Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

BACKGROUND: ErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity. METHODS: We isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients' response to trastuzumab-based treatments. RESULTS: We found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments. CONCLUSIONS: Upregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.

Correction to: Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity.

An amendment to this paper has been published and can be accessed via the original article.

The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity.

Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano-flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as "oncosomes". Indeed, oncogenic transformation alters the repertoire of EV-associated proteins, increases the presence of pro-invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.

Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content.

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here, we report that the oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell-derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases can be detected in EVs purified from plasma of tumor-bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804 but not with anti-EGFR antibody (Cetuximab) or etoposide, triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR, and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Tyr-845, Tyr-1068, and Tyr-1173), ERK, and AKT varies between cells and their corresponding EVs and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.

PML-RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells.

Oncogenic transformation is believed to impact the vascular phenotype and microenvironment in cancer, at least in part, through mechanisms involving extracellular vesicles (EVs). We explored these questions in the context of acute promyelocytic leukemia cells (NB4) expressing oncogenic fusion protein, PML-RARa and exquisitely sensitive to its clinically used antagonist, the all-trans retinoic acid (ATRA). We report that NB4 cells produce considerable numbers of EVs, which are readily taken up by cultured endothelial cells triggering their increased survival. NB4 EVs contain PML-RARa transcript, but no detectable protein, which is also absent in endothelial cells upon the vesicle uptake, thereby precluding an active intercellular trafficking of this oncogene in this setting. ATRA treatment changes the emission profile of NB4-related EVs resulting in preponderance of smaller vesicles, an effect that occurs in parallel with the onset of cellular differentiation. ATRA also increases IL-8 mRNA and protein content in NB4 cells and their EVs, while decreasing the levels of VEGF and tissue factor (TF). Endothelial cell uptake of NB4-derived EVs renders these cells more TF-positive and procoagulant, and this effect is diminished by pre-treatment of EV donor cells with ATRA. Profiling angiogenesis-related transcripts in intact and ATRA-treated APL cells and their EVs reveals multiple differences attributable to cellular responses and EV molecular packaging. These observations point to the potential significance of changes in the angiogenic signature and activity associated with EVs released from tumor cells subjected to targeted therapy.

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells.

Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

The contribution of tumor and host tissue factor expression to oncogene-driven gliomagenesis.

Glioblastoma multiforme (GBM) is an aggressive form of glial brain tumors, associated with angiogenesis, thrombosis, and upregulation of tissue factor (TF), the key cellular trigger of coagulation and signaling. Since TF is upregulated by oncogenic mutations occurring in different subsets of human brain tumors we investigated whether TF contributes to tumourigenesis driven by oncogenic activation of EGFR (EGFRvIII) and RAS pathways in the brain. Here we show that TF expression correlates with poor prognosis in glioma, but not in GBM. In situ, the TF protein expression is heterogeneously expressed in adult and pediatric gliomas. GBM cells harboring EGFRvIII (U373vIII) grow aggressively as xenografts in SCID mice and their progression is delayed by administration of monoclonal antibodies blocking coagulant (CNTO 859) and signaling (10H10) effects of TF in vivo. Mice in which TF gene is disrupted in the neuroectodermal lineage exhibit delayed progression of spontaneous brain tumors driven by oncogenic N-ras and SV40 large T antigen (SV40LT) expressed under the control of sleeping beauty transposase. Reduced host TF levels in low-TF/SCID hypomorphic mice mitigated growth of glioma subcutaneously but not in the brain. Thus, we suggest that tumor-associated TF may serve as therapeutic target in the context of oncogene-driven disease progression in a subset of glioma.

Mesenchymal glioma stem cells trigger vasectasia-distinct neovascularization process stimulated by extracellular vesicles carrying EGFR.

