The increasing prevalence of cancer coupled with approvals of new drugs and technologies used in therapy have brought increased scrutiny to the cost and value of treatments in oncology. To address the rising concern about oncology drug costs, several organizations have developed value frameworks to help assess the value of oncology regimens. The objective of this study was to assess oncologists’ perceptions, awareness, and knowledge of all oncology value frameworks in the United States and to understand oncologists’ perceptions of affordability in the context of National Comprehensive Cancer Network (NCCN) Evidence Blocks.To (a) assess oncologists’ awareness, knowledge, perceptions, and ratings of the American Society of Clinical Oncology Value Framework (AVF), the Institute for Clinical and Economic Review (ICER) value framework, NCCN Evidence Blocks, and Memorial Sloan Kettering Cancer Center’s DrugAbacus; (b) assess oncologists’ knowledge and perceptions of drug affordability as defined by the NCCN Evidence Blocks methodology; and (c) determine the factors that influence drug affordability ratings.Data were collected from an electronic cross-sectional survey of 200 U.S.-based oncologists from a variety of practice settings. Oncologists were asked about their knowledge and perceptions of 4 value frameworks—NCCN Evidence Blocks, AVF, the ICER value framework, and DrugAbacus. Using NCCN Evidence Blocks, oncologists were asked to rate a variety of hypothetical cancer therapies and assign costs (in U.S. dollars) to the 5 levels of affordability. Additional questions that assessed perceived patient out-of-pocket (OOP) costs and comfort level in assessing affordability were also included in the survey.Oncologists were most familiar with NCCN Evidence Blocks (90%), followed by the AVF (84%), ICER value framework (57%), and DrugAbacus (56%). Oncologists rated affordability higher (mean rating 3: moderately expensive) versus the actual NCCN panel affordability rating (mean rating 1: very expensive). The affordability rating was similar across a variety of hypothetical cancer therapies and tumor types (rating: 3). Oncologists estimated the costs for this rating of 3 to range from $4,600 to $6,000 per month, which was inconsistent with actual drug costs. Oncologists estimated the mean monthly OOP costs for patients with insurance to range from $1,260 for a new oral medication to $1,700 for a new infused medication. Only 26% of oncologists were comfortable or very comfortable with rating costs associated with affordability levels.Surveyed oncologists rated cancer therapies as more affordable (per NCCN Evidence Blocks criteria) than NCCN panel ratings. Costs associated with affordability were not consistent with actual treatment costs; however, most oncologists were not comfortable with rating affordability. Patient OOP costs had the biggest influence on affordability ratings; however, physicians overestimated patient OOP costs significantly. There is an opportunity to improve the value frameworks, especially with regard to affordability assessment. What is already known about this subjectTo address the rising concern about oncology drug costs, several stakeholders have developed frameworks to help assess the value of oncology regimens.Oncology value frameworks in the United States include the American Society for Clinical Oncology Value Framework (AVF), National Comprehensive Cancer Network (NCCN) Evidence Blocks, the Institute for Clinical and Economic Review value framework, and Memorial Sloan Kettering Cancer Center’s DrugAbacus.What this study addsCosts associated with affordability ratings of NCCN Evidence Blocks were not consistent with actual treatment costs and were underestimated by 39%-60% across treatments.Patient out-of-pocket (OOP) costs had the most influence on affordability ratings; however, physicians overestimated patient OOP costs for oral and infusion treatments.Affordability ratings did not differ based on oncologist specialty, practice site, or NCCN/National Cancer Institute membership and may be global impressions of what oncologists consider “affordable,” rather than being specific to a treatment or tumor type.1 The increasing prevalence of cancer coupled with the rise in expensive new drugs and technologies used in therapy have brought increased scrutiny to the cost and value of treatments in oncology. To address the rising concern about oncology drug costs, several stakeholders have developed frameworks to help assess the value of oncology regimens.2 In the United States, these include the American Society for Clinical Oncology Value Framework (AVF), National Comprehensive Cancer Network (NCCN) Evidence Blocks, the Institute for Clinical and Economic Review (ICER) value framework, and Memorial Sloan Kettering Cancer Center’s DrugAbacus.By 2020, cancer care costs are projected to reach approximately $158 billion, up from $125 billion in 2010.3 DrugAbacus is an online tool that allows users to set the weights for several factors, including novelty of the drug, to determine the value-based price of an oncology regimen. The ICER value framework assesses clinical benefit, cost-effectiveness, and budget impact of a drug or health intervention. It has a broader audience and is intended for use by payers, policymakers, patients, and physicians.2 It is not limited to oncology regimens and can be used to assess the value of any drug, device, or procedure.The AVF leverages head-to-head randomized clinical trials to compare 2 cancer treatment regimens. The framework generates a net health benefit score and presents this score along with the direct cost of treatment to form a summary assessment for the treatment.5In contrast to these value frameworks, NCCN Evidence Blocks generate 5 scores for a treatment’s efficacy, safety, quality and consistency of evidence, and affordability. Evidence Blocks are developed by analyzing the scores of NCCN’s expert panel members, which are then plotted onto a 5 × 5 matrix to produce a visual plot of the panel members’ responses.As cost pressures continue to rise in the United States, NCCN Evidence Blocks may be increasingly used by patients and providers to determine which regimens to prescribe and by payers to determine which regimens to reimburse as part of their health plans. Given that the Evidence Blocks could have considerable impact on the use and coverage of oncology regimens, it is important to gain a better understanding of the validity and key drivers of NCCN Evidence Block ratings. Further, assessment of cost and affordability play an important role in NCCN Evidence Blocks; however, the degree of oncologists’ understanding of the cost and affordability of cancer treatments and whether affordability ratings correspond to actual drug costs are unclear. In addition, it is not clear if the NCCN expert panel ratings of affordability differ from other practicing oncologists.The primary objectives of this study were as follows: To (a) assess oncologists’ awareness, knowledge, perceptions, and ratings of the AVF, the ICER value framework, NCCN Evidence Blocks, and DrugAbacus; (b) assess oncologists’ knowledge and perceptions of drug affordability as defined by the NCCN Evidence Blocks methodology; and (c) determine the factors that influence drug affordability ratings.http://www.medefield.com/). The Medefield panel is an opt-in panel, in which physicians join with the understanding that they will complete periodic surveys. All physicians in the United States have their credentials validated with the American Medical Association.This was an online cross-sectional study conducted in the United States between November 15, 2016, and January 15, 2017. Oncologists were recruited from the Medefield physician panel  would be ±6.86%, assuming a 95% confidence interval and the maximum variance of a binomial distribution .Physicians provided information about their age, sex, ethnicity, region, board certification status, and years in practice. Physicians provided information about their primary practice setting, including years in practice, type of practice site, patient load, state where practice is located, percentage of Medicaid patients seen in practice, and types of cancer treated.Oncologists provided information about their awareness and perceptions of the AVF, the ICER value framework, NCCN Evidence Blocks, and DrugAbacus. Oncologists ranked these 4 value frameworks on the following characteristics: ease of use, comprehensiveness, feasibility for use in the clinical setting, ability of patients to understand the framework, and most favorable overall.5 After rating the drug’s affordability, oncologists were asked to assign costs (in U.S. dollars) to the 5 levels of affordability. Additional questions assessing perceived patient out-of-pocket (OOP) costs, perceived drug acquisition costs, and comfort level in assessing affordability were also included in the survey.Using NCCN Evidence Blocks, oncologists were asked to rate affordability for 4 hypothetical cancer therapies: an anaplastic lymphoma kinase (ALK) inhibitor, 2 immunotherapy agents (1 for breast cancer and 1 for lung cancer), and an oral oncology treatment for leukemia/lymphoma. The NCCN affordability rating scale was used to measure oncologists’ perceptions of a drug’s affordability as follows: 5: very inexpensive, 4: inexpensive, 3: moderately expensive, 2: expensive, and 1: very expensive. Affordability (as defined by NCCN) refers to the overall cost of an intervention, including drug cost, required supportive care, infusions, toxicity monitoring, management of toxicity, and the probability of care being delivered in an inpatient hospital setting.5 NCCN does not provide panel members with information about the cost of the intervention; panel members are asked to rate the affordability based on their knowledge.5 For this study, we provided information on efficacy, safety, quality, and consistency, but not on affordability , consistent with NCCN practice.The NCCN Evidence Block evaluation is administered via survey, during which NCCN provides information to panel members on the efficacy, safety of the drug, and the quality and consistency of evidence.After rating the affordability of each of the drug products, oncologists were asked to select and then rank the factors that influenced their affordability rating. Factors included patient OOP costs, drug acquisition