MR-visible lipids and the tumor microenvironment

Abstract

MR-visible lipids or mobile lipids are defined as lipids that are observable using proton MRS in cells and tissues. These MR-visible lipids are composed of triglycerides and cholesterol esters that accumulate in neutral lipid droplets, where their MR visibility is conferred as a result of the increased molecular motion available in this unique physical environment. This review discusses the factors that lead to the biogenesis of MR-visible lipids in cancer cells and in other cell types, such as immune cells and fibroblasts. We focus on the accumulations of mobile lipids that are inducible in cultured cells by a number of stresses, including culture conditions, and in response to activating stimuli or apoptotic cell death induced by anticancer drugs. This is compared with animal tumor models, where increases in mobile lipids are observed in response to chemo- and radiotherapy, and to human tumors, where mobile lipids are observed predominantly in high-grade brain tumors and in regions of necrosis. Conducive conditions for mobile lipid formation in the tumor microenvironment are discussed, including low pH, oxygen availability and the presence of inflammatory cells. It is concluded that MR-visible lipids appear in cancer cells and human tumors as a stress response. Mobile lipids stored as neutral lipid droplets may play a role in the detoxification of the cell or act as an alternative energy source, especially in cancer cells, which often grow in ischemic/hypoxic environments. The role of MR-visible lipids in cancer diagnosis and the assessment of the treatment response in both animal models of cancer and human brain tumors is also discussed. Although technical limitations exist in the accurate detection of intratumoral mobile lipids, early increases in mobile lipids after therapeutic interventions may be useful as a potential biomarker for the assessment of treatment response in cancer.

Figure 1

One-dimensional 1H MR spectra of HBL-100 transformed human breast cells treated with (a) PBS (control cells), (b) 6.25 μM TPP, (c) 6.25 μM TPP and 25 μM chlorpromazine for 48 h. Spectrum (a) from control cells shows resonances only from mobile amino acids (the mobile proteome) and small molecular weight metabolites. Spectrum (b) from cells treated with the mitochondrial toxin TPP is dominated by resonances from MR-visible or mobile lipids at 0.9, 1.3, 1.6, 2.0, 2.8 and 5.3 ppm. Spectrum (c) shows that pretreatment with the lysomotropic agent chlorpromazine substantially reduced mobile lipid resonances indicating lysosomal involvement in drug-induced lipid accumulations. Abbreviations: cho: choline, PC: phosphocholine, GPC: glycerophosphocholine, Cr: creatine, PCr: phosphocreatine, lys: lysine, val: valine, ile: isoleucine, leu: leucine, PABA: para-aminobenzoic acid, a chemical shift and concentration reference that was added in an internal capillary. Adapted from [2] and reprinted with permission.

Figure 2

Metabolic flow chart showing the phosphatidylcholine cycle of phospholipid metabolism and the pathways leading to the production of triglycerides. The final step in triglyceride synthesis occurs in the endoplasmic reticulum and is the condensation of an activated fatty acid (fa-S-CoA) with diacylglycerol (DG) via the enzyme diacylglycerolacyltransferase (DGAT). The flow chart shows other key pathways in phosphatidylcholine catabolism. Most notably, during cell stress the catabolic production of GPC arises from the consecutive removal of fatty acyl chains from phosphatidylcholine by phospholipase A2 and lysosphospholipase. The fatty acids released can be reesterified into phospholipids or converted into triglycerides. Enzymes: DGAT, diacylglycerolacyltransferase (E.C. 2.3.1.20); GPC-PDE, GPC-phosphodiesterase (E.C. 3.1.4.2); PLA2, phospholipase A2 (E.C. 3.1.1.4); l-PL, lysophospholipase (E.C. 3.1.1.5); PLC, phospholipase C (E.C. 3.1.4.3); PLD, phospholipase D (E.C. 3.1.4.4); PPH, phosphatidate phosphohydrolase (E.C. 3.1.3.4). Metabolites: cho, choline; DG, 1,2-diacylglycerol; PtdOH, phosphatidic acid, PC, phosphocholine, PtdCho, phosphatidylcholine; TG, triacylglycerol (triglycerides). Adapted from [46] and reprinted with permission.

