A Tetrazine-Based Ratiometric Sensor Quantifying pH Gradient in Tumorspheres through Bio-Orthogonal Labeling

Area of Science:

• Biomedical Engineering
• Chemical Biology
• Oncology

Background:

• pH regulation is vital for biological processes, significantly impacting tumor development and the tumor immune microenvironment.
• Tumor pH exhibits complex spatiotemporal heterogeneity, posing challenges for accurate detection and analysis.
• Existing methods lack systematic and quantitative approaches for mapping tumor pH distribution.

Purpose of the Study:

• To develop a novel, stable, and precise ratiometric pH sensor for in situ quantification of pH within tumors.
• To enable visualization and tracking of pH dynamics associated with cellular processes like glycolysis.
• To provide a versatile tool for studying disease-related pH changes in complex biological systems.

Main Methods:

• Fabrication of a tetrazine-based ratiometric pH sensor (TzR-H) by linking a BODIPY donor and a rhodamine acceptor.
• Utilizing bio-orthogonal reactions and glycometabolism for stable anchoring of the sensor in live cells and organelles.
• Employing the sensor to quantify pH gradients in tumor cells and multicellular tumor spheroids under varying metabolic conditions.

Main Results:

• The TzR-H sensor demonstrated a pKa of 6.92 and high precision (0.02 pH units) across a pH range of 5.0-9.0.
• Successfully visualized and quantified glycolysis-associated pH fluctuations in tumor cells and spheroids.
• Revealed distinct pH gradients within tumor spheroids, with average pH values changing over time post-glycolysis and varying from core to surface.

Conclusions:

• The tetrazine-based TzR-H sensor offers a robust platform for stable cell labeling and precise, in situ pH measurement.
• This approach enables spatial and temporal quantitation of pH within complex tumor models.
• Provides a universal strategy for quantifying disease-associated pH variations, aiding in the study of tumor biology and microenvironments.