Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.

Figure 2. Comparison of size and detection limit between PalmGRET-and dye-labeled bEVs and sEVs. a) NTA of 4T1-bEVs and -sEVs demonstrating distinct size distributions (left; representative data of three independent experiments) and mean sizes (right; determined from three independent experiments). n.s., p > 0.05; *p < 0.05 with 2-tailed Student's t-test. b) Schematic of inner EV membrane labeling using PalmGRET (top) and outer EV membrane labeling using lipophilic fluorescent dyes (bottom). c) Dot blot analysis showing that PalmGRET labeled the inner membrane of bEVs and sEVs. The positive control was whole cell lysates (WCL). d) Representative TEM images of 4T1-PalmGRET-bEVs and -sEVs. The same imaging parameters were used to acquire bEV and sEV TEM images. Images on the right of each row depict the enlarged images of the boxed regions (red). Black scale bar: 200 nm; red scale bar: 100 nm. e) Mean particle size (left) and particle size distribution (right) of 4T1-PalmGRET-bEVs and -sEVs determined from TEM image analysis. The bEV and sEV particles (N = 400 per group) in the captured TEM images were analyzed using Fiji software (ImageJ, NIH) to measure individual particle sizes (left). Size distribution analysis of bEVs and sEVs (right) demonstrating that bEVs are largely composed of 201-400 nm particles, while sEVs mainly consist of 1-100 and 101-200 nm particles. ****p < 0.0001 with 2-tailed Student's t-test. f-h) NTA of PalmGRET-and dye-labeled 4T1-EVs. f) Size distribution and g) mean size of bEVs and sEVs labeled with PalmGRET or colabeled with PKH26, DiD, and DiR are shown. h) Analysis of particle size composition of the bEVs and sEVs showed that PalmGRET did not change the size distribution in all size divisions as compared to the WT EV controls. The 101-200 nm particles were decreased, while the 201-300 nm and 301-500 nm particles were increased among the lipophilic-dye-labeled bEVs. Similarly, the 101-200 nm particles were decreased and 201-300 nm particles were increased among the lipophilic-dye-labeled sEVs. Data are from three independent experiments. n.s., p > 0.05; *p < 0.05; **p < 0.01 with 1-way ANOVA followed by Dunnett's post hoc test versus the control.

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs [bEVs]; >200 nm, microvesicles) are less well explored. Here, we identify bEVs and sEVs as distinct EV populations, and determine that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence resonance energy transfer (BRET)-based EV reporter, reveals dose-dependent EV biodistribution at non-lethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. Remarkably, the bEVs and sEVs exhibit unique biodistribution profiles, yet individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass spectrometry (MS)-identified tumor progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include SLC29A1, CD9 and CD44. tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.