Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and miR-132-inducing bufalin synergistically arrests proliferation, induces differentiation, and prolongs the survival of differentiated MES SK-N-AS cells for 8 weeks. RNA- seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained other MES neuroblastoma, as well as glioblastoma cells, over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

HOXDeRNA activates a cancerous transcription program and super-enhancers genome-wide.

Background: The origin and genesis of highly malignant and heterogenous glioblastoma brain tumors remain unknown. We previously identified an enhancer-associated long non-coding RNA, LINC01116 (named HOXDeRNA here), that is absent in the normal brain but is commonly expressed in malignant glioma. HOXDeRNA has a unique capacity to transform human astrocytes into glioma-like cells. This work aimed to investigate molecular events underlying the genome-wide function of this lncRNA in glial cell fate and transformation. Results: Using a combination of RNA-Seq, ChIRP-Seq, and ChIP-Seq, we now demonstrate that HOXDeRNA binds in trans to the promoters of genes encoding 44 glioma-specific transcription factors distributed throughout the genome and derepresses them by removing the Polycomb repressive complex 2 (PRC2). Among the activated transcription factors are the core neurodevelopmental regulators SOX2, OLIG2, POU3F2, and SALL2. This process requires an RNA quadruplex structure of HOXDeRNA that interacts with EZH2. Moreover, HOXDeRNA-induced astrocyte transformation is accompanied by the activation of multiple oncogenes such as EGFR, PDGFR, BRAF, and miR-21, and glioma-specific super-enhancers enriched for binding sites of glioma master transcription factors SOX2 and OLIG2. Conclusions: Our results demonstrate that HOXDeRNA overrides PRC2 repression of glioma core regulatory circuitry with RNA quadruplex structure. These findings help reconstruct the sequence of events underlying the process of astrocyte transformation and suggest a driving role for HOXDeRNA and a unifying RNA-dependent mechanism of gliomagenesis.

Inhibition of the epigenetically activated miR-483-5p/IGF-2 pathway results in rapid loss of meningioma tumor cell viability.

PURPOSE: Meningioma is the most common primary central nervous system tumor often causing serious complications, and presently no medical treatment is available. The goal of this study was to discover miRNAs dysregulated in meningioma, and explore miRNA-associated pathways amenable for therapeutic interventions. METHODS: Small RNA sequencing was performed on meningioma tumor samples to study grade-dependent changes in microRNA expression. Gene expression was analyzed by chromatin marks, qRT-PCR and western blot. miRNA modulation, anti-IGF-2 neutralizing antibodies, and inhibitors against IGF1R were evaluated in a tumor-derived primary cultures of meningioma cells. RESULTS: Meningioma tumor samples showed high, grade-dependent expression of miR-483-5p, associated with high mRNA and protein expression of its host gene IGF-2. Inhibition of miR-483-5p reduced the growth of cultured meningioma cells, whereas a miR-483 mimic increased cell proliferation. Similarly, inhibition of this pathway with anti-IGF-2 neutralizing antibodies reduced meningioma cell proliferation. Small molecule tyrosine kinase inhibitor blockade of the IGF-2 receptor (IGF1R) resulted in rapid loss of viability of cultured meningioma tumor-derived cells, suggesting that autocrine IGF-2 feedback is obligatory for meningioma tumor cell survival and growth. The observed IGF1R-inhibitory IC50 for GSK1838705A and ceritinib in cell-based assays along with the available pharmacokinetics data predicted that effective drug concentration could be achieved in vivo as a new medical treatment of meningioma. CONCLUSION: Meningioma cell growth is critically dependent on autocrine miR-483/IGF-2 stimulation and the IGF-2 pathway provides a feasible meningioma treatment target.

Secreted PGK1 and IGFBP2 contribute to the bystander effect of miR-10b gene editing in glioma.

MicroRNA-10b (miR-10b) is an essential glioma driver and one of the top candidates for targeted therapies for glioblastoma and other cancers. This unique miRNA controls glioma cell cycle and viability via an array of established conventional and unconventional mechanisms. Previously reported CRISPR-Cas9-mediated miR-10b gene editing of glioma cells in vitro and established orthotopic glioblastoma in mouse models demonstrated the efficacy of this approach and its promise for therapy development. However, therapeutic gene editing in patients' brain tumors may be hampered, among other factors, by the imperfect delivery and distribution of targeting vectors. Here, we demonstrate that miR-10b gene editing in glioma cells triggers a potent bystander effect that leads to the selective cell death of the unedited glioma cells without affecting the normal neuroglial cells. The effect is mediated by the secreted miR-10b targets phosphoglycerate kinase 1 (PGK1) and insulin-like growth factor binding protein 2 (IGFBP2) that block cell-cycle progression and induce glioma cell death. These findings further support the feasibility of therapeutic miR-10b editing without the need to target every cell of the tumor.

Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma.

miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. We demonstrate that the entire miR-10b-hosting HOXD locus is activated in glioma via the cis-acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer. Knockdown of either lncRNA in glioma cells alters CTCF and cohesin binding, abolishes chromatin looping, inhibits the expression of all genes within HOXD locus, and leads to glioma cell death. Conversely, in cortical astrocytes, enhancer activation is sufficient for HOXD/miR-10b locus reorganization, gene derepression, and neoplastic cell transformation. LINC01116 RNA is essential for this process. Our results demonstrate the interplay of two lncRNAs in the chromatin folding and concordant regulation of miR-10b and multiple HOXD genes normally silenced in astrocytes and triggering the neoplastic glial transformation.

A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing.

BACKGROUND: miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA's poorly investigated and largely unconventional properties hamper the clinical translation. METHODS: We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing. RESULTS: We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. CONCLUSIONS: We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

Tumor-Derived Cell Culture Model for the Investigation of Meningioma Biology.

Meningioma is the most common primary central nervous system tumor. Although mostly nonmalignant, meningioma can cause serious complications by mass effect and vasogenic edema. While surgery and radiation improve outcomes, not all cases can be treated due to eloquent location. Presently no medical treatment is available to slow meningioma growth owing to incomplete understanding of the underlying pathology, which in turn is due to the lack of high-fidelity tissue culture and animal models. We propose a simple and rapid method for the establishment of meningioma tumor-derived primary cultures. These cells can be maintained in culture for a limited time in serum-free media as spheres and form adherent cultures in the presence of 4% fetal calf serum. Many of the tissue samples show expression of the lineage marker PDG2S, which is typically retained in matched cultured cells, suggesting the presence of cells of arachnoid origin. Furthermore, nonarachnoid cells including vascular endothelial cells are also present in the cultures in addition to arachnoid cells, potentially providing a more accurate tumor cell microenvironment, and thus making the model more relevant for meningioma research and high-throughput drug screening.

Repression of the stress granule protein G3BP2 inhibits immune checkpoint molecule PD-L1.

Mounting evidence suggests that cancer stemness and immunosuppression are related, but the underlying mechanisms behind these are not clear. We previously reported that the stress granule-associated protein G3BP2 is involved in the regulation of tumor-initiating (stem) cells. In this study, we show that this protein also upregulates the immune checkpoint molecule PD-L1 under conditions of stress in breast and glioblastoma cancer cells, revealing a previously unknown connection between stemness programs, stress responses, and immune checkpoint control. We also identified a significant correlation between G3BP2 and PD-L1 co-expression in tumor tissues from cancer patients. To assess the targetability of G3BP2, we employed a small molecule (C108) that binds G3BP2 and interferes with the stress response. Tumors treated with C108 had increased CD8 T-cell proliferation and infiltration. Moreover, treatment of breast tumor-bearing mice with C108 resulted in a significant survival benefit and long-term cures. Cancer cells treated with C108 or cancer cells with genetically repressed G3BP2 had decreased PD-L1 expression due to enhanced mRNA degradation. Our study provides a compelling mechanism linking stress granule formation and immune checkpoint program of cancer, suggesting this link may provide new opportunities for improving anticancer immunotherapy.

Glioblastoma-Derived Extracellular Vesicles Facilitate Transformation of Astrocytes via Reprogramming Oncogenic Metabolism.

Glioblastoma (GBM) may arise from astrocytes through a multistep process involving a progressive accumulation of mutations. We explored whether GBM-derived extracellular vesicles (EVs) may facilitate neoplastic transformation and malignant growth of astrocytes. We utilized conditioned media (CM) of cultured glioma cells, its sequential filtration, diverse cell-based assays, RNA sequencing, and metabolic assays to compare the effects of EV-containing and EV-depleted CM. GBM EVs facilitated the neoplastic growth of pre-transformed astrocytes but not normal human or mouse astrocytes. They induced proliferation, self-renewal, and colony formation of pre-transformed astrocytes and enhanced astrocytoma growth in a mouse allograft model. GBM EVs appear to reprogram astrocyte metabolism by inducing a shift in gene expression that may be partly associated with EV-mediated transfer of full-length mRNAs encoding ribosomal proteins, oxidative phosphorylation, and glycolytic factors. Our study suggests an EV/extracellular RNA (exRNA)-mediated mechanism that contributes to astrocyte transformation via metabolic reprograming and implicates horizontal mRNA transfer.

