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

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.