Plasma exosome microRNAs are indicative of breast cancer

Abstract

Background: microRNAs 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.

Methods: Exosomes 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.

Results: Small 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.

Conclusions: Our 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.

Keywords: Biomarker; Breast cancer; Exosomes; Patient-derived xenograft; microRNA.

Fig. 1

Characterization of exosomes from breast cancer cell lines. Exosomes were isolated from the conditioned media of MCF10A, MCF7, and MDA-MB-231 cells. a Nanoparticle tracking analysis of 50X diluted MCF10A, MCF7 and MDA-MB-231 exosomes. b MCF7 and MDA-MB-231 exosomes were visualized by electron microscopy (×30,000). c MCF7 and MDA-MB-231 exosomes were immunogold-labeled and visualized by electron microscopy of human CD63-gold particles (10 nM gold) (right × 40,000; left × 100,000). d Western blot of CD63 (30–60 kDa) under non-reducing conditions

Fig. 2

Small RNA next generation sequencing analysis of microRNAs in mammary epithelial cells and breast cancer cell lines versus exosomes. Cellular and exosome RNA was isolated from normal mammary cells (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). microRNAs were differentially expressed in MCF10A (a), MCF7 (c) and MDA-MB-231 (e) exosomes with a fold-change ≥6.0 (exosome vs. cell, p < 0.05) with mapped reads ≥ 200 are plotted. The abundance of the microRNAs highly expressed in exosomes vs. the cellular RNA in MCF10A (b), MCF7 (d) and MDA-MB-231 (f) are plotted by their mean number of reads per million reads

Fig. 3

qRT-PCR analysis of microRNAs in breast cancer cell line exosomes versus mammary epithelial cell exosomes. qRT-PCR analysis of selected microRNAs that are abundantly present in MCF7, ZR-75-1, T47D, MDA-MB-231, BT-20, BT-474, and SK-BR-3 exosomes vs. MCF10A exosomes. Fold-change in expression is shown for the exosome microRNAs relative to their cellular microRNA levels and normalized to the spike-in cel-miR-54 control

Fig. 4

Characterization and microRNA expression analysis of human exosomes isolated from the plasma of patient-derived xenograft (PDX) mice. Exosomes were isolated from the plasma of Huntsman Cancer Institute human breast cancer orthotopic xenograft (HCI-PDX) and nod scid gamma (NSG) mice. a Exosomes were characterized by nanoparticle tracking analysis of 500X diluted exosomes (n = 3). b-c Exosomes were isolated from the mouse plasma exosome sample by magnetic-bead based immunoaffinity isolation using an antibody against human CD63, the total RNA was extracted and the expression of miR-1246 and miR-122 were evaluated by qRT-PCR analysis (absolute quantitation) in the immunoaffinity isolated human CD63-positive exosomes from the plasma of three NSG (n = 3, in triplicate) and nine HCI-PDX models (with available biological replicates as indicated in c, in triplicate); Student’s t test; ***p < 0.001. The corresponding clinical biomarkers including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) amplification status and the PAM50 subtype of the human tumor (if known) are indicated and are available in Additional file 2

Fig. 5

qRT-PCR analysis of exosome microRNA expression in normal plasma and plasma from patients with breast cancer. Plasma samples were collected under an Institutional Review Board approved protocol from healthy women with no history of cancer (n = 16, mean age 42 years, age range 27–66) and women with breast cancer (n = 16, mean age 59.6 years, range 42–80; see Table 3). Exosomes were isolated from plasma samples using the Exoquick reagent and total RNA was extracted. a Human plasma exosomes were visualized by electron microscopy (×30,000). b Candidate microRNA expression was measured in normal plasma (n = 16) and plasma from patients with breast cancer (n = 16) by qRT-PCR. Student’s t test, *p < 0.05

Fig. 6

Receiver operator characteristic (ROC) analysis of microRNA expression in plasma exomes from normal subjects and patients with breast cancer. ROC curves for classifying plasma exosomes (breast cancer vs. normal) were produced using each microRNA expression value for miR-21 (a) and miR-1246 (b) separately, and for miR-21 and miR-1246 combined (c). For each patient sample and each microRNA, the average of three replicate expression values was computed. The resulting microRNA expression values were standardized for the statistical analysis. The area under the curve (AUC) with 95 % CI were computed for each ROC curve. The Wilcoxon-Mann-Whitney test was used to test the null hypothesis that the AUC is equal to 0.5 (i.e., no predictive power)