Glioma stem cell (GSC)-derived autoschizis-like products confer GSC niche properties involving M1-like tumor-associated macrophages.

Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.

A Synthetic Polymer Scaffold Reveals the Self-Maintenance Strategies of Rat Glioma Stem Cells by Organization of the Advantageous Niche.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.

Follicular dendritic cells activate HIV-1 replication in monocytes/macrophages through a juxtacrine mechanism mediated by P-selectin glycoprotein ligand 1.

Follicular dendritic cells (FDCs) are located in the lymphoid follicles of secondary lymphoid tissues and play a pivotal role in the selection of memory B lymphocytes within the germinal center, a major site for HIV-1 infection. Germinal centers are composed of highly activated B cells, macrophages, CD4(+)T cells, and FDCs. However, the physiological role of FDCs in HIV-1 replication remains largely unknown. We demonstrate in our current study that FDCs can efficiently activate HIV-1 replication in latently infected monocytic cells via an intercellular communication network mediated by the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction. Upon coculture with FDCs, HIV-1 replication was significantly induced in infected monocytic cell lines, primary monocytes, or macrophages. These cocultures were found to synergistically induce the expression of P-selectin in FDCs via NF-kappaB activation and its cognate receptor PSGL-1 in HIV-1-infected cells. Consistent with this observation, we find that this response is significantly blocked by antagonistic Abs against PSGL-1 and almost completely inhibited by PSGL-1 small interfering RNA. Moreover, a selective inhibitor for Syk, which is a downstream effector of PSGL-1, blocked HIV-1 replication in our cultures. We have thus elucidated a novel regulatory mechanism in which FDCs are a potent positive bystander that facilitates HIV-1 replication in adjacent infected monocytic cells via a juxtacrine signaling mechanism.

Follicular dendritic cell sarcoma with microtubuloreticular structure and virus-like particle production in vitro.

Neoplasm of follicular dendritic cells (FDC), follicular dendritic cell sarcoma (FDCS), is a rare tumor of intermediate to high-grade malignancy in lymph nodes and visceral organs. Reported herein is a case of FDCS arising from cervical lymph nodes in a 16-year-old Japanese boy, who died of the disease 3 years after diagnosis. The tumor cells were pale eosinophilic and elongated with euchromatic nuclei and were positive for CD21, clusterin, and CNA-42 on immunohistochemistry, as well as desmosome-like junctions on electron microscopy. The presence of microtubuloreticular structures (MTRS) in the tumor cells and associated lymphocytes characterized this case, suggesting some viral infection, although qualitative polymerase chain reaction of genomic and complementary DNA obtained from the tumor failed to demonstrate any viral infection at the laboratory level. The stimulation of dispersed tumor cells and peripheral blood mononuclear cells with mAb to CD3 and interleukin-2 was attempted; and the cell line established by the authors (FDCS-Sa) was stimulated with iododeoxyuridine. Virus-like particles (VLP) were successfully induced from each cellular source. The VLP, 100 nm in diameter, showed an electron-dense thorny envelope and granular core. This is the first case of FDCS with MTRS accompanying VLP production in vitro.

A new humanized mouse model of Epstein-Barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses.

The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (

Humanized NOD/SCID/IL2Rgamma(null) mice transplanted with hematopoietic stem cells under nonmyeloablative conditions show prolonged life spans and allow detailed analysis of human immunodeficiency virus type 1 pathogenesis.

In a previous study, we demonstrated that humanized NOD/SCID/IL2Rgamma(null) (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.

Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses.

Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.

Efficient intervention of growth and infiltration of primary adult T-cell leukemia cells by an HIV protease inhibitor, ritonavir.

Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.

Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-kappaB activity in unconditioned NOD/SCID/gammac(null) mice.

As there are very few reproducible animal models without conditioning available for the study of human B-cell-type Hodgkin's lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/gammac(null) (NOG) mice. Four human Epstein-Barr virus-negative cell lines (KM-H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B-cell-type, but not T-cell-type, HL cells correlated with an elevation in serum human interleukin-6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)-kappaB DNA binding activity, and the induced NF-kappaB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B-cell-type and T-cell-type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies.

Mouse serum factor(s) down-modulate the CD4 and CXCR4 molecules on human T cells conferring resistance to HIV infection in NOG mice.

Human cells have developed innate immunity, exploiting several means to block virus infection, and viruses have evolved diverse strategies to resist these. We show here that the human immunodeficiency virus 1 (HIV-1) could neither progressively infect engrafted human leukemic T cells nor repress their growth in NOG mice. However, ED-40515(-) cells infected with HIV-1 before inoculation were found to significantly delay the onset of tumor growth and increased the survival period of NOG mice. ED-40515(-) tumor cells showed resistance to HIV-1 which was apparently correlated with the down-regulation of CD4 and CXCR4 molecules in NOG mice. Serum from three different mouse strains, including NOG, retained a suppressive effect on the CD4 molecule of ED-40515(-) cells in vitro. ED-40515(-) cells obtained from mice re-expressed CD4 and CXCR4 molecules upon in vitro culture and were again successfully infected with HIV-1. These findings indicate that HIV-1 may initially successfully delay or regress tumor growth in NOG mice, but eventually fails to do so because of the evolution of HIV-resistant cells due to a rapid down-modulation of CD4 and CXCR4. Our data also demonstrated that some unknown soluble factor(s) present in mouse serum was responsible for conferring resistance to HIV infection to human T cells.

Prompt tumor formation and maintenance of constitutive NF-kappaB activity of multiple myeloma cells in NOD/SCID/gammacnull mice.

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM-1 and U-266 in an in vivo cell proliferation model using NOD/SCID/gammacnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post-auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM-1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human lambda-chain in various organs of all NOG mice, while the U-266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human lambda Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL-6R and gp130. Tumor cells sustained a strong NF-kappaB activity in vivo and induced NF-kappaB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

Rapid tumor formation of human T-cell leukemia virus type 1-infected cell lines in novel NOD-SCID/gammac(null) mice: suppression by an inhibitor against NF-kappaB.

We established a novel experimental model for human T-cell leukemia virus type 1 (HTLV-1)-induced tumor using NOD-SCID/gammac(null) (NOG) mice. This model is very useful for investigating the mechanism of tumorigenesis and malignant cell growth of adult T-cell leukemia (ATL)/lymphoma, which still remains unclear. Nine HTLV-1-infected cell lines were inoculated subcutaneously in the postauricular region of NOG mice. As early as 2 to 3 weeks after inoculation, seven cell lines produced a visible tumor while two transformed cell lines failed to do so. Five of seven lines produced a progressively growing large tumor with leukemic infiltration of the cells in various organs that eventually killed the animals. Leukemic cell lines formed soft tumors, whereas some transformed cell lines developed into hemorrhagic hard tumors in NOG mice. One of the leukemic cell lines, ED-40515(-), was unable to produce visible tumors in NOD-SCID mice with a common gamma-chain after 2 weeks. In vivo NF-kappaB DNA binding activity of the ED-40515(-) cell line was higher and the NF-kappaB components were changed compared to cells in vitro. Bay 11-7082, a specific and effective NF-kappaB inhibitor, prevented tumor growth at the sites of the primary region and leukemic infiltration in various organs of NOG mice. This in vivo model of ATL could provide a novel system for use in clarifying the mechanism of growth of HTLV-1-infected cells as well as for the development of new drugs against ATL.