Illinois researchers developed a method to detect microRNA cancer markers with single-molecule resolution, a technique that could be used for liquid biopsies.

From left: Taylor Canady, postdoctoral scholar; Andrew Smith, professor of bioengineering; Nantao Li, graduate student; Lucas Smith, postdoctoral scholar; and Brian Cunningham – professor of Electrical and Computer Engineering; director of Micro and Nanotechnology Laboratory.
Photo by L. Brian Stauffer

Thanks to the University of Illinois News Bureau for allowing us to share this article here.

CHAMPAIGN, Ill. — A fast, inexpensive yet sensitive technique to detect cancer markers is bringing researchers closer to a liquid biopsy – a test using a small sample of blood or serum to detect cancer, rather than the invasive tissue sampling routinely used for diagnosis.

Researchers at the University of Illinois developed a method to capture and count cancer-associated microRNAs, or tiny bits of messenger molecules that are exuded from cells and can be detected in blood or serum, with single-molecule resolution. The team published its results in the Proceedings of the National Academy of Science.

“Cancer cells contain gene mutations that enable them to proliferate out of control and to evade the immune system, and some of those mutations turn up in microRNAs,” said study leader Brian Cunningham, an Illinois professor of electrical and computer engineering. Cunningham also directs the Holonyak Micro and Nanotechnology Lab at Illinois.

“There are specific microRNA molecules whose presence and concentration is known to be related to the presence and aggressiveness of specific types of cancer, so they are known as biomarkers that can be the target molecule for a diagnostic test,” he said.

Cunningham’s group developed a technique named Photonic Resonator Absorption Microscopy to capture and count microRNA biomarkers. In collaboration with professor Manish Kohli at the Moffitt Cancer Center in Florida, they tested PRAM on two microRNAs that are known markers for prostate cancer.

They found it was sensitive enough to detect small amounts that would be present in a patient’s serum, yet also selective enough to detect the marker among a cocktail of molecules that also would be present in serum.

“One of the main challenges of biosensing is to maintain sensitivity and selectivity at the same time,” said Nantao Li, a graduate student and co-first author. “You want it to be sensitive enough to detect very small amounts, but you don’t want it to pick up every RNA in the blood. You want this specific sequence to be your target.”
 

Each dot seen in this PRAM image represents one microRNA that has bound to the sensor.
Image courtesy of Nantao Li

 

PRAM achieves both qualities by combining a molecular probe and a photonic crystal sensor. The probe very specifically pairs to a designated microRNA and has a protective cap that comes off when it finds and binds to the target biomarker. The exposed end of the probe can then bind to the sensor, producing a signal visible through a microscope.

Each individual probe that binds sends a separate signal that the researchers can count. This means researchers are able to detect much smaller amounts than traditional methods like fluorescence, which need to exceed a certain threshold to emit a measurable signal. Being able to count each biomarker also carries the added benefit of allowing researchers to monitor changes in the concentration of the biomarker over time.

“With PRAM, we squirt a sample into a solution and get a readout within two hours,” said postdoctoral researcher Taylor Canady, a co-first author of the study. “Other technologies that produce single-molecule readouts require extra processing and additional steps, and they require a day or more of waiting. PRAM seems like something that could be much more feasible clinically. In addition, by using an optical signal instead of fluorescence, we could one day build a miniaturized device that doesn’t need a trained laboratory technician.”

The PRAM approach could be adapted to different microRNAs or other biomarkers, the researchers say, and is compatible with existing microscope platforms.

“This approach makes the idea of performing a ‘liquid biopsy’ for low-concentration cancer-related molecules a step closer to reality,” Cunningham said. “This advance demonstrates that it is possible to have an inexpensive and routine method that is sensitive enough to require only a droplet of blood. The results of the test might tell a physician whether a regimen of chemotherapy is working, whether a person’s cancer is developing a new mutation that would make it resistant to a drug, or whether a person who had been previously treated for cancer might be having a remission.”

The Carl R. Woese Institute for Genomic Biology at the U. of I. and the National Institutes of Health supported this work. Illinois chemistry professor Yi Lu and bioengineering professor Andrew Smith were coauthors of the work.

Reference
Canady TD, Li N, Smith LD, Lu Y, Kohli M, Smith AM & Cunningham BT. Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy. Proc Natl Acad Sci U S A. (2019) 116:19362-19367. doi: 10.1073/pnas.1904770116 PMID: 31501320

Thanks to Eileen Leahy from Elsevier and Chhavi Chauhan, Director of Scientific Outreach for the Journal of Molecular Diagnostics, for sharing this post here.

A novel non-invasive technique may detect human papilloma virus-16, the strain associated with oropharyngeal cancer, in saliva samples, reports The Journal of Molecular Diagnostics.

Philadelphia, December 13, 2019 – Unfortunately, cancers that occur in the back of the mouth and upper throat are often not diagnosed until they become advanced, partly because their location makes them difficult to see during routine clinical exams. A report in The Journal of Molecular Diagnostics, published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of co published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of confirmed OPC patients.

“OPC has an approximate incidence of 115,000 cases per year worldwide and is one of the fastest-rising cancers in Western countries due to increasing HPV-related incidence, especially in younger patients. It is paramount that surveillance methods are developed to improve early detection and outcomes,” explained co-lead investigator Tony Jun Huang, PhD, Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA.

“Considering these factors, the successful detection of HPV from salivary exosomes isolated by our acoustofluidic platform offers distinct advantages, including early detection, risk assessment, and screening,” added Dr. Huang. This technique may also help physicians predict which patients will respond well to radiation therapy or achieve longer progression-free survival.

Exosomes are tiny microvesicles originating within cells that are secreted into body fluids. They are believed to play a role in intercellular communication and their numbers are elevated in association with several types of cancers. Acoustofluidics is an advanced technology that fuses acoustics and microfluidics. Fluid samples are analyzed using a tiny acoustofluidic chip developed to isolate salivary exosomes by removing unwanted particles based on size, leaving exosome-rich concentrated samples that make it easier to detect tumor-specific biomarkers.

Acoustofluidic exosome isolation chip
Acoustofluidic exosome isolation chip for salivary exosome isolation. The microfluidic channels are shown by red dye, and the coin demonstrates the size of the chip. Two pairs of gold interdigital transducers are deposited along the channel, which separates particles according to size.

In this study investigators analyzed saliva samples from 10 patients diagnosed with HPV-OPC using traditional methods. They found that the technique identified the tumor biomarker HPV-16 DNA in 80 percent of the cases when coupled with droplet digital PCR. Since this method is independent of sample variability arising from changes in saliva viscosity and collection method, it may prove ideal for use in clinical settings.

Dr. Huang highlighted some of the technique’s features, including automated and fast exosome isolation (less than five minutes of processing time compared to approximately eight hours of processing time using benchmark technologies). Analyses can be performed at relatively low cost and at points of care. Also, it is suitable for repeated and continuous monitoring of tumor progression and treatment, unlike traditional biopsy.

“With these features, the acoustofluidic technology has the potential to significantly exceed current industry standards, address unmet needs in the field, help expedite exosome-related biomedical research, and aid in the discovery of new exosomal biomarkers,” commented Dr. Huang.

“The saliva exosome liquid biopsy is an effective early detection and risk assessment approach for OPC,” said co-lead investigator David T.W. Wong, DMD, DMSc, of the Center for Oral/Head and Neck Oncology Research, School of Dentistry at the University of California Los Angeles, CA, USA. “The acoustofluidic separation technique provides a fast, biocompatible, high-yield, high-purity, label-free method for exosome isolation from saliva.” According to the researchers, this technology can also be used to analyze other biofluids such as blood, urine, and plasma.

The study was an international collaboration between Duke University, UCLA, and University of Birmingham (UK). According to Prof Hisham Mehanna, Director of the Institute of Head and Neck Studies and Education, University of Birmingham, Birmingham, UK, “The results are a testament to the power of interdisciplinary research and international collaboration.”

Reference
Wang Z et al. Acoustofluidic salivary exosome isolation: A liquid biopsy compatible approach for human papillomavirus—associated oropharyngeal cancer detection. Journal of Molecular Diagnostics v22, January 2020. doi: 10.1016/j.jmoldx.2019.08.004.

This work was supported by the National Institutes of Health (D.T.W.W.: UG3/UH3 TR002978, UH3 TR000923, U01 CA233370, UH2 CA206126), (T.J.H.: R01GM132603, R01 HD086325), (D.TW.W. and F.L.: R21 CA239052) and Canadian Institute of Health (CIHR) Doctoral Foreign Student Award (J.C.), Tobacco Related Disease Research Program (TRDRP) Predoctoral Fellowship (J.C.). Funding was also provided by the Queen Elizabeth Hospital Birmingham (QEHB) Charity UK and the Get-A-Head charity UK.

Malignant gliomas are highly aggressive brain tumors. Surgical removal and chemoradiation of the tumor are the standard of care. Recently, the U.S. Food and Drug Administration (FDA) approved a compound called 5-aminolevulinic acid (5-ALA) as an imaging agent to aid in differentiating tumor from normal tissue during surgery. 5-ALA is a precursor in the heme biosynthesis pathway, which is inefficient in glioma cells because their strongly rewired metabolism does not rely on heme. When patients with malignant glioma ingest 5-ALA prior to surgery, the glioma cells fluoresce pink under a blue light due to their preferential uptake and conversion of 5-ALA to the final precursor in heme biosynthesis, the fluorescent molecule protoporphyrin IX (PpIX). We sought to investigate whether extracellular vesicles (EVs) released from PpIX-enriched glioma cells would fluoresce and be detectable in the blood of these patients.

We employed Amnis® Imaging Flow, which combines flow cytometry and microscopy to detect PpIX-positive EVs. We first determined the optimal 5-ALA dose to maximize fluorescence and minimize cell death. We used a combination of beads of different size (100-500nm) and liposomes with different emission spectra to ensure that the signal emitted in Channel 11 (~640nm) of the Amnis® output was indeed from PpIX, and that all other channels reported no signal. Controls also included lysis with Triton-X of liposomes and EVs.

Importantly, we showed that glioma cells released a significantly higher number of PpIX-positive EVs (247-fold increase) than normal endothelial cells (6-fold increase) after 5-ALA ingestion. We also used xenograft mouse models to show that the presence of PpIX-positive EVs in circulating plasma after 5-ALA ingestion correlated strongly with the presence of a primary brain tumor, while the signal from the plasma of normal control mice remained below background both before and after 5-ALA ingestion.