cost, duration of treatment, cost to manage drugrelated toxicities, administration costs, site of care, costs of other treatment options, supportive care costs, patient socioeconomic status, efficacy and safety of the drug, potential for hospitalization, or other factors.Physician demographic and practice site characteristics, knowledge, and opinions were reported descriptively using frequencies and percentages for categorical variables. Continuous variables were reported using means, standard deviations (SDs), medians, and interquartile range. Mean values were calculated for affordability ratings and rounded to the nearest integer, consistent with NCCN practice. T-tests and analysis of variance and Kruskal-Wallis tests were used to assess differences in means and medians for continuous variables. Chi-square tests were used to assess differences in familiarity and perceptions of value frameworks by physician demographic/practice characteristics. All analyses were conducted in Stata software package 13  or R software package 3.3 .A total of 200 oncologists completed the survey; 122 (61%) were medical oncologists and 78 (39%) were hematologists/oncologists . PhysiciOncologists were most familiar with NCCN Evidence Blocks (90%), followed by the AVF (84%), ICER value framework (57%), and DrugAbacus  for its ease of use, comprehensiveness, feasibility for use in the clinical setting, ability of patients to understand, and most favorable overall. NCCN Evidence Blocks was ranked highest on all measures, while the AVF was consistently ranked second, the ICER value framework was consistently ranked third, and DrugAbacus ranked fourth on all measures.The NCCN Evidence Block affordability rating by oncologists was similar across a variety of hypothetical cancer therapies and tumor types , despite differing monthly drug costs and OOP costs .P > 0.05 for all comparisons).These ratings were higher (more affordable) than those given by the NCCN expert panel for drugs in a similar therapy class, such as cancer immunotherapies (CITs) in non-small cell lung cancer (NSCLC) or ALK inhibitors. Affordability ratings did not differ based on oncologist specialty, practice site, or NCCN or National Cancer Institute (NCI) membership . Mean OOP costs were also calculated by the percentage of Medicaid patients seen in practice; however, there were no significant differences for infusion or oral product costs (P > 0.05 for both comparisons).To further understand the top 2 factors influencing affordability ratings, we assessed oncologists’ perceptions of patient OOP costs and drug acquisition costs. Oncologists’ estimates for patient OOP costs differed by mode of administration, with estimated mean monthly OOP costs for patients with insurance ranging from $1,260  for a new oral medication to $1,666  for a new infused medication. Estimated OOP costs did not vary significantly by oncologist’s specialty, practice site, or comfort level in estimating costs . Estimated monthly drug acquisition costs associated with affordability ratings by treatment and tumor type are shown in P = 0.06 for ALK+ inhibitor, P = 0.83 for CIT in NSCLC, P = 0.07 for CIT in breast cancer, and P = 0.29 for oral hematology/oncology drug).Only 52 (26%) oncologists were “comfortable/very comfortable” with rating costs associated with affordability levels; 94 (47%) were “somewhat comfortable”; and 54 (27%) were “uncomfortable/very uncomfortable” . Afforda6The majority of oncologists were familiar with value frameworks and had used them in the past. Of the 4 oncology value frameworks, NCCN Evidence Blocks were perceived most favorably by oncologists, followed by the AVF. Since NCCN Evidence Blocks and the AVF are intended for use by the oncologist and patient as part of the treatment decision-making process, they may be the most directly relevant and hence, may be considered most useful by oncologists.5NCCN Evidence Blocks use a simplified metric to assess affordability that ranges from 1 = very expensive to 5 = very inexpensive. However, the scoring criteria are subjective and are based on panel members’ knowledge and clinical experience. Additionally, NCCN does not provide panel members with any information pertaining to the cost categories that go into determining the affordability rating—drug cost, supportive care, administration and monitoring costs, and toxicity management costs. Rather, affordability is rated using the panel members’ knowledge of overall cost of the treatment regimen. Further, it is not clear if there are experts in health economics on the panel who may provide a deeper understanding of treatment costs.There are no clearly defined ranges or thresholds for determining the affordability rating, and it is not clear if oncologists are aware of the true costs associated with each of the above categories, given the opaque nature of drug pricing in the United States. In our study, oncologists’ ratings of affordability were higher than those of the NCCN expert panel—an indication that oncologists underestimated treatment costs. Affordability ratings did not differ by the respondents’ practice characteristics, their specialty, NCCN/NCI membership, or comfort level in rating affordability, suggesting that these ratings are independent of practice characteristics.8 Thus, oncologists in the study overestimated patient OOP costs for oral treatments by 140%-1,400%, whereas costs for infusion treatments were overestimated by 700%-1,960%.The main drivers of the affordability ratings were patient OOP costs and drug acquisition costs. Oncologists estimated the mean monthly OOP costs for patients with insurance, ranging from $1,260 for a new oral medication to $1,666 for a new infused medication. However, actual mean monthly OOP costs for a new oral treatment are estimated to be in the range of $90-$900, whereas those for an infused medication are estimated to be $85-$238.Drug acquisition costs were the second biggest factor driving oncologists’ affordability ratings. In this study, oncologists’ estimated drug acquisition costs did not differ by treatment or tumor type. Ranges were consistent for each affordability rating irrespective of treatment type, whereas costs associated with each affordability rating were underestimated by 39%-60%. This suggests that oncologists’ perceptions of affordability are not affected by the treatment type or cancer type; rather, they are global impressions of what oncologists consider “affordable.”2 However, some aspects of their methodology, especially pertaining to affordability assessment, have not been tested or validated.Oncology value frameworks can be useful, and their methods to assess efficacy and safety are largely based on known and accepted standards.9This study presents the first assessment of oncologists’ perceptions of affordability using NCCN Evidence Blocks’ approach to affordability and its associated cost ranges. Oncologists in this study overestimated patient OOP costs and underestimated drug acquisition costs; however, only 26% reported feeling comfortable estimating costs associated with affordability. Given that no information currently exists to assess the reliability and validity of NCCN Evidence Blocks’ approach to affordability, the results of this study raise a potential issue of whether the affordability ratings accurately reflect costs. Further, the scale used to assess affordability has not been tested or validated. Of the first 10 tumor types to have Evidence Blocks published, none have received a rating of 5, and only 4.79% have received a rating of 1, suggesting that ratings may not span the full spectrum of the scale.As oncologists in this study indicated, most of them are not comfortable with rating the construct of affordability as defined by NCCN. Currently, NCCN Evidence Blocks do not specify the perspective or the context for the construct of affordability. Additionally, the components of the affordability metric are largely undefined. For example, one of the components is drug acquisition cost, but it is not clear from whose perspective . Supportive care costs, administration, toxicity, and management costs are all part of the affordability construct, but they are hard to estimate and define.It is unclear whether oncologists are considering the costs of all of these components when rating affordability and, if so, what sources of information on the costs of these components they are using. Patient OOP costs are not a component of the affordability metric as defined by NCCN; however, oncologists indicated that is the main factor driving their affordability ratings. Future research should assess oncologists’ knowledge of costs and cost components as defined by NCCN. Future research should also assess the reliability and validity of the affordability metric of the Evidence Blocks and determine if ratings align with actual costs. As value frameworks evolve, such as with the newly released NCCN Preference Strategy Guidelines, it will be important to refine and validate their metrics.This study has some limitations to consider. The study was descriptive in nature without any adjustment for how confounding variables could have influenced affordability ratings. Only members of an Internet panel were eligible to participate; these physicians might differ from the broader oncologist population or from NCCN panel members in their experience and understanding of drug costs and affordability. Since this was an online survey, it is possible that oncologists could have accessed pricing references when ranking affordability. However, given that the average length of time required to complete the survey was about 20 minutes, and there were no large deviations from this time, it is unlikely that this occurred.Surveyed oncologists rated cancer therapies as more affordable than did NCCN panel ratings. Costs associated with affordability were not consistent with actual treatment costs; however, most oncologists were not comfortable rating affordability. Patient OOP costs had the biggest influence on affordability ratings; however, physicians overestimated patient OOP costs significantly. Given these results, there is an opportunity to both improve oncologists’ knowledge of affordability to enable them to have a discussion with their patients as well as improve the validity of the NCCN Evidence Block framework, especially with regard to the affordability assessment.


Pomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA).MDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging.Cells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract.Overall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.The online version contains supplementary material available at 10.1186/s12906-023-04134-1. Punica granatum L.) has been used since ancient times in the traditional medicine of several cultures, particularly in the Middle East. Pomegranate fruit (arils), juice and peel extracts are rich sources of polyphenols such as tannins, anthocyanins, and flavonoids and hence possess potent antioxidant properties [Pomegranate  as a byproduct of the respiratory chain-mediated transfer of electrons to oxygen and its incomplete reduction. The respiratory chain complexes are themselves very sensitive targets for ROS with significant damaging effects . AccordiGiven the beneficial properties as therapeutic potential of pomegranate, and considering that the fruits are highly susceptible to weight loss and decay during postharvest handling and storage , 17, detThe overall aim of this study was to investigate the biological effect of PJ, on breast cancer cells reporting its effect on: (i) reducing oxidative stress, (ii) reducing proliferation rate of cancer cell, (iii) increasing mitochondrial function. Moreover, we determined the optimum storage conditions able to preserve PJ efficiency in modulating the mitochondrial oxidative metabolism.All PJ extracts, with the exception of thermal pasteurized PJ (TP-PJ) and high pressure processed PJ (HPP-PJ), were obtained from fresh pomegranate arils removed from fruits, variety ‘Wonderful’, grown in southern Italy were provided by “Masseria Fruttirossi srl”, from its orchards located in Castellaneta, Italy, in accordance with the relevan guidelines/regulations/legislation.2 + 10 kPa CO2 (Controlled Atmosphere), or (ii) air at both 4 and 8 °C.Pomegranate fruits were stored for 30 and 60 days in 50L-containers connected to a continuous humidified flow of (i) 3 kPa Ohttps://shop.lomesuperfruit.com/).Initially and at each sampling fruit were evaluated for quality and PJ was extracted homogenizing about 100 g of arils in an Ultraturrax  for 1 min; the homogenate was filtered through cheesecloth and immediately transferred for cell treatment. In some cases, arils from stored fruit were held in modified-atmosphere packaging for 5 days at 5 °C to simulate the ready-to-eat product before extracting the PJ. Frozen PJ was obtained holding fresh extracted PJ at -20 °C for 30 days before cell treatment. TP-PJ and HPP-PJ was obtained from commercially available fruit juices from Lome Superfruit (2 humidified atmosphere in complete DMEM High Glucose medium (25 mM glucose), supplemented with 10% fetal bovine serum, penicillin–streptomycin (100 U/mL) and 2 mM L-glutamine. For PJ treatment, 0.5–2.0 × 106 cells/mL were supplemented with graded concentration of filtered PJ  for 24 h.MDA-MB-231 (MDA) cells, human caucasian breast adenocarcinoma were purchased from American Type Culture Collection . MDA is a highly aggressive, invasive and poorly differentiated triple-negative breast cancer cell line as it lacks oestrogen and progesterone receptor expression, as well as HER2  amplification . The celOxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in adherent MDA cells with a XFe96 Extracellular Flux Analyzer  as described in . 6000 ceOCR was measured under basal conditions and after sequential injection of oligomycin (1 μM), FCCP , and rotenone + antimycin A (1 μM + 1 μM) to evaluate the ATP-linked respiration, the maximal respiratory capacity, and the non-mitochondrial oxygen consumption, respectively. For ECAR analysis, at the end of running, 50 mM 2-deoxyglucose was added in order to determine the glycolysis-independent acidification.OCR and ECAR values were normalized to protein content in each well, determined using the BCA assay .The xCELLigence System Real-Time Cell Analyzer  was used for monitoring proliferation of cancer cells. MDA cells were seeded at a cell density of 6000 cells/well in 16-well E-plates, connected to the RTCA platform placed into a cell incubator at 37 °C and 5% CO2. When cells reached log growth phase, approximately 24 h after seeding, medium was removed, and cells were treated for 24 h with pomegranate juice or PBS as vehicle. Cell proliferation was monitored every 30 min for a period of up to 96 h, measuring changes of electrical impedance as a dimensionless parameter termed cell index (CI). CI was normalized just before treatment and converted into normalized cell index (NCI), by the RTCA-integrated software . Normalized cell index was calculated as described in [Confocal microscopy Imaging was performed as described in . Brieflyp-value < 0.05 was accepted as statistically significant. All analyses were performed using GraphPad Prism Software Version 8 .Data are reported as mean ± standard error mean (SEM) of at least three independent experiments and compared by one-way or two-way ANOVA, followed by Bonferroni post-hoc test; a *The antioxidant effect of PJ is widely reported in literature both in vitro and in vivo studies . To veriTo rule out that the above observed effects were partly due to pH changes of the culturing medium following supplementation of the PJ we carried out control experiments with the pH of the medium buffered at the lowest pH attained with 10% v/v PJ. No significant effects were observed neither on the ROS scavenging activity or the cell growth Fig S. SimilarAs mitochondrial respiration plays an important role in producing energy by OxPhos and it is also a major source of cellular ROS production, we tested on it the effect of fresh PJ treatment. To this aim we performed a systematic analysis using the Seahorse technology to assess the two major cellular metabolic fluxes, the mitochondrial respiratory activities driving the OxPhos, measured as oxygen consumption rate (OCR), and glycolysis, measured as extracellular acidification rate (ECAR), mainly due to conversion of pyruvate to lactate, as previously described . Using tFreshly made PJ can be immediately used for fresh market, but its quality lasts only a few days in a cold storage. Therefore, alternative methods of processing and storage are needed which can increase microbiological stability and preserve nutritional and bioactive properties . ThermalIt has been reported that controlled atmosphere (CA) storage is a better option for pomegranate fruit storage than regular air (RA) in prolonging the shelf life of fresh products . In ordeFreezing is one of the common procedure to preserve food materials such as fruit juices, but it is unknown whether the juice’s nutritional value is preserved during prolonged frozen storage. To address this issue, we analyzed ROS content and proliferation in MDA cells treated with frozen PJ (FZ-PJ) prepared from fresh pomegranate. Figure Natural products have been used for the treatment of various diseases and are becoming an important research area for drug discovery in cancer. These products have been extensively studied and have exhibited anti-carcinogenic activities by interfering with the initiation, development and progression of cancer through the modulation of various mechanisms including cellular proliferation, differentiation, apoptosis, angiogenesis, and metastasis. This concept is gaining attention because it is a cost-effective alternative to cancer treatment. Accumulating scientific evidences show that daily consumption of a diet rich in fruits and vegetables reduces the risk of cancer  and growIn this context, pomegranate fruits is gaining high considerations for its strong antioxidant and anti-inflammatory properties due to their high content of hydrolysable tannins and anthocyanins  that havHowever, the commonly accepted correlation between maintenance of a pro-oxidative tone and control of cell growth and proliferation is over-simplified and requires deepening as negative outcomes on cancer cell growth have been also reported. Indeed, several evidence suggests that ROS, or more generally a pro-oxidative state, within cells act as second messengers in intracellular signaling cascades which induce and maintain the oncogenic phenotype of cancer cells, due to their