Lipid profiles of prostate cancer cells

Abstract

Lipids are important cellular components which can be significantly altered in a range of disease states including prostate cancer. Here, a unique systematic approach has been used to define lipid profiles of prostate cancer cell lines, using quantitative mass spectrometry (LC-ESI-MS/MS), FTIR spectroscopy and fluorescent microscopy. All three approaches identified significant difference in the lipid profiles of the three prostate cancer cell lines (DU145, LNCaP and 22RV1) and one non-malignant cell line (PNT1a). Specific lipid classes and species, such as phospholipids (e.g., phosphatidylethanolamine 18:1/16:0 and 18:1/18:1) and cholesteryl esters, detected by LC-ESI-MS/MS, allowed statistical separation of all four prostate cell lines. Lipid mapping by FTIR revealed that variations in these lipid classes could also be detected at a single cell level, however further investigation into this approach would be needed to generate large enough data sets for quantitation. Visualisation by fluorescence microscopy showed striking variations that could be observed in lipid staining patterns between cell lines allowing visual separation of cell lines. In particular, polar lipid staining by a fluorescent marker was observed to increase significantly in prostate cancer lines cells, when compared to PNT1a cells, which was consistent with lipid quantitation by LC-ESI-MS/MS and FTIR spectroscopy. Thus, multiple technologies can be employed to either quantify or visualise changes in lipid composition, and moreover specific lipid profiles could be used to detect and phenotype prostate cancer cells.

Keywords: FTIR; LC-ESI-MS/MS; lipid dyes; lipid profiles; prostate cancer.

Figure 1. Quantitative LC-ESI-MS/MS data analysis of the relative abundances of lipids in prostate cell lines

(a) Comparison of average concentrations [nmol mg^-1 protein] of (CE) cholesteryl esters, (SM) sphingomyelin, (FC) free cholesterol, (PE) phosphatidylethanolamine, (PC) phosphatidylcholine and (GM) gangliosides in prostate cell lines for six parallel samples with their standard error. One-way ANOVA and Tukey's multiple comparison tests showed significant differences among the means for the samples (depicted by different letters on the bars, p < 0.05). (b) The PCA scores plot comparing non-malignant PNT1a (black circles) and prostate cancer cell lines DU145 (green squares), 22RV1 (blue triangles) and LNCaP (red diamonds), using identified lipid species. (c) Loadings plot of PCA for PC-1 (equal to 78%). (d) Comparison of average concentrations [nmol mg^-1 protein] of lipids that allowed the differentiation of non-malignant PNT1a and prostate cancer cell lines, DU145, 22RV1 and LNCaP (p < 0.05). Data presented as mean ± SEM of six independent biological replicates for each of the four prostate cell lines.

Figure 2. FTIR spectroscopy on prostate cancer cells

(a-d) Optical and (a^/-d^/) FTIR images collected by integrating the area under the ν_s(CH₂) band (2862–2847 cm^-1) and generated from (a, a^/) PNT1a and prostate cancer cell lines, (b, b^/) DU145, (c, c^/) 22RV1 and (d, d^/) LNCaP. (e) Average FTIR spectra from PNT1a (black) and prostate cancer cell lines, DU145 (green), 22RV1 (blue) and LNCaP (red), acquired over the 3600–900 cm^-1 spectral region, where (A) C–H and (B) C=O bands were located. (f) Averaged spectra of the C–H stretching region (3000–2800 cm^-1) with band assignment. (g) Second-derivative spectra of the C–H stretching region shown in f. (h) Second-derivative spectra of the C=O stretching region.

Figure 3. Distribution of lipids in prostate cancer cells

(a-l) Micrographs of cross-sections through prostate cells that show the intracellular location of neutral and polar lipids. Cholesterol was depicted by staining cells with Filipin III (a-d). Neutral lipids such as triglycerides and cholesteryl esters were detected by staining cells with BODIPY^® 493/503 (e-h). ReZolve-L1™ (i-l) was used for staining polar lipids. Representative images from non-malignant PNT1a (a, e, i) and prostate cancer DU145 (b, f, j), 22RV1 (c, g, k) and LNCaP (d, h, l) cell lines. Prostate cells were fixed with 4% PFA (a-h) or imaged live (i-l). Scale bars, 20 μm.