Table 2 Discussion of in vitro and in vivo methods for studying the role of EVs in TME

For investigating the intricate interplay between EVs and the TME cells, whether in vitro or in vivo, the primary approach revolves around employing fluorescence labeling of EVs through liposomal bilayers or using fluorescently tagged cargos embedded within these vesicles. Typically, EVs are labeled using lipophilic dyes such as PKH67 (green fluorescence)/PKH26 (red fluorescence) and Di-series dyes. Alternatively, specific proteins like CD63 and the green fluorescent protein (GFP) expression elements within exosomes can be engineered into plasmids and subsequently packaged into lentiviruses. These lentiviruses are then used to infect cells, resulting in the secretion of exosomes adorned with green fluorescence [54]. Depending on the objectives, models, and TME analyses, EVs can be administered through subcutaneous injection, intravenous injection, or intraperitoneal injection. After a certain duration, live animal imaging systems are employed for in vivo tracking, or the experimental animals are euthanized, and target tissues are collected for imaging observations, thereby assessing whether EVs have been taken up by the target cells within the TME. In contrast, in vitro experiments typically involve co-culturing these exosomes with the cells under study for a certain period. Fluorescence signals are observed using imaging instruments like laser confocal microscopes

The functional analysis of the intricate interplay between EVs and the TME is contingent upon the specific research questions at hand. In both in vivo and in vitro experiments, methods such as immuno-electron microscopy and enzyme-linked immunosorbent assay (ELISA) can be employed to investigate whether extracellular structures bind to target cells [55], particularly when studying the membrane surface proteins of EVs. For studying the proteins and lipids contained within exosomes, proteomic and lipidomic analyses are conducted to trace the protein and lipid components, their types, and their interactions with other biomolecules within target cells. Immunoprecipitation methods can also be used for proteins [55]. In the case of non-coding RNAs encapsulated within EVs, RNA immunoprecipitation is an applicable technique. The distinction between in vitro and in vivo research lies primarily in the analysis of target cells or tissues. In vitro experiments involve co-culturing EVs with target cells, and upon confirming the internalization of EVs by target cells, functional changes such as cytokine expression, cell proliferation, migration and invasion, apoptosis, and luminal formation experiments are observed. In vivo experiments, on the other hand, encompass assessing tumor volume, mass, or conducting single-cell RNA sequencing analysis to elucidate cellular functional changes within animal tissues