Finally, we tested the optimized assay in the plasma of patients with gliomas undergoing 5-ALA fluorescence guided surgery at the Massachusetts General Hospital. Samples were collected prior to 5-ALA intake as well as at the time of surgery, prior to tumor removal. Pre- and post-5-ALA plasma samples were kept in the dark to avoid bleaching of the PpIX signal, as were the patients for 24 hours post 5-ALA. We collected samples from 4 patients whose tumors were avidly fluorescent during surgery and 2 patients whose tumors showed minimal fluorescence. Interestingly, we detected PpIX-positive EVs only in the plasma samples from patients whose tumors were avidly fluorescent. Finally, when we compared the fold increase (pre/post-5-ALA) in PpIX-positive signal to the size of the tumor, we found a clear correlation, suggesting that the detected events are likely coming directly from the tumor. This is the first time intracranially derived EVs have been quantified in circulating plasma, and this development opens the door for many exciting studies that can shed light on brain-derived EV dynamics and half-life. For example, we detected between 3,000 and 8,000 PpIX-positive events per mL of plasma. Assuming each 1 mL of plasma contains roughly 1010 EV/mL, we can deduct that only 0.00008% of EVs in blood are of glioma tumor origin. Furthermore, this assay allows us to study EV dynamics in tumor patients undergoing therapy as well as determine the effects of medications such as dexamethasone on the release of EVs into the bloodstream.

Clinically, there is a major need for minimally invasive diagnosis of brain cancer, and characterizing circulating tumor-specific fluorescent EVs provides a window into the primary tumor’s presence and status. Detecting and characterizing fluorescent EVs after administering 5-ALA allows for diagnosis and potentially monitoring of malignant gliomas over time.

Reference

Jones PS, et al. Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma. (2019) eBioMedicine 48:23-35. doi: 10.1016/j.ebiom.2019.09.025. PMID: 31628025.

Rett Syndrome Research TrustCline laboratory at Scripps ResearchThis blog was first published on the Rett Syndrome Research Trust (RSRT) website. Thanks to Pranav Sharma and the Cline lab at Scripps Research for allowing us to share it here.

I am a scientist at Scripps Research Institute in La Jolla, California working in the lab of Professor Hollis Cline. A thirst for knowledge is what originally attracted me to science. The potential to contribute, even in a small way, to alleviating suffering drives that thirst and passion even more.

Human biology has always fascinated me. Imagine for a moment how the human body is created. It starts with a single cell that multiplies to create a complex organism of trillions of cells. The human brain alone is estimated to contain more than 150 billion cells, 86 billion neurons and about an equal number of non-neuronal cells, all of a wide variety of specializations. It is mind boggling to imagine that a few founder cells contain the programming information that, through a series of cell fate decisions, produces a complex organ like the brain. What kind of communication and logistics are required to orchestrate the development and function of this behemoth?

One requirement is a package delivery mechanism; think of it as the body’s UPS, which allows information and material transfer between cells. Over the past decade, researchers have discovered that our bodies employ amazing inter-cellular couriers called exosomes or extracellular vesicles to transport fundamental biomolecules like proteins, nucleic acids and lipids. Exosomes can also perform additional duties, such as scouting and laying the path for a growing axon or migrating cells. For example, cancer cells use exosomes to lay the foundation of their migration out of a tumor, leading to metastasis.

Our work has uncovered a fundamental role of exosome communication in brain development. We show that exosomes secreted by neurons contain signals to direct the development and function of neural circuits. Importantly we have discovered that exosomes have the potential to become therapeutics for neurodevelopmental disorders, including Rett Syndrome.

Our brain works like a musical ensemble. The neurons fire to produce a pattern of activity very much like an ensemble of musicians playing together to produce a melody. Historically, a vast majority of studies directed towards understanding brain function focused on the skills of the individual neurons or their training together in producing a melody. We found that when these musicians in our brain called neurons, hang together and socialize, they use exosomes to communicate between themselves. These exosomes contained messages that provided them great collective motivation and were extremely helpful in their training and performance. Extending this analogy to the case of Rett Syndrome, Rett neurons practice very hard but are unable to play together and produce a melody. Rett neurons not only lacked some music skills, they had problems coordinating their music with each other. We found that the Rett exosome no longer contained motivating messages to help the neurons with their music skills and coordination.

We thought that maybe if we take exosomes from healthy neurons and give them to Rett neurons, it will provide them the message they are lacking and help motivate them to play a melody. Remarkably, the exosome message from healthy neurons let Rett Syndrome neurons overcome their shortcomings and fire together in a synchronous way to produce a melody.

For the scientifically inclined readers I’ll provide a more scientific description. All cells in the brain secrete exosomes. However, it was not very clear what function the exosomes perform in the brain. We purified exosomes from functional neural cultures and asked, could these exosomes contain a bioactivity to perform any function in a developing neural circuit? We observed that exosome treatment led to an increase in neuronal number. This led to a further question – if exosomes have a role in developing neural circuits, what happens when the neural development is deficient? A good way to find that out is to compare exosomes from healthy neurons to exosomes from neurons with a neurodevelopmental disorder.

We decided to explore this question by experimenting with induced pluripotent stem cells (iPSC) from a Rett Syndrome patient. Rett is caused by disruption of a single gene, MECP2. We restored the function of the MECP2 gene in the iPSCs using CRISPR gene editing. We therefore had two human iPSC neural cultures that are identical to each other genetically except in the function of just one protein, MECP2. This was an ideal setup to study the fundamental role of exosomes in normal neural circuit development and compare it to a condition where neural circuit development is deficient.

The Rett patient iPSC derived neural cultures displayed cellular and circuit manifestations of Rett Syndrome, whereas CRISPR corrected controls were normal. We then purified exosomes secreted by each culture, yielding normal control exosomes and Rett exosomes, and compared them. Our results were so remarkable that it took us a while to appreciate them.

First, exosomes were full of proteins that are important in development of neurons and formation and maintenance of synapses. Synapses are conduits of electrochemical information flow between neurons, and are critical to proper brain function.

Second, the Rett exosomes displayed specific alterations in their signaling capacities, like proliferation, neural development, and synaptic function. In short, we found that normal exosomes could potentially guide proliferation, neuron development, and synapse function, and Rett exosomes are somewhat deficient in that function.

