Reprogramming Tumor-Associated Dendritic Cells In Vivo Using miRNA Mimetics Triggers Protective Immunity against Ovarian Cancer

miRNAs are small endogenous noncoding RNAs implicated in the posttranscriptional control of gene expression in developmental, physiologic, and pathologic processes. Biologically active/mature miRNAs bind to partially complementary sequences [miRNA recognition element (MRE)] in hundreds of mRNAs, which diminish protein production via mRNA degradation and/or translational repression. miRNA-mediated regulation therefore constitutes a major mechanism to control global gene expression patterns (1–3).

miRNAs are quickly challenging our understanding of genetic regulation in health and disease, including cancer etiology (4) and the generation and inhibition of antitumor immune responses (5–9). Biologically active miRNAs bind to MREs on multiple mRNAs and simultaneously silence multiple target genes. This process can directly or indirectly modulate global gene expression and eventually determines transcriptional programs associated with a specific phenotype.

Because immune responses–including those against tumor antigens–depend on rapid phenotypic changes, it is not surprising that miRNAs have emerged as critical regulators of virtually all immune cell types (5, 7). miR-155 epitomizes the role of miRNAs in the immune system. miR-155 is basally expressed at low levels in B cells (7, 8), T cells (10), macrophages (11), dendritic cells (DC; ref. 7), and progenitor/stem cell populations (10). Activation signals such as antigen, toll-like receptor (TLR) stimulation and inflammatory cytokines, rapidly increase miR-155 expression in various leukocytic subsets, including bone marrow DCs (BMDC) and macrophages (7, 8, 11). Interestingly, BMDCs matured in the absence of miR-155 upregulate MHC-II and costimulatory molecules but are incapable of effectively activating T cells (7).

We previously showed that leukocytes with predominant phenotypic attributes of regulatory DCs (including the expression of CD11c and DEC205) home to perivascular locations in the ovarian cancer microenvironment, where they express multiple immunosuppressive mediators (12–14). From their position around blood vessels, these regulatory DCs inhibit the protective function of antitumor T cells infiltrating the tumor from the blood. Although specific delivery of RNA oligonucleotides to cancer cells is challenging because of low bioavailability, poor cellular uptake, and abundant phagocytic activity of other cell types in the tumor microenvironment (15), the enhanced endocytic pathways and relative accessibility of ovarian cancer–associated myeloid leukocytes makes them ideal targets for nanocomplex-mediated delivery. Thus, we previously showed that polyethylenimine (PEI)-based nanocomplexes are selectively and avidly taken up by DCs at ovarian cancer locations, in the absence of any targeting motif (12). Using this optimized system, we now show that activity of mature miR-155 can be augmented in tumor-associated DCs by delivering novel Dicer substrate RNA duplexes that mimic the structure of endogenous precursor miR-155 hairpin (Dmi155) and that are efficiently processed in vivo. Replenishing miR-155 levels in tumor-associated DCs reprogrammed their immunosuppressive phenotype by modulating the expression of nearly half of the mRNAs in their transcriptome. Synthetic enhancement of miR-155 signaling in DCs boosted potent antitumor immune responses that abrogated the progression of established ovarian cancers. Our results show the feasibility of supplementing/replenishing miRNAs in vivo to boost antitumor immunity against aggressive, advanced, and frequently lethal tumors.

Endotoxin-free PEI for in vivo experiments “in vivo-jetPEI” was purchased from PolyPlus Transfection. Dicer substrates (Dsi) were synthesized at Integrated DNA Technologies (IDT) using the following chimeric sequences:

• Control GFP-specific Dicer substrate (GFP Dsi):

  • Plus: 5′ rUrGrCrArGrArUrGrArArCrUrUrCrArGrGrGrUrCrArGrCTT 3′

  • Minus: 5′ rArArGrCrU rGrArC rCrCrU rGrArA rGrUrU rCrArUrCrUrG rCrArUrU 3′

• Control GFP-specific “bulged” Dicer substrate:

  • Plus: 5′ rUrGrCrArGrArUrGrArArCrUrUrCrArGrGrGrUrCrArGrCTT 3′

  • Minus: 5′ rArArGrCrU rGrArC rCrCrU rG rGrUrU rCrArU rCrUrGrCrArUrU 3′

• siRNA-like miR-155 Dicer substrate (Dsi155):

