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

The Journal of steroid biochemistry and molecular biology (2016-02-16)
Hanne Roberg-Larsen, Kaja Lund, Kristina Erikstad Seterdal, Stian Solheim, Tore Vehus, Nina Solberg, Stefan Krauss, Elsa Lundanes, Steven Ray Wilson
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Millipore
ANTI-FLAG® antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
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Anti-Actin antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
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25-Hydroxycholesterol, ≥98%
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1,3-Propylene sulfite, 99%