Taking cues from these results, we compared the bioactivity and found that normal exosomes boosted proliferation of neural stem cells and Rett exosomes did not. In addition, normal exosome treatment led to a big increase in neural progeny and modest increase in astrocyte progeny; astrocytes are another cell type in the brain that have a range of ancillary functions. In comparison, Rett exosome treatment, while it lacked the capability to increase neural progeny, still directed the modest increase of astrocyte progeny. This result shows that Rett exosomes retain some functions, but their neural specific functions are lacking.

However, the most important question was still nagging us. Could treatment with normal control exosomes rescue deficits in Rett Syndrome neural cultures? After an onerous journey of problem solving and establishment of assays, we successfully demonstrated that treatment of Rett neural cultures with normal control exosomes could increase neuron number, boost the number of synapses, and make neurons fire in a more synchronized way. Importantly, exosome treatment showed improvements at the cellular, synaptic, and functional level.

While a very exciting result, we wanted to take this a step further into live animals. So we took healthy exosomes and injected them into the brain of developing mice and monitored neuronal proliferation in hippocampus, a brain area important for learning and memory. Exosome injections led to a remarkable boost in neuronal proliferation in hippocampus, just like human in vitro disease models. This showed that if delivered to the brain in live animals, the exosomes can deliver the promised bioactivity.

I belive exosomes have immense therapeutic potential as they have inherent advantages. Unlike stem cells, there is no possibility that they can go rogue and form tumors. Importantly, exosomes do not elicit an immunune response when injected into the patient. Exosomes can be sourced from cultured neurons made from the patient’s own cells, providing personalized medicine.

Neural exosomes are thought to contain signals that guide the exosome to the brain. They can be loaded with any therapeutic drugs or molecules developed for Rett Syndrome and delivered to the brain. Our future work will focus on optimizing exosomes for specific and efficient delivery to the brain; finding the least invasive way of delivering exosomes to the brain; and showing that exosomes can be used to rescue disease in a mouse model of Rett Syndrome.

Acknowledgements: This symphony would have been impossible without our musical ensemble of Hollis T. Cline, Alysson R. Muotri, John R. Yates III, Pinar Mesci, Cassiano Carromeu, Daniel B. McClatchy and Lucio Schiapparelli. I sincerely thank Monica Coenraads for help in providing better voice to my words.

This blog originated as a press release from the University of Sussex.

New research by scientists at the University of Sussex could be the first step towards developing a blood test to diagnose the most aggressive type of brain tumour, known as Glioblastoma.

A team from Professor Georgios Giamas’ lab at the University of Sussex has identified novel biomarkers within bodily fluids, which signal the presence of the tumour. Dr. Giamas is Professor of Cancer Cell Signalling in the School of Life Sciences.

Cancer biomarkers are molecules that are either exclusively found or over-expressed in cancer cells, as compared to ‘normal’, healthy cells. Biomarkers can be considered as biological signatures for a disease, as they indicate the presence of cancer in the body.

In a new paper published in the Nature journal Communications Biology, Professor Giamas and his team describe particular biomarkers that are associated with extracellular vesicles – small ’packages’ released by cells into bodily fluids so cells can communicate with each other.

The discovery suggests that bodily fluids like blood could be a simpler way to test for glioblastoma, rather than a biopsy, which is both invasive and painful for the patient as well as taking considerable time to get the results.

Giamas said, “At the moment, the outlook for glioblastoma patients is bleak. As the most aggressive type of brain tumour, survival rate is low.

“Our research provides more information about the markers which can signal the presence of glioblastoma – and the fact we’ve been able to identify ones that are associated with extracellular vesicles suggests that there could be a way to use bodily fluids to test for the tumour in future.”

Currently, a growing body of research is looking into the possibility of developing liquid biopsies like blood tests to spot other types of cancers (e.g. pancreatic). Rather than taking a piece of tissue from the relevant organ, liquid biopsies would allow doctors to take a small sample of blood and test for a range of biomarkers which will help identify the subtype of tumour.

Dr Thomas Simon, co-author of this study, highlighted that: “Liquid biopsies mean a less invasive procedure for patients, and arguably quicker results – something which is invaluable for those with an aggressive tumour that severely cuts life expectancy.

“But it could also mean better patient follow-up care, as a simple test can be carried out to check for the efficacy of existing treatments or for monitoring relapse.

“The more we know about biomarkers the better, so this is a step which should provide hope for anyone whose lives have been impacted by glioblastoma.”

There are three sub-types of glioblastoma which all have biomarkers containing different information. The more researchers find out about these signatures, the more work can be done to improve the accuracy of diagnosis and to personalise treatment depending on the sub-type of cancer.

Rosemary Lane, a PhD student in Professor Giamas’ lab and co-author of the study, added: “Glioblastoma subtyping is crucial for patient prognosis and personalised therapies. The fact that we can identify these molecular differences in extracellular vesicles is very exciting and will be of huge importance for discovering new biomarkers in the future.”

Marian Vintu, a neurosurgeon and co-author, said: “Clinical research in brain cancer is such a powerful tool to expand our knowledge in this terrible disease and improve our patient’s outcome.”

The next step for Professor Giamas’ team will be to test and validate the presence of these newly described biomarkers in glioblastoma patients.

The research, funded by the charity Action Against Cancer, suggests that this technique could ultimately become an option for diagnosing glioblastoma.

Tulane University

This blog post originated as a press release from Tulane University.

