Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity

Abstract

Background/objectives: Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors' expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated.

Methods: CD56⁺ CD3^- NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10× magnification for 24 h.

Results: Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation.

Conclusions: Our findings point to the role of Siglec-sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies.

Keywords: NK cells; hypoxia; immune synapses; nanoparticles; sialoglycan ligands; siglec receptors.

Figure 1

The effect of hypoxia on the surface expression of Siglec-7 and Siglec-9 receptors on immune cells. Histograms of one representative experiment show the effect of hypoxia (1% oxygen) for 24 h in comparison with normoxia (21% oxygen) on the surface expression of Siglec-7 and Siglec-9. (A,B) NK-92; (C,D) primary NKs; (E,F) U937 monocytes cell line. The bar charts represent the mean of the Median Fluorescent Intensity (MFI) of Siglec-7 Receptor (G) and Siglec-9 Receptor (H) on the surface of the indicated effector cells. U937 monocytes were used as a positive control while unstained samples for each experiment were used as negative controls. Error bars represent SEM of three independent experiments.

Figure 2

The effect of hypoxia on the surface expression of Siglec-7 and Siglec-9 ligands on tumor cell lines. The bar charts represent the Median Fluorescent Intensity (MFI) of (A) Siglec-7 ligand and (B) Siglec-9 ligand on the surface of K562 and MCF-7 cell lines in hypoxia and normoxia. Error bars represent SEM of three independent experiments. (**) p value ≤ 0.01 based on unpaired t-test.

Figure 3

The effect of hypoxia on the regulation of Siglec-7 and Siglec-9 ligands on the surface of the K562 and MCF-7 cell lines. The bar charts show the percentages of K562 cells that express Siglec-7 ligand (A) and Siglec-9 ligand (B), as well as MCF-7 cells that express Siglec-7 ligand (C) and Siglec-9 ligand (D) at different levels: low, medium, and high expression levels, as assessed by flow cytometry. Error bars represent SEM of three independent experiments. (*) p value ≤ 0.05 based on unpaired t-test.

Figure 4

Immunological synapse formation. (A) Inhibition of Siglec-7/Fc chimera and Siglec-9/Fc chimera binding to K562 cells by Sig-7/9 NPs. Representative histogram shown from three independent experiments. (B) Confocal imaging of the accumulation of granzyme B, actin, and phospho-tyrosine between Sig-7/9 NPs and Control NPs treated and non-treated peripheral NK and MCF-7 cells representing immunological synapse formation and/or recognition. Scale bar = 10 µm. (C) Quantification of the treated and non-treated peripheral NK cells recognizing and/or forming an immunological synapse with MCF-7. Error bars represent SEM of three independent experiments. Statistical significance was determined using two-way ANOVA. (***) p value ≤ 0.001; (****) p ≤ 0.0001.

Figure 5

The effect of nanoparticles on primary NK-mediated cytotoxicity. Target tumor cell lines (A) K562 and (B) MCF-7 were co-cultured with treated primary NK cells and assessed using LDH cytotoxicity assay. Error bars represent SEM of three independent experiments. Statistical significance was determined using two-way ANOVA. (*) p value ≤ 0.05; (**) p value ≤ 0.01.

Figure 6

Live-cell imaging of peripheral NK cytotoxicity treated with Sig-7/9 NPs, Control NPs and non-treated peripheral NK cytotoxicity on MCF-7 cells. MCF-7 cells labeled with CMFDA green co-cultured with primary NK labeled with CMTMR orange. Dead cells shown in red. (A) Representative confocal images taken at different time points (0, 4 and 8 h). Scale bar = 100 µm. Orange arrows are pointing at the pNK cells forming immunological synapses with the target cells. (B) The percentages of killed MCF-7 cells by the treated and non-treated peripheral NK. Error bars represent SEM of three independent experiments. Statistical significance was determined using two-way ANOVA. (*) p value ≤ 0.05.