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Breast cancer has a high risk of metastasis; however, no effective treatment has been established. We developed a novel immunotherapy for breast cancer to enhance cytotoxic T lymphocytes against cancer cells using N1-type neutrophils with anti-tumor properties. For this purpose, we combined CXCL2 (CXC chemokine ligand 2) plasmid DNA with inactivated Sendai virus (hemagglutinating virus of Japan)-envelope (HVJ-E). The combination of CXCL2 DNA and HVJ-E (C/H) suppressed the growth of murine breast cancers in orthotopic syngeneic models by enhancing cytotoxic T lymphocytes and inhibited lung metastasis of breast cancer from primary lesions. N1-type neutrophils (CD11b+ Ly6G+ FAS+) increased in the tumor microenvironment with C/H treatment, and tumor suppression and cytotoxic T lymphocyte activation from C/H was blocked after administrating anti-neutrophil antibodies, which indicates the role of N1-type neutrophils in cancer immunotherapy. We also demonstrated that the anti-tumor activities of C/H treatment were enhanced by the administration of anti-PD-1 antibodies through neutrophil-mediated cytotoxic T lymphocyte activation. Thus, the triple combination of C/H and anti-PD-1 antibody C/H treatment may provide an improvement in cancer immunotherapy., Graphical Abstract, The combination of CXCL2 plasmid DNA and HVJ-E treatment polarized neutrophils to N1 neutrophils, which activated CTL to suppress both primary and metastatic murine breast cancer legions. The anti-tumor effect of the combination therapy using CXCL2 plasmid DNA and HVJ-E was enhanced by anti-PD-1 antibody treatment.

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

Background Subcellular localization of a photosensitizer is known to determine the therapeutic efficacy of photodynamic therapy (PDT). Cell membrane is an optimal target that promises an effective treatment outcome. Objectives We previously developed a novel photosensitizer named porphyrus envelope (PE) by combining hemagglutinating virus of Japan envelope (HVJ-E) with lipidated protoporphyrin IX (PpIX lipid). In the current study, the cellular localization of PE and its ability to induce multiple anti-tumor effect were characterized. Materials and Methods The localization and uptake of PpIX lipid in cells were evaluated with confocal laser scanning microscopy and a cell-based fluorescent assay, respectively. The ability of PE to suppress the migration and proliferation of cancer cells was assessed using a scratch-wound assay. The synergistic effect of PDT and HVJ-E treatment was evaluated using an in vitro experiment with PC-3 cells. Results PE localized along the cell membrane and PpIX lipid accumulated selectively in the prostate cancer cells within 10 min. Also, PE maintained the ability to undergo fusion and induce cancer cell death even after light irradiation at the dose for PDT. Incubation with PE resulted in delayed migratory and proliferative activity of PC-3 cells. PE-mediated PDT was twice as effective when cells were further incubated with PE following PDT. Conclusions PE allows rapid drug delivery targeting the cell membrane. Because the cytotoxicity of HVJ-E was maintained, synergistic effect of HVJ-E and the photochemical reactions resulted in highly effective killing of prostate cancer cells in vitro and thus represents a promising treatment for prostate cancer.

