Yasushi Kiyono

Cytotoxic effects of γδ T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy

Nitrogen containing‐bisphosphonates (N‐BPs), widely used to treat bone diseases, have direct antitumor effects via the inactivation of Ras proteins. In addition to the direct antitumor activities, N‐BPs expand gdγδT cells, which exhibit major histocompatibility complex‐unrestricted lytic activity. BPs accumulate intermediate metabolites which may be tumor antigens in target cells. The purpose of our study was to clarify the cytotoxicity of gdγδ T cells expanded ex vivo by the most potent N‐BP, zoledronate (ZOL). Especially, we focused on the importance of pretreatment against target cells also with ZOL; 1 mμM ZOL plus IL‐2 increased the absolute number of gdγδT cells 298–768 fold for 14 days incubation. The small cell lung cancer and fibrosarcoma cell lines pretreated with 5 mμM ZOL showed a marked increase in sensitivity to lysis by gdγδT cells. While, untreated cell lines were much less sensitive to lysis by gdT cells. Video microscopy clearly demonstrated that gdγδT cells killed target cells pre‐treated with ZOL within 3 hr. Pretreatment with 80 mμg/kg ZOL also significantly enhanced the antitumor activity of gdγδT cells in mice xenografted with SBC‐5 cells. These findings show that ZOL significantly stimulated the proliferation of gdγδT cells and that gdγδT cells required pre‐treatment with ZOL for cytotoxic activity against target cells.

V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity

Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis, and exerts important physiological regulation through three distinct receptors, V1a, V1b, and V2. Although AVP is used clinically as a potent vasoconstrictor (V1a receptor-mediated) in patients with circulatory shock, the physiological role of vasopressin V1a receptors in blood pressure (BP) homeostasis is ill-defined. In this study, we investigated the functional roles of the V1a receptor in cardiovascular homeostasis using gene targeting. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a−/−) was significantly (P < 0.001) lower compared to the wild-type mice (V1a+/+) without a notable change in heart rate. There was no significant alteration in cardiac functions as assessed by echocardiogram in the mutant mice. AVP-induced vasopressor responses were abolished in the mutant mice; rather, AVP caused a decrease in BP, which occurred in part through V2 receptor-mediated release of nitric oxide from the vascular endothelium. Arterial baroreceptor reflexes were markedly impaired in mutant mice, consistent with a loss of V1a receptors in the central area of baroreflex control. Notably, mutant mice showed a significant 9% reduction in circulating blood volume. Furthermore, mutant mice had normal plasma AVP levels and a normal AVP secretory response, but had significantly lower adrenocortical responsiveness to adrenocorticotropic hormone. Taken together, these results indicate that the V1a receptor plays an important role in normal resting arterial BP regulation mainly by its regulation of circulating blood volume and baroreflex sensitivity.

The use of nanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cell migration and well-regulated spheroid formation.

Two-dimensional (2D) cell cultures are essential for drug development and tumor research. However, the limitations of 2D cultures are widely recognized, and a better technique is needed. Recent studies have indicated that a strong physical contact between cells and 2D substrates induces cellular characteristics that differ from those of tumors growing in vivo. 3D cell cultures using various substrates are then developing; nevertheless, conventional approaches have failed in maintenance of cellular proliferation and viability, uniformity, reproducibility, and/or simplicity of these assays. Here, we developed a 3D culture system with inorganic nanoscale scaffolding using nanoimprinting technology (nano-culture plates), which reproduced the characteristics of tumor cells growing in vivo. Diminished cell-to-substrate physical contact facilitated spontaneous tumor cell migration, intercellular adhesion, and multi-cellular 3D-spheroid formation while maintaining cellular proliferation and viability. The resulting multi-cellular spheroids formed hypoxic core regions similar to tumors growing in vivo. This technology allows creating uniform and highly-reproducible 3D cultures, which is easily applicable for microscopic and spectrophotometric assays, which can be used for high-throughput/high-content screening of anticancer drugs and should accelerate discovery of more effective anticancer therapies.