Figure 3

Induction of MR-visible lipids by differentiating agents in prostate cancer cells. The upper left panel shows ¹H MR spectra of DU145 cells grown on Biosilon beads and perfused in the magnet. From the top, the three traces are: treated with 10 mM PB for 16 h, treated with 10 mM phenylacetate for 16 h, control. The top right panel shows the relative increase of the methylene to water ratio (1.3 ppm / 4.7 ppm). The relative increase in MR-visible lipid is greater when cells are treated with PB compared to phenylacetate or control. The bottom left panel shows oil red O and electron micrographs of control cells (left) and cells treated with PB (right), demonstrating the increase in cytoplasmic lipid droplets that accompany drug treatment. The bottom right panel plots number of lipid droplets per cell (circles) and the cellular volume fraction occupied by lipid droplets (squares) as a function of the methylene to water ratio showing a linear relationship for both measures for control, phenylacetate and PB-treated cells. Adapted from [46] and [45] and reprinted with permission.

Figure 4

MR-visible lipid spectra can be modulated by external serum levels. On the left, ¹H MR 1D spectra obtained from neutrophils incubated in Hanks' balanced salt solution (control), AB serum containing low levels of fatty acids, lipopolysaccharide (LPS), and CSL serum containing high levels of fatty acids. The levels of lipid accumulation are higher in the CSL serum than in cells stimulated with LPS. The arrows indicate the lipid methylene peak at 1.3 ppm, the methyl peak at 0.9 ppm, and the taurine/choline peak at 3.2 ppm. The internal standard, p-aminobenzoic acid produces two doublets, one at 6.83 and the other at 7.83 ppm. On the right hand side, the level of mobile lipids in human mixed peripheral blood lymphocyte cultures is directly dependent on the amount of human serum in the culture medium. Note in both sets of spectra the presence of resonances at 3.2 and 3.4 ppm indicating high levels of taurine as well as choline. Taurine is present in the cytoplasm of immune cells at high concentrations where it acts as an osmolyte and radioprotectant.

Figure 5

Sequential single voxel MR spectra from an HSV-tk positive rat BT4C glioma. The spectrum from the tumor before treatment (day 0), shows strong resonances from the mobile lipid methylene resonance at 1.3 ppm and tCho at 3.2 ppm. Treatment with ganciclovir leads to increases in the PUFA resonances (shaded peaks) at 2.8 and 5.3 ppm, as well as in the mobile lipid resonances at 0.9 and 1.3 ppm. These increases were highest at 4 days, which correlated with maximum apoptosis. A decrease in all metabolite resonances was seen after 8 days of treatment, which correlated with formation of scar tissue in the tumor. Reprinted with permission from [50].

Figure 6

MR images of a rat brain containing an HSV-tk positive BT4C glioma undergoing ganciclovir treatment. The top row shows T₂-weighted coronal plane images with the region of interest for the CSI studies (box). The spatial change in tCho, mobile lipids (0.9 and 1.3 ppm) and PUFA (5.3 ppm) after treatment is shown by the CSI maps. An increase in mobile lipid concentration was observed for 8 days followed by reduction to baseline levels. This correlated with a decrease in tCho levels. Reprinted with permission from [89].

Figure 7

Representative MR spectra and histological sections from a subcutaneously implanted human diffuse large B cell lymphoma xenograft showing changes in ML resonances after radiation therapy. (a) The top left panel shows spectra from a tumor before treatment and at 24 h and 72 h after 15 Gy irradiation. Labeled resonances are from: 1: lipid methyl (CH₃ at 0.9 ppm); 2: methylene ((-CH₂-)_n at 1.3 ppm); 3: polyunsaturated fatty acid (-CH=CH-CH₂-CH=CH- at 2.8 ppm) and 4: unsaturated fatty acids (-CH=CH- at 5.3 ppm). The ratio of the mobile lipid resonance at 1.3 ppm to unsuppressed water area (upper right panel) shows a significant increase in mobile lipids in treated tumors (grey bars) in comparison to untreated control tumors (black bars). The histological sections stained with hematoxylin and oil red O in the bottom panel shows the increase in perinuclear lipid droplets (red) after 72 h treatment (right) compared to controls (left). Adapted from [49] and reprinted with permission.

Figure 8

Fluid attenuated inversion recovery (FLAIR, A) and post contrast enhanced T₁-weighted MRI image (B) from a patient with primary cerebral diffuse large B cell lymphoma (PCL). Boxes in (B) show voxels from contrast-enhancing and peritumoral regions. The MR spectra from the contrast enhanced area (C) as well as the peritumoral region (D) show elevated choline and mobile lipid resonances indicating the infiltrative nature of the neoplasm. The mobile lipid resonances are probably due to the presence of infiltrative lymphocytes. Reprinted with permission from [137].