Cell Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages

Abstract

Cell labeling and tracking are important processes in understanding biologic mechanisms and the therapeutic effect of inoculated cells in vivo. Numerous attempts have been made to label and track inoculated cells in vivo; however, these methods have limitations as a result of their biological effects, including secondary phagocytosis of macrophages and genetic modification. Here, we investigated a new cell labeling and tracking strategy based on metabolic glycoengineering and bioorthogonal click chemistry. We first treated cells with tetra-acetylated N-azidoacetyl-D-mannosamine to generate unnatural sialic acids with azide groups on the surface of the target cells. The azide-labeled cells were then transplanted to mouse liver, and dibenzyl cyclooctyne-conjugated Cy5 (DBCO-Cy5) was intravenously injected into mice to chemically bind with the azide groups on the surface of the target cells in vivo for target cell visualization. Unnatural sialic acids with azide groups could be artificially induced on the surface of target cells by glycoengineering. We then tracked the azide groups on the surface of the cells by DBCO-Cy5 in vivo using bioorthogonal click chemistry. Importantly, labeling efficacy was enhanced and false signals by phagocytosis of macrophages were reduced. This strategy will be highly useful for cell labeling and tracking.

Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking

“…Additionally, many studies of cell or virus labeling and tracking used over 50 μM Ac4ManNAz 3, 6, 40. In our study, however, cells treated with 50 μM Ac4ManNAz showed a decrease of proliferation, migration and invasion ability.…”

Mesenchymal Stem Cells As Guideposts for Nanoparticle-Mediated Targeted Drug Delivery in Ovarian Cancer

“…The second step is the introduction of DBCO-functionalized drug-loaded nanoparticles that have a high affinity for the azide groups. The click reaction between azide receptors and DBCO is relatively fast, highly specific, catalyst-free, and can proceed under physiological conditions [23,35,36]. Additionally, azide and DBCO moieties are relatively small and biologically inert, facilitating the incorporation of multiple azide groups on the cell surface and subsequent click reaction [37].…”

“…To address these problems, we developed a novel two-step targeting strategy that relies on the delivery of mesenchymal stem cells (MSCs) labeled with synthetic functional receptors (azides) to tumor tissues, followed by delivery of drug-loaded nanocarriers targeted to the functional receptors [22]. Previous studies have shown the feasibility of utilizing glycoengineering approaches for tumor targeting [23,24]. These studies also utilized the principle that a synthetic azide sugar is accepted by the CMP-sialic acid biosynthesis machinery in mammalian cells, leading to the biosynthesis and cell surface expression of azido sialic acid containing N 3 -linked glycoproteins [25].…”

Metabolisches Glykoengineering mit N‐Acyl‐Seiten‐ ketten‐modifizierten Mannosaminen

“…Kürzlich wurde ein “Doppelklick”‐Ansatz etabliert, der die Staudinger‐Ligation, oder SPAAC, mit nachfolgender DARinv‐Konjugation kombiniert, um O ‐Glykane in Organen oder Tumorgeweben durch SPECT zu visualisieren . In einem anderen Experiment wurden Zellen mit peracetyliertem ManNAz vorbehandelt und danach in die Leber von Mäusen transplantiert . Im Anschluss wurden die Zellen mithilfe von SPAAC mit einem fluoreszierenden Farbstoff markiert, wodurch ihre Lokalisierung innerhalb der Leber in vivo kenntlich gemacht wurde.…”