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 2

Engulfment of PEI-based nanocomplexes induces maturation of tumor-associated DCs in vivo and in situ.

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Engulfment of PEI-based nanocomplexes induces maturation of tumor-associ...
(A) Expression of CD80 and MHC-II on tumor-associated DCs from mice bearing ID8-Defb29/Vegf-A tumors for 3 weeks and injected with PBS. (B) Rhodamine-labeled NTsiRNA-PEI or (C) equivalent amounts of rhodamine-labeled PEI alone (see Methods) were intraperitoneally injected into mice bearing ID8-Defb29/Vegf-A tumors for 3 weeks. Expression of CD80, MHC-I, and MHC-II on tumor DCs that engulfed the nanoparticles or PEI was analyzed by FACS 3 days later. Rhod, rhodamine-labeled PEI. Filled histograms represent expression on DCs that did not engulf nanoparticles or PEI. Open histograms indicate expression by DCs that engulfed nanoparticles or PEI in the same host. Data are representative of 3 mice per group in 4 independent experiments. In A–C, the percentage of tumor-associated DCs coexpressing MHC-II and CD80 is indicated. (D) siRNA-PEI nanoparticles induced maturation of human tumor DCs. Leukocytes from 6 unselected dissociated stage III human ovarian tumors were enriched by Ficoll, 5 × 106 cells were incubated in 5 ml RPMI containing 100 μl PBS (dotted histograms) or 100 μl NTsiRNA-PEI (open histograms; see Methods), and the levels of CD80 on CD45+DEC205+CD3–CD20–CD14– tumor DCs were analyzed by FACS 18 hours later. Filled histograms represent staining with isotype control antibodies. MFI, mean fluorescence intensity of CD80 staining. Data points and horizontal bars denote individual values and means, respectively. **P < 0.01 (Mann-Whitney).