This blog originated as a press release from ISGlobal, the Barcelona Institute for Global Health. Thanks to ISGlobal for permission to post it here.
A new study shows that extracellular vesicles from the malaria parasite Plasmodium vivax promote parasite adhesion to spleen cells
Extracellular vesicles (EVs) play a role in the pathogenesis of malaria vivax, according to a study led by researchers from the Barcelona Institute for Global Health (ISGlobal), an institution supported by the ”la Caixa” Foundation, and the Germans Trias i Pujol Research Institute (IGTP). The findings, published in Nature Communications, indicate that EVs from P. vivax patients communicate with spleen fibroblasts promoting the adhesion of parasite-infected red blood cells. These data provide important insights into the pathology of vivax malaria. The study was carried out at the Can Ruti Campus, with the participation of the IGTP Genomics platform, the Nephrology service of the Germans Trias i Pujos Hospital, and researchers from the Irsicaixa AIDS Research Institute.
Plasmodium vivax is the most widely distributed human malaria parasite, mostly outside sub-Saharan Africa, and responsible for millions of clinical cases yearly, including severe disease and death. The mechanisms by which P. vivax causes disease are not well understood. Recent evidence suggests that, similar to what has been observed with the more lethal P. falciparum, red blood cells infected by the parasite may accumulate in internal organs and that this could contribute to the pathology of the disease. In fact, the team led by Hernando A. del Portillo and Carmen Fernández-Becerra, recently showed that P. vivax-infected red blood cells adhere to human spleen fibroblasts thanks to the surface expression of certain parasite proteins, and that this expression is induced by the spleen itself. “These findings indicate that the spleen plays a dual role in malaria vivax,” says ICREA researcher Hernando A del Portillo. “On one hand, it eliminates infected red blood cells. On the other hand, it may serve as a “hiding” place for the parasite.” This could explain why P. vivax can cause severe disease in spite of low peripheral blood parasitemia.
Hernando A. del Portillo and Carmen Fernández-Becerra
To understand the molecular mechanisms responsible for this adhesion process, the research team turned its attention to something they have been working on for the last few years: extracellular vesicles. These small particles surrounded by a membrane are naturally released from almost any cell and play a role in communication between cells. There is increasing evidence that they could be involved in a wide range of pathologies, including parasitic diseases such as malaria. “Our new findings reveal, for what we believe is the first time, a physiological role of EVs in malaria,” says del Portillo, last author of the study.
The research team isolated EVs from the blood of patients with acute P. vivax infection or from healthy volunteers and showed a very efficient uptake of the former by human spleen fibroblasts. Furthermore, this uptake induced the expression of a molecule (ICAM-1) on the surface of the fibroblast which in turn serves as an “anchor” for the adherence of P. vivax-infected red blood cells.
“Our study provides insight into the role of extracellular vesicles in malaria vivax and supports the existence of parasite populations adhering to particular cells of the spleen, where they can multiply while not circulating in the blood” says Fernández-Becerra, senior co-author of the study. “Importantly, these hidden infections could represent an additional challenge to disease diagnosis and elimination efforts as they might be the source of asymptomatic infections,” she adds.
Reference Toda H, Diaz-Varela M, Segui-Barber J. Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence. Nat Commun11:2761. doi: 10.1038/s41467-020-16337-y PMID: 32487994.
Researchers at the University of California San Diego discovered that high blood levels of RNA produced by the PHGDH gene could serve as a biomarker for early detection of Alzheimer’s disease. The work could lead to the development of a blood test to identify individuals who will develop the disease years before they show symptoms.
The PHGDH gene produces RNA and proteins that are critical for brain development and function in infants, children, and adolescents. As people get older, the gene typically ramps down its production of these RNAs and proteins. The new study, led by Sheng Zhong, a professor of bioengineering at the UC San Diego Jacobs School of Engineering in collaboration with Dr. Edward Koo, a professor of neuroscience at the UC San Diego School of Medicine, suggests that overproduction of extracellular RNA (exRNA) by the PHGDH gene in the elderly could provide an early warning sign of Alzheimer’s disease.
“Several known changes associated with Alzheimer’s disease usually show up around the time of clinical diagnosis, which is a little too late. We had a hunch that there is a molecular predictor that would show up years before, and that’s what motivated this study,” Zhong said.
The discovery was made possible thanks to a technique developed by Zhong and colleagues that is sensitive enough to sequence tens of thousands of exRNAs in less than one drop of blood. The method, dubbed SILVER-SEQ, was used to analyze the exRNA profiles in blood samples of 35 elderly individuals 70 years and older who were monitored up to 15 years prior to death. The subjects consisted of 15 patients with Alzheimer’s disease; 11 “converters,” which are subjects who were initially healthy then later developed Alzheimer’s; and 9 healthy controls. Clinical diagnoses were confirmed by analysis of post-mortem brain tissue.
The results showed a steep increase in PHGDH exRNA production in all converters approximately two years before they were clinically diagnosed with Alzheimer’s. PHGDH exRNA levels were on average higher in Alzheimer’s patients. They did not exhibit an increasing trend in the controls, except for in one control that became classified as a converter.
The researchers note some uncertainty regarding the anomalous converter. Since the subject died sometime during the 15-year monitoring, it is unclear whether that individual would have indeed developed Alzheimer’s if he or she lived longer, Zhong said.
The team acknowledges additional limitations of the study.
“This is a retrospective study based on clinical follow-ups from the past, not a randomized clinical trial on a larger sample size. So we are not yet calling this a verified blood test for Alzheimer’s disease,” said co-first author Zixu Zhou, a bioengineering alumnus from Zhong’s lab who is now at Genemo Inc., a startup founded by Zhong. “Nevertheless, our data, which were from clinically collected samples, strongly support the discovery of a biomarker for predicting the development of Alzheimer’s disease.”
In addition to randomized trials, future studies will include testing if the PHGDH biomarker can be used to identify patients who will respond to drugs for Alzheimer’s disease.
The team is also open to collaborating with Alzheimer’s research groups that might be interested in testing and validating this biomarker.
“If our results can be replicated by other centers and expanded to more cases, then it suggests that there are biomarkers outside of the brain that are altered before clinical disease onset and that these changes also predict the possible onset or development of Alzheimer’s disease,” Koo said. “If this PDGDH signal is shown to be accurate, it can be quite informative for diagnosis and even treatment response for Alzheimer’s research.”
This study was performed in collaboration with Genemo Inc.
Reference Yan Z*, Zhou Z*, Wu Q*, et al. Presymptomatic increase of an extracellular RNA in blood plasma associates with the development of Alzheimer’s disease. Curr Biol (2020) AOP. doi: 10.1016/j.cub.2020.02.084 PMID: 32220323.
Extracellular vesicles (EVs) regulate many processes in the healthy body. They also play a role in cancer, sending signals between cells in the tumor microenvironment. EVs can stimulate tumor cell migration, invasion, blood vessel growth, immune response, and cell survival, as well as metastasis. However, we know little about the cargo of these EVs that play such diverse roles. Analysis of vesicle cargo can shed light on the molecular mechanisms of vesicle biology and be helpful in disease diagnosis and prognosis.
I am lucky to be a member in Jan Lötvall’s lab in Gothenburg, Sweden, which pioneered the field of extracellular vesicles with the early discovery of exosomes shuttling RNA between cells. An exciting collaboration with Yong Song Gho from POSTECH in South Korea led us to develop a new approach to isolate vesicles from human tumor tissues. Using this technology, we were able to isolate and characterize subpopulations of extracellular vesicles from melanoma metastatic tissue. We just published our findings in the Journal of Extracellular Vesicles. Jan Lötvall also discussed them in a recent ERCC webinar.
Finding extracellular vesicles in tumor tissue with TEM
Our first challenge was to find vesicles in metastatic melanoma tumor tissues. Using transmission electron microscopy, we showed that the tumor microenvironment is a complex world composed of different types of cells and structures with vesicles present between them.
Transmission electron micrograph of melanoma metastatic tissue showing a large tumor cell and two lymphocytes. Black stain, possibly melanin, is clearly visible inside the melanoma cells, which are recognizable from their characteristic cell membrane. The higher magnification image shows vesicles (red arrows) in the extracellular space.
In this study, we performed a detailed proteomics analysis of EVs isolated from metastatic melanoma tissues from 27 patients. We identified numerous new EV proteins, including potential biomarkers for metastatic melanoma.
Tumor tissue vs. Cell lines
Studying extracellular vesicles in tumor tissues is important, because, compared to cell lines, tumor tissues more closely approximate the situation in vivo. EVs from tumor tissue are more likely to represent the full array of vesicle behaviors and populations in the tumor microenvironment. Furthermore, to develop a non-invasive test for cancer, we must use biofluids such as circulating plasma, where vesicles from all over the body intermingle. A proteomic snapshot of vesicles isolated directly from tumor tissue can help target the search for disease-specific biomarker in that complex mixture. We trust the tools and experiments developed in this work will contribute to our field’s understanding of EV function in complicated tissues such as the metastatic melanoma tumor.
Reference Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög J.L, Johansson I, Fuchs J, Thorsell A, Gho YS, R, Olofsson Bagge R, Lötvall J Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. Journal of Extracellular Vesicles9:1, 1722433 doi: 10.1080/20013078.2020.1722433.
This work was supported by the Swedish Research Council (K2014-85X-22504-01-3), the Swedish Heart and Lung Foundation (20120528), the Swedish Cancer Foundation (CAN2014/844), and the Knut och Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden).
Flow cytometry (FC) is a powerful method for counting single cells and measuring their molecular components. There is increasing interest in applying flow cytometry to the analysis of extracellular vesicles (EV), but EVs are orders of magnitude smaller than the cells for which FC instruments and protocols were originally designed. To catalyze the development of new instruments and assays for EV flow cytometry, three scientific societies came together to form the EV Flow Cytometry Working Group (evflowcytometry.org):
ISEV, the International Society of Extracellular Vesicles
ISAC, the International Society for Advancement of Cytometry, and
ISTH, the International Society for Thrombosis and Haemostasis.
The working group first performed two standardization studies, distributing standards and samples to EV-FC laboratories worldwide to enable an objective comparison of methods, instruments, controls, and analytical tools. Those initial studies led to the realization that a standard framework for reporting experimental results is essential.
The working group has now published that standard in the Journal of Extracellular Vesicles. It is called MIFlowCyt-EV, the Minimum Information to report for Flow Cytometry studies of Extracellular Vesicles. The MIFlowCyt-EV reporting framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a Flow Cytometry experiment (MIFlowCyt) standard.
The figure above outlines the 7 main categories of information included in the framework. Not all EV-FC experiments will involve all seven areas, but any area touched on by an experiment should follow the MIFlowCyt-EV reporting guidelines.
MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not mandate the use of specific instruments or protocols, since the field of EV flow cytometry is still rapidly evolving. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Consistent reporting of the results of EV flow cytometry studies will improve the ability to quantitatively compare results from different laboratories and support the development of new instruments and assays for improved measurement of EVs.
Reference Welsh JA, et al. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J Extracell Vesicles (2020) doi: 10.1080/20013078.2020.1713526
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 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.
Jones PS, et al. Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma. (2019) eBioMedicine48:23-35. doi: 10.1016/j.ebiom.2019.09.025. PMID: 31628025.
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.
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.
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.
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.