IJC International Journal of Cancer
Lung tumours reprogram pulmonary dendritic cell immunogenicity at the microRNA level Lotte Pyfferoen1,2,3, Pieter Mestdagh4, Karl Vergote2, Nancy De Cabooter1, Jo Vandesompele4, Bart N. Lambrecht2,3,5 and Karim Y. Vermaelen1 1
Department of Respiratory Medicine, Tumor Immunology Laboratory, Ghent University Hospital, Ghent, Belgium VIB Inflammation Research Center, Ghent, Belgium 3 Department of Respiratory Medicine, Ghent University, Ghent, Belgium 4 Center for Medical Genetics, Ghent University, Ghent, Belgium 5 Department of Pulmonary Medicine, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands 2
The host response against tumors is characterized by a complex interplay between transformed cells and the stromal compartment, the latter including various immune cell types. DCs appear involved in anti-tumoural immunity in several preclinical models.1,2 In this process, DCs are thought to
Key words: dendritic cells, lung cancer, microRNAs, Dicer Abbreviations: BMDC: bone marrow-derived DC; DC: dendritic cell; ILN: inguinal LN; i.v.: intravenous; i.t.: intratracheal; LLC: Lewis lung carcinoma; LN: lymph node; LNDC: lymph node dendritic cell; migr-DC: migratory DC; miR(NA): microRNA; MLN: mediastinal LN; resid-DC: resident DC; RT-qPCR: reverse transcription quantitative PCR; Tc1: T cytotoxic 1; TDLN: tumourdraining LN; Th: T helper; (t)OVA: (truncated) ovalbumin; Treg: regulatory T cell Additional Supporting Information may be found in the online version of this article. Grant sponsors: FWO Research Project G.A015.11, IWT and IUAP grant P7/03; Grant number: SB/083359; Grant sponsor: FWO Senior Clinical Investigator Award DOI: 10.1002/ijc.28945 History: Received 26 Nov 2013; Accepted 15 Apr 2014; Online 2 May 2014 Correspondence to: Karim Y. Vermaelen, Ghent University Hospital, 7K12IE, De Pintelaan 185, 9000 Ghent, Belgium, Tel.: 13293322815, E-mail: [email protected]
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transport tumour-derived antigens to draining lymph nodes (LN), where anti-tumoural adaptive immunity is primed, more specifically cytotoxic CD81 T cell responses (Tc1) and CD41 T helper (Th) responses polarized towards IFN-g secreting Th1 cells, whose concerted action can lead to tumour cell eradication.3 However, cancerous lesions develop several strategies to resist immune defences and reprogram immune cells to directly or indirectly promote tumour growth and spread. Evading immune attack is now acknowledged as one of the “emerging hallmarks” of cancer.4 The impact of research into these mechanisms is highlighted by the recent emergence of compounds targeting immune checkpoint receptors, showing remarkable clinical benefit in several solid tumours including lung cancer.5 The immunosuppressive tumour microenvironment can interfere with DC maturation and function, generating both a regulatory T cell (Treg) and a Th2 profile, which are known for their immune suppressing and tumour-promoting capacities via secretion of IL-10, TGF-b, and IL-4, IL-13.6–8 Besides, a tumour-promoting effect for Th17-polarized CD41 T cells has been demonstrated in preclinical studies.9,10 Reprogramming of the immune response involves rapid coordinated changes at the level of the transcriptome and/or proteome. A novel layer of cell regulation consists of microRNAs (miRNAs), a family of small noncoding RNAs individually capable of regulating multiple biological pathways, including those involved in cancer and immune responses.
Tumor Immunology
Lung cancer arises in a context of tumour-induced immune suppression. Dendritic cells (DCs) are central players in the induction of anti-tumoural immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. Meanwhile, microRNAs are associated with tumour development as well as immune regulation. We postulated that lung tumours escape immune control by reprogramming DC immunogenicity at the microRNA level. Using an orthotopic model of lung cancer, we first identified the DC population responsible for transport and cross-presentation of lung tumour-derived antigens to na€ıve T cells in the draining mediastinal lymph nodes (LNs). Profiling the full microRNA repertoire of these DCs revealed a restricted set of microRNAs that was consistently dysregulated in the presence of lung tumours, with miR-301a as one of the top upregulated transcripts. Overexpression of miR-301a in DCs suppressed IL-12 secretion, decreased IFN-c release from antigenspecific cytotoxic T cells, and shifted antigen-specific T helper cytokine profile away from IFN-c towards IL-13 and IL-17Asecreting T cells. Strikingly, DC-selective Dicer1 gene deletion resulted in delayed lung tumour growth and a survival benefit. Taken together, our data reveal that lung tumours induce an immunosuppressive microRNA signature in pulmonary DCs. Interfering with the DC-intrinsic capacity to remodel microRNA repertoires affects lung tumour outcome.
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microRNA plasticity in lung cancer-associated DCs
Tumor Immunology
What’s new? MicroRNAs (miRNAs) regulate many biological pathways, including those involved in tumor development and immune regulation. Dendritic cells (DCs) are central players in antitumoral immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. In this study, the authors found that a group of immunomodulatory miRNAs produced by DCs is consistently dysregulated in the presence of lung tumors. When miRNA biogenesis in these DCs was blocked, tumor growth was significantly delayed in vivo. These results support the hypothesis that lung tumors escape immune control by reprogramming miRNA expression in DCs.
MiRNA profiling studies on tumour samples are generally performed on bulk tumour samples, with expression patterns interpreted as arising from cancer cells.11 However, several studies suggest that the dominant miRNA signatures at different stages of tumourigenesis can be attributed to infiltrating leukocytes rather than arising from the transformed cells themselves.12 This remarkable observation, combined with the implication of miRNAs in immunomodulation and the crucial role of DCs in the anti-tumoural immune response, led us to hypothesize that pulmonary tumour growth is associated with active immune reprogramming via modulation of miRNA expression in DCs. We addressed this hypothesis in a preclinical model of lung cancer, in which lung tumours grow orthotopically in immunocompetent hosts. Our studies uncovered a tumour-induced immunomodulatory miRNA signature in the DC population responsible for presentation of lung tumour antigen in the pulmonary lymph nodes (LNs). Interfering with the intrinsic capacity of DCs to reprogram their miRNA repertoire significantly delayed tumour growth.
Material and Methods
day 28 to 32 post-transfer. The lung metastasis melanoma tumour model was induced by intravenous (i.v.) injection of 0.4 3 106 B16 cells and mice were examined 18 days after injection. Antigen-specific T-cell proliferation
To measure the presentation of tumour antigen in mediastinal LN in situ, 2 3 106 immunomagnetically purified (MACS beads, Miltenyi) and CFSE-labelled (10 mM) CD81 OT-I or CD41 OT-II T cells were administered i.v. into lung tumourbearing hosts 28 days after transfer of tumour cells. In vitro antigen-specific T cell proliferation was assessed by fluorescence-activated cell sorting (FACS Aria II, BD Biosciences) of MHCIIhighCD11c1CD32CD192 lymph node DCs (LNDCs), and co-culturing 10,000 purified LNDCs with 100,000 MACS-purified, CFSE-labelled (5 mM) OT-I or OTII T cells. In some experiments 10 mg/mL ovalbumin (OVA) was added to the cultures. In both in situ and in vitro protocols CFSE dilutions of OT-I or OT-II T cells were measured by flow cytometry after three or four days, respectively. The percentage of divided T cells was subsequently calculated using FlowJo’s Proliferation Tool (Treestar Inc.).
Mice and cell lines
C57BL/6 mice were obtained from Harlan. B6.CgDicer1tm1Bdh/J mice (The Jackson Laboratory) were intercrossed with Cd11c Cre mice13 to generate Dicerfl/flCd11cCre mice. OT-II and OT-I OVA-TCR transgenic mice14 and CCR7 knockout mice (The Jackson Laboratory) were bred at Ghent University (Ghent, Belgium). The Animal Ethics Committee of Ghent University approved all in vivo manipulations. The murine Lewis lung carcinoma (LLC-A) cell line (C57Bl/6 background) was kindly provided by Prof. M. Bracke (Ghent University, Belgium). LL2-Thy1.1-OVA cells expressing a truncated, cytoplasmic form of ovalbumin15 were a gift from Prof. D. Fearon (Cambridge University, UK). Murine melanoma cells (B16-MO4, C57Bl/6 background) were provided by Prof. D. Elewaut (Ghent University, Belgium). Transplantable tumour models
To generate orthotopically growing lung tumours, 1 3 106 LLC cells (C57BL/6-compatible) in sterile saline solution (0.9% NaCl) were instilled intratracheally (i.t.) in anesthetized C57BL/6 mice. Mice were routinely sacrificed and analysed at
Generation of bone marrow-derived dendritic cells and functional assays
Bone marrow-derived DCs (BMDCs) were generated as described earlier.16 To determine T cell priming capacities, 100,000 MACS-purified CD41 OT-II or CD81 OT-I cells were labelled with 5 mM CFSE (Invitrogen) and co-cultured with 10,000 Flt3L-DCs. CFSE profiles were determined after three days for OT-I and after four days for OT-II cells. Cytokines in the supernatants of DC cultures and co-cultures were measured using ELISA kits (eBioscience). MicroRNA profiling of FACSorted DCs
Total RNA, containing miRNA, was extracted from isolated DCs using the miRNeasy kit (Qiagen) according to manufacturer’s instructions. Quantity and quality checks were performed on a NanoDrop spectrophotometer (NanoDrop Technologies) and Experion automated electrophoresis system (RNA Quality Indicator, Bio-Rad). Total RNA was reverse-transcribed using two murine-specific Megaplex stemloop primer pools in combination with the miRNA reverse transcription kit (Applied Biosystems) according to our C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
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Ambion) were transfected using 1 lL RNAiMax lipofectamine (Invitrogen). Cy3-labeled control anti-miRTM miRNA inhibitor (Ambion) was used to determine transfection efficiency. After 24 h the medium was replaced and 6 to 24 h later, cells were harvested for flow cytometry, RNA isolation or co-culture with T cells, while the supernatant was collected for ELISA. Statistical analysis
For all experiments, the differences between the two groups were analysed using Student’s t-test. If data did not follow Gaussian distribution, non-parametric tests were used (Mann-Whitney U for unpaired data). Kaplan-Meier survival curves were analysed by the log-rank test. Differences were considered statistically significant if p value 0.05, Supporting Information Fig. E3). MiR-301a overexpression in DCs profoundly impairs antigen-specific Tc1 and Th1 polarization
The impact of miR-301a on DC biology is unknown, in contrast to the other significantly upregulated miRNA species we detected in DCs of tumour-bearing hosts. To dissect the role of miR-301a in DC-mediated immune responses, we used Flt3L-supplemented bone marrow cultures, generating conventional DC (cDC) subsets analogous to the cDCs found in C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
LN: CD24highCD11b2 DCs represent analogues of the migratory CD11b2 DCs of the lung and resident CD8a1 LNDCs, whereas CD24lowCD11bhigh DCs correspond to the CD11b1 migratory and resident cDCs in vivo.16,24 DCs were transiently transfected with miR-301a-specific miRNA mimics or inhibitors. DC subset-specific transfection efficiency was assessed using fluorescently labelled control oligomers (Fig. 4a). Baseline levels of miR-301a were very low in DCs, regardless of activation status (data not shown), and we could not detect suppression after antagomir transfection (Fig. 4b). A strong upregulation of miR-301a transcripts was confirmed by RT-qPCR after transfection with mimic molecules (Fig. 4b). Transfected DCs were pulsed with soluble OVA prior to co-culture with OT-I and OT-II cells. Overexpression of miR-301a in DCs led to a striking repression of IFN-g release from antigen-specific CD81 responders (Fig. 4c). Moreover, miR-301a-overexpressing DCs skewed the antigen-specific CD41 Th cytokine profile away from IFN-g-secretion, in favour of the induction of IL-13 and IL-17A-production (Fig. 4d). DC-driven CD41 and CD81 T cell proliferation was however not affected by miR-301a overexpression in DCs (Fig. 4e). As expected, transfection of miR-301a inhibitors in DCs did not bias the Th cytokine profile or proliferation (Figs. 4c–4e). To elucidate how miR-301a overexpression in DCs altered the quality of CD41 and CD81 T cell responses, we assessed DC co-stimulatory molecule expression and cytokine
Tumor Immunology
Figure 3. Differential expression of miRNAs in the pulmonary migr-LNDCs of lung tumour bearing mice. (a) Workflow of the miRNA expression profiling. (b) Heatmap of normalized RT-qPCR values showing mature miRNA transcripts differentially expressed in migr-LNDCs from tumour-draining nodes versus control mice or control LN. There are 11 replicates from four independent experiments (I, II, III, and IV), each replicate consisting of a pool of LN from 3 to 16 mice. Rows represent significantly modulated miRNAs (t-test, p < 0.05). Colours denote log2 fold expression levels. (c) Table showing fold changes and 95 % confidence intervals (CI) of the differentially expressed miRNAs relative to 2 different control categories (as in b).
Tumor Immunology
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Figure 4. Effect of miR-301a modulation in DCs on T cell priming. (a) DCs generated from Flt3L-supplemented BM cultures were transfected with Cy3-labeled scrambled miRNA constructs. Histograms show transfection efficiency in (CD11c1B2202)CD241 (left) and (CD11c1B2202SIRPa1)CD11b1 BMDCs (right) 24 h post-transfection, representative of five independent experiments. (b) RT-qPCR of miR301a expression relative to snoRNA202 in day 8 BMDCs transfected with miR-301a mimics versus inhibitors or control mimic versus inhibitor oligonucleotides. Additional controls include transfection with lipofectamine only (“mock”) or control medium (“no transf."). (c) IFN-g secretion by OT-I cells in response to miR-301a-modulated DCs. Log2 fold levels are shown relative to control DC transfection conditions. (d) Th polarization in response to miR-301a-modulated DCs. Skewing is expressed as log2 fold change of IL-13, IL-17A, and IL-10 protein levels normalized to responses evoked by control oligonucleotide-transfected DCs, and relative to IFN-g levels. (E) Proliferation of CFSElabelled OT-I or OT-II cells co-cultured with miR-301a-transfected DCs (filled histograms) or control miRNA-transfected DCs (unfilled histograms) pulsed overnight with 100 mg/mL OVA and 0.5 mg/mL CpG. Data are pooled (c,d) or representative (e) from three independent experiments, each with two biological replicates. Error bars represent the interquartile range (*p < 0.05).
production. We could not detect any significant modulation of MHCII or co-stimulatory molecule (CD40, CD86) surface expression in miR-301a mimic versus antagomir-treated DCs (data not shown). However, dramatic effects were seen at the level of polarizing cytokine output: overexpression of miR301a led to a profound suppression of DC-derived IL-12 activity, whether measured as bioactive IL-12p70 release in DC supernatant, intracellular IL-12p40 protein expression (with both cDC subsets affected), or IL-12p35 and IL-12p40 mRNA levels (Figs. 5a–5c). In addition, DC-derived IL-6 and TNF-a (and to a lesser extent IL-10) secretion was also suppressed by miR-301a (Fig. 5c). DC-intrinsic impairment in reshaping miRNA repertoires affects tumour outcome
Next we aimed to determine whether interfering with a shift in the LNDC microRNome might affect the host response to a tumour. Alteration of the miRNA repertoire in a given cell
can either arise from cell-intrinsic processes, or be the result of transfer/uptake of exogenous miRNAs e.g. via exosomes. We studied the contribution of the first hypothesis (and possible uptake of immature miRNAs), by generating mice in which DCs lack DICER, the RNAseIII enzyme involved in processing pre-miRNAs into mature miRNAs (Fig. 6a). Dicerfl/flCd11cCre1 mice were generated by intercrossing B6.Cg-Dicer1tm1Bdh/J and Cd11c Cre mice. Cre-mediated recombination led to a 122-fold decrease of Dicer mRNA expression in splenic DC and 18-fold decrease in BMDC (Fig. 6b). Remarkably, in the orthotopic lung cancer model cohorts of transgenic mice with DICER-insufficient DCs showed a significant survival advantage over control littermates (Fig. 6c). Also, in a different tumour model, i.e. haematogenous dissemination of B16 melanoma to the lungs, mice with DICER-insufficient DCs showed a significantly lower pulmonary metastatic tumour load (Fig. 6d).
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Finally, analysis of miR-301a expression within purified mediastinal lymph node DC subpopulations revealed strongly decreased levels of this immunosuppressive miRNA in Dicerfl/flCd11cCre1 compared to Cd11cCre2 lung tumourbearing hosts (Fig. 6e).
Discussion This is the first study revealing a lung tumour-induced imprinting on the microRNA repertoire of dendritic cells in vivo, with potential consequences on the quality of downstream anti-tumoural immune responses. We used an orthotopic model of lung cancer as a convenient preclinical tool to dissect the impact of lung tumours on
the pulmonary immune compartment, with a specific emphasis on DCs. Non-invasive inoculation of Lewis lung carcinoma cells (LLC) into the airways of C57Bl/6 mice led to the development of orthotopic lung tumors, appearing as solitary masses bearing strong histological similarity to adenosquamous nonsmall cell lung cancer in humans.25 We hypothesized that the general paradigm of DCs as initiators of immune responses against tumour antigens would apply in this model as well. We found the same MHCIIhighCD11c1 lymph node DC subpopulation (migr-DC), previously implicated in the acquisition and presentation of antigens from the airways,19 to be exclusively responsible for cross-priming T cell responses to lung tumour antigens as well. Our data are in line with a report showing DC-mediated cross-presentation of antigen from apoptotic cells deposited in the lung.26 Successful cross-presentation but inefficient initiation of CD41 T cell responses of tOVAderived antigens has been observed in other mouse tumour models as well.27,28 However, in vivo CD41 T cell responses against LL2 antigens other than tOVA are not excluded. The discrepancy we observed between intact in vivo T-cell proliferation in response to cross-presented tumour antigen, versus unopposed tumour growth suggested a failure in the induction of adequate effector T cell function. Particularly for CD81 T cells, the provision of a “third signal” (e.g. IL-12 or type I interferon) by DCs governs the acquisition of effective cytotoxic functions versus tolerance, irrespective of the capacity to induce proliferation.29 Tumour-exposed DCs may thus effectively present antigens but also convey immunomodulatory signals that could undermine the quality of T-cell effector responses. Rather than focusing on individual candidate factors, we hypothesized that this third signal emanates from a higher level of regulation, and looked for tumour-induced
Figure 5. Effect of miR-301a modulation in DCs on cytokine secretion by DCs. (a) IL-12p35 (left) and IL-12p40 (right) mRNA expression (normalized to Hprt) in miR-301a-modulated BMDCs stimulated for 6 h with 1 mg/mL CpG. Values pooled from two independent experiments each consisting of one to two biological replicates. (b) Intracellular staining for IL-12p40 in BMDCs stimulated for 6 h with 1 mg/ mL CpG, the last 4 h in the presence of 10 mg/mL Brefeldin A. Cytokine levels are shown for CD241 and CD11b1 miR-301a-modulated DC subsets. Filled histograms represent miR-301a-mimic or inhibitor-transfected BMDCs. Unfilled histograms show control miRNA-transfected BMDCs. Numbers indicate fraction of IL-12p40positive DCs as well as MFI. Data are representative of three independent experiments, with two biological replicates in each experiment. (c) Modulation of IL-12p70, IL-10, IL-6, and TNF-a secretion as measured in the supernatant of CpG-matured BMDCs transfected with miR-301a mimics or inhibitors. Median secretion levels (interquartile range) in DCs transfected with control mimics are 3732.0 pg/mL (3151.4-7764.3) IL-12p70, 157.4 pg/mL (108.8-258.5) IL-10, 3356.4 pg/mL (2783.1-5794.6) IL-6 and 3636.7 pg/mL (2660.25662.6) TNF-a. Data in graph are expressed as log2 fold change relative to the corresponding control miRNA-treated DCs (mimic or inhibitor). Data are pooled from three independent experiments, each with two biological replicates. Error bars represent the interquartile range (*p < 0.05).
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Tumor Immunology
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Tumor Immunology
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microRNA plasticity in lung cancer-associated DCs
Figure 6. Tumour growth in Dicerfl/flCd11cCre mice. (a) Left: Gene targeting strategy for the deletion of DICER activity in CD11c1 cells. LoxP sites flank exon 23, containing the catalytic domain. F and R designate the positions of forward and reverse primer for genomic PCR. Right: Genomic PCR for Dicer deletion in sorted CD11c1MHCII1 conventional splenic DC and GM-CSF-supplemented BMDC. (b) Dicer mRNA expression relative to Hprt as assessed by RT-qPCR in Dicer wild-type (WT/WT) and deleted (D/D) splenic DC (n 5 7) and BMDC (n 5 2–4). Error bars represent interquartile range (***p < 0.001). (c) Kaplan–Meier survival curves for WT/WT and D/D mice bearing LLC lung tumours. Representative of two independent experiments with similar results (n 5 9–15 per group). (d) Quantification of lung metastatic tumour load after i.v. injection of B16 melanoma cells in Dicer WT/WT vs. D/D mice, taking into account both nodule size and number (n 5 12–13 mice per group). Error bars represent SEM (*p < 0.05). (E) RT-qPCR of miR-301a expression relative to snoRNA202 in the migratory DC population from lung tumour-bearing Dicer WT/WT vs. D/D mice. Each replicate is the result of pooling LN from three mice.
disturbances in the miRNA repertoire of DCs. A key feature of miRNAs is their capacity to broadly regulate multiple components in a given cellular response pathway, or across different pathways. We developed a workflow allowing us to detect changes in the microRNome of highly purified migratory DC populations from lung tumour draining mediastinal LNs. Intriguingly, the six miRNAs emerging as significanty regulated in tumour-exposed DCs have also been described as oncomirs in recent reports. Moreover, the direction of change of these miRNAs in DCs follows the pattern observed in several tumours. MiR-301 is oncogenic in breast CA,30 while miR-21 is associated with poor prognosis in non-small-cell lung carcinoma.31 Moreover, miR-21-overexpressing transgenic mice have enhanced lung tumourigenesis.32 MiR-222 is oncogenic as well in many cancer types,33 while much less is known about miR28 in this setting. MiR-146a has been described as a tumour suppressor and miR-30a-3p was shown to be downregulated in colorectal34 and bladder cancer.35 However, as already pointed out, these studies are typically performed on biopsies, precluding any conclusions on the source of the observed miRNA disturbances (i.e. cancer cells vs. stroma, including immune infiltrates). Indeed, each of the miRNAs in the signature has been described in the setting of immunomodulation as well, with some of them specifically implicated in DC biology. For miR-301a, a detrimental role in anti-tumoural immune
responses can be inferred. It was shown to favour Th17 differentiation of naive CD41 T cells.36 In addition, miR-301a can augment signal transducer and activator of transcription 3 (STAT3) activity by targeting its negative regulator, PIAS3 (protein inhibitor of activated STAT3).36 STAT3 signalling drives an immunosuppressive cascade that propagates from tumour cells to dendritic cells, involving inhibition of DC maturation, which includes repressed IL-12 production.37 In this report, we describe for the first time the direct impact of increased miR-301a levels in DCs and observed a potent suppression of IL-12 secretion and impaired IFN-g secretion by DC-primed CD81 T cells. Also, miR-301a-overexpressing DCs skewed the CD41 T cell cytokine profile away from IFN-g, towards IL-13 and IL-17A-secreting cells. MiR-21 has been reported as a suppressor of the IL-12/IFN-g axis as well, directly by targeting IL12p35 in DCs,38 or indirectly by targeting the NF-jB activator PDCD4, resulting in an increase in the immunosuppressive IL-10.39 However, our experiments with miR-21 overexpression only showed a trend in line with previously reported effects, with miR-301a effects dominating those of miR-21 when both microRNAs were overexpressed in DCs (data not shown). MiR-28 modulation had no impact on costimulatory molecule expression or cytokine output of DCs (data not shown), and we did not investigate the impact of miR-222 as it was already shown to promote the development of C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
conventional over plasmacytoid DCs from c-kit1 bone marrow progenitors.40 Among the significantly downregulated miRNAs in DCs, miR-146a has already been described as a known counterregulator of NF-jB activity by directly targeting IRAK1 and TRAF6. Interestingly, successful phenotypical and functional DC maturation has been shown to be associated with a high miR-146a/low miR-21 signature.41 The reverse pattern we observed in the pulmonary lymph node DCs of lung tumourbearing hosts further suggests impairment in DC-driven immune responses. A recent report described modulation of miRNA expression in DCs exposed to mouse melanoma and breast cancer cell lines, however the observed miRNA signature was only examined in terms of effects on DC survival.42 In contrast, the miRNA species we found to be specifically modulated in LNDCs from lung tumour-bearing hosts would act in concert to impair the induction of a Th1/Tc1-polarized response, which in itself is a fundamental prerequisite for effective tumour control.3 Future studies should address whether in vivo interference with miR-301a and/or miR-21 induction in DCs could correct the anti-tumoural immune response and eventually affect host survival. In this study, we aimed to determine whether interfering with the global capacity of DCs to remodel their microRNome would impact on the host response to a lung tumour. Alteration of the miRNA repertoire in a given cell can either arise from cell-intrinsic processes, or be the result of transfer/uptake of exogenous miRNAs e.g. via exosomes. We evaluated the contribution of the first mechanism, by generating mice in which DCs lack DICER, the RNAseIII enzyme involved in processing pre-miRNAs into mature miRNAs, and observed a significantly delayed lung tumour development. This is the first report showing that miRNA processing in the immune compartment, and in DCs in particular, affects tumour development, both in a model of primary lung cancer as well as in a pulmonary metastasis model. This is in contrast to studies indicating that cancer cell-intrinsic impairment in miRNA processing enhances lung tumourigenesis (presumably by decreasing the pool of tumour-suppressor miRNAs).43 While we can clearly demonstrate impaired upregulation of miR-301a in tumour-draining lymph node DCs of Dicerfl/flCd11cCre1 hosts, our preliminary data on DC biology and downstream immune functions do not yet point to a clear-cut mechanistic basis for the survival advantage in these transgenic animals. Our use of the CD11c-Cre transgene raises the question of possible Cre-activity (and hence Dicer excision) in other immune cells of these mice. However it has already been extensively verified in a previous study that biologically rele-
vant Cre activity is overwhelmingly detected in CD11chigh cells after DC lineage commitment, with
Lung tumours reprogram pulmonary dendritic cell immunogenicity at the microRNA level Lotte Pyfferoen1,2,3, Pieter Mestdagh4, Karl Vergote2, Nancy De Cabooter1, Jo Vandesompele4, Bart N. Lambrecht2,3,5 and Karim Y. Vermaelen1 1
Department of Respiratory Medicine, Tumor Immunology Laboratory, Ghent University Hospital, Ghent, Belgium VIB Inflammation Research Center, Ghent, Belgium 3 Department of Respiratory Medicine, Ghent University, Ghent, Belgium 4 Center for Medical Genetics, Ghent University, Ghent, Belgium 5 Department of Pulmonary Medicine, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands 2
The host response against tumors is characterized by a complex interplay between transformed cells and the stromal compartment, the latter including various immune cell types. DCs appear involved in anti-tumoural immunity in several preclinical models.1,2 In this process, DCs are thought to
Key words: dendritic cells, lung cancer, microRNAs, Dicer Abbreviations: BMDC: bone marrow-derived DC; DC: dendritic cell; ILN: inguinal LN; i.v.: intravenous; i.t.: intratracheal; LLC: Lewis lung carcinoma; LN: lymph node; LNDC: lymph node dendritic cell; migr-DC: migratory DC; miR(NA): microRNA; MLN: mediastinal LN; resid-DC: resident DC; RT-qPCR: reverse transcription quantitative PCR; Tc1: T cytotoxic 1; TDLN: tumourdraining LN; Th: T helper; (t)OVA: (truncated) ovalbumin; Treg: regulatory T cell Additional Supporting Information may be found in the online version of this article. Grant sponsors: FWO Research Project G.A015.11, IWT and IUAP grant P7/03; Grant number: SB/083359; Grant sponsor: FWO Senior Clinical Investigator Award DOI: 10.1002/ijc.28945 History: Received 26 Nov 2013; Accepted 15 Apr 2014; Online 2 May 2014 Correspondence to: Karim Y. Vermaelen, Ghent University Hospital, 7K12IE, De Pintelaan 185, 9000 Ghent, Belgium, Tel.: 13293322815, E-mail: [email protected]
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transport tumour-derived antigens to draining lymph nodes (LN), where anti-tumoural adaptive immunity is primed, more specifically cytotoxic CD81 T cell responses (Tc1) and CD41 T helper (Th) responses polarized towards IFN-g secreting Th1 cells, whose concerted action can lead to tumour cell eradication.3 However, cancerous lesions develop several strategies to resist immune defences and reprogram immune cells to directly or indirectly promote tumour growth and spread. Evading immune attack is now acknowledged as one of the “emerging hallmarks” of cancer.4 The impact of research into these mechanisms is highlighted by the recent emergence of compounds targeting immune checkpoint receptors, showing remarkable clinical benefit in several solid tumours including lung cancer.5 The immunosuppressive tumour microenvironment can interfere with DC maturation and function, generating both a regulatory T cell (Treg) and a Th2 profile, which are known for their immune suppressing and tumour-promoting capacities via secretion of IL-10, TGF-b, and IL-4, IL-13.6–8 Besides, a tumour-promoting effect for Th17-polarized CD41 T cells has been demonstrated in preclinical studies.9,10 Reprogramming of the immune response involves rapid coordinated changes at the level of the transcriptome and/or proteome. A novel layer of cell regulation consists of microRNAs (miRNAs), a family of small noncoding RNAs individually capable of regulating multiple biological pathways, including those involved in cancer and immune responses.
Tumor Immunology
Lung cancer arises in a context of tumour-induced immune suppression. Dendritic cells (DCs) are central players in the induction of anti-tumoural immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. Meanwhile, microRNAs are associated with tumour development as well as immune regulation. We postulated that lung tumours escape immune control by reprogramming DC immunogenicity at the microRNA level. Using an orthotopic model of lung cancer, we first identified the DC population responsible for transport and cross-presentation of lung tumour-derived antigens to na€ıve T cells in the draining mediastinal lymph nodes (LNs). Profiling the full microRNA repertoire of these DCs revealed a restricted set of microRNAs that was consistently dysregulated in the presence of lung tumours, with miR-301a as one of the top upregulated transcripts. Overexpression of miR-301a in DCs suppressed IL-12 secretion, decreased IFN-c release from antigenspecific cytotoxic T cells, and shifted antigen-specific T helper cytokine profile away from IFN-c towards IL-13 and IL-17Asecreting T cells. Strikingly, DC-selective Dicer1 gene deletion resulted in delayed lung tumour growth and a survival benefit. Taken together, our data reveal that lung tumours induce an immunosuppressive microRNA signature in pulmonary DCs. Interfering with the DC-intrinsic capacity to remodel microRNA repertoires affects lung tumour outcome.
2
microRNA plasticity in lung cancer-associated DCs
Tumor Immunology
What’s new? MicroRNAs (miRNAs) regulate many biological pathways, including those involved in tumor development and immune regulation. Dendritic cells (DCs) are central players in antitumoral immunity, providing critical signals that drive the induction of cytotoxic T-cell responses. In this study, the authors found that a group of immunomodulatory miRNAs produced by DCs is consistently dysregulated in the presence of lung tumors. When miRNA biogenesis in these DCs was blocked, tumor growth was significantly delayed in vivo. These results support the hypothesis that lung tumors escape immune control by reprogramming miRNA expression in DCs.
MiRNA profiling studies on tumour samples are generally performed on bulk tumour samples, with expression patterns interpreted as arising from cancer cells.11 However, several studies suggest that the dominant miRNA signatures at different stages of tumourigenesis can be attributed to infiltrating leukocytes rather than arising from the transformed cells themselves.12 This remarkable observation, combined with the implication of miRNAs in immunomodulation and the crucial role of DCs in the anti-tumoural immune response, led us to hypothesize that pulmonary tumour growth is associated with active immune reprogramming via modulation of miRNA expression in DCs. We addressed this hypothesis in a preclinical model of lung cancer, in which lung tumours grow orthotopically in immunocompetent hosts. Our studies uncovered a tumour-induced immunomodulatory miRNA signature in the DC population responsible for presentation of lung tumour antigen in the pulmonary lymph nodes (LNs). Interfering with the intrinsic capacity of DCs to reprogram their miRNA repertoire significantly delayed tumour growth.
Material and Methods
day 28 to 32 post-transfer. The lung metastasis melanoma tumour model was induced by intravenous (i.v.) injection of 0.4 3 106 B16 cells and mice were examined 18 days after injection. Antigen-specific T-cell proliferation
To measure the presentation of tumour antigen in mediastinal LN in situ, 2 3 106 immunomagnetically purified (MACS beads, Miltenyi) and CFSE-labelled (10 mM) CD81 OT-I or CD41 OT-II T cells were administered i.v. into lung tumourbearing hosts 28 days after transfer of tumour cells. In vitro antigen-specific T cell proliferation was assessed by fluorescence-activated cell sorting (FACS Aria II, BD Biosciences) of MHCIIhighCD11c1CD32CD192 lymph node DCs (LNDCs), and co-culturing 10,000 purified LNDCs with 100,000 MACS-purified, CFSE-labelled (5 mM) OT-I or OTII T cells. In some experiments 10 mg/mL ovalbumin (OVA) was added to the cultures. In both in situ and in vitro protocols CFSE dilutions of OT-I or OT-II T cells were measured by flow cytometry after three or four days, respectively. The percentage of divided T cells was subsequently calculated using FlowJo’s Proliferation Tool (Treestar Inc.).
Mice and cell lines
C57BL/6 mice were obtained from Harlan. B6.CgDicer1tm1Bdh/J mice (The Jackson Laboratory) were intercrossed with Cd11c Cre mice13 to generate Dicerfl/flCd11cCre mice. OT-II and OT-I OVA-TCR transgenic mice14 and CCR7 knockout mice (The Jackson Laboratory) were bred at Ghent University (Ghent, Belgium). The Animal Ethics Committee of Ghent University approved all in vivo manipulations. The murine Lewis lung carcinoma (LLC-A) cell line (C57Bl/6 background) was kindly provided by Prof. M. Bracke (Ghent University, Belgium). LL2-Thy1.1-OVA cells expressing a truncated, cytoplasmic form of ovalbumin15 were a gift from Prof. D. Fearon (Cambridge University, UK). Murine melanoma cells (B16-MO4, C57Bl/6 background) were provided by Prof. D. Elewaut (Ghent University, Belgium). Transplantable tumour models
To generate orthotopically growing lung tumours, 1 3 106 LLC cells (C57BL/6-compatible) in sterile saline solution (0.9% NaCl) were instilled intratracheally (i.t.) in anesthetized C57BL/6 mice. Mice were routinely sacrificed and analysed at
Generation of bone marrow-derived dendritic cells and functional assays
Bone marrow-derived DCs (BMDCs) were generated as described earlier.16 To determine T cell priming capacities, 100,000 MACS-purified CD41 OT-II or CD81 OT-I cells were labelled with 5 mM CFSE (Invitrogen) and co-cultured with 10,000 Flt3L-DCs. CFSE profiles were determined after three days for OT-I and after four days for OT-II cells. Cytokines in the supernatants of DC cultures and co-cultures were measured using ELISA kits (eBioscience). MicroRNA profiling of FACSorted DCs
Total RNA, containing miRNA, was extracted from isolated DCs using the miRNeasy kit (Qiagen) according to manufacturer’s instructions. Quantity and quality checks were performed on a NanoDrop spectrophotometer (NanoDrop Technologies) and Experion automated electrophoresis system (RNA Quality Indicator, Bio-Rad). Total RNA was reverse-transcribed using two murine-specific Megaplex stemloop primer pools in combination with the miRNA reverse transcription kit (Applied Biosystems) according to our C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
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Ambion) were transfected using 1 lL RNAiMax lipofectamine (Invitrogen). Cy3-labeled control anti-miRTM miRNA inhibitor (Ambion) was used to determine transfection efficiency. After 24 h the medium was replaced and 6 to 24 h later, cells were harvested for flow cytometry, RNA isolation or co-culture with T cells, while the supernatant was collected for ELISA. Statistical analysis
For all experiments, the differences between the two groups were analysed using Student’s t-test. If data did not follow Gaussian distribution, non-parametric tests were used (Mann-Whitney U for unpaired data). Kaplan-Meier survival curves were analysed by the log-rank test. Differences were considered statistically significant if p value 0.05, Supporting Information Fig. E3). MiR-301a overexpression in DCs profoundly impairs antigen-specific Tc1 and Th1 polarization
The impact of miR-301a on DC biology is unknown, in contrast to the other significantly upregulated miRNA species we detected in DCs of tumour-bearing hosts. To dissect the role of miR-301a in DC-mediated immune responses, we used Flt3L-supplemented bone marrow cultures, generating conventional DC (cDC) subsets analogous to the cDCs found in C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
LN: CD24highCD11b2 DCs represent analogues of the migratory CD11b2 DCs of the lung and resident CD8a1 LNDCs, whereas CD24lowCD11bhigh DCs correspond to the CD11b1 migratory and resident cDCs in vivo.16,24 DCs were transiently transfected with miR-301a-specific miRNA mimics or inhibitors. DC subset-specific transfection efficiency was assessed using fluorescently labelled control oligomers (Fig. 4a). Baseline levels of miR-301a were very low in DCs, regardless of activation status (data not shown), and we could not detect suppression after antagomir transfection (Fig. 4b). A strong upregulation of miR-301a transcripts was confirmed by RT-qPCR after transfection with mimic molecules (Fig. 4b). Transfected DCs were pulsed with soluble OVA prior to co-culture with OT-I and OT-II cells. Overexpression of miR-301a in DCs led to a striking repression of IFN-g release from antigen-specific CD81 responders (Fig. 4c). Moreover, miR-301a-overexpressing DCs skewed the antigen-specific CD41 Th cytokine profile away from IFN-g-secretion, in favour of the induction of IL-13 and IL-17A-production (Fig. 4d). DC-driven CD41 and CD81 T cell proliferation was however not affected by miR-301a overexpression in DCs (Fig. 4e). As expected, transfection of miR-301a inhibitors in DCs did not bias the Th cytokine profile or proliferation (Figs. 4c–4e). To elucidate how miR-301a overexpression in DCs altered the quality of CD41 and CD81 T cell responses, we assessed DC co-stimulatory molecule expression and cytokine
Tumor Immunology
Figure 3. Differential expression of miRNAs in the pulmonary migr-LNDCs of lung tumour bearing mice. (a) Workflow of the miRNA expression profiling. (b) Heatmap of normalized RT-qPCR values showing mature miRNA transcripts differentially expressed in migr-LNDCs from tumour-draining nodes versus control mice or control LN. There are 11 replicates from four independent experiments (I, II, III, and IV), each replicate consisting of a pool of LN from 3 to 16 mice. Rows represent significantly modulated miRNAs (t-test, p < 0.05). Colours denote log2 fold expression levels. (c) Table showing fold changes and 95 % confidence intervals (CI) of the differentially expressed miRNAs relative to 2 different control categories (as in b).
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Figure 4. Effect of miR-301a modulation in DCs on T cell priming. (a) DCs generated from Flt3L-supplemented BM cultures were transfected with Cy3-labeled scrambled miRNA constructs. Histograms show transfection efficiency in (CD11c1B2202)CD241 (left) and (CD11c1B2202SIRPa1)CD11b1 BMDCs (right) 24 h post-transfection, representative of five independent experiments. (b) RT-qPCR of miR301a expression relative to snoRNA202 in day 8 BMDCs transfected with miR-301a mimics versus inhibitors or control mimic versus inhibitor oligonucleotides. Additional controls include transfection with lipofectamine only (“mock”) or control medium (“no transf."). (c) IFN-g secretion by OT-I cells in response to miR-301a-modulated DCs. Log2 fold levels are shown relative to control DC transfection conditions. (d) Th polarization in response to miR-301a-modulated DCs. Skewing is expressed as log2 fold change of IL-13, IL-17A, and IL-10 protein levels normalized to responses evoked by control oligonucleotide-transfected DCs, and relative to IFN-g levels. (E) Proliferation of CFSElabelled OT-I or OT-II cells co-cultured with miR-301a-transfected DCs (filled histograms) or control miRNA-transfected DCs (unfilled histograms) pulsed overnight with 100 mg/mL OVA and 0.5 mg/mL CpG. Data are pooled (c,d) or representative (e) from three independent experiments, each with two biological replicates. Error bars represent the interquartile range (*p < 0.05).
production. We could not detect any significant modulation of MHCII or co-stimulatory molecule (CD40, CD86) surface expression in miR-301a mimic versus antagomir-treated DCs (data not shown). However, dramatic effects were seen at the level of polarizing cytokine output: overexpression of miR301a led to a profound suppression of DC-derived IL-12 activity, whether measured as bioactive IL-12p70 release in DC supernatant, intracellular IL-12p40 protein expression (with both cDC subsets affected), or IL-12p35 and IL-12p40 mRNA levels (Figs. 5a–5c). In addition, DC-derived IL-6 and TNF-a (and to a lesser extent IL-10) secretion was also suppressed by miR-301a (Fig. 5c). DC-intrinsic impairment in reshaping miRNA repertoires affects tumour outcome
Next we aimed to determine whether interfering with a shift in the LNDC microRNome might affect the host response to a tumour. Alteration of the miRNA repertoire in a given cell
can either arise from cell-intrinsic processes, or be the result of transfer/uptake of exogenous miRNAs e.g. via exosomes. We studied the contribution of the first hypothesis (and possible uptake of immature miRNAs), by generating mice in which DCs lack DICER, the RNAseIII enzyme involved in processing pre-miRNAs into mature miRNAs (Fig. 6a). Dicerfl/flCd11cCre1 mice were generated by intercrossing B6.Cg-Dicer1tm1Bdh/J and Cd11c Cre mice. Cre-mediated recombination led to a 122-fold decrease of Dicer mRNA expression in splenic DC and 18-fold decrease in BMDC (Fig. 6b). Remarkably, in the orthotopic lung cancer model cohorts of transgenic mice with DICER-insufficient DCs showed a significant survival advantage over control littermates (Fig. 6c). Also, in a different tumour model, i.e. haematogenous dissemination of B16 melanoma to the lungs, mice with DICER-insufficient DCs showed a significantly lower pulmonary metastatic tumour load (Fig. 6d).
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Finally, analysis of miR-301a expression within purified mediastinal lymph node DC subpopulations revealed strongly decreased levels of this immunosuppressive miRNA in Dicerfl/flCd11cCre1 compared to Cd11cCre2 lung tumourbearing hosts (Fig. 6e).
Discussion This is the first study revealing a lung tumour-induced imprinting on the microRNA repertoire of dendritic cells in vivo, with potential consequences on the quality of downstream anti-tumoural immune responses. We used an orthotopic model of lung cancer as a convenient preclinical tool to dissect the impact of lung tumours on
the pulmonary immune compartment, with a specific emphasis on DCs. Non-invasive inoculation of Lewis lung carcinoma cells (LLC) into the airways of C57Bl/6 mice led to the development of orthotopic lung tumors, appearing as solitary masses bearing strong histological similarity to adenosquamous nonsmall cell lung cancer in humans.25 We hypothesized that the general paradigm of DCs as initiators of immune responses against tumour antigens would apply in this model as well. We found the same MHCIIhighCD11c1 lymph node DC subpopulation (migr-DC), previously implicated in the acquisition and presentation of antigens from the airways,19 to be exclusively responsible for cross-priming T cell responses to lung tumour antigens as well. Our data are in line with a report showing DC-mediated cross-presentation of antigen from apoptotic cells deposited in the lung.26 Successful cross-presentation but inefficient initiation of CD41 T cell responses of tOVAderived antigens has been observed in other mouse tumour models as well.27,28 However, in vivo CD41 T cell responses against LL2 antigens other than tOVA are not excluded. The discrepancy we observed between intact in vivo T-cell proliferation in response to cross-presented tumour antigen, versus unopposed tumour growth suggested a failure in the induction of adequate effector T cell function. Particularly for CD81 T cells, the provision of a “third signal” (e.g. IL-12 or type I interferon) by DCs governs the acquisition of effective cytotoxic functions versus tolerance, irrespective of the capacity to induce proliferation.29 Tumour-exposed DCs may thus effectively present antigens but also convey immunomodulatory signals that could undermine the quality of T-cell effector responses. Rather than focusing on individual candidate factors, we hypothesized that this third signal emanates from a higher level of regulation, and looked for tumour-induced
Figure 5. Effect of miR-301a modulation in DCs on cytokine secretion by DCs. (a) IL-12p35 (left) and IL-12p40 (right) mRNA expression (normalized to Hprt) in miR-301a-modulated BMDCs stimulated for 6 h with 1 mg/mL CpG. Values pooled from two independent experiments each consisting of one to two biological replicates. (b) Intracellular staining for IL-12p40 in BMDCs stimulated for 6 h with 1 mg/ mL CpG, the last 4 h in the presence of 10 mg/mL Brefeldin A. Cytokine levels are shown for CD241 and CD11b1 miR-301a-modulated DC subsets. Filled histograms represent miR-301a-mimic or inhibitor-transfected BMDCs. Unfilled histograms show control miRNA-transfected BMDCs. Numbers indicate fraction of IL-12p40positive DCs as well as MFI. Data are representative of three independent experiments, with two biological replicates in each experiment. (c) Modulation of IL-12p70, IL-10, IL-6, and TNF-a secretion as measured in the supernatant of CpG-matured BMDCs transfected with miR-301a mimics or inhibitors. Median secretion levels (interquartile range) in DCs transfected with control mimics are 3732.0 pg/mL (3151.4-7764.3) IL-12p70, 157.4 pg/mL (108.8-258.5) IL-10, 3356.4 pg/mL (2783.1-5794.6) IL-6 and 3636.7 pg/mL (2660.25662.6) TNF-a. Data in graph are expressed as log2 fold change relative to the corresponding control miRNA-treated DCs (mimic or inhibitor). Data are pooled from three independent experiments, each with two biological replicates. Error bars represent the interquartile range (*p < 0.05).
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Figure 6. Tumour growth in Dicerfl/flCd11cCre mice. (a) Left: Gene targeting strategy for the deletion of DICER activity in CD11c1 cells. LoxP sites flank exon 23, containing the catalytic domain. F and R designate the positions of forward and reverse primer for genomic PCR. Right: Genomic PCR for Dicer deletion in sorted CD11c1MHCII1 conventional splenic DC and GM-CSF-supplemented BMDC. (b) Dicer mRNA expression relative to Hprt as assessed by RT-qPCR in Dicer wild-type (WT/WT) and deleted (D/D) splenic DC (n 5 7) and BMDC (n 5 2–4). Error bars represent interquartile range (***p < 0.001). (c) Kaplan–Meier survival curves for WT/WT and D/D mice bearing LLC lung tumours. Representative of two independent experiments with similar results (n 5 9–15 per group). (d) Quantification of lung metastatic tumour load after i.v. injection of B16 melanoma cells in Dicer WT/WT vs. D/D mice, taking into account both nodule size and number (n 5 12–13 mice per group). Error bars represent SEM (*p < 0.05). (E) RT-qPCR of miR-301a expression relative to snoRNA202 in the migratory DC population from lung tumour-bearing Dicer WT/WT vs. D/D mice. Each replicate is the result of pooling LN from three mice.
disturbances in the miRNA repertoire of DCs. A key feature of miRNAs is their capacity to broadly regulate multiple components in a given cellular response pathway, or across different pathways. We developed a workflow allowing us to detect changes in the microRNome of highly purified migratory DC populations from lung tumour draining mediastinal LNs. Intriguingly, the six miRNAs emerging as significanty regulated in tumour-exposed DCs have also been described as oncomirs in recent reports. Moreover, the direction of change of these miRNAs in DCs follows the pattern observed in several tumours. MiR-301 is oncogenic in breast CA,30 while miR-21 is associated with poor prognosis in non-small-cell lung carcinoma.31 Moreover, miR-21-overexpressing transgenic mice have enhanced lung tumourigenesis.32 MiR-222 is oncogenic as well in many cancer types,33 while much less is known about miR28 in this setting. MiR-146a has been described as a tumour suppressor and miR-30a-3p was shown to be downregulated in colorectal34 and bladder cancer.35 However, as already pointed out, these studies are typically performed on biopsies, precluding any conclusions on the source of the observed miRNA disturbances (i.e. cancer cells vs. stroma, including immune infiltrates). Indeed, each of the miRNAs in the signature has been described in the setting of immunomodulation as well, with some of them specifically implicated in DC biology. For miR-301a, a detrimental role in anti-tumoural immune
responses can be inferred. It was shown to favour Th17 differentiation of naive CD41 T cells.36 In addition, miR-301a can augment signal transducer and activator of transcription 3 (STAT3) activity by targeting its negative regulator, PIAS3 (protein inhibitor of activated STAT3).36 STAT3 signalling drives an immunosuppressive cascade that propagates from tumour cells to dendritic cells, involving inhibition of DC maturation, which includes repressed IL-12 production.37 In this report, we describe for the first time the direct impact of increased miR-301a levels in DCs and observed a potent suppression of IL-12 secretion and impaired IFN-g secretion by DC-primed CD81 T cells. Also, miR-301a-overexpressing DCs skewed the CD41 T cell cytokine profile away from IFN-g, towards IL-13 and IL-17A-secreting cells. MiR-21 has been reported as a suppressor of the IL-12/IFN-g axis as well, directly by targeting IL12p35 in DCs,38 or indirectly by targeting the NF-jB activator PDCD4, resulting in an increase in the immunosuppressive IL-10.39 However, our experiments with miR-21 overexpression only showed a trend in line with previously reported effects, with miR-301a effects dominating those of miR-21 when both microRNAs were overexpressed in DCs (data not shown). MiR-28 modulation had no impact on costimulatory molecule expression or cytokine output of DCs (data not shown), and we did not investigate the impact of miR-222 as it was already shown to promote the development of C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
conventional over plasmacytoid DCs from c-kit1 bone marrow progenitors.40 Among the significantly downregulated miRNAs in DCs, miR-146a has already been described as a known counterregulator of NF-jB activity by directly targeting IRAK1 and TRAF6. Interestingly, successful phenotypical and functional DC maturation has been shown to be associated with a high miR-146a/low miR-21 signature.41 The reverse pattern we observed in the pulmonary lymph node DCs of lung tumourbearing hosts further suggests impairment in DC-driven immune responses. A recent report described modulation of miRNA expression in DCs exposed to mouse melanoma and breast cancer cell lines, however the observed miRNA signature was only examined in terms of effects on DC survival.42 In contrast, the miRNA species we found to be specifically modulated in LNDCs from lung tumour-bearing hosts would act in concert to impair the induction of a Th1/Tc1-polarized response, which in itself is a fundamental prerequisite for effective tumour control.3 Future studies should address whether in vivo interference with miR-301a and/or miR-21 induction in DCs could correct the anti-tumoural immune response and eventually affect host survival. In this study, we aimed to determine whether interfering with the global capacity of DCs to remodel their microRNome would impact on the host response to a lung tumour. Alteration of the miRNA repertoire in a given cell can either arise from cell-intrinsic processes, or be the result of transfer/uptake of exogenous miRNAs e.g. via exosomes. We evaluated the contribution of the first mechanism, by generating mice in which DCs lack DICER, the RNAseIII enzyme involved in processing pre-miRNAs into mature miRNAs, and observed a significantly delayed lung tumour development. This is the first report showing that miRNA processing in the immune compartment, and in DCs in particular, affects tumour development, both in a model of primary lung cancer as well as in a pulmonary metastasis model. This is in contrast to studies indicating that cancer cell-intrinsic impairment in miRNA processing enhances lung tumourigenesis (presumably by decreasing the pool of tumour-suppressor miRNAs).43 While we can clearly demonstrate impaired upregulation of miR-301a in tumour-draining lymph node DCs of Dicerfl/flCd11cCre1 hosts, our preliminary data on DC biology and downstream immune functions do not yet point to a clear-cut mechanistic basis for the survival advantage in these transgenic animals. Our use of the CD11c-Cre transgene raises the question of possible Cre-activity (and hence Dicer excision) in other immune cells of these mice. However it has already been extensively verified in a previous study that biologically rele-
vant Cre activity is overwhelmingly detected in CD11chigh cells after DC lineage commitment, with
