Fluorescence microscopic analysis of checkpoint protein expression is capable of predicting clinical outcomes for checkpoint blockade immunotherapy. However, accurate detection of their expression levels is hindered by fluorophore photobleaching and cell autofluorescence. We now develop a sensitive and robust fluorescence microscopy method that uses antifade graphite-structured carbon dots (GCDs) on a plasmonic Ag substrate (named ACPAS) for the accurate detection of checkpoint proteins in immunotherapy. In ACPAS, a Ag substrate is used to enhance the fluorescence of GCDs while a continuous illumination is implemented to quench cell autofluorescence, thus enabling a dramatic improvement in the signal-to-background ratio by up to 33-fold. We use ACPAS to monitor programmed death ligand-1 (PD-L1) expression levels on various tumor cells and finely differentiate their microscopic changes in combination with chemokine receptor CXCR4-targeted treatments. ACPAS analysis reveals for the first time that CXCR4 agonist (SDF-1α) and antagonist (AMD3100) can potentiate PD-L1 expression by down-regulating CXCR4 expression on tumor cells, which provides valuable information on the development of anti-PD-L1 and anti-CXCR4 combination therapy. We envision that ACPAS will become a broadly useful tool for protein expression studies in biomedicine and life sciences.
Keywords: antiphotobleaching; carbon dots; checkpoint protein; fluorescence enhancement; plasmon substrate.