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.
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.
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:
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.”
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.
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.
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.
University of Alabama at Birmingham researchers have found a novel, previously unreported pathogenic entity that is a fundamental link between chronic inflammation and tissue destruction in the lungs of patients with chronic obstructive pulmonary disease, or COPD. COPD is the fourth-leading cause of death in the world.
This pathogenic entity — exosomes from activated polymorphonuclear leukocytes, or PMNs — caused COPD damage when the small, subcellular particles, collected from purified PMNs, were instilled into the lungs of healthy mice. Remarkably, the UAB researchers also collected exosomes from the lung fluids of human patients with COPD and the lung fluids of neonatal ICU babies with the lung disease bronchopulmonary dysplasia; when those human-derived exosomes were instilled into the lungs of healthy mice, they also caused COPD lung damage. Damage was primarily from PMN-derived exosomes from the human lungs.
“This report seems to provide the first evidence of the capability of a defined non-infectious subcellular entity to recapitulate disease phenotype when transferred from human to mouse,” said J. Edwin Blalock, Ph.D., professor of pulmonary, allergy and critical care medicine in the UAB Department of Medicine. “I think this could be a very profound discovery. A lot of what we have found here will apply in other tissues, depending on the disease.”
Other diseases marked by immune cell inflammation and tissue destruction include heart attacks, metastatic cancer, and chronic kidney disease. The activated PMN exosomes may also contribute to lung damage in other lung diseases that have excessive PMN-driven inflammation, such as cystic fibrosis. The study is reported in the journal Cell.
“These findings highlight a novel role of the innate immune response in chronic lung diseases and could be used for the development of new diagnostics and therapeutics for COPD and possibly cystic fibrosis,” said James Kiley, Ph.D., director of the Division of Lung Diseases at the National Heart, Lung, and Blood Institute, part of the National Institutes of Health.
Background COPD, a smoking-associated disease, is marked by PMN-driven inflammation in the lungs. Damage to the lung tissue leads to airway obstruction, shortness of breath, and respiratory failure. PMN immune cells, also known as neutrophils, are part of the body’s white blood cell defense against infections and tissue damage. They comprise 60 percent of the body’s white blood cells, or about 2.5 billion PMNs in each pint of blood. PMNs are voracious eaters of microbes or damaged human cells after activation by a signal of infection.
All cells shed exosomes. These tiny extracellular membrane-bound vesicles can be mediators of cell-to-cell communication, and they can ferry a diverse cargo of proteins, lipids, and nucleic acids from cell to cell. The UAB research focused on a recently found third role for exosomes — the ability to harbor protease enzymes.
Activated PMNs are known to release neutrophil elastase, or NE, a protease that can degrade type I collagen and elastin. The collagen and elastin proteins help form the extracellular matrix that glues cells together. In the lungs, the extracellular matrix and lung cells are sheets of tissue that help form the tiny alveoli, where the lung exchanges oxygen and carbon dioxide. In COPD, the damaged alveoli enlarge, reducing oxygen exchange and forcing the heart to pump harder to push blood through the lungs.
NE and other proteases from PMNs can attack microbes. Healthy lungs are protected by anti-proteases that can inhibit the proteases. Normally, NE is inhibited by a robust barrier of alpha1-antitrypsin in the lung.
The research Blalock and fellow researchers investigated whether NE might exist in an exosomal form and whether such exosomes might bypass alpha1-antitrypsin inhibition to contribute to inflammatory lung disease.
They found that exosomes from quiescent PMNs did not cause COPD when transferred to healthy mice. In contrast, exosomes from activated PMNs did cause COPD, as measured by histologic changes of the alveoli, increased pulmonary resistance and enlargement of the right heart ventricle that pumps blood to the lung.
The activated PMN exosomes were covered with enzymatically active surface-bound NE, while quiescent PMN exosomes had none. This surface NE was resistant to alpha1-antitrypsin inhibition; the exosomes from activated PMNs degraded collagen, they caused emphysema when put into mouse lungs, and they carried the PMN cell-surface markers CD63 and CD66b that identify them as coming from PMNs. Human COPD lung-derived exosomes carrying those PMN cell-surface markers conferred COPD to mice.
A very large dose of purified NE — enough to overwhelm the alpha1-antitrypsin barrier — can cause alveolar enlargement in mice. Because the exosome-bound NE was protected against apha1-antitrypsin inhibition, researchers found that the dose of activated PMN exosomes needed to cause the same damage as purified NE was 10,000 times less.
The activated PMN exosomes had another cause for their aggressive proteolysis — they carried integrin Mac-1 on their surface. Integrin Mac-1 allowed the exosomes to bind directly to collagen fibrils, a second mechanism besides protected NE for why the proteolytic exosomes exert an outsized degradative capacity in relation to their size and protease load.
“This investigation reveals an entirely unappreciated aspect of the interplay between inflammation, proteolysis and matrix remodeling with far-reaching implications for future research,” Blalock said. “Our report significantly expands the biological repertoire of the exosome, demonstrating potent biological effects of these particles ex cellula.”
Looking ahead The study also suggests therapeutic strategies to interrupt pathogenic aspects of PMN exosome function: 1) disrupting the ionic binding of the NE to the exosome, to dislodge the NE and make it susceptible to alpha1-antitrypsin; 2) inhibiting the exosomal integrin Mac-1 to block collagen binding; and 3) directly inhibiting the exosomal NE with small-molecule compounds.
Blalock is also interested in another big question — exosome activity in healthy smokers.
“Only one in seven or one in eight smokers gets COPD,” he said. “It would be an amazing outcome if we found activated PMN exosomes in a subpopulation of people who smoke.” Those people could then be warned of the risk they faced.
This Cell study took six years of work.
Significant research was done by co-first authors Kristopher Genschmer, Ph.D., and Derek W. Russell, M.D., who were NIH T32 grant trainees with Blalock. Both are assistant professors in the UAB Division of Pulmonary, Allergy and Critical Care Medicine. Amit Gaggar, M.D., Ph.D., a professor of pulmonary, allergy and critical care medicine, is co-senior author with Blalock, and he is a former trainee who did his Ph.D. with Blalock. Co-author Charitharth Vivek Lal, M.D., assistant professor in the UAB Pediatrics Division of Neonatology, is the physician who collected the lung fluid from neonates and performed all of the bronchopulmonary dysplasia work.
Co-authors with Genschmer, Russell, Gaggar, Lal and Blalock of the paper “Activated PMN exosomes: Pathogenic entities causing matrix destruction and disease in the lung” are Tomasz Szul, Mojtaba Abdul Roda, Xin Xu, Liliana Viera, Tarek H. Abdalla, Robert W. King, J. Michael Wells and Mark T. Dransfield, UAB Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine; Preston E. Bratcher, National Jewish Medical Center, Denver, Colorado; Brett D. Noerager, University of Montevallo, Montevallo, Alabama; Gabriel Rezonzew, UAB Department of Pediatrics; Brian S. Dobosh, Camilla Margaroli and Rabindra Tirouvanziam, Department of Pediatrics, Emory University, Atlanta, Georgia; and Carmel M. McNicholas, UAB Department of Cell, Developmental and Integrative Biology.
This study was supported by National Institutes of Health grants HL135710, HL077783, HL114439, HL110950, HL126596, HL102371, HL126603, HL123940, HL105346-07 and HL105346-05; American Heart Association grant 17SDG32720009; and Veterans Affairs grant BX001756.
Blalock is a distinguished professor in the UAB School of Medicine, and he holds the Nancy E. Dunlap, M.D., Endowed Chair in Pulmonary Disease.
Reference Genschmer KR, Russell DW, et al. Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung. Cell (2019) 176: 113-126. doi: 10.1016/j.cell.2018.12.002. PMID: 30633902.
Pharmacodynamic Biomarkers and DM There is now strong support for the concept that a panel of splicing events may serve as a pharmacodynamic biomarker for go/no go decisions in drug development for myotonic dystrophy type 1 (DM1) and Duchenne muscular dystrophy (DMD). Data establishing splicing event sensitivity to free MBNL levels has converged with the natural history of alternative splicing patterns in DM patients to yield a subset of splicing events with the sensitivity and reproducibility to evaluate candidate therapeutics in early stage clinical trials. Quantitative pharmacodynamic biomarkers are invaluable in de-risking industry drug discovery and development, as they facilitate early stage assessment of molecular target engagement and modulation and may inform dose ranging studies. The only caveat is the dependence of these measures upon repeated muscle biopsies (a risk reduced, but not eliminated, by more tolerable needle biopsies). The identification and validation of a non-invasive assay of patient splicing status would be a valuable step forward for clinical trials in DM.
Early Support for a Non-Invasive Biomarker for DM1 Dr. Thurman Wheeler and colleagues at Massachusetts General, Harvard Medical, and Boston Children’s have explored the concept that a subset of extracellular RNAs (exRNAs) released into blood or urine may: (a) reflect alternative splicing status in DM-affected tissues and (b) thereby serve as an easily accessible pharmacodynamic biomarker platform for DM1 (Antoury et al., 2018). These studies were supported in part by a grant to facilitate “Development of Biomarkers for Myotonic Studies” from Myotonic Dystrophy Foundation/Wyck Foundation.
The research team initially found that > 30 transcripts that are alternatively spliced in DM1 muscle biopsies were detectable in human blood and urine samples; follow-up studies confirmed the presence of RNAs in extracellular fluids/exosomal particles. Normalized DMPK expression levels in urine from DM1 patients, by droplet digital PCR, were ~50% of unaffected controls. Assessments of DM1-established alternative splicing events showed that a subset (10/33) also occurred in urine exRNA, including being conserved in longitudinal (6-26 month) studies of the same patients. Assessments of alternative splicing events in blood exRNA did not yield the same value.
Using principal component analysis of 10 alternative splicing events observed in urine exRNA, the research team then generated a putative composite biomarker panel for DM1. The ensuing predictive model of alternative splicing in DM1 proved to be 100% accurate in comparisons of training and independent validation data sets to distinguish DM1 from unaffected controls and in distinguishing disease status of subsequently enrolled subjects. The research team also linked alternative splicing patterns in urine exRNA to variation in DM1 clinical phenotypes, suggesting that modeling of urine exRNA alternative splicing may allow both the tracking of disease progression and the impact of candidate therapeutics.
Finally, to address questions as to the source of urine exRNA, the team assessed alternative splicing in urinary tract cells of DM1 mouse models (the ubiquitous Mbnl1 ko and the tissue-specific HSALR). While kidney and bladder cells of the Mbnl1 ko reflected patterns in skeletal muscle, assessments of the same tissues in the HSALR showed no differences from control mice. These data strongly suggested that the exRNAs assessed in urine reflect exosomes released from urinary tract cells. Some of the alternatively spliced transcripts in urine exRNA also were shown to be altered by antisense oligonucleotide drugs previously shown to correct splicing patterns in DM1 mouse models. The research team’s parallel studies of Duchenne muscular dystrophy also supported the concept that urine exRNA has utility as a pharmacodynamic biomarker in drug intervention studies.
Towards a Non-Invasive Biomarker for DM1 Taken together, these data provide compelling proof of concept that a panel of alternative splicing events assessed in urine may serve as a robust composite biomarker of DM1 progression and as a tool for assessment of candidate therapeutics. A non-invasive biomarker such as this would greatly extend the ability to perform repeated measurements in longitudinal natural history studies (as a disease progression biomarker) and in interventional clinical trials (as patient stratification and pharmacodynamic biomarkers), including making assessment of pediatric DM1 patient cohorts feasible. Although it is not essential to formally qualify a biomarker, existing regulatory agency guidance documents (see References below) provide a valuable evidentiary framework for moving non-invasive biomarker work towards an accepted clinical tool for DM1.
References Antoury L, Hu N, Balaj L, Das S, Georghiou S, Darras B, Clark T, Breakefield XO, Wheeler TM. Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies. Nat Commun. (2018) 9: 3906. doi: 10.1038/s41467-018-06206-0. PMID: 30254196
Framework for Defining Evidentiary Criteria for Biomarker Qualification. Foundation for the National Institutes of Health (FNIH) Evidentiary Criteria Writing Group. October 2016. (announcement) (pdf)
Guidance for Industry and FDA Staff: Qualification Process for Drug Development Tools. (pdf)