Journal Club

Glioblastoma multiforme is the most common type of malignant brain tumor. These tumors actively divide and send invasive cells throughout the brain. This migration complicates patient treatment, because simply removing the tumor does not clear the brain of migrating cells, which can initiate new tumors elsewhere. This problem has nearly halted progress in prognosis or treatment of glioblastoma over the past 20 years, and has afflicted many of our most important political figures, including Ted Kennedy and John McCain. This astonishing lack of improvement in treatment has motivated Dr. Xandra Breakefield and her team to explore an alternative pathway for treatment. Dr. Breakefield discussed her work in a recent Wednesday afternoon lecture at NIH.

Saboteurs

To start their investigation, Dr. Breakefield gathered a “fresh” sample of glioblastoma multiforme that came straight out of the operating room. The idea of studying a fresh sample was novel; previously, samples were a few hours old or from mouse models. Examining fresh samples in a living condition revealed that glioblastomas are extremely physically active, constantly extending protrusions from the surface of the tumor cells. These protrusions turn into extracellular vesicles (EVs) of many sizes, released at the rate of about 10,000 vesicles per cell per day. When profiled, these EVs were found to contain RNAs, enzymes, transcription factors, other proteins, and many other cellular components. The team hypothesized that the EVs released by the tumor must somehow promote tumor progression.

Using a fluorescent tag that labels both cell and vesicle membranes, they tracked the tumor EVs in the living brain of mice and found that many of them are taken up by surrounding healthy myeloid cells — microglia and macrophages. Macrophages are the primary form of defense in the central nervous system. Normally functioning microglia are sentinels, warriors, and nurturers. As sentinels, they actively scan the brain for damage, then rush in to nurture injured areas to repair the damage. In their warrior role they kill and eat invading cells. Ironically, a higher density of microglia and macrophages in a tumor results in a worse prognosis. They are attracted to but then subjugated by the glioblastoma, and are coerced into an abnormal function of supporting tumor growth. Understanding how glioblastoma sabotages these immune cells and co-opts them to support tumor growth is key to ultimately finding a cure for the disease.

miR-21

Glioblastomas and their EVs typically have very high levels of miR-21, a microRNA affiliated with various cancer pathways. Dr. Breakefield and her team found that microglia grown near a glioblastoma and exposed to its EVs had higher levels of miR-21, higher proliferation rates, and lower expression of proteins from pathways involved in sensing and attacking invaders. They hypothesized that miR-21 transferred from the tumor to surrounding benign microglia by EVs plays a key role in recruiting and transforming them. Then they asked whether microglia that have taken up many tumor vesicles have a different phenotype than microglia that haven’t taken up as many. To test this question, the Breakefield lab implanted fluorescently (GFP) labelled gliomas into the brains of mice and developed a flow cytometry method to sort cells extracted from that environment based on their level of GFP. The more tumor vesicles the surrounding normal cells have taken up, the more GFP they should have. The researchers used single-cell RNA sequencing to compare the expression profile of mRNAs in microglia and macrophages with high vs. low levels of GFP, indicating uptake of tumor vesicles. They found significant differences in mRNA expression in brain microglia but not in macrophages.

The genetic pathways responsible for microglia’s sentinel role have been called the “microglial sensome” (Hickman et al., 2013). The mRNA for most sensome genes were down-regulated in microglia that had taken up more tumor EVs – thus, the tumor EVs “blinded” the microglia. Pathways for immune suppression were up-regulated – showing that uptake of the tumor EVs compromised the microglias’ warrior function. Finally, pathways for tissue repair were up-regulated – indicating that the tumor EVs co-opted the microglias’ nurturing function to support the tumor instead of normal brain cells.

Biomarkers

EVs are a promising biomarker for earlier diagnosis of disease. They are released by all cells and found in all biofluids. They can contain a wide variety of different information, including cell specific proteins and RNAs.

Dr. Breakefield was interested in finding biomarkers for glioblastoma that could be found in the bloodstream or other accessible biofluids. She targeted EGFRvIII, a deletion in the EGFR gene quite common in glioblastomas, and IDH1/2, a single point mutation that results in lower grade tumors with better prognosis. Her team is examining cerebrospinal fluid (CSF) and serum from patients to identify the presence or absence of EGFRvIII with promising success. Examining exRNA and free circulating DNA in plasma, her team was able to differentiate IDH1/2 mutant carriers from healthy volunteers. That work is being developed into a clinical tool for earlier diagnosis of low-grade gliomas which have a better prognosis and different treatment options than non-IDH1/2 gliomas.

Therapy EVs are proven, effective therapies for a variety of diseases. They can be obtained from many cell types and protect their fragile molecular cargo when administered into the body. They can also be efficiently taken up by specific target cells in vivo. EVs have been used therapeutically in immune modulation, tissue repair, and antigen presentation for vaccination.

