Songji Zhao

Dominant-Negative Hypoxia-Inducible Factor-1α Reduces Tumorigenicity of Pancreatic Cancer Cells through the Suppression of Glucose Metabolism

In the tumor cells exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1)-mediated adaptation responses such as angiogenesis and anaerobic metabolism are induced for their survival. We have recently reported that the constitutive expression of HIF-1 alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and glucose deprivation. We then established dominant-negative HIF-1 alpha (dnHIF-1 alpha) transfectants and examined their susceptibility to apoptosis and growth inhibition induced by hypoxia and glucose deprivation in vitro and their tumorigenicity in SCID mice. We further examined the expressions of aldolase A and Glut-1 in vitro and Glut-1 expression and glucose uptake in the tumor tissues and microvessel counts in the tumor tissues. As a result, dnHIF-1 alpha rendered the pancreatic cancer cells sensitive to apoptosis and growth inhibition induced by hypoxia and glucose deprivation. Also it abrogated the enhanced expression of Glut-1 and aldolase A mRNAs under hypoxia and reduced the expression of Glut-1 and the glucose uptake in the tumor tissues and consequently in vivo tumorigenicity. We found no significant difference in the microvessel counts among the tumor tissues. From these results, we suggest that the disruption of the HIF-1 pathway might be effective in the treatment of pancreatic cancers.

All-trans Retinoic Acid Enhances Murine Dendritic Cell Migration to Draining Lymph Nodes via the Balance of Matrix Metalloproteinases and Their Inhibitors

Cancers escape immune surveillance through the manipulation of the host’s immune system. Sequestration of dendritic cells (DCs) within tumor tissues and the subsequent inhibition of their migration is one of the several mechanisms by which tumors induce immunosuppression. In view of recent findings depicting the improvement of tumor immune responses in cancer patients following all-trans retinoic acid (ATRA) treatment, we sought to identify the effects of ATRA on DC mobility in the context of tumor immunotherapy. Our results demonstrate that ATRA, added to differentiating murine bone marrow progenitor cells, enhances the invasive capacity of the resulting DCs. Immature DCs injected intratumorally in mice show increased accumulation in draining lymph nodes, but not in nondraining lymph nodes and spleens, when differentiated in the presence of ATRA. The in vitro migration of mature DCs through the basement membrane matrix toward the lymphoid chemokines CCL19 and CCL21 is enhanced in these cells, albeit not in the presence of a matrix metalloproteinase (MMP) inhibitor. An increase in MMP production with a simultaneous decrease in the production of their inhibitors (tissue inhibitors of matrix metalloproteinase or TIMPs) is provoked by ATRA. This affects the MMP/TIMP balance in DCs, in particular that of MMP-9 and TIMP-1, favoring protease activity and thus allowing for enhanced DC mobilization. In conclusion, this study demonstrates that ATRA is capable of improving DC trafficking in a tumor milieu and, in view of the encouraging results obtained in the clinic, further supports the notion that ATRA might be a valuable chemical adjuvant to current immunotherapeutic strategies for cancer.

Usefulness of 11C-Methionine for Differentiating Tumors from Granulomas in Experimental Rat Models: A Comparison with 18F-FDG and 18F-FLT

