Assessment of lysosomal drug sequestration and release using fluorescence microscopy

Lysosomotropism refers to the ability of certain basic lipophilic compounds to accumulate in lysosomes via pH partitioning. Various drugs including anticancer agents are trapped in lysosomes, and this process can prevent such drugs from reaching their primary target, thereby limiting their effectiveness. Strategies aimed at preventing drug sequestration or inducing drug release from lysosomes have garnered considerable interest. Chloroquine is a widely used anti-malarial drug that triggers lysosome membrane permeabilization (LMP) to liberate sequestered drugs from these organelles. In this study, we first evaluate the lysosomotropism of various fluorescent anticancer agents in silico. Next, we outline a simple, fast and robust method for the visualization and quantification of their lysosomal sequestration and release by fluorescence microscopy. The method is used on live cells and consists of two steps: (i) visualization of the compounds in lysosomes by analyzing their colocalization with a specific fluorescent lysosomal marker, and (ii) assessment of drug release from lysosomes. Furthermore, we present fluorescence microscopy protocols for monitoring LMP by analyzing the subcellular localization of LGALS3 (Galectin-3), which normally distributes diffusely in the cytoplasm but translocates into lysosomes upon LMP. This can be achieved on fixed cells by detecting endogenous LGALS3 with immunostaining or by the visualization of a transgenic LGALS3-mCherry fusion protein on live cells. Altogether, these methods facilitate qualitative and quantitative fluorescence imaging of lysosomal sequestration and liberation of lysosomotropic drugs.