Chika Sakamoto

Coxsackievirus B3 Is an Oncolytic Virus with Immunostimulatory Properties That Is Active against Lung Adenocarcinoma

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC.

Inhibition of PTEN tumor suppressor promotes the generation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.

Absence of LTB4/BLT1 axis facilitates generation of mouse GM-CSF-induced long-lasting antitumor immunologic memory by enhancing innate and adaptive immune systems.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.

2-D inversion of VES data in Saqqara archaeological area, Egypt

The interpretation of actual geophysical field data still has a problem for obtaining a unique solution. In order to investigate the groundwater potentials in Saqqara archaeological area, vertical electrical soundings with Schlumberger array have been carried out. In the interpretation of VES data, 1D resistivity inversion has been performed based on a horizontally layered earth model by El-Qady (1995). However, some results of 1D inversion are not fully satisfied for actual 3D structures such as archaeological tombs. Therefore, we have carried out 2D inversion based on ABIC least squares method for Schlumberger VES data obtained in Saqqara area. Although the results of 2D cross sections were correlated with the previous interpretation, the 2D inversion still shows a rough spatial resistivity distribution, which is the abrupt change in resistivity between two neighboring blocks of the computed region. It is concluded that 3D interpretation is recommended for visualizing ground water distribution with depth in the Saqqara area.

TLR7 ligand augments GM-CSF-initiated antitumor immunity through activation of plasmacytoid dendritic cells

Narusawa and colleagues found that type 1 IFNs and plasmacytoid dendritic cells in the tumor-draining lymph nodes mediate GM-CSF–induced antitumor immunity in immunocompetent mice, and they report that the synthetic TLR7 ligand imiquimod could overcome tolerance and enhance autologous GM-CSF antitumor effects. Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)–transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF–sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)–related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF–induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR−/−) mice were used. Importantly, in both LLC and CT26 colon cancer–bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4+CD25+FoxP3+ regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF–based cancer immunotherapy. Cancer Immunol Res; 2(6); 568–80. ©2014 AACR.

Therapeutic vaccination based on side population cells transduced by the granulocyte-macrophage colony-stimulating factor gene elicits potent antitumor immunity

Among cancer immunotherapies, granulocyte–macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccine (GVAX) therapies appear promising and have been shown to be safe and effective in multiple clinical trials. However, the antitumor efficacies of GVAX therapy alone are in some cases limited. Here we showed that GVAX therapy targeting cancer stem cells (CSCs) substantially suppressed tumor development in syngeneic immunocompetent mice recapitulating normal immune systems. CSCs were isolated as side population (SP) cells from 4T1 murine breast carcinoma cell line and transduced with GM-CSF gene delivered by non-transmissible Sendai virus (4T1-SP/GM). Impaired tumorigenicity of subcutaneously injected 4T1-SP/GM depended on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Mice therapeutically vaccinated with irradiated 4T1-SP/GM cells had markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with those treated with irradiated cells of non-transduced 4T1-SP cells or non-SP (4T1-NSP/GM) cells. Tumor suppression was accompanied by the robust accumulation of mature dendritic cells at vaccination sites and T-helper type 1-skewed systemic cellular immunity. Our results suggested that CSC cell-based GVAX immunotherapy might be clinically useful for inducing potent tumor-specific antitumor immunity.

5. Newly Developed Measles Virus Vector Can Simultaneously Transfer Multiple Genes Into Human Hematopoietic Cells and Induce Ground State Like Pluripotent Stem Cells