Imaging-AMARETTO: An Imaging Genomics Software Tool to Interrogate Multiomics Networks for Relevance to Radiography and Histopathology Imaging Biomarkers of Clinical Outcomes.

PURPOSE: The availability of increasing volumes of multiomics, imaging, and clinical data in complex diseases such as cancer opens opportunities for the formulation and development of computational imaging genomics methods that can link multiomics, imaging, and clinical data. METHODS: Here, we present the Imaging-AMARETTO algorithms and software tools to systematically interrogate regulatory networks derived from multiomics data within and across related patient studies for their relevance to radiography and histopathology imaging features predicting clinical outcomes. RESULTS: To demonstrate its utility, we applied Imaging-AMARETTO to integrate three patient studies of brain tumors, specifically, multiomics with radiography imaging data from The Cancer Genome Atlas (TCGA) glioblastoma multiforme (GBM) and low-grade glioma (LGG) cohorts and transcriptomics with histopathology imaging data from the Ivy Glioblastoma Atlas Project (IvyGAP) GBM cohort. Our results show that Imaging-AMARETTO recapitulates known key drivers of tumor-associated microglia and macrophage mechanisms, mediated by STAT3, AHR, and CCR2, and neurodevelopmental and stemness mechanisms, mediated by OLIG2. Imaging-AMARETTO provides interpretation of their underlying molecular mechanisms in light of imaging biomarkers of clinical outcomes and uncovers novel master drivers, THBS1 and MAP2, that establish relationships across these distinct mechanisms. CONCLUSION: Our network-based imaging genomics tools serve as hypothesis generators that facilitate the interrogation of known and uncovering of novel hypotheses for follow-up with experimental validation studies. We anticipate that our Imaging-AMARETTO imaging genomics tools will be useful to the community of biomedical researchers for applications to similar studies of cancer and other complex diseases with available multiomics, imaging, and clinical data.

Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells.

Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers.

Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21.

Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.

Physical and Molecular Landscapes of Mouse Glioma Extracellular Vesicles Define Heterogeneity.

Cancer extracellular vesicles (EVs) are highly heterogeneous, which impedes our understanding of their function as intercellular communication agents and biomarkers. To deconstruct this heterogeneity, we analyzed extracellular RNAs (exRNAs) and extracellular proteins (exPTNs) from size fractionation of large, medium, and small EVs and ribonucleoprotein complexes (RNPs) from mouse glioblastoma cells by RNA sequencing and quantitative proteomics. mRNA from medium-sized EVs most closely reflects the cellular transcriptome, whereas small EV exRNA is enriched in small non-coding RNAs and RNPs contain precisely processed tRNA fragments. The exPTN composition of EVs and RNPs reveals that they are closely related by vesicle type, independent of their cellular origin, and single EV analysis reveals that small EVs are less heterogeneous in their protein content than larger ones. We provide a foundation for better understanding of segregation of macromolecules in glioma EVs through a catalog of diverse exRNAs and exPTNs.

Co-cultures of Glioma Stem Cells and Primary Neurons, Astrocytes, Microglia, and Endothelial Cells for Investigation of Intercellular Communication in the Brain.

Intercellular communication within complex biological and pathological systems via extracellular vesicles (EVs) and secreted factors is a highly attractive area of research. However, cell models enabling investigation of such communication in vitro are limited. Commonly utilized is the supplementation of hyper-concentrated EVs or other extracellular factors to the recipient cell cultures. This approach requires purification of the secreted complexes and is confounded by the contamination of media components. Two-chamber co-cultures of donor and recipient cells separated by a pore membrane may represent a more physiological and better-controlled system for the investigation of intercellular communication. Yet, distinct culture conditions for different neural cell types often make them incompatible for co-culturing. Here we optimized short-term co-cultures of patient-derived low-passage glioma-initiating stem cells with normal cells of the brain microenvironment, such as primary neurons, astrocytes, microglia, and brain endothelial cells. We demonstrate the culture compatibility of these cell types and internalization of glioma-derived extracellular RNA by the normal recipient cells. The presented protocols are valuable for the investigation of intercellular communication between glioma brain tumor and cells of its microenvironment, including but not limited to the EVs-mediated communication. RESEARCH IN CONTEXT: Cell-to-cell communication is essential in normal physiology and implicated in disease; however, experimental systems for its modeling in vitro are limited. Particularly, the investigation of communication between brain tumors and normal cells of the brain microenvironment has been challenged by the lack of adequate culture models. Here we developed co-cultures of glioma stem cells with various types of normal brain cells, including primary neurons, astrocytes, microglia, and brain endothelial cells, and demonstrated their utility for the study of intercellular communication. Detection of proposed markers in the recipient cells confirmed RNA transfer in these co-cultures.

Oligonucleotide Therapeutics as a New Class of Drugs for Malignant Brain Tumors: Targeting mRNAs, Regulatory RNAs, Mutations, Combinations, and Beyond.

Malignant brain tumors are rapidly progressive and often fatal owing to resistance to therapies and based on their complex biology, heterogeneity, and isolation from systemic circulation. Glioblastoma is the most common and most aggressive primary brain tumor, has high mortality, and affects both children and adults. Despite significant advances in understanding the pathology, multiple clinical trials employing various treatment strategies have failed. With much expanded knowledge of the GBM genome, epigenome, and transcriptome, the field of neuro-oncology is getting closer to achieve breakthrough-targeted molecular therapies. Current developments of oligonucleotide chemistries for CNS applications make this new class of drugs very attractive for targeting molecular pathways dysregulated in brain tumors and are anticipated to vastly expand the spectrum of currently targetable molecules. In this chapter, we will overview the molecular landscape of malignant gliomas and explore the most prominent molecular targets (mRNAs, miRNAs, lncRNAs, and genomic mutations) that provide opportunities for the development of oligonucleotide therapeutics for this class of neurologic diseases. Because malignant brain tumors focally disrupt the blood-brain barrier, this class of diseases might be also more susceptible to systemic treatments with oligonucleotides than other neurologic disorders and, thus, present an entry point for the oligonucleotide therapeutics to the CNS. Nevertheless, delivery of oligonucleotides remains a crucial part of the treatment strategy. Finally, synthetic gRNAs guiding CRISPR-Cas9 editing technologies have a tremendous potential to further expand the applications of oligonucleotide therapeutics and take them beyond RNA targeting.

Multidimensional communication in the microenvirons of glioblastoma.

Glioblastomas are heterogeneous and invariably lethal tumours. They are characterized by genetic and epigenetic variations among tumour cells, which makes the development of therapies that eradicate all tumour cells challenging and currently impossible. An important component of glioblastoma growth is communication with and manipulation of other cells in the brain environs, which supports tumour progression and resistance to therapy. Glioblastoma cells recruit innate immune cells and change their phenotype to support tumour growth. Tumour cells also suppress adaptive immune responses, and our increasing understanding of how T cells access the brain and how the tumour thwarts the immune response offers new strategies for mobilizing an antitumour response. Tumours also subvert normal brain cells - including endothelial cells, neurons and astrocytes - to create a microenviron that favours tumour success. Overall, after glioblastoma-induced phenotypic modifications, normal cells cooperate with tumour cells to promote tumour proliferation, invasion of the brain, immune suppression and angiogenesis. This glioblastoma takeover of the brain involves multiple modes of communication, including soluble factors such as chemokines and cytokines, direct cell-cell contact, extracellular vesicles (including exosomes and microvesicles) and connecting nanotubes and microtubes. Understanding these multidimensional communications between the tumour and the cells in its environs could open new avenues for therapy.

Coding and noncoding landscape of extracellular RNA released by human glioma stem cells.

Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.

Genome Editing Reveals Glioblastoma Addiction to MicroRNA-10b.

Glioblastoma (GBM) brain tumor remains among the most lethal and incurable human diseases. Oncogenic microRNA-10b (miR-10b) is strongly and universally upregulated in GBM, and its inhibition by antisense oligonucleotides (ASOs) reduces the growth of heterogeneous glioma cells; therefore, miR-10b represents a unique therapeutic target for GBM. Here we explored the effects of miR-10b gene editing on GBM. Using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, we investigated effects of miR-10b gene editing on the growth of cultured human glioma cells, tumor-initiating stem-like cells, and mouse GBM xenografts, as well as the oncogene-induced transformation of normal astrocytes. We show that GBM is strictly "addicted" to miR-10b and that miR-10b gene ablation is lethal for glioma cell cultures and established intracranial tumors. miR-10b loss-of-function mutations lead to the death of glioma, but not other cancer cell lines. We have not detected escaped proliferative clones of GBM cells edited in the miR-10b locus. Finally, neoplastic transformation of normal astrocytes was abolished by the miR-10b-editing vectors. This study demonstrates the feasibility of gene editing for brain tumors in vivo and suggests virus-mediated miR-10b gene ablation as a promising therapeutic approach that permanently eliminates the key regulator essential for tumor growth and survival.

Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA.