Despite the evidence for calibrated cardiac monitored devices to determine fluid responsiveness, there is minimal evidence that the use of cardiac output monitor devices leads to an overall change in IV fluid use. We sought to investigate the feasibility of performing a randomised controlled study using calibrated cardiac output monitoring devices in shocked ICU patients and whether the use of these devices led to a difference in total volume of IV fluid administered.We performed a single-centre non-blinded randomised controlled study which included patients who met the clinical criteria for shock on admission to ICU. Patients were divided into two groups (cardiac output monitors or standard) by block randomisation. Patients allocated to the cardiac output monitor all received EV1000 with Volume View sets. Daily intravenous fluid administration and cumulative fluid balance was recorded for 3 days. The primary outcome assessed was the difference in daily intravenous fluid administration and cumulative fluid balance at 72 h between the two groups. We also assessed how often the clinicians used the cardiac monitor to guide fluid therapy and the different reasoning for initiating further intravenous fluids.p = 0.96). There was no significant difference between the groups for daily intravenous fluid administration on days 1, 2 or 3. In the cardiac monitored group, only 43% of the time was the EV1000 output incorporated into the decision to give further intravenous fluids.Eighty patients were randomised and 37 received calibrated cardiac output monitors. We found no adverse outcomes in the use of calibrated cardiac output monitoring devices and that was feasible to perform a randomised controlled trial. There was no significant difference between the standard care group vs the cardiac monitoring group for cumulative fluid balance  contains supplementary material, which is available to authorized users. A pillar of critical care medicine that has come under examination recently is the use of intravenous (IV) fluids for volume resuscitation in critically ill patients. Over the past 18 years, multiple studies have illustrated the association between positive fluid balance and morbidity and mortality. One proThe challenge of determining the optimum fluid volume to be administered to critically ill patients is well known. Usually such patients receive liberal administration of fluids following presentation to hospital yet accurately evaluating ongoing fluid requirements can be difficult in the face of ongoing hypotension and oliguria. Due to the complex haemodynamic changes in the state of shock, judging fluid balance by conventional means is highly challenging , 9.As illustrated by the FENICE trial in 2015, despite evidence to the contrary, most clinicians will still base their decision to give fluids on the presence of hypotension or static parameters, including single measurement central venous pressures (CVP). The useSeveral dynamic modalities have been used to assess fluid responsiveness in the critically ill patient. The use of devices employing transpulmonary thermodilution to assess fluid responsiveness such as cardiac output/index (CO/CI), stroke volume variation (SVV) and other derived haemodynamic parameters have gained in popularity –16. The Despite the evidence for calibrated cardiac monitored devices to determine fluid responsiveness, there is minimal support that the use of cardiac output monitor devices leads to an overall change in IV fluid use. Furthermore, it is still to be determined if the ability to more accurately predict fluid responsiveness correlates with improved fluid administration.We sought to perform a pilot study to assess (i) feasibility of performing a randomised controlled study in our unit using calibrated cardiac output monitoring devices, (ii) whether the use of these devices led to a difference in volume of IV fluid used and (iii) if there is a difference in clinician rationale for giving fluid boluses when a cardiac output monitor device was present.We conducted a non-blinded, randomised controlled study at the Nepean Hospital Intensive Care Unit (ICU), Sydney, NSW, Australia from December 2016 to January 2018. The study was approved by the Nepean Blue Mountains Local Health District Research Governance Office (Study 16/54–HREC/16/NEPEAN/89). Consent for participation was waived as cardiac output monitoring does not require additional invasive procedures to establish, and its use is part of standard therapy in our unit.Inclusion criteria were adult patients (18 years or older) admitted to the ICU with, or developed, shock defined as ongoing hypotension, despite fluid resuscitation, requiring vasopressors to maintain mean arterial blood pressure (MAP) ≥ 65 mmHg + and a serum lactate ≥ 2 mmol/l.Exclusion criteria were patients in hypovolemic/haemorrhagic shock or atrial fibrillation (AF), pre-existing need for dialysis, pregnancy and patients who were unlikely to remain in the ICU for at least 72 h. Additionally, patients were excluded if advanced haemodynamic monitoring was considered essential by the treating clinician.Those randomised to the cardiac output monitoring group received an EV1000 in combination with Volume View . Volume View allows for continuous and intermittently calibrated measurements of CO, CI and SVV. The calibration is performed by transpulmonary thermodilution, requiring a subclavian or internal jugular central line and a femoral arterial line equipped with a thermistor. While the EV1000 can be setup to utilise both inflatable finger cuff monitoring (Nexfin) and non-calibrated arterial waveform analysis (FloTrac), both methods have been shown to be unreliable in the critically ill (11). Therefore, Volume View was mandatory for all patients randomised to the cardiac output monitor group.Patients identified for inclusion were randomised within the first 24 h of development of shock to either the standard care group or to the cardiac output monitoring group. Block randomisation was performed via an independent statistician with block size of 4 and 1:1 allocation. Patients allocated to the cardiac output monitor all received EV1000 with Volume View sets. For patients allocated to standard monitoring, the treating ICU team could use any method of assessing fluid status/responsiveness commonly utilised in ICU. In the cardiac output monitoring group, despite the presence of an EV1000, the treating team were not mandated to utilise the EV1000 parameters and were free to use non-EV1000 data to guide fluid use. An independent investigator (TS) recorded total IV fluids given and total fluid balance at 24, 48 and 72 h. Clinicians who decided a fluid bolus was necessary during the 72-h study period were asked to record the following: reason, volume and type of fluid given, and also what parameters the clinician used to