Dendritic cells are transformed to become immunosuppressive during ovarian cancer progression.

The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression.Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1-ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5 -/-littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor-associated DCs. In ovarian carcinoma-bearing mice, this induced T cell-mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.

Boosting therapeutically relevant immunity against lethal epithelial tumors may require targeting tumor-induced immunosuppression on an individualized basis. Here, we show that, in the ovarian carcinoma microenvironment, CD11c + MHC-II + dendritic cells spontaneously engulf tumor materials but, rather than enhancing antitumor immunity, suppress T-cell function. In situ costimulation of CD40 and Toll-like receptor (TLR) 3 on tumor-infiltrating dendritic cells decreased their L-arginase activity, enhanced their production of type I IFN and interleukin-12 (p70), augmented their capacity to process antigens, and up-regulated costimulatory molecules in vivo in mice and in vitro in human dissociated tumors. Synergistic CD40/TLR activation also induced the migration of activated dendritic cells to lymphatic locations and promoted their capacity to present antigens. Correspondingly, without exogenous antigen, combined CD40/TLR agonists boosted measurable T-cell-mediated antitumor immunity and induced the rejection of otherwise lethal i.p. ovarian carcinomas. Our results highlight the potential of transforming tumor-infiltrating dendritic cells (the most abundant leukocyte subset in the solid ovarian carcinoma microenvironment) from an immunosuppressive to an immunostimulatory cell type. Combined administration of synergistic CD40 and TLR3 agonists could enhance their individual therapeutic effects against ovarian and other lethal epithelial cancers.

Dendritic cells (DC) and cytokines that expand myeloid progenitors are widely used to treat cancer. Here, we show that CD11c + DEC205 + DCs coexpressing A-smooth muscle actin and VE-cadherin home to perivascular areas in the ovarian cancer microenvironment and are required for the maintenance of tumor vasculature. Consequently, depletion of DCs in mice bearing established ovarian cancer by targeting different specific markers significantly delays tumor growth and enhances the effect of standard chemotherapies. Tumor growth restriction was associated with vascular apoptosis after DC ablation followed by necrosis, which triggered an antitumor immunogenic boost. Our findings provide a mechanistic rationale for selectively eliminating tumorassociated leukocytes to promote antitumor immunity while impeding tumor vascularization and to develop more effective DC vaccines based on a better understanding of the tumor microenvironment. [Cancer Res 2008;68(18):7684-91]

Modulating the activity of microRNAs (miRNAs) provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DCs) to selectively supplement the immunomostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RISC. Endogenous processing of synthetic miR-155 favored Ago2- and, to a lesser extent, Ago4-loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent anti-tumor responses that abrogated the progression of established ovarian cancers. Our results demonstrate both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using non-viral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design and a mechanistic rationale for the clinical testing of non-viral miRNA mimetics.