hour, 24, 72, and 168 hours following the first bevacizumab injection. For each mouse study, one batch of ICG was used and its degradation was measured at each time points.
(a) (b) (c) Figure 2. (a) Bioluminescence signal on white light image of mouse inside the Maestro 2 In Vivo imaging system chamber (b) Processed dynamic fluorescence imaging data (brain:magenta, lungs-yellow, liver-red, healthy kidney-green, intestine –light blue, tumor-dark blue (c) time trace of ICG uptake and washout in different organs and the tumor (same color code as Fig 2b)
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Figure 3.(A) Signal changes in the ROI at different time points. Bevacizumab was injected after 0 and 72 hour measurement. (B) Col IV stained image of excised tumor of untreated mouse showing tumor vasculature. (C) Col IV stained image of excised tumor of treated mouse (96 hours after first Avastin injection) showing vascular normalization.!
! 3. Result Since the Ewing sarcoma cells were engineered to express luciferase, the bioluminescence images were used to ! the tumor (Fig. 2a). However, since luciferase is express by all tumor cells, the bioluminescence locate measurements cannot be used to image the vascular response to the drug therapy. Indeed the size of the tumor as determined by bioluminescence remained virtually constant over the 7-day treatment period. To study the vascular response, we used the DyCE software package developed by Hillman et al [9]. Using this software we selected a region of interest (ROI) provided by the bioluminescence image (Fig. 2a) and generated an ICG uptake and washout curve from all pixels in that region (blue curve in Fig. 2b). In addition, by selecting estimated areas of other organs, such as brain, lung, liver, intestines, kidney etc, additional organ specific ICG uptake traces were produced (Fig. 2b). Based on these uptake and washout time traces, the DyCE software generated a color-coded organ and tumor map, in which regions are identified with similar uptake characteristics. As an
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JMA74.pdf example, Fig. 2c shows such a map for a tumor -bearing mouse and a tumor area marked blue overlaps with the ROI identified with the bioluminescence imaging. This tumor has completely replaced the left kidney. The healthy kidney can be seen in green contra-laterally. In all tumor-bearing mice we found marked difference in the ICG dynamics between the tumor-bearing kidney and the healthy kidney. The ICG uptake in the tumor is consistently slower than in the unaffected contralateral kidney. Furthermore, following the treatment with bevacizumab we observed an increase in the ICG-uptake speed in the tumor region within 72 hours. We also integrated the fluorescence intensity over the area identified by bioluminescence imaging and plotted the resulting total fluorescence signal as a function of time. Unlike the bioluminescence signal, which varied little over the 7-day period, we observed a marked drop in the total fluorescence signals (Fig. 3a). These observations are in agreement with a so-called vascular normalization that occurs as remodeling of larger vessels and pruning of smaller ones is induced by loss of VEGF signaling [10]. We confirmed that vascular remodeling occurs with well-established immunostaining methods using anti-PECAM-1, "SMA antibodies, and Col IV for endothelial and vascular mural cells (Fig. 3b,c). 4. Summary We investigated the potential of dynamic fluorescence imaging for monitoring tumor vasculature during antiangiogenic therapy. Using an Ewing sarcoma model in NCR mice, we examined the ICG fluorescence signal changes over a 7 day period following treatment initiation with bevacizumab. We observed changes in the ICG uptake and washout dynamics in the intrarenally implanted human tumor as well as changes in the total fluorescence signal over time. This work was supported in part by a Herbert Irving Comprehensive Cancer Center inter-programmatic pilot project grant funded by the National Cancer Institute NCI (2 P30 CA013696-35), and as several other NCI grants (4R33CA118666 [AHH], K08CA107077 [JH], R21 CA139173[JK], R01CA124644 [DY]). 5. References [1]C. Sessa, A. Guibal, G. D. Conte, C. Rüegg, “Biomarkers of angiogenesis for the development of antiangiogenic therapies in oncology:tools or decorations? ” Nature Clinical Practice Oncology, Vol 5, No 7(2008) [2] Y.R. Kim, A. Yudina, J. Figueiredo, W. Reichardt, D. Hu-Lowe, A. Petrovsky, H.W. Kang, D Torres, U. Mahmood, R. Wissleder, A. A. Bogdanov, Jr. “Detection of Early Antiangiogenic Effects in Human Colon Adenocarcinoma Xenografts: In vivo Changes of Tumor Blood Volume in Response to Experimental VEGFR Tyrosine Kinase Inhibitor ” Cancer Res 2005; 65: (20) (2005) [3]F. Kabbinavar, H.I. Hurwitz, L.Fehrenbacher, N.. Meropol, W.F. Novotny, G. Lieberman, S. Griffing, E. Bergsland, “Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer,” J. Clin. Oncol. 21,pp. 60–65 (2003). [4]H. Hurwitz, L. Fehrenbacher, W. Novotny, T. Cartwright, J. Hainsworth, W. Heim, J. Berlin, A. Baron, S. Griffing, E. Holmgren, N. Ferrara, G. Fyfe, B. Rogers, R. Ross, F. Kabbinavar, “Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer,” N. Engl. J. Med. 350, pp. 2335–2342 (2004). [5]K.J. Kim, B. Li, J. Winer, M. Armanini, N. Gillett, H.S. Phillips, N. Ferrara, “Inhibition of vascular endothelial growth factorinduced angiogenesis suppresses tumour growth in vivo,” Nature 362, pp. 841–844 (1993). [6]N.R. Smith, N.H. James, I. Oakley, A. Wainwright, C. Copley, J. Kendrew, et al, “Acute pharmacodynamic and antivascular effects of the vascular endothelial growth factor signaling inhibitor AZD2171 in Calu!6 human lung tumor xenografts,” Molecular Cancer Therapeutics 6, pp. 2198!2208 (2007). [7]D.W. Miller, S. Vosseler, N. Mirancea, D.J. Hicklin, P. Bohlen, H.E. Volcker, F.G. Holz and N.E. Fusenig, “Rapid Vessel Regression, Protease Inhibition, and Stromal Normalization upon Short-Term Vascular Endothelial Growth Factor Receptor 2 Inhibition in Skin Carcinoma Heterotransplants,” American Journal of Pathology 167(5), pp. 1389!1403 (2005). [8]V. Saxena, M. Sadoqu, J. Shao “Polymeric nanoparticulate delivery system for indocyanine green: Biodistribution in healthy mice” international journal of Pharmaceutics 308, 200-203 (2006). [9]E.M.C. Hillman, A. Moore “All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast ” Nature photonics, vol 1(2007) [10]R.T. Tong, Y. Boucher, S.V. Kozin, F. Winkler, D.J. Hicklin, R.K. Jain, "Vascular Normalization by Vascular Endothelial Growth Factor Receptor 2 Blockade Induces a Pressure Gradient Across the Vasculature and Improves Drug Penetration in Tumors", Cancer Research 64, 3731-3736 (2004).
