Size Dependent Lipidomic Analysis of Urinary Exosomes from Patients with Prostate Cancer by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Exosomes are membrane-bound extracellular vesicles involved in intercellular communication and tumor cell metastasis. In this study, flow field-flow fractionation (FlFFF) was utilized to separate urinary exosomes by size, demonstrating a significant difference in exosome sizes between healthy controls and patients with prostate cancer (PCa). Exosome fractions of different sizes were collected for microscopic analysis during an FlFFF run and evaluated with exosome marker proteins using Western blot analysis. The results indicated that exosomes of different sizes originated from different types of cells. Collected exosome fractions were further examined using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for lipidomic analysis. A total of 162 lipids (from 286 identified) were quantified using a selected reaction monitoring (SRM) method. The overall amount of lipids increased by 1.5- to 2-fold in patients with PCa and degree of increase was more significant in the smaller fractions (diameter <150 nm) than in the larger ones (diameter >150 nm) some classes of lipids. In addition, neutral lipids like diacylglycerol (DAG) and triacylglycerol (TAG) decreased in all exosomes without size dependency. Moreover, a dramatic increase in 22:6/22:6-phosphatidylglycerol (PG) was observed and significant decrease in (16:0,16:0)- and (16:1, 18:1)-DAG species (nearly 5-fold) and high abundant TAG species (>2.5-fold) was observed in patients with PCa. The results of this study indicate that FlFFF can be employed for the high-speed screening of urinary exosome sizes in patients with PCa and lipidomic analysis of the fractionated exosomes has potential for developing and distinguishing biomarkers of PCa.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

“…[103]). Such methods include tangential flow filtration and variations thereon [21,[104][105][106][107][108][109][110], field-flow fractionation (FFF) [111], asymmetric flow field-flow fractionation (AFFF, A4F, or AF4) [112][113][114], field-free viscoelastic flow [115], alternating current electrophoretics [116,117], acoustics [118], variations on size exclusion chromatography (SEC) [100,[119][120][121], ion exchange chromatography [122][123][124], microfiltration [125], fluorescenceactivated sorting [126,127] (especially for larger EVs including large apoptotic bodies [128] and large oncosomes [129]), deterministic lateral displacement (DLD) arrays [130], novel immunoisolation or other affinity isolation technologies [131][132][133][134][135][136][137][138], including lipid affinity [139], novel precipitation/combination techniques [140][141][142], hydrostatic filtration dialysis [143], ...…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…In TFF the fluid flows tangentially across the surface, avoiding the filter clogging. Other filtration methods, such as field flow fractionation (FFF) [ 99 ] and AF4 [ 29 ], were successfully used to separate exosomes by size. Recently, a “hydrostatic filtration dialysis” method was also developed to enrich human urinary EVs, and characterize the integral transmembrane proteins present [ 100 ].…”

Extracellular vesicles in the tumor microenvironment: Therapeutic resistance, clinical biomarkers, and targeting strategies

“…In addition to the frequently used MS‐based proteomic platforms, quantitative analysis via isobaric tags for relative and absolute quantification (iTRAQ) provides another option, as illustrated by a new study that detected approximately 700–800 EV proteins per serum sample of cancer patients, some of which are implicated in metastasis and treatment resistance . A flow field‐flow fractionation (FlFFF) approach was employed to separate urinary EVs of PCa patients, revealing the significant difference in EV sizes between these patients and healthy controls . Nanoflow ultrahigh performance liquid chromatography‐electrospray ionization‐tandem mass spectrometry (nUPLC‐ESI‐MS/MS) was subsequently used to examine the collected EV fractions for lipidomic analysis, indicating a 1.5‐ to twofold increase of overall lipid amount in PCa patients with the increase more significant in the smaller fractions than in the larger ones (a 150 nm diameter borderline) .…”

“…137 Nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) was subsequently used to examine the collected EV fractions for lipidomic analysis, indicating a 1.5-to twofold increase of overall lipid amount in PCa patients with the increase more significant in the smaller fractions than in the larger ones (a 150 nm diameter borderline). 137 As the unique nature of the EV bilayer confers its RNA molecules with remarkable resistance against degradation by cellular RNAses even after a storage of extended time period, EV-specific miRNA signatures can be developed via qRT-PCR and QuantStudio TM (a three-dimensional real-time digital PCR system designed by Applied Biosystems) integrated with a 20K Chip. 138 The techniques are recently documented in a protocol that described the full procedure of capturing EV samples from cell culture medium via standard ultracentrifugation or OptiPrep density gradient fractionation, which is also adaptable for analysis of miRNAs upon EV isolation from biofluids including plasma, serum, or urine.…”