Regucalcin-containing extracellular vesicles suppress M2 macrophage polarization and attenuate tumor progression in vivo

Regucalcin (RGN) plays diverse roles in cell biology, highlighting its importance in both physiological and pathological conditions. Prostate cancer patients with higher RGN expression exhibited significantly longer disease-free survival. Although RGN is a cell signaling suppressor, the molecular mechanisms underlying tumor suppression by RGN in the tumor microenvironment through cell-cell communication remain unclear. PC3 prostate cancer cell lines stably expressing RGN or a control vector were generated for this study. Extracellular vesicles (EVs) were isolated from these cell lines using differential ultracentrifugation. The murine macrophage cell line J7441 was treated with isolated EVs, and effects on M2 polarization were evaluated using qRT-PCR and western blot analysis. To assess the potential anti-tumor effects of EVs, PC3 parental cells were subcutaneously implanted at two sites per mouse, followed by intratumoral injection of the respective EVs. Tumor volume was monitored. Harvested fresh frozen tumor tissues underwent immunofluorescence staining for CD206, an M2 macrophage marker. RGN was detected in EVs from RGN-expressing cells, and treatment with these RGN-containing EVs was associated with reduced tumor growth and reduced M2 macrophage polarization in vitro and in vivo. Furthermore, recombinant RGN protein reduced the levels of p-AKT1 and p-ERK1/2. Moreover, the suppression of M2 macrophage polarization by RGN-containing EVs was accompanied by decreased p-AKT1 and p-ERK1/2 in vitro. This study describes an EV-associated mechanism that may contribute to the regulation of macrophage polarization and indicates that RGN-containing EVs merit further evaluation as a candidate approach for cancer treatment. Causal validation, such as macrophage depletion or CD206 knockdown, and evaluation in additional models remain to be addressed in future studies.