**7. A successful TEX capture from plasma of patients with melanoma**

Compelling evidence indicates the immune capture method to isolate TEX from patients' plasma yields excellent results when essential requirements are met. For example, we have reported separation of TEX from non-TEX in plasma of patients with melanoma [20] that have allowed for extensive characterization of the molecular cargo and functional repertoire of these sEV fractions [27]. The immune capture of TEX was performed using chondroitin sulfate peptidoglycan 4 (CSPG4)-specific mAbs developed by one of us [28]. These mAbs recognize CSPG4 which is selectively expressed on melanoma cells (and on the EVs these cells produce) but is not detectable on any other non-malignant cells in the body except for activated pericytes in the TME [29, 30]. Immunohistochemical staining with mAbs of more than 2000 melanoma lesions has showed that CSPG4 is expressed on about 80% of all investigated melanoma specimens [29, 30]. In melanoma tissues, CSPG4-specific mAbs decorate the surface of malignant cells; flow cytometric analysis of EVs stained with mAbs visualizes CSPG4 on their membrane [31]. Monoclonal Abs recognizing distinct CSPG4 epitopes are available to be selectively used for the capture of TEX, which is isolated and "purified" by SEC, and for subsequent antigen detection by flow cytometry confirmed that CSPG4 is expressed on TEX but is not detectable on non-TEX [7]. As with all immune capture experiments, titrations of the capture and detection Abs are critical for success as are the vesicle/Ab ratios, and these must be determined a priori and strictly adhered to during capture. As described in detail elsewhere [7, 20], all immunocaptured melanoma TEX are positive for CSPG4 and for melanomaassociated antigens (MAA), while non-TEX are negative. The exosome recovery ranged from 60 to 100 μg protein/mL plasma, and the ratio of TEX/total exosomes in plasma varied among melanoma patients from 0.2 to 0.6. The separation by immune capture of melanoma TEX from non-TEX yielded sufficient numbers of both exosome fractions for studies of their protein content by on-bead flow cytometry and by highresolution mass spectrometry (HRMS) as well as of their function in co-incubation assays with immune cell subsets [7]. TEX were enriched in immunosuppressive and non-TEX in immunostimulatory proteins, and co-incubation of the fractionated exosomes with immune cells confirmed their distinct immunoregulatory functions [7]. Melanoma TEX carrying CD39, CD73, FasL, PD-L1, TGF-β and TRAIL, among other suppressive proteins consistently inhibited functions of immune cells, while non-TEX were stimulatory in co-incubation assays. Using LC–MS/MS-based proteomics, we identified a profile of 16 proteins highly overexpressed in TEX which discriminated TEX from non-TEX. These proteins were components of molecular pathways mediating cellular events such as vesicle transport, immune reactivity, signal transduction, and disease activity [27]. Further, by dividing the analyzed melanoma patients into two groups of 7 patients with no evident disease (NED) and 8 with progressive

disease (PD) at the time of phlebotomy for exosome isolation from plasma, we were able to identify a signature of 12 proteins significantly and consistently overexpressed in TEX of patients with PD relative to TEX of patients with NED [27]. This ability of TEX bearing the signature of 5/12 most significantly (*p* < 0.0003) overexpressed proteins to discriminate melanoma patients with PD from those with NED within a very small patient cohort emphasizes the potential of TEX to serve as a biomarker of disease activity in melanoma [27]. In addition, this immunocapture-based study was the first to show that melanoma TEX, which are especially abundant in plasma of patients with advanced disease, are largely responsible for immune suppression that potentially promotes immune escape and tumor progression.