// Che-Hsin Lee 1, 2 , Tomoyuki Nishikawa 3 , Yasufumi Kaneda 3 1 Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan 2 Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan 3 Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Osaka, Japan Correspondence to: Che-Hsin Lee, email: chlee@mail.nsysu.edu.tw Yasufumi Kaneda, email: kaneday@gts.med.osaka-u.ac.jp Keywords: Salmonella, targeted therapy, polymer, hemagglutinating virus of Japan-envelope Received: December 08, 2016 Accepted: March 30, 2017 Published: April 11, 2017 ABSTRACT Salmonella can target to tumor microenvironments after systemic treatment. The hemagglutinating virus of Japan-envelope (HVJ-E) induced apoptosis in tumor cells without toxicity in normal cells. Current HVJ-E therapeutic strategies, aimed at using HVJ-E for intratumor treatment, have shown great promise in animal models but have achieved only limited systemic treatment. The purpose of this study was to investigate the modulation of the anti-tumor efficiency of HVJ-E by coating the particles with poly (allylamine hydrochloride) (PAH), designated as P-HVJ-E. Treatment with P-HVJ-E resulted in decreased hemagglutinating activity and maintained tumor cell-selective apoptosis and anti-tumor immunity. The use of Salmonella as a coating for P-HVJ-E (PHS) enhanced the antitumor activity and maintained the tumor-targeting activity. Treatment with PHS resulted in delayed tumor growth in tumor-bearing mice. Furthermore, a Western blot assay of the tumors revealed that HVJ-E targeted to the tumor after systemic treatment with PHS. These results indicate that Salmonella coating viral particles may provide a new approach for tumor therapy.

Inactivated Sendai virus particle (or hemagglutinating virus of Japan envelope; HVJ-E) has been previously reported to possess antitumour properties that activate antitumour immunity. Two glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN), are present on the surface of HVJ-E. HN is necessary for binding to receptors such as acidic gangliosides, and F induces membrane fusion by associating with membrane lipids. We previously reported that liposomes reconstituted with F but not HN showed antitumour activity by inducing IL-6 secretion in dendritic cells (DCs), suggesting that F protein is capable of eliciting antitumour activity. Here, we attempted to deliver F gene into tumour tissue in mice by electroporation and demonstrated that F gene therapy retarded tumour growth, increased CD4+ and CD8+ T-cell infiltration into tumours and induced tumour-specific IFN-γ T-cell response. However, neutralisation of IL-6R signalling did not impact F plasmid-mediated antitumour effect. Instead, we found that F plasmid treatment resulted in a significant increase in the secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) by tumour-infiltrating T cells. Neutralising antibody against RANTES abolished the antitumour effect of F plasmid treatment in a dose-dependent manner. Thus, F gene therapy may show promise as a novel therapeutic for single or combined cancer immunotherapy.

BACKGROUND: Photodynamic therapy (PDT), a minimally invasive cancer treatment involving the activation of photosensitizer by a specific wavelength of light, is considered to be a promising treatment option for drug-resistant prostate cancer. Hemagglutinating virus of Japan envelope (HVJ-E) has the potential to serve as a highly effective cancer therapy through selective drug delivery and enhancement of the anti-tumor immune response. OBJECTIVES: To improve therapeutic efficacy and selective accumulation of photosensitizer into tumor cells, we developed a novel photosensitizer, Laserphyrin(®)-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin(®), Meiji Seika Pharma) into HVJ-E. MATERIALS AND METHODS: The therapeutic effect of PDT with Laserphyrin(®) or L-HVJ-E was evaluated in the human prostate cancer cell line PC-3 in vitro. The subcellular localizations of Laserphyrin(®) and L-HVJ-E were observed by confocal microscopy. Apoptosis or necrosis following PDT was detected by annexin V-fluorescein/propidium iodide double staining. RESULTS: The cytotoxic effect of Laserphyrin(®)- and L-HVJ-E-mediated PDT were determined by evaluating cell survival rate and production of reactive oxygen species. The cytotoxicity of L-HVJ-E-mediated PDT was dependent on drug concentration and light dose. Laserphyrin(®) and L-HVJ-E gradually entered cells as incubation time increased, and both agents tended to be distributed in lysosomes rather than mitochondria. Time and dose dependent increase in ROS production was observed, and induction of both apoptotic and necrotic cell death was confirmed. CONCLUSIONS: Laserphyrin(®) and L-HVJ-E were distributed mainly in lysosomes and induced cell death by both apoptosis and necrosis. Furthermore, L-HVJ-E-mediated PDT effectively killed cultured PC-3 cells and exerted higher photocytotoxicity than Laserphyrin(®)-mediated PDT.

