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. 2021 Nov 18:12:748895.
doi: 10.3389/fphys.2021.748895. eCollection 2021.

A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential

Simona Lobasso  1 , Paola Tanzarella  1 , Francesco Mannavola  2   3 , Marco Tucci  2   3 , Francesco Silvestris  2   3 , Claudia Felici  2   3   4 , Chiara Ingrosso  5 , Angela Corcelli  1 , Patrizia Lopalco  1
Affiliations

A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential

Simona Lobasso et al. Front Physiol. .

Abstract

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

Keywords: MALDI-TOF/MS; lipids; melanoma; membrane vesicles; osteotropism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Lipidomics workflow for melanoma cells and exosomes. Lipid extract from melanoma cells has been analyzed by MALDI-TOF/MS and coupled TLC and MALDI-TOF/MS. After exosomes have been purified from supernatants of LCP and SK-Mel28 cells, small amounts of intact exosomes and cells were loaded on the MALDI target and lipids were directly analyzed by MS in order to perform statistical analysis.
FIGURE 2
FIGURE 2
MALDI-TOF/MS analysis of individual lipid bands isolated from melanoma cells by TLC. The total lipid extract of cells (LCP cell line) was loaded on the plate (120 μg per each lane); TLC was stained with iodine vapors (temporary staining of all classes of lipids). Six lipid bands (B1–B6) were marked with a pencil, and silica was scraped; lipids were extracted from silica and analyzed by MALDI-TOF/MS. The same TLC plate is shown on the left of both panels. (A) shows the MALDI-TOF/MS spectra of the lipid bands acquired in positive ion mode, while (B) shows the mass spectra of the lipid bands acquired in negative ion mode.
FIGURE 3
FIGURE 3
Negative ion mode MALDI-TOF/MS lipid profiles of two melanoma cell lines and the derived exosomes. The upper spectra show the typical lipid profiles of LCP (A) and SK-Mel28 (B) cells by the intact method. The lower spectra show the typical lipid profiles of intact exosomes derived from LCP (C) and SK-Mel28 (D) cells. The enlargements of range m/z 1,100–1,500 of mass spectra are also shown. GPs, glycerophospholipids; SPs, sphingolipids; GSLs, glycosphingolipids; CLs, cardiolipins.
FIGURE 4
FIGURE 4
Significant differences of (–) MALDI signals in lipid profiles of two cell lines (upper panels) and the derived exosomes (lower panels). The histograms (A–G) show the significant differences in intensity between the lipid peaks present in the four series of (–) mass spectra. p-value < 0.05 was set as the threshold to define significant differences. Significant differences between the signals detected in the cell lines profiles (shown in the upper panels) are also highlighted by an asterisk. Data are reported as the average value of intensity ± SD. The numbers reported between upper and lower panels indicate the MALDI m/z signals. Lipid assignments for each signal are also indicated.
FIGURE 5
FIGURE 5
Positive ion mode MALDI-TOF/MS lipid profiles of two melanoma cell lines and the derived exosomes. The upper spectra show the typical lipid profiles of LCP (A) and SK-Mel28 (B) cells by the intact method. The lower spectra show the typical lipid profiles of intact exosomes derived from LCP (C) and SK-Mel28 (D) cells. The enlargements of range m/z 480–560 of mass spectra are also shown.
FIGURE 6
FIGURE 6
Significant differences of (+) MALDI signals in lipid profiles of two cell lines (upper panels) and the derived exosomes (lower panels). The histograms (A–C) show the significant differences in intensity between the lipid peaks present in the four series of (+) mass spectra. p-value < 0.05 was set as the threshold to define significant differences. Significant differences between the signals detected in the cell lines profiles (shown in the upper panels) are also highlighted by an asterisk. Data are reported as the average value of intensity ± SD. The numbers reported between upper and lower panels indicate the MALDI m/z signals. Lipid assignments for each signal are also indicated.
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