Jeffrey R. Tseng

Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long‐term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV‐Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843±28) to passage 20 (250±10) to passage 40 (44±3) to passage 60 (3±1 RLU/μg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5azacytidine treatment led to higher levels of firefly luciferase mRNA (RT‐PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5‐azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene‐based imaging of stem cell transplantation.

Reproducibility of 18F-FDG microPET Studies in Mouse Tumor Xenografts

18F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of 18F-FDG small-animal PET studies. Methods: Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq 18F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of 18F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first 18F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values. Results: The COV for the mean %ID/g between 18F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% ± 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans. Conclusion: 18F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.

Preclinical Efficacy of the c-Met Inhibitor CE-355621 in a U87 MG Mouse Xenograft Model Evaluated by 18F-FDG Small-Animal PET

The purpose of this study was to evaluate the efficacy of CE-355621, a novel antibody against c-Met, in a subcutaneous U87 MG xenograft mouse model using 18F-FDG small-animal PET. Methods: CE-355621 or control vehicle was administered intraperitoneally into nude mice (drug-treated group, n = 12; control group, n = 14) with U87 MG subcutaneous tumor xenografts. Drug efficacy was evaluated over 2 wk using 18F-FDG small-animal PET and compared with tumor volume growth curves. Results: The maximum %ID/g (percentage injected dose per gram of tissue) of 18F-FDG accumulation in mice treated with CE-355621 remained essentially unchanged over 2 wk, whereas the %ID/g of the control tumors increased 66% compared with the baseline. Significant inhibition of 18F-FDG accumulation was seen 3 d after drug treatment, which was earlier than the inhibition of tumor volume growth seen at 7 d after drug treatment. Conclusion: CE-355621 is an efficacious novel antineoplastic chemotherapeutic agent that inhibits 18F-FDG accumulation earlier than tumor volume changes in a mouse xenograft model. These results support the use of 18F-FDG PET to assess early tumor response for CE-355621.

A strategy for blood biomarker amplification and localization using ultrasound.

Blood biomarkers have significant potential applications in early detection and management of various diseases, including cancer. Most biomarkers are present in low concentrations in blood and are difficult to discriminate from noise. Furthermore, blood measurements of a biomarker do not provide information about the location(s) where it is produced. We hypothesize a previously undescribed strategy to increase the concentration of biomarkers in blood as well as localize the source of biomarker signal using ultrasound energy directly applied to tumor cells. We test and validate our hypothesis in cell culture experiments and mouse tumor xenograft models using the human colon cancer cell line LS174T, while measuring the biomarker carcinoembryonic antigen (CEA) before and after the use of ultrasound to liberate the biomarker from the tumor cells. The results demonstrate that the application of low-frequency ultrasound to tumor cells causes a significant release of tumor biomarker, which can be measured in the blood. Furthermore, we establish that this release is specific to the direct application of the ultrasound to the tumor, enabling a method for localization of biomarker production. This work shows that it is possible to use ultrasound to amplify and localize the source of CEA levels in blood of tumor-bearing mice and will allow for a previously undescribed way to determine the presence and localization of disease more accurately using a relatively simple and noninvasive strategy.

Pattern of 18F-FDG uptake in the spinal cord in patients with non-central nervous system malignancy.

Study Design. Retrospective review. Objective. To (1) propose a standard method to quantitate 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) uptake in the spinal cord and (2) use this methodology to retrospectively characterize the pattern of uptake within the entire spinal cord using whole-body positron emission tomography/computed tomography (PET/CT) imaging. Summary of Background Data. A physiologic understanding of glucose metabolism within the spinal cord may provide insight regarding infectious, inflammatory, vascular, and neoplastic spinal cord diseases. Methods. Institutional review board approval was obtained. A total of 131 consecutive whole-body PET/CT studies from July to August 2004 were reviewed, and using exclusionary criteria of: (1) severe spinal arthropathy or curvature, (2) motion artifact, (3) canal hardware, (4) spinal tumor, and (5) marrow hyperplasia, 92 studies of neurologically intact patients (49 men and 43 women) were selected for a retrospective review of spinal cord 18F-FDG activity. The transaxial CT was used to define the canal and circular regions of interests were placed within the canal at the level of the vertebral body midpoint from C1 to L3. Region of interest total count, area, and maximum standardized uptake value (SUVmax) were recorded. Measurements at L5 served as an internal control. For comparative analysis, the cord-to-background (CTB) ratio was defined as spinal cord SUVmax to L5 SUVmax. Results. Mean CTB decreased along each spinal level from cranial to caudal (P < 0.001). Significant relative increases were observed at the T11-T12 vertebral body levels (P < 0.001). Although insignificant, a relative increase was observed at C4. No significant interactions of age or sex on CTB were observed. Conclusion. The pattern of 18F-FDG uptake within the spinal cord, observed in patients with non-central nervous system malignancy, may be helpful in understanding glucose physiology of spinal cord diseases and warrants further research.

18F and 18FDG PET imaging of osteosarcoma to non-invasively monitor in situ changes in cellular proliferation and bone differentiation upon MYC inactivation

Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor’s response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest, and differentiate into mature bone. We reasoned that we could use our model system to develop non-invasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed Positron Emission Tomography (PET) combining the use of both 18F-fluorodeoxyglucose (18FDG) and 18F-flouride (18F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of 18FDG and a significant increase in the uptake of 18F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate 18FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that 18F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both 18FDG and 18F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation