Saturated and mono-unsaturated lysophosphatidylcholine metabolism in tumour cells: a potential therapeutic target for preventing metastases

Abstract

Background: Metastasis is the leading cause of mortality in malignant diseases. Patients with metastasis often show reduced Lysophosphatidylcholine (LysoPC) plasma levels and treatment of metastatic tumour cells with saturated LysoPC species reduced their metastatic potential in vivo in mouse experiments. To provide a first insight into the interplay of tumour cells and LysoPC, the interactions of ten solid epithelial tumour cell lines and six leukaemic cell lines with saturated and mono-unsaturated LysoPC species were explored.

Methods: LysoPC metabolism by the different tumour cells was investigated by a combination of cell culture assays, GC and MS techniques. Functional consequences of changed membrane properties were followed microscopically by detecting lateral lipid diffusion or cellular migration. Experimental metastasis studies in mice were performed after pretreatment of B16.F10 melanoma cells with LysoPC and FFA, respectively.

Results: In contrast to the leukaemic cells, all solid tumour cells show a very fast extracellular degradation of the LysoPC species to free fatty acids (FFA) and glycerophosphocholine. We provide evidence that the formerly LysoPC bound FFA were rapidly incorporated into the cellular phospholipids, thereby changing the FA-compositions accordingly. A massive increase of the neutral lipid amount was observed, inducing the formation of lipid droplets. Saturated LysoPC and to a lesser extent also mono-unsaturated LysoPC increased the cell membrane rigidity, which is assumed to alter cellular functions involved in metastasis. According to that, saturated and mono-unsaturated LysoPC as well as the respective FFA reduced the metastatic potential of B16.F10 cells in mice. Application of high doses of liposomes mainly consisting of saturated PC was shown to be a suitable way to strongly increase the plasma level of saturated LysoPC in mice.

Conclusion: These data show that solid tumours display a high activity to hydrolyse LysoPC followed by a very rapid uptake of the resulting FFA; a mechanistic model is provided. In contrast to the physiological mix of LysoPC species, saturated and mono-unsaturated LysoPC alone apparently attenuate the metastatic activity of tumours and the artificial increase of saturated and mono-unsaturated LysoPC in plasma appears as novel therapeutic approach to interfere with metastasis.

Fig. 1

LysoPC removal and FA incorporation by solid tumour cells and leukaemic cells. a: Removal of exogenously added LysoPC 17:0 (450 μM) in cell culture supernatants of solid tumour cells (n = 3 measurements). b: Time course of cellular FA C17:0 ratio of LysoPC 17:0 treated solid tumour cells (450 μM). c: Time course of the degradation of LysoPC 17:0 (450 μM) from cell culture supernatant of leukaemic cells (n = 6 measurements). d: Incorporation of FA C17:0 into cellular lipids due to LysoPC treatment of leukaemic cells (n = 3 measurements). Changes in FA C17:0 contents are shown in % of total FA in time course experiments

Fig. 2

Metabolism of saturated and unsaturated LysoPC and FFA species by solid tumour cell. a: Time course removal of exogenously added saturated and unsaturated LysoPC species (18:0 and 18:1) in cell culture supernatants of solid tumour cell lines B16.F10, MV3, and AsPC1 cells. b: Changes of cellular lipid FA ratios of FA C18:0 and FA C18:1 (in % of total FA) in LysoPC (C18:0 and C18:1) treated cells. c: FA composition in B16.F10 cells after treatment with two LysoPC species simultaneously in different ratios (for 48 h). d: Release of FFA to cell culture supernatants, analysed by an enzymatic FFA assay. B16.F10 mouse melanoma cells were incubated with LysoPC 17:0, C18:1, and BSA medium. Media without contact to cells were analysed as control. e: Comparison of incorporation of different LysoPC species and the corresponding FFA into B16.F10 mouse melanoma cells. Increases of the respective FA are shown in time course experiments. f: Comparison of LysoPC degradation in B16.F10 tumour cells (green) with the degradation in supernatant after removal of the tumour cells (blue), and LysoPC containing medium without cell contact (red). B16.F10 cells were cultivated in LysoPC 17:0 medium. After 6 h, supernatant was separated from the cells and further incubated (blue)

Fig. 3

FA incorporation into PL and neutral lipid fraction and LD formation. Changes in the FA distribution of B16.F10 tumour cells treated with LysoPC 17:0 over 5 days. a: Lipid fractions, neutral lipids and PL were individually analysed and total FA are displayed as area counts of gas chromatography measurement (n = 4 measurements). b/c: Fluorescence Laser Scanning Microscope images of B16.F10 melanoma cells after staining with DAPI (blue) and BODIPY (green). b: Cells cultivated in control medium DMEM-BSA, c: Cells treated with LysoPC 17:0 (450 μM) for 16 days

Fig. 4

Effects of saturated and unsaturated LysoPC species on membrane fluidity and cell migration. a: Measurement of half-life of fluorescence recovery of B16.F10 cells with NBD-PE C18:1 as the fluorescent probe at a temperature of 37 °C. Higher values indicate slower lateral movement and lower membrane fluidity. Control cells in DMEM (n = 17), BSA cells, LysoPC 18:0, and LysoPC 18:1 (n = 17-18), mean ± SD; * p ≤ 0.05, *** p ≤ 0.001. b: Speed of two dimensional cell migration detected by scratching assay onto an uncoated or collagen-coated surface of B16.F10 cells untreated or pre-incubated with BSA containing LysoPC 18:0 or LysoPC 18:1 media (450 μmol/l); (n = 2)

Fig. 5

In vivo effects of pre-incubated B16.F10 cells with saturated and unsaturated LysoPC and FFA species. Luciferase activity of homogenised mouse lungs analysed by luciferase assay (day 18). a: mean values ± SEM of mice which received different pre-treated B16.F10 cells (n = 10 for each group). b: single values of each mouse ± SEM. Values compared by ANOVA: n.s. p ≥ 0.05, * p ≤ 0.05, ** p ≤ 0.01. c: Pictures of mouse’s lungs after necroscopy, examples of two mice per group are shown

Fig. 6

LysoPC plasma level in healthy mice and tumour bearing mice. Total LysoPC plasma levels of healthy mice (n = 4) and mice intravenously injected with B16.F10 melanoma cells; one (n = 3), two (n = 3) and three (n = 3) weeks after injection of tumour cells. LysoPC levels were determined by HPLC MS/MS analysis. Values compared by ANOVA: * p ≤ 0.05, ** p ≤ 0.01

Fig. 7

Manipulation of the LysoPC plasma levels and its effects on metastatic spreading: a: Luciferase activity of mouse lung homogenates from mice without tumour cell injection (healthy control mice, n = 5) and mice injected with B16.F10 tumour cells receiving different treatments: mice receiving supplemented chow starting one month before tumour cell injection (EPC3pro, n = 4) and starting after tumour cell injection (EPC3ther, n = 5); mice with LysoPC s.c. injections (n = 5) and tumour control mice (no therapy, n = 5). b: Mean values ± SD of plasma LysoPC levels before (n = 9) and after (n = 7) i.v. injection of liposomes, plasma LysoPC levels determined by HPLC MS/MS analysis

Fig. 8

LysoPC removal and FA incorporation by different tumour cell lines. Correlation of LysoPC elimination from cell culture supernatant and incorporation of the respective FA into cellular lipids after 24 h of incubation. Blue: leukaemic cell lines, red: solid tumour cell lines, (including human melanoma, breast cancer, prostate cancer, and pancreatic cancer cells)

Fig. 9

Proposed uptake/metabolism of LysoPC in tumour cells. The majority of LysoPC is extracellularly degraded to GlyceroPC and FFA by a LysoPC degrading factor. This factor – probably a LysoPLA – is partly released into the supernatant of the tumour cells. The resulting extracellular FFA can subsequently be taken up and incorporated into membrane PL and neutral lipids. Excess of neutral lipids can be stored as LD. Another possible uptake route for LysoPC is its incorporation into the cellular membrane as a whole molecule, where it becomes part of the Lands’ Cycle: the deacylation and reacylation process