Palmitic Acid, As Verified Using Lipid Profiling By Secondary Ion Mass Spectrometry, Demonstrates Anti-Tumor Activity Against Multiple Myeloma
Authors: Nagata Yasuyuki , Ishizaki Itsuko , Waki Michihiko , Ide Yoshimi , Hossen Md Amir , Ohnishi Kazunori , Setou Mitsutoshi 
Published: October 2019

Abstract
Introduction Many recent studies have examined lipid metabolic changes in multiple myeloma (MM). Changes in lipid metabolism affect the survival of MM cells. Developments in imaging mass spectrometry (IMS) have facilitated research on the lipid profiles of tumors. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an IMS technique that uses a focused ion beam as the primary source for ionization. TOF-SIMS imaging is used to analyze the surface of specimens at sub-micrometer resolution, enabling analyses of the subcellular distribution of molecules in individual cells. TOF-SIMS analysis has enabled the detection of multiple fatty acid groups from single cells. Therefore, we applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. Using the different lipid profiles for MM cells and normal plasma cells (PCs), we conducted a cytocidal assay with MM cell lines supplemented with the fatty acids screened out by the profiles to assess lipotoxicity against MM. The molecules demonstrating distinct differences among cell types (i.e., MM and PC) were considered candidates for which supplementation leads to imbalanced lipid metabolism and cell death in a tumor-specific manner. We further evaluated the induction of apoptosis.
Methods Primary patient MM cells and normal PCs were isolated from the bone marrow aspirates of two patients and two healthy volunteers using fluorescence-activated cell sorting. These separated cells were analyzed with PHI TRIFT V (ULVAC-PHI, Inc.). Analyses were performed in negative ion mode, and signals in the mass range of m/z 0 to 1850 were monitored. We performed pairwise comparisons of mean signal intensities for five types of fatty acids between MM cells and PCs.
MM cell lines (U266 and RPMI-8226) were treated with 0–1000 µM of palmitic acid, palmitoleic acid, linoleic acid, oleic acid, and stearic acid. The number of viable cells in suspension at 72 hours after treatment was determined by the trypan blue exclusion test. HS-5, a human bone marrow stromal cell line, was used in the co-culture experiment. Healthy volunteers’ normal peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density-gradient centrifugation. The distribution of apoptotic and necrotic cells were analyzed by measuring AnnexinV binding and propidium iodide uptake.
Results The amounts of MM cells and PCs relative to the total nucleated cells were 3.38%, 35.9% for MM cells, 0.0368% and 0.246% for PCs. Multiple ions, including phosphoric acid, and five species of fatty acids (palmitoleic acid, palmitic acid, linoleic acid, oleic acid, and stearic acid) were detected. The mean signal intensities of palmitoleic acid and palmitic acid of MM cells were significantly lower than those of normal PCs (P = .00081 and .0018, respectively). These results were replicated in a second pairwise comparison. We did not observe statistically significant differences in intensities for linoleic acid, oleic acid, or stearic acid.
In the cytocidal assay, palmitic acid reduced U266 cell viability dose-dependently for doses of 50–1000 μM. High concentrations of the other fatty acids also reduced cell viability; however, the effect on cell death was not observed at the low dose of 50–100 µM, as it was for palmitic acid. Even in co-culture experiments, palmitic acid decreased the viability of MM cells. Moreover, the proportions of both apoptotic and necrotic cells increased and the proportion of viable cells decreased 24 hours after palmitic acid treatment in MM cells. Palmitic acid also reduced the viability of RPMI-8226 cell lines. Meanwhile, cell viabilities of normal PBMCs were not affected by palmitic acid, even at 100–500 µM.
Conclusion We applied the single-cell TOF-SIMS lipid analysis effectively to a very small population of cells. Significantly smaller intensities of palmitoleic acid and palmitic acid were observed in MM cells compared to normal cells. We also demonstrated an inhibitory effect of palmitic acid on the survival of MM cells. Palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM and should be considered in future studies of MM in a lipid biology framework.

Disclosures
No relevant conflicts of interest to declare.