ability to increase cell proliferation, survival, cellular migration participating in the Ras-Raf-MEK1/2-ERK1/2 oncogenic signaling, and also by inducing DNA damage leading to genetic lesions that initiate tumorigenicity and sustain subsequent tumor progression . In thisIndeed, our results show that relatively low concentrations of PJ, that effectively remove basal reactive species content, have practically no effect on the growth of MDA cancer cell line after 48–72 h post treatment. Significant inhibition of cell growth is observed at higher concentration of PJ or after much longer treatment. Given the multiplicity of bioactive compounds present in the PJ it is possible that some of those, present at high concentration, are responsible for the antioxidant effect whereas other, present at lower concentration, are responsible for the slowdown/arrest of cell growth. Anthocyanidins, ellagic acid, ursolic acid, luteolin, punicalagin among the others are all PJ constituent proved to inhibit cell proliferation when used as pure compound. Although it would be informative to assess the compositional profile of the major bioactive compounds present in the PJ extract this was not the scope of the present study.Mitochondria are a major intracellular source of ROS as a byproduct of respiration. Leak of electrons from the respiratory chain complexes directly to di-oxygen causes its incomplete reduction to the superoxide anion radical, that is the germinal species generating further reactive oxygen as well as nitrogen species. Because of the proximity to the ROS generating sites the respiratory complexes are themselves potential targets of oxidative modification . This woAlthough the link herein shown between the observed PJ-mediated pro-oxidative metabolic shift and the dampening of the cell proliferation is at the correlative level nevertheless it is insightful. Most cancer cells are known to rely on aerobic glycolysis even when oxygen availability is not limiting (Warburg effect) . AerobicIn line with this notion our group has provided in vitro evidence that utilization of inhibitors of metabolic pathways differentially affects the viability of a given cancer cell line depending on their specific metabolism , 47. RelThe above described results were obtained with PJ extract from pomegranate immediately harvested. As pomegranate is a seasonal fruit, determining optimum storage conditions for fruit or its juice aiming at preserving its valuable nutritional components that may be reduced during storage, is important. In fact, during post harvesting storage, pomegranate displays important quality loss as a result of several physiological and enzymatic disorders with the major storage problem being water loss, leading to browning symptoms in both peel and arils , 49. TheThermal processing (pasteurization) is the most used preservation technique to extend the shelf life of juices. However, this process may have adverse effects on sensory and nutritional values of juices therefore alternative preservation methods, which can destroy undesirable microorganisms with less adverse effects on product quality, urge. High Pressure Processing (HPP) is another technology shown to have several benefits such extension of food shelf-life, superior organoleptic quality and better nutrient retention to keep its original freshness, flavor, taste and color changes, which are minimal.However, the results provided in this study show that when MDA cells were treated with PJ processed either with pasteurization or HPP technology the impact on the metabolic fluxes was completely different from that attained with fresh PJ. Indeed, we found a significant about 50% decrease of the mitochondrial oxidative metabolism with no significant effect on the glycolytic flux. So, even if it has been previously established that HPP treatments showed only a slight reduction in antioxidant capacity during storage time , this stStorage under controlled atmosphere extends storage life of several agricultural products. It has been used successfully for maintaining quality of many tropical and sub-tropical fruit. Storage of fruit under controlled atmosphere and cold temperature reduces fruit respiration rate, ethylene production, suppresses or delays senescence processes and eventually increases postharvest shelf life . When weHowever, the above reported effect of CA is completely lost after 60 days of storage with significant depression of both mitochondrial respiration and glycolysis. Intriguingly, and somehow surprising, storage under RA resulted even after 60 days in limited effects on the metabolic fluxes as compare with untreated cells.To note PJ extract from packaged arils of pomegranate stored for 30 days in CA or RA, did not elicit the same positive effect on respiration, even if packaged arils stored at low temperatures retained appreciable nutritional and bioactive compound levels with low microbial count during the entire storage period , 51.Freezing is another procedure used to preserve food products such as fruit juices. In the food industry, a storage temperature of − 18 °C effectively reduces chemical and microbial spoilage of foods. Nevertheless, frozen storage, even at − 25 °C, does not appear to preserve all the nutritional value of PJ. In fact evaluation of the total anthocyanin and phenolic contents in PJ stored at − 25 °C unveiled its decrease after 20 days’ frozen storage with consequent reduction of antioxidant activity . HoweverHowever, treatment of MDA-MB-231 with frozen PJ was unable to recapitulate the metabolic shift observed with fresh PJ though conserving the capability to inhibit MDA proliferation. Treatment with frozen PJ resulted in inhibition of mitochondrial respiration and enhanced glycolysis at low and intermediate concentrations of PJ thereby causing also in this case a metabolic shift but promoting a more glycolytic/quiescent phenotype. Understanding the reasons for such conflicting results would require a “metabolomic” approach to assess the bioactive compounds present in the PJ before and after freezing to identify relevant changes in its composition. This was however beyond the aim of the presented study.Based on what is reported in this study one is tempted to speculate that perhaps a balanced contribution of catabolic pathways such as glycolysis and mitochondrial oxidative routes is necessary beyond their bioenergetic outcome. Indeed, both glycolysis and tricarboxylic acid provide intermediates for biosynthesis of lipids, aminoacids, nucleotides as well as reducing equivalents. Therefore, it is possible that perturbing an established balance between different metabolic pathways might turn in dampening of the proliferative efficiency irrespective of the direction of the metabolic shift underwent (i.e. pro-glycolytic vs pro-oxidative).This study confirms the potential of pomegranate juice extract as a promising support in preventing cancer or even in assisting conventional onco-therapy as previously reported –11. ThisHowever, at the same time, this study underlines the intrinsic difficulties in providing mechanistic insights when a multeplicity of effects are elicited by a mixture of bio-active compounds as those present in crude extracts. Untangling the specific contribution of the extract constituents or of their dosed mixture on selected biochemical cellular processes urges to improve the beneficial utilization of nature-provided compounds for preserving health.Additional file 1: Fig. S1. Effect of PJ from pomegranate stores in Controlled atmosphere (CA) on mitochondrial function in MDA cells. Fig. S2. Effect of PJ extracted from arils packaged for 5 days, previously extracted from pomegranates stored for 30 days in CA or RA, on mitochondrial function in MDA cells. Fig. S3. Effect of frozen PJ processed with thermal pasteurization (PT) and high hydrostatic pressure (HHP) on mitochondrial function in MDA cells. Fig. S4. Effect of frozen PJ from pomegranate stores at 4°C in Controlled atmosphere (CA) and in regular air (RA) on mitochondrial function in MDA cells. Fig. S5. Effect of frozen PJ from pomegranate stores at 8°C in Controlled atmosphere (CA) on mitochondrial function in MDA cells. Fig. S6. Effects of pH on cell viability, ROS production and metabolic fluxes in MDA cells.