Asim Abdel-Mageed, DVM, PhD, professor of urology and Marguerite Main Zimmerman Professor of Cancer Research at the Tulane School of Medicine, was recently honored by the journal Scientific Reports for authoring one of the top 100 accessed oncology papers for the journal in 2018.

His publication, “High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: a drug repurposing strategy for advanced cancer”, received 3,154 article views, placing it seventh on the list, which features authors from around the world whose papers highlight valuable research in oncology.

The article reveals the results of research supported by a $4.2 million National Institutes of Health grant awarded to Abdel-Mageed in 2014. His project involved using a rapid high-volume robotic screening technique to investigate drugs already approved by the Food and Drug Administration (FDA) to treat a large variety of diseases or conditions to see which, if any, could also be effective in preventing prostate cancer metastasis.

Targeting Metastasis

For cancer cells to spread to other places in the body — or metastasize — they need to communicate with resident and recruited cells, such as stem cells. One way they do this is through biomolecular messages delivered in exosome cargos. Exosomes are molecules that carry information from cell to cell. “They are routinely biosynthesized and released by cancer cells, including prostate cancer, and are implicated in cancer progression,” said Abdel-Mageed.

Currently there are no known drugs that selectively target and inhibit the biosynthesis and release of exosomes by tumor cells. To accelerate the discovery of effective drugs, Abdel-Mageed and his team, in partnership with investigators at the National Center for Advancing Translational Science (NCATS), investigated 4,580 known pharmacologically active compounds and found that 22 — including antibiotics, antifungal medicines and anti-inflammatory agents — were effective in preventing advanced prostate tumor cells from releasing exosomes or in blocking their production.

Future Work

Since the Scientific Reports publication, subsequent research by Abdel-Mageed’s team has further narrowed their investigation to five of these agents, and he hopes in the near future to receive additional funding to support this work.

“Drug repurposing is a golden opportunity,” said Abdel-Mageed. “Because drug discovery from concept to market takes an average time of 12 years, our identified drugs, which are already human approved, could be repurposed for the treatment of advanced prostate cancer within a relatively short period of time. It represents a quick way of adding an adjuvant therapy to existing therapies that might curb the progression of cancer.”

As a steering committee member of the National Institutes of Health Extracellular RNA Communication Consortium (ERCC), a summary of Abdel-Mageed’s study was also published as part of the ERCC leading-edge perspective paper in Cell.

This blog post originated as a press release from Vanderbilt University Medical Center.

A report by researchers at Vanderbilt University Medical Center has shattered conventional wisdom about how cells, including cancer cells, shed DNA into the bloodstream: they don’t do it by packaging the genetic material in tiny vesicles called exosomes.

Their findings, reported April 4 in the journal Cell, have important implications for the development of “liquid biopsies” that could speed cancer diagnosis and improve treatment by detecting tumor-specific genetic material in the blood.

“It’s been a big deal that there is supposedly DNA in exosomes … (and) you could isolate these exosomes in a relatively simple way,” said the paper’s first author, Dennis Jeppesen, PhD. The problem is exosomes don’t contain DNA.

“Exosomes are not your target,” said Jeppesen, a research fellow in the lab of Robert Coffey, Jr., MD, who is internationally known for his studies of colorectal cancer. Instead, Jeppesen and his colleagues propose a new model for how DNA is actively secreted by cells.

Research by Robert Coffey, MD, left, Dennis Jeppesen, PhD, and colleagues has revealed a new way cells shed DNA into the bloodstream.
(Photo by Steve Green / Vanderbilt University)

Greater precision in determining how DNA, RNA, and proteins are packaged and secreted from cells “is crucial for identification of biomarkers and design of future drug interventions,” the researchers concluded.

Coffey, Ingram Professor of Cancer Research in the Vanderbilt University School of Medicine, predicted the paper will “set the field on a firmer foundation” to understand what’s in exosomes and what’s not, and how exosomes might be used as biomarkers or therapeutic targets.

Coffey’s team is part a nationwide consortium funded by the National Institutes of Health (NIH) to study the role of extracellular RNA in diseases including diabetes, glaucoma, muscular dystrophy, and cancer.

Last month the NIH announced the publication of what it called a “landmark collection” of scientific papers in the Cell family of journals on the biology and possible clinical applications of extracellular RNA. Two of those papers, including the paper about exosomes, came from Coffey’s lab.

Virtually every cell in the body releases DNA, RNA, proteins, lipids, and other particles. These so-called “nanoparticles” are thought to be a way that cells communicate with each other. But they also might signal cancer to spread — or metastasize — to other parts of the body.

Cancer by its very nature is constantly evolving. That trait enables many tumors to escape or to become resistant to chemotherapy and other efforts to destroy them.

The genetic material released by cancer cells, however, may also reveal their points of vulnerability. A simple blood test that picks up these circulating cancer clues therefore could lead to earlier diagnoses and more effective treatments.

The problem is how to snag cancer-specific genetic material from the sea of circulating nanoparticles. Each milliliter of blood (it takes 5 milliliters to fill a teaspoon) contains a quadrillion (a thousand trillion) nanoparticles, and at least 100 million small extracellular vesicles.

Many of these vesicles are exosomes, which are known to carry extracellular RNA. Exosomes can be identified by the proteins (called antigens) that sprout from their surfaces. Identifying specific exosomes is one thing; determining what they carry is quite another.

The traditional way is to use high-speed centrifugation to spin exosomes out of a blood sample and into a “pellet” at the bottom of a test tube. Biochemical methods are then used to characterize the RNA, DNA, and proteins in the pellet.

The assumption was that the pellet contained only exosomes and their cargoes. But Jeppesen, who earned his PhD in Molecular Medicine at Aarhus University in Denmark, was skeptical. He decided to take a different approach.

