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n2:pmcid: PMC0
bibo:doi: 10.1038%2Fnbt.1564
n5:contains: _:vb6007677 _:vb6007672 _:vb6007639

Subject Item: _:vb6007639
rdf:type: n5:Section
dc:title: results
n5:contains: _:vb6007664 _:vb6007665 _:vb6007666 _:vb6007667 _:vb6007668 _:vb6007669 _:vb6007670 _:vb6007671 _:vb6007656 _:vb6007657 _:vb6007658 _:vb6007659 _:vb6007660 _:vb6007661 _:vb6007662 _:vb6007663 _:vb6007648 _:vb6007649 _:vb6007650 _:vb6007651 _:vb6007652 _:vb6007653 _:vb6007654 _:vb6007655 _:vb6007640 _:vb6007641 _:vb6007642 _:vb6007643 _:vb6007644 _:vb6007645 _:vb6007646 _:vb6007647

Subject Item: _:vb6007640
rdf:type: n2:Context
rdf:value: Synthesis of the antisense strand of the siRNA (27mer) was followed by CpG1668 ODN synthesis>>31<<,32, producing a single stranded oligonucleotide connected through a carbon chain linker (Fig.
n2:mentions: n3:8610135

Subject Item: _:vb6007641
rdf:type: n2:Context
rdf:value: Synthesis of the antisense strand of the siRNA (27mer) was followed by CpG1668 ODN synthesis31,>>32<<, producing a single stranded oligonucleotide connected through a carbon chain linker (Fig.
n2:mentions: n3:7700380

Subject Item: _:vb6007642
rdf:type: n2:Context
rdf:value: The asymmetric 25/27mer siRNA was optimized for specific processing by Dicer and was more potent in target gene silencing>>33<<,34. Adding either CpG1688 alone or CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resulted in a similar increase in expression of co-stimulatory CD40 and CD80 molecules, suggesting that CpG-Stat3 siRNA conjugate retains its
n2:mentions: n3:15619617

Subject Item: _:vb6007643
rdf:type: n2:Context
rdf:value: The asymmetric 25/27mer siRNA was optimized for specific processing by Dicer and was more potent in target gene silencing33,>>34<<. Adding either CpG1688 alone or CpG-Stat3 siRNA conjugate to cultured DC2.4 dendritic cells resulted in a similar increase in expression of co-stimulatory CD40 and CD80 molecules, suggesting that CpG-Stat3 siRNA conjugate retains its
n2:mentions: n3:16049023

Subject Item: _:vb6007644
rdf:type: n2:Context
rdf:value: This uptake pattern reflects the known distribution of TLR9 expression in murine leukocyte subsets>>35<<,26. Intracellular staining of TLR9 in fixed splenocytes by flow cytometry CD11c+ DCs confirmed TLR9 expression (Fig. 2a, bottom right). Unconjugated Stat3 siRNA was not efficiently incorporated into DCs even after 24 h incubation,
n2:mentions: n3:11130078

Subject Item: _:vb6007645
rdf:type: n2:Context
rdf:value: This uptake pattern reflects the known distribution of TLR9 expression in murine leukocyte subsets35,>>26<<. Intracellular staining of TLR9 in fixed splenocytes by flow cytometry CD11c+ DCs confirmed TLR9 expression (Fig. 2a, bottom right). Unconjugated Stat3 siRNA was not efficiently incorporated into DCs even after 24 h incubation,
n2:mentions: n3:15454922

Subject Item: _:vb6007646
rdf:type: n2:Context
rdf:value: 3) and reported previously>>36<<. By 60 min, greater than 80% DC 2.4 cells were positive for uptake of the conjugate, which was confirmed by confocal microscopic analysis.
n2:mentions: n3:14716310

Subject Item: _:vb6007647
rdf:type: n2:Context
rdf:value: Previous studies have demonstrated that binding of the Dicer nuclease to the siRNA oligonucleotide is required for further siRNA processing to shorter 21mer fragments that mediate RISC complex-dependent mRNA degradation>>37<<. We observed transient colocalization of the CpG-Stat3 siRNA with Dicer within 2 h after adding the oligonucleotide to cultured DCs (Fig. 2c, two bottom rows and Supplementary Fig. 4). The colocalization of CpG-Stat3-siRNA and Dicer
n2:mentions: n3:15973356

Subject Item: _:vb6007648
rdf:type: n2:Context
rdf:value: GpC-conjugated Stat3 siRNA, which binds but does not activate TLR>>936<<, failed to silence Stat3, suggesting a possible requirement of TLR9 activation for the CpG-siRNA to be processed.
n2:mentions: n3:14716310

Subject Item: _:vb6007649
rdf:type: n2:Context
rdf:value: Note that, as indicated above, stimulation using CpG itself also resulted in Stat3 activation>>25<<,38 (Fig. 2f), thereby complicating the EMSA analysis for detection of Stat3 silencing. None-the-less, at higher concentrations, CpG-Stat3 siRNA diminished Stat3 DNA-binding activity relative to the conjugate scrambled RNA controls. To
n2:mentions: n3:19258507

Subject Item: _:vb6007650
rdf:type: n2:Context
rdf:value: Note that, as indicated above, stimulation using CpG itself also resulted in Stat3 activation25,>>38<< (Fig. 2f), thereby complicating the EMSA analysis for detection of Stat3 silencing. None-the-less, at higher concentrations, CpG-Stat3 siRNA diminished Stat3 DNA-binding activity relative to the conjugate scrambled RNA controls. To
n2:mentions: n3:17238832

Subject Item: _:vb6007651
rdf:type: n2:Context
rdf:value: Mice over-expressing firefly luciferase under control of the β-actin promoter>>39<< were challenged with tumor cells, followed by repeated peritumoral injections of CpG-Luc siRNA.
n2:mentions: n3:14688412

Subject Item: _:vb6007652
rdf:type: n2:Context
rdf:value: Both DCs and macrophages in the tumor microenvironment are known to promote immune tolerance>>11<<,17. Our previous work demonstrated that Stat3 is persistently-activated in myeloid cells in the tumor milieu and that genetic ablation of Stat3 in the myeloid compartment elicits potent antitumor immunity10.
n2:mentions: n3:15776005

Subject Item: _:vb6007653
rdf:type: n2:Context
rdf:value: Both DCs and macrophages in the tumor microenvironment are known to promote immune tolerance11,>>17<<. Our previous work demonstrated that Stat3 is persistently-activated in myeloid cells in the tumor milieu and that genetic ablation of Stat3 in the myeloid compartment elicits potent antitumor immunity10.
n2:mentions: n3:17186030

Subject Item: _:vb6007654
rdf:type: n2:Context
rdf:value: Our previous work demonstrated that Stat3 is persistently-activated in myeloid cells in the tumor milieu and that genetic ablation of Stat3 in the myeloid compartment elicits potent antitumor immunity>>10<<. Furthermore, both CpG and LPS treatment activates Stat338,25,40, which acts as a negative feedback mechanism to constrain Th1 immune responses. Therefore, we assessed whether the CpG-Stat3-siRNA conjugates could reverse the
n2:mentions: n3:16288283

Subject Item: _:vb6007655
rdf:type: n2:Context
rdf:value: Furthermore, both CpG and LPS treatment activates Stat>>338<<,25,40, which acts as a negative feedback mechanism to constrain Th1 immune responses.
n2:mentions: n3:17238832

Subject Item: _:vb6007656
rdf:type: n2:Context
rdf:value: Furthermore, both CpG and LPS treatment activates Stat338,>>25<<,40, which acts as a negative feedback mechanism to constrain Th1 immune responses.
n2:mentions: n3:19258507

Subject Item: _:vb6007657
rdf:type: n2:Context
rdf:value: Furthermore, both CpG and LPS treatment activates Stat338,25,>>40<<, which acts as a negative feedback mechanism to constrain Th1 immune responses.
n2:mentions: n3:10903771

Subject Item: _:vb6007658
rdf:type: n2:Context
rdf:value: CpG-Stat3 siRNA oligonucleotides inhibited growth of both a poorly immunogenic variant of K1735 melanoma, C>>441<<, and CT26 colon carcinomas in C3H and BALB/c mice, respectively (Fig.
n2:mentions: n3:15116091

Subject Item: _:vb6007659
rdf:type: n2:Context
rdf:value: Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines>>10<<,20,42,43, which can be greatly amplified by CpG treatment25.
n2:mentions: n3:16288283

Subject Item: _:vb6007660
rdf:type: n2:Context
rdf:value: Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines10,>>20<<,42,43, which can be greatly amplified by CpG treatment25.
n2:mentions: n3:19185846

Subject Item: _:vb6007661
rdf:type: n2:Context
rdf:value: Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines10,20,>>42<<,43, which can be greatly amplified by CpG treatment25.
n2:mentions: n3:10023769

Subject Item: _:vb6007662
rdf:type: n2:Context
rdf:value: Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines10,20,42,>>43<<, which can be greatly amplified by CpG treatment25.
n2:mentions: n3:12571365

Subject Item: _:vb6007663
rdf:type: n2:Context
rdf:value: Lack of Stat3 in DCs has been shown to upregulate expression of Th1 cytokines/chemokines10,20,42,43, which can be greatly amplified by CpG treatment>>25<<. As shown in Fig. 5a, injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation10,20,42,43. It has also been documented that DCs
n2:mentions: n3:19258507

Subject Item: _:vb6007664
rdf:type: n2:Context
rdf:value: 5a, injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation>>10<<,20,42,43. It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance44, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in
n2:mentions: n3:16288283

Subject Item: _:vb6007665
rdf:type: n2:Context
rdf:value: 5a, injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation10,>>20<<,42,43. It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance44, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in
n2:mentions: n3:19185846

Subject Item: _:vb6007666
rdf:type: n2:Context
rdf:value: 5a, injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation10,20,>>42<<,43. It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance44, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in tumor-associated
n2:mentions: n3:10023769

Subject Item: _:vb6007667
rdf:type: n2:Context
rdf:value: 5a, injection of the CpG-Stat3 siRNA at tumor site resulted in upregulation of several Th1 cytokines and chemokines, which were shown to be upregulated by Stat3 ablation10,20,42,>>43<<. It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance44, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in tumor-associated
n2:mentions: n3:12571365

Subject Item: _:vb6007668
rdf:type: n2:Context
rdf:value: It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance>>44<<, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in tumor-associated DCs10.
n2:mentions: n3:11208863

Subject Item: _:vb6007669
rdf:type: n2:Context
rdf:value: It has also been documented that DCs with low expression levels of co-stimulatory molecules mediate immune tolerance44, which is one of the proposed mechanisms for tumor immune evasion induced by Stat3 activation in tumor-associated DCs>>10<<. We therefore analyzed expression of co-stimulatory molecules by DCs enriched from TDLNs.
n2:mentions: n3:16288283

Subject Item: _:vb6007670
rdf:type: n2:Context
rdf:value: Stat3 ablation in myeloid cells followed by local treatment has been shown to induce potent antitumor innate immune responses that involve neutrophils>>10<<. We therefore assessed whether CpG-Stat3 siRNA conjugate treatment could lead to neutrophil-associated tumor cell apoptosis. Co-staining B16 tumor tissue sections with antibodies specific to activated caspase-3 and neutrophils revealed
n2:mentions: n3:16288283

Subject Item: _:vb6007671
rdf:type: n2:Context
rdf:value: The ratio of effector to regulatory T cells within the tumor microenvironment is considered indicative of the effect of adaptive immune responses on tumor progression and metastasis>>45<<. We investigated the numbers of tumor infiltrating T cell populations in subcutaneously growing B16 tumors treated locally for 2 weeks with CpG-Stat3 siRNA, CpG-scrambled RNA control or PBS only.
n2:mentions: n3:16849580

