Hideki Tsutsumi

Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice

The application of carbon nanotubes (CNTs) as biomaterials is of wide interest, and studies examining their application in medicine have had considerable significance. Biological safety is the most important factor when considering the clinical application of CNTs as biomaterials, and various toxicity evaluations are required. Among these evaluations, carcinogenicity should be examined with the highest priority; however, no report using transgenic mice to evaluate the carcinogenicity of CNTs has been published to date. Here, we performed a carcinogenicity test by implanting multi-walled CNTs (MWCNTs) into the subcutaneous tissue of rasH2 mice, using the carbon black present in black tattoo ink as a reference material for safety. The rasH2 mice did not develop neoplasms after being injected with MWCNTs; instead, MWCNTs showed lower carcinogenicity than carbon black. Such evaluations should facilitate the clinical application and development of CNTs for use in important medical fields.

Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD/Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products

The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγnull (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD50) values of HeLa cells were stable at 1.3 × 104 and 4.0 × 105 cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD50 value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 106 or 107 human mesenchymal stem cells as well as Matrigel, the TPD50 values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs.

Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate in an Ultra-Short-Term Skin Carcinogenesis Bioassay Using rasH2 Mice

Assessment of the skin tumor–promoting potential of 12-O-tetradecanoylphorbol-13-acetate (TPA) after initiation with 7,12-dimethylbenz[a]anthracene (DMBA) was conducted using rasH2 transgenic (Tg) mice and their nontransgenic (non-Tg) littermates. Mice were treated with DMBA (50 μg/100 μL acetone) on clipped back skin at the commencement of the study, and 1 week thereafter, TPA was applied at 8 μg/200 μL or 4 μg/200 μL acetone, once or twice weekly, for 7 weeks. Skin nodules were observed in the rasH2 Tg mice from week 4, and the incidence reached 100% at weeks 5 and 6. The number of skin nodules (multiplicity) in the 8-μg twice-weekly, 8-μg once-weekly, 4-μg twice-weekly, and 4-μg once-weekly groups was 62.4, 46.2, 62.6, and 36.9, respectively. The non-Tg mice also developed skin nodules, but the sensitivity to induction in the rasH2 Tg mice was higher. No nodules were observed in the acetone groups, but single nodules were apparent in the no-treatment rasH2 Tg and non-Tg groups. In conclusion, skin promotion effects could be detected within only 8 weeks in the rasH2 mice, and the concentration of 4 μg TPA once weekly was sufficient as a positive control. This short-term skin carcinogenesis bioassay using rasH2 mice could represent a useful tool for the assessment of drug and chemical safety with cutaneous treatment.

Tumorigenicity-associated characteristics of human iPS cell lines

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61–77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.

CORRIGENDUM: Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice

CORRIGENDUM: Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice