This blog originated as a press release from Cedars-Sinai. Thanks to them for allowing us to repost it here.

Analysis of particles shed by tumors points to new, less invasive way to diagnose malignancies

A recent study sheds light on proteins in particles called extracellular vesicles, which are released by tumor cells into the bloodstream and promote the spread of cancer. The findings suggest how a blood test involving these vesicles might be used to diagnose cancer in the future, avoiding the need for invasive surgical biopsies.

Extracellular vesicles Extracellular vesicles
Credit: NIH

The research is a large-scale analysis of what are known as palmitoylated proteins inside extracellular vesicles, according to Dolores Di Vizio, MD, PhD, professor of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine at Cedars-Sinai. Di Vizio is co-corresponding author of the study, published online June 10 in the Journal of Extracellular Vesicles.

Extracellular vesicles have gained significant attention in the last decade because they contain proteins and other biologically important molecules whose information can be transferred from cell to cell. They are known to help cancer metastasize to distant sites in the body, but exactly how this happens is not clear.

To learn more about this process, the research team looked into a process called palmitoylation, in which enzymes transfer lipid molecules onto proteins. Palmitoylation can affect where proteins are located within cells, their activities, and their contribution to cancer progression.

The investigators examined two types of extracellular vesicles, small and large, in samples of human prostate cancer cells. Using centrifuges, they separated the extracellular vesicles from the other cell materials and analyzed the levels of palmitoylation and the types of proteins present.

The team found extracellular vesicles derived from the cancer cells contained palmitoylated proteins that are associated with the spread of cancer. Further, when the team chemically suppressed the palmitoylation process, the level of some of these proteins went down in the extracellular vesicles.

“Our results suggest that protein palmitoylation may be involved in the selective packaging of proteins to different extracellular vesicle populations in the body,” Di Vizio said. “This finding raises the possibility that by examining these proteins in extracellular vesicles in the bloodstream, we may be able to detect and characterize cancer in a patient in the future without performing a surgical biopsy.”

Dolores Di Vizio Dolores Di Vizio, MD, PhD
Professor of Surgery, Biomedical Sciences and Pathology
and Laboratory Medicine at Cedars-Sinai.

Di Vizio said the next step in the research is to conduct a study in collaboration with her Cedars-Sinai colleagues and industry partners that will use advanced technologies, including mass spectrometry and flow cytometry, with the goal of identifying clinically significant prostate cancer at diagnosis.

In addition to Di Vizio, Wei Yang, PhD, associate professor of Surgery at Cedars-Sinai, and Andries Zijlstra, PhD, are co-corresponding authors for the study. Zijlstra completed the research while working at Vanderbilt University Medical Center in Nashville. Javier Mariscal, PhD, a postdoctoral scientist in Di Vizio’s laboratory, is the study’s first author.

Reference
Mariscal J et al. Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles. (2020) J Extracell Vesicles 9:1764192. doi: 10.1080/20013078.2020.1764192. PMID: 32944167.

Funding: Research reported in this publication was supported by the National Institutes of Health under award number R01CA218526 and by the U.S. Department of Defense.

This blog originated as a press release from the University of Sussex. Thanks to them for allowing us to repost it here.

Scientists at the University of Sussex have identified a potential pattern within blood which signals the presence of motor neuron disease; a discovery which could significantly improve diagnosis.

Currently, it can take up to a year for a patient to be diagnosed with amyotrophic lateral sclerosis (ALS), more commonly known as motor neuron disease (MND).

But after comparing blood samples from patients with ALS, those with other motor-related neurological diseases, and healthy patients, researchers were able to identify specific biomarkers which act as a diagnostic signature for the disease.

Researchers hope that their findings, published in the journal Brain Communications, and funded by the Motor Neurone Disease Association (MNDA), could lead to the development of a blood test which will identify the unique biomarker, significantly simplifying and speeding up diagnosis.

With patients living, on average, just 2-5 years after diagnosis, this time could be crucial.

Professor Majid Hafezparast, a professor of Molecular Neuroscience at the University of Sussex, led the research in collaboration with Professors Nigel Leigh and Sarah Newbury from the Brighton and Sussex Medical School, Martin Turner from the University of Oxford, Andrea Malaspina from Queen Mary, University of London, and Albert Ludolph from the University of Ulm.

He said: “In order to effectively diagnose and treat ALS, we are in urgent need of biomarkers as a tool for early diagnosis and for monitoring the efficacy of therapeutic interventions in clinical trials.

“Biomarkers can indicate the disease is present and help us to predict its progression rate.

“In our study, we compared serum samples taken from the blood of 245 patients and controls, analysing their patterns of non-coding ribonucleic acids (ncRNA).

“We found a biomarker signature for motor neurone disease that is made up of a combination of seven ncRNAs. When these ncRNA are expressed in a particular pattern, we are able to classify whether our samples come from ALS patients or controls.”

Biomarker discovery pipeline

Dr Greig Joilin, the research fellow who undertook this work in Professor Hafezparast’s team said: “We hope that, with further work to validate these biomarkers, a blood test could be developed to help improve diagnosis of motor neuron disease.

“We are now looking to see whether they can predict prognosis to give patients and their families some insight as they begin to understand the disease. Our work could also help other scientists to measure the effectiveness of potential drug treatments against the ncRNA levels. Further, it provides new insight into the cellular and molecular events that contribute to the disease.”

ALS is a group of conditions which affects the nerves in the brain and spinal cord leading to weakness in the muscles and rapid deterioration.

Doctors still don’t know why this happens and there is currently no cure, although existing drug treatments can help patients with daily life and extend life expectancy – but only by two to four months on average.

Stephen Hawking is perhaps one of the most famous cases of motor neuron disease, but more recently Geoff Whaley and his wife Ann brought to light the troubling situation of patients in the UK who wish to end their life before the final phase of the disease takes hold.

Professor Hafezparast hopes that his team’s discovery will improve the outlook for patients by improving diagnosis and giving other researchers a valuable tool to test potential treatments. The researchers are now looking to validate this biomarker signature in a larger cohort of patients and begin to understand why these ncRNAs change in ALS patients.

Reference
Joilin G, et al. Identification of a potential non-coding RNA biomarker signature for Amyotrophic Lateral Sclerosis. (2020) Brain Commun. 2: fcaa053. doi: 110.1093/braincomms/fcaa053 PMID: 32613197.

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
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 Commun 11:2761. doi: 10.1038/s41467-020-16337-y PMID: 32487994.

With a focus on screening local healthcare workers and first responders, the epidemiological study seeks to understand the prevalence of coronavirus infections in the community. The lab of ERCC2’s Louise Laurent is part of the core research team.

LA JOLLA, CA—A consortium that includes many of San Diego’s top medical and scientific research institutes has launched a large-scale COVID-19 screening effort to better understand the spread and prevalence of the virus in the local community, with an initial focus on evaluating healthcare workers and first responders.

Known as the San Diego Epidemiology and Research for COVID Health (SEARCH) alliance, the cross-institutional collaboration is co-led by scientists and clinical researchers at Rady Children’s Hospital-San Diego, Rady Children’s Institute for Genomic Medicine, Scripps Research, and University of California San Diego.

As part of the SEARCH study, San Diego fire fighters are screened for SARS-CoV-2, the virus that causes COVID-19. .
Credit: Don Boomer

The research project is applying innovative technologies and screening strategies to paint a more comprehensive picture of how widely COVID-19 has spread—and continues to spread—throughout the San Diego area. All data collected will contribute to an epidemiological study that will encompass active cases of COVID-19 as well as its “silent spread” to people who never developed symptoms.

“For health officials to gain the upper hand on a virus in our community, they need more complete information about how it’s moving through the population,” says Lauge Farnaes, MD, PhD, assistant medical director at Rady Children’s Institute for Genomic Medicine. “Our goal is to fill those gaps of knowledge by leveraging San Diego’s unique expertise in science and medicine.”

As COVID-19 cases in San Diego began to rapidly increase in late March, the collaborators sprang into action. Through emails and Zoom meetings, they formulated a research proposal and created a scalable testing framework that would enable them to screen symptomatic individuals as well as people who may have COVID-19 without showing symptoms.

In the initial phase of the program, nasopharyngeal swabs are used to collect samples from study participants at a local drive-up site and the samples are screened at research laboratories at Scripps Research and UC San Diego. Any positive results are then confirmed by Rady Children’s Institute of Genomic Medicine’s nationally accredited and certified clinical laboratory.

In addition, the researchers are conducting “serosurvey” studies that look for antibodies to the virus. Serosurveys, short for serological surveys, involve finger-prick blood tests of people who haven’t been diagnosed with COVID-19 to gauge the extent to which SARS-CoV-2 has spread undetected. The program relies heavily on automation for screening, with the capacity to screen thousands of individuals daily while keeping costs low.

Since the study launched, SEARCH has enrolled more than 10,000 participants who are asymptomatic or mildly symptomatic. Thus far, researchers have found that an average of two participants per every 1,000 enrolled had a positive result for the SARS-CoV-2 virus.

Participation is voluntary but currently limited to invited healthcare workers from participating hospitals, firefighters and other first responders.

“The majority of our personnel are firefighters and lifeguards who regularly interact with the public and are at a greater risk of exposure to COVID-19. Our goal is for each and every employee to be screened,” says San Diego Fire-Rescue Chief Colin Stowell. “We appreciate the opportunity to participate in the SEARCH study, which benefits our employees and the communities we serve.”

SEARCH is also conducting large-scale SARS-CoV-2 genomic studies, analyzing changes in the virus genome from patient samples for clues to how the disease moved from city to city and person to person. All genomic data gathered by SEARCH is deidentified and then made publicly available to the scientific community to expedite discoveries that will help end the pandemic.

SEARCH’s core research team consists of the following members of the San Diego scientific and medical communities:

  • Kristian Andersen, PhD, Professor of Immunology and Microbiology at Scripps Research
  • Lauge Farnaes, MD, PhD, Assistant Medical Director at Rady Children’s Institute for Genomic Medicine
  • Rob Knight, PhD, Professor of Pediatrics, Bioengineering and Computer Science & Engineering, and Founding Director of the Center for Microbiome Innovation at UC San Diego
  • Louise Laurent, MD, PhD, Professor of Obstetrics, Gynecology, and Reproductive Sciences at UC SanDiego School of Medicine
  • Gene Yeo, PhD, MBA, Professor of Cellular and Molecular Medicine and Co-Director Bioinformatics and Systems Biology Graduate Program at UC San Diego School of Medicine

The research is made possible by a dedicated team of laboratory staff, postdoctoral researchers, graduate students, nurses, physicians, and volunteers across the partner institutions.

For more information, visit searchcovid.info.

This blog originated as a press release from Scripps Research. Thanks to Scripps and the SEARCH team for permission to post it here.

This blog originated as a press release from Notre Dame News.

As testing for the coronavirus continues throughout the United States, researchers have been closely watching results, particularly reported rates of false negatives.

According to the Radiological Society of North America, a reported 40 to 70 percent of coronavirus tests from throat swab samples returned false negatives at the onset of the epidemic. Given the highly infectious nature of this particular coronavirus, individuals receiving false negative results — told they do not carry the virus when in fact they do — could continue to infect others.

“It is very concerning,” said Hsueh-Chia Chang, the Bayer Professor of Chemical and Biomolecular Engineering at the University of Notre Dame. “In an overcrowded hospital, where there is only room to quarantine the COVID-19 carriers, false negatives would mean some carriers can continue to infect other patients and healthcare workers. This, unfortunately, is also true for other infectious viral diseases such as dengue and malaria, when there is an epidemic. False negatives are usually not an urgent problem, when every symptomatic patient can be quarantined and there are fewer people to infect — until an epidemic overcrowds our hospitals and we have only enough space to sequester the carriers.”

At Notre Dame, Chang’s research lab focuses on the development of new diagnostic and micro/nanofluidic devices that are portable, sensitive and fast. His work includes diagnostics with applications to DNA/RNA sensing. Current coronavirus tests are RNA-based.

Chang said technology his lab developed for other uses could easily be extended to apply to testing for the coronavirus.

“I had developed the technology for isolating cellular material such as vesicles and exosomes during liquid biopsies. They turn out to be the same size as the virus,” he said.

Dr. Chang at the ERCC2 kickoff meeting in September 2019 discussing his work developing technology to isolate extracellular vesicles.

The tests combine nanofiltration with immersed AC Electrospray (iACE) digital droplet isothermal polymerase chain reaction (PCR) technology. The nanofiltration part of the test would work to wash away inhibitors while the iACE would allow detection of a very small number of the coronavirus viral particles per sample, improving sensitivity during testing.

Detection can be inhibited at the molecular level, Chang explained. The current tests for coronavirus are PCR-based, a common method that replicates a small sample of RNA — from a nose or throat swab, for example —increasing the number of RNA exponentially in order to identify the presence of the virus and determine the stage of infection.

“The inhibitors, in this case molecules and ions, prevent the reaction from occurring even when the target virus is there, resulting in a false negative,” said Chang. “Our technology removes these inhibitors. There is also the question of yield. In removing the inhibitors, you do not want to lose the target virus as well, so they escape detection. Our technology achieves higher yield in retaining the virus. It extracts the target virus with higher yield and purity than current technology.”

His size-based nanotechnology is especially useful in this case. The coronavirus is between 60 and 140 nm in size. The inhibiting molecules, Chang explained, are smaller than 60 nm, which means he can effectively wash away those particles while retaining the virus.

“The issue is that such small particles often cause clogging and produce high pressure during tests, and break up virus particles, so they’re lost to detection. This is one cause for false negatives,” Chang said. “We already have a patented design that allows filtration of the virus from inhibitors without clogging and without breaking the target virus particles.”

Notre Dame has suspended laboratory research operations across campus with the exception of coronavirus-related research. Chang’s lab is one that received approval to remain operational. Researchers in his lab are not currently working with samples that contain the coronavirus, rather they are testing the technology against a lentivirus serum — a virus that is similar but safe to work with.

“I’m fortunate to have very passionate and capable postdoctoral and Ph.D. students that believe in these technologies and are willing to be in the lab during these trying times,” he said. “Their presence is completely voluntary. In fact, we reduced the number of researchers to three essential people even though several more had volunteered. They abide by very stringent social distancing and lab hygiene rules. They also work in shifts to minimize contact. Another research professor and I are in constant email and cellular communication with them. They are currently testing lentivirus in saliva samples and trying to get more data to back up the numbers.”

The numbers, so far, show that Chang’s test combining nanofiltration with iACE technology are 1,800 times more sensitive in tests run with the lentivirus.

If additional grant funding is approved for his research, Chang said he intends to work with the Centers for Disease Control and Prevention or other Food and Drug Administration approved labs to validate the technology with actual samples containing the coronavirus.

In a white paper outlining the research, Chang set milestones for the work with hopes —if approved — to begin manufacturing devices in six months. However, given the current state of the pandemic, Chang said realistically the technology would be used in cases of future epidemics and outbreaks.

“I think the country is realizing the need for better control of infectious epidemics,” he said. “We hope to develop technology that will help control future epidemics involving any virus or bacteria, not just in the U.S., but especially in the developing world.”

This blog originated as a press release from UCSD News.

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 team published their findings in Current Biology.

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.

*These authors contributed equally

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.

TEM of extracellular vesicles in tumor tissue
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 Vesicles 9:1, 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
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.