Subject Item: _:vb6007672
rdf:type: n5:Section
dc:title: methods
n5:contains: _:vb6007676 _:vb6007673 _:vb6007674 _:vb6007675

Subject Item: _:vb6007673
rdf:type: n2:Context
rdf:value: The generation of transgenic C57BL/6.CEA mice and MC-38.CEA cell line was previously described>>50<<.
n2:mentions: n3:9537250

Subject Item: _:vb6007674
rdf:type: n2:Context
rdf:value: The sequence of firefly luciferase-specific 25/27mer siRNA (Luc1 R 25D/27), used for the CpG1668-Luc siRNA conjugate molecule, was published>>34<<. The correct formation of siRNA duplex was confirmed by in vitro Dicer cleavage assays.
n2:mentions: n3:16049023

Subject Item: _:vb6007675
rdf:type: n2:Context
rdf:value: EMSA and Western blot analysis to detect Stat3 DNA-binding and protein expression were performed as described previously>>18<<. The protein levels of Stat3 detected by Western blotting were later quantified by densitometry using AlphaEase FC software (Alpha Innotech).
n2:mentions: n3:14702634

Subject Item: _:vb6007676
rdf:type: n2:Context
rdf:value: We prepared single-cell suspensions of spleen, lymph node or tumor tissues by mechanic dispersion followed by collagenase D/DNase I treatment as described before>>10<<. For extracellular staining of immune markers 1 × 106 of freshly prepared cells suspended in PBS/2% FCS/0.1% w/v sodium azide was preincubated with FcγIII/IIR-specific antibody to block non-specific binding and stained with different
n2:mentions: n3:16288283

Subject Item: _:vb6007677
rdf:type: n5:Section
dc:title: discussion
n5:contains: _:vb6007678 _:vb6007679 _:vb6007680 _:vb6007681 _:vb6007682 _:vb6007683 _:vb6007684 _:vb6007685 _:vb6007686 _:vb6007687 _:vb6007688 _:vb6007689 _:vb6007690 _:vb6007691 _:vb6007692

Subject Item: _:vb6007678
rdf:type: n2:Context
rdf:value: Although it has been established that binding to TLR9 is necessary for CpG-mediated immune activation, it remains to be fully explored how CpG ODN enter cells>>36<<. Our results indicated that cellular uptake of both CpG ODN and the CpG-siRNA constructs can occur in the absence of TLR9. However, TLR9, at least in mouse cells, is essential for CpG-siRNA-mediated gene silencing. While the exact
n2:mentions: n3:14716310

Subject Item: _:vb6007679
rdf:type: n2:Context
rdf:value: Nevertheless, while the highest levels of constitutive TLR9 expression is observed on human plasmacytoid DCs and B cells, it is also expressed at lower levels on human monocytes and macrophages>>26<<. These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity27,29,46. Furthermore, it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor
n2:mentions: n3:15454922

Subject Item: _:vb6007680
rdf:type: n2:Context
rdf:value: These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity>>27<<,29,46. Furthermore, it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor effects of siRNA by stimulating antitumor immune responses, likely through intracellular RNA sensors such as
n2:mentions: n3:17479101

Subject Item: _:vb6007681
rdf:type: n2:Context
rdf:value: These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity27,>>29<<,46. Furthermore, it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor effects of siRNA by stimulating antitumor immune responses, likely through intracellular RNA sensors such as RIG-I
n2:mentions: n3:18176597

Subject Item: _:vb6007682
rdf:type: n2:Context
rdf:value: These immune cells can serve as antigen-presenting cells and induce innate, adaptive or humoral immunity27,29,>>46<<. Furthermore, it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor effects of siRNA by stimulating antitumor immune responses, likely through intracellular RNA sensors such as RIG-I or
n2:mentions: n3:18430787

