# MALDI-TOF/MS Analysis of Extracellular Vesicles Released by Cancer Cells
## 2 Applications
### Breast Cancer  
Breast cancer is the leading cause of cancer-related death in women worldwide [22]. It has six molecular subtypes that differ in terms of clinical relevance, therapeutic approaches, responses to treatment, and prognosis. Breast cancer has a relatively high (80-92%) five-year survival rate that strongly decreases (\(\prec\)25%) in the case of metastasis, making its early diagnosis extremely important. The most used approach for prevention is mammography, characterized by some known limitations such as high rates of false negatives and false positives. Therefore, the discovery of novel approaches for its screening and follow-up is highly required. Extracellular vesicles' characterization by MALDI-TOF has been useful for the identification of biomarkers for breast cancer. A proteomic study was conducted on exosome-like vesicles isolated by ultracentrifugation from MDA-MB-231 breast cancer cell cultures, proteins were then separated by 2-DE, and eventually analyzed by MALDI-TOF [23] searching for possible specific biomarkers. Among the 179 identified proteins, 32 isoforms were upregulated in the vesicles in comparison to whole cell lysates, suggesting that the observed distinctive proteome could represent the signature of breast cancer vesicles that play key roles in cancer progression. MALDI-TOF and 2-DE and were also used [24] to compare the proteins secreted by two different breast cancer cell lines (BT474 and SKBR3) with those released by normal human mammary epithelial cells (184A1) and with the proteomes of the corresponding whole cell lysates, to understand how cell-to-cell communication becomes altered in the presence of cancer, hypothesizing that the tumor secretome could reflect the editing process associated with oncogenesis. Similar secretome profiles were found for BT474 and SKBR3 cells, whereas 184A1 cells exhibited different patterns. The results implied that all the investigated cell lines released exosomes and that those secreted by tumor cells presented higher amounts of proteins involved in antigen-processing and presentation and glycolytic metabolism, pathways associated with the evasion of tumor immunosurveillance and the deregulating of cellular energetics, two of the emerging cancer signatures. In another study, plasma concentrations of exosomes were found to be higher in breast cancer patients than in healthy women [25]. Electrophoresis separation showed exosomal proteins between 10 and 250 kDa and differences between healthy and cancer subjects in the expression level and number of proteins that were planned to be identified by MALDI TOF in view of their potential utility as breast cancer markers. [PERSON] et al. isolated total blood exosomes (plasma exosomes and those associated with blood cells) from healthy donors and breast cancer patients by ultrafiltration followed by ultracentylation in two different works, focused on the search for new protein tumor markers for breast cancer [26] and the assessment of the role of blood exosomes in tumor dissemination [27], respectively. In the first work, the maps of the proteome contained in the vesicles were obtained by 2D-E and compared between the two sets of samples, permitting the individuation of remarkable differences between normal and pathological subjects. A MALDI-TOF analysis allowed the identification of 99 proteins in the blood exosomes of both the investigated groups and, according to the exosome database Exocarta, some of them were observed for the first time. Interestingly, proteins associated with breast cancer were specifically detected in the total blood exosomes of cancer patients, even if further experiments were required to validate their role as biomarkers for the disease. In the second study, a MALDI analysis allowed the identification of 111 and 146 proteins in the blood exosomes (48 for the first time) of the healthy and pathological samples, respectively.