Selective Glycopolymer Inhibitors of Galectin-3: Supportive Anti-Cancer Agents Protecting Monocytes and Preserving Interferon-Gamma Function

Abstract

Introduction: The immunosuppressive roles of galectin-3 (Gal-3) in carcinogenesis make this lectin an attractive target for pharmacological inhibition in immunotherapy. Although current clinical immunotherapies appear promising in the treatment of solid tumors, their efficacy is significantly weakened by the hostile immunosuppressive tumor microenvironment (TME). Gal-3, a prominent TME modulator, efficiently subverts the elimination of cancer, either directly by inducing apoptosis of immune cells or indirectly by binding essential effector molecules, such as interferon-gamma (IFNγ).

Methods: N-(2-Hydroxypropyl)methacrylamide (HPMA)-based glycopolymers bearing poly-N-acetyllactosamine-derived tetrasaccharide ligands of Gal-3 were designed, synthesized, and characterized using high-performance liquid chromatography, dynamic light scattering, UV-Vis spectrophotometry, gel permeation chromatography, nuclear magnetic resonance, high-resolution mass spectrometry and CCK-8 assay for evaluation of glycopolymer non-toxicity. Pro-immunogenic effects of purified glycopolymers were tested by apoptotic assay using flow cytometry, competitive ELISA, and in vitro cell-free INFγ-based assay.

Results: All tested glycopolymers completely inhibited Gal-3-induced apoptosis of monocytes/macrophages, of which the M1 subtype is responsible for eliminating cancer cells during immunotherapy. Moreover, the glycopolymers suppressed Gal-3-induced capture of glycosylated IFNγ by competitive inhibition to Gal-3 carbohydrate recognition domain (CRD), which enables further inherent biological activities of this effector, such as differentiation of monocytes into M1 macrophages and repolarization of M2-macrophages to the M1 state.

Conclusion: The prepared glycopolymers are promising inhibitors of Gal-3 and may serve as important supportive anti-cancer nanosystems enabling the infiltration of proinflammatory macrophages and the reprogramming of unwanted M2 macrophages into the M1 subtype.

Keywords: carbohydrate; galectin-3; glycopolymer; interferon-gamma; monocyte; tumor microenvironment.

Graphical abstract

Scheme 1

Sequential enzymatic synthesis of functionalized tetrasaccharide ligands: 4 (LN1-LN2), 5 (LN2-LN2), 6 (LDN-LN2) followed by the synthesis of polymer precursors and glycopolymers G1–G9.

Figure 1

Competitive inhibition of binding of Gal-3 to ASF by glycopolymers determined by ELISA. Dose-response inhibition curves for LN1-LN2-based (A), LN2-LN2-based (B), and LDN-LN2-based (C) glycopolymers. IC₅₀ values are stated in Table 1.

Figure 2

Inhibition of Gal-3-induced apoptosis of macrophages by glycopolymers. Addition of recombinant Gal-3 induces apoptosis to ca 15–25% of macrophages. This process is dose-dependently prevented by the addition of glycopolymers that capture Gal-3 and do not allow it to bind to macrophages. (A) Representative flow cytometry scatter plots and gating strategy of J774A.1 macrophages treated with 10 µM Gal-3 only or in combination with 20 µM glycopolymer G2, G3 (upper row), G5, G6 (middle row), or G8, G9 (bottom row), respectively. Early apoptotic and late apoptotic macrophage cells are present in the Q3 or Q2 quadrant, respectively. Decrease in percentage of cells especially in Q2 quadrant in the absence (Gal-3 column) and in the presence of all glycopolymers (Gal-3 + G2 – G9 column) is clearly visible. (B) Flow cytometry analysis of apoptosis (sum of early and late apoptosis, ie percentage of apoptotic cells in Q3 and Q2 quadrant) of J774A.1 macrophages treated with 10 µM Gal-3 only or in combination with 0–20 µM glycopolymers G2, G3 (upper row), G5, G6 (middle row), or G8, G9 (bottom row), respectively. Bar graphs indicate the mean ± SEM (n = 2, statistical analysis by one-way ANOVA with Dunnett’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control).

Figure 3

Competitive inhibition of Gal-3-mediated IFNγ capture by glycopolymers. (A) Dose-response decrease in IFNγ concentration after incubation with Gal-3-coated magnetic beads. (B–D) Concentration of residual IFNγ after incubation with Gal-3-coated beads (Gal-3/IFNγ molar ratio 1/10) with glycopolymer G3 (B), G6 (C) or G9 (D) as determined by ELISA. Graphs represent means ± SEM (n = 3; statistical analysis by one-way ANOVA with Dunnett’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001 versus control). Black columns - uncoated magnetic beads, blue columns – Gal-3-coated magnetic beads.