An innovative spectroscopic method that allows to chemically and structurally characterize viruses directly in suspension within few minutes was developed. A library of five different plant viruses was obtained combining dielectrophoresis (DEP), performed with a device specifically designed to capture and agglomerate virus particles, and Raman spectroscopy to provide a chemical fingerprint of virions. The tested viruses, purified from infected plants, were chosen for their economic impact on horticultural crops and for their different morphological and structural features. Using the Raman-DEP device, specific profiles for each virus were successfully obtained, relying on chemical differences occurring even with genetically similar viruses belonging to the same taxonomic species and morphologically indiscernible by transmission electron microscopy (TEM). Moreover, we investigated the potentiality of Raman-DEP to follow dynamic changes occurring upon heat treatment of tobacco mosaic virus (TMV) particles. Raman peak deviations linked to TMV coat protein conformation were observed upon treatment at temperatures equal or higher than 85°C, substantiating the rod-to-spherical shape transitions observed by TEM and the concomitant drastic loss of infectivity following plant inoculation. Overall, the Raman-DEP method can be useful for the characterization of virus (nano)particles, setting the basis to create a database suitable for the study of viruses or virus derived-nanoparticles relevant for the agricultural, medical, or biotechnological fields. Recently, a dramatic increase in viral outbreaks is occurring worldwide, provoking severe consequences on human, animal, and plant health . DespiteRaman spectroscopy (RS) relies on the phenomenon of inelastic light scattering by the molecules present in a sample after their excitation to higher rotational or vibrational slates. RS offers interesting advantages in the analytical sector, thanks to its versatility, specificity, rapidity, and simplicity in sample preparation, allowing to perform dynamic analysis and providing structural and chemical information at the molecular level , with anMoreover, RS-based approaches have been used to characterize directly viruses  and planThe combination of Raman and DEP was already successfully applied for the rapid characterization of bacteria and the detection of antibiotic resistance, in alternative to classical microbiological techniques . Raman-DHere, a new DEP cell was developed for the analysis of viruses, and the Raman-DEP approach was applied for the first time to characterize plant-infecting viruses, selected as safer candidates compared to animal-infecting ones. To set up the procedure and test its performances, five different viruses were used, i.e., tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tomato brown rugose fruit virus (TBFRV), cucumber mosaic virus (CMV), and tomato spotted wilt virus (TSWV). These viruses were selected not only for their worldwide economic importance , but alsTobamovirus, the largest taxonomic group within the Virgaviridae family, and are characterized by rigid virions with a predominant length of 300–310 nm and a diameter of 18 nm. Tobamoviruses possess a non-segmented positive linear single-stranded RNA (ssRNA) genome, encoding three non-structural proteins which are required for viral replication and movement, and a structural capsid protein (CP) (17–18 kDa). TMV is the most well-characterized virus from the structural point of view; its virions consist of 2,130 identical copies of the monomeric CP, assembled in a helical arrangement. In nature, tobamoviruses are transmitted mechanically during agricultural practices or through seeds.TMV, ToMV, and TBRFV belong to the genus Cucumovirus, family Bromoviridae) is a multicomponent virus whose genome consists of three ssRNA molecules individually encapsidated in isometric icosahedral particles of 28 nm, made of a single CP (CMV (genus ingle CP . Beside ingle CP .Orthotospovirus family Tospoviridae) has an extremely wide host range, infecting up to 1,100 different crop and weed species. Virions appear as spherical particles with a diameter of 80–120 nm, surrounded by a membrane. Two glycoproteins (G1 and G2) protrude from the virion surface and govern the transmission by thrip vectors. The TSWV genome consists of three RNA segments  and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were used. Following Raman-DEP analysis, tentative assignment of the chemical meaning of each spectral signal was obtained, revealing a very high spectral specificity.In addition, the potential of Raman-DEP was tested to monitor dynamic changes occurring in TMV particles subjected to different temperature treatments. TMV virions can withstand extremely high temperatures  and, upoOverall, the method described in this work can be useful for a direct characterization of viruses, including animal and human pathogens. DEP has already been applied to detect and discriminate viruses, but in most cases it was applied to indirectly measure the different DEP responses of virus-infected and non-infected cells grown in culture . Here, tFurther details on the methods used in this work are presented in the Nicotiana benthamiana, N. tabacum, and N. clevelandii were maintained in a greenhouse at 23°C (14/10 h light/dark). The list of isolates of TMV, ToMV, TBRFV, CMV, and TSWV used in this study is presented in Plants of N. benthamiana, the purification method of Chapman was essentially followed  operating at 60 kV. Micrograph films were developed and digitally acquired at high resolution with a D800 Nikon camera. Images were trimmed and adjusted for brightness and contrast using Fiji software.Virions were analyzed by negative staining with 0.5% aqueous uranyl acetate. Appropriate dilutions of purified virions were adsorbed onto PelcoThe protein composition of purified virion preparations was analyzed by SDS-PAGE. Each virion suspension was dissolved in 2× SDS-PAGE gel loading buffer and incubated at 95°C for 3 min. Clarified protein extracts were separated on Mini-Protean TGX acrylamide gradient (4–20%) gels  in Tris-glycine buffer and gels stained with Coomassie brilliant blue, according to standard procedures.The thermal stability of purified TMV particles was assessed on 50-μl aliquots of purified TMV suspension (7.5 mg/mL). Following incubation in ice for 10 min, virion aliquots were moved separately to thermal blocks set at either 75°C, 85°C, or 90°C for 3 min, or left in ice as control. Afterwards, each treated sample was cooled back in ice for 10 min and then either loaded onto EM grids for TEM observation, used for infectivity assays or for Raman analysis.−2, 10−3, and 10−4 dilutions with silicon carbide onto N. clevelandii plants . At 5 days post-inoculation, the number of chlorotic lesions developed on leaves was counted.Purified TMV virion suspensions (7.5 mg/mL) subjected to different thermal treatments (see above) were mechanically inoculated at 10A custom-made DEP sample holder was manufactured as previously detailed  and adap−1, spectral resolution 5 cm−1). A 60× water immersion, cover-glass corrected objective with 1.1 NA (Olympus LUMFLN60XW) and 1.5 mm working distance was employed in a 180° backscattering collection geometry; transmission white-lamp illumination for bright-field microscopy was used for positioning and for general control of the system.For all experiments, aliquots of 5 μL of each purified virus, resuspended in phosphate buffer saline solution  were injected into the Raman-DEP device and analyzed with a Thermo Scientific™ DXRxi dispersive Raman microscope, equipped with a 532 nm Nd:YAG excitation laser and an Andor EMCCD spectrograph with a 900 lines/mm grating , using an excitation power of 20 mW (measured at the sample). Wavelength and relative intensity were calibrated before each experiment using a neon low-pressure emission lamp and NIST SRM 2242a .TEM analysis of virion preparations showed that each sample consisted of highly homogenous populations of intact particles. As shown in When separated in denaturing gel and stained with Coomassie, protein extracts obtained from each virion sample appeared to consist only of the structural proteins characteristic of each virus, with no detectable contaminants in the range from 250,000–10 kDa. Specifically, the tobamovirus CPs migrated next to the 20 kDa marker, according to their calculated molecular mass (MM), i.e., 17.7 kDa for ToMV, 17.6 kDa for TMV, and 17.5 kDa for TBRFV . The CMV−1, negative DEP was achieved for all virion preparations. After sample injection, the electric field was generated until the confocal volume of the microscope was filled, resulting in a stabilized signal-to-noise ratio of the Raman spectra. The agglomeration time varied according to each virus, i.e., 1 min for TSWV, 3 min for TMV and TBRFV, and 5 min for ToMV and CMV.In the electric conditions applied, considering an estimated medium conductivity of approximately 1.6 S m−1 were considered, and a chemical assignment was made for each signal (−1 and amide III at 960–964 cm−1) and the phenylalanine and other