Takashi Kishimoto

Immune activation during pregnancy in mice leads to dopaminergic hyperfunction and cognitive impairment in the offspring: a neurodevelopmental animal model of schizophrenia.

BACKGROUND Maternal viral infection is associated with increased risk for schizophrenia. It is hypothesized that the maternal immune response to viruses may influence fetal brain development and lead to schizophrenia. METHODS To mimic a viral infection, the synthetic double strand RNA polyriboinosinic-polyribocytidilic acid (poly I:C) was administered into pregnant mice. Behavioral evaluations (thigmotaxis, methamphetamine [MAP]-induced hyperactivity, novel-object recognition test [NORT]), sensorimotor gating (prepulse inhibition [PPI]), and biochemical evaluation of the dopaminergic function of the offspring of phosphate-buffered saline (PBS)-treated dams (PBS-mice) and that of poly I:C-treated dams (poly I:C-mice) were examined. RESULTS In juveniles, no difference was found between the poly I:C-mice and PBS-mice. However, in adults, the poly I:C-mice exhibited attenuated thigmotaxis, greater response in MAP-induced (2 mg/kg) hyperlocomotion, deficits in PPI, and cognitive impairment in NORT compared with the PBS-mice. Cognitive impairment in the adult poly I:C-mice could be improved by subchronic administration of clozapine (5.0 mg/kg) but not haloperidol (.1 mg/kg). Increased dopamine (DA) turnover and decreased receptor binding of D2-like receptors, but not D1-like receptors, in the striatum were found in adult poly I:C-mice. CONCLUSIONS Prenatal poly I:C administration causes maturation-dependent increased subcortical DA function and cognitive impairment in the offspring, indicating a neurodevelopmental animal model of schizophrenia.

Rapamycin, a specific inhibitor of the mammalian target of rapamycin, suppresses lymphangiogenesis and lymphatic metastasis

Tumor lymphangiogenesis is now known to play a causal role in lymph node metastasis, and thus its inhibition would have great significance for the prevention of lymph node metastasis in cancer therapy. VEGF‐C has recently been identified as a key molecule that involved in tumor lymphangiogenesis and lymphatic metastasis. However, the expressional regulation of VEGF‐C is not fully understood. We investigated the role of mTOR, which is a downstream kinase of the phosphatidylinositol 3‐kinase/Akt pathway, and the MAPK family (MEK1/2, p38, and JNK) in the regulation of VEGF‐C and VEGF‐A expression in B13LM cells, a lymphatic metastasis‐prone pancreatic tumor cell line. We also investigated the antilymphangiogenic effect of rapamycin, a specific inhibitor of mTOR in vivo using male BALB/c nu/nu mice. VEGF‐C expression was inhibited by the inhibitors for mTOR, p38, and JNK, but not by the inhibitor for MEK1/2, whereas VEGF‐A expression was inhibited by all four of these inhibitors. The serum starvation‐induced expression of VEGF‐C was inhibited by rapamycin, whereas that of VEGF‐A was incompletely inhibited. The metastatic experiment in vivo demonstrated that the number and the area of lymphatic vessels in the primary tumors were significantly decreased by rapamycin. Finally, the lymph node metastasis was significantly suppressed in rapamycin‐treated mice. Our results suggest that mTOR, p38, and JNK play important roles in VEGF‐C expression, and that rapamycin has an antilymphangiogentic effect and exerts the expected inhibition of lymphatic metastasis. (Cancer Sci 2007; 98: 726–733)

Non-Invasive Detection of a Small Number of Bioluminescent Cancer Cells In Vivo

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Development of autoimmune hepatitis–like disease and production of autoantibodies to nuclear antigens in mice lacking B and T lymphocyte attenuator

OBJECTIVE B and T lymphocyte attenuator (BTLA), a coreceptor expressed on lymphocytes, was recently described as an inhibitory coreceptor that negatively regulates lymphocyte activation. The purpose of this study was to investigate the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity. METHODS We examined the levels of immunoglobulins and autoantibodies to nuclear antigens and the activation status of T cells in BTLA(-/-) mice. We also examined histopathologic changes in the organs of BTLA(-/-) mice. RESULTS We observed that BTLA(-/-) mice gradually developed hypergammaglobulinemia, antinuclear antibodies, anti-SSA antibodies, anti-double-stranded DNA antibodies, and an increased number of activated CD4+ T cells in the periphery with age. Lack of BTLA led to spontaneous development of autoimmune hepatitis-like disease characterized by an elevation in the level of transaminases, interface hepatitis, and spotty necrosis of the liver. BTLA(-/-) mice also showed inflammatory cell infiltration of multiple organs, including the salivary glands, lungs, and pancreas; these features are similar to those of Sjögrens syndrome, which is a frequent complication of autoimmune hepatitis. Furthermore, the survival rate of BTLA(-/-) mice was significantly reduced after the age of 7 months. CONCLUSION Our results indicate that BTLA plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases.