judge the success of the fluids given . The daily IV fluid use was calculated to ensure any observed difference in fluid balance between the two groups was due to fluids used as a therapy and not a confounder from higher rates of other IV fluids.Sample size estimation using data from our unit by Pittard et al. 2017) with an effect size of 300 ml and standard deviation of 2000 ml, determined that 697 patients would be required for a full study 7 with an).t test was used for normally distributed data and a Mann-Whitney U test for non-parametric data. Probability values are considered two-sided and a p value < 0.05 was considered significant. Statistical analysis was performed on an intention to treat basis.Statistical analysis was performed with SPSS version 24 . Continuous variables are reported as mean ± standard deviation (SD) or median ± interquartile range (IQR) if not normally distributed. Categorical variables are expressed as number of patients and percentage of group. Comparisons for categorical data was made by Pearson’s chi-squared test or Fisher’s exact test if less than five patients were in a specific group. For continuous variables, independent From December 2016 to January 2018, 86 patients were recruited into this study. The flow chart for enrolment is shown in Fig. ).The median age of patients in the study was 64 years old and 49% of the cohort was male. The majority of shock was due to sepsis (73%) and cardiogenic 17%), with the remaining 10% due to other causes. Twenty-three patients died while in the ICU . Median length of stay was 9 days (IQR 1 to 17 days). There was no significant difference between the standard and cardiac monitored group in any baseline parameter. There was also no significant difference in MAP or lactate at 24, 48 or 72 h between the two groups .When comparing fluid balance at time of discharge, death or at 72 h (whichever came first), there was no significant difference between the standard care group vs the cardiac monitoring group  and a trend in serum lactate . In contrast, the cardiac output monitoring group clinicians utilised the EV1000 parameters  and increasing vasopressor requirements  as a guide for further fluid boluses more frequently than the standard monitoring group. The justification of whether a fluid bolus had been successful was similar to the initial reason to give fluids within each group. Change in blood pressure was used for 34% of the standard monitoring group and only 9% of the cardiac output monitoring group (p < 0.001). The response to fluid bolus in the cardiac output monitoring group was judged by change in EV1000 parameters and change in vasopressor requirements at a significantly higher rate than in the standard monitoring group. However, for 43% of fluid boluses over both groups, there was no recorded assessment of a parameter of interest following fluid bolus administration.The significant differences between the two groups were the standard monitoring group had a significantly higher percentage of fluid boluses given for hypotension  and hypotension (35%). Over one third of cardiac monitoring cases still focused on hypotension as a reason to give fluid when a cardiac monitor was present. A similar picture is seen when looking at how doctors judged the success of their fluid bolus, with hypotension and change in vasopressor requirement the leading reasons between the two groups.This underlines that clinicians continue to target blood pressure as an endpoint for fluid boluses despite the evidence that fluid bolus therapy with an aim to resolve hypotension has limited use . Using iThere are several possible explanations for why there was no difference between the two groups with regard to fluid balance. The most likely explanation is a lack of physician interest and comprehension of dynamic parameters of fluid responsiveness, resulting in non-compliance. This is well illustrated in Table Higher cumulative fluid balances have been reported in other studies. Sadaka 2014) reported a day 1 fluid balance of 6.5 l and Boyd (2011) had average cumulative 96-h fluid balance of 11 l 014 repor. This maFinding no observed difference in total IV fluid use or overall fluid balance when a cardiac output monitoring device was used may be due to the underlying principle of how we define fluid responsiveness being flawed. As shown by multiple studies, while patients who are deemed to be fluid responsive have a significant increase in cardiac output at 1-min post IV fluid challenge, over time this effect will dissipate and after 90 min, there will be no difference between fluid responders and non-responders , 22. ThiStrengths of our study include a large sample size for a pilot study and the randomisation process. In addition, the ratio of septic shock to cardiogenic and other forms of shock in this trial is in agreement with other studies, which have found similar proportions of causes of shock . This shFuture studies should take note of the non-compliance issue regarding fluid responsiveness assessment, and study designs should be designed to minimalize this effect. Ultimately, how we monitor fluid balance on the macro haemodynamic level may not change the amount of fluid given to the shocked patient. Instead, re-education is needed for how fluids are viewed, from a harmless intervention to one that has risk if too much or too little is provided. A paradigm shift into how the shocked patient is managed is required to solve the issue of how best to determine the optimal volume of IV therapy required.In this pilot study, we found that it was feasible to perform a randomised controlled trial using these devices. In addition, there was no trend to suggest that the use of an EV1000 and Volume View device leads to lower IV fluid use in the shocked patient. A follow up trial with a design to optimise the use of a cardiac output monitor is necessary to determine the utility of these devices in the critically ill.Additional file 1:Example of data sheet completed by clinicians to assess reasons for fluid boluses. (PDF 116 kb)Additional file 2:Comparison of fluid balance and clinical outcomes between patients where haemodynamic monitoring was actively used and all other patients where haemodynamic montioring was not actively used. (DOCX 18 kb)


