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 3

siRNA-PEI nanoparticles enhance antigen presentation and induce tumoricidal activity by ovarian cancer DCs.

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siRNA-PEI nanoparticles enhance antigen presentation and induce tumorici...
(A) Improved antigen-processing capacity of tumor DCs engulfing PEI-based nanocomplexes in vivo. Mice bearing ID8-Defb29/Vegf-A tumors were intraperitoneally injected with DQ-OVA (see Methods), and 24 hours later received a single intraperitoneal injection of PBS, PEI, or NTsiRNA-PEI (3 mice per group, 2 independent experiments). Percentages denote the proportion of peritoneal tumor-associated DCs that efficiently processed the probe (FITC+), determined by FACS 48 hours later. (B) Enhanced antigen-presenting ability of OVA-pulsed tumor DCs (tDCs) engulfing siRNA-PEI nanoparticles in vivo (see Methods). Shown are representative FACS analysis of CFSE dilution and graphical representation of percent proliferating cells in triplicate for each condition. (C) Uptake of siRNA-PEI nanocomplexes induced tumoricidal DCs. Tumor-bearing mice were intraperitoneally injected with PBS, PEI, or NTsiRNA-PEI; 36 hours later, CD11c+MHC-II+CD3–NK1.1– tumor DCs from each group were sorted and incubated with 51Cr-labeled ID8-Defb29/Vegf-A cells. Release of 51Cr was measured in cell supernatants 5 hours later. Radiolabeled tumor cells were also cocultured with C57BL/6 splenocytes preincubated with 1,000 U/ml IL-2 for 5 days as a positive control of lysis. (D) Quantification of TRAIL mRNA levels by real-time RT-PCR in the same tumor DCs as in C. Error bars in B–D denote SEM. **P < 0.01.