Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication under both normal and pathological conditions. While small EVs (sEVs; <100–200 nm, commonly referred to as exosomes) are well-characterized, the properties and functions of larger EVs—here termed big EVs (bEVs; >200 nm, often called microvesicles)—remain less understood, particularly in cancer biology. 

This study aimed to develop a versatile bioluminescence resonance energy transfer (BRET)-based EV reporter, PalmGRET, to enable real-time, high-resolution imaging and tracking of EVs in vitro and in vivo. Using this tool, we sought to characterize the distinct biodistribution and functional roles of sEVs and bEVs in the context of breast cancer progression. 

EV subpopulations were isolated and characterized using nanoparticle tracking analysis (NTA), revealing distinct size profiles and release patterns from triple-negative breast cancer (TNBC) cells. PalmGRET allowed for dynamic visualization of EV biodistribution at physiologically relevant, non-lethal doses—providing greater specificity than traditional lipophilic dyes. Functional assays and proteomic profiling were conducted using EVs derived from TNBC cells, and their effects were evaluated in a syngeneic, immunocompetent TNBC mouse model. 

TNBC cells released a higher ratio of bEVs to sEVs. Biodistribution analysis showed that bEVs and sEVs followed distinct trafficking patterns in vivo, yet both contributed to tumor growth promotion. Proteomic analysis identified shared tumor-promoting surface membrane proteins (i.e., SLC29A1, CD9, and CD44) present on both EV types. Depletion of these surface proteins significantly altered EV biodistribution, reduced lung organotropism, and attenuated their protumorigenic effects.

Our findings reveal that sEVs and bEVs possess distinct in vivo behaviors and contribute differentially to tumor progression. The underrecognized role of bEVs highlights their potential as diagnostic markers and therapeutic targets. This work advances our understanding of EV-mediated mechanisms in cancer and opens new avenues for EV-based therapeutic delivery.