Radiology Molecular Imaging

[Molecular Imaging] Human Breast Cancer Tumor Models: Molecular Imaging of Drug Susceptibility and Dosing during HER2/neu-targeted Therapy

2008-08-18

Purpose: To use near-infrared (NIR) optical imaging to assess the therapeutic susceptibility and drug dosing of orthotopic human breast cancers implanted in mice treated with molecularly targeted therapy.

Materials and Methods: This study was approved by the institutional animal care and use committee. Imaging probes were synthesized by conjugating the human epidermal growth factor receptor type 2 (HER2)-specific antibody trastuzumab with fluorescent dyes. In vitro probe binding was assessed with flow cytometry. HER2-normal and HER2-overexpressing human breast cancer cells were orthotopically implanted in nude mice. Intravital laser scanning fluorescence microscopy was used to evaluate the in vivo association of the probe with the tumor cells. Mice bearing 3–5-mm-diameter tumors were intravenously injected with 0.4 nmol of HER2 probe before or after treatment. A total of 123 mice were used for all in vivo tumor growth and imaging experiments. Tumor fluorescence intensity was assessed, and standard fluorescence values were determined. Statistical significance was determined by performing standard analysis of variance across the imaging cohorts.

Results: HER2 probe enabled differentiation between HER2-normal and HER2-overexpressing human breast cancer cells in vitro and in vivo, with binding levels correlating with tumor trastuzumab susceptibility. Serial imaging before and during trastuzumab therapy revealed a significant reduction (P < .05) in probe binding with treatment and thus provided early evidence of successful HER2 inhibition days before the overall reduction in tumor growth was apparent.

Conclusion: NIR imaging with HER2-specific imaging probes enables evaluation of the therapeutic susceptibility of human mammary tumors and of drug dosing during HER2-targeted therapy with trastuzumab. This approach, combined with tomographic imaging techniques, has potential in the clinical setting for determining patient eligibility for and adequate drug dosing in molecularly targeted cancer therapies.

[Molecular Imaging] Dual-targeted Contrast Agent for US Assessment of Tumor Angiogenesis in Vivo

2008-08-18

Purpose: To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and _vβ₃ integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB_D) with that from single-targeted MBs (MB_S) in a murine model of tumor angiogenesis.

Materials and Methods: Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti–_vβ₃ integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G–labeled control MBs (MB_C) and nontargeted nonlabeled MBs (MB_N) were tested with VEGFR2-positive and _vβ₃ integrin–positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material–enhanced US by using anti-VEGFR2–targeted MBs (MB_V), anti-_vβ₃ integrin–targeted MBs (MB_I), MB_D, and MB_C were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, _vβ₃ integrin, and CD31.

Results: Attachment of MB_D to SVR cells (mean, 0.74 MBs per cell ± 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 ± 0.03), and attachment to SVR cells was higher for MB_D than for MB_V (mean, 0.58 ± 0.09), MB_I (mean, 0.42 ± 0.21), MB_C (mean, 0.11 ± 0.13), and MB_N (mean, 0.01 ± 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB_D (mean, 16.7 ± 7.2) than for MB_V (mean, 11.3 ± 5.7), MB_I (mean, 7.8 ± 5.3), MB_C (mean, 2.8 ± 0.9), and MB_N (mean, 1.1 ± 0.4). Immunofluorescence confirmed expression of VEGFR2 and _vβ₃ integrin on tumor vasculature.

Conclusion: Dual-targeted contrast-enhanced US directed at both VEGFR2 and _vβ₃ integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice.

Supplemental material: http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1

[Molecular Imaging] Imaging Hypoxia in Orthotopic Rat Liver Tumors with Iodine 124-labeled Iodoazomycin Galactopyranoside PET

2008-07-18

Purpose: To evaluate iodine 124 (¹²⁴I)-labeled iodoazomycin galactopyranoside (IAZGP) positron emission tomography (PET) in the detection of hypoxia in an orthotopic rat liver tumor model by comparing regions of high ¹²⁴I-IAZGP uptake with independent measures of hypoxia and to determine the optimal time after injection to depict hypoxia.

Materials and Methods: The institutional animal care and use committee approved this study. Morris hepatoma tumors were established in the livers of 15 rats. Tumor oxygenation was measured in two rats with a fluorescence fiberoptic oxygen probe. ¹²⁴I-IAZGP was coadministered with the established hypoxia markers pimonidazole and EF5 in nine rats; 12-hour PET data acquisition was performed 24 hours later. Tumor cryosections were analyzed with immunofluorescence and autoradiography. In the four remaining rats, serial 20- and 60-minute PET data acquisition was peformed up to 48 hours after tracer administration.

Results: Oxygen probe measurements showed severe hypoxia (<1 mm Hg) distributed evenly throughout tumor tissue. Analysis of cryosections showed diffuse homogeneous uptake of ¹²⁴I-IAZGP throughout all tumors. The ¹²⁴I-IAZGP distribution correlated positively with pimonidazole (r = 0.78) and EF5 (r = 0.76) distribution. Tracer uptake in tumors was detectable with PET after 24 hours in seven of nine rats. In rats that underwent serial PET, tumor-to-liver contrast was sufficient to enable detection of hypoxia between 6 and 48 hours after tracer administration. The optimal ratio between signal intensity and tumor-to-liver contrast occurred 6 hours after tracer administration.

Conclusion: Regions of high ¹²⁴I-IAZGP uptake in orthotopic rat liver tumors are consistent with independent measures of hypoxia; visualization of hypoxia with ¹²⁴I-IAZGP PET is optimal 6 hours after injection.

[Molecular Imaging] In Vivo Near-infrared Fluorescence Imaging of Carcinoembryonic Antigen-expressing Tumor Cells in Mice

2008-05-16

Purpose: To prospectively depict carcinoembryonic antigen (CEA)-expressing tumors in mice with a high-affinity probe consisting of a near-infrared (NIR) fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab.

Materials and Methods: This study was approved by the regional animal committee. By coupling a NIR fluorescent (NIRF) cyanine dye (DY-676) to a specific antibody fragment directed against CEA (arcitumomab) and a nonspecific IgG Fab fragment, a bio-optical high-affinity fluorescent probe (anti-CEA–DY-676) and a low-affinity fluorescent probe (FabIgG–DY-676) were designed. The dye-to-protein ratios were determined, and both probes were tested for NIRF imaging in vitro on CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells by using a bio-optical NIR small-animal imager. In vivo data of xenografted LS-174T and A-375 tumors in mice (n = 10) were recorded and statistically analyzed (Student t test).

Results: The dye-to-protein ratios were determined as 3.0–3.5 for both probes. In vitro experiments revealed the specific binding of the anti-CEA–DY-676 probe on CEA-expressing cells as compared with CEA-nonexpressing cells; the FabIgG–DY-676 probe showed a markedly lower binding affinity to cells. In vivo LS-174T tumors xenografted in all mice could be significantly distinguished from A-375 tumors with application of the anti-CEA–DY-676 but not with that of the FabIgG–DY-676 at different times (2–24 hours, P < .005) after intravenous injection of the probes. Semiquantitative analysis revealed maximal fluorescence signals of anti-CEA–DY-676 to CEA-expressing tumors about 8 hours after injection.

Conclusion: Findings of this study indicate the potential use of the high-affinity probe anti-CEA–DY-676 for specific NIRF imaging in in vivo tumor diagnosis.

[Molecular Imaging] Postinfarction Myocardial Scarring in Mice: Molecular MR Imaging with Use of a Collagen-targeting Contrast Agent

2008-05-16

Purpose: To prospectively evaluate a gadolinium-based collagen-targeting contrast agent, EP-3533, for in vivo magnetic resonance (MR) imaging of myocardial fibrosis in a mouse model of healed myocardial infarction (MI).

Materials and Methods: All procedures were performed in accordance with protocols approved by the animal care and use committee. MI was induced in eight mice by means of occlusion of the left anterior descending coronary artery followed by reperfusion. Four MR examinations were performed in each animal: one examination before, one examination 1 day after, and two examinations 6 weeks after the MI. For the latter two examinations, electrocardiographically gated inversion-recovery gradient-echo MR images were acquired before and serially (every 5 minutes) after the intravenous injection of either gadopentetate dimeglumine or EP-3533. The image enhancement kinetic properties of the postinfarction scar, normal myocardium, and blood were compared.

Results: Dynamic T1-weighted MR imaging revealed the washout time constants for EP-3533 to be significantly longer than those for gadopentetate dimeglumine in regions of postinfarction scarring (mean, 194.8 minutes ±116.8 [standard deviation] vs 25.5 minutes ± 4.2; P < .05) and in normal myocardium (mean, 45.4 minutes ± 16.7 vs 25.1 minutes ± 9.7; P < .05). Findings on postmortem histologic sections stained for collagen correlated well with EP-3533–enhanced areas seen on inversion-recovery MR images. Fifty minutes after EP-3533 injection, the postinfarction scar tissue samples, as compared with the normal myocardium, had a twofold higher concentration of gadolinium.

Conclusion: Use of the gadolinium-based collagen-targeting contrast agent, EP-3533, enabled in vivo molecular MR imaging of fibrosis in a mouse model of healed postinfarction myocardial scarring.