aromatic amino acids vibrations . Specifically, a different Raman intensity ratio could be observed between bands related to amide I  and to aromatic amino acids ; furthermore, the peaks at 1004 cm−1 and 621 cm−1, typical of phenylalanine, appeared narrower and more intense in the TMV and TBFRV spectra compared to ToMV. Moreover, the band at 960–964 cm−1, attributable to amide III, tyrosine and ribose, was very pronounced in the TMV and ToMV spectra, but only barely visible for TBRFV. These features, obtained from viruses which are morphologically and structurally very similar  of the three tobamoviruses here considered , the amiPlant RNA viruses have the tendency to mutate during vector transmission or host infection, with relevant consequences from the adaptation and evolutionary point of view . To eval-1 and 1,002 cm-1  which are the most representatives of lipids, whose peaks appear narrower and higher for TSWV.TSWV, the only virus tested surrounded by a lipidic shell on its surface showed a very different Raman profile. Compared to the non-enveloped viruses here considered, most of the differences rely on the CH bonds vibrational regions  the C-O and C-N bonds signals, absent in the TSWV spectrum and (ii) a very sharp and intense peak due to P-O vibrations at 809 cm-1, present only in TSWV. This peak suggests a possible phosphorylation of the TSWV virion-associated proteins. Following a predictive search for phosphorylation sites of the TSWV N protein using NetPhos – 3.1, kinases . Accordi kinases .Furthermore, Raman signals related to phenylalanine and other aromatic residues were reduced or even absent in the TSWV spectrum compared to non-enveloped viruses; this could result from a partial masking by the prominent CH signals relevant to the higher lipids/proteins ratio. Considering the number of aromatic residues in the structural proteins of the viruses tested, we observed that the TSWV N and G1-G2 proteins have 7.4% and up to 13% aromatic residues, respectively , supportTMV particles maintained at different temperatures were subjected to Raman-DEP analysis, to ascertain whether differences linked to chemical and/or structural alterations occurring in the transition from intact to disrupted virions could be ascertained. For this, the same batch of purified TMV virions used above was incubated at temperatures ranging from 75°C to 90°C, based on published reports .-1). This was particularly evident for virions subjected to temperatures ≥85°C. A specific change occurs in a spectral region known to be related to the O=C–NH peptide bond and, ultimately, refers to structural modifications involving inter- and intramolecular H bond interactions, with possible consequences on protein folding. In particular, the Raman signal of amide I results from the sum of two components, one at 1640–1666 cm-1 which is mainly due to the β-sheet structural conformation of the proteins and one at 1675–1680 cm-1, linked to structural conformation with turns. A specific inversion of these two peaks was manifest in TMV samples heated at temperatures ≥85°C, showing an increase in the peak linked to protein conformation with turns (more disordered) at the expenses of the peak linked to β-sheet . This technique allowed for the first time to build a plant virus library within a few hours, measuring purified samples directly in aqueous suspension. Moreover, this method successfully revealed spectral differences associated with structural modifications of TMV virions occurring at temperatures not smaller than 85°C, monitoring the rod-to-spherical transition of this virus, one of the most studied virus-based nanotechnological tools (With the Raman-DEP method here developed, profiles specific for each of the tested plant viruses were gathered, discriminating chemical differences even among viruses which are morphologically and structurally indiscernible and belong to the same taxonomic group (al tools .To explore the usefulness of Raman-DEP for diagnostic purposes, future investigations with partially purified virion preparations could be conducted, aiming to simplify the purification procedures. By fine-tuning the electrical conductivity of the suspension liquids and the electric field frequency applied, selective accumulation of virions in the DEP cell and reduction of contaminating components are sought.In sum, Raman-DEP allows a fine characterization of virions directly on purified aqueous preparations, making a turning point in the development of analytical techniques useful in virology. In this line, Raman-DEP could be adopted, for example, to test the efficacy of disinfectant or virucidal substances, allowing real-time investigation of structural and chemical alterations of virions. Finally, structural changes derived from genetic manipulation, chemical modification, or thermal treatment of virions or virion-like particles could be investigated, with important biotechnological implications .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AS: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. GB: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. SM: Formal analysis, Methodology, Writing – review & editing. AG: Formal analysis, Validation. AR: Conceptualization, Supervision, Funding acquisition, Writing – review & editing. CD’E: Formal analysis, Methodology, Writing – review & editing. MV: Formal analysis, Methodology. MC: Formal analysis, Methodology. EN: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing, Data curation. CP: Conceptualization, Funding acquisition, Project administration.


In the past decade, there has been little progress in the treatment of malignant glioma. Recently, oncolytic virus has made great progress in glioma treatment, and a number of clinical trials have shown their potential of prolonging the survival time of glioma patients. Our objective is to evaluate effectiveness and safety of oncolytic virus (OV) in malignant glioma treatment.Based upon PRISMA, we collected relevant published clinical trials by searching medical databases up to January 16, 2023, applying the language restrictions in English and Chinese. We cross-searched the terms: ‘glioma’, ‘glioblastoma’, ‘oncolytic viruses’, ‘oncolytic virotherapy’ with filter ‘clinical trial’. Two researchers independently extracted the data regarding case definitions, published years, trial phase, characteristics of patients, administration of drug, overall survival (OS), and adverse events.19 published clinical trials in OV treatment of malignant glioma were included in the further systematic review analysis. None of them induced irresistible adverse effects attributing to OV treatment, median overall survival varied from 3.25 to 20.2 months after treatments. According to trials providing patient’s detailed molecular diagnosis, we find that the effectiveness of OV treatment has no significant difference in patients with different IDH or MGMT status.Current clinical trials have initially shown the potential of oncolytic virotherapy as a new treatment for malignant glioma. Besides development of virus types, the strategy of OV use is an urgent problem to be solved in future clinical application, such as repeated administrations, innovative drug delivery systems, and biomarkers. Malignant glioma accounts for nearly 80% of all malignant primary brain tumors . The annAmong these immunotherapies, oncolytic virus showed great potential for clinical application. Currently, several virus species were performed in the trials of malignant glioma treatment, such as Herpes Simplex Virus‑1, Adenovirus, Reovirus, Parvovirus, Polio/Rhinovirus Recombinant, Newcastle Disease Virus, and so on. Oncolytic viruses can be natural or genetically modified viruses which has the capability to replicate in the tumor, and their antitumor activity involves a variety of mechanisms . FirstlyConsidering the effectiveness of oncolytic virus therapy varies greatly, the purpose of this review is to systematically analyze the published clinical trials based on the evaluation of virus types, administration, efficacy and safety.This systematic review is conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 Statement . We collWe regarded studies as eligible for inclusion done in clinical trials studying oncolytic therapy in treatments of malignant glioma no matter in adults or children. Exclusive criteria were as follows: the study therapy that virus was only used as the vector to deliver therapeutic agent was excluded; the case reports, case series, and conference papers were excluded as well. Study titles and abstracts were reviewed by two independent investigators. Then the inclusion criteria were received after a full-text assessment. In case of disagreement, a third reviewer independently assesses the search methods, and any issue was resolved by consensus.Under the guidance of the established research protocol, two researchers independently extracted the data regarding case definitions, published years, trial phase, characteristics of patients, administration of drug, overall survival (OS), and adverse events. Notably, we did not sum up the median progression-free survival