Mass-spectrometric characterization of peroxidized and hydrolyzed lipids in plasma and dendritic cells of tumor-bearing animals

Abstract

Dendritic cells are the most potent antigen presenting cells responsible for the development of immune responses in cancer. However, it is known that the function of dendritic cells in tumor-bearing hosts is severely compromised. Our previous studies demonstrated that the defects in dendritic cell function are due, to a large extent, to the accumulation of high amounts of lipids--predominantly triglycerides--in a substantial proportion of dendritic cells in tumor-bearing mice and patients with cancer. The dendritic cells accumulation of lipids is likely associated with their up-regulation of a scavenger receptor A. This receptor is primarily responsible for uptake of modified lipids. Here, by using different versions of liquid chromatography-mass spectrometry, we identified several molecular species of oxygenated lipids in plasma of tumor-bearing animals that may be responsible for their uptake and accumulation by dendritic cells via scavenger receptor A-dependent pathway--the effect that may be associated with the loss of dendritic cell's immune surveillance function in cancer.

Figure 1

LC/ESI-MS analysis of FFA molecular species isolated from plasma and DC from C57BL/6 naïve tumor-free and EL-4 tumor-bearing mice. Typical LC/ESI-MS profile of FFA (A) and oxidized FFA (FFAox) (A, insert) from plasma of EL-4 tumor-bearing mice. Content of non-oxidized FFA in mouse plasma (B) and DCs (C). Open bars – naïve mice, closed bars - tumor bearing mice. Data are mean ± SD of at least three experiments. *P<0.05 in samples from tumor bearing mice vs naïve mice.

Figure 2

Identification of oxidized FFA. LC/ESI-MS reconstructed profiles of hydroxy-FFA isolated from plasma of EL-4 tumor-bearing mice (left panels) and their MS/MS spectra (right panels). Possible structures of hydroxy-FFA are presented as inserts. A - molecular ion with m/z 295 corresponding to C_18:2–OH (a mixture of 13S-HODE and 9S-HODE). B - molecular ion with m/z 319 corresponding to C_20:4–OH (12S-HETE). C - molecular ion with m/z 265 corresponding to tetranor-C_20:4–OH (tetranor-12S-HETE). D - molecular ion with m/z 343 corresponding to C_22:6–OH (16-HDoHE).

Figure 3

Levels of oxygenated FFA in DC isolated from C57BL/6 naïve and C57BL6/Msr1^−/− mice. DCs were maintained either in complete medium supplemented with GM-CSF or in the same medium containing TES as described in the Methods section.

Figure 4

LC/ESI-MS analysis of TAG molecular species in DC isolated from C57BL/6 naïve tumor-free and EL-4 tumor-bearing mice. LC/ESI-MS reconstructed profiles (A) of TAG molecular species with m/z 848 from naïve and EL-4 tumor bearing (TB) mice. MS/MS spectrum (in the range of m/z from 530 to 600) of TAG with m/z 848 from DC isolated from TB (insert). Quantitative assessment of non-oxidized (B) and oxidized (C) TAG with m/z 848 in DC. Note that oxidized TAG molecular species with m/z 848 corresponding to C_16:1/C_18:2-OH/C_15:0 were detected in DC from EL-4 tumor bearing mice only. Data are mean ± SD. *P<0.05 vs DC from naïve mice.