We reports newly developed virus vector, measles virus vector can transfer multiple genes into human hematopoietic cells effectively and induce ground state pluripotent stem cells from somatic cells without affecting the host genome.Measles virus (MV) which belongs to negative single strand RNA viruses has been known to have high affinity for human peripheral immune cells including monocytes, B cells and T cells. We recently have developed novel MV gene transfer vector which is non-transmissible, can transfer multiple genes simultaneously. The MV vector which carries 5 genes (GFP, human OCT3/4, SOX2, KLF4, and L-MYC) (MV-dF-OSKL-EGFP) could express these genes in various human cells with differential expression levels depending on the arrangement of the gene in the vector. Especially, MV-dF-OSKL-EGFP was able to transduce genes into more than 80% of hematopoietic cells besides natural killer cells. Naive and stem cell memory T cells were also transduced by MV-dF-OSKL-EGFP.These results indicated that the newly developed MV vector had the significant character as the new gene transfer vector compared with conventional viral gene transfer vectors including Sendai virus vector, which belongs to the same RNA virus vector.We could successfully generated induced pluripotent stem cells (iPS cells) from human fibroblasts or peripheral blood T cells using MV-dF-OSKL-EGFP. These iPS cells expressed the pluripotent markers of NANOG and Tra-1-60 and were demethylated. These iPS cells also differentiated into three germ line tissues in vitro and in vivo. Importantly, we also could establish the ground state like pluripotent cell (GSL-iPS cells) from hematopoietic cells by using MV-dF-OSKL-EGFP. In the presence of human leukemia inhibitory factor (LIF), GSK-3 inhibitor (CHIR99021), and MEK inhibitor (PD0325901), GSL-iPS cells were able to be cultured from a dissociated single cell with rapid cell growth. GSL-iPS cells also expressed pluripotent markers of NANOG and Tra-1-60, and were able to differentiate into three germ line cells. In conclusion, our newly developed MV vector may induce revolutional advance in the field of gene and cell therapy using iPS cells.

Abstract 2814: Therapeutic vaccination with GM-CSF gene-transduced iPS cells induces potent T cells-mediated antitumor immunity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic similarities with embryonic stem (ES) cells and the vaccination using ES cells could generate antitumor immunity. However, the use of ES cells raises potential immunological and ethical issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed to induced pluripotent stem (iPS) cells that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPS cells genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells and induce long term antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells. Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/GM-CSF) was effective to produce abundant GM-CSF in vitro and iPS/GM-CSF cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. Prophylactic iPSCs vaccine studies revealed that wild-type female mice subcutaneously vaccinated with irradiated iPS (ir.iPS) cells on weeks 1, 2, and 3 before the tumor challenge with LLC cells significantly suppressed the LLC tumor growth compared with untreated mice (p<0.05). Importantly, mice carrying pre-established LLC tumor treated with ir.iPS/GM-CSF cells showed significantly suppressed tumor growth compared with mice treated with ir.iPS/GFP cells (p<0.05). No serious adverse events were observed without any organ damages of liver and kidney. Furthermore, the antitumor effects observed in mice treated with ir.iPS/ GM- CSF cells were significantly abrogated when CD4+ T or CD8+ T cells were depleted, showing their capacity to incite T cells-mediated antitumor immunity. Lastly, we performed a cDNA microarray analysis to identify commonly upregulated genes of the putative CSCs-associated antigens as a target of iPS cells-based vaccines among LLC cells, iPS vaccine cells. Several sperm- or cell surface- specific antigens were predominantly expressed and shared between the 2 distinctive cell fractions. Taken together, our results demonstrated that iPS cells-based vaccine could induce both prophylactic and therapeutic antitumor immunity in syngeneic mouse models, and suggested that this novel vaccine strategy may be a promising modality for cancer immunotherapy. Citation Format: Hiroyuki Inoue, Ayumi Watanabe, Megumi Narusawa, Chika Sakamoto, Takafumi Hiramoto, Shohei Miyamoto, Makoto Inoue, Koichi Takayama, Mamoru Hasegawa, Yoichi Nakanishi, Tomoki Todo, Kenzaburo Tani. Therapeutic vaccination with GM-CSF gene-transduced iPS cells induces potent T cells-mediated antitumor immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2814. doi:10.1158/1538-7445.AM2014-2814