Abstract
Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”. EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy.
Highlights
Extracellular vesicles (EVs) are released by cells into the extracellular space and are classified according to their size and biogenesis [1, 2]
EVs derived from oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC), and melanoma brain metastasis cell lines were isolated from the cell culture supernatant combining UF and size-exclusion chromatography (SEC), and were characterized by nanoparticle tracking analysis (NTA), western blot (WB), immunoaffinity capture, and transmission electron microscopy (TEM) (Fig 1)
In the present study we used liquid chromatography-mass spectrometry (LC-Mass spectrometry (MS)) to characterize the protein content of small EVs derived from OSCC, PDAC, and melanoma brain metastasis cell lines
Summary
Extracellular vesicles (EVs) are released by cells into the extracellular space and are classified according to their size and biogenesis [1, 2]. EVs are quite abundant in biofluids as they are continuously released by cells [2]. E.g., cancer, the amount of EVs in the biofluids increases [5]. The EVs in the blood of cancer patients are released both by normal and cancer cells, and their number is estimated to be twice of that found in the blood of healthy individuals [6,7,8]. Oncogenes in cancer-derived EVs can modulate normal host cells, e.g. fibroblasts and macrophages, as well as local cancer cells and metastatic cells [9,10,11]. Tumor-derived EVs can contribute to and maintain the Hallmarks of cancer, a panel of acquired abilities of malignant tumors such as cancer cell proliferation, evasion of growth suppressors, resistance to cell death, migration, and invasion as well as modulating normal cells to favor tumor progression by transforming the microenvironment into a more permissive one [12,13,14,15]
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