Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity
Juan R. Cubillos-Ruiz, … , Ross Kedl, Jose R. Conejo-Garcia
Juan R. Cubillos-Ruiz, … , Ross Kedl, Jose R. Conejo-Garcia
Published July 13, 2009
Citation Information: J Clin Invest. 2009;119(8):2231-2244. https://doi.org/10.1172/JCI37716.
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Research Article Oncology

Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity

Abstract

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.

Authors

Juan R. Cubillos-Ruiz, Xavier Engle, Uciane K. Scarlett, Diana Martinez, Amorette Barber, Raul Elgueta, Li Wang, Yolanda Nesbeth, Yvon Durant, Andrew T. Gewirtz, Charles L. Sentman, Ross Kedl, Jose R. Conejo-Garcia

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Figure 4

siRNA-PEI nanoparticles stimulate multiple TLRs in vitro and in vivo.

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siRNA-PEI nanoparticles stimulate multiple TLRs in vitro and in vivo. (A...
(A) siRNA-PEI nanocomplexes dose-dependently stimulated TLR3, TLR5, and TLR7 in vitro. Cotransfected HEK293 cells were stimulated with increasing amounts of siRNA-PEI nanoparticles or positive control agonists (+) as described in Methods. Data are representative of 4 independent experiments. (B) PEI was sufficient to stimulate TLR5 in vitro in a dose-dependent manner. HEK293 cells expressing TLR5 and harboring an NF-κB–dependent luciferase reporter plasmid were stimulated with increasing amounts of PEI, proteinase K (Prot K), or PEI treated overnight at 55°C with proteinase K (1 mg/ml), and luciferase activity was measured as described in Methods. Data are representative of 3 independent experiments. (C) PEI induced rapid cytokine secretion in vivo in a TLR5-dependent manner. Naive WT or Tlr5–/– mice were intraperitoneally injected with 5% glucose (gluc) or linear PEI, and serum levels of KC and IP-10 were determined 2 hours later by cytokine assay (see Methods). Data are representative of 4 mice per group, 2 independent experiments. (D) PEI-complexed siRNA induced MyD88-dependent secretion of IFN-β at tumor locations. Ascites from Myd88+/– or Myd88–/– mice bearing advanced ID8-Defb29/Vegf-A ovarian tumors were collected prior to (Basal) and 3 hours after intraperitoneal administration of PEI or NTsiRNA-PEI, and levels of IFN-β were analyzed by ELISA. Data are representative of 4 mice per group, 2 independent experiments. (E) CD45+CD11c+MHC-II+ tumor DCs were sorted from the peritoneal cavity of mice bearing ID8-Defb29/Vegf-A tumors, and TLR expression was confirmed by RT-PCR. Error bars in A–D denote SEM. *P < 0.05; **P < 0.01.