Therefore, perfluorocarbon materials may have great potential for reigniting clinical research on hypoxia-based drugs.Hypoxia-based agents (HBAs), such as anaerobic bacteria and bioreductive prodrugs, require both a permeable and hypoxic intratumoural environment to be fully effective. To solve this problem, herein, we report that perfluorocarbon nanoparticles (PNPs) can be used to create a long-lasting, penetrable and hypoxic tumour microenvironment for ensuring both the delivery and activation of subsequently administered HBAs. In addition to the increased permeability and enhanced hypoxia caused by the PNPs, the PNPs can be retained to further achieve the long-term inhibition of intratumoural O Hypoxia-based agents need permeable and hypoxic intratumour environment to be effective. Here, the authors show that perfluorocarbon nanoparticles promote increased permeability and sustained hypoxia to improve accumulation of hypoxia-based agents, and inhibit intratumour oxygen reperfusion. For example, the bioreductive prodrug tirapazamine (TPZ) can be reductively metabolized to toxic reductants in hypoxic tumours; Salmonella and other facultative anaerobic bacteria can selectively damage hypoxic cancer cells via pathogenicity island 2 and the immune response of the host11. However, these anaerobic therapies ultimately failed due to unsatisfactory efficacy in clinical studies. TPZ, for instance, clinically failed due to restricted penetration into hypoxic tumours13. Since the abnormal vasculature and dense tissue of tumours severely limit intratumoural penetration, those anaerobic drugs are generally restricted to the normoxic tumour periphery rather than allowed to reach the hypoxic core16. As a result, HBAs have little effect on hypoxic or aerobic cells in whole tumours.Hypoxia-based agents (HBAs) exert hypoxia-based cytotoxic effects against solid tumours16; therefore, the penetration of HBAs can be optimized by improving tumour permeability for treating whole tumours. However, increased tumour permeability achieved using antiangiogenic agents17 and other methods are also associated with the reperfusion of O222, which further weakens the effect of HBAs. Conversely, other methods that aim to increase the hypoxia of tumours, such as blocking tumour capillaries, are associated with low drug penetration23. As such, photodynamic therapy (PDT), which both consumes intratumoural oxygen and enhances tumour permeability, has recently been utilized to enhance the efficacy of HBAs; however, the efficacy could not be maintained after the laser was removed24. Therefore, there is still an urgent need for new approaches to enhance the efficacy of HBAs.Both the periphery and hypoxic core of tumours are less permeable29, to simultaneously enhance tumour permeability and intratumoural hypoxia to match the requirements of HBAs. First, tumour permeability can be enhanced by PFTBA due to its platelet inhibition effects33. Subsequently, tumour deoxygenation is achieved by PFTBA absorbing oxygen and by PDT. Third, in contrast to previous PDT agents, retained PFTBA can further maintain the favourable environmental conditions for over 24 h to enable maximal HBA delivery and activation via the oxygen-absorbing and platelet-inhibiting activities of PFTBA TBA Fig. . The enh36. Briefly, PFTBA was ultrasonically emulsified with an IR780 (a near-infrared (NIR) photosensitizer)-loaded human serum albumin (HSA) to form PNPs. IR780 nanoparticles (NPs) and oil nanoparticles (ONPs) were also prepared by replacing PFTBA with water or olive oil as controls. The structure of the PNPs was clearly visualized as a uniform sphere-like structure using transmission electron microscopy  and thus may meet this goal. To verify whether PNP can serve as such an oxygen nanosponge, we added deoxygenated PNPs to water to simulate the process of O2 absorption , but only slight Δ [O2] in the ONP and NP solutions  39 , it is critical for such deoxygenated PNPs to enhance permeability and hypoxia for a long time.44. We have previously reported that inhibiting platelet aggregation can increase tumour permeability33. Considering that one of the side effects of perfluorocarbon-based blood substitutes is the inhibition of platelet activity45, we speculated whether tumour permeability could be increased by PFTBA, which has a strong inhibitory effect on platelets  in vivo. Real-time changes in the intratumoural DO level were recorded after mice were locally injected with deoxygenated PNPs  than the controls , further indicating that deoxygenated PNPs could more efficiently absorb the surrounding O2 to create a more hypoxic microenvironment.In addition, we examined whether ONPs Fig. . The DO 2, resulting in long-lasting tumour hypoxia.Hypoxia-inducible factor-1α (HIF-1α) staining was performed 24 h after injection. Significant green fluorescence was observed in tumours treated with deoxygenated PNPs, whereas cells treated with ONPs still showed negligible fluorescence Fig. . Quantit1O2) under different O2 environments was confirmed, and the cellular 1O2 level was determined   apoptosis detection was first performed to measure the apoptosis-inducing capability of the HBA plus the PNPs in vitro. All cells showed green fluorescence before laser irradiation, indicating that both the PNPs and TPZ were non-toxic , PNP, ONP, TPZ, ONP plus TPZ and PNP plus TPZ group. At 24 h post injection, all tumours were exposed to an 808 nm laser for 5 min at a density of 400 mW cmved Fig. , 24. Theved Fig. . The bodved Fig. , indicatH&E and TUNEL staining was also performed to investigate the level of tumour apoptosis ex vivo. H&E staining showed prominent tumour cell necrosis after treatment with PNPs plus TPZ, whereas moderate damage was achieved by PNPs and TPZ alone Fig. . The TUN7. Some studies have shown that Sa. only distributes in the necrotic areas of hypoxic tumours, which are far from tumour vessels5. In this case, sustained whole-tumour hypoxia is quite important for ensuring that injected bacteria accumulate as much as possible throughout the entire tumour, extensively distributing and proliferating in time and space.After successfully demonstrating the effect of TPZ combined with PNPs, Sa. was chosen as another example for combination with PNP pretreatment. A phase I clinical study of VNP20009 failed because of the lack of therapeutic efficacyTo confirm whether pretreatment with PNPs could enhance the efficacy of the bacteria, we first investigated the intratumoural distribution of VNP20009 by performing ex vivo immunofluorescence staining with glut-1 protein antibody (red) and Salmonella antibody (green). At day 3, sections of PNP-treated tumours showed obviously increased hypoxia levels and VNP20009 distributions compared with the control groups, and VNP20009 was exactly distributed in the hypoxic areas exhibiting greater glut-1 protein expression Fig. . SemiquaNext, we evaluated the intratumoural VNP20009 level by colony-forming assay (CFU). Approximately 12.7 times as much VNP20009 accumulated in tumours treated with PNPs plus VNP20009 as in tumours treated with VNP20009 alone Fig. . In addi3 in size were randomly divided into the following six groups: the saline, PNP, ONP, Sa., ONP plus Sa., and PNP plus Sa. groups. At 24 h post injection, Sa. was intravenously injected if needed; then, all mice were irradiated with an 808 nm laser (400 mW cm−2) for 5 min. PNPs plus Sa. achieved the most effective tumour inhibition  using matrix metalloproteinases and relaxin), the accompanied reperfusion of blood oxygen will ease the hypoxia and limit drug efficacy. Lastly, the intratumoural distribution of hypoxic drugs often takes hours or even days (such as for Sa.); even if the hypoxic environment and drug penetration are simultaneously increased (i.e. by photodynamic O2 to cytotoxic 1O2 conversion), additional strategies are still needed to maintain these environmental conditions for long periods of time.HBAs  alone usually cannot achieve satisfactory antitumour effects2/1O2 and PFTBA, with irradiation, the PNPs can efficiently convert tumour O2 into cytotoxic 1O2, resulting in oxygen consumption, vascular perforation, tissue necrosis and apoptosis for improved HBA penetration.In this study, we construct a perfluorocarbon-based nanotherapeutic that can enhance anaerobic therapies in a two-step manner. First, drug permeation and tumour hypoxia were improved via PDT. Synergism between the co-encapsulated photosensitizer and PFTBA enable PNPs to exert a superior photodynamic effect compared to ordinary photosensitizers. Based on the unique affinities between O33, which plays a crucial role in limiting the penetration of drugs into tumours44. Other assays revealed that intravenously injected Evans blue, TPZ and VNP20009 accumulated more in PNP-treated tumours, even without any laser stimulation.Second, the PNPs maintained the increased intratumoural drug permeability and hypoxia for over 24 h after laser irradiation, which is long enough to allow both the delivery and activation of HBAs. This function is achieved by the PFTBA rather than the photodynamic treatment. The oxygen capacity of PFTBA is 20 times higher than that of water, which enables the PNPs to serve as buffers to restrict tumour oxygen recovery. Pimonidazole and HIF-1α staining showed that PNP-treated tumours were more hypoxic than tumours in the control groups even at 24 h after irradiation. In addition, PFTBA has an inhibitory effect on platelets56. additionally, increased tumour penetration may provide more opportunities for tumour spreading57. However, at the same time, recent studies have reported that the pro-progression effects of pre-hypoxia treatments could be reversed by HBAs59. HBAs may suppress hypoxia-stimulated tumour metastasis and down-regulate the expression of Snail transcription factors, which are responsible for cancer invasion and metastasis60. In addition, increased tumour penetration may enhance the infiltration and response of immune cells61. In conclusion, the advantages and disadvantages of using PNPs to regulate the tumour microenvironment need to be further verified. Additionally, it should be emphasized that particle diameter is a critical aspect of extravascular penetration. Although we achieved therapeutic efficacy against CT26 solid tumours with 160 nm PNPs, it is possible to achieve better results by adjusting the particle size to adapt to tumours of different types and stages16.A possible problem with using PNPs to regulate the tumour environment is the potential pro-progression effects. Enhanced hypoxia may promote tumour aggression and immune suppression and further promote tumour spreading62. Currently, many technical solutions have been reported to solve this problem, and some good progress has been made. For example, using invasive fibre optics for internal irradiation63, using upconversion nanoparticles64 and triplet fusion upconversion65 and so on to convert penetrating NIR light into photodynamic visible light in situ. It is worth expecting that new PDT with additional device will be used for non-superficial diseases soon.Another issue worth discussing is the potential challenge of light source in treating non-superficial diseasesIn summary, we successfully constructed a biocompatible nanotherapeutic that can specifically accumulate in tumours and simultaneously increase the tumour permeability and hypoxia to activate hypoxia-based therapies such as bioreductive and bacterial cancer therapies. Considering that the PNPs were formed by combining clinically approved blood substitutes and photosensitizers, these PNPs have significant potential for clinical translation in combination with HBAs for the treatment of solid tumours.2DCFDA were obtained from Molecular Probes. The O2 Consumption Rate Assay Kit was supplied by Cayman Chemical Company, and the Hypoxia/Oxidative Stress Detection Kit was purchased from Enzo. Cell Counting Kit 8 was supplied by Dojindo Laboratories (Japan). For western blot detection of HIF-1α, anti-HIF-1α antibody was obtained from Abcam (ab51608); anti-β-actin antibody was supplied by Seville (GB13001-1). For ex vivo immunofluorescence staining, anti-Salmonella antibody (FITC) was purchased from Abcam (ab69253), Glut-1 polyclonal antibody was obtained from Proteintech (21829-1-AP) and anti-CD31 antibody was provided by BioLegend (102508). The mouse CT26 colorectal cancer cells and 4T1 breast cancer cells were purchased from China Type Culture Collection obtained from the American Type Culture Collectio. BALB/c mice were purchased from the Yangzhou University Medical Centre ; the mice weighed ~20–25 g.HSA solution was purchased from CSL Behring AG. Olive oil was obtained from Aladdin Industrial Corporation. PFTBA was supplied by Meryer Chemical Technology Co., Ltd. . HIF-1α antibody was obtained from Thermal Fisher (MA5-16009), Pimonidazole  and its antibody , IR780, calcein-AM and propidium iodide (PI) were provided by Sigma-Aldrich Chemical Corporation. Alamar Blue cell viability reagent was supplied by Thermo Fisher. Singlet Oxygen Sensor Green and carbon-Hg, 5 min) was used to remove large aggregates of IR780 post ultrafiltration.NPs were formed according to protocols described below. Briefly, 120 mg of HSA was added to 60 mL of deionized water and stirred for 10 min. Then, IR780, which was dissolved in ethanol, was slowly added to the HSA solution to form NPs. To remove free IR780, the NPs were hyper-filtrated by a 30 kDa Millipore filter. Centrifugation  for 10 min.To prepare ONPs and PNPs, an unfolding/self-assembling method was adoptedDLS  was employed to measure the particles size. The structure of the different samples was characterized by TEM . To measure the amount of IR780, the NPs were dissolved in DMSO and then detected by UV–vis absorption spectra .2 microelectrode (Unisense) was used to explore the hypoxic conditions mediated by PNPs in vitro. Briefly, 1 mL of water was added to a vial and covered with mineral oil for to separate it from air. Then, 0.06 mL of PNPs was mixed into the water, with a final IR780 concentration of 2 µg mL−1  and 4 µg mL−1 (2% v/v PFTBA). DO was monitored using the microelectrode before and after 10 min of laser irradiation .An O2 absorption, the sample  was deoxygenated  and then (step 