Identification of miRNA-7 by genome-wide analysis as a critical sensitizer for TRAIL-induced apoptosis in glioblastoma cells.

Glioblastoma (GBM) is still one of the most lethal forms of brain tumor despite of the improvements in treatments. TRAIL (TNF-related apoptosis-inducing ligand) is a promising anticancer agent that can be potentially used as an alternative or complementary therapy because of its specific antitumor activity. To define the novel pathways that regulate susceptibility to TRAIL in GBM cells, we performed a genome-wide expression profiling of microRNAs in GBM cell lines with the distinct sensitivity to TRAIL-induced apoptosis. We found that the expression pattern of miR-7 is closely correlated with sensitivity of GBM cells to TRAIL. Furthermore, our gain and loss of function experiments showed that miR-7 is a potential sensitizer for TRAIL-induced apoptosis in GBM cells. In the mechanistic study, we identified XIAP is a direct downstream gene of miR-7. Additionally, this regulatory axis could also exert in other types of tumor cells like hepatocellular carcinoma cells. More importantly, in the xenograft model, enforced expression of miR-7 in TRAIL-overexpressed mesenchymal stem cells increased apoptosis and suppressed tumor growth in an exosome dependent manner. In conclusion, we identify that miR-7 is a critical sensitizer for TRAIL-induced apoptosis, thus making it as a promising therapeutic candidate for TRAIL resistance in GBM cells.

[Systemic delivery of complexes of melanoma RNA with mannosylated liposomes activates highly efficient murine melanoma-specific cytotoxic T cells in vivo].

The efficiency of the antitumor immune response triggered by dendritic cell (DC)-based vaccines depends predominantly on the efficiency of delivering tumor antigen-coding nucleic acids into DCs. Mannosylated liposomes were used to deliver tumor total RNA into DCs both ex vivo and in vivo, and the cytotoxic T-lymphocyte (CTL) antitumor response was assayed. The liposomes contained the mannosylated lipid conjugate 3-[6-(α-D-mannopyranosyloxy)hexyl]amino-4-{6-[rac-2,3-di(tetradecyloxy)prop-1-yl oxycarbonylamino]hexyl}aminocyclobut-3-en-1,2-dione), the polycationic lipid 2X3 (1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), and the zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 1: 3: 6 and were used as a transfection agent. Total RNA isolated from B16-F10 mouse melanoma cells served as a source of tumor antigens. Systemic administration of mannosylated liposomes-tumor RNA complexes into circulation of melanoma-bearing mice induced an efficient CTL response, which reduced the melanoma cell index in vitro with the same efficiency (by a factor of 2.8) as CTLs activated via an inoculation of DCs loaded with complexes of the same composition ex vivo. Complexes of tumor RNA with control liposomes, which lacked the mannosylated lipid conjugate, or DCs transfected with these complexes ex vivo were less efficient and reduced the melanoma cell count by a factor of only 1.6-1.8.

Actively personalized cancer vaccines--the step into clinical application.

Cancer vaccine development enters a new phase of innovation based on the development of modern sequencing technologies and novel RNA-based synthetic drug formats which enable the analysis and therapeutic targeting of every patient's tumor genome. By applying and combining these innovations, we have brought the concept of "actively personalized cancer vaccines" to clinical testing. Synthetic RNA is used as the drug format, allowing affordable, individual "on demand" manufacturing of tumor-optimized vaccines.

Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model.

Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates D-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids. ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.

A novel personalized vaccine approach in combination with targeted therapy in advanced renal cell carcinoma.

The historical treatment paradigm for metastatic renal cell carcinoma has focused on immunomodulatory agents, such as IFN-α and IL-2, which provide good clinical outcomes in only a subset of patients. The development of therapies that target the VEGF and mTOR pathways have significantly altered the treatment landscape for this disease, with novel inhibitors providing substantial improvements in progression-free and overall survival over previous standards of care. Despite these advances, toxicity from targeted therapy and the development of resistance results in disease progression. By contrast, vaccine-based immunotherapy represents a promising new approach for the treatment of patients with metastatic renal cell carcinoma; however, tumor-induced immunosuppression has limited the clinical efficacy of this modality until recently. Some evidence suggests that certain targeted therapies, such as sunitinib, may reduce this immunosuppression and enhance the tumor microenvironment to promote synergy with autologous dendritic cell vaccines.

