Comprehensive gene profiling of the metabolic landscape of humanized livers in mice.

BACKGROUND & AIMS: The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. METHODS: We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. RESULTS: Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. CONCLUSION: Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). IMPACT AND IMPLICATIONS: Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans.

A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Microarray analysis on germfree mice elucidates the primary target of a traditional Japanese medicine juzentaihoto: acceleration of IFN-α response via affecting the ISGF3-IRF7 signaling cascade.

BACKGROUND: The traditional Japanese medicine juzentaihoto (JTX) is a pharmaceutical grade multi-herbal medicine widely used for the prevention of cancer metastasis and infection in immuno-compromized patients in Japan. The effect of JTX has been supposed to be intimately affected by the immunological properties of host and enteric microflora. The influence of JTX on the gene expression profile in the large and small intestines was investigated by microarray analyses using mice of different strains with or without enteric microflora. RESULTS: In all types of mice, including germfree (GF) animals, the genes most affected by two-week oral JTX treatment were the type 1 interferon (IFN)-related genes including Stat1, Isgf3g and Irf7, which play a critical role in the feedback loop of IFN-α production cascade. In IQI specific pathogen free (SPF) mice JTX increased the steady state level of the expression of IFN-related genes, but had the opposite effect in IQI GF and BALB/c SPF mice. Promoter analysis suggests that tandem repeated $IRFF (the promoter sequences for interferon regulatory factors) may be a primary target for JTX action. Pre-treatment of JTX accelerated the effects of an oral IFN "inducer" 2-amino-5-bromo-6-methyl-4-pyrimidinol (ABMP) (up-regulation of IFN-α production in IQI strain and down-regulation in BALB/c mice), which is in good accordance with the effect of JTX on gene expression of type 1 IFN-related genes. CONCLUSIONS: Microarray analysis revealed that the target of JTX might be the transcription machinery regulating the steady-state level of genes involved in the ISGF3-IRF7 cascade, whose effect is bi-directional in a strain- and microbiota-dependent manner.

A microarray analysis of gnotobiotic mice indicating that microbial exposure during the neonatal period plays an essential role in immune system development.

BACKGROUND: Epidemiological studies have suggested that the encounter with commensal microorganisms during the neonatal period is essential for normal development of the host immune system. Basic research involving gnotobiotic mice has demonstrated that colonization at the age of 5 weeks is too late to reconstitute normal immune function. In this study, we examined the transcriptome profiles of the large intestine (LI), small intestine (SI), liver (LIV), and spleen (SPL) of 3 bacterial colonization models-specific pathogen-free mice (SPF), ex-germ-free mice with bacterial reconstitution at the time of delivery (0WexGF), and ex-germ-free mice with bacterial reconstitution at 5 weeks of age (5WexGF)-and compared them with those of germ-free (GF) mice. RESULTS: Hundreds of genes were affected in all tissues in each of the colonized models; however, a gene set enrichment analysis method, MetaGene Profiler (MGP), demonstrated that the specific changes of Gene Ontology (GO) categories occurred predominantly in 0WexGF LI, SPF SI, and 5WexGF SPL, respectively. MGP analysis on signal pathways revealed prominent changes in toll-like receptor (TLR)- and type 1 interferon (IFN)-signaling in LI of 0WexGF and SPF mice, but not 5WexGF mice, while 5WexGF mice showed specific changes in chemokine signaling. RT-PCR analysis of TLR-related genes showed that the expression of interferon regulatory factor 3 (Irf3), a crucial rate-limiting transcription factor in the induction of type 1 IFN, prominently decreased in 0WexGF and SPF mice but not in 5WexGF and GF mice. CONCLUSION: The present study provides important new information regarding the molecular mechanisms of the so-called "hygiene hypothesis".

Pancreatic enlargement in a patient receiving therapy with vasodilators for pulmonary arterial hypertension: a case report.

