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 6

PG545 enhances CpG-mediated activation of TLR9.

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PG545 enhances CpG-mediated activation of TLR9. (A) HEK 293T cells stabl...
(A) HEK 293T cells stably expressing human TLR9 were treated with increasing concentrations of CpG (ODN 2395) in the presence (+PG545) or absence (No PG545) of PG545 (5 μg/ml). As a control, 293T cells not expressing human TLR9 were assayed at the highest CpG concentration (10 μg/ml). 18 hours later, culture supernatants were assayed for IL-8 production by ELISA. (B) Quantitative RT-PCR was performed on cDNA obtained from BMDCs incubated for 18 hours with low-dose (0.3 μg/ml) or high-dose (3 μg/ml) CpG in the presence of absence of PG545 (5 μg/ml). Fold change in expression over the no-treatment reference sample, normalized to reference genes Gapdh and β-actin, is shown for triplicate samples. (C–E) WT and Tlr9–/– BMDCs were treated with increasing concentrations of CpG in the presence or absence of PG545 (5 μg/ml). 18 hours later, DCs were analyzed for IL-12 production by intracellular staining. (C) The percentage of IL-12–producing DCs among CD11c+ DCs is shown. Culture supernatants were assayed for the secretion of (D) IL-6 and (E) TNF-α by ELISA. Data are representative of 3 independent experiments. *P < 0.05, 2-tailed Student’s t test.