Activatable Bioorthogonal Imaging of Radiotherapy-Induced Glucose Flux with Cell-Lineage Resolution

Abstract

Abstract Glucose uptake in the tumor microenvironment is closely linked to both malignant progression and immune function. Radiotherapy reshapes glucose utilization across different cell populations, yet distinguishing cell-lineage-specific uptake changes remains a major technical challenge. Here we report a two-step bioorthogonal near-infrared fluorescent system based on the Staudinger ligation, consisting of an azide-modified glucose reporter (GAz4) and a triphenylphosphine-masked hemicyanine probe (HCYP). The tetrafluorophenyl azide motif in GAz4 enhances electrophilicity, conferring a fast Staudinger ligation rate constant of 30.0 ± 3.0 M−1 s−1. HCYP first accumulates in cells in a masked, weakly fluorescent state; subsequent GAz4 delivery triggers intracellular Staudinger ligation to produce an activated fluorescence signal reporting glucose uptake. Using sequential administration, we show that fractionated radiotherapy significantly increases glucose-uptake-related signals in tumor-bearing mice compared with non-irradiated controls. Ex vivo flow cytometry reveals that these signals rise in both CD45- tumor cells and CD3+ T cells after radiotherapy, with trends consistent across cell populations. Importantly, our system demonstrates that the post-radiotherapy increase in bulk tumor glucose uptake arises from combined contributions of tumor and immune cells, not from a single lineage. This bioorthogonal approach provides a quantitative, cell-lineage-resolved tool for dissecting metabolic dynamics in the tumor microenvironment.