Isolating Exosomes for Cancer Diagnostics
Article Nov 08, 2017 | By Anna MacDonald, Editor for Technology Networks
Once considered cellular rubbish, exosomes are now known to play important roles in cellular communication. These small messengers are defined as “extracellular vesicles that are released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body, with the plasma membrane.” (Edgar 2016) Although they were first described in 1983, widespread interest in exosomes did not take hold until the last decade, when their roles in a range of biological functions became more apparent.
Seeing exosomes in a new light
In 2007, research by Dr Jan Lotvall demonstrated that contrary to previous belief, exosomes contained not only proteins and lipids, but also RNA. Further research by Dr Johan Skog showed that exosomes released from glioblastoma tumor cells transport mRNA and miRNA to recipient cells. This opened a new avenue of using exosomes as a proxy to study cancer, with the potential to identify individual mutations and personalize treatment via liquid biopsies.
Dr Skog speaks more about what lead to this discovery and the importance of it in this video.
Challenges of exosome isolation
Before exosomes can be analyzed for diagnostic purposes, they must first be detected and isolated from the blood, urine, saliva or other bodily fluid they are residing in.
This can often be a complex process, with their small size presenting challenges. As Dr Tony Jun Huang, Professor of Mechanical Engineering and Materials Science, Duke University tells us, “exosomes are very tiny, typically ranging from 30nm to 150nm, which is thousands of times smaller than the diameter of hair. It is much more challenging to precisely manipulate nano-objects than micro-objects.”
“Isolation efficiency, purity, speed and cost are all challenges for an exosome isolation technology. On the biological side, the heterogeneity of vesicle populations and exosome isolation from complex samples (e.g. body fluids) are often barriers. For clinical samples, the sample volume, exosome concentration and isolation of specific cell-derived exosomes are all demanding tasks and affect the choice of available technologies,” adds Dr Siyang Zheng, Associate Professor, Penn State University.
There are currently a number of methods for isolating exosomes available, each with their own advantages and disadvantages.
Overcoming limitations of ultracentrifugation
In an effort to overcome limitations of one of the most commonly used methods, ultracentrifugation, researchers from MIT and Duke University have recently developed an acoustofluidic unit that can separate extracellular vesicles from undiluted whole blood using tilted-angle standing surface acoustic waves.
The new method offers a host of advantages, including biohazard containment, high reproducibility, and low turnaround time, taking only 25 minutes to complete the process of isolation, compared to the 8 hours that differential centrifugation needs. The method is also expected to achieve high exosome purity and yield, with prototypes demonstrating a purity of 67% and yield of ~82%. Importantly, the acoustofluidic technology can isolate biologically active and content-intact exosomes, thanks to its gentle, clean and label-free nature. “We believe that it offers the best opportunity to preserve the integrity of the isolated exosomes,” states Professor Huang. Finally, the platform is compact and inexpensive, making it well suited as a point-of-care device for use in under-served populations.
“With the aforementioned advantages, the proposed acoustofluidic technology has the potential to significantly exceed current standards in the clinic and experimental setting, address unmet needs in the market, and help expedite exosome-related cancer research to aid in the discovery of new exosomal biomarkers for cancer diagnosis and treatment,” Professor Huang adds.
Professor Huang and researchers are planning to develop “an all-in-one, easy-to-use prototype that enables researchers to perform rapid, high-yield, high-purity, biocompatible exosome isolation through a very simple procedure.”
Magnetically isolating exosomes
An alternative method recently developed at Penn State University, relies on nanoprobes to magnetically isolate exosomes. The new lipid-nanoprobe system is comprised of two nanomaterial components: a labeling probe that spontaneously and instantly inserts into the lipid bilayer of exosomes, and a capture probe that extracts the labeled exosomes using a magnet.
“It’s a simple solution that can isolate exosomes from clinical samples in 15 minutes, which is much faster than conventional methods. It provides good isolation efficiency (~50 - 80%) and is especially suitable for clinical samples of relatively small sample volume. It is also highly compatible with downstream detection assays,” explains Dr Siyang Zheng.
In the current paper, the team demonstrated that the lipid-nanoprobe system enables identification of EGFR and KRAS mutations in NSCLC patients, highlighting the potential of the technology to improve the process of diagnosing and treating this cancer. Dr Zhang adds that “we are actively working towards developing new cancer diagnostic technology (liquid biopsy) based on this exosome isolation technology.”
The future of exosomes in cancer diagnostics
Exosomes offer a promising avenue for the minimally invasive diagnosis and monitoring of a range of cancers; future work to identify more biomarkers could help improve enrichment and detection. Overcoming the challenges of isolating these small vesicles from biofluids could lead to wider adoption of exosome technologies in the clinic, and eventually translate into earlier detection and improved treatment plans for cancer patients.
The Importance of Aflatoxin Discrimination in Food TestingArticle
Aflatoxins have been an issue in food and feed since the 1970’s, but the first global effort to regulate them took place in 2003. Aflatoxin B1 is by far the most prevalent and potent sub-type. Accordingly, maximum levels for B1 are set lower than total aflatoxin levels which highlights the need to test these two measures independently.READ MORE