2) irradiated at the same intensity as above with O2 microelectrode detection. Repeated O2 consumption profiles were measured using the same method, except the samples were alternately exposed to irradiation or kept in the dark every 10 min. The irradiated PNPs were then combined with air-saturated water as an O2 supply before the next exposure.To evaluate O2 absorption in vivo, 50 µL of deoxygenated PNPs  were injected into the tumour. Dissolved O2 was monitored using the microelectrode before and after the injection. To measure laser-triggered O2 alterations, the tumour was subjected to laser irradiation  for 20 s immediately after the intratumoural injection of 50 μL of PNPs . These mice were exposed to the laser three times.To detect the PNP-mediated O−2) was used to increase the tumour hypoxia in vivo. At 24 h after the injection of 200 μL of PNPs , the mice were irradiated  for 5 min. The O2 microelectrode was placed ~1 mm into the tumour to monitor the changes in O2.Then, low-intensity laser irradiation  was intravenously injected into the CT26 tumour-bearing mice; the mice were then irradiated  for 5 min at 24 h post injection. The mice then received an injection of pimonidazole hydrochloride intravenously at a dose of 60 mg kg−1. Tumour sections were sequentially stained for HSA, treated with Triton X-100 and stained with anti-pimonidazole antibody . Finally, the sections were imaged using a digital microscope . To measure the expression of HIF-1α, treatments similar to pimonidazole staining were performed, and the mice were sacrificed 24 h later. The tumours were removed and treated according to the instructions of the HIF-1α Immunocytochemistry Kit (anti-HIF-1α antibody was diluted with 1:200). Deoxygenated PNPs (N2 flow rate = 0.01 L min−1 for 1 min) were intratumourally injected into mice for this evaluation of HIF-1α expression. Images of the HIF-1α expression in tumour sections were captured by microscopy. The hypoxia status, as indicated by the staining intensity of each section, was statistically analysed using the ImageJ software.Ex vivo immunofluorescence staining of the hypoxyprobe (pimonidazole hydrochloride) and HIF-1α were employed to evaluate the hypoxic conditions in the tumours. Briefly, 200 µL of the samples  were injected intravenously into the mice when the CT26 tumour diameter reached 6–8 mm. Then, the tumours were irradiated  for 5 min at 24 h post administration. Evans blue dye (20 mg kg−1 in 0.1 mL of PBS) was also injected intravenously 1 h before laser irradiation; 3 h later, the mice were sacrificed, and the tumours were removed and weighed. Evans blue dye in the tumour was extracted with formamide and quantified using a UV–vis spectrometer . Ex vivo images of tumour sections stained with Evan blue were also obtained after the same treatments. To explore PNP-mediated changes in tumour permeability without laser irradiation, similar treatments were performed without laser irradiation.Evans blue was employed to evaluate changes in tumour penetration mediated by PNPs under laser irradiation. Briefly, 200 µL of the samples (PNPs [1.4 mg kg−2) for 5 min, and then those tumours were washed with ice-cold PBS and ground in 4 mL extract buffer on ice for 30 min. Using a micro-centrifuge, the extract was spun down at a rate of 10,000 × g for 5 min at 4 °C, and the lysate was acquired for further analysis of corresponding proteins. Then, proteins were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The membrane was then incubated with a dilute solution (1:1000) of anti-HIF-1α and anti-actin antibody, which was used to detect the actin proteins as a loading control for whole-cell lysate after blocking with 0.05% Tween-20 and 5% skimmed milk in PBS. All the antibodies were purchased from Wuhan Servicebio Technology Co., Ltd. The results of western blot were performed to visualize using the chemiluminescence technology. Unprocessed scans of the most important blots were supplied in the Source Data file. In addition, corresponding quantifications were measured by the Alpha software.Tumour-bearing mice injected with different samples intravenously were exposed laser , and the animals were cared for and used in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. During the study, these animals had free access to food and water. Before treatment, the hair on the flanks of the mice was removed; then, anaesthesia was induced with an intraperitoneal injection of 1% pentobarbital sodium (100 µL) anaesthesia. To establish the subcutaneous tumour models, 1 × 10During the study, animals were observed every day for any clinically relevant abnormalities. If any mouse was moribund due to treatment-induced toxicity, the appearance of severe ulceration around the ectopic tumour or the loss of 20% of body weight compared with the start of study before scheduled killing, it was euthanized. Mice were treated in accordance with NJU-IACUC-approved animal use guidelines.3) were randomly divided into six groups (n ≥ 6). The treatment scheme was as follows: 1. saline; 2. TPZ; 3. ONPs; 4. PNPs; 5. ONPs plus TPZ; and 6. PNPs plus TPZ. Both PNPs  and TPZ (20 mg kg−1) were injected intravenously into mice on days 1, 3 and 5. Tumours in groups 3, 4, 5 and 6 were irradiated three times  for 5 min at 24 h after the administration of the PNPs. Then, the tumour length (L) and width (W) were recorded every day using a digital calliper, and the volume (V) was calculated according to the following formula: V = L × W2/2. Mice in each group were sacrificed on day 13, and the tumours were isolated and weighed. The therapeutic efficacy of the different treatments was also evaluated by H&E and TUNEL staining on day 8.Mice (20–25 g) bearing CT26 tumours (60–80 mmn = 3) were used in this study. When the tumour size reached 60–100 mm3, VNP20009  or PBS was intravenously injected into the mice. At different time points (1 and 7 days), blood and serum samples were obtained. Haematologic indexes were measured using an autohaematology analyser (Sysmex XE2100), and blood biochemical parameters were evaluated using corresponding kits according to their instructions.CT26 tumour-bearing mice . A total of nine mice bearing CT26 tumours were divided into three groups. The mice received 200 µL of PNPs , ONPs or saline intravenously. VNP20009 (5 × 106 CFU mouse−1) was intravenously injected into the mice 1 h before laser irradiation. All tumours were irradiated  for 5 min at 24 h after the injection of VNP20009. The mice were sacrificed 3 days after irradiation, and the heart, liver, spleen, lungs, kidneys and tumour were removed under sterile conditions. After being weighed, these tissues were homogenized in pre-cooled PBS, diluted and coated onto LB plates. The number of VNP20009 colonies was calculated after 12–16 h of culture. After similar tumour treatments, ex vivo immunofluorescence