An official website of the United States government
Extracellular vesicles (EVs)-mediated cellular communication plays a role in cancer development and progression. This study focuses on identifying glioblastoma-specific EV protein markers through a comparative mass spectrometry bottom-up proteomic analysis of the LN-229 cell line and human neurons, astrocytes, and endothelial brain cells (HEBCs) using timsTOF Pro 2 instrument. The statistically significant upregulated proteins with fold change greater than 2 in the glioblastoma-derived EVs were clustered based on physical and functional interactions using the STRING database and analyzed using Gene Ontology enrichment. LN229-derived EVs contained an average of 2,635 proteins, while human astrocytes, neurons, and HEBC encapsulated 2,647, 716, and 2285 proteins, respectively. NanoParticle Tracking Analysis indicated that glioblastoma-derived EVs exhibited greater size variability compared to EVs from healthy cells. Statistical analysis identified 25 statistically significant proteins with increased levels in LN229 EVs relative to at least two healthy cell lines suggesting their potential as glioblastoma markers. Functional clustering using the STRING database and GO analysis indicated involvement in epigenetic regulation, metastasis, angiogenesis, and protein folding. Post-translational modification analysis identified a subset of 17 proteins unique to the cancer-derived EVs involved in chromatin regulation, extracellular matrix remodeling, and basement membrane organization pathways, highlighting their role in tumor progression.
more »
« less
Extraction of redox extracellular vesicles using exclusion-based sample preparation
Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.
more »
« less
- Award ID(s):
- 2154934
- PAR ID:
- 10586770
- Publisher / Repository:
- Springer Nature
- Date Published:
- Journal Name:
- Analytical and Bioanalytical Chemistry
- Volume:
- 416
- Issue:
- 28
- ISSN:
- 1618-2642
- Page Range / eLocation ID:
- 6317 to 6331
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
High-grade serous ovarian cancer (HGSOC) constitutes the majority of all ovarian cancer cases and has staggering rates of both refractory and recurrent disease. While most patients respond to the initial treatment with paclitaxel and platinum-based drugs, up to 25% do not, and of the remaining that do, 75% experience disease recurrence within the subsequent two years. Intrinsic resistance in refractory cases is driven by environmental stressors like tumor hypoxia which alter the tumor microenvironment to promote cancer progression and resistance to anticancer drugs. Recurrent disease describes the acquisition of chemoresistance whereby cancer cells survive the initial exposure to chemotherapy and develop adaptations to enhance their chances of surviving subsequent treatments. Of the environmental stressors cancer cells endure, exposure to hypoxia has been identified as a potent trigger and priming agent for the development of chemoresistance. Both in the presence of the stress of hypoxia or the therapeutic stress of chemotherapy, cancer cells manage to cope and develop adaptations which prime populations to survive in future stress. One adaptation is the modification in the secretome. Chemoresistance is associated with translational reprogramming for increased protein synthesis, ribosome biogenesis, and vesicle trafficking. This leads to increased production of soluble proteins and extracellular vesicles (EVs) involved in autocrine and paracrine signaling processes. Numerous studies have demonstrated that these factors are largely altered between the secretomes of chemosensitive and chemoresistant patients. Such factors include cytokines, growth factors, EVs, and EV-encapsulated microRNAs (miRNAs), which serve to induce invasive molecular, biophysical, and chemoresistant phenotypes in neighboring normal and cancer cells. This review examines the modifications in the secretome of distinct chemoresistant ovarian cancer cell populations and specific secreted factors, which may serve as candidate biomarkers for aggressive and chemoresistant cancers.more » « less
-
This study investigates the therapeutic effect of astrocyte-derived extracellular vesicles (EVs) in mitigating neurotoxicity-induced transcriptome changes, mitochondrial function, and base excision repair mechanisms in human brain endothelial cells (HBECs). Neurodegenerative disorders are marked by inflammatory processes impacting the blood–brain barrier (BBB) that involve its main components- HBECs and astrocytes. Astrocytes maintain homeostasis through various mechanisms, including EV release. The effect of these EVs on mitigating neurotoxicity in HBECs has not been investigated. This study assesses the impact of astrocyte-derived EVs on global transcriptome changes, cell proliferation, cytotoxicity, oxidative DNA damage, and mitochondrial morphology in HBECs exposed to the neurotoxic reagent Na2Cr2O7. Exposure to Na2Cr2O7 for 5 and 16 h induced oxidative DNA damage, measured by an increase in genomic 8OHdG, while the EVs reduced the accumulation of the adduct. A neurotoxic environment caused a non-statistically significant upregulation of the DNA repair enzyme OGG1 while the addition of astrocyte-derived EVs was associated with the same level of expression. EVs caused increased cell proliferation and reduced cytotoxicity in Na2Cr2O7-treated cells. Mitochondrial dysfunction associated with a reduced copy number and circular morphology induced by neurotoxic exposure was not reversed by astrocyte-derived EVs. High-throughput RNA sequencing revealed that exposure to Na2Cr2O7 suppressed immune response genes. The addition of astrocyte-derived EVs resulted in the dysregulation of long noncoding RNAs impacting genes associated with brain development and angiogenesis. These findings reveal the positive impact of astrocytes-derived EVs in mitigating neurotoxicity and as potential therapeutic avenues for neurodegenerative diseases.more » « less
-
Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic and stem cell-like. Biophysical stresses such as FSS are also known to cause the production of extracellular vesicles (EVs) that can facilitate cell-cell communication by carrying biomolecular cargos such as microRNAs. Here, we hypothesized that physiological FSS will impact the yield of EV production, and that these EVs will have biomolecules that transform the recipient cells. The EVs were isolated using direct flow filtration with and without FSS from the MDA-MB-231 cancer cell line, and the expression of key stemness-related genes and microRNAs was characterized. There was a significantly increased yield of EVs under FSS. These EVs also contained significantly increased levels of miR-21, which was previously implicated to promote metastatic progression and chemotherapeutic resistance. When these EVs from FSS were introduced to MCF-7 cancer cells, the recipient cells had a significant increase in their stem-like gene expression and CD44+/CD24− cancer stem cell-like subpopulation. There was also a correlated increased proliferation along with an increased ATP production. Together, these findings indicate that the presence of physiological FSS can directly influence the EVs’ production and their contents, and that the EV-mediated transfer of miR-21 can have an important role in FSS-existing contexts, such as in cancer metastasis.more » « less
-
null (Ed.)One of the challenges of exploiting extracellular vesicles (EVs) as a disease biomarker is to differentiate EVs released by similar cell types or phenotypes. This paper reports a high-throughput and label-free EV microarray technology to differentiate EVs by simultaneous characterization of a panel of EV membrane proteins. The EsupplV microarray platform, which consists of an array of antibodies printed on a photonic crystal biosensor and a microscopic hyperspectral imaging technique, can rapidly assess the binding of the EV membrane proteins with their corresponding antibodies. The EV microarray assay requires only a 2 μL sample volume and a detection time of less than 2 h. The EV microarray assay was validated by not only quantifying seven membrane proteins carried by macrophage-derived EVs but also distinguishing the EVs secreted by three macrophage phenotypes. In particular, the EV microarray technology can generate a molecular fingerprint of target EVs that can be used to identify the EVs' parental cells, and thus has utility for basic science research as well as for point-of-care disease diagnostics and therapeutics.more » « less