Genetically modified dendritic cells in cancer immunotherapy: a better tomorrow?

IMPORTANCE OF THE FIELD: Dendritic cells (DC) are powerful antigen-presenting cells that induce and maintain primary cytotoxic T lymphocyte (CTL) responses directed against tumor antigens. Consequently, there has been much interest in their application as antitumor vaccines. AREAS COVERED IN THIS REVIEW: A large number of DC-based vaccine trials targeting a variety of cancers have been conducted; however, the rate of reported clinically significant responses remains low. Modification of DC to express tumor antigens or immunostimulatory molecules through the transfer of genes or mRNA transfection offers a logical alternative with potential advantages over peptide- or protein antigen-loaded DC. In this article, we review the current results and future prospects for genetically modified DC vaccines for the treatment of cancer. WHAT THE READER WILL GAIN: Genetically-modified dendritic cell-based vaccines represent a powerful tool for cancer therapy. Numerous preclinical and clinical studies have demonstrated the potential of dendritic cell vaccines alone or in combination with other therapeutic modalities. TAKE HOME MESSAGE: Genetically modified DC-based anti-cancer vaccination holds promise, perhaps being best employed in the adjuvant setting with minimal residual disease after primary therapy, or in combination with other antitumor or immune-enhancing therapies.

Complete in vivo reversal of the multidrug resistance phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.

Triggering the RNA interference (RNAi) pathway by inducing the expression of short hairpin RNA (shRNA) molecules has become a promising tool for efficient silencing of a given gene in gene therapy applications. In this study, shRNA encoding DNA was utilized to reverse the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype in vivo. For the first time, the nonviral jet-injection technology was applied for delivering naked shRNA-vector constructs for direct intratumoral in vivo transfer. The highly efficient anti-MDR1 shRNA expression vectors were applied twice in the human MDR1/P-gp overexpressing MaTu/ADR cancer xenograft-bearing mice, and twice in the corresponding drug-sensitive parental MaTu tumor xenograft bearing mice as well. Two days after anti-MDR1 shRNA vector injection, the expression level of the MDR1 messenger RNA (mRNA) was decreased by more than 90% and the corresponding MDR1/P-gp protein was no longer detectable in the tumors. Two jet-injections of anti-MDR1 shRNA vectors into the tumors, combined with two intravenous (IV) administrations of doxorubicin, were sufficient to achieve complete reversal of the drug-resistant phenotype. The data show that jet-injection delivery of shRNA-expressing vectors is effective in reversing MDR1/P-gp-mediated MDR in vivo, and is therefore a promising strategy for making tumors with an MDR1/Pgp-dependent MDR phenotype revert to a drug-sensitive state.

Dendritic cell-based immunotherapy of malignant gliomas.

The failure of conventional treatment modalities for gliomas, in spite of tremendous progress in research in the past two decades, has led to increasing interest in alternative treatment strategies, including immunotherapy. It has become evident that vaccination with dendritic cells (DC), designed to express tumor antigens, is a potent strategy to elicit anti-tumor immune response in both pre-clinical and clinical settings. Various methods have been applied in order to induce DC to express tumor antigens including: pulsing with isolated tumor peptides or whole tumor lysate; fusion with tumor cells; and pulsing with apoptotic tumor cells. Herein, we review the recent progress in DC biology with regard to tumor immunity and discuss current DC-based strategies and future prospects in immunotherapy for malignant gliomas.

Progress in the development of immunotherapy of cancer using ex vivo-generated dendritic cells expressing multiple tumor antigen epitopes.

