Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples.

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system ("AFFL"). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.

Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes.

Exosomes from cancer cells are rich sources of biomarkers and may contain elevated levels of lipids of diagnostic value. 27-Hydroxycholesterol (27-OHC) is associated with proliferation and metastasis in estrogen receptor positive (ER+) breast cancer. In this study, we investigated the levels of 27-OHC, and other sidechain-hydroxylated oxysterols in exosomes. To study both cytoplasmic and exosomal oxysterol samples of limited size, we have developed a capillary liquid chromatography-mass spectrometry platform that outperforms our previously published systems regarding chromatographic resolution, analysis time and sensitivity. In the analyzed samples, the quantified level of cytoplasmic 27-OHC using this platform fitted with mRNA levels of 27-OHC's corresponding enzyme, CYP27A1. We find clearly increased levels of 27-OHC in exosomes (i.e., enrichment) from an ER+ breast cancer cell line (MCF-7) compared to exosomes derived from an estrogen receptor (ER-) breast cancer cell line (MDA-MB-231) and other control exosomes (non-cancerous cell line (HEK293) and human pooled serum). The exosomal oxysterol profile did not reflect cytoplasmic oxysterol profiles in the cells of origin; cytoplasmic 27-OHC was low in ER+ MCF-7 cells while high in MDA-MB-231 cells. Other control cancer cells showed varied cytoplasmic oxysterol levels. Hence, exosome profiling in cancer cells might provide complementary information with the possibility of diagnostic value.