Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity
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
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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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 5

siRNA-PEI nanocomplexes prolong survival in a T cell–mediated, MyD88-dependent manner.

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siRNA-PEI nanocomplexes prolong survival in a T cell–mediated, MyD88-dep...
(A and B) Mice bearing aggressive ID8-Defb29/Vegf-A (A) or luciferase-expressing parental ID8 (B) tumors were treated intraperitoneally with PBS or NTsiRNA-PEI nanoparticles at days 8, 13, 18, 23, and 27 after tumor challenge, and survival was monitored over time. Data are representative of 2 independent experiments with a total of 12 mice per group. (C) Mice bearing preestablished luciferase-expressing parental ID8 ovarian tumors were intraperitoneally treated with PBS or NTsiRNA-PEI as described above, and luciferin was injected 70 days after tumor challenge to monitor tumor burden in vivo. (D) Quantification of the tumor burden shown in C. (E) Nanoparticle-mediated increase in survival was completely MyD88 dependent. Myd88+/– or Myd88–/– mice bearing aggressive ID8-Defb29/Vegf-A ovarian tumors were treated with PBS or NTsiRNA-PEI as described above, and survival was monitored over time. Data are representative of 2 independent experiments with a total of 10 mice per group. (F) Treatment with siRNA-PEI nanocomplexes induced T cell–mediated antitumor protective immunity. CD3+ T cells (3 × 106) purified from the spleens of mice treated with PBS or NTsiRNA-PEI were intravenously transferred into naive C57BL/6 mice previously irradiated with 3 Gy (n = 5 per group); after 24 hours, mice were challenged in the flank with ID8-Defb29/Vegf-A ovarian carcinoma cells. Tumor pictures were taken 2 months later. (G) Quantification of tumor size from F. In D and G, data points and horizontal bars denote individual values and means, respectively. *P < 0.05; **P < 0.01 versus respective PBS control, log-rank test (A, B, and E) or Mann-Whitney test (D and G).