After working on glioblastomas for an extended period, Dr. Breakefield decided to study schwannomas, a related but benign class of tumor. There are three types of hereditary diseases that stem from schwannomas: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. These diseases cause motor dysfunction, pain, and potential hearing loss.

In NF2, tumors form along nerve fibers, including those at the base of the spine. The current treatment is surgical removal of the tumor, but that can cause irreparable nerve damage. Because these tumors are not malignant, reducing their size could be an effective alternative treatment.

Dr. Breakefield and colleagues made a model system using human Schwann cells from an NF2 patient. They implanted those cells in the sciatic nerve of mice, forming tumors. They then created an adenovirus-associated virus (AAV) vector containing the pro-inflammatory enzyme caspase 1 attached to a promoter (P0) exclusively active in Schwann cells. They then injected this vector into the tumors to try to shrink them.

In this model system, the tumor grew when the AAV-GFP was injected. It regressed when AAV-P0-ICE was injected. P0-ICE creates a “bystander” effect, in which cells surrounding the injected cell are also killed. Surprisingly, Schwann cells appeared completely normal after treatment with P0-ICE, while the tumor (schwannoma) shrunk after injection of the tumor with AAV-P0-ICE. Although the EVs’ role in this finding is not known, Dr. Breakefield theorizes that caspase-1 is incorporated into the EVs and transferred to surrounding schwannoma cells.

There is still much to be discovered about EVs’ roles in tumors, both benign and malignant. We have not yet found glioblastoma’s Achilles’ heel, but further research may yet do so. Dr. Breakefield looks forward to continuing her work and potentially creating better treatment options for those afflicted by these devastating tumors. The previously unrecognized role of the tumor-derived EVs in transferring genetic information and co-opting other cells in their microenviroment may be an important key.

Early detection of viral infections is extremely important for control of disease transmission, prompt initiation of treatment, and prevention of infection-related complications. Because of our hypothesis that viral DNA, messenger RNA, and proteins cannot be detected in all infected individuals, we wanted to determine whether detection of exogenous miRNAs encoded by viruses represents a more sensitive assay of the true prevalence of infection by viruses including latent Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV). Therefore, we measured plasma miRNAs using RT-qPCR and compared it to the current standard method for detection of viral infection, an enzyme-linked immunosorbent assay (ELISA) of blood plasma, which detects antibodies generated by the host against the infecting virus. Our study population was 214 Caucasian patients from the United States and Romania, separated into four independent patient cohorts. We examined a total of 300 plasma samples from this population. This study enabled us to develop an approach to detect infection by KSHV using multiplexed RT-qPCR of multiple viral miRNAs.

We found that our method had clear advantages over the current ELISA-based approach. It detected a significantly higher prevalence of KSHV infection than that determined by seropositivity, with the difference most pronounced in immuno-depressed patients. When applied to EBV, our new method based on plasma viral miRNA quantification proved that EBV infection is ubiquitous. This strategy has the potential to become a gold standard method in clinical practice to detect latency of viruses and viremia — viral infection of the bloodstream — in both general and immune-compromised populations.

Detecting viral infection by amplifying viral miRNA has clear advantages over the current ELISA-based approach.

Fuentes-Mattei E, Giza DE, Shimizu M, Ivan C, Manning JT, Tudor S, Ciccone M, Kargin OA, Zhang X, Mur P, do Amaral NS, Chen M, Tarrand JJ, Lupu F, Ferrajoli A, Keating MJ, Vasilescu C, Yeung SJ, Calin GA. Plasma viral miRNAs indicate a high prevalence of occult viral infections. EBioMedicine. (2017) 20:182-192. doi: 10.1016/j.ebiom.2017.04.018. Pubmed: 28465156.

Extracellular vesicles, such as exosomes and microvesicles, are small vesicular particles that are constantly being produced and shed by cells. Due to their natural origin, and their ability to efficiently deliver their cargo to target cells and alter biological functions, exosomes attracted researchers to study their potential use as drug delivery systems. In the past few years, numerous studies have reported the effective use of exosomes to deliver therapeutic cargo ranging from miRNA, siRNA and even small molecule drugs in both in vitro cell models and in vivo animal models. However, since exosomes are cell-derived vesicles, it is unclear how these natural carriers of biomolecules may induce immune responses or induce toxicity either in animal models of disease or eventually in humans as we progress toward clinical evaluation of exosomes in healthy volunteers or in patients. Furthermore, what can we conclude about the presence or lack of immunogenicity or toxicity in our animal models as we work toward delivery of exosomes in humans?

Our lab has been studying the production of therapeutic exosomes using genetically engineered HEK293T cells for treatment of hepatocellular carcinoma (HCC). We developed engineered HEK293T cells that endogenously package miR-199a-3p, a miR commonly downregulated in HCC, into exosomes, and we are evaluating these and also exosomes exogenously loaded with therapeutic miRs in vitro and in vivo. Although demonstrating in vivo efficacy is a major milestone for all drug development efforts, understanding the potential toxicities and immunogenic responses associated with exosome therapy is equally important. The ability to identify and characterize adverse responses in preclinical models is critical to the drug development process and a necessary component of an Investigational New Drug (IND) application. Therefore, approaches to characterizing potential toxicities and immune responses induced by exosomes will be a necessary component of any effort to develop therapeutic exosomes.

In our article, “Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells,” that was just published online in the Journal of Extracellular Vesicles, we provide a general template process for comprehensively evaluating toxicity and immunogenicity of therapeutic exosomes in preclinical animal models. We started by dosing mice with wild type or engineered HEK293T-derived exosomes over a period of three weeks. Mice received 10 doses via intraperitoneal and intravenous routes of injection, and blood samples were collected at various times throughout the 3-week study. Animals were euthanized 24 hours after the last dose, and blood and all organs were collected from each animal for gross necropsy and evaluation of various markers of immune response and potential exosome-induced toxicity markers.

This study demonstrates one approach to immunogenicity and toxicity evaluations of human-derived exosomes in mice, and it highlights some of the variables that must be considered during these evaluations. For example, what are appropriate animal models in which to study immunogenicity and toxicity? What doses and dose regimens should be evaluated? How might the cell type from which the exosomes were harvested impact immunogenicity and toxicity? Just as with efficacy evaluations in animals, comprehensive study of these other factors will be necessary for safely moving therapeutic exosomes into human trials.

Acute liver failure is a potentially fatal consequence of severe liver injury. Liver transplantation may be necessary for survival if the liver injury exceeds the ability of the liver to regenerate (also called fulminant liver failure). New therapeutic interventions are needed to enhance tissue regeneration and improve the outcome of acute or fulminant liver failure. Previous studies have reported that mesenchymal stem cell (MSC) transfusions can improve function in liver facing acute failure. Stem cells can grow into multiple cell types and may support the replacement of functional liver tissue. In addition, paracrine effects — signalling between nearby cells — resulting from the release of soluble factors and extracellular vesicles (EV) may contribute to some of the beneficial effects observed with stem cell therapies.

A new study by Haga et al. reports on the beneficial effects of EV derived from stem cells. EV were isolated using classical ultracentrifugation methods. To mimic liver injury, D-galactosamine and recombinant tumor necrosis factor-α were injected into male mice. Subsequently, systemic administration of EV was shown to result in a dramatic improvement in survival. Whereas control animals receiving placebo showed complete lethality within 12 hours of D-galactosamine/TNF-α injection, the mice injected with mouse stem-cell-derived EV had a 57% survival at 24 hrs. When human MSC-EV were administered, a 37.5% survival was noted. Also noteworthy, survival was observed even with EV that had been cryopreserved. The EV reduced hepatic inflammation, likely by protecting the hepatocytes from apoptosis and recruiting Kupffer cells that protect from liver injury. The figure shows an overview of this process. Some of the beneficial effects were shown to be mediated by Y-RNA-1, a long non-coding RNA that is enriched within MSC-EV.

Stem-cell-derived extracellular vesicles repair tissue after acute liver failure

The beneficial effects of stem cells, mediated through EV and their RNA content, in severe injury models provides new avenues for investigation of the pathophysiology of liver injury and inflammation. These observations provide a very compelling justification for the future use of MSC-EV as therapeutics for severe liver injury.

Reference:
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Stem Cells Transl Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.

The role of extracellular vesicles (EVs) in cancer has recently become a promising area of research. A primary function of EVs is to deliver molecules from a donor cell to regulate cellular processes in a target cell. Little research has investigated what effect departing EVs may have on the donor cell.

In cancer, EVs from tumor cells can deviate from their original purpose. Altering vesicular content could benefit tumors, for example by increasing tumor proliferation or strengthening drug resistance.
 

Exosomal Packaging
Could donor tumor cells selectively shunt cancer-fighting molecules into secreted vesicles to escape their effects? Teng et al. thought this might be the case. They hypothesized that tumor cells specifically secrete tumor suppressor miRNA, namely miR-193a, into exosomes, a class of EV secreted via the endocytic membrane transport pathway, while oncogenic miRNAs are kept.

To test this theory, they validated miR-193a’s function in a mouse model. Specifically, they found that miR-193a targets Caprin1, a cell-cycle-associated protein, and arrests the cell cycle in phase G1. Thus, secreting miR-193a from the cell in exosomes would restart the cell cycle and enable a tumor cell to proliferate.

After studying miR-193a’s primary function, the authors explored what might facilitate its secretion. They showed that MVP (major vault protein) complexes with miR-193a and that knock-down of MVP leads to higher levels of miR-193a in the cell and less miR-193a in exosomes. They concluded that MVP mediates the sorting of miR-193a into exosomes. They also found that a higher level of MVP in the cell correlates with lower levels of miR-193a and higher levels of Caprin1, indicating that MVP aids in cell proliferation. Lastly, the researchers determined in a mouse model that export of miR-193a by MVP promotes metastasis of colon cancer to the liver.

Figure 1 shows a model of these interactions. In the pre-metastatic cell, miR-193a is freely expressed. In the tumor metastatic cell, MVP has complexed with miR-193a, driving it into exosomes to be secreted.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.


 

Cancer Biomarker
This finding implicates exosomal miR-193a as a potential biomarker for colon cancer. Could higher levels of exosomal miR-193a indicate a more aggressive disease? To answer these questions, the authors applied their findings in the clinical setting. Teng et al. examined the livers of mice with metastatic colon cancer, performing an extensive characterization of the levels of tumor suppressive and oncogenic miRNAs in normal and tumor cells and in the exosomes secreted by both. From that extensive analysis, they chose three miRNAs upregulated (miR-193a, miR-126 and miR-148a) and one miRNA downregulated (miR-196b) in exosomes from tumor cells and examined the levels of those miRNAs in exosomes purified from the blood (plasma) of 40 colon cancer patients, 15 with metastasis to the liver and 25 without. They found the same upregulation and downregulation of the 4 miRNAs in the patient population, with higher levels in the population with liver metastasis.

This exciting study identifies potential biomarkers for colon cancer. The two main findings highlight the importance of exosomes in cancer proliferation. First, the authors found that tumor suppressing miRNAs are packed into exosomes, while oncogenic miRNAs remain in the cell. Next, they found that there are higher amounts of tumor suppressing miRNAs in tumor-derived exosomes relative to exosomes from healthy cells. They also found that MVP acts as a mediator of these differences between metastatic and healthy cells. Teng et al. believe that once we develop a dependable method to purify exosomes, scientists may uncover further roles for exosomes in cancer progression.

Recent research from members of the ExRNA Communication Consortium (ERCC) suggests that extracellular RNAs (exRNAs) circulating in plasma play an active role in insulin resistance (IR). Insulin resistance is an incurable but manageable syndrome where the body stops reacting efficiently to the insulin hormone, which stimulates the uptake of glucose in the blood into cells and inhibits the body from using fat for energy, resulting in high blood sugar levels. The study, by Shav et al., points to certain exRNAs, particularly miR-122 and miR-192, as indicators and active players in IR regardless of the age, sex, or BMI of a person, which suggests that they may serve as more than metabolic markers and that they perhaps have functional, trans-organ roles in mediating IR.

Previous research has demonstrated that exRNAs have different functions in pathways relating to metabolic syndrome, which is a series of conditions that increase the risk of heart disease, stroke, and diabetes. For example, there is measurable miRNA dysregulation in obesity and in the progression of cardiometabolic disease. Other miRNAs are involved in brown/white fat specification, adipose tissue inflammation (Karbiener and Scheideler, 2014), and hepatic steatosis (fatty liver disease, Becker et al., 2015). Although these studies have identified specific exRNAs and miRNA networks that also have roles in IR, they have had either small sample sizes and lack validation in large populations or have been carried out in non-human models. In order to validate these data, the authors carried out a large-scale human translational study in which they analyzed detailed obesity-related phenotypic data from over 2,500 participants (most of whom were non-diabetic) from the Framingham Heart Study (FHS), an unrelated cardiovascular disease study (Feinleib et al., 1975).

To begin, the investigators analyzed blood samples from 2,317 non-diabetic study participants and quantified the plasma extracellular circulating exRNAs. They looked at RNAs [including piwi-interacting RNA (piRNA) and small nucleolar RNA (snoRNA)] expressed above a threshold level and excluded RNAs that were not found in at least 100 participants. From the resulting panel of 391 exRNAs, the investigators identified 16 microRNAs (miRNA), 1 piRNA, and 1 snoRNA that were associated with insulin after controlling for age, sex, and BMI. Of note, the abundance of miR-122 was shown to increase in a stepwise fashion as levels of insulin increased across the population. Higher levels of both miR-122 and miR-192 in the plasma were also consistently associated with a series of metabolic phenotypes, such as greater BMI and waist circumference, visceral fat quantity and quality, and liver attenuation. On the other hand, neither miRNA was associated with subcutaneous fat. These results were consistent whether the analysis included only the non-diabetic participants or the entire FHS population.

miRNAs function to regulate gene expression, so the authors conducted a pathway analysis to determine the targets of the 16 identified miRNAs. Almost unsurprisingly, all 16 miRNAs target insulin signaling pathways such that there is ample crosstalk and targeting of multiple IR-related genes by multiple miRNAs. This analysis validated findings from previous studies that implicated several miRNA target genes in the pathogenesis of IR, notably protein tyrosine phosphatase, nonreceptor type 1 (PTP1B) (Stull et al., 2012), mitogen-activated protein kinases (MAPKs) (Wang, Goalstone & Draznin, 2004), and 5′ adenosine monophosphate-activated protein kinase (AMPK) (Ruderman et al., 2013).

For the second part of the study, the investigators determined whether the miR-122 and miR-192 associations to age, sex, and BMI held true in a separate study population, a cohort of 90 overweight or obese young adults involved in the POOL study. Analyses of the youths’ plasma samples indicated that miR-122 (but not miR-192) was associated with greater IR after adjusting for age, sex, and BMI, and that this association remained even after the miRNA was analyzed independently of age, sex, BMI, or metabolite profile.

This study provides additional evidence and translational support for the role of exRNAs in IR. The findings indicate not only an association of exRNAs with insulin levels, but that the exRNAs may be playing an active role in the development or sustainment of IR. It is therefore critical to conduct further mechanistic investigations into the role of exRNAs in the metabolic architecture of IR.

The study of RNAs that do not produce proteins, so-called noncoding RNAs, has been an active area of research for many years. Recently, new kinds of non-coding RNAs have been described that have poorly defined activities. Circular RNA (circRNAs) are one of these more enigmatic biomolecules. They are formed when the 5′ head and 3′ tail of a messenger RNA precursor are spliced together. Next-generation sequencing studies have recently shown that circRNAs are abundant and widely expressed in mammals. While other non-coding RNAs have been shown to play critical roles in cancer, the association between circRNAs and cancer is largely unknown. In addition, the degree to which circRNAs are secreted outside the cell has not been well explored.

To study the presence and regulated release of circRNAs during colorectal cancer (CRC) progression, we used three related colon cancer cell lines that differ only in the mutation status of KRAS, an enzyme that acts at the beginning of a wide array of cellular signaling pathways. The parental cell line (DLD-1) contains both wild-type and G13D mutant KRAS alleles, whereas the derivative cell lines contain only a mutant KRAS (DKO-1) or wild-type KRAS (DKs-8) allele (Shirasawa et al. 1993). The G13D mutation locks KRAS into an active state. KRAS mutations occur in approximately 34–45% of CRCs and have been associated with a wide range of tumor-promoting effects (Vogelstein et al. 1988, Wong and Cunningham 2008). We performed deep RNA-Seq analysis of ribosomal RNA-depleted total RNA libraries to characterize circRNA expression in these cell lines and in the exosomes they release. The results from this study were recently published in the journal Scientific Reports (Dou et al. 2016).

Using a unique pipeline developed by our group, we identified hundreds of high-quality candidate circRNAs in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in the cell lines with mutant KRAS alleles (DLD-1 and DKO-1) compared to wild type (DKs-8), indicating a widespread effect of mutant KRAS on circRNA abundance (see Figure 1). This finding was confirmed in another pair of cell lines. In all of these cell lines, circRNAs were found associated with secreted exosomes, and circRNAs were more abundant there than in cells. Although circRNAs were down-regulated in cell lines with mutant KRAS alleles, it is difficult to conclude that KRAS directly regulates circRNAs. Nevertheless, our analysis did show that down-regulation of circRNAs in KRAS mutant cells was not caused by their increased export to exosomes.
 

Figure 1.

Figure 1. The blue highlight shows that expression of most circRNAs is lower in the KRAS-mutant cell lines than in the KRAS wild-type cell line.
FDR = False Discovery Rate; a higher number indicates a more confident prediction of a difference in expression.

There are complex regulatory mechanisms for expression of both circRNA and the host genes from which they derive. Figure 2 shows that lower expression of circRNA in the mutant KRAS vs. wild-type cell lines was not matched by a similar lower expression of host gene mRNA. We found a similar lack of correlation in circRNA and host gene mRNA expression level in all the exosome populations we studied. These results imply that regulation of circRNAs can occur independent of their host genes, and different regulatory processes might direct secretion of circRNA and host gene mRNA.
 

Figure 2.

Figure 2. The blue highlight shows that while expression of most circRNAs is lower in the KRAS-mutant than in the wild-type cell line, host gene mRNA expression shows no such pattern.

To further delineate how circRNA biogenesis could be affected by mutant KRAS, we also examined the expression levels of the RNA-editing enzyme ADAR and the RNA-binding protein QKI, which have been reported as circRNA regulators (Ivanov et al. 2015, Conn et al. 2015) (see Figure 3). Here we obtained contradictory results. The level of ADAR was decreased in the KRAS mutant cells; reduced ADAR activity could lead to an increase of circRNAs. QKI was also down-regulated in KRAS mutant cells, which could lead to a decrease of circRNAs.
 

Figure 3. Effect of ADAR and QKI on pre-mRNA circularization

Figure 3. Effect of ADAR and QKI on pre-mRNA circularization

More broadly, we studied the expression levels of all RNA-binding proteins within the RBPDB database (Cook et al. 2011). Six were found to be significantly differentially expressed in KRAS mutant cell lines compared with wild-type KRAS cell lines (ELAVL2, RBMS3, BICC1, MSI1, RBM44, and LARP6). These genes may serve as candidate circRNA regulators. However, our previous work shows that the correlation between mRNA and protein expression level is low for RNA-binding proteins (Zhang et al. 2014), and thus RNA levels for these RNA-binding proteins might not reflect their true protein levels. Further investigation will be needed to precisely define how circRNAs are regulated. Nevertheless, our results show that oncogenic mutations can change circRNA composition in cells and exosomes and suggest that circRNAs may serve as promising cancer biomarkers.

References

Conn, S.J., et al. The RNA binding protein Quaking regulates formation of circRNAs. Cell (2015) 160: 1125-1134. PMID 25768908.

Cook, K.B., et al. RBPDB: a database of RNA-binding specificities. Nucleic Acids Res (2011) 39: D301-D308. PMID 21036867.

Dou, Y., et al. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci Rep (2016) 6: 37982. PMID 27892494.

Ivanov, A., et al. Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. Cell Rep (2015) 10:170-177. PMID 25558066.

Shirasawa et al. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science (1993) 260:85-88. PMID 8465203.

Vogelstein, B., et al. Genetic alterations during colorectal-tumor development. N Engl J Med (1988) 319:525-532. PMID 2841597.

Wong, R. and Cunningham, D. Using predictive biomarkers to select patients with advanced colorectal cancer for treatment with Epidermal Growth Factor Receptor antibodies. J Clin Oncol (2008) 26:5668-5670. PMID 19001346.

Zhang, B., et al. Proteogenomic characterization of human colon and rectal cancer. Nature (2014) 513:382-387. PMID 25043054.

Alzheimer’s Disease (AD) accounts for a large number of dementia cases resulting in impaired memory, thinking, and behavior. Risk factors for AD include age and family history, but unfortunately there is not yet a definitive way to predict if an individual will develop the disease. There are reference biomarkers that can indicate a higher risk of developing AD, such as APOE genotype. Carriers of the APOE4 allele, present in ~20% of the population, are at increased risk for AD. Cerebrospinal fluid (CSF) is a body fluid found in the brain and spine that cushions and protects the brain from injury. CSF protein biomarkers, such as Aβ42, tau and phospho-tau, are important in screening for brain disease, but these reference markers often lack the sensitivity and specificity necessary for clinical utility.

Extracellular RNA, specifically microRNA (miRNA), has been found in CSF and may serve as a useful resource for improved AD biomarkers. In a recently published study, the Saugstad lab from Oregon Health and Science University examined CSF from a large group of living donors to identify unique miRNA biomarkers enriched in AD patients. In the study, miRNA expression levels from 50 AD and 49 control subjects were assessed using TaqMan Low Density Arrays containing probes for 754 validated miRNAs. Each miRNA was given a “Multitest Score” combining the results of four statistical tests, and miRNAs that passed two or more of the tests were considered for further analyses.

Two statistical tests, log-rank and logistic regression, were used to identify candidates that were twice as likely to be associated with AD status as not. The other tests were two variants of random forest classifier, CART and CHAID, designed to select biomarker candidates able to reliably distinguish AD from non-AD status when grouped with random subsets of other miRNAs. 36 miRNA biomarker candidates were identified by at least two of these analyses. The researchers found that linear combinations of subsets of miRNA, and the addition of ApoE genotyping status, further increased the sensitivity and specificity of AD detection (Figure 1).

 

Figure 1. CSF miRNA biomarkers and APOE genotype predict AD status better together. AUC - Area Under the Curve; higher AUC indicates higher predictive power.

Figure 1. CSF miRNA biomarkers and APOE genotype predict AD status better together. AUC – Area Under the Curve; higher AUC indicates higher predictive power.

Reprinted with permission from IOS Press.


 

This study shows the potential use of miRNAs isolated from CSF as AD biomarkers. The stringent statistical analyses and large sample size together provided strength to these initial studies. These 36 candidate biomarkers are currently being tested in further validation studies in CSF from a new group of 120 donors, which will also include APOE genotyping and Aβ42 and tau protein levels. Ultimately, a combination of miRNA CSF biomarkers with existing reference biomarkers (APOE, Aβ42, tau) may provide a specific and sensitive tool for the diagnosis of AD in the clinic.

Citation:
MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer’s Disease
Lusardi T, Phillips J, Wiedrick, J, Harrington C, Lind B, Lapidus J, Quinn J, Saugstad J. Journal of Alzheimer’s Disease (2017) 55: 1223-1233. doi: 10.3233/JAD-160835

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Currently, magnetic resonance imaging (MRI) is the most commonly used method to diagnose and monitor MS, but there is a poor correlation between MRI disease measures and clinical disability or disease progression in MS. MRI is also an expensive tool that might carry potential risks due to brain accumulation of contrast material (Kanda et al., 2015). In the last few years, a lot of effort has been invested in the identification of biomarkers for MS; however, to date, few of these findings have proven clinically useful. Thus, there is a strong unmet clinical need for objective body fluid biomarkers to assist in early diagnosis, predicting long-term prognosis, monitoring treatment response, and predicting potential adverse effects in MS.

Circulating miRNAs have been detected in several body fluids (Cortez et al., 2011) where they are highly stable as they are resistant to circulating ribonucleases (Mitchell et al., 2008). Their stability, along with the development of sensitive methods for their detection and quantification (Guerau-de-Arellano M. et al., 2012), makes them ideal candidates for biomarkers. We previously reported changes in circulating plasma miRNAs in MS patients (Gandhi R. et al., 2013). In a new study, our group investigated serum miRNAs as biomarkers in MS as part of an NCATS-funded UH2 initiative. We found that several serum miRNAs were differentially expressed in MS, were associated with disease stage, and correlated with disability.

Study Design (Figure 1): Serum from 296 participants including patients with MS, other neurologic diseases (Alzheimer’s disease and amyotrophic lateral sclerosis), inflammatory diseases (rheumatoid arthritis and asthma), and healthy controls (HC) were tested. miRNA profiles were determined using LNA (locked nucleic acid) based qPCR. MS patients were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured from the serum of 26 MS patients and 20 healthy controls. Those miRNAs from the discovery set that were significantly differentially expressed (p <0.05) in cases vs controls were validated using qPCR in 58 MS patients and 30 healthy controls.

 

Serum miRNA biomarkers in MS - Figure 1

 

Note: Results in the current study were normalized to the four most stably expressed miRNA across all the subjects. We agree with other blogs posted on exRNA.org suggesting that there is an immediate need to identify reference miRNA/exRNA that could be used for data normalization.

 

Figure 2: Differentially expressed circulating miRNAs as biomarkers in Multiple Sclerosis (MS). Up to top five miRNAs with p<0.05 are represented for each group comparison; a) MS, b) relapsing remitting MS (RRMS) and secondary progressive (SPMS) compared to the healthy control (HC), c) RRMS vs. SPMS, and d) the correlation of miRNA with the expanded disease severity scale (EDSS).

 

Results: We found 7 miRNAs (p<0.05 in both discovery phase and validation) that differentiate MS patients from healthy controls; miR-320a up-regulation was the most significantly changing serum miRNA in MS patients. We found 8 miRNAs that differentiated relapsing-remitting MS (RRMS) from HC. Among these, miR-484 up-regulation in RRMS patients showed the strongest association. When comparing secondary progressive MS (SPMS) patients to HC, 34 miRNAs significantly differentiated between the groups in both phases, with miR-320a up-regulation showing the strongest link. We also identified two miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with degree of disability according to the Kurtzke Expanded Disability Status Scale (EDSS), of which miR-199a-5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS, but not in serum. Kegg Pathway Analysis showed that significant and differentially expressed miRNAs target important immune functions and are related to the maintenance of neuronal homeostasis. For example, miR-27a-3p, the strongest miRNA to distinguish RRMS from SPMS and progressive MS (PMS) (up-regulated in the relapsing form as compared to the progressive forms) shows a strong link to both the neurotrophin signaling pathway and the T cell receptor signaling pathway. Other studies have shown that miR-27a-3p targets multiple proteins of intracellular signaling networks that regulate the activity of NF-κB and MAPKs 6. As a consequence, miR-27a inhibits differentiation of Th1 and Th17 cells and promotes the accumulation of Tr1 and Treg cells (Min S. et al., 2012). It has also been shown that miRa-27-3p is up-regulated in MS active brain lesions and that the level of miR-27a-3p in CSF is reduced in patients with dementia due to Alzheimer’s disease (AD) (Frigerio C.S. et al., 2013). Of all the miRNAs, miR-486-5p was identified in the largest number of comparisons. It correlates with EDSS and is up-regulated in MS compared to HC, to other neurological diseases, and to other inflammatory diseases. This particular miRNA was found to be associated with TGF-beta signaling pathways and is a known tumor suppressor (Oh H.K. et al., 2011). miR-320a has been previously described to be highly expressed in B cells of MS patients and was suggested to contribute to increased blood-brain barrier permeability due to regulation of MMP-9 (Aung L.L. et al., 2015). Pathway analysis links this miRNA to cell-to-cell adhesion pathways, another indication that it may be linked to blood-brain barrier permeability.