Using a technique called high-resolution density gradient fractionation, Jeppesen and his colleagues separated blood components based on their buoyant density. They found that exosomes floated at a relatively low density while higher-density proteins, including those that bind DNA, sank to a lower gradient.

“We showed this for multiple cancer cell lines,” he said. “We also see the same kind of thing in normal cells.”

To isolate and study exosomes apart from the traditional pellet, Jeppesen and his colleagues developed another technique they called direct immunoaffinity capture. They coated magnetic beads with a “capture antibody” that targeted one of the known proteins, or antigens, on the exosome surface.

That’s how they were able to determine that exosomes don’t carry DNA.

The DNA found in pellets must be secreted by the cell in other ways. One way, the Vanderbilt researchers reported, is through the formation of novel hybrid organelles termed amphisomes.

“We can actually see these amphisomes traffic to the cell surface,” Jeppesen said. “Now it’s possible to say with greater precision what’s in the exosome and what’s in these other vesicles. Now you have an idea of what is the target you’re looking for.”

This blog post was adapted from a press release by the Baylor College of Medicine. See this related video from the ERCC Webinar Series for a discussion of the exceRpt pipeline used in the analysis presented here.

Scientists have improved their understanding of a new form of cell-cell communication that is based on extracellular RNA (exRNA). RNA, a molecule that was once thought to function only inside cells, is now known to participate in a cell-cell communication system that delivers messages throughout the body. To better understand this system, the Extracellular RNA Communication Consortium (ERCC), which includes researchers from Baylor College of Medicine, created the exRNA Atlas resource, the first detailed catalog of exRNAs in human bodily fluids. They also developed web-accessible computational tools other researchers can use to analyze exRNAs from their own data. The study (Murillo, Thistlethwaite, et al. 2019), published in the journal Cell, contributes the first ‘map of the terrain’ that will enable scientists to study the potential roles exRNA plays in health and disease.

“About 10 years ago, scientists began discovering a new communication system between cells that is mediated by exRNA,” said corresponding author Dr. Aleksandar Milosavljevic, professor of molecular and human genetics and co-director of the Computational and Integrative Biomedical Research Center at Baylor College of Medicine. “The system seems to work in normal physiological conditions, as well as in diseases such as cancer.”

The Milosavljevic lab worked with other members of the ERCC to analyze human exRNAs from 19 studies. They soon realized that the system was significantly more complex than initially assumed. Due to that unanticipated complexity, existing laboratory methods failed to reproducibly isolate exRNAs and their carriers. To help create the first map of this complex system of communication, Milosavljevic and his colleagues used computational tools to deconvolute the complex experimental data. Deconvolution refers to a mathematical method and a computational algorithm that separates complex information into components that are easier to interpret.

“Using computational deconvolution, we discovered six major types of exRNA cargo and their carriers that can be detected in bodily fluids, including serum, plasma, cerebrospinal fluid, saliva, and urine,” said co-first author Oscar D. Murillo, a graduate student in Baylor’s Molecular and Human Genetics Graduate Program working in the Milosavljevic lab. “The carriers act like molecular vessels moving their RNA cargo throughout the body. They include lipoproteins – one of the major carriers is High-Density Lipoprotein (HDL or the “good cholesterol”) – a variety of small protein-containing particles, and small vesicles, all of which can be taken up by cells.”

The researchers found that the computational method helps reveal biological signals that could not previously be detected in individual studies due to the naturally complex variation in the biological system. For example, in an exercise challenge study their computational approach revealed differences before and after exercise in the proportions of the exRNA cargo in HDL particles and vesicles in human plasma.

“Exercise increased a proportion of RNA molecules involved in regulating metabolism and muscle function, suggesting adaptive response of the organism to exercise challenge,” Milosavljevic said. “This finding opens the possibility that in other conditions, both in health or disease, the computational method might identify signals that could have physiological and clinical relevance.”

To help researchers around the world with their analyses, Murillo, Milosavljevic and their colleagues have made a computational tool available online (https://exRNA-Atlas.org).

“We anticipate that it will take a combination of scientific knowledge, enhanced experimental techniques to isolate cargo and carriers in bodily fluids, and advanced computational methods to deconvolute and interpret the complexity of the exRNA communication system,” Murillo said.

Other contributors to this work from Baylor College of Medicine include William Thistlethwaite, Matthew E. Roth, Sal Lakshmi Subramanian, Rocco Lucero, Neethu Sha, and Andrew R. Jackson. See the full article for details about the numerous other contributors from the consortium.

This work is part of the NIH Extracellular RNA Communication Consortium paper package and was supported by the NIH Common Fund Extracellular RNA Communication Program (grant U54 DA036134).

Reference
Murillo OD, Thistlethwaite W, et al. exRNA Atlas analysis reveals distinct extracellular RNA cargo types and their carriers present across human biofluids. (2019) Cell 177:463-477. doi: 10.1016/j.cell.2019.02.018. PMID: 30951672.


This blog post originated as a press release from UCSF.

Discovery May Help Explain Immunotherapy Resistance, Hints at New Therapies

Immunotherapy drugs known as checkpoint inhibitors have revolutionized cancer treatment: many patients with malignancies that until recently would have been considered untreatable are experiencing long-term remissions. But the majority of patients don’t respond to these drugs, and they work far better in some cancers than others, for reasons that have befuddled scientists. Now, UC San Francisco researchers have identified a surprising phenomenon that may explain why many cancers don’t respond to these drugs, and hints at new strategies to unleash the immune system against disease.