Cytosolic acetyl-CoA synthetase affected tumor cell survival under hypoxia: the possible function in tumor acetyl-CoA/acetate metabolism

Understanding tumor‐specific metabolism under hypoxia is important to find novel targets for antitumor drug design. Here we found that tumor cells expressed higher levels of cytosolic acetyl‐CoA synthetase (ACSS2) under hypoxia than normoxia. Knockdown of ACSS2 by RNA interference (RNAi) in tumor cells enhanced tumor cell death under long‐term hypoxia in vitro. Our data also demonstrated that the ACSS2 suppression slowed tumor growth in vivo. These findings showed that ACSS2 plays a significant role in tumor cell survival under hypoxia and that ACSS2 would be a potential target for tumor treatment. Furthermore, we found that tumor cells excreted acetate and the quantity increased under hypoxia: the pattern of acetate excretion followed the expression pattern of ACSS2. Additionally, the ACSS2 knockdown led to a corresponding reduction in the acetate excretion in tumor cells. These results mean that ACSS2 can conduct the reverse reaction from acetyl‐CoA to acetate in tumor cells, which indicates that ACSS2 is a bi‐directional enzyme in tumor cells and that ACSS2 might play a buffering role in tumor acetyl‐CoA/acetate metabolism. (Cancer Sci 2009; 100: 821–827)

Pathophysiologic Correlation Between 62Cu-ATSM and 18F-FDG in Lung Cancer

The purpose of this study was to delineate the differences in intratumoral uptake and tracer distribution of 62Cu-diacetyl-bis(N4-methylthiosemicarbazone) (62Cu-ATSM), a well-known hypoxic imaging tracer, and 18F-FDG in patients with lung cancer of pathohistologically different types. Methods: Eight patients with squamous cell carcinoma (SCC) and 5 with adenocarcinoma underwent 62Cu-ATSM and 18F-FDG PET within a 1-wk interval. For 62Cu-ATSM PET, 10-min static data acquisition was started at 10 min after a 370- to 740-MBq tracer injection. After image reconstruction, 62Cu-ATSM and 18F-FDG images were coregistered, and multiple small regions of interest were drawn on tumor lesions of the 2 images to obtain standardized uptake values (SUVs). The regression lines were determined between SUVs for 62Cu-ATSM and 18F-FDG in each tumor. The slope values were compared between SCC and adenocarcinoma to observe pathohistologic differences in intratumoral distribution of the tracers. Results: SUVs for 62Cu-ATSM were lower than those for 18F-FDG in both SCC and adenocarcinoma. SCC tumors showed high 62Cu-ATSM and low 18F-FDG uptakes in the peripheral region of tumors but low 62Cu-ATSM and high 18F-FDG uptakes toward the center (spatial mismatching). The relationship of SUVs for the 2 tracers was negatively correlated with a mean regression slope of −0.07 ± 0.05. On the other hand, adenocarcinoma tumors had a spatially similar distribution of 62Cu-ATSM and 18F-FDG, with positive regression slopes averaging 0.24 ± 0.13. The regression slopes for 62Cu-ATSM and 18F-FDG differed significantly between SCC and adenocarcinoma (P < 0.001). Conclusion: The intratumoral distribution patterns of 62Cu-ATSM and 18F-FDG were different between SCC and adenocarcinoma in lung cancers, indicating that intratumoral regions of high glucose metabolism and hypoxia could differ with the pathohistologic type of lung cancer. The identification of regional biologic characteristics in tumors such as hypoxia, energy metabolism, and proliferation could play a significant role in the clinical diagnosis and therapy planning for non–small cell lung cancer patients.

Fatty Acid Synthase Is a Key Target in Multiple Essential Tumor Functions of Prostate Cancer: Uptake of Radiolabeled Acetate as a Predictor of the Targeted Therapy Outcome

Fatty acid synthase (FASN) expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In addition, how FASN inhibition affects tumor progression remains unclear. Here, we showed the method to predict FASN-targeted therapy outcome using radiolabeled acetate uptake and presented mechanisms of FASN inhibition with human prostate cancer cell lines, to provide the treatment strategy of FASN-targeted therapy. We revealed that tumor uptake of radiolabeled acetate reflected the FASN expression levels and sensitivity to FASN-targeted therapy with orlistat in vitro and in vivo. FASN-targeted therapy was noticeably effective against tumors with high FASN expression, which was indicated by high acetate uptake. To examine mechanisms, we established FASN knockdown prostate cancer cells by transduction of short-hairpin RNA against FASN and investigated the characteristics by analyses on morphology and cell behavior and microarray-based gene expression profiling. FASN inhibition not only suppressed cell proliferation but prevented pseudopodia formation and suppressed cell adhesion, migration, and invasion. FASN inhibition also suppressed genes involved in production of intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression. Collectively, our data demonstrated that uptake of radiolabeled acetate is a useful predictor of FASN-targeted therapy outcome. This suggests that [1-11C]acetate positron emission tomography (PET) could be a powerful tool to accomplish personalized FASN-targeted therapy by non-invasive visualization of tumor acetate uptake and selection of responsive tumors. FASN-targeted therapy could be an effective treatment to suppress multiple steps related to tumor progression in prostate cancers selected by [1-11C]acetate PET.