Immunotherapy with tumor-associated antigen-pulsed, ex vivo-generated dendritic cells (DCs) is a promising approach for the treatment of cancer that has shown efficacy in animal models and is now being tested in the clinic. The majority of studies performed to date make use of a single tumor-associated epitope. However, because of the high rate of mutation in tumor cells allowing for loss of expression of a single antigen, it is likely that use of multiple antigenic epitopes will induce a broader, longer-lasting, and effective tumor-specific immune response. Multiple vehicles for loading DCs with multiple antigenic epitopes are under investigation to determine the most effective method for vaccination, with many of these methods showing promise. These loading methods, as well as other critical considerations for making DC vaccination as efficacious as possible, are discussed in this article.

Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA.

OBJECTIVE: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. METHODS: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. RESULTS: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccination with DC/extract or DC/RNA, either prior to G422 tumor challenge or in tumor-harboring mice, significantly prolonged survival compared with other control groups (P<0.01). CONCLUSION: DCs pulsed with tumor extracts or RNA derived from autologous tumors has potential antitumor effects via activation of cell-mediated immunity. Our results suggest a useful therapeutic strategy against gliomas.

Results of a phase 1 study utilizing monocyte-derived dendritic cells pulsed with tumor RNA in children and young adults with brain cancer.

We conducted a phase 1 study of 9 pediatric patients with recurrent brain tumors using monocyte-derived dendritic cells pulsed with tumor RNA to produce antitumor vaccine (DCRNA) preparations. The objectives of this study included (1) establishing safety and feasibility and (2) measuring changes in general, antigen-specific, and tumor-specific immune responses after DCRNA. Dendritic cells were derived from freshly isolated monocytes after 7 days of culture with IL-4 and granulocyte-macrophage colony-stimulating factor, pulsed with autologous tumor RNA, and then cryopreserved. Patients received at least 3 vaccines, each consisting of an intravenous and an intradermal administration at biweekly intervals. The study showed that this method for producing and administering DCRNA from a single leukapheresis product was both feasible and safe in this pediatric brain tumor population. Immune function at the time of enrollment into the study was impaired in all patients tested. While humoral responses to recall antigens (diphtheria and tetanus) were intact in all patients, cellular responses to mitogen and recall antigens were below normal. Following DCRNA vaccine, 2 of 7 patients showed stable clinical disease and 1 of 7 showed a partial response. Two of 7 patients who were tested showed a tumor-specific immune response to DCRNA. This study showed that DCRNA vaccines are both safe and feasible in children with tumors of the central nervous system with a single leukapheresis.

Synergy between tumor immunotherapy and antiangiogenic therapy.

This study tested the hypothesis that combination of antiangiogenic therapy and tumor immunotherapy of cancer is synergistic. To inhibit angiogenesis, mice were immunized with dendritic cells (DCs) transfected with mRNA that encode products that are preferentially expressed during neoangiogenesis: vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie2 expressed in proliferating endothelial cells, and vascular endothelial growth factor (VEGF) expressed in the angiogenic stroma as well as the tumor cells used in this study. Immunization of mice against VEGF or VEGFR-2 stimulated cytotoxic T lymphocyte (CTL) responses and led to partial inhibition of angiogenesis. Antiangiogenic immunity was not associated with morbidity or mortality except for a transient impact on fertility seen in mice immunized against VEGFR-2, but not VEGF. Tumor growth was significantly inhibited in mice immunized against VEGF, VEGFR-2, and Tie2, either before tumor challenge or in the setting of pre-existing disease in murine B16/F10.9 melanoma and MBT-2 bladder tumor models. Coimmunization of mice against VEGFR-2 or Tie2 and total tumor RNA exhibited a synergistic antitumor effect. Synergism was also observed when mice were coimmunized with various combinations of defined tumor-expressed antigens, telomerase reverse transcriptase (TERT) or TRP-2, and VEGF or VEGFR-2. This study shows that coimmunizing mice against angiogenesis-associated and tumor-expressed antigens can deliver 2 compatible and synergistic cancer treatment modalities via a common treatment, namely immunization.

Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA.