Many clinical PET studies have shown that increased 18F-FDG uptake is not specific to malignant tumors. 18F-FDG is also taken up in inflammatory lesions, particularly in granulomatous lesions such as sarcoidosis or active inflammatory processes after chemoradiotherapy, making it difficult to differentiate malignant tumors from benign lesions, and is the main source of false-positive 18F-FDG PET findings in oncology. These problems may be overcome by multitracer studies using 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) or l-11C-methionine. However, 18F-FLT or 11C-methionine uptake in granulomatous lesions remains unclarified. In this study, the potentials of 18F-FLT and 11C-methionine in differentiating malignant tumors from granulomas were compared with 18F-FDG using experimental rat models. Methods: Dual-tracer tissue distribution studies using 18F-FDG and 3H-FLT (groups I and III) or 18F-FDG and 14C-methionine (groups II and IV) were performed on rats bearing both granulomas (Mycobacterium bovis bacillus Calmette-Guérin [BCG]–induced) and hepatomas (KDH-8–induced) (groups I and II) or on rats bearing both turpentine oil–induced inflammation and hepatomas (groups III and IV). One hour after the injection of a mixture of 18F-FDG and 3H-FLT or of 18F-FDG and 14C-methionine, tissues were excised to determine the radioactivities of 18F-FDG, 3H-FLT, and 14C-methionine (differential uptake ratio). Results: Mature epithelioid cell granuloma formation and massive lymphocyte infiltration were observed in the granuloma tissue induced by BCG, histologically similar to sarcoidosis. The granulomas showed high 18F-FDG uptake comparable to that in the hepatomas (group I, 8.18 ± 2.40 vs. 9.13 ± 1.52, P = NS; group II, 8.43 ± 1.45 vs. 8.91 ± 2.32, P = NS). 14C-Methionine uptake in the granuloma was significantly lower than that in the hepatoma (1.31 ± 0.22 vs. 2.47 ± 0.60, P < 0.01), whereas 3H-FLT uptake in the granuloma was comparable to that in the hepatoma (1.98 ± 0.70 vs. 2.30 ± 0.67, P = NS). Mean uptake of 18F-FDG, 3H-FLT, and 14C-methionine was markedly lower in the turpentine oil–induced inflammation than in the tumor. Conclusion: 14C-Methionine uptake was significantly lower in the granuloma than in the tumor, whereas 18F-FDG and 3H-FLT were not able to differentiate granulomas from tumors. These results suggest that 14C-methionine has the potential to accurately differentiate malignant tumors from benign lesions, particularly granulomatous lesions, providing a biologic basis for clinical PET studies.

Evaluating performance of a pixel array semiconductor SPECT system for small animal imaging

ObjectivesSmall animal imaging has recently been focused on basic nuclear medicine. We have designed and built a small animal SPECT imaging system using a semiconductor camera and a newly designed collimator. We assess the performance of this system for small object imaging.MethodsWe employed an MGC1500 (Acrorad Co.) camera including a CdTe semiconductor. The pixel size was 1.4 mm/pixel. We designed and produced a parallel-hole collimator with 20-mm hole length. Our SPECT system consisted of a semiconductor camera with the subject holder set on an electric rotating stage controlled by a computer. We compared this system with a conventional small animal SPECT system comprising a SPECT-2000H scanner with four Anger type cameras and pinhole collimators. The count rate linearity for estimation of the scatter was evaluated for a pie-chart phantom containing different concentrations of99mTc. We measured the FWHM of the99mTc SPECT line source along with scatter. The system volume sensitivity was examined using a flood source phantom which was 35 mm long with a 32-mm inside diameter. Additionally, anin vivo myocardial perfusion SPECT study was performed with a rat.ResultsWith regards to energy resolution, the semiconductor camera (5.6%) was superior to the conventional Anger type camera (9.8%). In the count rate linearity evaluation, the regression lines of the SPECT values werey = 0.0 9x+ 0.031 (r2 = 0.999) for our system andy = 0.018* + 0.060 (r2 = 0.997) for the conventional system. Thus, the scatter count using the semiconductor camera was less than that using the conventional camera. FWHMs of our system and the conventional system were 2.9 ± 0.1 and 2.0 ± 0.1 mm, respectively. Moreover, the system volume sensitivity of our system [0.51 kcps/(MBq/ ml)/cm] was superior to that of the conventional system [0.44 kcps/(MBq/m/)/cm]. Our system provided clear images of the rat myocardium, sufficient for practical use in small animal imaging.ConclusionsOur SPECT system, utilizing a semiconductor camera, permits high quantitative analysis by virtue of its low scatter radiation and high sensitivity. Therefore, this system may contribute to molecular imaging of small animals and basic medical research. Key words: semiconductor detectors, small animal imaging, single photon emission computed tomography

Comparison of 99mTc-annexin A5 with 18F-FDG for the detection of atherosclerosis in ApoE−/− mice

