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 1

siRNA-PEI nanoparticles are preferentially engulfed by tumor-associated DCs.

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siRNA-PEI nanoparticles are preferentially engulfed by tumor-associated ...
(A) NTsiRNA-PEI were stained with uranyl acetate and visualized using transmission electron microscopy. Original magnification, ×145,000 (left); ×285,000 (right). Scale bars: 20 nm (left); 100 nm (right). Average nanoparticle size was 40–60 nm. (B) Selective engulfment of NTsiRNA-PEI by peritoneal tumor-associated CD11c+ DCs. Rhodamine-labeled NTsiRNA-PEI were intraperitoneally injected into mice bearing ID8-Defb29/Vegf-A ovarian carcinoma, and peritoneal wash samples were analyzed by FACS after 3 days. (C) Time-course analysis of nanocomplex uptake by peritoneal CD11c+ DCs in tumor-bearing mice after a single intraperitoneal injection. The percentage of cells retaining the nanoparticles is indicated for each time point. Data are representative of 3 mice analyzed per time point in 2 independent experiments. (D) Biodistribution of intraperitoneally injected siRNA-PEI nanoparticles. Ovarian carcinoma–bearing mice received a single intraperitoneal injection of rhodamine-labeled NTsiRNA-PEI, and multiple organs were collected at different time points after injection. Fluorescence microscopy was performed on histological sections from different organs to determine the presence of nanoparticles. Red indicates rhodamine-labeled nanocomplexes. Blue denotes nuclei. Data are representative of at least 3 independent experiments. Original magnification, ×200; ×40 (inset). (E) Nanoparticle uptake by DCs infiltrating solid ovarian tumors. Ovaries from tumor-bearing mice were collected at different time points after a single intraperitoneal injection with rhodamine-labeled NTsiRNA-PEI. Shown is the percentage of ovarian tumor–resident DCs engulfing nanoparticles in situ, determined by FACS. Data are representative of 2 independent experiments. SSC-A, side-scattered light.