Cell-cell interactions via non-covalent click chemistry

Abstract

Metabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both β-cyclodextrins (β-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell-cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions.

Fig. 1. Principle of cell surface engineering with complementary artificial recognition markers based on host–guest pairs. Step 1: metabolic glycan labelling with Ac₄ManNAz resulting in azide tag incorporation at cell surfaces. Step 2: introduction of the complementary artificial markers Tri-β-CD and Tri-Adam on the cell membranes via the bioorthogonal SPAAC ligation reaction. Step 3: cell–cell adhesion through non-covalent click chemistry.

Scheme 1. Synthesis of the artificial cell surface markers Tri-β-CD and Tri-Adam. (a) 2, Et₃N, DMF, r.t., 12 h, 78%; (b) TFA/CH₂Cl₂ (20/80), 0 °C then r.t., 1 h, 95%; (c) 3, DMF, r.t., 12 h, 76%; (d) 4, Et₃N, DMSO, r.t., 12 h, 77%; (e) TFA/CH₂Cl₂ (20/80), 0 °C then r.t., 3 h, 97%; (f) 5, Cu(MeCN)₄PF₆, tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA), DMSO, r.t., 4 h, 66%; (g) 3, Et₃N, DMSO, r.t., 2 h, 84%.

Fig. 2. Confocal microscopy imaging of cellular recognition. (a) A549 adherent tumor cells (green) and Jurkat T cells (red) (1 : 5 ratio) were incubated together for 10 minutes and washed with PBS prior to imaging; (b) 3D imaging of cell–cell interactions.

Fig. 3. Electron microscopy imaging of cellular recognition. (a) Imaging of A549 cells bearing Tri-β-CD artificial surface markers; (b) imaging of Jurkat T cells functionalized with Tri-Adam markers; (c) and (d) imaging of Jurkat T cell on the surface of A549 cells at two different magnifications, showing the appearance of filaments (arrows).

Fig. 4. (a) Potential cell interaction network engendered by an unnatural cell recognition leading to the destruction of tumor cells; (b) Concentration of IL-2 secreted by Jurkat T cells when incubated with A549 cells in the presence of PHA and PMA (GI: glycoengineered cells, WT: wild-type cells); (c) NK cells-mediated cytotoxicity for A549 cells directed by cell–cell interactions.