  • Plus: 5′ rUrUrA rArUrG rCrUrA rArUrU rGrUrG rArUrA rGrGrGrGrUT T 3′

  • Minus: 5′ rArArA rCrCrC rCrUrA rUrCrA rCrArArUrUrArGrCrArUrUrA rArUrU 3′

• miRNA-like bulged miR-155 Dicer substrate (Dmi155):

  • Plus: 5′ rUrUrA rArUrG rCrUrA rArUrU rGrUrG rArUrA rGrGrGrGrUT T 3′

  • Minus: 5′ rArArA rCrCrC rCrUrA rUrCrA rA rUrUrA rGrCrArUrUrA rArUrU 3′

In all cases, “r” represents a ribonucleotide and the absence of an “r” indicates a deoxynucleotide. The “plus” strand contains 2 terminal deoxynucleotides that resemble the loop of endogenous pre-miRNA and that function as cleavage signal for Dicer. The “plus” strand refers to the strand that will give rise to the mature miRNA after Dicer processing and preferential incorporation into the RNA-induced silencing complex (RISC).

To generate PEI-based nanoparticles encapsulating Dsi, 50 to 100 μg of each annealed duplex were complexed with “in vivo-jetPEI” at an N/P ratio of 6, following the recommendations of the manufacturer (PolyPlus Transfection). For biodistribution experiments, Dsi were fluorescently labeled in the 3′ end of the plus strand using Cy3 (IDT). Biotinylated Dsi were also chemically synthesized at IDT and include a Biotin group in the 5′ end of the “plus” strand. Thus, after intracellular processing of the Dsi, the mature form of the miRNA remains biotinylated in vivo.

Lipofectamine 2000 (Invitrogen) was used for in vitro transfection of Dsi into HEK293 cells in 96-well plates, following the recommendations of the manufacturer. For in vivo biodistribution, phenotypic and gene silencing experiments, mice bearing ID8-Defb29/Vegf-A tumors (12) for 3 to 4 weeks were intraperitoneally injected with PEI-Dsi nanoparticles (50 μg of Dsi complexed with “in vivo-jetPEI” at N/P 6, per mouse). In all phenotypic and functional experiments, tumor-associated DCs from mice injected with nanoparticles were sorted from ascites or peritoneal wash samples by flow cytometry on the basis of CD45, CD11c, and MHC-II positive expression.

Wild-type C57BL/6 mice were intraperitoneally injected with 2 × 10⁶ parental ID8 (kindly provided by K. Robby, University of Kansas Medical Center, Kansas City, KS; ref. 16) and treatments started 15 days posttumor injection. A total of 2 × 10⁶ aggressive ID8-Defb29/Vegf-A ovarian carcinoma cells were injected intraperitoneally and treatments started after 8 days. In all cases mice received 50 μg of Dsi complexed with “in vivo-jetPEI” at N/P 6 in glucose 5% at the indicated time points. Some experimental groups were also intraperitoneally injected with 50 μg anti-CD40 antibody (clone FGK4.5) 3 hours before administration of PEI-based nanoparticles containing Dsi.

For tumor rechallenge protection experiments, 3 × 10⁶ CD3⁺ T cells negatively immunopurified from the spleens of tumor-bearing mice treated with PBS (day 32 after tumor challenge) or αCD40 Ab plus Dmi155-PEI nanoparticles (day 61 after tumor challenge; treatments at days 8, 13, 18, 23, 27, and 60) were intravenously transferred into naive C57BL/6 mice previously irradiated with 300γ (5 mice per group). Twenty-four hours later mice were challenged in the flank with ID8-Defb29/Vegf-A ovarian carcinoma cells, as described (14). Tumor pictures were taken 25 days later. Tumor volumes were calculated by the formula V = 0.5 (L × W²), in which L is length and W is width.

miR-155 plays an important role in oncogenesis (9) but is also required for optimal antigen presentation and T-cell activation by mature DCs (7). We found that immunosuppressive CD45⁺CD11c⁺MHC-II⁺ DCs (12, 13, 17–19) sorted from advanced orthotopic ID8-Defb29/Vegf-A tumors, an aggressive model of ovarian cancer that recapitulates the inflammatory microenvironment of human ovarian carcinomas (13, 14, 20, 21), showed significantly reduced levels of mature miR-155 (Fig. 1A). However, in vivo administration of CD40 plus TLR3 agonists, which synergistically transform tumor-associated DCs from immunosuppressive to immunostimulatory (13), induced a dramatic upregulation of mature miR-155 (Fig. 1B). We therefore hypothesized that miR-155 upregulation in DCs in vivo at tumor locations could be the crucial event promoting their capacity to elicit therapeutic antitumor immunity.