The skin is well-suited to serve as a substrate for vaccination strategies. In this regard, epidermal cells exposed to granulocyte-macrophage colony-stimulating factor can, upon subcutaneous injection into naïve mice, present a soluble extract of tumor as a source of tumor-associated antigens for the induction of in vivo anti-tumor immunity. Use of RNA for immunization has a potential advantage over this technique. Because RNA can be amplified, only a small amount of tumor is needed for antigen preparation and, as with a soluble extract, it is not necessary to know the molecular nature of the antigen(s) relevant to immunity. To test the hypothesis that RNA-pulsed epidermal cells can induce anti-tumor immunity, total cellular RNA was isolated from the S1509a spindle cell tumor and used to pulse CAF1 epidermal cells enriched for Langerhans cell content and pre-exposed to granulocyte-macrophage colony-stimulating factor. These cells were then injected subcutaneously into naïve CAF1 mice three times at weekly intervals followed by challenge with living S1509a cells. Tumor growth was significantly less than in control animals immunized in an identical fashion but with irrelevant RNA. Digestion of S1509a RNA with RNase prior to pulsing of epidermal cells prevented the development of immunity. In separate experiments, intradermal injection of S1509a RNA into naïve mice three times at weekly intervals also induced immunity to challenge with the tumor. Digestion of S1509a RNA with RNase also prevented development of immunity in this system. Effective anti-tumor immunity can be induced in mice utilizing RNA-pulsed epidermal cells for in vivo immunization or by injecting RNA intradermally into naïve mice.

Immunotherapy of metastases with lymphocytes treated with exogenous RNA in mice bearing B16 melanoma.

Subline B16-F10, a variant cell line of B16 melanoma, is highly metastatic to the lung when injected intravenously into C57BL/6 mice. This experimental metastasis model was used to test the anti-tumor effect of exogenous RNA extracted from the lymphoid organs of immunized animals with B16-F10 cells. This RNA preparation is referred to as B16-RNA. Adoptive immunotherapy with lymphocytes treated with B16-RNA was effective in reducing significantly the number of pulmonary metastatic nodules. Lymphocytes incubated with medium alone or with RNA from non-immunized animals (N-RNA) were used as controls. The ability of B16-RNA in modulating antimetastatic activity of normal lymphocytes is abolished by hydrolysis with KOH. This finding indicates that the integrity of the polynucleotide chain is essential for the activity of B16-RNA. The anti-tumor effect of lymphocytes treated with B16-RNA was enhanced by incubation with a low dose of interleukin-2 (IL-2). A possible role of the double-stranded RNA dependent protein kinase in this phenomenon is discussed.

Ca2+ oscillations induced by fibroblast growth factor 2 in Xenopus oocytes expressing fibroblast growth factor receptors.

Double electrode voltage clamp technique was used to follow precisely the calcium signalling pathway activated by FGF receptors from a normal and a carcinogenous cell environment. Functional FGF receptors were expressed in Xenopus oocytes following either the injection of PFR1 cRNA from Pleurodeles, an homologue of the human FGFR1 mRNA, or breast cancer MCF7 cells total mRNA. Cytosolic calcium oscillations were monitored through the endogenous Ca(2+)-dependent Cl- channel activity from both RNA injected systems, under FGF2 treatment. The Ca(2+)-dependent Cl- channel was demonstrated using the Cl- channel blocker SITS (250 microM) and by the determination of the reversal potential of the Cl- ions close to -20 mV. The FGF2-evoked Ca(2+)-dependent Cl- current was abolished by external application of genistein (10 microM, tyrosine kinase inhibitor), neomycin (10 mM, phosphatidylinositol turnover inhibitor), caffeine (10 mM, inhibitor of Ins(1,4,5)P3-mediated release of intracellular calcium), and injection of BAPTA (50 microM, calcium chelator) or heparin (2 micrograms/ml, inhibitor of the binding of Ins(1,4,5)P3). The recorded current was independent of extracellular Ca2+ but involved tyrosine kinase phosphorylation and intracellular Ins(1,4,5)P3 sensitive stores. External application of heparin enhanced the oscillatory Ca2+ rise, suggesting a role for the heparan sulfates in the regulatory mechanism of the FGF receptors. The similarities in the Ca(2+)-dependent Cl- current obtained in PFR1 and total MCF7 FGF receptors expressing oocytes are discussed.

Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.