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.