Targeting neovascularization in glioblastoma (GBM) is hampered by poor understanding of the underlying mechanisms and unclear linkages to tumour molecular landscapes. Here we report that different molecular subtypes of human glioma stem cells (GSC) trigger distinct endothelial responses involving either angiogenic or circumferential vascular growth (vasectasia). The latter process is selectively triggered by mesenchymal (but not proneural) GSCs and is mediated by a subset of extracellular vesicles (EVs) able to transfer EGFR/EGFRvIII transcript to endothelial cells. Inhibition of the expression and phosphorylation of EGFR in endothelial cells, either pharmacologically (Dacomitinib) or genetically (gene editing), abolishes their EV responses in vitro and disrupts vasectasia in vivo. Therapeutic inhibition of EGFR markedly extends anticancer effects of VEGF blockade in mice, coupled with abrogation of vasectasia and prolonged survival. Thus, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new therapeutic target in a subset of GBMs.

Cancer cells induced to express mesenchymal phenotype release exosome-like extracellular vesicles carrying tissue factor.

Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding.

MoS2-Plasmonic Nanocavities for Raman Spectra of Single Extracellular Vesicles Reveal Molecular Progression in Glioblastoma.

Extracellular vesicles (EVs) are continually released from cancer cells into biofluids, carrying actionable molecular fingerprints of the underlying disease with considerable diagnostic and therapeutic potential. The scarcity, heterogeneity and intrinsic complexity of tumor EVs present a major technological challenge in real-time monitoring of complex cancers such as glioblastoma (GBM). Surface-enhanced Raman spectroscopy (SERS) outputs a label-free spectroscopic fingerprint for EV molecular profiling. However, it has not been exploited to detect known biomarkers at the single EV level. We developed a multiplex fluidic device with embedded arrayed nanocavity microchips (MoSERS microchip) that achieves 97% confinement of single EVs in a minute amount of fluid (<10 µL) and enables molecular profiling of single EVs with SERS. The nanocavity arrays combine two featuring characteristics: (1) An embedded MoS2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS2 edge sites and the lipid bilayer; and (2) A layered plasmonic cavity that enables sufficient electromagnetic field enhancement inside the cavities to obtain a single EV level signal resolution for stratifying the molecular alterations. We used the GBM paradigm to demonstrate the diagnostic potential of the SERS single EV molecular profiling approach. The MoSERS multiplexing fluidic achieves parallel signal acquisition of glioma molecular variants (EGFRvIII oncogenic mutation and MGMT expression) in GBM cells. The detection limit of 1.23% was found for stratifying these key molecular variants in the wild-type population. When interfaced with a convolutional neural network (CNN), MoSERS improved diagnostic accuracy (87%) with which GBM mutations were detected in 12 patient blood samples, on par with clinical pathology tests. Thus, MoSERS demonstrates the potential for molecular stratification of cancer patients using circulating EVs.

Quantitative proteomics and biological activity of extracellular vesicles engineered to express SARS-CoV-2 spike protein.

SARS-CoV-2 viral infection led to the devastating COVID-19 pandemic, where illness stemmed from interactions between virions and recipient host cells resulting in multi-layered pathological consequences. The role of the infection portal is now understood to be the cellular angiotensin converting enzyme-2 (ACE2) receptor, which binds to viral spike (S) protein initiating virion internalisation process. Since SARS-CoV-2 virions bear some resemblance to endogenously produced small extracellular vesicles (sEVs) we reasoned that EVs engineered to express S protein (viral mimics) may interfere with viral infection. Here, we report generation of HEK293T cells producing sEVs enriched for transmembrane S-protein tagged with green fluorescent protein (S/GFP). Strikingly, S protein drove the GFP tag to the membrane of sEVs, while GFP alone was not efficiently included in the sEV cargo. High-throughput quantitative proteomics revealed that S/GFP sEVs contained over 1000 proteins including canonical components of the exosomal pathway such as ALIX, syntenin-1, and tetraspanins (CD81, CD9), but depleted for calnexin and cytochrome c. We found that 84 sEV proteins were significantly altered by the presence of S/GFP. S protein expressing EVs efficiently adhered to target cells in an ACE2-dependent manner, but they were poorly internalised. Importantly, prolonged administration of S/GFP EV to K18-hACE2 mice provided a significant protection against SARS-CoV-2 infection. Thus, the generation of sEV containing S protein can be considered as a novel therapeutic approach in reducing the transmission of SARS-CoV-2.