Epoprostenol is a prostacyclin (prostaglandin I2) analog that causes vasodilation and inhibits platelet aggregation and is used in the management of severe pulmonary arterial hypertension (PAH). We herein report a patient with PAH who developed pancreatic enlargement after the initiation of therapy including epoprostenol. Although it is well known that thyroid enlargement occurs in patients with PAH receiving epoprostenol therapy, the pancreatic findings associated with epoprostenol therapy have not been well described. Although the size of the pancreas was clearly increased, there was no blood data or symptoms suggestive of abnormal pancreatic function and pancreatitis, and the patient's abdominal complaints improved quickly, despite the continuation of epoprostenol therapy. Eleven months after the start of continuous intravenous epoprostenol infusion therapy, the pancreatic enlargement was still evident on imaging, but there were no abdominal symptoms or elevated pancreatic enzymes. This case highlights the fact that epoprostenol therapy may cause pancreatic changes that mimic acute or autoimmune pancreatitis.

Identification of human long noncoding RNAs associated with nonalcoholic fatty liver disease and metabolic homeostasis.

A growing number of long noncoding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are nonconserved and highly tissue specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we established a pipeline to identify putative hLMRs that are metabolically sensitive, disease relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and respond to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one nonconserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcame the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.

Interferon-alpha 2b-induced thrombocytopenia is caused by inhibition of platelet production but not proliferation and endomitosis in human megakaryocytes.

Human interferon (IFN)-alpha is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-alpha and often leads to dose reduction or treatment discontinuation. However, there is little information on how IFN-alpha inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-alpha did not inhibit colony formation of megakaryocytes from human CD34(+) hematopoietic stem cells. IFN-alpha did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-alpha suppressed the expression of transcription factors regulating late-stage megakaryopoiesis, such as GATA-1, p45(NF-E2), MafG. IFN-alpha also significantly reduced the number of human platelets but not megakaryocytes, and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2R gamma(null) (NOG) mice transplanted with human CD34(+) cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic, NIP-004, was effective for treating IFN-alpha-induced thrombocytopenia in hu-NOG mice. From ultrastructural study, IFN-alpha inhibited the maturation of demarcation membranes in megakaryocytes, although NIP-004 prevented the inhibitory effects of IFN-alpha. These results defined the pathogenesis of IFN-alpha-induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics.

Whole-exome sequencing of 79 xenografts as a potential approach for the identification of genetic variants associated with sensitivity to cytotoxic anticancer drugs.

Chemotherapy response remains unpredictable in most patients with cancer. In this study, we performed whole-exome sequencing of 79 cancer xenografts derived from human cancer tissues to identify genetic predictors of chemosensitivity to nine cytotoxic anticancer drugs. Xenografts were harvested from 12 organs with cancer and implanted into nude mice. The mice were exposed to one of nine cytotoxic anticancer drugs (5-fluorouracil, nimustine, adriamycin, cyclophosphamide, cisplatin, mitomycin C, methotrexate, vincristine, and vinblastine) to assess the correlation between chemosensitivity response and variant allele frequency. We found 162 candidate variants that were possibly associated with chemosensitivity to one or more of the nine anticancer drugs (P < 0.01). In a subgroup analysis of breast and gastric cancer xenografts, 78 and 67 variants, respectively, were possibly associated with chemosensitivity. This approach may help to contribute to the development of personalized treatments that may allow for the prescription of optimal chemotherapy regimens among patients with cancer.

Effects of Supplementation with Anti-Inflammatory Compound Extracted from Herbs in Healthy and Obese Cats.

