BackgroundmicroRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring.MethodsExosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis.ResultsSmall RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels.ConclusionsOur results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification.

Introduction: microRNAs are promising candidate biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. The key to identifying breast cancer-derived microRNAs relies on capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. Methods: Exosomes were collected from the conditioned media of breast cancer cell lines, breast ductal fluids, mouse plasma from patient-derived breast tumor orthotopic xenograft models (PDX), and from human plasma samples. Exosomes were verified by electron microscopy and western blot analysis. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome populations were selected by immunoaffinity isolation utilizing antibodies against CD63 and MUC1. Exosome microRNA expression was measured by qRT-PCR. Results: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively expressed in breast cancer exosomes. Importantly, human breast cancer specific microRNAs were detectable in PDX mouse plasma. The microRNA expression patterns in the MUC1-precipitated plasma exosomes differed between breast cancer patients and control subjects. These results provide a potential new strategy to selectively analyze plasma breast cancer microRNAs that may be indicative of the presence of breast cancer. Conclusions: Several microRNAs are selectively enriched in breast cancer exosomes, which can be detected in the plasma of PDX mice and breast cancer patients. MUC1 is a viable membrane protein candidate for selective capture of circulating breast cancer specific exosomes from the plasma. These results suggest that selective capture and molecular analysis of breast cancer specific circulating exosomes is a promising strategy for breast cancer biomarker development. Citation Format: Bethany N. Hannafon, Yvonne D. Trigoso, David H. Lum, Alana L. Welm, William C. Dooley, Wei-Qun Ding. Exosome-associated microRNAs as plasma biomarkers for breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A55.