Expression of an activated mammalian target of rapamycin (mTOR) in gastroenteropancreatic neuroendocrine tumors

AimsGastroenteropancreatic neuroendocrine tumors are rare, and the current WHO classification divides this tumor entity into well-differentiated (neuro)endocrine tumors, well-differentiated (neuro)endocrine carcinomas, and poorly differentiated (neuro)endocrine carcinomas. Poorly differentiated (neuro)endocrine carcinoma is extremely aggressive, and no appropriate therapeutic approach has been established. The mammalian target of rapamycin (mTOR), an important regulator of cell proliferation and protein translation, is activated in various malignancies. Recent phase II trial has revealed the efficacy of mTOR inhibitor (RAD001; everolimus) against low-to-intermediate grade neuroendocrine tumors. However, the beneficial role of mTOR inhibitor against poorly neuroendocrine carcinoma remains uncertain. The purpose of the present study was to determine the activation of mTOR in gastropancreatic neuroendocrine tumors, especially in poorly differentiated neuroendocrine carcinomas.MethodsExpression of p-mTOR(Ser2448) was assessed by immunohistochemistry in 20 gastropancreatic neuroendocrine tumors (seven well-differentiated neuroendocrine tumors, four well-differentiated neuroendocrine carcinomas, and nine poorly differentiated neuroendocrine carcinomas). Double immunohistochemistry was performed with p-Akt for patients with high p-mTOR expression.ResultsExpression of mTOR was seen in 9 (45%) of 20 gastroenteropancreatic neuroendocrine tumors. High expression of p-mTOR was seen in 6 (67%) of 9 poorly differentiated neuroendocrine carcinomas which was higher than the expression rate of well-differentiated neuroendocrine tumors and carcinomas, 3 (27%) of 11. All large cell neuroendocrine carcinomas showed high p-mTOR expression. Some tumor cells showed positive staining for p-mTOR co-expressed p-Akt.ConclusionsHigh expression rate of p-mTOR in poorly differentiated neuroendocrine carcinomas (large-cell type) may suggest the potential role of mTOR inhibitors as effective therapeutic agents for this highly malignant disease.

Hepatoid carcinoma of the ovary: a report of three cases admixed with a common surface epithelial carcinoma.

&NA; Hepatoid carcinoma of the ovary is an ovarian carcinoma that has phenotypic properties in common with hepatocellular carcinomas. However, the extent of the tumor cells’ similarity to and their difference from hepatocytes is largely unknown. In addition, the precursor cell of origin for hepatoid carcinoma of the ovary has not been identified. Three cases of alpha‐fetoprotein‐producing hepatoid carcinoma of the ovary that were admixed with an adenocarcinoma of common surface epithelial type are reported. The hepatoid carcinomas had a trabecular architecture with canaliculi detected by polyclonal (but not monoclonal) anticarcinoembryonic antigen antibodies. A hepatic phenotype in the hepatoid tumor cells was further supported by the production of albumin mRNA by in situ hybridization. The adenocarcinomas in the three cases were mucinous (Case 1), serous (Case 2), and endometrioid (Case 3), respectively. The cytokeratin (CK) profile in both the hepatoid and adenocarcinomatous components was CK18+/CK19+/CK20±, whereas normal and neoplastic hepatocytes were CK18+/CK19‐/CK20‐. Although this study supports a hepatic phenotype in ovarian hepatoid carcinoma, the CK profile of hepatoid carcinoma differs from that of normal and neoplastic hepatocytes but resembles that of the associated common epithelial adenocarcinoma. These findings suggest that hepatoid carcinoma of the ovary is probably derived from carcinomas of surface epithelial origin by a process of neometaplasia or transdifferentiation.

Predominance of IgG4+ plasma cells and CD68 positivity in sclerosing angiomatoid nodular transformation (SANT)

1. Young RH, Scully RE. Metastatic tumors of the ovary. In Kurman RJ ed. Blaustein’s pathology of the female genital tract, 5th edn. Chapter 22. New York: Springer-Verlag, 2002; 1067–1068. 2. Ricci R, Komminoth P, Bannwart F et al. PTEN as a molecular marker to distinguish metastatic from primary synchronous endometrioid carcinomas of the ovary and uterus. Diagn. Mol. Pathol. 2003; 12; 71–78. 3. Kaneki E, Oda Y, Ohishi Y et al. Frequent microsatellite instability in synchronous ovarian and endometrial adenocarcinoma and its usefulness for differential diagnosis. Hum. Pathol. 2004; 35; 1484– 1493. 4. Nowak NJ, Snijders A, Conroy JM et al. The BAC resource: Tools for array CGH and FISH. Curr. Protoc. Hum. Genet. 2005; 1–34. 5. Nowak NJ, Miecznikowski J, Moore SR et al. Challenges in array comparative genomic hybridization for the analysis of cancer samples. Genet. Med. 2007; 9; 585–595. 6. Le Levan K, Partheen K, Osterberg L, Helou K, Horvath G. Chromosomal alterations in 98 endometrioid carcinomas analyzed with comparative genomic hybridization. Cytogenet. Genome Res. 2006; 115; 16–22. 7. Sonoda G, Palazzo J, du Manoir S et al. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas. Genes Chromosomes Cancer 1997; 20; 320–328. 8. Bayani J, Brenton JD, Macgregor PF et al. Parallel analysis of sporadic primary ovarian carcinomas by spectral karyotyping, comparative genomic hybridization, and expression microarrays. Cancer Res. 2002; 62; 3466–3476. 9. Fishman A, Shalom-Paz E, Fejgin M, Gaber E, Altaras M, Amiel A. Comparing the genetic changes detected in the primary and secondary tumor sites of ovarian cancer using comparative genomic hybridization. Int. J. Gynecol. Cancer 2005; 15; 261– 266.

Inhibitory effects of the antiangiogenic agent TNP-470 on establishment and growth of hematogenous metastasis of human pancreatic carcinoma in SCID beige mice in vivo

Effects on the liver of the antiangiogenesis agent O-(chloroacetyl-carbamoyl) fumagillol (TNP-470) on hematogenous metastasis of a human pancreatic carcinoma cell line were examined. One million PCI-43 cells, a human pancreas carcinoma cell line, were injected into the spleen of SCID beige mice, then TNP-470 at 30 mg/kg was administered subcutaneously every other day. The mice were killed 6 or 10 weeks thereafter and metastatic nodules in the liver were counted and measured microscopically. Metastases were inhibited and an ∼10% loss of weight was evident in the TNP-470-administered mice. There was no suppression in maximal size of metastatic colonies in mice given the agent for 6 weeks, while inhibition was apparent in mice given the drug for 10 weeks. Suppression of proliferation and an increase in apoptosis were evident in metastatic nodules in the TNP-470-administered groups, following stainings for proliferative cell nuclear antigen and terminal deoxytransferase-mediated dUTP-biotin nick endlabeling, respectively. TNP-470 inhibited the proliferation of human umbilical vein endothelial cells but not PCI-43 in vitro. TNP-470 did not suppress production of vascular endothelial growth factor by PCI-43 cells. Neovascularization in vivo induced by PCI-43 cells was suppressed in the TNP-470-administered mice, using a diffusion chamber placed in subcutaneous tissues of SCID beige mice. These observations suggest that inhibition of angiogenesis is effective in suppressing establishment and subsequent growth of hematogenous micrometastases of pancreatic adenocarcinoma to the liver.

Hepatoid adenocarcinoma: a new clinicopathological entity and the hypotheses on carcinogenesis

Hepatoid adenocarcinoma is reviewed in its clinicopathological and oncogenetic aspects. This variant of adenocarcinoma has been found to be an alpha-fetoprotein (AFP) -producing carcinoma arising in extrahepatic organs, and it mimics hepatocellular carcinoma in terms of morphology and function. Vascular invasion, usually prominent, is often complicated by extensive liver metastases. A prompt and accurate diagnosis of hepatoid adenocarcinoma is important because the prognosis is very poor compared with that of common types of adenocarcinoma. The characteristic features of hepatoid adenocarcinoma are summarized on the basis of our own experiences and the literature. In addition, a possible molecular mechanism that under-lies the ectopic appearance of the hepatic phenotype is discussed.