Exosomes are small (∼30–140 nm) lipid bilayer-enclosed particles of endosomal origin. They are a subset of extracellular vesicles (EVs) that are secreted by most cell types. There has been growing interest in exosome research in the last decade due to their emerging role as intercellular messengers and their potential in disease diagnosis. Indeed, exosomes contain proteins, lipids, and RNAs that are specific to their cell origin and could deliver cargo to both nearby and distant cells. As a result, investigation of exosome cargo contents could offer opportunities for disease detection and treatment. Moreover, exosomes have been explored as natural drug delivery vehicles since they can travel safely in extracellular fluids and deliver cargo to destined cells with high specificity and efficiency. Despite significant efforts made in this relatively new field of research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization, and lack of specific biomarkers. In this review, we summarize the current knowledge in exosome biogenesis, their roles in disease progression, and therapeutic applications and opportunities in bioengineering. Furthermore, we highlight the established and emerging technological developments in exosome isolation and characterization. We aim to consider critical challenges in exosome research and provide directions for future studies.

The development of array comparative genomic hybridization (array CGH) at tiling-path resolution has enabled the detection of gene-sized segmental DNA copy number gains and losses. Here, we present the first application of whole genome tiling-path array CGH to archival clinical specimens for the detailed analysis of oral squamous cell carcinomas (OSCC). We describe the genomes of 20 OSCCs as well as a selection of matched normal DNA in unprecedented detail. Examination of their whole genome profiles enabled the identification of alterations ranging in size from whole-arm, segmental, to gene size alterations. Tiling-path resolution enabled the detection of many more alterations within each tumor than previously reported, many of which include narrow alterations found to be frequent events among the 20 OSCCs. We report the presence of several novel frequent submegabase alterations, such as the 0.58 Mb gain at 5p15.2 containing triple functional domain (TRIO), detected in 45% of cases. We also report the first coamplification of two gene clusters, by fine-mapping the precise base pair boundaries of the high-level amplification at 11q22.2-22.3 containing both matrix metalloproteinase and baculoviral IAP repeatcontaining protein 2 (BIRC) gene clusters. These results show the large improvement in detection sensitivity and resolution compared with genome interval marker arrays and the utility of tiling resolution array CGH for the detection of both submegabase and single copy gains and losses in cancer gene discovery. (Cancer Res 2005; 65(17): 7561-7)

William Lockwood and colleagues show that the focal amplification of a gene, BRF2, on Chromosome 8p12 plays a key role in squamous cell carcinoma of the lung.

Chromosomal regions harboring tumor suppressors and oncogenes are often deleted or amplified. Array comparative genomic hybridization detects segmental DNA copy number alterations in tumor DNA relative to a normal control. The recent development of a bacterial artificial chromosome array, which spans the human genome in a tiling path manner with >32,000 clones, has facilitated whole genome profiling at an unprecedented resolution. Using this technology, we comprehensively describe and compare the genomes of 28 commonly used non-small cell lung carcinoma (NSCLC) cell models, derived from 18 adenocarcinomas (AC), 9 squamous cell carcinomas and 1 large cell carcinoma. Analysis at such resolution not only provided a detailed genomic alteration template for each of these model cell lines, but revealed novel regions of frequent duplication and deletion. Significantly, a detailed analysis of chromosome 7 identified 6 distinct regions of alterations across this chromosome, implicating the presence of multiple novel oncogene loci on this chromosome. As well, a comparison between the squamous and AC cells revealed alterations common to both subtypes, such as the loss of 3p and gain of 5p, in addition to multiple hotspots more frequently associated with only 1 subtype. Interestingly, chromosome 3q, which is known to be amplified in both subtypes, showed 2 distinct regions of alteration, 1 frequently altered in squamous and 1 more frequently altered in AC. In summary, our data demonstrate the unique information generated by high resolution analysis of NSCLC genomes and uncover the presence of genetic alterations prevalent in the different NSCLC subtypes.

BackgroundMicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression by preventing the translation of specific mRNA transcripts. Recent studies have shown that miRNAs are stably expressed in human serum samples, making them good candidates for the non-invasive detection of disease. However, before circulating miRNAs can be used reliably as biomarkers of disease, the pre-measurement variables that may affect serum miRNA levels must be assessed.MethodsIn this study we used quantitative RT-PCR to examine the effect of hemolysis, fasting, and smoking on the levels of 742 miRNAs in the serum of healthy individuals. We also compared serum miRNA profiles of samples taken from healthy individuals over different time periods to assess normal serum miRNA fluctuations.ResultsWe have found that mechanical hemolysis of blood samples can significantly alter serum miRNA quantification and have identified 162 miRNAs that are significantly up-regulated in hemolysed serum samples. Conversely, fasting and smoking were demonstrated to not have a significant effect on the overall serum miRNA profiles of healthy individuals. The serum miRNA profiles of matched samples taken from individuals over varying time periods showed a high correlation and no miRNAs were significantly differentially expressed in these samples further suggesting the utility of serum miRNAs as biomarkers of disease. Taking the above results into consideration, we have identified miR-99a-5p and miR-139-5p as novel endogenous controls for serum miRNA studies due to their consistency across all sample sets.ConclusionThese results identify important pre-profiling factors that should be taken into consideration when identifying endogenous controls and candidate biomarkers for circulating miRNA studies.

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Packaging of small molecular factors, including miRNAs, into small extracellular vesicles (SEVs) may contribute to malignant phenotypes and facilitate communication between cancer cells and tumor stroma. The process by which some miRNAs are enclosed in SEVs is selective rather than indiscriminate, with selection in part governed by specific miRNA sequences. Herein, we describe the selective packaging and removal via SEVs of four miRNAs (miR-142-3p, miR-150-5p, miR-451a, and miR-223-3p) in a panel of oral dysplasia and oral squamous cell carcinoma cell lines. Inhibition of exosome export protein Rab27A increased intracellular concentration of these miRNA candidates and prevented their exclusion via SEVs. Increased intracellular miR-142-3p specifically was found to target TGFBR1, causing a decrease in TGFBR1 expression in donor cells and a reduction of malignant features such as growth and colony formation. Conversely, increased excretion of miR-142-3p via donor cell SEVs and uptake by recipient endothelial cells was found to reduce TGFBR1 activity and cause tumor-promoting changes in these cells in vitro and in vivo.

Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound structures that play key roles in intercellular communication. EVs are potent regulators of tumorigenesis and function largely via the shuttling of cargo molecules (RNA, DNA, protein, etc.) among cancer cells and the cells of the tumor stroma. EV-based crosstalk can promote proliferation, shape the tumor microenvironment, enhance metastasis, and allow tumor cells to evade immune destruction. In many cases these functions have been linked to the presence of specific cargo molecules. Herein we will review various types of EV cargo molecule and their functional impacts in the context of oncology.