Summary In the last years orthotopic mice models of brain tumors are becoming interesting tools to test innovative class of targeted therapeutics. RNA- mediated gene silencing holds particular interest for cancer research, due to the tumor suppressor action. Multi-modal imaging is now well-established in routine clinical practices as well as in preclinical research field. The high sensitivity of PET - in the picomolar range - allows detection of even minute amounts of radiolabeled markers in vivo, making PET the modality of choice for molecular imaging. The radiolabeled thymidine analog 3?-deoxy-3?-18F-fluorothymidine (18F-FLT) has been used for the diagnostic evaluation of brain tumor proliferation and to monitor radiotracer uptake in untreated and treated ones. This parameter has been shown to correlate with histopathologic proliferation markers, such as Ki67 or proliferative cell nuclear antigen, in preclinical models and patient studies. The combination of PET with microCT provides the combination of functional and structural information. The small size of the mouse brain pose significant challenges for imaging, requiring high spatial resolution, great sensitivity, expensive equipment and established expertise. This study assessed the complementary use of of 18F-FLT PET and microCT in orthotopic mice models of brain tumors to evaluate the therapeutic potential of different RNA-based gene silencing approaches, using a high-resolution animal PET and microCT system and dedicated images post-processing technology. Materials and Methods Four nude mice with orthotopic medulloblastoma and four with glioblastoma underwent PET/CT studies to assess the efficacy of several RNA interference therapies (miRNA 199/B, praja2-siRNA) on tumor metabolic activity and growth. Mice were injected in tail vein with 9.5 MBq of 18F-FLT and were kept awake in a ventilated cage (26°C) during a tracer uptake period of 60 minutes. Imaging was performed with the dedicated small-animal PET scanner eXplore Vista GE Healthcare, with a PET spatial resolution of 1.6 mm FWHM and a CT spatial resolution of 200 micron. The static PET data were acquired for 30 minutes. Image datasets were corrected for random coincidences, scatter and physical decay to the time of injection. The counting rates in the reconstructed images were converted to activity concentrations (SUV units) by use of a system calibration factor (1035 Bq/mL/cps/voxel) derived from the imaging of a mouse-size water-equivalent phantom containing 18F. PET/CT images were post-processed to obtain 3D volume rendering and fusion images using Osirix (with a MacOS operating system). Maximum (SUVmax) and Mean (SUVmean) Standardized Uptake Values (SUV = Tissue activity (MBq/cc) / [Injected dose (MBq)/body weight (g)] ) were calculated from PET studies with [18F] FLT with Explore Vista Analysis Tools in a ROI (region of interest) corresponding to the median transverse slice of the lesions. Quantitative data were obtained using GE eXplore Vista software, on the base of a "region growing" procedure, by adding all spatially connected voxels with SUV>50%. Measurements of length and volume of lesions were also calculated from PET and CT data using Osirix tools. Mice was euthanized and the brain was extracted and fixed in a 1:7 formaline/PBS solution for two days. The mouse brain was then soaked in high concentration iodinated contrast media (iomeprol - Iomeron, Bracco 400 mg I/ml) diluted 1:10 with PBS for a period of 5 days prior to microCT imaging. Subsequently, the brain was removed from iodine contrast media solution, placed in a plastic tube and imaged with air as the background media (GE Healthcare eXplore Locus, spatial resolution 27 micron). 2D and 3D reconstructions were obtained using MicroView (GE eXplore Locus). Results FLT uptake and "metabolical" volume in control and RNAi treated mice were compared (Figure 1-2). 18F-FLT tumor accumulation values were greater in control than in RNAi treated groups, like tumor volume. In addition, in vivo microCT showed in two mice with medulloblastoma a wide skull defect at the level of implant site, with corresponding FLT uptake at PET (Figure 2). Concordant length and volume measurements were obtained with ex vivo microCT and PET image analysis (Figure 3-4). Conclusions PET and microCT gave complementary information to characterize murine models of orthotopic brain cancer. Since the same animal can be imaged repeatedly and each animal can be its own control, the number of animals examined is significantly reduced and the variability caused by inter-individual differences is removed, according to the principle of "refinement, reduction, and replacement". Accurate and serial PET quantification may allow to test in vivo siRNAs, miRNAs, and shRNAs gene-knockdown capability in preclinical brain tumor models, and to optimize technical aspects, such as selectivity, stability, in vivo delivery, efficacy, and safety before RNAi can become a successful therapeutic strategy. References Garzia L, Andolfo I, Cusanelli E, Marino N, Petrosino G, De Martino D, Esposito V, Galeone ANavas L, Esposito S, Gargiulo S, Fattet S, Donofrio V, Cinalli G, Brunetti A, Vecchio LD, Northcott PA, Delattre O, Taylor MD, Iolascon A, Zollo M.- MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma. PLoS One. 2009;4(3):e4998. M. Gramanzini, S. Gargiulo, L. Lignitto, A. Greco, A. Feliciello, A. Brunetti.[18F]FLT preclinical PET to evaluate the role of a novel cancer-associated gene in the aggressiveness of human glioma. Simposio AISAL 2012, 4-6 ottobre 2012, Roma, Italia
18F-FLT PET and microCT in different murine models of brain tumors.
Matteo Gramanzini;Sara Gargiulo;
2012
Abstract
Summary In the last years orthotopic mice models of brain tumors are becoming interesting tools to test innovative class of targeted therapeutics. RNA- mediated gene silencing holds particular interest for cancer research, due to the tumor suppressor action. Multi-modal imaging is now well-established in routine clinical practices as well as in preclinical research field. The high sensitivity of PET - in the picomolar range - allows detection of even minute amounts of radiolabeled markers in vivo, making PET the modality of choice for molecular imaging. The radiolabeled thymidine analog 3?-deoxy-3?-18F-fluorothymidine (18F-FLT) has been used for the diagnostic evaluation of brain tumor proliferation and to monitor radiotracer uptake in untreated and treated ones. This parameter has been shown to correlate with histopathologic proliferation markers, such as Ki67 or proliferative cell nuclear antigen, in preclinical models and patient studies. The combination of PET with microCT provides the combination of functional and structural information. The small size of the mouse brain pose significant challenges for imaging, requiring high spatial resolution, great sensitivity, expensive equipment and established expertise. This study assessed the complementary use of of 18F-FLT PET and microCT in orthotopic mice models of brain tumors to evaluate the therapeutic potential of different RNA-based gene silencing approaches, using a high-resolution animal PET and microCT system and dedicated images post-processing technology. Materials and Methods Four nude mice with orthotopic medulloblastoma and four with glioblastoma underwent PET/CT studies to assess the efficacy of several RNA interference therapies (miRNA 199/B, praja2-siRNA) on tumor metabolic activity and growth. Mice were injected in tail vein with 9.5 MBq of 18F-FLT and were kept awake in a ventilated cage (26°C) during a tracer uptake period of 60 minutes. Imaging was performed with the dedicated small-animal PET scanner eXplore Vista GE Healthcare, with a PET spatial resolution of 1.6 mm FWHM and a CT spatial resolution of 200 micron. The static PET data were acquired for 30 minutes. Image datasets were corrected for random coincidences, scatter and physical decay to the time of injection. The counting rates in the reconstructed images were converted to activity concentrations (SUV units) by use of a system calibration factor (1035 Bq/mL/cps/voxel) derived from the imaging of a mouse-size water-equivalent phantom containing 18F. PET/CT images were post-processed to obtain 3D volume rendering and fusion images using Osirix (with a MacOS operating system). Maximum (SUVmax) and Mean (SUVmean) Standardized Uptake Values (SUV = Tissue activity (MBq/cc) / [Injected dose (MBq)/body weight (g)] ) were calculated from PET studies with [18F] FLT with Explore Vista Analysis Tools in a ROI (region of interest) corresponding to the median transverse slice of the lesions. Quantitative data were obtained using GE eXplore Vista software, on the base of a "region growing" procedure, by adding all spatially connected voxels with SUV>50%. Measurements of length and volume of lesions were also calculated from PET and CT data using Osirix tools. Mice was euthanized and the brain was extracted and fixed in a 1:7 formaline/PBS solution for two days. The mouse brain was then soaked in high concentration iodinated contrast media (iomeprol - Iomeron, Bracco 400 mg I/ml) diluted 1:10 with PBS for a period of 5 days prior to microCT imaging. Subsequently, the brain was removed from iodine contrast media solution, placed in a plastic tube and imaged with air as the background media (GE Healthcare eXplore Locus, spatial resolution 27 micron). 2D and 3D reconstructions were obtained using MicroView (GE eXplore Locus). Results FLT uptake and "metabolical" volume in control and RNAi treated mice were compared (Figure 1-2). 18F-FLT tumor accumulation values were greater in control than in RNAi treated groups, like tumor volume. In addition, in vivo microCT showed in two mice with medulloblastoma a wide skull defect at the level of implant site, with corresponding FLT uptake at PET (Figure 2). Concordant length and volume measurements were obtained with ex vivo microCT and PET image analysis (Figure 3-4). Conclusions PET and microCT gave complementary information to characterize murine models of orthotopic brain cancer. Since the same animal can be imaged repeatedly and each animal can be its own control, the number of animals examined is significantly reduced and the variability caused by inter-individual differences is removed, according to the principle of "refinement, reduction, and replacement". Accurate and serial PET quantification may allow to test in vivo siRNAs, miRNAs, and shRNAs gene-knockdown capability in preclinical brain tumor models, and to optimize technical aspects, such as selectivity, stability, in vivo delivery, efficacy, and safety before RNAi can become a successful therapeutic strategy. References Garzia L, Andolfo I, Cusanelli E, Marino N, Petrosino G, De Martino D, Esposito V, Galeone ANavas L, Esposito S, Gargiulo S, Fattet S, Donofrio V, Cinalli G, Brunetti A, Vecchio LD, Northcott PA, Delattre O, Taylor MD, Iolascon A, Zollo M.- MicroRNA-199b-5p impairs cancer stem cells through negative regulation of HES1 in medulloblastoma. PLoS One. 2009;4(3):e4998. M. Gramanzini, S. Gargiulo, L. Lignitto, A. Greco, A. Feliciello, A. Brunetti.[18F]FLT preclinical PET to evaluate the role of a novel cancer-associated gene in the aggressiveness of human glioma. Simposio AISAL 2012, 4-6 ottobre 2012, Roma, ItaliaI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