Subject Item: _:vb6007683
rdf:type: n2:Context
rdf:value: it has been demonstrated recently that adding triphosphate to the 5′ of siRNA can potentiate the antitumor effects of siRNA by stimulating antitumor immune responses, likely through intracellular RNA sensors such as RIG-I or MDA->>54<<. It is therefore conceivable to incorporate triphosphate to the CpG-siRNA to further amplify antitumor immunity.
n2:mentions: n3:18978796

Subject Item: _:vb6007684
rdf:type: n2:Context
rdf:value: In addition, a critical role of tumor stromal macrophages and B cells in promoting tumor development has been well documented>>47<<,48. Importantly, Stat3 and several other molecules produced by the tumor myeloid population, and possibly tumor-associated B cells, are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B
n2:mentions: n3:17476345

Subject Item: _:vb6007685
rdf:type: n2:Context
rdf:value: In addition, a critical role of tumor stromal macrophages and B cells in promoting tumor development has been well documented47,>>48<<. Importantly, Stat3 and several other molecules produced by the tumor myeloid population, and possibly tumor-associated B cells, are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B cells
n2:mentions: n3:17276050

Subject Item: _:vb6007686
rdf:type: n2:Context
rdf:value: Importantly, Stat3 and several other molecules produced by the tumor myeloid population, and possibly tumor-associated B cells, are critical for tumor immunosuppression>>17<<, and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival12,21,22,23.
n2:mentions: n3:17186030

Subject Item: _:vb6007687
rdf:type: n2:Context
rdf:value: B cells, are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival>>12<<,21,22,23. In addition to Stat3, other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies.
n2:mentions: n3:18776941

Subject Item: _:vb6007688
rdf:type: n2:Context
rdf:value: are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival12,>>21<<,22,23. In addition to Stat3, other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies.
n2:mentions: n3:19185844

Subject Item: _:vb6007689
rdf:type: n2:Context
rdf:value: are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival12,21,>>22<<,23. In addition to Stat3, other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies.
n2:mentions: n3:19185845

Subject Item: _:vb6007690
rdf:type: n2:Context
rdf:value: are critical for tumor immunosuppression17, and Stat3 activity in the myeloid compartment (possibly B cells as well) promotes Stat3 activity in tumor cells and endothelial cells in the tumor, enhancing tumor cell growth/survival12,21,22,>>23<<. In addition to Stat3, other oncogenic molecules produced by the tumor myeloid/B cell compartment are also critical in promoting cancer growth and resistance to various therapies.
n2:mentions: n3:19345327

Subject Item: _:vb6007691
rdf:type: n2:Context
rdf:value: In addition to normal immune cells, several types of tumor cells, including those of B cell origin, and some solid tumor cells, are also positive for TLR>>949<<. Our preliminary results suggested the feasibility of CpG-siRNA approach to silence genes in TLR9+ tumor cells. For example, treating human TLR9+ tumors in NOD/SCID/IL-2RγKO mice with CpG-Stat3 siRNA resulted in tumor cell apoptosis and
n2:mentions: n3:18176602

Subject Item: _:vb6007692
rdf:type: n2:Context
rdf:value: It is possible that in vivo repeated treatments allow accumulative gene silencing effects, and the crosstalk between various cells in the tumor microenvironment could lead to secondary inhibitory effects on Stat3 activity>>23<<. The half-life of the constructs at present is limited. Although the CpG ODN in the construct is phosphothioated, which should resist serum degradation, the siRNA in the chimeric construct is unmodified and negatively charged. Chemically
n2:mentions: n3:19345327

Subject Item: _:vb413690836
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n2:RelevantScore: 15
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Subject Item: _:vb413690837
rdf:type: n2:RelevantBibliographicResource
n2:RelevantScore: 15
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Subject Item: _:vb413690838
rdf:type: n2:RelevantBibliographicResource
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rdf:type: n2:RelevantBibliographicResource
n2:RelevantScore: 14
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Subject Item: _:vb413690841
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