Purpose99mTc-annexin A5, a marker of ongoing apoptosis, and 18F-FDG, a marker of the increased metabolism of inflammatory cells, are supposed to be useful in the detection of metabolically active atheroma. This study reports a comparison of the intralesional distribution of these tracers in relation to lesion development in ApoE−/− mice.MethodsMale ApoE−/− mice (n = 12–14/group) were maintained on a Western-type diet after the age of 5 weeks. At 25 weeks, 99mTc-annexin A5 or 18F-FDG was injected and the aortas were harvested for autoradiography (ARG) and Oil Red O staining. Regional radioactivity accumulation was compared in relation to the Oil Red O staining score (ranging from 0 to 3, a semiquantitative parameter for evaluating lesion development).ResultsBoth 99mTc-annexin A5 and 18F-FDG showed preferential uptake into atherosclerotic lesions, with higher uptake levels for 18F-FDG (mean, 56.07 %ID×kg/m2) than for 99mTc-annexin A5 (mean, 10.38 %ID×kg/m2). The regional uptake levels of each tracer correlated with the Oil Red O staining score (r = 0.65, p < 0.05 for 99mTc-annexin A5; r = 0.56, p < 0.05 for 18F-FDG). The uptake ratios of advanced lesions (score >0.5) to early lesions (score <0.5) were significantly higher for 99mTc-annexin A5 than for 18F-FDG (f = 4.73, p = 0.03).ConclusionBoth 99mTc-annexin A5 and 18F-FDG accumulate in atherosclerotic lesions and correlate with the severity of each lesion. The higher absolute uptake levels of 18F-FDG may be advantageous for lesion detection, whereas the preferential uptake of 99mTc-annexin A5 in advanced lesions may be a useful indicator of late-stage lesions or vulnerable plaque transformation.

A novel murine model for non-alcoholic steatohepatitis developed by combination of a high-fat diet and oxidized low-density lipoprotein

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome that is characterized by steatosis, inflammation, and fibrosis, and may progress to cirrhosis and carcinoma. To investigate its pathogenic processes, we established a novel murine model for NASH by combination of a high-fat diet (HFD) and oxidized low-density lipoprotein (oxLDL). Mice that received HFD for 23 weeks showed hepatic steatosis, slight fibrosis, and a high level of lipid peroxidation compared with a regular diet (RD)-fed mice. Hepatic injury and elevated tumor necrosis factor (TNF)-α mRNA expression were also detected in these mice. Moreover, oxLDL administration to HFD-fed mice during weeks 21–23 not only aggravated hepatic steatosis, fibrosis, and lipid metabolism, but also resulted in intense inflammation, including severe hepatic injury and inflammatory cell infiltration, which are the typical histological features of NASH. Inflammation was accompanied by increased gene expression of TNF-α and interleukin (IL)-6. Additionally, the livers of RD-fed animals treated with oxLDL during weeks 21–23 were characterized by foamy macrophages and inflammatory cell infiltration along with an elevated IL-6 mRNA level. These results suggest that an increased oxidative state, including HFD-induced intracellular lipid peroxidation and its extracellular source from oxLDL, is the actual trigger for hepatic inflammation in which liver injury is mediated by TNF-α and inflammatory cell accumulation is dependent on IL-6. HFD and oxLDL also induced insulin resistance in mice; additionally, oxLDL downregulated insulin secretion. In this model, CD36 overexpression was observed in the hepatocytes of HFD-fed mice and those treated with HFD and oxLDL, and in the hepatic macrophages of RD-fed mice immediately after oxLDL treatment. In vitro experiments indicated a rapid and transient elevation of CD36 on macrophage plasma membrane in response to oxLDL. Our findings demonstrate that CD36 expressed on hepatocytes and hepatic macrophages mediates the pathophysiology of NASH.