The current study is the most comprehensive evaluation to date of the role of serum miRNAs as biomarkers in MS, with the largest sample size and employing two independent cohort designs. One limitation of our study is that participant subject samples were collected from a single MS center. Further external validation of our results will require investigating samples from patients at other centers. We are currently performing such multicenter studies, which may also increase the power of our results. A second limitation of our study is the relatively small number of participants who contributed to each group comparison. Future work will require larger sample sizes to ensure that we have sufficient power to detect miRNAs with smaller effect sizes. Although miRNAs have been studied in cells and the CNS of MS patients, ours is the first comprehensive investigation of serum miRNAs.

Conclusions: Our findings identify circulating serum miRNAs (Figure 2) as potential biomarkers to diagnose and monitor disease status in MS. These findings are now being tested using patient samples obtained from other international MS centers. We are now investigating the role of miRNA as biomarkers for disease prognosis and treatment response in MS.

Acknowledgements: This study is a highly collaborative project, and I thank my whole team at the Ann Romney Center for Neurologic Diseases & MS Center for their contribution. The grant TR000890 is supported by the NIH Common Fund, through the Office of Strategic Coordination / Office of the NIH Director.

Cell culture is a staple of modern biology, and Fetal Bovine Serum (FBS) is an essential component of many cell culture protocols. A specific use for FBS is to supply nutrients to cells and to stimulate their growth. Another role of FBS in cell culture research is to represent the complexities and functionality of endogenous biological environments; however, precisely this complexity has long been a potential confounding factor for researchers. For example, cytokines in FBS can lead to the stimulation of cells, thus producing unintended experimental environments. Despite these disadvantages, FBS retains a prominent role in modern cell culture, with estimated sales as high as 700,000 liters per year. Because of its ubiquity in cell culture research, it is critical to investigate how the components of FBS may be influencing experiments and downstream analysis.

Variability and uncertainty in the composition of FBS is especially problematic for studies that evaluate cellular secretions. For example, to successfully determine the array of RNA secreted by cultured cells, we need to know the extent to which the medium is contaminated by exogenous RNA. Additionally, extracellular RNA (exRNA) is not only found distributed freely throughout the liquid medium, but it is also often found packaged inside of extracellular vesicles (EVs) or lipoprotein complexes. Therefore, in a paper released online yesterday, Wei et al. evaluated how the RNA composition of FBS might be confounding research.

The authors first evaluated exogenous RNA contamination. They grew cultures of a cell type known not to express a particular RNA, then evaluated the presence of that RNA in the culture media. If that RNA was found, its origin was probably the media itself. For example, the authors demonstrated that miR-122, a liver-specific miRNA, is present in media from cultured glioma cells, suggesting that its source is likely FBS itself. They then attempted to deplete RNA from FBS via ultracentrifugation, but despite a 24 hour spin at 100,000g, about 75% of total RNA remained in the supernatant. This result has also been found by researchers attempting to deplete FBS of RNA-containing EVs and emphasizes the difficulty of producing media truly free from contaminating RNA.

These results led the authors to ask whether existing studies have wrongly attributed the presence of exRNA to a particular experimental procedure or cell type, when it should be recognized as a component of the FBS in the cell culture media. To answer this question, the authors first broadly profiled the RNA composition of FBS using RNA sequencing. They determined that between 9% and 22% of FBS RNA mapped to the human genome, depending on the stringency of the mapping algorithm and FBS preparation. They also checked for the presence of bovine-specific RNA in existing human cell culture exRNA datasets, finding levels as high as 17%, with samples from exosomes (a type of EV) containing particularly high levels. Finally, they demonstrated experimentally that bovine-specific transcripts are taken up into cells, interfering not only with exRNA analysis but also with intracellular RNA studies.

Moving forward, a significant remaining issue is deciding how to treat conserved RNA known to be present in both FBS and the cell line under study. Switching from FBS to purely chemically defined media can help with this problem, but it is not possible for all cell types and experimental conditions. Alternatively, a quantitative analysis of the chemical composition of the media might make it possible to estimate which RNAs are secreted by the cells of interest by filtering out known FBS RNAs from the total RNA pool.

This research cautions us to be careful in the design and interpretation of experiments to identify extracellular RNAs that use FBS in culture media. The paper, Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA, released in Scientific Reports yesterday, is authored by Zhiyun Wei, Arsen O. Batagov, David R. F. Carter, and Anna M. Krichevsky.