Functional Images Reflect Aggressiveness of Endometrial Carcinoma: Estrogen Receptor Expression Combined with 18F-FDG PET

The grade of histologic differentiation is one of the most important prognostic factors in patients with endometrial carcinoma and postoperative staging. The aim of this study was to investigate whether 16α-18F-fluoro-17β-estradiol (18F-FES) and 18F-FDG PET reflect clinicopathologic features in patients with endometrial tumors. Methods: A total of 22 patients with endometrial adenocarcinoma and 9 with endometrial hyperplasia (mean age, 56.0 ± 15.3 y) underwent 18F-FES PET for estrogen receptor imaging and 18F-FDG PET. Regional values of tracer uptake were evaluated using standardized uptake value (SUV) and the SUV ratio of 18F-FDG to 18F-FES. The accuracy for predicting tumor aggressiveness defined as high-risk carcinoma (International Federation of Gynecology and Obstetrics [FIGO] stage ≥ Ic or histologic grade ≥ 2), low-risk carcinoma (FIGO stage ≤ Ib and grade 1), and hyperplasia was compared for each PET parameter using receiver-operating-characteristic (ROC) analysis. The diagnostic accuracy of MRI findings for clinical staging was also compared. Results: Although the SUV for 18F-FDG was significantly lower in endometrial hyperplasia than in carcinoma, a significant difference between high-risk and low-risk carcinoma was observed only in SUV for 18F-FES. High-risk carcinoma showed a significantly greater 18F-FDG–to–18F-FES ratio (3.6 ± 2.1) than did low-risk carcinoma (1.3 ± 0.5, P < 0.01) and hyperplasia (0.3 ± 0.1, P < 0.005). Low-risk carcinoma showed a significantly higher 18F-FDG–to–18F-FES ratio than hyperplasia (P < 0.0001). In ROC analysis, the most accurate diagnostic PET parameter for predicting high-risk and low-risk carcinoma was the 18F-FDG–to–18F-FES ratio. The optimal 18F-FDG/18F-FES cutoff value of 2.0, determined by ROC analysis, revealed 73% sensitivity, 100% specificity, and 86% accuracy, which was better than the 77% accuracy for MRI. The 18F-FDG–to–18F-FES ratio of 0.5 yielded a correct diagnosis for carcinoma from hyperplasia with 100% accuracy. Conclusion: Endometrial carcinoma reduces estrogen dependency with accelerated glucose metabolism as it progresses to a higher stage or grade. 18F-FES and 18F-FDG PET studies provide a new index of the 18F-FDG–to–18F-FES ratio, which is considered the most informative index reflecting tumor aggressiveness. This index will be useful for making noninvasive diagnoses and deciding the appropriate therapeutic strategy for patients with endometrial carcinoma.

18F-FES and 18F-FDG PET for Differential Diagnosis and Quantitative Evaluation of Mesenchymal Uterine Tumors: Correlation with Immunohistochemical Analysis

The aim of this study was to investigate the relationship between the tumor uptake of 16α-18F-fluoro-17β-estradiol (18F-FES) and 18F-FDG using PET and expressions of sex hormone receptors, such as estrogen receptor (ER), as well as glucose transporter 1 (GLUT-1) and Ki-67 analyzed by the immunohistochemistry method in mesenchymal uterine tumors. Methods: Forty-seven patients with mesenchymal uterine tumors were studied with 18F-FES and 18F-FDG PET. Postoperative pathologic diagnosis revealed 33 uterine leiomyomas and 14 uterine sarcomas. Tissue samples were assayed for expression of ERα, ERβ, progesterone receptor (PR), PR-B, GLUT-1, and Ki-67 by an immunohistochemistry method. Standardized uptake values (SUVs) for 18F-FES and 18F-FDG were compared with the semiquantitative immunoreactive score (0–12) and quantitative labeling index (LI) for Ki-67 in immunohistochemistry. Results: 18F-FES uptake was significantly lower (P < 0.001) and the 18F-FDG uptake and SUV ratio of 18F-FDG to 18F-FES (18F-FDG/18F-FES ratio) (P < 0.005 and P < 0.001, respectively) were significantly higher in uterine sarcomas than in leiomyomas. Immunohistochemistry analysis showed significantly higher expressions of ERα, PR, and PR-B in uterine leiomyomas than in sarcomas. The Ki-67 LI was significantly greater in uterine sarcomas than in leiomyomas. Correlation analysis for all tumors showed positive correlations between 18F-FES SUV and immunohistochemistry scores of ERα, PR (P < 0.001), and PR-B (P < 0.005) as well as between 18F-FDG SUV and GLUT-1 and Ki-67 (P < 0.001). However, the 18F-FDG/18F-FES ratio showed significantly negative correlations with ERα, PR (P < 0.001), and PR-B (P < 0.005) and a positive correlation with Ki-67 LI (P < 0.001). In uterine sarcomas, ERα and 18F-FES SUV showed a positive correlation (P < 0.001) in a low SUV range, and the 18F-FDG/18F-FES ratio showed positive correlations with ERβ and GLUT-1 expression (P < 0.005). Conclusion: 18F-FES and 18F-FDG PET showed correlations between tracer uptake and expressions of sex hormone receptors, GLUT-1, and Ki-67 in mesenchymal uterine tumors. The 18F-FDG/18F-FES ratio was correlated with Ki-67, GLUT-1, and ERβ in uterine sarcoma. Functional PET imaging and PET parameters would be useful noninvasive biomarkers for the assessment of tumor hormone receptor expression, glucose metabolism, and proliferation and for differential diagnosis of uterine leiomyoma and sarcoma.