Bone Marrow Stromal Cell Transplantation Enhances Recovery of Local Glucose Metabolism After Cerebral Infarction in Rats: A Serial 18F-FDG PET Study

This study aimed to assess whether 18F-FDG PET could serially monitor the beneficial effects of bone marrow stromal cells (BMSC) on cerebral glucose metabolism when transplanted into the infarct brain of rats. Methods: The BMSC from green fluorescent protein transgenic rats or vehicle was stereotactically transplanted into the ipsilateral striatum at 7 d after permanent middle cerebral artery occlusion of rats. Local glucose metabolism was semiquantitatively measured at 6 and 35 d after ischemia using 18F-FDG PET. Motor function was serially evaluated throughout the experiments. At 35 d after ischemia, immunohistochemistry was performed to evaluate the phenotype of BMSC and their effects on the expression of brain-type glucose transporters. Results: BMSC transplantation not only enhanced functional recovery but also promoted the recovery of glucose utilization in the periinfarct area when stereotactically transplanted at 1 wk after ischemia. The engrafted cells were widely distributed, and most expressed a neuron-specific protein, NeuN. BMSC transplantation also prevented the pathologic upregulation of glucose transporters in the periinfarct neocortex. Conclusion: The present findings strongly suggest that the BMSC may enhance functional recovery by promoting the recovery of local glucose metabolism in the periinfarct area when directly transplanted into the infarct brain at clinically relevant timing. The BMSC also inhibit the pathologic upregulation of brain-isoform glucose transporters type 1 and 3. 18F-FDG PET may be a valuable modality to scientifically prove the beneficial effects of BMSC transplantation on the host brain in clinical situations.

Prolonged High-Fat Feeding Enhances Aortic 18F-FDG and 99mTc-Annexin A5 Uptake in Apolipoprotein E-Deficient and Wild-Type C57BL/6J Mice

18F-FDG, a marker of the enhanced metabolism characteristic of activated inflammatory cells, and 99mTc-annexin A5, a marker of apoptosis, are both widely believed to be useful for the imaging of unstable atheroma (rupture-prone vulnerable plaques [VP]). Serum cholesterol functions as a proinflammatory factor, driving the formation of VP, and affects the immune responses of aortic tissues systemically. It is therefore reasonable to postulate that prolonged cholesterol loading may alter the aortic uptake of these tracers. Here, we evaluated the aortic uptake of 18F-FDG and 99mTc-annexin A5 in apolipoprotein E-deficient (apoE−/−) and wild-type mice placed on high-fat diets. Methods: Male apoE−/− and wild-type (C57BL/6J) mice were maintained on high-fat diets after the age of 5 wk. Wild-type mice fed regular chow were used as controls. At the ages of 10, 18, and 25 wk (5–15 mice per group at each time point), mice were injected with 18F-FDG or 99mTc-annexin A5 after 12 h of fasting. At 1 h after 18F-FDG injection (or 2 h after 99mTc-annexin A5 injection), mice were sacrificed, and the aortas were removed for well-type scintillation counting of radioactivity. The results were expressed as percentage injected dose per gram of tissue and normalized by animal body weight [(ID%/g) × kg]. En face staining was then performed to assess the location and size (surface area) of the lipid pool within each aortic specimen. Concurrent blood samples were obtained to determine the plasma lipid profile of each group. Results: No atherosclerotic lesions were found in wild-type mice regardless of the diet, whereas the lesion area progressively increased with age in apoE−/− mice. Mean plasma cholesterol levels remained stable with the regular diet in wild-type mice (73–78 mg/dL) but increased with cholesterol feeding in wild-type mice (143–179 mg/dL) and in apoE−/− mice (>1,300 mg/dL). Aortic tracer uptake [(ID%/g) × kg] remained stable with the regular diet in wild-type mice (0.054–0.053 and 0.021–0.023 for 99mTc-annexin A5) but increased with cholesterol feeding in wild-type mice (0.164 for 18F-FDG and 0.036 for 99mTc-annexin A5 at 25 wk) and in apoE−/− mice (0.249 for 18F-FDG and 0.047 for 99mTc-annexin A5 at 25 wk). Conclusion: The accumulation of 18F-FDG and 99mTc-annexin A5 in aortic tissues is influenced not only by the progression of atherosclerotic disease but also by cholesterol loading over time.

