Multimode chromatography-based techniques for high purity isolation of extracellular vesicles from human blood plasma.

Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation-based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography-based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography- and ultracentrifugation-based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.

Correction: Zhitkevich et al. HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype. Viruses 2024, 16, 193.

Author Juris Jansons requested his exclusion from the authors of the original publication [...].

Epidemiology of Orbital Inflammatory Disease: An AAO IRIS Registry Study.

PURPOSE: The current study queries the American Academy of Ophthalmology (AAO) Intelligent Research in Sight (IRIS) registry for data on the epidemiology, work-up, and management patterns of autoimmune orbital inflammation. METHODS: Analysis and description of patient data from the IRIS registry between 2013 and 2019 reviewing patients with autoimmune or idiopathic orbital inflammation with filters based on International Classification of Disease (ICD) and Current Procedural Terminology (CPT) codes. Patients with thyroid eye disease, orbital cellulitis, and orbital abscess were excluded. MAIN OUTCOME MEASURES: Demographic descriptions included gender, age, geographic region, and treatment. Sub-analysis was performed by assessing rates of imaging, biopsy, lab work-up, and diagnostic categories. RESULTS: In a final cohort of 20,584 patients, the mean age of onset of orbital inflammation was 51.7 years; 67% female; and 63% Caucasian, 21% unknown, 12% Black, 2.6% Asian, and 1.5% other. Only 49 had imaging, 78 had laboratory work-up, and 1,411 had biopsy codes. Treatment results showed 166 patients receiving antibiotics, 224 patients receiving steroids, and 35 patients receiving both. CONCLUSIONS: This study assessed the epidemiology, diagnostic patterns, and treatment patterns for orbital inflammation through the AAO IRIS registry. Practise patterns suggest a relatively low overall rate of imaging and laboratory studies compared to biopsies, although this certainly under-represents the actual number of imaging and laboratory studies and exemplifies the inherent imprecision of using a large database. However, the methodology of this study provides a framework of approaching the IRIS registry for oculoplastic research.

Fellow Eyes Conversion Rates in Patients With Unilateral Exudative Age-Related Macular Degeneration: An Academy IRIS® Registry Analysis.

BACKGROUND AND OBJECTIVE: This study aimed to examine conversion rates from non-exudative to exudative age-related macular degeneration (AMD) in the fellow eye of patients with unilateral exudative AMD using the Academy IRIS® Registry. PATIENTS AND METHODS: This study was a retrospective, cohort analysis from 2016 to 2019. Patient and disease characteristics including initial AMD stage were collected. Cox proportional-hazard (PH) and logistic regression modeling were performed. RESULTS: The risk of conversion was lower for men relative to women and for Asians and Blacks relative to Whites. Compared to never-smokers, active smokers were at increased risk of conversion, and compared to initially early non-exudative AMD eyes, intermediate and advanced non-exudative AMD eyes had higher rates of conversion. Compared to active choroidal neovascularization eyes, eyes with inactive choroidal neovascularization and inactive scars had lower rates of fellow eye conversion. CONCLUSIONS: In this cohort analysis of unilateral exudative AMD patients, women, Whites, and active smokers had higher rates of non-exudative to exudative AMD conversion in the fellow eye. [Ophthalmic Surg Lasers Imaging Retina 2024;55:220-226.].

HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

Chronic Viral Infections and Cancer, Openings for Therapies and Vaccines.

Infections are responsible for approximately one out of six cases of cancer worldwide [...].

Simultaneous determination of cystine and other free aminothiols in blood plasma using capillary electrophoresis with pH-mediated stacking.

We developed a method of sensitive capillary electrophoresis using UV detection for the determination of certain free aminothiols (reduced cysteinylglycine (rCysGly), cysteine (rCys), glutathione (rGln), and cystine (CysS) in human blood plasma. The reduced thiols were derivatized with N-ethylmaleimide. The plasma was purified from proteins via ultrafiltration. Electrophoretic separation was performed using 115 mM Na phosphate with 7.5% (v/v) polyethylene glycol 600, pH 2.3. The in-capillary concentration of the analytes was achieved with a pH gradient created via the preinjection of triethanolamine and postinjection of phosphoric acid. The separation was carried out using a silica capillary (50 µm i.d.; total/effective separation length 42/35 cm) at a 25 kV voltage. The total analysis/regeneration time was 18 min. The quantification limits varied from 1.3 µM (rCysGly) to 5.4 µM (CysS). The accuracy was 95%-99%, and the repeatability and reproducibility were approximately 1.8%-3.8% and 1.9%-5.0%, respectively. An analysis of plasma samples from healthy volunteers (N = 41) showed that the mean levels of rCysGly, rCys, rGln, and CysS were 1.64, 10.6, 2.58, and 46.2 µM, respectively.

Reining in Radium for Nuclear Medicine: Extra-Large Chelator Development for an Extra-Large Ion.

Targeted α therapy (TAT) of soft-tissue cancers using the α particle-emitting radionuclide 223Ra holds great potential because of its favorable nuclear properties, adequate availability, and established clinical use for treating metastatic prostate cancer of the bone. Despite these advantages, the use of 223Ra has been largely overshadowed by other α emitters due to its challenging chelation chemistry. A key criterion that needs to be met for a radionuclide to be used in TAT is its stable attachment to a targeting vector via a bifunctional chelator. The low charge density of Ra2+ arising from its large ionic radius weakens its electrostatic binding interactions with chelators, leading to insufficient complex stability in vivo. In this study, we synthesized and evaluated macropa-XL as a novel chelator for 223Ra. It bears a large 21-crown-7 macrocyclic core and two picolinate pendent groups, which we hypothesized would effectively saturate the large coordination sphere of the Ra2+ ion. The structural chemistry of macropa-XL was first established with the nonradioactive Ba2+ ion using X-ray diffraction and X-ray absorption spectroscopy, which revealed the formation of an 11-coordinate complex in a rare anti pendent-arm configuration. Subsequently, the stability constant of the [Ra(macropa-XL)] complex was determined via competitive cation exchange with 223Ra and 224Ra radiotracers and compared with that of macropa, the current state-of-the-art chelator for Ra2+. A moderate log KML value of 8.12 was measured for [Ra(macropa-XL)], which is approximately 1.5 log K units lower than the stability constant of [Ra(macropa)]. This relative decrease in Ra2+ complex stability for macropa-XL versus macropa was further probed using density functional theory calculations. Additionally, macropa-XL was radiolabeled with 223Ra, and the kinetic stability of the resulting complex was evaluated in human serum. Although macropa-XL could effectively bind 223Ra under mild conditions, the complex appeared to be unstable to transchelation. Collectively, this study sheds additional light on the chelation chemistry of the exotic Ra2+ ion and contributes to the small, but growing, number of chelator development efforts for 223Ra-based TAT.

The Diagnostic and Prognostic Roles Played by Homocysteine and Other Aminothiols in Patients with Chronic Kidney Disease.

We examined standard clinical and laboratory biochemical parameters, as well as the levels of aminothiols in the blood and urine (homocysteine (Hcy), cysteine (Cys), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)) via capillary electrophoresis in patients with CKD at stages II-V. Patient outcomes were assessed after five years. To complete forecasting, correlation and ROC analysis were performed. It was found that the levels of Cys and Hcy in blood plasma were earlier markers of CKD starting from stage II, while the levels of SAM and SAM/SAH in urine made it possible to differentiate between CKD at stages II and III. Blood plasma Hcy and urinary SAM and SAM/SAH correlated with mortality, but plasma Hcy concentrations were more significant. Thus, plasma Hcy, urine SAM, and SAM/SAH can be considered to be potential diagnostic and prognostic markers in patients with CKD.

Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications.

Ultralow flow LC employs ultra-narrow bore columns and mid-range pL/min to low nL/min flow rates (i.e., ≤20 nL/min). The separation columns that are used under these conditions are typically 2-30 µm in inner diameter. Ultralow flow LC systems allow for exceptionally high sensitivity and frequently high resolution. There has been an increasing interest in the analysis of scarce biological samples, for example, circulating tumor cells, extracellular vesicles, organelles, and single cells, and ultralow flow LC was efficiently applied to such samples. Hence, advances towards dedicated ultralow flow LC instrumentation, technical approaches, and higher throughput (e.g., tens-to-hundreds of single cells analyzed per day) were recently made. Here, we review the types of ultralow flow LC technology, followed by a discussion of selected representative ultralow flow LC applications, focusing on the progress made in bioanalysis of amount-limited samples during the last 10 years. We also discuss several recently reported high-sensitivity applications utilizing flow rates up to 100 nL/min, which are below commonly used nanoLC flow rates. Finally, we discuss the path forward for future developments of ultralow flow LC.

Neuroprotective Effect of Platinum Nanoparticles Is Not Associated with Their Accumulation in the Brain of Rats.

Platinum nanoparticles (nPts) have neuroprotective/antioxidant properties, but the mechanisms of their action in cerebrovascular disease remain unclear. We investigated the brain bioavailability of nPts and their effects on brain damage, cerebral blood flow (CBF), and development of brain and systemic oxidative stress (OS) in a model of cerebral ischemia (hemorrhage + temporary bilateral common carotid artery occlusion, tBCAO) in rats. The nPts (0.04 g/L, 3 ± 1 nm diameter) were administered to rats (N = 19) intraperitoneally at the start of blood reperfusion. Measurement of CBF via laser Doppler flowmetry revealed that the nPts caused a rapid attenuation of postischemic hypoperfusion. The nPts attenuated the apoptosis of hippocampal neurons, the decrease in reduced aminothiols level in plasma, and the glutathione redox status in the brain, which were induced by tBCAO. The content of Pt in the brain was extremely low (≤1 ng/g). Thus, nPts, despite the extremely low brain bioavailability, can attenuate the development of brain OS, CBF dysregulation, and neuronal apoptosis. This may indicate that the neuroprotective effects of nPts are due to indirect mechanisms rather than direct activity in the brain tissue. Research on such mechanisms may offer a promising trend in the treatment of acute disorders of CBF.

Influence of Coronary Artery Bypass Grafts on Blood Aminothiols in Patients with Coronary Artery Disease.

Coronary artery disease (CAD) and the coronary artery bypass graft (CABG) are associated with a decreased blood glutathione (bGSH) level. Since GSH metabolism is closely related to other aminothiols (homocysteine and cysteine) and glucose, the aim of this study was to reveal the associations of bGSH with glucose and plasma aminothiols in CAD patients (N = 35) before CABG and in the early postoperative period. Forty-three volunteers with no history of cardiovascular disease formed the control group. bGSH and its redox status were significantly lower in CAD patients at admission. CABG had no significant effect on these parameters, with the exception of an increase in the bGSH/hemoglobin ratio. At admission, CAD patients were characterized by negative associations of homocysteine and cysteine with bGSH. All these associations disappeared after CABG. An association was found between an increase in oxidized GSH in the blood in the postoperative period and fasting glucose levels. Thus, CAD is associated with the depletion of the intracellular pool and the redox status of bGSH, in which hyperhomocysteinemia and a decrease in the bioavailability of the extracellular pool of cysteine play a role. The present study indicates that CABG causes disruptions in aminothiol metabolism and induces the synthesis of bGSH. Moreover, glucose becomes an important factor in the dysregulation of GSH metabolism in CABG.

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells.

Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.

Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus.

APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4-800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%-99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.

Hepatitis Delta Virus Antigens Trigger Oxidative Stress, Activate Antioxidant Nrf2/ARE Pathway, and Induce Unfolded Protein Response.

Hepatitis delta virus (HDV) is a viroid-like satellite that may co-infect individuals together with hepatitis B virus (HBV), as well as cause superinfection by infecting patients with chronic hepatitis B (CHB). Being a defective virus, HDV requires HBV structural proteins for virion production. Although the virus encodes just two forms of its single antigen, it enhances the progression of liver disease to cirrhosis in CHB patients and increases the incidence of hepatocellular carcinoma. HDV pathogenesis so far has been attributed to virus-induced humoral and cellular immune responses, while other factors have been neglected. Here, we evaluated the impact of the virus on the redox status of hepatocytes, as oxidative stress is believed to contribute to the pathogenesis of various viruses, including HBV and hepatitis C virus (HCV). We show that the overexpression of large HDV antigen (L-HDAg) or autonomous replication of the viral genome in cells leads to increased production of reactive oxygen species (ROS). It also leads to the upregulated expression of NADPH oxidases 1 and 4, cytochrome P450 2E1, and ER oxidoreductin 1α, which have previously been shown to mediate oxidative stress induced by HCV. Both HDV antigens also activated the Nrf2/ARE pathway, which controls the expression of a spectrum of antioxidant enzymes. Finally, HDV and its large antigen also induced endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR). In conclusion, HDV may enhance oxidative and ER stress induced by HBV, thus aggravating HBV-associated pathologies, including inflammation, liver fibrosis, and the development of cirrhosis and hepatocellular carcinoma.

SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines.

Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.

Anticancer Drugs Paclitaxel, Carboplatin, Doxorubicin, and Cyclophosphamide Alter the Biophysical Characteristics of Red Blood Cells, In Vitro.

Red blood cells (RBCs) are the most numerous cells in the body and perform gas exchange between all tissues. During the infusion of cancer chemotherapeutic (CT) agents, blood cells are the first ones to encounter aggressive cytostatics. Erythrocyte dysfunction caused by direct cytotoxic damage might be a part of the problem of chemotherapy-induced anemia-one of the most frequent side effects. The aim of the current study is to evaluate the functional status of RBCs exposed to mono and combinations of widely used commercial pharmaceutical CT drugs with different action mechanisms: paclitaxel, carboplatin, cyclophosphamide, and doxorubicin, in vitro. Using laser diffraction, flow cytometry, and confocal microscopy, we show that paclitaxel, having a directed effect on cytoskeleton proteins, by itself and in combination with carboplatin, caused the most marked abnormalities-loss of control of volume regulation, resistance to osmotic load, and stomatocytosis. Direct simulations of RBCs' microcirculation in microfluidic channels showed both the appearance of a subpopulation of cells with impaired velocity (slow damaged cells) and an increased number of cases of occlusions. In contrast to paclitaxel, such drugs as carboplatin, cyclophosphamide, and doxorubicin, whose main target in cancer cells is DNA, showed significantly less cytotoxicity to erythrocytes in short-term exposure. However, the combination of drugs had an additive effect. While the obtained results should be confirmed in in vivo models, one can envisioned that such data could be used for minimizing anemia side effects during cancer chemotherapy.

Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS).

Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.

Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus.

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.