To augment miR-155 activity, we generated novel synthetic Dicer substrate (Dsi) RNA duplexes. To become functionally active, Dsi require processing by Dicer, the same RNAse type III enzyme that processes endogenous miRNA precursors and exogenous siRNAs. In addition, Dsi exhibit markedly enhanced silencing efficiency compared with conventional 21-mer siRNA oligonucleotides (22, 23). In all cases, we designed a forward (sense) RNA strand containing the sequence of endogenous mature miR-155 followed by 2 terminal deoxynucleotides in the 3′ end. We then generated 2 structural versions for miR-155 mimetic compounds by using different passenger (antisense) strands: An internally bulged complementary strand that recapitulates the precursor miRNA hairpin (Dmi155) and a perfectly matching, siRNA-like, complementary strand (Dsi155; Fig. 1C). Control irrelevant bulged or siRNA-like Dsi designed to target GFP were also produced in parallel.

Transfection of HEK293 cells with either Dsi155 or Dmi155 led to a dramatic dose-dependent increase in the intracellular levels of processed miR-155, as detected by mature miRNA-specific stem-loop quantitative reverse transcriptase PCR (qRT-PCR; Fig. 1D). Confirming the selective detection of processed miRNAs by the cellular machinery, negligible signal was detected when synthetic Dsi155 or Dmi155 were directly reversed transcribed and amplified before transfection (Fig. 1E). To determine the functionality of processed miR-155 RNA generated from synthetic RNA, we cotransfected HEK293 cells with a luciferase reporter construct containing the miR-155 MRE of Sfpi1, an experimentally validated target gene of miR-155 (24). As expected, Dmi155 and, to a significantly lesser extent, Dsi155, rapidly silenced luciferase protein expression, whereas control (GFP specific) Dsi had no effect (Fig. 1F). Importantly, duplexes did not alter luciferase expression when the reporter constructs lacked the cognate miR-155 MRE (Fig. 1G). Together, these data showed that synthetic Dsi RNA duplexes can be used to effectively generate functionally active mature miR-155 in the cell, and suggest that a bulged structure may be important for the functionality of the miRNA generated.

We have shown that intraperitoneally injected nanocomplexes of PEI and siRNA are avidly and selectively taken up by tolerogenic DCs residing at ovarian cancer locations (12). As expected, PEI-based nanoparticles encapsulating Cy3-labeled Dmi155 were also preferentially engulfed by CD45⁺CD11c⁺ DCs in the tumor (peritoneal) microenvironment (Fig. 2A and B). Less than 1% of tumor cells incorporated the nanoparticles and only 3% of other leukocytes (primarily myeloid-derived suppressor cells and canonical macrophages) showed rhodamine fluorescence (Fig. 2B). Synthetic miR-155 was rapidly compartmentalized in the perinuclear region, typical of endosome–lysosome vesicle formation (ref. 25; Supplementary Fig. S1A). Most importantly, tumor-associated DCs endocytozing Dsi155 or Dmi155 nanocomplexes in vivo showed a marked increase in the intracellular levels of mature miR-155, as detected by stem-loop qRT-PCR, compared with tumor-associated DCs in mice untreated or receiving control GFP-specific Dsi (Fig. 2C). Ectopic mature miR-155 did not saturate the cellular silencing machinery because other endogenous mature miRNAs were found at comparable levels in DCs incorporating various RNA duplexes (Fig. 2D and Supplementary Fig. S1B).

