Y-box binding protein 1 in small extracellular vesicles reduces mesenchymal stem cell differentiation to osteoblasts-implications for acute myeloid leukaemia.

Small extracellular vesicles (sEVs) released by acute myeloid leukaemia (AML) cells have been reported to influence the trilineage differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it remains elusive which biological cargo from AML-sEVs is responsible for this effect. In this study, sEVs were isolated from cell-conditioned media and blood plasma using size-exclusion chromatography and ultrafiltration and characterized according to MISEV2018 guidelines. Our results demonstrated that AML-sEVs increased the proliferation of BM-MSCs. Conversely, key proteins that are important for normal haematopoiesis were downregulated in BM-MSCs. Additionally, we revealed that AML-sEVs significantly reduced the differentiation of BM-MSCs to osteoblasts without affecting adipogenic or chondrogenic differentiation. Next, LC-MS/MS proteomics elucidated that various proteins, including Y-box-binding protein 1 (YBX1), were upregulated in both AML-sEVs and BM-MSCs treated with AML-sEVs. Clinically relevant, we found that YBX1 is considerably upregulated in most paediatric AML patient-derived sEVs compared to healthy controls. Interestingly, sEVs isolated after the downregulation of YBX1 in AML cells remarkably rescued the osteoblastic differentiation of BM-MSCs. Altogether, our data demonstrate for the first time that YBX1 containing AML-sEVs is one of the key players that disrupt the normal function of bone marrow microenvironment by reducing the osteogenic differentiation of BM-MSCs.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Single Molecule Localization Microscopy for Studying Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are 30-200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.

Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell-Cell Communication in Cancer.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell-cell communication in cancer.

Evaluation of Immunoregulatory Biomarkers on Plasma Small Extracellular Vesicles for Disease Progression and Early Therapeutic Response in Head and Neck Cancer.

Head and Neck Cancers (HNCs) have highly immunosuppressive properties. Small extracellular vesicles (sEVs), including exosomes, nanosized mediators of intercellular communication in the blood, carry immunosuppressive proteins and effectively inhibit anti-tumor immune responses in HNCs. This study evaluates immunosuppressive markers on sEVs from 40 HNC patients at different disease stages and 3- and 6-month follow-up after surgery and/or chemoradiotherapy. As controls, sEVs from normal donors (NDs) are examined. Immunoregulatory surface markers on sEVs were detected as relative fluorescence intensity (RFI) using on-bead flow cytometry, and their expression levels were monitored in the early and late stages of HNC and during follow-up. In parallel, the sEV-mediated apoptosis of CD8+ Jurkat cells was assessed. Together with TGF-ß1 and PD-L1 abundance, total sEV proteins are elevated with disease progression. In contrast, total sEV protein, including TGF-ß1, PD-1 and PD-L1, decrease upon therapy response during follow-up. Overall survival analysis implies that high sEV PD-1/PD-L1 content is an unfavorable prognostic marker in HNC. Consistently, the sEV-mediated induction of apoptosis in CD8+ T cells correlates with the disease activity and therapy response. These findings indicate that a combination of immunoregulatory marker profiles should be preferred over a single marker to monitor disease progression and therapy response in HNC.

DNA in extracellular vesicles: from evolution to its current application in health and disease.

Extracellular vesicle (EV) secretion is a highly conserved evolutionary trait in all organisms in the three domains of life. The packaging and release of EVs appears to be a bulk-flow process which takes place mainly under extreme conditions. EVs participate in horizontal gene transfer, which supports the survival of prokaryotic and eukaryotic microbes. In higher eukaryotes, almost all cells secrete a heterogeneous population of EVs loaded with various biomolecules. EV secretion is typically higher in cancer microenvironments, promoting tumor progression and metastasis. EVs are now recognized as additional mediators of autocrine and paracrine communication in health and disease. In this context, proteins and RNAs have been studied the most, but extracellular vesicle DNA (EV-DNA) has started to gain in importance in the last few years. In this review, we summarize new findings related to the loading mechanism(s), localization, and post-shedding function of EV-DNA. We also discuss the feasibility of using EV-DNA as a biomarker when performing a liquid biopsy, at the same time emphasizing the lack of data from clinical trials in this regard. Finally, we outline the potential of EV-DNA uptake and its interaction with the host genome as a promising tool for understanding the mechanisms of cancer evolution.

Glioma: molecular signature and crossroads with tumor microenvironment.

In patients with glioblastoma, the average survival time with current treatments is short, mainly due to recurrences and resistance to therapy. This insufficient treatment success is, in large parts, due to the tremendous molecular heterogeneity of gliomas, which affects the overall prognosis and response to therapies and plays a vital role in gliomas' grading. In addition, the tumor microenvironment is a major player for glioma development and resistance to therapy. Active communication between glioma cells and local or neighboring healthy cells and the immune environment promotes the cancerogenic processes and contributes to establishing glioma stem cells, which drives therapy resistance. Besides genetic alterations in the primary tumor, tumor-released factors, cytokines, proteins, extracellular vesicles, and environmental influences like hypoxia provide tumor cells the ability to evade host tumor surveillance machinery and promote disease progression. Moreover, there is increasing evidence that these players affect the molecular biological properties of gliomas and enable inter-cell communication that supports pro-cancerogenic cell properties. Identifying and characterizing these complex mechanisms are inevitably necessary to adapt therapeutic strategies and to develop novel measures. Here we provide an update about these junctions where constant traffic of biomolecules adds complexity in the management of glioblastoma.

Tumour Progression Stage-Dependent Secretion of YB-1 Stimulates Melanoma Cell Migration and Invasion.

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

Insights into the limitations of transient expression systems for the functional study of p53 acetylation site and oncogenic mutants.

Tumor suppressor protein p53 protects cells against malignant transformation mostly through transcriptional activation. Lysine acetylation is required to mediate activation of p53. The protein displays eight lysine residues and their evolutionary conservation argues for an essential role. The aim of this study was to investigate the significance of individual acetylation sites in mediating p53 functions. Differences in intracellular localization, protein expression levels, and transcriptional activity were investigated by overexpressing acetylation-deficient p53 variants in the colon carcinoma-derived p53 knock-out cell line HCT 116 p53(-/-). We found that not all lysine residues are equally capable of promoting p53's functions. Individual amino acid mutations or combinations thereof led to altered p53 expression levels, intracellular distribution, or transcriptional transactivation capacity, as compared to the wild-type protein. However, we observed that the choice of protein tag and expression vector could significantly alter obtained results on certain aspects of p53 function.

Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.

Challenges in the Isolation and Proteomic Analysis of Cancer Exosomes-Implications for Translational Research.

Exosomes belong to the group of extracellular vesicles (EVs) that derive from various cell populations and mediate intercellular communication in health and disease. Like hormones or cytokines, exosomes released by cells can play a potent role in the communication between the cell of origin and distant cells in the body to maintain homeostatic or pathological processes, including tumorigenesis. The nucleic acids, and lipid and protein cargo present in the exosomes are involved in a myriad of carcinogenic processes, including cell proliferation, tumor angiogenesis, immunomodulation, and metastasis formation. The ability of exosomal proteins to mediate direct functions by interaction with other cells qualifies them as tumor-specific biomarkers and targeted therapeutic approaches. However, the heterogeneity of plasma-derived exosomes consistent of (a) exosomes derived from all kinds of body cells, including cancer cells and (b) contamination of exosome preparation with other extracellular vesicles, such as apoptotic bodies, makes it challenging to obtain solid proteomics data for downstream clinical application. In this manuscript, we review these challenges beginning with the choice of different isolation methods, through the evaluation of obtained exosomes and limitations in the process of proteome analysis of cancer-derived exosomes to identify novel protein targets with functional impact in the context of translational oncology.

Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle-derived RNA.

Despite high remission rates, almost 25% of patients with AML will suffer relapse 3-5 years after diagnosis. Therefore, in addition to existing diagnostic and MRD detection tools, there is still a need for the development of novel approaches that can provide information on the state of the disease. Extracellular vesicles (EVs), containing genetic material reflecting the status of the parental cell, have gained interest in recent years as potential diagnostic biomarkers in cancer. Therefore, isolation and characterization of blood and bone marrow plasma-derived EVs from pediatric AML patients could be an additional approach in AML diagnostics and disease monitoring. In this study, we attempt to establish a plasma EV-RNA-based method to detect leukemia-specific FLT3-ITD and NPM1 mutations using established leukemia cell lines and primary pediatric AML plasma samples. We were successfully able to detect FLT3-ITD and NPM1 mutations in the EV-RNA using GeneScan-based fragment-length analysis and real-time PCR assays, respectively, in samples before therapy. This was corresponding to the gDNA mutational analysis from leukemic blasts, and supports the potential of using EV-RNA as a diagnostic biomarker in pediatric AML.

NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils.

Tumor-associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro- or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro-angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10-tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up-regulated in TANs from melanoma and head-and-neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro-angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy.

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis.

Tumor-secreted extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments. Accordingly, EVs play an essential role in both primary tumor growth and metastatic evolution. EVs orchestrate multiple systemic pathophysiological processes, such as coagulation, vascular leakiness, and reprogramming of stromal recipient cells to support pre-metastatic niche formation and subsequent metastasis. Clinically, EVs may be biomarkers and novel therapeutic targets for cancer progression, particularly for predicting and preventing future metastatic development.

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor ß secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Molecular transport machinery involved in orchestrating luminal acid-induced duodenal bicarbonate secretion in vivo.

The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1), the Na(+)/H(+) exchanger isoform 3 (NHE3), as well as the anion channels cystic fibrosis transmembrane conductance regulator (CFTR) and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9 or Slc9a3 (NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal bicarbonate secretory response (DBS-R) to luminal acid; and to compare it to their role in DBS-R elicited by the adenylyl cyclase agonist forskolin. While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS-R to forskolin was not altered. In contrast, the DBS-R to a 5 min exposure to luminal acid (pH 2.5) was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)-172] reduced the first phase of the acid-induced DBS-R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS-R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis in the later stage. We conclude that the long-lasting DBS-R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3.