Glycocalyx Engineering with a Recycling Glycopolymer that Increases Cell Survival In Vivo

Abstract

Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be employed to probe processes that occur on longer time scales. In this work, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA) anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least ten days, even while the cells are dividing. As with native mucins, the cell-surface display of CholA-anchored glycopolymers influenced the focal adhesion distribution. Furthermore, we show that these mimetics enhance the survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand the application of glycocalyx engineering in glycobiology.

Keywords: cancer; cell adhesion; glycoconjugates; glycopolymers; lipids.

Figure 1

Synthesis of a panel of lipid-anchored glycopolymers. a) Structure and representation of our glycopolymers with differing alkynyl lipids, R. b) Synthesis of dual-end-functionalized glycopolymers.

Figure 2

Glycopolymers with different lipid anchors have different cell-surface kinetics. a) Fluorescence of Jurkat cells incubated with 10 μM of AF488-capped polymer with the given lipid anchor. b) Fractional cell-surface retention of biotin-capped polymers relative to levels immediately following polymer loading. c) Absolute cell-surface abundance of biotin-capped CholA-anchored glycopolymers on long timescales. d) Effect of CholA-anchored glycopolymers on fluorescence over time of cells labeled with a cytosolic cell-tracking dye. For a, b, c and d, shown are mean +/− standard deviation of three replicate experiments.

Figure 3

CholA-anchored glycopolymers recycle from reservoirs inside the cell to the cell surface. a) Jurkats were labeled with 5 μM AF488-capped glycopolymers. b) Jurkats loaded with CholA glycopolymers and incubated with AF647-transferrin. c) Jurkats loaded with CholA-anchored glycopolymers, then treated with anti-AF488 antibody to quench cell surface fluorescence, then allowed to incubate at the indicated temperature. d) A model for the trafficking of CholA-anchored glycopolymers. Scale bars are 10 μm.

Figure 4

CholA-anchored glycopolymers are excluded from sites of focal adhesion formation and drive a resistance to anchorage-dependent cell death. a) A kinetic funnel model for glycocalyx-driven integrin clustering. b) Effect of polymers on viability of MCF-10A cells plated on soft substrates. Error bars are SEM for at least three independent experiments. c) TIRF imaging of mCherry-paxillin expressing MCF-10A breast epithelial cells incubated with biotin-capped CholA polymers and stained with AF488-anti-biotin. Scale bars are 5 μm. d) Manders’ Colocalization Coefficients for coincidence of mCherry-paxillin with AF488-anti-biotin antibody for cells treated as in (c). Error bars are SEM for at least five cells quantified from at least two independent experiments. **p<0.01, ***p<0.001.

Figure 5

Long CholA-anchored glycopolymers protect previously nonmalignant cells from apoptosis in vivo. a) Zebrafish embryos, 48 hpf, were injected with GFP-H2B expressing MCF-10A cells loaded with either long (90 nm) or short (3 nm) CholA-anchored glycopolymers and imaged at the indicated time points. b) Effect of polymers on viability of cells injected as in (a) after 27 hours. Scale bars are 100 μm. Error bars are SEM of five biological replicates. **p<0.01.