Yukie Yoshii

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)

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.

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.

Copper-64-diacetyl-bis (N4-methylthiosemicarbazone) accumulates in rich regions of CD133+ highly tumorigenic cells in mouse colon carcinoma

INTRODUCTION (64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a potential imaging agent of hypoxic tumor for use with PET. Recent literature demonstrated that cancer cells expressing CD133, which is a frequently used marker for so-called cancer stem cells or cancer stem cell-like cells (collectively referred to here as CSCs), contribute to tumors therapeutic resistance and metastasis ability. Culturing under hypoxia is also reported to enlarge the proportion of CD133(+) cells, which would indicate survival advantage of CD133(+) cells under hypoxia. Here, we investigated the relationships between (64)Cu-ATSM accumulation and existence of CD133(+) cells using mouse colon carcinoma (colon-26) tumor. METHODS Intratumor distribution of (64)Cu-ATSM and (18)F-fluorodeoxyglucose ((18)FDG) was compared with immunohistochemical staining for CD133 with a colon-26 model. In vitro characterization of CD133(+) colon-26 cells was also performed. RESULTS In colon-26 tumors, (64)Cu-ATSM localized preferentially in regions with a high density of CD133(+) cells. The percentage of CD133(+) cells was 11-fold higher in (64)Cu-ATSM high-uptake regions compared with (18)FDG high- (but (64)Cu-ATSM low-) uptake regions. CD133(+) colon-26 cells showed characteristics previously linked with CSCs in other cancer cell lines, such as high colony-forming ability, high tumor-initiating ability and enrichment under hypoxic cultivation. The proportion of CD133(+) cells was enlarged by culturing under glucose starvation as well as hypoxia, and (64)Cu-ATSM uptake was increased under such conditions. CONCLUSIONS Our findings showed that, in colon-26 tumors, (64)Cu-ATSM accumulates in rich regions of CD133(+) cells with characteristics of CSCs. Therefore (64)Cu-ATSM could be a potential imaging agent for rich regions of CD133(+) cells, associated with CSCs, within tumors.

Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation.

OBJECTIVES Radiolabeled Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) (*Cu-ATSM), including (60/62/64)Cu-ATSM, is a potential imaging agent of hypoxic tumors for positron emission tomography (PET). We have reported that *Cu-ATSM is trapped in tumor cells under intracellular overreduced states, e.g., hypoxia. Here we evaluated *Cu-ATSM as an indicator of intracellular overreduced states in mitochondrial disorders using cell lines with mitochondrial dysfunction. METHODS Mitochondrial DNA-less ρ(0)206 cells; the parental 143B human osteosarcoma cells; the cybrids carrying mutated mitochondria from a patient of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (2SD); and that carrying wild-type one (2SA) were used. Cells were treated under normoxia or hypoxia, and (64)Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH. RESULTS ρ(0)206 cells showed higher (64)Cu-ATSM uptake than control 143B cells under normoxia, whereas (64)Cu-ATSM uptake was not significantly increased under hypoxia in ρ(0)206 cells. Additionally, (64)Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased (64)Cu-ATSM uptake under normoxia as compared with the control 2SA, and (64)Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions. CONCLUSIONS (64)Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that (62)Cu-ATSM-PET can visualize stroke-like episodes maintaining oxygen supply in MELAS patients. Taken together, our data indicate that *Cu-ATSM uptake reflects overreduced intracellular states, despite oxygenic conditions; thus, *Cu-ATSM would be a promising marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinsons disease and Alzheimers disease.

Internal radiotherapy with copper-64-diacetyl-bis (N4-methylthiosemicarbazone) reduces CD133+ highly tumorigenic cells and metastatic ability of mouse colon carcinoma.

INTRODUCTION (64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is an imaging agent for positron emission tomography (PET) that targets hypoxic tumors. (64)Cu-ATSM is also reported to be a potential agent for internal radiotherapy. In a mouse colon carcinoma (Colon-26) model, we have shown that (64)Cu-ATSM preferentially localizes in intratumoral regions with a high density of CD133(+) cells, which show characteristics of cancer stem cells or cancer stem cell-like cells (collectively referred here as CSCs). In this study, we evaluated the therapeutic effect of (64)Cu-ATSM in relation to CD133 expression using this model. METHODS Systemic administration of 37 MBq (64)Cu-ATSM or saline was conducted twice within a 1-week interval to mice bearing 1-week-old Colon-26 tumors (days 0-7). At day 19, tumor size measurement, flow cytometry analysis and experimental lung metastatic assay were performed. The therapeutic effect of (64)Cu-ATSM on sorted CD133(+) and CD133(-) Colon-26 cells was also examined in vitro. RESULTS In vivo studies showed that (64)Cu-ATSM treatment inhibited tumor growth. The percentage of CD133(+) cells and metastatic ability in (64)Cu-ATSM treated tumors was decreased compared with that in control animals. In vitro studies demonstrated that (64)Cu-ATSM accumulated in cells under hypoxic conditions and incorporation of (64)Cu-ATSM under hypoxia caused cell death in both CD133(+) and CD133(-) cells in a similar extent. CONCLUSIONS (64)Cu-ATSM administration reduced tumor volume as well as the percentage of CD133(+) cells and the metastatic ability of Colon-26 tumors. Together with our data, it is suggested that (64)Cu-ATSM accumulates in regions high in CD133(+) highly tumorigenic cells and kills such regions by radiation, resulting in a decrease of the percentage of CD133(+) cells.

Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: Overview and application

Understanding cancer-specific metabolism is important for identifying novel targets for cancer diagnosis and therapy. Induced acetate/acetyl CoA metabolism is a notable feature that is related to fatty acid synthesis supporting tumor growth. In this review, we focused on the recent findings related to cancer acetate/acetyl CoA metabolism. We also introduce [1-¹¹C]acetate positron emission tomography (PET), which is a useful tool to visualize up-regulation of acetate/acetyl CoA metabolism in cancer, and discuss the utility of [1-¹¹C]acetate PET in cancer diagnosis and its application to personalized medicine.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells.

Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as β-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease.