Inhibition of Cholesterol Transport from Lysosomes by Itraconazole Repolarizes Tumor-associated Macrophages to...

Targeting tumor-associated macrophages (TAMs) represents a promising strategy for next-generation immunotherapy. This study investigates the underlying mechanisms of TAM repolarization from the pro-tumorigenic M2 phenotype to the anti-tumorigenic M1 phenotype induced by itraconazole (ITZ).

M2 macrophages derived from THP-1 cells were used in all experiments, and ITZ was administered at a concentration of 10^-5 M. Morphological changes were monitored via time-lapse imaging. Single-cell RNA sequencing (scRNA-seq) and triple-color immunostaining for organelles, cholesterol, and the M1 marker interleukin (IL)-1β were performed with and without ITZ treatment. For inhibition studies, β-cyclodextrin polyrotaxane (βCD-PRX), a compound that removes cholesterol from lysosomes, was used. The effects were assessed by time-lapse imaging and western blot analysis of the M2 marker CD163.

Following ITZ treatment, a subpopulation of M2 macrophages exhibited morphological changes, shedding dendrites and migrating, indicative of an M1-like phenotype. Additionally, intracellular lipid droplets enlarged and swelled. scRNA-seq analysis revealed that M2 macrophages with reduced lysosomal vesicle biogenesis transitioned to an M1-like phenotype and identified 1,142 significantly enriched pathways, including M1-related signaling activation and cholesterol metabolism and transport pathways. Immunofluorescence analysis confirmed that macrophages shifting toward an M1-like phenotype expressed IL-1β, with enlarged intracellular lipid droplets identified as cholesterol-containing lysosomes. Notably, M2 macrophages that had adopted an M1-like morphology in response to ITZ reverted to their original M2-like shape and exhibited increased CD163 expression following βCD-PRX treatment.

Inhibition of lysosomal cholesterol release by ITZ reprogrammed M2 macrophages into an M1-like phenotype, revealing a novel mechanism that may serve as a foundation for developing innovative TAM-targeted immunotherapies.