Oncogenic RAS drives the CRAF-dependent extracellular vesicle uptake mechanism coupled with metastasis.

Oncogenic RAS impacts communication between cancer cells and their microenvironment, but it is unclear how this process influences cellular interactions with extracellular vesicles (EVs). This is important as intercellular EV trafficking plays a key role in cancer invasion and metastasis. Here we report that overexpression of mutant RAS drives the EV internalization switch from endocytosis (in non-transformed cells) to macropinocytosis (in cancer cells) resulting in enhanced EV uptake. This process depends on the surface proteoglycan, fibronectin and EV engulfment mechanism regulated by CRAF. Both mutant RAS and activated CRAF expression is associated with formation of membrane ruffles to which they colocalize along with actin, sodium-hydrogen exchangers (NHEs) and phosphorylated myosin phosphatase (pMYPT). RAS-transformed cells internalize EVs in the vicinity of ruffled structures followed by apparent trafficking to lysosome and degradation. NHE inhibitor (EIPA) suppresses RAS-driven EV uptake, along with adhesion-independent clonal growth and experimental metastasis in mice. Thus, EV uptake may represent a targetable step in progression of RAS-driven cancers.

Plasmonic nanobowtiefluidic device for sensitive detection of glioma extracellular vesicles by Raman spectrometry.

Cancer cells shed into biofluids extracellular vesicles (EVs) - nanoscale membrane particles carrying diagnostic information. EVs shed by heterogeneous populations of tumor cells offer a unique opportunity to access biologically important aspects of disease complexity. Glioblastoma (GBM) exemplifies cancers that are incurable, because their temporal dynamics and molecular complexity evade standard diagnostic methods and confound therapeutic efforts. Liquid biopsy based on EVs offers unprecedented real-time access to complex tumour signatures, but it is not used clinically due to inefficient testing methods. We report on a nanostructured microfluidic-device that employs SERS for unambiguous identification of EVs from different GBM cell populations. The device features fabless plasmonic nanobowties for label-free and non-immunological SERS detection of EVs. This nanobowtiefluidic device combines the advanced characteristics of plasmonic nanobowties with a high throughput sample-delivery system for concentration of the analytes in the vicinity of the detection site. We showed theoretically and experimentally that the fluidic device assists the monolayer distribution of the EVs, which dramatically increase the probability of EV's existence in the laser illumination area. In addition, the optimized fabless nanobowtie structures with an average electric field enhancement factor of 9 × 105 achieve distinguishable and high intensity SERS signals. Using the nanobowtiefluidic and micro-Raman equipment, we were able to distinguish a library of peaks expressed in GBM EV subpopulations from two distinct glioblastoma cell lines (U373, U87) and compare them to those of non-cancerous glial EVs (NHA) and artificial homogenous vesicles (e.g. DOPC/Chol). This cost-effective and easy-to-fabricate SERS platform and a portable sample-delivery system for discerning the sub-population of GBM EVs and non-cancerous glial EVs may have broader applications to different types of cancer cells and their molecular/oncogenic signature.

Rational Development of Liquid Biopsy Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is known for its variable clinical behavior and outcome, including heterogeneity in developing relapse or metastasis. Recent data highlighted the potential of somatic mutations as promising biomarkers for risk stratification in RCC. Likewise, the analysis of circulating tumor DNA (ctDNA) for such informative somatic mutations (liquid biopsy) is considered an important advance for precision oncology in RCC, allowing to monitor molecular disease evolution in real time. However, our knowledge about the utility of ctDNA analysis in RCC is limited, in part due to the lack of RCC-appropriate assays for ctDNA analysis. Here, by interrogating different blood compartments in xenograft models, we identified plasma cell-free (cf) DNA and extracellular vesicles (ev) DNA enriched for RCC-associated ctDNA. Additionally, we developed sensitive targeted sequencing and bioinformatics workflows capable of detecting somatic mutations in RCC-relevant genes with allele frequencies ≥ 0.5%. Applying this assay to patient-matched tumor and liquid biopsies, we captured tumor mutations in cf- and ev-DNA fractions isolated from the blood, highlighting the potentials of both fractions for ctDNA analysis. Overall, our study presents an RCC-appropriate sequencing assay and workflow for ctDNA analysis and provides a proof of principle as to the feasibility of detecting tumor-specific mutations in liquid biopsy in RCC patients.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.