To confirm the functional activity of miR-155 generated in vivo upon synthetic RNA processing, we analyzed the expression of 3 different experimentally validated targets of miR-155. Strikingly, the expression of C/ebpβ (10, 26, 27) and Socs1 (28) was rapidly and potently silenced only in tumor-associated DCs engulfing nanoparticles of bulged Dmi155, but not perfectly matching Dsi155 or irrelevant Dsi (Fig. 2E and F). In addition, although Dsi155 induced a significant decrease in the expression of Sfpi1 (10, 24), the silencing effect elicited by bulged Dmi155 was significantly greater (Fig. 2G). Therefore, although both Dsi155 and Dmi155 are biologically processed into mature miR-155, these data suggested that the structural features of the RNA duplex more closely mimicking the endogenous pre-miRNA hairpin are important for optimal silencing of target genes.

After Dicer processing, mature miRNAs are loaded by various Argonaute proteins (Ago1-4) into the RISC, a process that guides this multiprotein system to silence target mRNAs via cleavage, translational repression, or deadenylation (29). However, only Ago2 has slicer activity (30). Notably, we detected significantly greater amounts of mature miR-155 generated from both Dsi155 and Dmi155 by stem-loop qRT-PCR in Ago2 immunoprecipitates of peritoneal microenvironmental cells, compared with precipitation using Ago4 or, to an even lesser extent, Ago1 antibodies (Fig. 3B). Most importantly, significantly higher amounts of mature miR-155 processed from Dmi155 versus Dsi155 were found in slicer activity-endowed Ago2 pull-downs (Fig. 3B). Correspondingly, superior recovery of various known miR-155 targets was evidenced in Ago2-immunoprecipitated RNA only upon in vivo delivery of PEI-Dmi155, compared with administration of PEI-Dsi155 (Fig. 3C and D). Together, these results indicated that a bulged structure, similar to that of endogenous pre-miR-155, facilitates the efficient incorporation of mature miR-155 into the RISC via optimal loading onto Ago2 and, to a lesser extent, Ago4 and Ago1 proteins. There is no reliable Ago3 antibody for immunoprecipitation experiments and consequently Ago3 association studies could not be realized at this time.

Because miR-155 is critical for DC-mediated antigen presentation (7) and its expression increases in response to CD40/TLR agonists, we hypothesized that delivery of miR-155 to CD40/TLR-stimulated tumor-associated DCs could further improve their antigen-presenting capacity at tumor locations. As expected, the proliferation of CFSE-labeled OT-1 T cells in situ in the ovarian cancer microenvironment was significantly enhanced in mice pulsed with full-length OVA when anti-CD40 plus (irrelevant) GFP Dsi-PEI nanocomplexes were administered (Fig. 4A and B). However, delivery of bulged Dmi155 induced a stronger antigen-specific T-cell proliferation at tumor locations (Fig. 4A and B), indicating that ectopic supplementation of miR-155 robustly enhances the immunostimulatory capacity of tumor-associated DCs beyond TLR activation.

Consistent with improved antigen presentation, higher proportions of antigen-experienced (CD44⁺) CD4⁺ and CD8⁺ T cells were found in the tumor microenvironment of mice treated 4 times with CD40 agonists plus GFP Dsi-PEI nanocomplexes, compared with untreated mice (Fig. 4C and D and Supplementary Fig. S1C). The proportion of antigen-experienced T cells at tumor (peritoneal) locations was again further increased by treatment with PEI-complexed (bulged) Dmi155. Consistent with ineffective loading onto Ago2, the effect of perfectly matching Dsi155 was not superior to control GFP Dsi formulations (Fig. 4C and D and Supplementary Fig. S1C). Correspondingly, the number of tumor (peritoneal) T cells secreting Granzyme B in ELISPOT analyses in response to tumor antigens was also significantly increased upon administration of bulged Dmi155, whereas treatment with siRNA-like Dsi155 did not enhance tumor antigen–specific T-cell responses more than control GFP Dsi (Fig. 4E). Likewise, mice treated with Dmi155 also showed a marked increase in the numbers of splenic T cells secreting Granzyme B upon restimulation with tumor antigens (Fig. 4F). These responses were tumor specific because they were significantly diminished when antigen-presenting cells (APC) were pulsed with irrelevant (3T3) cells in independent experiments (Supplementary Fig. S1D). Furthermore, Dmi155 treatment resulted in a significant increase in the proportions of total splenic CD8⁺ T cells exhibiting central memory attributes (CD44⁺ CD62L⁺; Fig. 4G and Supplementary Fig. S1E). Finally, in vivo production of Th1 cytokines with antitumor potential such as TNFα, IL-12, IFNγ, and CCL5 (19, 31) was significantly enhanced at tumor locations in mice receiving Dmi155, compared with Dsi155 or control RNA (Fig. 4H). Together, these results indicated that delivery of bulged pre-miR-155 mimetic RNA, but not siRNA-like reagents, enhances the capacity of otherwise regulatory DCs at tumor locations to effectively present antigen, boost T-cell–dependent antitumor immunity, and induce the secretion of immunostimulatory cytokines beyond the sequence-independent, nonspecific activation of CD40 and TLRs (12, 13).

Because tumor-associated DCs harboring increased levels of mature miR-155 exhibited functional properties of highly immunostimulatory APCs, we next determined the immunotherapeutic potential of delivering miR-155 mimetic RNA to ovarian cancer DCs, along with synergistic CD40 agonists (13). Mice growing orthotopic established ID8 ovarian tumors (16) were treated with agonistic anti-CD40 antibodies plus PEI-complexed control Dsi RNA (GFP specific), Dsi155, or Dmi155. Importantly, no obvious toxicity or secondary tumor growth in distant organs derived from the uptake of miR-155 mimetic RNA by cancer cells was observed in any case. As we previously reported (12), the intrinsic immunostimulatory activity of PEI-complexed RNA induced a significant increase of approximately 50% in the median survival of tumor-bearing mice treated with irrelevant (GFP specific) Dsi, compared with untreated mice (Fig. 5A). Consistent with the limited immunostimulatory effects on DCs, survival of mice treated with PEI-complexed Dsi155 was not superior to that elicited by the TLR5 agonist PEI alone (12), or by CD40 agonists plus the TLR5 agonist PEI, and was similar to that in mice treated with anti-CD40 antibodies plus irrelevant GFP Dsi (Fig. 5A). In contrast, treatment with bulged Dmi155 induced significantly superior effects and even abrogated disease progression in 33% of mice, which remained alive 80 days after controls succumbed to the disease (Fig. 5A). These results showed the therapeutic potential of supplementing miR-155 to tumor-infiltrating DCs in vivo using bulged RNA that mimics the structure of endogenous miR-155 and are consistent with the deficient silencing activity of miR-155 processed from perfectly matching oligonucleotides that merely include the sequence of mature miRNAs.

In addition, when treatments were administered to mice growing more aggressive ID8-Defb29/Vegf-A ovarian tumors, CD40 agonists synergized with PEI-complexed irrelevant double-stranded RNA oligonucleotides to induce a marked increase of approximately 43% in the median survival, and the effect of Dsi155 was again indistinguishable (Fig. 5B). Confirming our previous observations (13), agonistic anti-CD40 antibodies alone induce no therapeutic benefit against these tumors (Fig. 5B), unless they were combined with TLR agonists such as PEI (a TLR5 agonist; ref. 12) or double-stranded RNA. Notably, irrelevant bulged and siRNA-like Dsi targeting GFP induced identical effects (Supplementary Fig. S1F) and were, therefore, indistinctively used in subsequent experiments. Most importantly, mice receiving only 4 additional injections (days 13, 18, 23, and 27) of CD40 agonists plus PEI-complexed bulged Dmi155 exhibited a significant overall survival increase of approximately 65% compared with untreated mice. This therapeutic effect was significantly stronger than that induced by an identical schedule of GFP Dsi-PEI or Dsi155-PEI treatments (Fig. 5B).

To confirm the antitumor effects of miR-155 mimetics in the absence of the mRNAs upregulated by CD40 activation, we finally treated aggressive tumor-bearing mice with an identical regimen of only control or miR-155 mimicking compounds. As shown in Fig. 5C, corresponding, although obviously weaker effects were observed. Notably, survival increases resulting from miR-155 supplementation were associated with T-cell–dependent protection because T cells from CD40/Dmi155-treated mice restrained tumor growth upon rechallenge, compared with T cells from untreated mice (Fig. 5D). Together, these results showed that only Dmi155 mimicking the bulged structure of endogenous pre-miR-155 is able to induce therapeutic benefits and synergize with the in situ activation of CD40 to extend survival in hosts bearing established aggressive ovarian carcinomas.

