**5. Rationale for sEV fractionation into TEX and non-TEX**

In cancer patients, total exosomes isolated from plasma by SEC contain various proportions of TEX. In patients with melanoma, 20–70% of total plasma exosomes are tumor cell-derived [7]. While total plasma exosomes with a high content of TEX might largely reflect the TEX characteristics, non-TEX present in the mix might influence the estimates of effects plasma exosomes to exert in recipient cells. Thus, the separation of TEX from a mix of other vesicles in plasma is a necessary step to evaluate their unique phenotypic, molecular and functional characteristics. This step may be especially important when TEX account for only a small fraction of total sEVs in plasma. Immunoaffinity capture of TEX from plasma has been introduced as an approach to the pulldown of TEX based on the use of Abs specific for the antigens selectively expressed or markedly overexpressed by cancer cells and carried by TEX [20]. Immunocapture-based exosome isolation from body fluids has been extensively used in diseases other than cancer, including neurological diseases, where Ab-based capture is broadly used for the isolation of neuron-derived L1CAM bearing EVs (NDEVs) [21]. In cancer, TEX separated from non-TEX are expected to serve as a liquid tumor biopsy that faithfully recapitulates molecular and genetic features of parental cancer cells.