Modulation of Lgl1 by steroid, retinoic acid, and vitamin D models complex transcriptional regulation during alveolarization.

Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA), and vitamin D (VitD). Bronchopulmonary dysplasia, a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of bronchopulmonary dysplasia, reduced levels of late gestation lung 1 (Lgl1) normalize during recovery. We show that GC (100 nM) stimulates (7- to 115-fold) and VitD (100 microM) suppresses (twofold) Lgl1 expression. RA (all-trans/9-cis, 10 microM) effects are biphasic. From postnatal days 7-10, RA was stimulatory (twofold) at 24 h, after which effects were inhibitory (3- to 15-fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared with 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.

Extracellular vesicles from genetically unstable, oncogene-driven cancer cells trigger micronuclei formation in endothelial cells.

Oncogenic transformation impacts cancer cell interactions with their stroma, including through formation of abnormal blood vessels. This influence is often attributed to angiogenic growth factors, either soluble, or associated with tumor cell-derived extracellular vesicles (EVs). Here we examine some of the cancer-specific components of EV-mediated tumor-vascular interactions, including the impact of genetic driver mutations and genetic instability. Cancer cells expressing mutant HRAS oncogene exhibit aberrations of chromatin architecture, aneuploidy, cytoplasmic chromatin deposition and formation of micronuclei with a non-random chromosome content. EVs released from such HRAS-driven cells carry genomic DNA, including oncogenic sequences, and transfer this material to endothelial cells while inducing abnormal formation of micronuclei, along with cell migration and proliferation. Micronuclei were also triggered following treatment with EVs derived from glioma cells (and stem cells) expressing EGFRvIII oncogene, and in both endothelial cells and astrocytes. EVs from HRAS and EGFRvIII-driven cancer cells carry 19 common proteins while EVs from indolent control cells exhibit more divergent proteomes. Immortalized endothelial cell lines with disrupted TP53 pathway were refractory to EV-mediated micronuclei induction. We suggest that oncogenic transformation and intercellular trafficking of cancer-derived EVs may contribute to pathological vascular responses in cancer due to intercellular transmission of genomic instability.

Mapping Subpopulations of Cancer Cell-Derived Extracellular Vesicles and Particles by Nano-Flow Cytometry.

The elusive complexity of membranous extracellular vesicle (EV) and membrane-less extracellular particle (EP) populations released from various cellular sources contains clues as to their biological functions and diagnostic utility. In this study, we employed optimized multicolor nano-flow cytometry, structured illumination (SIM), and atomic force microscopy (AFM) to bridge sensitive detection at the single EV/EP level and high-throughput analysis of cancer cell secretomes. We applied these approaches to particles released from intact cells driven by several different transforming mechanisms or to cells under therapeutic stress imposed by pharmacological inhibition of their oncogenic drivers, such as epidermal growth factor receptor (EGFR). We demonstrate a highly heterogeneous distribution of biologically relevant elements of the EV/EP cargo, including oncoproteins (EGFR), clotting factors (tissue factor), pro-metastatic integrins (ITGA6, ITGA4), tetraspanins (CD63), and genomic DNA across the entire particulate secretome of cancer cells. We observed that targeting EGFR activity with irreversible kinase inhibitors (dacomitinib) triggers emission of DNA containing EP/EV subpopulations, including particles (chromatimeres) harboring both EGFR and DNase-resistant chromatin. While nano-flow cytometry enables quantification of these changes across the entire particular secretome, SIM reveals individual molecular topography of EV/EP subsets and AFM exposes some of their physical properties, including the presence of nanofilaments and other substructures. We describe differential uptake rates of distinct EV subsets, resulting in preferential internalization of exosome-like small EVs by cancer cells to the exclusion of larger EVs. Thus, our study illustrates the potential of nano-flow cytometry coupled with high-resolution microscopy to explore the cancer-related EV/EP landscape.