Limits...
Plasma exosome microRNAs are indicative of breast cancer

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5016889&req=5

Fig4: 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

Mentions: We reasoned that mice bearing human breast tumors should have circulating human exosomes and sought to determine whether selectively secreted human exosome microRNAs are detectable in the plasma of these mice. Plasma was collected from nine Huntsman Cancer Institute-PDX (HCI-PDX) mice models [20] and three non-tumor-bearing nod-scid gamma (NSG) mice at the time of killing (see PDX information in Additional file 2). Whole exosomes were isolated from the plasma of a PDX mouse and analyzed by nanoparticle tracking analysis (Fig. 4a). The exosomes were further purified by magnetic-bead-based immunoaffinity isolation using an antibody against human CD63. Total RNA was extracted from the bead-bound exosomes and the expression of miR-1246, miR-451 and miR-122 were measured by qRT-PCR.Fig. 4


Plasma exosome microRNAs are indicative of breast cancer
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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016889&req=5

Fig4: 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
Mentions: We reasoned that mice bearing human breast tumors should have circulating human exosomes and sought to determine whether selectively secreted human exosome microRNAs are detectable in the plasma of these mice. Plasma was collected from nine Huntsman Cancer Institute-PDX (HCI-PDX) mice models [20] and three non-tumor-bearing nod-scid gamma (NSG) mice at the time of killing (see PDX information in Additional file 2). Whole exosomes were isolated from the plasma of a PDX mouse and analyzed by nanoparticle tracking analysis (Fig. 4a). The exosomes were further purified by magnetic-bead-based immunoaffinity isolation using an antibody against human CD63. Total RNA was extracted from the bead-bound exosomes and the expression of miR-1246, miR-451 and miR-122 were measured by qRT-PCR.Fig. 4

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus