Low Vacuum Filtration Method as an Alternative Extracellular Vesicle Concentration Method: Comparison with Ultracentrifugation and Differential Centrifugation

Drożdż, Anna; Kamińska, Agnieszka; Surman, Magdalena; Gonet-Surówka, Agnieszka; Jach, Robert; Huras, Hubert; Przybyło, Małgorzata; Stępień, Ewa

doi:10.20944/preprints202007.0655.v1

2020

DOI: 10.20944/preprints202007.0655.v1

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Low Vacuum Filtration Method as an Alternative Extracellular Vesicle Concentration Method: Comparison with Ultracentrifugation and Differential Centrifugation

Anna Drożdż¹,

Agnieszka Kamińska²,

Magdalena Surman³

et al.

Abstract: Recent years brought great focus in the field of development of extracellular vesicles (EVs) based drug-delivery systems. Considering possible applications of EVs as a drug carriers the isolation process is a crucial step. To solve problems related with EV isolation, we created and validated a new EVs isolation method – Low Vacuum Filtration (LVF) and compared it with two commonly applied procedures - differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cells c… Show more

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Cited by 4 publications

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Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry

Surman

Jankowska

Wilczak

et al. 2023

Cancers

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Intercellular transport of proteins mediated by extracellular vesicles (EVs)—exosomes and ectosomes—is one of the factors facilitating carcinogenesis. Therefore, the research on protein cargo of melanoma-derived EVs may provide a better understanding of the mechanisms involved in melanoma progression and contribute to the development of alternative biomarkers. Proteomic data on melanoma-derived EVs are very limited. The shotgun nanoLC-MS/MS approach was applied to analyze the protein composition of primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) cutaneous melanoma cells and exosomes released by them. All cells secreted homogeneous populations of exosomes that shared a characteristic set of proteins. In total, 3514 and 1234 unique proteins were identified in melanoma cells and exosomes, respectively. Gene ontology analysis showed enrichment in several cancer-related categories, including cell proliferation, migration, negative regulation of apoptosis, and angiogenesis. The obtained results broaden our knowledge on the role of selected proteins in exosome biology, as well as their functional role in the development and progression of cutaneous melanoma. The results may also inspire future studies on the clinical potential of exosomes.

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Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry

Surman

Jankowska

Wilczak

et al. 2023

Cancers

View full text Add to dashboard Cite

show abstract

Comparative Proteomic Profiling of Ectosomes Derived from Thyroid Carcinoma and Normal Thyroid Cells Uncovers Multiple Proteins with Functional Implications in Cancer

Surman

Kędracka–Krok

Wilczak

et al. 2022

Cells

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Proteins carried by tumor-derived ectosomes play an important role in cancer progression, and are considered promising diagnostic markers. In the present study, a shotgun nanoLC–MS/MS proteomic approach was applied to profile and compare the protein content of ectosomes released in vitro by normal human thyroid follicular epithelial Nthy-ori 3-1 cells and human anaplastic thyroid carcinoma (TC) 8305C cells. Additionally, the pro-migratory and pro-proliferative effects of Nthy-ori 3-1- and 8305C-derived ectosomes exerted on the recipient cells were assessed in wound closure and Alamar Blue assays. A total of 919 proteins were identified in all replicates of 8305C-derived ectosomes, while Nthy-ori 3-1-derived ectosomes contained a significantly lower number of 420 identified proteins. Qualitative analysis revealed 568 proteins present uniquely in 8305C-derived ectosomes, suggesting their applicability in TC diagnosis and management. In addition, 8305C-derived ectosomes were able to increase the proliferation and motility rates of the recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. Our description of ectosome protein content and its related functions provides the first insight into the role of ectosomes in TC development and progression. The results also indicate the applicability of some of these ectosomal proteins for further investigation regarding their potential as circulating TC biomarkers.

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The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

Surman,

Wilczak,

Bzowska

et al. 2024

Cells

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Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvβ3 and αvβ5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvβ3 and αvβ5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvβ3 and αvβ5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvβ5 integrin/VEGF than the αvβ3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvβ3 and αvβ5 integrins. It also provided a baseline understanding of ectosome-mediated αvβ3 integrin/TNF-α and αvβ5 integrin/VEGF signaling in angiogenesis.

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Low Vacuum Filtration Method as an Alternative Extracellular Vesicle Concentration Method: Comparison with Ultracentrifugation and Differential Centrifugation

Cited by 4 publications

References 0 publications

Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry

Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry

Comparative Proteomic Profiling of Ectosomes Derived from Thyroid Carcinoma and Normal Thyroid Cells Uncovers Multiple Proteins with Functional Implications in Cancer

The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

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