To understand how mature miR-155 processed from delivered Dmi155 promotes the immunostimulatory phenotype of tumor-associated DCs in such striking manner, we next analyzed transcriptional changes in treated mice. Strikingly, deep sequencing analysis of the transcriptome of tumor-associated CD45⁺CD11c⁺MHC-II⁺ DCs revealed that Dmi155, directly or indirectly, induced the silencing of thousands of transcripts, including multiple genes associated with an immunosuppressive phenotype (Supplementary File S1). Overall, 48% of total genes detected in tumor-associated DCs were downregulated 2-fold or more at the mRNA level by Dmi155 treatment. Those included known immunosuppressive targets of miR-155 such as C/epbβ, recently described as critical regulator of the immunosuppressive environment created by growing cancers (32); multiple mediators of Tgf-β signaling pathway, including Tgfβ1, Smad1, Smad6, and Smad7; and Ccl22, which recruits regulatory T cells to the tumor microenvironment (18). Unexpectedly, we also found that Satb1, a master genomic organizer (33), is expressed in tumor-associated DCs and silenced by miR-155.

In addition, we found downregulation of multiple transcripts not previously associated with miR-155. We focused on Cd200, a know mediator of DC-induced tolerance (34). Supporting that Cd200 is indeed a bona fide immunosuppressive target of miR-155, luciferase activity was silenced by Dmi155, but not by irrelevant Dsi, in the presence of the 3′-untranslated region (3′-UTR) of Cd200 (Fig. 6). The specificity of the analysis is supported by the parallel silencing of Satb1, recently confirmed as a target of miR-155, but not of Pdcd4, the expression of which is not significantly altered in vivo (Fig. 6).

Interestingly, Cd200 is not a predicted target of miR-155 in any major databases. This is not surprising because 56% of published targets of miR-155 are also not contained in any major databases, including Miranda, Targetscan, DianaMT, miRDB, Mirwalk, PITA, RNA22, and PicTar.

Together, these data indicated that the transformation of plastic DCs at tumor locations into immunostimulatory cells by synthetic miR-155 is the result of complex genome-wide transcriptional changes rather than the silencing of a limited set of targets. In addition, our optimization of miRNA mimetics and delivery system provides multiple experimental hints for new targets of individual miRNAs, which should help to improve bioinformatical predictions by providing new clues for the design of more reliable algorithms.

Here we show for the first time the feasibility of modulating miRNA activity selectively in ovarian cancer microenvironmental leukocytes using a nonviral approach, which promotes their capacity to elicit protective immunity.

Although expression of noncoding RNA in cancer cells can be achieved with viral vectors, the therapeutic use of viruses remains a clinical challenge. In addition, low bioavailability, poor cellular uptake, and preferential uptake by abundant phagocytic cells (15) are still major hurdles for specific delivery of genetic materials stabilized in nano- or microparticles to tumor cells. In contrast, the enhanced endocytic pathways and massive infiltration of the myeloid leukocytes that systematically accumulate in solid tumors make them ideal targets for nanocomplex-mediated delivery. Because of its relative accessibility, ovarian cancer–associated leukocytes are an ideal target for this approach.

We selected supplementing miR-155 because silencing of a nonredundant set of targets by this miRNA seems to be required for proper antigen presentation (7). However, miR-155 expression is frequently detected at high levels human cancer, both in solid tumors including breast, colorectal, lung, pancreatic, and thyroid carcinomas and in liquid tumors including lymphomas and some acute myeloid leukemias (9, 35). The association between oncogenesis and effective immunity is not surprising as robust adaptive immune responses entail rapid expansion of leukocytes. Furthermore, artificial upregulation of mir-155 leading to oncogenic conditions involves sustained overexpression in hematopoietic progenitors. In our study, the transient increase of mir-155 in lineage-committed myeloid cells such as tumor-associated DCs did not enhance ovarian cancer progression and did not result in generation of any secondary tumors. Instead, miR-155 delivery elicited robust antitumor immune responses that prolonged survival in mice bearing aggressive established ovarian cancer. Thus, miR-155 processed from endocytosed Dmi155 induced genome-wide transcriptional changes in DCs in situ at tumor locations, which significantly enhanced their immunostimulatory capacity. Because the expression of nearly half of the transcriptome was affected by synthetic miR-155 delivery, this significant phenotypic transformation was the result of complex coordinated transcriptional changes, rather than the silencing of a limited set of targets. Consequently, many genes known to promote tolerance were downregulated, including C/ebpβ, crucial immunosuppressive factor in cancer microenvironmental cells (32), and multiple mediators of the Tgfβ signaling pathway. In addition, we confirmed that other unpredicted targets of miR-155 such as Cd200 were indeed silenced in standard luciferase assays.