analysis was also performed for glut-1 and Sa. Anti-glut-1 and anti-Sa. antibodies were diluted with 1:200.The VNP20009 strain was transferred to a fresh LB medium. After the OD value reached 0.8, the VNP20009-containing medium was centrifuged at 3500 × 3 in size were randomly divided into six groups. The treatment scheme was as follows: 1. saline; 2. PNPs; 3. ONPs; 4. Sa.; 5. ONPs plus Sa.; and 6. PNPs plus Sa. The mice were treated and sacrificed on day 10; then, the tumours in each group were weighed.To explore the enhancement of bacterial cancer therapy mediated by the PNPs, male BALB/c mice bearing CT26 tumours 60–80 mmt test for two groups and one-way analysis of variance for multiple groups. P values: *p < 0.05, **p < 0.01, ***p < 0.001 . P values < 0.05 were considered statistically significant.Statistical analysis was performed using two-tailed Student’s Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Correction to: BMC Systems Biology 20149 13:1https://doi.org/10.1186/s12918-018-0674-7Following publication of the original article , it was Correct:https://diacomp.org/; DK076169; sub-award #25034–75 to J.H.), and University of North Dakota Post-Doc Pilot Grant (to K.G.), and the North Dakota EPSCoR through National Science Foundation grant #OIA-1355466 (to B.A.M.). Funding bodies had no role in the design, execution, or analysis of this work.The National Institute of Diabetes and Digestive and Kidney Diseases , the NIDDK Diabetic Complications Consortium Pilot Grant , and University of North Dakota Post-Doc Pilot Grant (to K.G.). Funding bodies had no role in the design, execution, or analysis of this work.The National Institute of Diabetes and Digestive and Kidney Diseases , the NIDDK Diabetic Complications Consortium Pilot Grant (DiaComp,


The molecular mechanisms regulating the endothelial response to high doses of radiation are suggested to be initiated via translocation of endothelial asmase from the inner to the outer leaflet of the plasma membrane. Further, asmase catalyzes the hydrolysis of sphingomyelin to generate the lipid second messenger ceramide that leads to the transmembrane signaling of apoptosis 5]. S[5]. Sasm. S[5]. Spathways . The mecw-dose 1.-3 Gy fraw-dose 1.-3 Gy fraasmase activation causes vascular collapse followed by endothelial apoptosis to be mandatory for tumour growth inhibition. Contrarily pretreating endothelial cells with the ceramide antagonist sphingosine-1-phosphate (S1P), reverses the asmase/ceramide-induced cell death process [Several studies have now confirmed radiation-induced  process . Multipl process .et al. investigated the role of asmase-ceramide mechanotransduction by combining USMB with radiation (2 and 8 Gy) in vivo. Experiments were conducted in asmase wild-type, as well as chemically treated (SIP) and genetically modified endothelial apoptosis-resistant mice (asmase knockout) implanted with fibrosarcoma xenografts. Tumour response to treatment was monitored using quantitative 3D Doppler ultrasound and immunohistochemistry at 0, 3, 24, and 72 hours after treatment. Results from experiments with asmase wild-type mice, evaluated 24 hours after treatments demonstrated a decrease in tumour perfusion of up to 46% by three hours following radiation and USMB. The drop in tumour perfusion persisted for up to 72 hours and hence was directly linked to an increase in tumour cell death by 53% followed combination of radiation and USMB. In contrary, mice treated with S1P and asmase knockout mice revealed no statistically significant decrease in tumour perfusion and no concomitant changes in cell death [In a recent study published in the Journal of the National Cancer Institute, El Kaffas ll death .et al. provide preclinical evidence that the combination of radiation and USMB resulted in significant increases in ceramide generation followed with tumour growth delay in asmase wild-type mice. In contrast, S1P-treated or asmase knockout mice were reported with minimal ceramide staining and growth delay. It has been confirmed previously that the paradigm of a tumour vascular disruption leading to endothelial apoptosis is directly dependent on ceramide generation [et al. also reported the changes in the concentration of plasma asmase and different ceramide metabolites (ceramide synthase and ceramide kinase). Interestingly, the results revealed high levels of plasma asmase and ceramide synthase in asmase wild-type mice but not in groups treated with S1P followed radiation and USMB. In opposition, ceramide kinase, an enzyme that metabolizes ceramide to ceramide-1-phosphate was found to be elevated only in asmase knockout groups which correlates with the restoration of radioresistance in asmase knockout animals [El Kaffas neration . Additio animals . Further animals .et al. study demonstrate for the first time the mechano-acoustic activation of asmase/ceramide pathway followed radiation and USMB using genetic and chemical approaches. The paper also highlights some of the limitations of the study. The authors mention that the study lacks the usage of ideal techniques to distinguish between perfused and nonperfused vessels in histopathology. Incorporating perfusion assays as a gold standard histological assessment will help overcome this problem. Secondly, the study addresses the relatively poor resolution of small blood vessel imaging detected by 3D Doppler high-frequency ultrasound and how implementing contrast-enhanced ultrasound or optical imaging could provide with better resolution of blood vessels. Finally, to translate this study in clinical research requires proper planning to maximize the combined effect of radiation and USMB on tumours with minimum effects on healthy tissues. That can be achieved by the selective focussing of ultrasound to only stimulate microbubbles within a tumour target. Collectively, the findings of El Kaffas et al provide a basis for understanding the importance of asmase/ceramide in radiotherapy and mechanobiology.To summate, the results of El Kaffas


