Glioblastoma is one of the most aggressive forms of brain cancer, characterized by rapid metastasis [1], treatment resistivity [2], and low survival rates [3]. Only 3 % of patients survive the five-year mark post-diagnosis, [3] a statistic that underscores the inadequacy of current treatments that include invasive surgery and chemotherapy [1]. Glioblastoma is a malignancy that originates from astrocytes, which play various supportive roles in the central nervous system, including maintaining the integrity of the blood-brain barrier (BBB) [4]. The BBB is a protective barrier that regulates the passage of substances between the bloodstream and the brain. Glioblastoma affects astrocyte-vascular interactions within the BBB and the brain's microenvironment which contributes to the challenges in treating this type of brain cancer [5]. Glioblastoma poses a diagnostic challenge due to the rapid rate of metastasis that quickly manifests in more pronounced symptoms, including seizures and hearing loss [6]. The aggressive nature of glioblastoma, combined with its origin in cells that contribute to the BBB, makes it difficult to remove the tumor without affecting surrounding healthy brain tissue. This complexity underscores the need for early detection strategies and targeted therapeutic approaches to glioblastoma. Factors that increase glioblastoma risk include age-dependent changes in the central nervous system, such as decreased immune function [7], genetic mutations in the p53 tumor suppressor gene [8], and exposure to high-level radiation [9]. Strategies focused on reducing metastasis include immunotherapy [10] and oncolytic virotherapy [11]. Due to the diverse genetic nature of the glioblastoma malignancy, the therapeutic translation of these promising approaches has not been successful in clinical trials [12].

Glioblastoma-derived extracellular vehicles (EVs) play a significant role in intercellular communication, encapsulating proteins, nucleic acids, and lipids, which can serve as potential biomarkers for the disease. Glioblastomas have diverse molecular characteristics, and the identification of EV-derived proteomic markers may contribute to the development of less invasive and personalized medical treatment by providing a more comprehensive understanding of the specific molecular profile of each patient's tumor [13]. Proteomic analysis indicates that the content of the EVs mirrors the phenotypic signature of the respective glioblastoma cells [14]. Previous work identified the circular RNA EV-derived circSMARCA5 and circHIPK3 as promising prognostic serum indicators [15]. High-throughput proteomic analysis of six cell glioblastoma cell lines identified ANXA1, ITGB1, ACTR3, ALIX, CALR, and IGF2R as potential markers of an aggressive tumor [16]. Mass spectrometry analysis of EVs derived from five glioblastoma cell lines identified a subset of 133 proteins associated with resistance to treatment, invasion, angiogenesis, and the modulation of the immune response [17].

While these studies provide a good map of EV-derived proteomic markers across different glioblastoma cell lines, they don't include a comparison of the expression profiles with healthy cells. This study aims to conduct a mass spectrometry analysis of EVs derived from the LN-229 glioblastoma cell line, human astrocytes, neurons, and human endothelial brain cells (HEBCs) (Fig. 1). This approach involves creating an in vitro model that includes a diverse EVs derived from several cell types as a more accurate representation of the complex environment in the brain. This work aims to identify glioblastoma-specific EV-derived proteomic markers that advance the understanding of cellular communication of brain cancer cells.