Evaluation of changes in the tumor microenvironment after sorafenib therapy by sequential histology and 18F-fluoromisonidazole hypoxia imaging in renal cell carcinoma

The mechanistic dissociation of ‘tumor starvation’ versus ‘vascular normalization’ following anti-angiogenic therapy is a subject of intense controversy in the field of experimental research. In addition, accurately evaluating changes of the tumor microenvironment after anti-angiogenic therapy is important for optimizing treatment strategy. Sorafenib has considerable anti-angiogenic effects that lead to tumor starvation and induce tumor hypoxia in the highly vascularized renal cell carcinoma (RCC) xenografts. 18F-fluoromisonidazole (18F-FMISO) is a proven hypoxia imaging probe. Thus, to clarify early changes in the tumor microenvironment following anti-angiogenic therapy and whether 18F-FMISO imaging can detect those changes, we evaluated early changes in the tumor microenvironment after sorafenib treatment in an RCC xenograft by sequential histological analysis and 18F-FMISO autoradiography (ARG). A human RCC xenograft (A498) was established in nude mice, for histological studies and ARG, and further assigned to the control and sorafenib-treated groups (80 mg/kg, per os). Mice were sacrificed on Days 1, 2, 3 and 7 in the histological study, and on Days 3 and 7 in ARG after sorafenib treatment. Tumor volume was measured every day. 18F-FMISO and pimonidazole were injected intravenously 4 and 2 h before sacrifice, respectively. Tumor sections were stained with hematoxylin and eosin and immunohistochemically with pimonidazole and CD31. Intratumoral 18F-FMISO distribution was quantified in ARG. Tumor volume did not significantly change on Day 7 after sorafenib treatment. In the histological study, hypoxic fraction significantly increased on Day 2, mean vessel density significantly decreased on Day 1 and necrosis area significantly increased on Day 2 after sorafenib treatment. Intratumoral 18F-FMISO distribution significantly increased on Days 3 (10.2-fold, p<0.01) and 7 (4.1-fold, p<0.01) after sorafenib treatment. The sequential histological evaluation of the tumor microenvironment clarified tumor starvation in A498 xenografts treated with sorafenib. 18F-FMISO hypoxia imaging confirmed the tumor starvation. 18F-FMISO PET may contribute to determine an optimum treatment protocol after anti-angiogenic therapy.

18p-FDG PET is superior to67Ga SPECT in the staging of non-Hodgkin’s lymphoma

ObjectiveOur study aims to compare diagnostic accuracy between18F-FDG PET and67Ga SPECT in the staging of non-Hodgkin’s lymphoma.MethodsTwenty-eight patients with non-Hodgkin’s lymphoma, underwent18F-FDG PET,67Ga SPECT and CT for the pretreatment staging of malignant lymphoma between August 1999 and March 2002.18F-FDG PET imaging was obtained 60 minutes after the intravenous administration of 185 MBq of18F-FDG.67Ga SPECT imaging was obtained 2 days after the intravenous administration of 148 MBq of67Ga.18F-FDG PET and 67Ga SPECT were performed within one month. Both imagings were performed on the area from the neck to the pelvis. The18F-FDG PET and67Ga SPECT findings were compared with the CT findings and the clinical course.ResultsSixty-six nodal lesions were clinically confirmed. Of these, 32 were identified by both18F-FDG PET and67Ga SPECT. The remaining 34 lesions were identified only by18F-FDG PET. The mean (± SD) sizes of the nodes were 34.7 ± 32.4 mm for18F-FDG-positive and67Ga-positive lesions and 15.7 ± 8.3 mm for18F-FDG-positive and67Ga-negative lesions (p >; 0.001). Of the 23 extranodal lesions, 12 were identified by both18F-FDG PET and67Ga SPECT, whereas 6 lesions were identified by only18F-FDG PET. Five lesions were not identified by either technique. No18F-FDG-negative but67Ga-positive nodal or extranodal lesions were observed. The difference in findings between the two studies is related to the difference in the size but not in the histology or site of the lesions.Conclusion18F-FDG PET detected significantly more lesions particularly small lesions than67Ga SPECT. Thus,18F-FDG PET is considered to be superior to67Ga SPECT in the staging of non-Hodgkin’s lymphoma.