because the main indicator of tumor progression was imaging evidence. While because of the intratumoral injection, the entire tumor enlargement might confuse the judgment of tumor progression. Moreover, Tomoki Todo et al. assumed that post-administration MRI features (injection site contrast-enhancement clearing and entire tumor enlargement) likely reflected tumor cell destruction via viral replication and lymphocyte infiltration towards tumor cells based on biopsies . Finally118 records were searched out from the medical databases mentioned above and 34 of them were excluded due to the duplication. After titles and abstracts screened, there were 30 published articles relevant and retrieving for full-text assessments: 10 of these studies were excluded according to the reasons listed in Fig. The majority of articles published to report the results of their clinical trials used HSV-1  and adenovirus . There were 2 trials utilizing reovirus (10%), while Polio/Rhinovirus Recombinant virus , H-1 Parvovirus , and New Castle Disease virus  has only one completed trial, respectively , with the increasing repeats of virus injections from 2 to 6, the median overall survival improved significantly from 7.2 months to 20.2 months  and O6-methylguanine DNA methyltranferase (MGMT) Table . Todo etBased on the available data, it is evident that oncolytic therapy for malignant gliomas exhibits a favorable safety profile irrespective of the viral type employed. Six virus types have been genetically modified to enhance their oncolytic potential, and they can be broadly categorized into two groups: RNA viruses and DNA viruses. Among these, HSV-1 and adenovirus have predominantly featured in clinical trials of oncolytic virotherapy due to their superior safety profiles and the ease of gene editing technology. In recent years, there has been an increasing focus on developing and utilizing various virus species, particularly RNA viruses such as reovirus, H-1 Parvovirus, PVSRIPO, and NDV. The most notable characteristic of these viruses lies in their ability to traverse the blood–brain barrier, thereby expanding the potential for intravenous administration. However, further evidence is required to substantiate the safety of these novel viral species, address challenges associated with genetic modification, and evaluate their actual efficacy. Moreover, it should be noted that oncolytic viral drugs are typically designed to target a wide range of solid tumors rather than specifically gliomas. Consequently, limited investment has been made in developing oncolytic viruses capable of efficiently crossing the blood–brain barrier.Based on the analysis of published clinical data, our opinion is that repeated administrations are more effective than single administration in treating malignant glioma with oncolytic virus therapy. The conditionally approved oncolytic product G47Δ has shown significantly increasing efficacy with up to six times of virus injection . HoweverFor neurosurgeons, intraventricular administration is a relatively mature method in clinical practice, and there are also widely-used drug delivery products such as Ommaya . HoweverThe clinical trials, such as Desjardins et al. and Todo et al., presented some impressive long-term survivors from recurrent GBM. And according to the clinical test results of subjects after treatment, the active viruses do not last more than a few months in the body , 24, 43.The idealized tumor immune-microenvironment following oncolytic virus treatment can be divided into two distinct phases. Firstly, it is crucial to suppress the anti-viral immunoreaction in order to facilitate the replication and dissemination of the oncolytic virus. This challenge is particularly evident in viruses such as adenovirus or herpes simplex virus, which may have a high prevalence within the population , 50. FurAs oncolytic virotherapy is still in its early stages, two practical issues for future clinical use are biomarkers and timing of administration. Unlike other chemotherapy drugs, there is a lack of suitable markers to evaluate the efficacy potential of oncolytic therapy . As we pThe past 20 years witness the rapid development of OV for malignant glioma, and the first oncolytic product conditional approved in Japan brightens the future of OV developments. Besides direct tumor lysis, oncolytic therapy arouses the anti-tumor immunity and shows the potential of long-term treatment for tumor. According to the systematic review of clinical trials for malignant glioma treatments, we glanced the safety and developments of OV. In the future, the repeated administrations and the combination with other immunotherapies are required more explorations. The next generation of the existing virus by gene editing or the stem cell delivery system were urged to be developed. Meanwhile, we advocate for the attention and disclosure of tumor molecular or genotyping information in subsequent clinical trials on ovarian cancer (OV) to facilitate the analysis of biomarkers that can predict treatment response, a critical factor for the successful implementation of OV therapy.In a nutshell, OV for malignant glioma shows great prospects in the future and promotes significantly development of cancer therapy.


Sulfolobales have been identified, most of which are from hot springs distributed in the United States, Russia, Iceland, Japan, and Italy. To further expand the reservoir of viruses infecting strains of Sulfolobaceae, we investigated virus diversity through cultivation-dependent approaches in hot springs in Tengchong, Yunnan, China. Eight different virus-like particles were detected in enrichment cultures, among which five new archaeal viruses were isolated and characterized. We showed that these viruses can infect acidophilic hyperthermophiles belonging to three different genera of the family Sulfolobaceae, namely, Saccharolobus, Sulfolobus, and Metallosphaera. We also compared the lipid compositions of the viral and cellular membranes and found that the lipid composition of some viral envelopes was very different from that of the host membrane. Collectively, our results showed that the Tengchong hot springs harbor highly diverse viruses, providing excellent models for archaeal virus-host studies.Archaeal viruses are one of the most mysterious parts of the virosphere because of their diverse morphologies and unique genome contents. The crenarchaeal viruses are commonly found in high temperature and acidic hot springs, and the number of identified crenarchaeal viruses is being rapidly increased in recent two decades. Over fifty viruses infecting the members of the order  Like bacteriophages , archaeal viruses are also one of the most abundant and diverse biological entities on Earth. Viruses of archaea shared very few features with those of bacteria or eukaryotes in that they exhibited exceptionally diverse morphologies, remarkably distinct genomic contents, and unique infection mechanisms. For example, the Sulfolobus spindle-shaped viruses (SSVs) represent one of the most well-studied virus prototypes infecting crenarchaeal hosts and the spindle-shaped viral particle morphology has only been observed in Archaea . In termAcidianus, Metallosphaera, and Sulfolobus have been identified by culture-independent approaches  belonging to the family Rudiviridae  and Sulfolobus islandicus filamentous virus (SIFV) were shown to selectively acquire host membrane lipids GDGT-0 (glycerol dibiphytanyl glycerol tetraether) or archaeol 2SO4, 0.28 g l−1KH2PO4, 0.25 g l−1 MgSO4·7H2O, 0.07 g l−1 CaCl2·2H2O, 1.8 mg l−1 MnCl2·7H2O, 4.5 mg l−1 Na2B4O7·107H2O, 0.22 mg l−1 ZnSO4·7H2O, 0.05 mg l−1 CuCl2·2H2O, 0.0 3 mg l−1 Na2MoO4·2H2O, 0.03 mg l−1 VOSO4·n7H2O, 0.01 mg l−1 CoSO4·7H2O], 0.2% (wt/vol) sucrose, 0.1% (wt/vol) tryptone, 0.1% (wt/vol) yeast extract, and 0.1% (wt/vol) amine. The pH was adjusted to 3.0 with 10 N H2SO4. The environmental samples were incubated for 10 days at 75°C with 200 rpm shaking.Eleven environmental samples were collected from the hot springs of Tengchong in Yunnan, China , and 1 gk medium  with prog for 15 min at 15°C. The supernatant was filtered through a 0.22-μm filter (Merck Millipore), and the VLPs were precipitated from the supernatant by the addition of NaCl to 1 M and PEG 6000 to 10% (wt/vol) and incubated overnight at 4°C. The pellet was collected by centrifugation at 12,000 × g for 30 min at 4°C, and then suspended in 100 μl SM buffer containing: 0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris–HCl, 0.005% (wt/vol) glycerol, pH 6.0 (To observe the virus-like particles (VLPs), the enrichment cultures were centrifuged at 6, 500 × , pH 6.0 .Negatively stained cells were prepared for transmission electron microscopy (TEM) by spotting 10 μl of culture or viral solutions onto carbon copper grids  for 2–5 min and air-dried. Samples were stained with 2% (wt/vol) uranyl acetate for 30 s, and then observed under an electron microscope  operated at 120 kV.The hot spring sediments were diluted with liquid Brock medium. Serial dilutions were directly spread onto plates’ medium supplemented with 1.0% Gelrite (wt/vol) and incubated under aerobic conditions for 14 days at 75°C. After incubation, single colonies were picked and inoculated onto plates by scribing to obtain purified strains. Then the purified strains were incubated and prepared for plaque assay. The cell culture was incubated for 1 h at 75°C to allow the adsorption of the virus to the host cells. Immediately the 600 μl cultures mixed with 4.4 ml modified Brock’s medium and Gelrite with final concentration 1.0%, and then poured onto the prepared bottom layer with modified Brock’s medium and 1.0% Gelrite. The plates were incubated for 3–5 days at 75°C to allow plaques to form. Zones of inhibition were cut out from the Gelrite plates and inoculated into exponentially growing cultures of the corresponding isolates. After incubation, the culture supernatant was examined by TEM. Meanwhile, pure viral strains were obtained by three rounds of single-plaque purification.16S rRNA gene was amplified using archaeal-specific primers 0023aF (5′-CTCCGGTTGATCCTGCC-3′) and 1406R (5’-GACGGGCGGTGTGTGC-3′) . Sanger Purified viral particles were collected, and viral DNA was extracted with the MiniBEST Viral RNA/DNA Extraction Kit . Viral DNA was sequenced by using the Illumina HiSeq-PE150 system. An Illumina shotgun library using the Illumina TruSeq Nano DNA Sample Prep Kit was reconstructed and sequenced in paired ends 150 bp × 2 using the Illumina HiSeq platform. Raw sequencing data were generated by Illumina base-calling software CASAVA v1.8.2Open reading frames (ORFs) were predicted on the assembled genomes using a combination of Prodigal , GeneiouMultilocus sequence analysis was performed for all viral genomes. The phylogenetic analysis of viral whole genomic and proteome sequences was carried out by the VICTOR online resource, using the distance formula D6 .g for 5 min at 4°C. The cellular and viral lipids were hydrolyzed with 5 ml hydrochloric acid (HCl): methanol (MeOH) =1:9 (vol/vol) in 40 ml capped glass vials at 70°C for 8 h. After acid hydrolysis, 10 ml of dichloromethane (DCM) and 10 ml of Nanopure water were added to the mixture, and lipids were fully extracted twice by vortexing with another 5 ml of DCM in a 50-mL Teflon tube. After centrifugation for 5 min at 2, 800 × g to separate the aqueous and organic phases, the bottom organic phase layer was transferred to a new collection glass vial. Total extracts of 20 ml organic phases were combined and filtered through a 0.45-μm PTFE filter and dried by N2 gas flow.For lipid analyses, virions were first precipitated and collected by the addition of NaCl to 1 M and PEG 6000 to 10% (wt/vol), and then purified by CsCl density gradient  ultracentrifugation . The opalescent virus-containing band was collected, pelleted, and resuspended in the SM buffer . The virvia two BEH HILIC columns  in tandem at 30°C. The flow rate was 0.2 ml/min and the total run time was 120 min and 10 μl of each sample was injected. The elution gradient was set with solvent A of n-hexane and solvent B of n-hexane: iso-propanol = 9:1 (vol: vol). Compounds were eluted with 18% B in the first 25 min, and then linearly increased to 35% B in the next 25 min, and subsequently 100% B in another 30 min. Finally, the gradient returned to 18% B in 10 min and re-equilibrated for 30 min. APCI settings with parameters as shown below: Nebulizer pressure 60 psi, vaporizer temperature 400°C, drying gas (nitrogen) flow 6 l/min at 200°C, capillary voltage 3 kV, and corona 5 μA. Archaeal ether lipids were detected using a single ion monitoring (SIM) mode .Lipid analysis was performed on an Agilent 1,290 Infinity II high-performance liquid chromatography (HPLC) system coupled with Agilent 6,495 quadrupole mass spectrometer under positive ionization mode with an atmospheric pressure chemical ionization (APCI) source. Compounds were separated Sulfolobaceae were the one of most abundant, within which isolates of Sulfolobus, Saccharolobus, and Metallosphaera account for about 73.7–94.0% of microbial community  were collected from supernatants and analyzed by transmission electron microscopy (TEM). Eight different VLPs were detected in the nine enrichment cultures  belong to family lobaceae , isolateosphaera . The 16SSulfolobus sp. 1,201–2 and Sulfolobus sp. 1,201–4 are found, both of which displayed 99% identity to the corresponding 16S rRNA gene sequences of Sulfolobus tengchongensis RT8-4. One virus, temporarily named as STSV3 (Sulfolobus tengchongensis spindle-shaped virus 3), has a spindle-shaped morphology (143 ± 20 × 85 ± 10 nm) with a tail of variable length (98 ± 10 nm) at one end   . Like ot virions  that are virions . Another virions , was foup. D4-HJ . Negativp. D4-HJ . The fivp. D4-HJ .Sulfolobales including three conserved lipothrixviral proteins, two virion structural proteins, and a glycosyltransferase  to sequences in the non-redundant protein database and six of these ORFs have homologs in MTIVs   and two incomplete virus genomes (STSV3 and STSV4). The genomes of new filamentous virus (SIFV3) and spherical viruses (MTIV3 and STIV3) show low similarity (<1%) on the nucleotide sequence level to the sequences in the public databases. The genes of those viruses show little similarity to genes of known function. However, analysis of the protein sequences encoded in the viral genomes showed that some putative proteins were homologous. The genome of SIFV3 is 30,153 bp in length and contains 56 ORFs, of which 18 ORFs have homologs in rod-shaped or filamentous viruses of the order nsferase , suggestl virus) . The STIl virus) . Althougl virus) . The incSulfolobus and together with the members of the family Lipothrixviridae. MTIV3 and STIV3 fall into the same clade and related with MTIVs  and STIVs  which are the known spherical dsDNA viruses  are dominant. By contrast, the envelope of SIFV3 is strongly enriched in diether lipid archaeol. The lipid archaeol is less than 1% in the host membrane but reaches over 70% of lipids in the viral envelope. Thus, like other filamentous viruses AFV1, SFV1, and SIFV which selectively acquired archaeol or GDGT-0 from the pool of host lipids  was used to analyze lipids extracted from mature virions and host cells. The lipid composition of the filamentous SIFV3 was found to be substantially different from that of the host membrane . The Sact lipids , the lipt lipids . Recent t lipids . While, t lipids .Sulfolobaceae were the one of most abundant. These dominated organisms thus could be the major hosts for the archaeal viruses in Tengchong hot springs. We also explored the virus diversity by establishing the enrichment culture and isolating crenarchaeal viruses that infect the members belonging to the family Sulfolobaceae. Finally, eight different particles were found in Sulfolobus enrichment cultures grown at 75°C and pH 3.0 for 10 days, and five viruses were isolated and identified. Two of the viral particle (STSV3 and STSV4) morphologies are similar to viruses infecting Sulfolobus species previously. Three viral particles  morphologies have not been previously reported from Tengchong hot springs. The results showed there are many crenarchaeal viruses with diverse morphologies in the Tengchong hot spring, which probably play key roles to drive microbial evolution and shape population structure. The five isolated viruses further expand the reservoir of already existed crenarchaeal viruses and provide new resources to study archaeal virus-host interaction and co-evolution.We surveyed the microbial diversity in hot and acidic springs of Tengchong by high-throughput sequencing of the PCR-amplified 16S rRNA genes and found the members of family Sulfolobus growth. Thus, it is reasonable to assume that a large number of potential viral hosts belonging to other families exist in Tengchong hot springs. We envision that viral metagenome (virome) analysis will be powerful to explore the viral diversity in acidic hot springs of Tengchong; and the analysis of CRISPR  spacer match between virus and host will gain some new insights into virus-host interactions.Despite some of virus-like particles isolated from Tengchong have nearly identical morphologies to MTIVs, SBFV3, and SPV1 isolated from Yellowstone National Park of United States and Beppu of Japan, the analysis of their genome sequences indicates that they are genetically distinct. The use of enrichment cultures was proved to be an effective way to isolate archaeal viruses in this study; however, this cultivation-dependent approach greatly limits the growth of other potential viral hosts as the culturing condition were selected in a bias way to support de novo  can be found in the article/XF, CZ, and ZZ conceived and designed the experiments. XF performed most of the experiments. YL, WY, and XL helped with the virus isolation. XF wrote the draft with substantial input from CZ and ZZ. CZ and ZZ supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 32170041 and No. 92051112), the Science, Technology, and Innovation Commission of Shenzhen Municipality (No. 20200925154325002), and the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (No. K19313901).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.