The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.

The biology, function, and biomedical applications of exosomes

The sustained growth, invasion, and metastasis of cancer cells depend upon bidirectional cell-cell communication within complex tissue environments. Such communication predominantly involves the secretion of soluble factors by cancer cells and/or stromal cells within the tumour microenvironment (TME), although these cell types have also been shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell-cell communication. These particles are known as extracellular vesicles (EVs) and include species of exosomes and shed microvesicles. EVs carry molecules such as oncoproteins and oncopeptides, RNA species (for example, microRNAs, mRNAs, and long non-coding RNAs), lipids, and DNA fragments from donor to recipient cells, initiating profound phenotypic changes in the TME. Emerging evidence suggests that EVs have crucial roles in cancer development, including pre-metastatic niche formation and metastasis. Cancer cells are now recognized to secrete more EVs than their nonmalignant counterparts, and these particles can be isolated from bodily fluids. Thus, EVs have strong potential as blood-based or urine-based biomarkers for the diagnosis, prognostication, and surveillance of cancer. In this Review, we discuss the biophysical properties and physiological functions of EVs, particularly their pro-metastatic effects, and highlight the utility of EVs for the development of cancer diagnostics and therapeutics.

Extracellular vesicles in cancer - implications for future improvements in cancer care.

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

Liquid biopsy refers to the sampling and molecular analysis of the biofluids of circulating tumor cells, extracellular vesicles, nucleic acids, and so forth. Exosomes are small extracellular vesicles with sizes between 30–150 nm. They are secreted by multivesicular bodies through exocytosis in live cells and can participate in intercellular communication due to their contents, including nucleic acids, proteins, and lipids. Herein, we investigate publication frequencies on exosomes over the past 10 years, and review recent clinical studies on liquid biopsy of exosomes in the fields of oncology, pregnancy disorders, cardiovascular diseases, and organ transplantation. We also describe the advantages of exosomes as an effective liquid biopsy tool and the progression of exosome extraction methods. Finally, we depict the commercial development of exosome research and discuss the future role of exosomes in liquid biopsy.

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Application of exosomes as liquid biopsy in clinical diagnosis.

Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for noninvasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic microbeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome comple...

Single-Exosome-Counting Immunoassays for Cancer Diagnostics.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1+ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1+ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR-96-5p and miR-149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1+ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR-96-5p and miR-149 significantly decreased GPC1 expression in HT-29 and HCT-116 cells, xenograft tumours, plasma in mice bearing HT-29 and HCT-116 tumours, and the secretion of GPC1+ exosomes from the HT-29 and HCT-116 cells and xenograft tumours. Overexpression of miR-96-5p and miR-149 significantly decreased cell viability and increased cell apoptosis in HT-29 and HCT-116 cells, and inhibited the growth of xenograft HT-29 and HCT-116 tumours. In conclusion, the increased plasma GPC1+ exosomes and reduced plasma miR-96-5p and miR-149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jcmm.12941

GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

Nasopharyngeal carcinoma (NPC) is one of the most prevailing cancers in southern China and southern Asia. Because of the nonspecific symptoms and lack of effective biomarker, most patients are diagnosed at advanced stages, resulting in poor 5-year survival rate. To identify a novel NPC biomarker facilitating early detection and effective therapy of NPC, a two-step strategy consisting of cancer cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) procedure and aptamer-based purification approach was developed. Using cell-SELEX procedure, four aptamers (S3, S5, S12 and S27) differentiating the molecular differences between NPC cells and NP cells were successfully screened. Then, using aptamer-based protein purification, membrane protein CD109 was identified as the target of aptamer S3. CD109 protein was further identified to be over-expressed in NPC cell lines and clinic tissues, but not or low in NP cell line and clinic NP tissues, detected by western blot and immunohistochemistry experiments. Our study demonstrated that CD109 identified by cell-SELEX and aptamer-based purification strategy might be used as a potential NPC biomarker for early diagnosis and targeted therapy.

/pdf/cd109-is-identified-as-a-potential-nasopharyngeal-carcinoma-49dko1878z.pdf

CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX.

Human embryonic stem (hES) cells are capable of unlimited cell proliferation yet maintain the potential to differentiate into many cell types. Here we reported an Epstein-Barr virus (EBV)-based vector system used to improve transfection efficiency in hES cells. Plasmids containing oriP, the latent replication origin of EBV, can be propagated stably as episomal DNA in human cells that express the EBV nuclear antigen 1 (EBNA1), which binds to oriP and functions as the trans-acting replication initiator. It was reported that the EBV replicon could harbor a DNA fragment of up to 330 kilobase pairs. Plasmids containing an enhanced green fluorescent protein (EGFP)/puromycin resistance gene cassette along with or without oriP were used to transfect hES cells that stably express EBNA1. The presence of oriP moderately increased the transient transfection efficiency and more importantly it elevated the stable transfection efficiency by approximately 1,000-fold as compared with oriP-minus plasmids. The oriP plasmid as episomal DNA and green fluorescent protein expression in hES cells was maintained for months in the presence of drug selection and gradually lost (2%-4% per cell doubling) in the absence of selection. The presence of EBNA1 did not interfere with the hES cell properties or differentiation we tested and could maintain stable EGFP expression during differentiation. In addition to transgene expression, the EBV vector system could effectively enhance the RNA interference efficiency in hES cells. Thus, the EBV vector system that allows a large DNA insert and sustained expression of transgene or small hairpin RNA will enhance basic and translational research using hES cells.

Establishment and applications of epstein-barr virus-based episomal vectors in human embryonic stem cells.

Objective(s):The potential roles of serum microRNAs (miRNAs), as biomarkers, in noninvasive diagnosis of endometriosis have been reported by microarray analysis. However, microarray analysis cannot...

Analysis of Serum microRNA Profile by Solexa Sequencing in Women With Endometriosis.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Recent study found an increased level of glypican-1 positive (GPC1+) plasma exosomes in patients with stage II CRC, but decreased levels of plasma miR-96-5p and miR-149. This study further investigated the clinical significance of plasma GPC1+ exosomes and plasma miR-96-5p and miR-149 levels in stage III CRC patients. To study the effect of these microRNAs on GPC1+ plasma exosomes, we isolated and purified exosomes and overexpressed human GPC1 and the microRNAs miR-96-5p and miR-149 by adenovirus vectors. Overexpression of GPC1 activated epithelial-mesenchymal transition (EMT) which then increased invasion and migration in HT29 and HCT-116 colon cancer cells. In contrast, silencing GPC1 expression and overexpressing miR-96-5p and miR-149 significantly inactivated EMT and decreased invasion and migration of HT29 and HCT-116 cells. miR-96-5p and miR-149 inhibitors significantly increased invasion and migration of HT29 and HCT-116 cells. Our results indicate that high levels of circulating GPC1 positive exosomes before and after surgery as well as low circulating miR-96-5p and miR-149 before surgery indicated a severe clinical status and poor prognosis in stage III colon cancer patients. We conclude that GPC1 can be a biomarker for relapse of stage III CRC and may be involved in EMT activation, invasion, and migration of colorectal cancer cells.

Lei Wang

Papers

GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX.

Establishment and applications of epstein-barr virus-based episomal vectors in human embryonic stem cells.

Analysis of Serum microRNA Profile by Solexa Sequencing in Women With Endometriosis.

The clinical significance of circulating GPC1 positive exosomes and its regulative miRNAs in colon cancer patients.