// Chin Yang Chang 1 , Jiayu A. Tai 1 , Sumin Li 1 , Tomoyuki Nishikawa 1 , Yasufumi Kaneda 1 1 Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan Correspondence to: Yasufumi Kaneda, email: kaneday@gts.med.osaka-u.ac.jp Keywords: HVJ-E, tumor associated neutrophils (TANs), anti-tumor immunity, tumor microenvironment (TME), cytotoxic T lymphocytes (CTLs) Received: November 20, 2015 Accepted: May 22, 2016 Published: May 31, 2016 ABSTRACT The tumor microenvironment (TME) fosters tumors by attenuating anti-tumor immunity, reinforcing tumor cell survival and increasing angiogenesis. Among the constituents of the TME, here, we focused on tumor-associated neutrophils (TANs). First, we found that the combination of poly I:C and inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) synergistically suppressed tumor growth in the B16-F10 melanoma mouse model. In this model, poly I:C contributed to the recruitment of CD11b + Ly6G + neutrophils to the TME, and co-injection of poly I:C and HVJ-E increased CD11b + Ly6G + FAS + TAN in the TME. Depletion of neutrophils abolished the synergistic anti-tumor effect of HVJ-E and poly I:C in B16-F10 tumors. We revealed that C-X-C motif chemokine ligand 2 (CXCL2) is produced in the TME by poly I:C, but HVJ-E enhanced neutrophil infiltration of the TME does not occur. An anti-CXCL2 antibody inhibited the tumor suppression by HVJ-E+poly I:C. HVJ-E in combination with recombinant CXCL2 protein or CXCL2 pDNA suppressed mouse melanoma by increasing cytotoxic T lymphocyte activity against B16-F10 melanoma, which was abolished by an anti-Ly6G antibody. HVJ-E directly and indirectly increased FAS and ICAM-1 expression in cultured bone marrow-derived naive neutrophils. Thus, HVJ-E activates anti-tumor immunity via anti-tumorigenic neutrophils in the TME. An HVJ-E vector containing the CXCL2 gene may be applicable as a novel cancer gene therapy strategy.

We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ‐E) has multiple anticancer effects, including induction of cancer‐selective cell death and activation of anticancer immunity. The HVJ‐E stimulates dendritic cells to produce cytokines and chemokines such as β‐interferon, interleukin‐6, chemokine (C‐C motif) ligand 5, and chemokine (C‐X‐C motif) ligand 10, which activate both CD8+ T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ‐E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ‐E induced the production of intercellular adhesion molecule‐1 (ICAM‐1, CD54), a ligand of lymphocyte function‐associated antigen 1, in several cancer cell lines through the activation of nuclear factor‐κB downstream of retinoic acid‐inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM‐1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM‐1 in MDA‐MB‐231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM‐1‐depleted MDA‐MB‐231 cells. In addition, HVJ‐E suppressed tumor growth in MDA‐MB‐231 tumor‐bearing SCID mice, and the HVJ‐E antitumor effect was impaired when NK cells were depleted by treatment with the anti‐asialo GM1 antibody. Our findings suggest that HVJ‐E enhances NK cell sensitivity against cancer cells by increasing ICAM‐1 expression on the cancer cell surface.

Tumor microenvironments include a number of fibrin clots due to the microbleeding caused by cancer cell invasion into blood vessels, which suggests the potential utility of a platelet vector for systemic cancer treatment. We previously reported that inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) activates anti-tumor immunity and induces cancer cell-selective apoptosis. The hemagglutination activity that blocks the systemic administration of HVJ-E was dramatically attenuated by incorporation into platelets. Platelets incorporating HVJ-E (PH complex) were then injected into the tail veins of B16F10 melanoma-bearing mice. The PH complex primarily accumulated in tumor tissues and caused the significant accumulation of various immune cells in the tumor bed. Injections of the PH complex to the melanoma-bearing mouse significantly reduced the tumor size, and the tumor growth was ultimately arrested. Secretion of the chemokine regulated upon activation normal T-expressed and presumably secreted (RANTES) was upregulated following PH stimulation. The RANTES-depletion in melanoma-bearing mice significantly attenuated the cytotoxic T lymphocyte activity and led to a dramatic abrogation of the mouse melanoma suppression induced by the PH complex. Thus, a platelet vector incorporating viral particles, a Trojan horse for cancer treatment, will provide a new approach for cancer therapy using oncolytic viruses.

Tumours constitute unique microenvironments where various blood cells and factors are exposed as a result of leaky vasculature. In the present study, we report that thrombin enrichment in B16F10 melanoma led to platelet aggregation and this property was exploited to administer an anticancer cytokine, interferon-gamma induced protein 10 (IP10), through the formation of a platelet-IP10 complex. When intravenously infused, the complex reached platelet microaggregates in the tumour. The responses induced by the complex were solely immune-mediated and tumour cytotoxicity was not observed. The complex suppressed the growth of mouse melanoma in vivo, while both platelets and the complex suppressed the accumulation of FoxP3+ regulatory T cells in the tumour. These results demonstrated that thrombin-dependent platelet aggregation in B16F10 tumours defines platelets as a vector to deliver anticancer cytokines and provide specific treatment benefits.

Dual-targeted therapy for antiangiogenesis and antilymphangiogenesis represents a potentially effective strategy for the treatment of various malignancies. Therefore, the goal of the present study was to identify genes that encode inhibitors of both angiogenesis and lymphangiogenesis. Using a cDNA library obtained from Lewis lung carcinoma (LL/2), a candidate gene was identified by the evaluation of growth inhibition in aortic and lymphatic endothelial cells (EC) as that coding for the mouse cold shock domain protein A (mCSDA). Overexpression of mCSDA significantly repressed cell proliferation and c-fos promoter activity in aortic, venous and lymphatic ECs. CSDA is a DNA-binding protein that binds to the hypoxia response element (HRE). Furthermore, of importance, we revealed that CSDA could directly bind to the serum response element (SRE) sequence, resulting in the inhibition of SRE activity, which may lead to growth inhibition in ECs. In an LL/2-inoculated mouse model, tumor growth was significantly repressed in an mCSDA-injected group. Histopathological analysis revealed that expression of blood and lymphatic EC markers was significantly decreased in mCSDA-injected groups. In conclusion, these data suggest that expression of CSDA can repress angiogenesis and lymphangiogenesis via direct binding to SRE in addition to HRE.

Background— Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. Methods and Results— Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs) were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c- fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF- and vascular endothelial growth factor 165–injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. Conclusions— These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.

Purpose: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppresses the growth of human cancer cells as effectively as replication-competent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E. Experimental Design: The molecules responsible for HVJ-E–induced cancer cell death were elucidated in prostate cancer cell lines, and the effect of HVJ-E on orthotopic prostate cancers was evaluated in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Results: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells but not the viability of the noncancerous prostate epithelium. Knockdown experiments using siRNAs showed that the cancer cell–selective killing induced by HVJ-E was mediated by retinoic acid–inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were upregulated by HVJ-E in the castration-resistant prostate cancer cell line PC3 but not in the noncancerous prostate epithelial cell line PNT2. TRAIL siRNA but not Noxa siRNA significantly inhibited HVJ-E–induced cell death in PC3 cells. However, Noxa siRNA effectively suppressed HVJ-E–induced cell death in DU145 cells, another castration-resistant prostate cancer cell line, in which Noxa but not TRAIL was upregulated by HVJ-E. Furthermore, the orthotopic prostate cancers were dramatically eradicated in immunodeficient mice injected with HVJ-E. Conclusion: The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy. Clin Cancer Res; 18(22); 6271–83. ©2012 AACR.

Objective— The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue–derived cells (ADSC) on angiogenic cell therapy. Method and Results— ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of Sca I and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and α-smooth muscle actin-positive cells in ischemic tissue. Conclusion— These adipose tissue–derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.