Tumor uptake of radiolabeled acetate reflects the expression of cytosolic acetyl-CoA synthetase: implications for the mechanism of acetate PET

INTRODUCTION [1-(11)C]Acetate positron emission tomography (PET) is used for myocardial studies. In the myocardium, mitochondrial acetyl-CoA synthetase (ACSS1) mainly contributes to the radiopharmaceutical uptake. [1-(11)C]Acetate PET is also used for tumor diagnosis; however, the uptake mechanism of radiolabeled acetate in tumors remains unclear. Our previous study reported that cytosolic acetyl-CoA synthetase (ACSS2) was expressed in tumor cells and up-regulated under hypoxia, whereas expression of ACSS1 was negligible regardless of the oxygen conditions. We also indicated that ACSS2 is a bidirectional enzyme that controls acetyl-CoA/acetate metabolism in tumor cells. In this study, to elucidate the basic mechanism of tumor acetate uptake, we focused on ACSS2 and investigated the role of ACSS2 in the uptake of radiolabeled acetate in tumor cells. METHODS [1-(14)C]Acetate uptake and ACSS2 expression were examined in four tumor cell lines under normoxia or hypoxia. An ACSS2 knockdown study was also performed. RESULTS [1-(14)C]Acetate uptake was increased in the tumor cells under hypoxia. This pattern followed that of ACSS2 expression. The incorporated (14)C was mostly distributed in the lipid-soluble fractions, and this tendency increased under hypoxia. ACSS2 knockdown led to a corresponding reduction in [1-(14)C]acetate uptake in all tumor cell lines examined under normoxia and hypoxia. CONCLUSIONS ACSS2 plays an important role in the uptake of radiolabeled acetate in tumor cells, which is different from that in the myocardium, which mainly involves ACSS1. The uptake of radiolabeled acetate in tumors increased under hypoxia along with up-regulation of ACSS2 expression. This suggests a possible mechanism for acetate PET for tumors.

Development of [90Y]DOTA-conjugated bisphosphonate for treatment of painful bone metastases

INTRODUCTION Based on the concept of bifunctional radiopharmaceuticals, we have previously developed (186)Re-complex-conjugated bisphosphonate analogs for palliation of painful bone metastases and have demonstrated the utility of these compounds. By applying a similar concept, we hypothesized that a bone-specific directed (90)Y-labeled radiopharmaceutical could be developed. METHODS In this study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as the chelating site, and DOTA was conjugated with 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. [(90)Y]DOTA-complex-conjugated bisphosphonate ([(90)Y]DOTA-HBP) was prepared by coordination with (90)Y, and its biodistribution was studied in comparison to [(90)Y]citrate. RESULTS In biodistribution experiments, [(90)Y]DOTA-HBP and [(90)Y]citrate rapidly accumulated and resided in the bone. Although [(90)Y]citrate showed a higher level of accumulation in the bone than [(90)Y]DOTA-HBP, the clearances of [(90)Y]DOTA-HBP from the blood and from almost all soft tissues were much faster than those of [(90)Y]citrate. As a result, the estimated absorbed dose ratios of soft tissues to osteogenic cells (target organ) of [(90)Y]DOTA-HBP were lower than those of [(90)Y]citrate. CONCLUSIONS [(90)Y]DOTA-HBP showed superior biodistribution characteristics as a bone-seeking agent and led to a decrease in the level of unnecessary radiation compared to [(90)Y]citrate. Since the DOTA ligand forms a stable complex not only with (90)Y but also with lutetium ((177)Lu), indium ((111)In), gallium ((67/68)Ga), gadolinium (Gd) and so on, complexes of DOTA-conjugated bisphosphonate with various metals could be useful as agents for palliation of metastatic bone pain, bone scintigraphy and magnetic resonance imaging.