Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation
Todd V. Brennan, … , Xiaopei Huang, Yiping Yang
Todd V. Brennan, … , Xiaopei Huang, Yiping Yang
Published December 7, 2015
Citation Information: J Clin Invest. 2016;126(1):207-219. https://doi.org/10.1172/JCI76566.
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Research Article Oncology

Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation

Abstract

Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell–based antitumor therapies.

Authors

Todd V. Brennan, Liwen Lin, Joshua D. Brandstadter, Victoria R. Rendell, Keith Dredge, Xiaopei Huang, Yiping Yang

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Figure 7

PG545 promotes the accumulation of CpG in the lysosomal compartment of DCs.

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PG545 promotes the accumulation of CpG in the lysosomal compartment of D...
(A) BMDCs were incubated with various concentrations of FITC-labeled CpG in the presence (CpG+PG545) or absence (CpG only) of PG545 (5 μg/ml), and the uptake of FITC-labeled CpG was analyzed by FACS 6 hours later. Cells without CpG treatment (Untreated) were used as a control. Representative plots from 3 independent experiments are shown. (B and C) DCs were treated with Cy5-labeled (red) CpG (0.3 μg/ml) for 90 minutes in the presence or absence of PG545 (5 μg/ml). Cells were then stained with (B) LysoTracker (green), a lysosome marker, or (C) Alexa Fluor 488–labeled dextran (green), an endosome marker (C), as well as nuclear staining with Hoechst 33342 (blue) and imaged by confocal microscopy. Representative images of DCs with CpG, dextran, or LysoTracker as well as merged images of CpG with dextran or LysoTracker are shown. (D) DC2.4 cells were transfected with LAMP-1-GFP and treated with CpG-Cy5 (0.3 μg/ml) for 90 minutes in the presence or absence of PG545 (5 μg/ml). Representative images of LAMP-1-GFP–transfected (green) DCs with CpG (red) as well as merged images with nuclear staining are shown. Scale bar: 5 μm.