“In the best-case scenarios, like melanoma, only 20 to 30 percent of patients respond to immune checkpoint inhibitors, while in other cases, like prostate cancer, there is only a single-digit response rate,” said Robert Blelloch, MD, PhD, professor of urology at UCSF and senior author of the new study, published April 4 in Cell. “That means a majority of patients are not responding. We wanted to know why.”

In malignant tissue, a protein called PD-L1 functions as an “invisibility cloak”: by displaying PD-L1 on their surfaces, cancer cells protect themselves from attacks by the immune system. Some of the most successful immunotherapies work by interfering with PD-L1 or with its receptor, PD-1, which resides on immune cells. When the interaction between PD-L1 and PD-1 is blocked, tumors lose their ability to hide from the immune system and become vulnerable to anti-cancer immune attacks.

One reason that some tumors may be resistant to these treatments is that they do not produce PD-L1, meaning that there is nowhere for existing checkpoint inhibitors to act — that is, they may avoid the immune system using other checkpoint proteins yet to be discovered. Scientists have previously shown the PD-L1 protein to be present at low levels, or completely absent, in tumor cells of prostate cancer patients, potentially explaining their resistance to the therapy.

But in their new paper Blelloch’s group is suggesting a very different answer to this puzzle: PD-L1 is being mass-produced by these tumors, they found, but instead of displaying the protein on their surface, cancer cells export PD-L1 in molecular freighters known as exosomes. These PD-L1–packed exosomes sprout from cancer cells and travel through the lymphatic system or bloodstream to lymph nodes, the sites where immune cells are activated to protect the body. There, the PD-L1 proteins act as itinerant molecular saboteurs, remotely disarming immune cells and preventing them from locating tumors to mount an anti-cancer offensive.

So rather than shutting down the immune response at the tumor surface, exosomal PD-L1 can inhibit immune cells before they even arrive there. And unlike PD-L1 found on the tumor’s surface, exosomal PD-L1, for unclear reasons, is resistant to existing checkpoint inhibitors.

“The standard model says that PD-L1 acts on immune cells that travel to the tumor niche, where they encounter this immune-suppressing protein,” Blelloch said. “Our data suggests that this isn’t true for many immunotherapy-resistant tumors. These tumors evade the immune system by delivering exosomal PD-L1 to lymph nodes, where they inhibit the activation of immune cells remotely. These findings represent a break from dogma.”

Blelloch’s group decided to explore exosomes when they noticed something strange that suggested the standard model of PD-L1 presentation was flawed. Like scientists that came before, they found low levels of PD-L1 protein in resistant cancers. But when they looked at messenger RNA (mRNA), the molecular precursor of all proteins, they observed an odd discrepancy: there was far too much PD-L1 mRNA for the scant amount of PD-L1 protein that they measured in the cells.

“We saw the difference between mRNA and protein levels and wanted to figure out what was happening,” Blelloch said. “Our experiments also showed that the protein was in fact being made at some point, and that it wasn’t being degraded. That’s when we looked at exosomes and found the missing PD-L1.”

Exosomal PD-L1 Hampers Immune Response, Promotes Cancer Growth

To show that exosomal PD-L1 was responsible for imparting immune invisibility, the researchers turned to a mouse prostate cancer model that’s resistant to checkpoint inhibitors. When they transplanted these cancer cells into healthy mice, tumors rapidly sprouted. But when the scientists used the gene-editing tool CRISPR to delete two genes required for exosome production, the edited cancer cells were unable to form tumors in genetically identical mice. Though both edited and unedited cells were producing PD-L1, only those unable to create exosomes were visible and vulnerable to the immune system when PD-L1 was blocked.

“The importance of this discovery was immediately evident,” said postdoctoral fellow Mauro Poggio, PhD, lead author of the new study. “Currently in the clinic, there are no drugs available that are capable of counteracting the destructive power of exosomal PD-L1, so understanding the biology of exosomal PD-L1 is the first fundamental step that might lead to novel therapeutic approaches for patients.”

In a complementary experiment, the same CRISPR-edited cancer cells were transplanted into healthy mice, immediately followed by a series of injections of exosomes carrying PD-L1. Unable to produce exosomes, the CRISPR-edited cancer cells should have fallen victim to the immune system. Instead, the injected exosomes were able to neutralize the immune response on behalf of the cancer, which allowed the exosome-deficient cancer cells to form tumors.

To figure out how exosomal PD-L1 was interfering with the immune system, the researchers inspected the lymph nodes of mice that received either CRISPR-edited or unadulterated cancer cells. Mice that received the edited cells showed increased immune cell proliferation and had higher numbers of activated immune cells in their lymph nodes, the central command hubs of the immune system.

In a separate mouse model — a colorectal cancer that’s only partially responsive to immunotherapy — the researchers identified two distinct pools of PD-L1: one on the surface of tumor cells that’s sensitive to PD-L1 inhibitors, and another in exosomes that’s resistant. When they treated the cancer with a combination therapy that involved both preventing exosome formation and administering PD-L1 inhibitors, the mice survived longer than those treated with either approach alone.

“These data from two very different cancer models suggest a novel therapeutic approach, where suppressing the release of PD-L1 in exosomes, either alone or in combination with current checkpoint inhibitors, could overcome resistance in a large fraction of patients currently resistant to treatment with checkpoint inhibitors alone,” Blelloch said.

Exosome-Deficient Tumor Cells Can Act as ‘Vaccine’ Against Immune Resistance

In a surprising result from the new paper, the researchers found that they could use CRISPR-edited, exosome-deficient cancer cells to induce an anti-cancer immune response that targeted tumors that normally resist immune attack.

The researchers first transplanted CRISPR-edited cancer cells unable to produce exosomes into normal mice and waited 90 days. They then transplanted unedited — and presumably immune-evading — cancer cells into the same mice. After having exposed the immune system to the CRISPR-edited, exosome-deficient cancer cells, the unedited cells were no longer invisible. Instead of ignoring these cells, the immune system mounted a vigorous response that targeted these formerly immune-evading cancer cells and prevented them from proliferating.

“The immune system develops an anti-tumor memory after being exposed to cancer cells that can’t produce exosomal PD-L1. Once the immune system has developed memory, it is no longer sensitive to this form of PD-L1 and thus targets exosomal PD-L1–producing cancer cells as well,” Blelloch said.

Another surprising result was achieved when both unedited and CRISPR-edited, exosome-deficient cancer cells were simultaneously transplanted into opposite sides of the same mouse. Though they were introduced at the same time, the CRISPR-edited cells proved dominant — they were able to activate the immune system, which then launched an attack that destroyed the unedited, supposedly immune-resistant tumors growing on the other side.

These results suggest that even the temporary inhibition of the release of PD-L1 in exosomes could lead to long-term, body-wide suppression of tumor growth. Furthermore, they hint at the possibility of a new kind of immunotherapy, one in which a patient’s cancer cells can be edited and reintroduced in order to activate the immune system and goad it into attacking immune-resistant cancers. Suppressing the release of PD-L1 in exosomes or the introduction of the “tumor cell vaccine” devised by the Blelloch team may one day offer hope to patients whose tumors don’t respond to today’s treatment options.

“Much more needs to be uncovered about PD-L1’s function in cancer,” Poggio said. “We are just scratching the surface of what could be a new mechanism that, if blocked, has the potential to suppress many aggressive tumors that don’t currently respond to treatment.”


Authors: Additional authors on the paper include TJ Hu, Chien-Chun Pai, Brandon Chu, Cassandra D. Belair, Anthony Chang, Ursula E. Lang, Qi Fu, and Lawrence Fong of UCSF; Elizabeth Montabana of UC Berkeley.

Funding: Research was supported by the National Institutes of Health Common Fund Extracellular RNA Consortium, the George and Judy Marcus Innovation Fund, and an NIH training grant.

Conflicts: The authors declare no competing financial interests.

About UCSF: UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises three top-ranked hospitals – UCSF Medical Center and UCSF Benioff Children’s Hospitals in San Francisco and Oakland – as well as Langley Porter Psychiatric Hospital and Clinics, UCSF Benioff Children’s Physicians and the UCSF Faculty Practice. UCSF Health has affiliations with hospitals and health organizations throughout the Bay Area. UCSF faculty also provide all physician care at the public Zuckerberg San Francisco General Hospital and Trauma Center, and the SF VA Medical Center. The UCSF Fresno Medical Education Program is a major branch of the University of California, San Francisco’s School of Medicine. Please visit ucsf.edu/news.

This commentary originally appeared as an Editor’s Choice in Science Translational Medicine. Thanks to STM and Steven Jay for permission to reprint here.

Abstract
Combining established techniques enables large-scale production of potentially therapeutic extracellular vesicles enriched with specific miRNAs.

Extracellular vesicles (EVs) are currently being intensively studied for their therapeutic potential following promising clinical results and recent regulatory approvals of cell-based therapies. However, for the excitement surrounding EVs to ultimately yield useful therapies, critical challenges remain to be overcome. Specifically, although microRNA (miRNA) is often cited as a critical component of EV therapeutic activity, specific miRNA amounts in native EVs can be quite low (far less than one miRNA per one EV on average in many cases), raising concern about the potency of EV-based therapies. Further, scalable biomanufacturing of therapeutic EVs is nontrivial and could present a barrier to translation.

To address these issues, Yoo et al. used a combination of established, commercially available technologies to define a method for producing large quantities of EVs enriched with specific miRNAs. First, they used lentiviral vectors to generate stable HEK293 cell lines capable of producing EVs with more than 2000-fold enrichment of specific miRNAs. Then, a hollow fiber bioreactor was employed for continuous production of EVs from the same stable cell lines for up to 30 days, with additional gains in miRNA levels observed compared with EVs harvested from cells grown in conventional cell culture flasks. Last, tangential flow filtration was used to concentrate miRNA-enriched EVs by ~200-fold without precipitate formation. To validate the potential therapeutic utility of EVs produced through this scheme, miR-133a-3p–enriched EVs were injected intraperitoneally in mice. The result was an increase in the level of circulating miR-133a-3p after four hours. The broad applicability of the techniques used in this process suggests that it could be used to increase blood levels of any desired miRNA via EV association.

Further optimization of this method will be necessary to enable production of EVs from different primary cell types, and this production scheme still contains potential manufacturing bottlenecks, such as lentiviral transfection. The ultimate therapeutic potential of miRNA delivery via EVs produced by the process still remains to be established. However, the general approach described is widely applicable to platform production of miRNA-enriched EVs. More importantly, all the technologies employed are commercially available and should be within reach for a majority of academic labs and small companies to access or acquire. Thus, this process could serve as an important template for advancing research and overcoming the lack of method standardization in development of EV therapeutics, taking the entire field closer to clinical translation.

Highlighted Article
K. Yoo, N. Li, V. Makani, R. Singh, A. Atala, & B. Lu. Large-scale preparation of extracellular vesicles enriched with specific microRNA. Tissue Eng. Part C: Methods (2018) 24: 637-644. doi: 10.1089/ten.TEC.2018.0249 PMID: 30306827.