New insights into size, protein and lipid composition may help in characterizing functionality of Extracellular Vesicles (EVs) and infer with their fast development as delivery tools. Unfortunately, despite latest size-based classifications which divide EVs in small (50-80nm) or large (80-120nm) exosomes, microvesicles (<1000nm) and the new smallest (<50nm) population (exomeres), overlapping of different EV populations and unproper separation methods impair the comprehension of their biological role. In addition (EVs) are a very attractive pharmacological target, due to their involvement in cell-cell communication in physiological and pathological conditions. Among lipids, cholesterol and BisMonoacylGlyceroPhosphate (BMP) play a paramount role in EV biogenesis since their interplay drive endosomes towards the secretory or recycling pathway. We used as model a lymph node metastatic cell line (LM-16). 1)We set up a reproducible ultracentrifugation (UC) method for a size-based separation of different EV populations by 5 UC steps, in which physical and dynamic parameters are determined by an algorithm developed by Livshits et al. 2) In vitro treatment LM-16 cells with simvastatin and/or KT182, respectively inhibitors of cholesterol biosynthesis and of BMP degradation, was carried for 3 days with 10% FCS and for 3 days in serum free conditions. In this case we only isolated two fractions according to canonical EVs isolation method, namely 10K (microvesicles) and 100K (exosomes). EVs were further characterized by Nanosight, Zetasizer, Confocal and Electron microscopy. Quantitative proteomic analysis was performed by mass spectrometry and results analyzed by Ingenuity Pathway Analysis (IPA) while lipid analysis was carried out by gas-liquid chromatography. 1) Zetasizer and TEM analysis documented the existence of 5 different EV populations, whose relative % in saturated fatty acids gradually and continuously increased from larger to smaller EV (from 37.21±0.21 to 64.79±9.47). Proteomics identified a total of 2003 proteins differentially distributed (or even unique) among the 5 EV populations (n=697, 819, 1079, 1621 and 1654, respectively). IPA analysis of these distributions revealed different characteristics signaling pathways. 2) Treatment with simvastatin (0.1µM) and KT182 (50nM) alone or in combination did not affect cell proliferation, and EVs size and counts. Proteomics identified 1294 and 1192 proteins respectively for 100K and 10K, showing different significant expression patterns, compared to control, with several proteins up- and down regulated. IPA showed that treatments specifically decreased the expression of proteins involved in cellular movement, migration, proliferation and cytoskeleton arrangement, both in 100K and 10K fractions, with typical and striking differences and patterns. Melanoma-released EVs include vesicles of different size, fatty acid and protein composition. These differences may translate into distinct behaviors and functions in biological fluids and help to define the role of specific EV populations in physiological and pathological processes. Cholesterol and BMP modulation of melanoma cells dramatically alters the protein content of released EVs, possibly leading to altered EVs functionality, suggesting the potential of lipid modulation in reshaping tumor cell-released EVs to develop new therapeutic approaches. Finally, these results pave the road to new pharmacological treatments to modulate EVs functions or to use EVs as pharmaceutical tools or melanoma biomarkers.

MAY A PHARMACOLOGICAL MODULATION OF LIPIDS ALTER PROTUMORIGENIC SIGNALING OF EXTRACELLULAR VESICLES? A LONG JOURNEY IN EVS DIMENSIONAL, LIPIDIC AND PROTEIC CHARACTERIZATION FROM A METASTATIC MELANOMA CELL LINE' / F.m. Accattatis ; coordinatore: A. L.Catapano ; coordinatore: G. D. Norata ; tutor: A. Llorente, A. Corsini ; co-tutor: L. Arnaboldi. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Apr 23. ((33. ciclo, Anno Accademico 2020. [10.13130/accattatis-felice-maria_phd2021-04-23].

MAY A PHARMACOLOGICAL MODULATION OF LIPIDS ALTER PROTUMORIGENIC SIGNALING OF EXTRACELLULAR VESICLES? A LONG JOURNEY IN EVS DIMENSIONAL, LIPIDIC AND PROTEIC CHARACTERIZATION FROM A METASTATIC MELANOMA CELL LINE'

F.M. Accattatis
2021

Abstract

New insights into size, protein and lipid composition may help in characterizing functionality of Extracellular Vesicles (EVs) and infer with their fast development as delivery tools. Unfortunately, despite latest size-based classifications which divide EVs in small (50-80nm) or large (80-120nm) exosomes, microvesicles (<1000nm) and the new smallest (<50nm) population (exomeres), overlapping of different EV populations and unproper separation methods impair the comprehension of their biological role. In addition (EVs) are a very attractive pharmacological target, due to their involvement in cell-cell communication in physiological and pathological conditions. Among lipids, cholesterol and BisMonoacylGlyceroPhosphate (BMP) play a paramount role in EV biogenesis since their interplay drive endosomes towards the secretory or recycling pathway. We used as model a lymph node metastatic cell line (LM-16). 1)We set up a reproducible ultracentrifugation (UC) method for a size-based separation of different EV populations by 5 UC steps, in which physical and dynamic parameters are determined by an algorithm developed by Livshits et al. 2) In vitro treatment LM-16 cells with simvastatin and/or KT182, respectively inhibitors of cholesterol biosynthesis and of BMP degradation, was carried for 3 days with 10% FCS and for 3 days in serum free conditions. In this case we only isolated two fractions according to canonical EVs isolation method, namely 10K (microvesicles) and 100K (exosomes). EVs were further characterized by Nanosight, Zetasizer, Confocal and Electron microscopy. Quantitative proteomic analysis was performed by mass spectrometry and results analyzed by Ingenuity Pathway Analysis (IPA) while lipid analysis was carried out by gas-liquid chromatography. 1) Zetasizer and TEM analysis documented the existence of 5 different EV populations, whose relative % in saturated fatty acids gradually and continuously increased from larger to smaller EV (from 37.21±0.21 to 64.79±9.47). Proteomics identified a total of 2003 proteins differentially distributed (or even unique) among the 5 EV populations (n=697, 819, 1079, 1621 and 1654, respectively). IPA analysis of these distributions revealed different characteristics signaling pathways. 2) Treatment with simvastatin (0.1µM) and KT182 (50nM) alone or in combination did not affect cell proliferation, and EVs size and counts. Proteomics identified 1294 and 1192 proteins respectively for 100K and 10K, showing different significant expression patterns, compared to control, with several proteins up- and down regulated. IPA showed that treatments specifically decreased the expression of proteins involved in cellular movement, migration, proliferation and cytoskeleton arrangement, both in 100K and 10K fractions, with typical and striking differences and patterns. Melanoma-released EVs include vesicles of different size, fatty acid and protein composition. These differences may translate into distinct behaviors and functions in biological fluids and help to define the role of specific EV populations in physiological and pathological processes. Cholesterol and BMP modulation of melanoma cells dramatically alters the protein content of released EVs, possibly leading to altered EVs functionality, suggesting the potential of lipid modulation in reshaping tumor cell-released EVs to develop new therapeutic approaches. Finally, these results pave the road to new pharmacological treatments to modulate EVs functions or to use EVs as pharmaceutical tools or melanoma biomarkers.
CORSINI, ALBERTO
NORATA, GIUSEPPE DANILO
Extracellular Vesicles; EV; exosome; microvesicle; BMP; lipid; cholesterol; pharmacology; cancer; melanoma; metastasis; proteomics.
Settore BIO/14 - Farmacologia
MAY A PHARMACOLOGICAL MODULATION OF LIPIDS ALTER PROTUMORIGENIC SIGNALING OF EXTRACELLULAR VESICLES? A LONG JOURNEY IN EVS DIMENSIONAL, LIPIDIC AND PROTEIC CHARACTERIZATION FROM A METASTATIC MELANOMA CELL LINE' / F.m. Accattatis ; coordinatore: A. L.Catapano ; coordinatore: G. D. Norata ; tutor: A. Llorente, A. Corsini ; co-tutor: L. Arnaboldi. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Apr 23. ((33. ciclo, Anno Accademico 2020. [10.13130/accattatis-felice-maria_phd2021-04-23].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/831719
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