Although PEI-siRNA nanocomplexes stimulate multiple TLRs on tumor-associated DCs (12), the robust enhancement in antigen presentation, production of Th1 cytokines, and expansion of tumor-reactive T cells selectively elicited by Dmi155 was significantly superior, compared with the nonspecific activation of DCs elicited by control sequences. Importantly, phenotypic transformation of DCs at tumor locations was not the result of the saturation of the RISC complex, because other mature miRNAs were clearly detected in Dmi155-treated cells. It is therefore very unlikely that the significant immunostimulatory effects, which abrogate the progression of established tumors, are the result of the sequestration of all Ago variants.

Most importantly for the clinical testing miRNA mimetics, we found that perfectly matching (siRNA-like) and bulged (miRNA-like) duplexes were both processed by tumor-associated DCs to generate mature miR-155. However, miR-155 generated from multiple batches of siRNA duplexes exhibited deficient silencing activity toward target genes in vivo, compared with Dmi155. Correspondingly, significantly higher amounts of mature miR-155 processed from Dmi155 versus Dsi155 were found in pull-downs of Ago2, the only Ago variant with slicer activity. miRNAs first associate with Agos as RNA duplexes that require activation, defined as conversion of the RNA duplex into a single-stranded miRNA. This activation process is the rate-limiting step in Ago loading and crucially depends on the thermodynamic instability of RNA duplexes (36). However, the cleavage activation pathway specific to Ago2 seems to be the only one insensitive to RNA thermostability in embryonic fibroblasts (36). It is possible that immune cells behave differently, so that Dmi155 and Dsi155 bind to Ago2 variants with similar affinity, but bulged duplexes with weaker thermodynamic stability are more efficiently processed and activated. Not mutually exclusive, it is also possible that chaperone proteins regulating the upload of small hairpin RNAs onto the RISC complex recognize the difference between a bulged versus a matching structure in DCs, so that different compositions are incorporated with distinct efficiency.

In summary, our results show the feasibility of delivering synthetic miRNAs to tumor microenvironmental cells as a novel cancer intervention and provide fundamental clues for the optimization of this approach.

No potential conflicts of interest were disclosed.

Conception and design: J.R. Cubillos-Ruiz, J.R. Baird, S.N. Fiering, L.F. Sempere, and J.R. Conejo-Garcia.

Development of methodology: J.R. Cubillos-Ruiz, J.R. Baird, S.N. Fiering, L.F. Sempere, and J.R. Conejo-Garcia.

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.R. Cubillos-Ruiz, J.R. Baird, A.J. Tesone, M.R. Rutkowski, A.L. Camposeco-Jacobs, J. Anadon-Arnillas, N.M. Harwood, M. Korc, and J.R. Conejo-Garcia.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.R. Cubillos-Ruiz, J.R. Baird, A.J. Tesone, M.R. Rutkowski, J. Anadon-Arnillas, N.M. Harwood, M. Korc, S.N. Fiering, and J.R. Conejo-Garcia.

Writing, review, and/or revision of the manuscript: J.R. Cubillos-Ruiz, A.J. Tesone, M.R. Rutkowski, U.K. Scarlett, S.N. Fiering, L.F. Sempere, and J.R. Conejo-Garcia.

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.R. Baird.

Generated data: U.K. Scarlett.

Study supervision: S.N. Fiering and J.R. Conejo-Garcia.

The authors thank the Bioinformatics, Genomics, Flow Cytometry, and Animal Facilities at The Wistar Institute and the Genomics and Microarray Laboratory at Dartmouth.

This study was supported by NCI grants CA157664, CA124515, CA124515S, CA132026, CA141017 and P30CA010815, and by DoD grant OC100059 (to J.R. Conejo-Garcia). J.R. Cubillos-Ruiz was supported by the 2009–2010 John H. Copenhaver, Jr. and William H. Thomas, MD 1952 Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.