BACKGROUND: Obesity has become a serious public health problem all over the world, and prevalence of obesity has increased in cats. Obesity is characterized by continuous low-grade inflammation based on oxidative stress by excessively produced reactive oxygen species (ROS). Supplementation with anti-oxidant and anti-inflammatory compounds is very effective to relieve the obesity condition. A plant extract mixture containing Rhus verniciflua and some other herbs, Rv-PEM01-99, shows anti-oxidant and anti-inflammatory effects in animals. The aim of this study was to evaluate the effects of supplementation with Rv-PEM01-99 as an anti-inflammatory compound in healthy and obese cats. MATERIALS AND METHODS: Ten healthy mix breed cats and four obesity disease cats were used. The healthy cats were randomly divided into control and test groups. Anti-inflammatory compound, Rv-PEM01-99, in which quercetin derivative is the main component, was supplemented to the healthy test group and the obesity disease cats at the dose of 100-120 mg/kg/day (2.5-3.0 mg/kg/day as quercetin) for 4 weeks. Metabolites, hormones and enzymes were measured before and after the compound supplementation. RESULTS: The anti-inflammatory compound supplementation decreased serum amyloid A (SAA) concentrations as inflammatory markers in both healthy and obesity disease cats. In obesity disease cats, plasma total cholesterol concentrations and AST and ALT activities decreased significantly after the compound supplementation. CONCLUSION: Quercetin derivative seems to have strong anti-inflammatory activities. In the healthy cats, anti-inflammatory compound supplementation decreased plasma NEFA and SAA concentrations. In the obesity disease cats, the compound supplementation may have alleviated obesity disease by relieving inflammation and improvement of lipid metabolism in livers.

In vivo functional analysis of non-conserved human lncRNAs associated with cardiometabolic traits.

Unlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) are considered non-conserved. Although lncRNAs have been shown to function in diverse pathophysiological processes in mice, it remains largely unknown whether human lncRNAs have such in vivo functions. Here, we describe an integrated pipeline to define the in vivo function of non-conserved human lncRNAs. We first identify lncRNAs with high function potential using multiple indicators derived from human genetic data related to cardiometabolic traits, then define lncRNA's function and specific target genes by integrating its correlated biological pathways in humans and co-regulated genes in a humanized mouse model. Finally, we demonstrate that the in vivo function of human-specific lncRNAs can be successfully examined in the humanized mouse model, and experimentally validate the predicted function of an obesity-associated lncRNA, LINC01018, in regulating the expression of genes in fatty acid oxidation in humanized livers through its interaction with RNA-binding protein HuR.

Spontaneous thymic lymphomas in the non-obese diabetic/Shi-scid, IL-2R gamma (null) mouse.

The NOD/Shi-scid, IL-2Rgamma(null) (NOG) mouse is a severely immunodeficient mouse used for the engraftment of human tissues and cells. In this study, 2406 mice (8-62 weeks old, 503 males and 1903 females) were subcutaneously engrafted with human tissues. In 16 mice (12-26 weeks old, 1 male and 15 females), a mass was seen in the anteroventralis of the thorax on gross examination with an incidence of 0.7%. Histologically, the masses were composed of sheets of lymphoblastic cells. A 'starry sky' pattern was observed with numerous mitoses. Immunohistochemically the lymphoblastic cells were positive for Thy 1. The lymphoblastic cells were also seen in the spleen, lung, liver, kidney and heart. The gross and histopathological findings led to the diagnosis of spontaneous thymic lymphoma in NOG mice.

Importance of the interferon-alpha system in murine large intestine indicated by microarray analysis of commensal bacteria-induced immunological changes.

BACKGROUND: Although microbiota play a critical role in the normal development and function of host immune systems, the underlying mechanisms, especially those involved in the large intestine (LI), remain unknown. In the present study, we performed transcriptome analysis of the LI of germ-free (GF) and specific pathogen-free (SPF) mice of the IQI strain, an inbred strain established from ICR mice. RESULTS: GeneChip analysis, quantitative real-time RT-PCR, and reconfirmation using bacteria-inoculated GF mice revealed differences in the expression levels of several immune-related genes, such as cryptdin-related sequences (CRS), certain subsets of type 1 interferon (IFN)-related genes, class Ib MHC molecules, and certain complements. LI expressed no authentic cryptdins but predominantly expressed CRS2, 4, and 7. The mRNA levels of IFN-related genes, including Irf7, Isgf3g, Ifit1 and Stat1, were lower in SPF- and flora-reconstituted mice. When an oral IFN-alpha inducer tilorone analog, R11567DA, was administered to SPF mice, IFN-alpha was induced rapidly in the LI at 4 h, whereas no IFN-alpha protein was detected in the small intestine (SI) or blood. In situ hybridization and immunohistochemistry suggested that the IFN-alpha production originated from Paneth cells in the SI, and portions of lamina proprial CD11b- or mPDCA1-positive cells in the LI. CONCLUSION: The present study suggests that microbial colonization, while inducing the expression of anti-microbial peptides, results in the down-regulation of certain genes responsible for immune responses, especially for type I IFN synthesis. This may reflect the adaptation process of the immune system in the LI to prevent excessive inflammation with respect to continuous microbial exposure. Further, the repertoire of anti-microbial peptides and the extraordinary role of interferon producing cells in the LI have been found to be distinct from those in the SI.

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice.

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.

The effect of a novel, small non-peptidyl molecule butyzamide on human thrombopoietin receptor and megakaryopoiesis.

BACKGROUND: Thrombocytopenia is a common problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin. DESIGN AND METHODS: We screened our chemical library and created a novel non-peptidyl thrombopoietin receptor, Mpl activator named butyzamide. We evaluated the effect of butyzamide on megakaryopoiesis in vitro using Ba/F3 cells expressing Mpl and human hematopoietic stem cells. For the evaluation of its in vivo effect, we administered butyzamide orally to immunodeficient NOD/Shi-scid,IL-2R gamma(null) (NOG) mice transplanted with human fetal liver-derived CD34(+) cells and investigated the production of human platelets. RESULTS: Butyzamide specifically reacted with human Mpl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyzamide did not react with murine Mpl and was shown to require the histidine residue in the transmembrane domain of Mpl for its agonistic activity. Butyzamide induced colony-forming unit-megakaryocytes and polyploid megakaryocytes from human CD34(+) hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was administered orally at the doses of 10 and 50 mg/kg for 20 days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, the human platelet count increased by 6.2- and 22.9-fold, respectively. CONCLUSIONS: Butyzamide is an orally bioavailable human Mpl activator, and appears to have potential for clinical development as a therapeutic agent for patients with thrombocytopenia.

Establishment and characterization of in vivo human tumor models in the NOD/SCID/gamma(c)(null) mouse.

Immunodeficient mice are widely used for xenografts of human cells and tissue. The purpose of the present study was to investigate the characteristics of xenograft human tumor models using engraftment of various non-hematopoietic tumors in the NOD/SCID/gamma(c) (null) mouse. For tumor models, human solid tumor tissues were serially passaged three or more times to establish tissue lines. A total of 326 fresh tumor specimens, mainly gastrointestinal and female genital tissue, were engrafted with 54 established tissue lines. The types of tissue lines varied and included tumor tissue of both epithelial and mesenchymal origin. In some cases the original surgical specimen was replaced with large mononuclear cells. In the established tumor tissue lines, differentiation and tumor structure were similar to that of the original surgical specimen. The interstitium of the xenograft tissue in the tissue lines was relatively well preserved although slightly decreased and replaced by host tissue. These results indicate that human solid tumors can be successfully engrafted into the NOD/SCID/gamma(c) (null) mouse and that tissue lines with the characteristics of the original tumors can be established. Investigators in the field of tumor research will benefit from the availability of tissue lines that allow the establishment of more relevant in vivo human tissue models.

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background.

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).

Targeted sequencing reveals genetic variants associated with sensitivity of 79 human cancer xenografts to anticancer drugs.

Although there has been progress moving from a 'one-size-fits-all' cytotoxic approach to personalized molecular medicine, the majority of patients with cancer receive chemotherapy using cytotoxic anticancer drugs. The sequencing analysis of 409 genes associated with cancer was conducted in the present study using 59 DNA sequences extracted from human cancer xenografts implanted into nude mice, of which sensitivity to 9 cytotoxic anticancer drugs [5-fluorouracil, nimustine, adriamycin, cyclophosphamide, cisplatin, mitomycin C (MMC), methotrexate, vincristine (VCR), and vinblastine] was examined. The present study investigated the association between the sensitivities of the xenografts to the 9 anticancer drugs and the frequency of single nucleotide variants (SNV). The correlation between the expression level of the genes and sensitivities to the 9 drugs in the above xenografts was also estimated. In the screening study using 59 xenografts, 3 SNVs (rs1805321, rs62456182 in PMS1 Homolog 2, Mismatch Repair System Component and rs13382825 in LDL Receptor Related Protein 1B), were associated with sensitivity to VCR and MMC, respectively (P<0.001). A replication study of 596 SNVs was subsequently performed, which indicated P<0.05 in the screening study using independent samples of 20 xenografts. A combined result of the screening and replication studies indicated that 35 SNVs were potentially associated with sensitivities to one or more of the nine anticancer drugs (Pcombined=0.0011-0.035). Of the 35 SNVs, rs16903989 and rs201432181 in Leukemia Inhibitory Factor Receptor α and Adhesion G Protein-Coupled Receptor A2 were commonly associated with sensitivity to 2 or 4 anticancer drugs, respectively. These findings provide novel insights which may benefit the development of personalized anticancer therapy for patients with cancer in the future.

Identification of a key molecular regulator of liver metastasis in human pancreatic carcinoma using a novel quantitative model of metastasis in NOD/SCID/gammacnull (NOG) mice.

We developed a reliable new model system for assaying liver metastasis using NOD/SCID/gamma(c)(null) (NOG) mice. Seven human pancreatic cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOD/SCID and NOG mice. Capan-2 and PL45 showed no metastasis when seeded at up to 10(5) cells in both strains, and no BxPC-3 metastasis was observed in NOD/SCID mice. The NOD/SCID mouse model detected liver metastasis only in the AsPC-1 cell line when inoculated with >10(3) cells. In contrast, when inoculated with only 10(2) MIA PaCa-2, AsPC-1 and PANC-1 cells, liver metastasis was evident in 71.4% (5/7), 57.1% (4/7) and 37.5% (3/8) of the NOG mice, respectively. Capan-1 and BxPC-3 cells metastasized when seeded at 10(3) cells in 50% (5/10) and in 12.5% (1/8) of the mice, respectively. Using the NOG mouse model system, we established a highly metastatic cell line, liver metastasized-BxPC-3 (LM-BxPC-3), from liver metastatic foci formed by the relatively poorly metastatic parental BxPC-3 cell line. The gene expression profiles of parental and LM-BxPC-3 cells were compared, and we identified forty-five genes that were either upregulated or downregulated >4-fold in the LM-BxPC-3 cell line. We validated 9 candidate protein-coding sequences, and examined the correlation between their expression pattern and the in vivo liver metastatic potential of all 7 pancreatic cancer cell lines. Only S100A4 expression correlated with the ability to form liver metastases, as evaluated in our quantitative model of metastasis in NOG mice. These results suggested that S100A4 is a key regulator of liver metastasis in pancreatic cancer, and demonstrated the feasibility of using the quantitative metastasis model to search for and develop new anti-cancer therapies and novel drugs against this and other key molecules.

Phenotypes of IRS-2 deficient mice produced by reproductive technology are stable.

We studied the impact of "IVF - ET" on the glucose tolerance test (GTT), insulin tolerance test (ITT) and adiponectin to investigate differences in the phenotypes of B6J- Irs2(-/-) mice. The B6J-Irs2(-/-) mice (KO-Nat group) were prepared by natural mating. Other mice were produced by IVF-ET used ICR strain recipients and surrogate mothers (KO-IVF group). Measurement of body weight, GTT, ITT and blood sampling were performed at the ages of 6, 14 and 24 weeks after birth. Body weights, impaired glucose tolerance, insulin resistance and plasma adiponectin concentrations did not differ for each gender between the KO-IVF and KO-Nat groups. Therefore, we concluded that phenotypes of Irs2(-/-) mice produced by reproductive technology are stable.