Protein Profiling of Extracellular Vesicles Associated With Cisplatin Resistance in Lung Cancer

Abstract

Background/Aim: Extracellular vesicles (EVs) can mediate drug resistance within the tumor microenvironment by delivering bioactive molecules, including proteins. Here, we performed a comparative proteomic analysis of EVs secreted by A549 lung cancer cells and their cisplatin-resistant counterparts in order to identify proteins involved in drug resistance. Materials and Methods: Cells were co-cultivated using a transwell system to evaluate EV exchange. EVs were isolated by ultracentrifugation and analyzed using microscopy and nanoparticle tracking. EV proteome was analyzed by mass spectrometry. Results: EV-mediated communication was observed between co-cultured A549 and A549/CDDP cells. EVs isolated from both cells were mainly exosome-like structures. Extracellular matrix components, cell adhesion proteins, complement factors, histones, proteasome subunits and membrane transporters were found enriched in the EVs released by cisplatin-resistant cells. Conclusion: Proteins identified in this work may have a relevant role in modulating the chemosensitivity of the recipient cells and could represent useful biomarkers to monitor cisplatin response in lung cancer.Lung cancer is the most common cancer worldwide and the leading cause of cancer death in both men and women (1). Nonsmall cell lung cancer (NSCLC) is the most prevalent type of lung cancer, accounting for approximately 85% of all lung cancer cases (2). Most patients with NSCLC have non operable, advanced-stage disease at the time of diagnosis and platinumbased chemotherapy is the first-line treatment for these patients (3). However, the high incidence of tumor chemoresistance limits treatment success (4). Although numerous mechanisms have been associated with cisplatin (CDDP) resistance in lung cancer (5, 6), the cellular events within the tumor microenvironment that facilitate the acquisition and maintenance of chemoresistance are still poorly understood.Recently, extracellular vesicles (EVs) have emerged as key players for cell-cell communication in the tumor microenvironment (7). EVs are a heterogeneous group of membrane-bound structures secreted by cells into the extracellular space. Based on their size and biogenesis, EVs can be broadly classified into two main categories: exosomes and microvesicles (8). Exosomes are small membrane vesicles (30-100 nm in diameter) formed as intraluminal vesicles within endosomal multivesicular bodies (MVBs), and secreted upon the fusion of MVBs with the plasma membrane. Microvesicles (MVs) range in size from 50 nm to 1000 nm in diameter, but the cancer-derived MV population termed oncosomes can be much larger (up to 10 μm) (9). MVs are generated by the outward budding and fission of the plasma membrane.EVs mediate cellular crosstalk by transporting bioactive cargo between cells, including proteins, lipids, and nucleic acids (10). The EV cargo composition determine its biological effects and varies depending on the cell type and physiological/pathological state (11). In the tumor microenvironment, EVs have been shown to med...

Complement in Tumourigenesis and the Response to Cancer Therapy

“…Increasing evidence suggests that exosomes play a role in treatment resistance, both by acting as a decoy for immunotherapies and by exporting drugs from cancer cells [244,245]. A recent proteomic analysis has demonstrated that complement components including C3 and CD55 are among the proteins enriched in exosomes derived from cisplatin resistant lung cancer cells, relative to cisplatin sensitive cells [241]. As previously discussed, emerging evidence has indicated that CD55 is associated with resistance to chemotherapy [176,179].…”

“…Complement components are among the many proteins associated with exosomes, a subject recently reviewed in detail [237]. Briefly, complement proteins have been identified in exosomes secreted from numerous cell types including antigen-presenting cells [238,239] and cancer cells [240,241]. Tumour cells can also eliminate surface MAC complexes by shedding microvesicles, allowing them to escape from complement-mediated lysis [242,243].…”

Multi-Omics Data Integration in Extracellular Vesicle Biology—Utopia or Future Reality?

“…Most often, proteins are extracted using a lysis buffer with or without detergents and are digested before the MS analysis [62]. Such attempts have been useful in evaluating the differential protein expression of EV cargo in normal cells as compared to cancer cells [82], map chemotherapy-induced variations [83], mediate tumor education and metabolic reprogramming [84], chemoresistance, and other functional adaptations of the recipient cells [85].…”

Proteomic Signature of Extracellular Vesicles for Lung Cancer Recognition

“…In a recent study using high-resolution mass spectrometry, it was found that proteins such as extracellular matrix components, cell-adhesion proteins, complement factors, histones, proteasome subunits, and membrane transporters were upregulated in EVs originating from cisplatin-resistant A549 lung cancer cells, as compared to their wild-type counterparts [8]. On the basis of shotgun mass spectrometry followed by Western blot (WB) and ELISA assays, lipopolysaccharide-binding proteins (LBPs) allowed EVs derived from the blood of metastatic and non-metastatic NSCLC patients to be distinguished [9].…”