Medication non-adherence is a major contributor to poor outcomes in diabetes. Previous research has shown an association between use of mail order pharmacy delivery and better medication adherence, but little is known about the barriers and facilitators to mail order pharmacy use in diabetes patients. This qualitative study examined factors related to mail order pharmacy use versus traditional “brick and mortar” pharmacies to refill prescriptions.We conducted four 90-min focus groups in 2016 among 28 diabetes patients in the Hawaii and Northern California regions of Kaiser Permanente, a large integrated health care delivery system. We queried participants on their preferred mode for refilling prescriptions and perceived barriers and facilitators of mail order pharmacy use. One researcher independently coded each focus group transcript, with two of these transcripts double-coded by a second researcher to promote reliability. We employed thematic analysis guided by the Capability, Opportunity, Motivation, and Behavior (COM-B) framework using NVivo 11 software.A total of 28 diabetes patients participated. Participants’ average age was 64.1 years; 57% were female; and racial/ethnic backgrounds included Asian/Native Hawaiian/Pacific Islander (36%), Black/African-American (21%) Hispanic/Latino (7%), and non-Hispanic White (36%). Analysis uncovered 26 themes related to the decision to use mail order pharmacy, with each theme representing a barrier or facilitator mapped to the COM-B framework. Most themes (20/26) fell into the COM-B category of ‘Opportunity.’ Opportunity barriers to mail order pharmacy use included unpredictability of medication delivery date, concerns about mail security, and difficulty coordinating refill orders for multiple prescriptions. In contrast, facilitators included greater access and convenience  compared to traditional pharmacies. Motivational facilitators to mail order pharmacy use included receiving a pharmacy benefit plan incentive of a free one-month supply of prescriptions.This study found that while patients with diabetes may benefit from mail order pharmacy use, they perceive numerous barriers to using the service. These findings will inform the design of interventions and quality improvement initiatives to increase mail order pharmacy use, which in turn may improve medication adherence and outcomes in diabetes patients, across health care systems.The online version of this article (10.1186/s12913-019-4250-7) contains supplementary material, which is available to authorized users. Over 30 million Americans have type 2 diabetes , a condiGood medication adherence is associated with improved health outcomes, fewer hospitalizations, and lower mortality in patients with diabetes –6. HowevUse of mail order pharmacy services to deliver medications, or mail order pharmacy use, accounts for at least an estimated 25% of pharmacy sales in the US, and rates are even higher among Americans ages 65 and up . Growth trial.gov registration number NCT02621476).Despite the evidence that mail order pharmacy may be an important strategy for improving diabetes care and outcomes, there is little to no published evidence identifying factors that influence patients’ uptake of this service. This qualitative study sought to identify and describe facilitators and barriers to mail order pharmacy use among diverse diabetes patients across two health systems. The purpose of this study was to inform patient outreach strategies for a pragmatic clinical trial designed to increase mail order pharmacy use among diabetes patients  is a non-profit, integrated, group-model health care delivery system that in 2016 served close to 4 million members in a 13-county area of Northern California. This population is more than 30% non-white, 20% have attained a high school or lower level of education, and almost 50% have a household income <$50 K annually. Kaiser Permanente Hawaii (KPHI) is a non-profit, mixed model health maintenance organization that in 2016 served nearly 230,000 members throughout Hawaii. This population is 70% non-white, representing one of the most ethnically and racially diverse populations in the United States; 16% have attained a high school or lower level of education, and about 45% have a household income <$50 K annually. The patient membership of both health systems is representative of the demographic characteristics of the geographic areas the health systems serve.kp.org) or a toll-free pharmacy refill telephone service. KP patients have telephone access to a pharmacist to answer any medication-related questions one-on-one regardless of whether a refill was dispensed via mail order pharmacy or a local KP pharmacy. There is no minimum days’ supply required for mail order delivery. KP typically dispenses 90–100 day medication supplies for most oral diabetes prescriptions through both mail order and local KP pharmacies. Some patients have a financial incentive to use mail order pharmacy through their health insurance pharmacy benefit structure. This is typically receiving 3 copayments’ worth of medication for the price of 2 copayments  when the medications are refilled by mail.KPNC and KPHI have over 120 local “brick and mortar” pharmacies, located on-site within outpatient clinics and hospital facilities. KPNC and KPHI both maintain KP-run mail-order pharmacy distribution systems in coordination with the local KP pharmacies. Although most new prescriptions are filled in the local KP pharmacy, KP patients can elect to receive their prescription refills sent via mail order pharmacy, ordered either through the online patient portal , were adults ages 18–89, and had filled an oral diabetes medication prescription  receptor agonist, meglitinide, sodium-glucose cotransporter-2 (SGL2) inhibitor, sulfonylurea, thiazolidinedione (TZD)) between 7/1/2014 and 6/30/2015. Insulin users who had filled one of these oral diabetes medication prescriptions were included; insulin-only users were excluded. Since the study was conducted in English, non-English speakers were excluded. To identify influences on the use of both mail order and local pharmacies, we sought the perspectives of both current mail order pharmacy users and non-users. We defined mail order pharmacy users as those who had used the mail order pharmacy at least once during the 12-month period prior to recruitment using KP prescription refill databases; the 12-month window was used to reduce recall bias when patients conveyed their experiences with the service.Patients were recruited via mailed letter and telephone outreach and offered $50 as an incentive for participation. We invited patients to participate in one of four 90-min focus groups in February and March of 2016, in either Oakland, CA (KPNC) or Honolulu, HI (KPHI). We used a 2 (site: KPNC vs. KPHI) × 2 (mail order pharmacy user vs. non-user) design to examine patient perspectives across health systems and across levels of familiarity with mail order pharmacy. We selected focus group methodology to elicit a broad range of unique ideas (rather than promote consensus) and to identify concepts that may inform future interventions promoting uptake of mail order pharmacy services. Participants provided written informed consent.The research team included professionals from health services, public health, epidemiology, medicine, behavioral science, pharmacy, clinical administration, and project management backgrounds. This purposefully interdisciplinary team fostered our ability to identify a range of potential influences on mail order pharmacy use. Focus groups were conducted by three trained facilitators, one in KPNC and two in KPHI. The researchers, including interviewers, were unknown to research participants prior to data collection. To improve neutrality and consistency of responses across groups , we usedWe employed thematic analysis  to inducn = 28). Slightly more than half were female (57%); the average age of participants was 64.1 years. Thirty-six percent of patients were Asian, Native Hawaiian or other Pacific Islander; 21% were Black/African-American; 7% were Hispanic/Latino, and 36% were non-Hispanic White.Table You don’t know when you going to get it. They cannot give you a delivery date. […] I can’t do it, because I’m too scared about that, […] it cost me too much, too expensive to take that chance with insulin [of failing to receive it on time]. (Non-user) KPNC, 6.So there’s a little apprehension on my part, because right now I currently live in a business district [where] the mail gets tampered with. (Non-user) KPHI, 2.And then, if your mailbox is not in a safe place – because there’s been mail theft – you could lose your drugs through mail theft. And if you paid for the drug, I don’t know that they’re going to, you know, replenish it even though it was stolen and even though you paid for it. So those are the drawbacks. (User) KPHI, 6.I have eight prescriptions, and because of the change of the medicines here and there, they’ve gotten all out of sync. So, I’m ordering eight different times in a three-month period, and that means I have to process eight different claims with the insurance company to be reimbursed for them. (User) KPNC, 1.Table What’s good is that, once you’ve put in all your information, you can just sort of hit the, you know, the basic control [to automate the process]. So, like, your credit card, you don’t have to put in numbers each time. (User) KPHI, 2.Kaiser’s website is very well organized and put together, and it’s a breeze, really. (User) KPHI, 8.In contrast, ‘opportunity’ facilitators included greater access and convenience  compared to traditional pharmacies, and finding the mail order pharmacy system easy to use. For example:Which is why I would rather use the mail-order service, because then you don’t have to deal with these overworked clerks. (User) KPHI, 7.Social opportunity factors—specifically, the ability to avoid interactions with pharmacy staff— emerged as a facilitator of mail order pharmacy use:I have gone into the pharmacy, and they’ve, like, just recently, they gave me a prescription…and I look at it when I get it – it was different. Okay? Now, this particular time, I was able to ask about it, and they said, “Oh, they changed manufacturers, and it’s the same medication.” But a couple of years ago, they had actually given me the wrong one. And if that happened with a mail order, and it’s something that I needed, you couldn’t, like, right then fix it. (Non-user) KPNC,5.Others described positive interactions with pharmacy staff, and therefore viewed lack of interaction as a barrier to using mail order pharmacy:So I was kind of concerned about that, if it sat in the mailbox too long. Is it really effective, or is it ineffective? (User) KPHI, 10.Also, I don’t know how good it would be for insulin to be shipped, because I don’t know how long it might sit in my mailbox before I get to it…. because it’s supposed to be kept cool…. And if it’s sitting in this tin mailbox in the beating sun… – (Non-user) KPNC, 5.Within the ‘motivation’ category, barriers to using mail order pharmacy included worry that prescriptions, including diabetes-specific medications such as insulin, would be negatively impacted  if left outside upon delivery:Well, previously, when you had the mail order, I did participate. And, at that time, they offered, if you ordered 90 days, you’d get 30 days free. I liked that benefit. (Non-user) KPHI,2.In contrast, facilitators included confidence in one’s ability to use the mail order pharmacy ordering system and receiving an incentive of a free one-month supply of medication each time a 3-month prescription was ordered:…if they just going to approximate the way you’re going to save money from parking or a bus or whatever, things like that, it does – for me, it’s not enough marketing to convince me….But if it’s real savings, like it’s a lower cost to me, then I might look at it. (Non-user) KPNC, 16.“I think the best reason that nobody has mentioned [within the focus group] yet is that you get the third month free. And it’s a tremendous savings.” (User)KPHI,2.I started mail order for the free month. And when I started, I was taking only two medications. Now I take six, so if you listed how much savings, that, you know, it would be in the hundreds of dollars that getting that extra, you know, month for each medication. You know, because I’m a type 2 diabetic. I’m a heart patient. You know, so I take six medications a day. So the savings for me is tremendous, you know? That’s why I do mail order. And now, online, it’s so easy, you know? It’s so easy that I can’t see another way to do it, you know, because it would cost, you know, like I said, a couple hundred dollars at least. (User) KPHI, 12.Further probing for the above patient confirmed that they had used mail order pharmacy more than 12 months ago but were non-users during the 12 month look back window, and that repeated address and PO box changes had discouraged them from using the service more recently.If you need that certain medication that you have to go down and sign for it, how are you going to get that through the mail? [...] there’s some that they say is like a [controlled] substance, like, pain and stuff like that. To refill that, you have to go down to the pharmacy to get it signed. How would you be able to get that through the mail? The same, you know, for refills. (Non-user) KPHI, 5.I didn’t even know that you [could] call and [order] over the phone…(User) KPHI, 2.Not everybody is computer literacy, you know. So, like, at my house, we have a computer, but I will say, “Hon, can you do this for me? Hon, can you do that for me?” But it’s where – if I go on a computer and I look for prescriptions, I wouldn’t know which one to press. (Non-user) KPHI, 8.Finally, within the ‘capability’ category, barriers included lack of knowledge about how mail order pharmacy works  and lack of technological literacy:waiting times for receiving medication compared to in-person refills, and concerns that insulin delivery via mail would compromise the safety and efficacy of the medication. One survey study of patients between the ages of 65 and 79 found that seniors’ concerns about mail order pharmacy use included the potential for lost or stolen medications, receiving the correct medication, and concerns that outdoor exposure may harm the prescription medication’s efficacy [Mail order pharmacy use is associated with better medication adherence and health care outcomes in diabetes patients, but little is known about patients’ barriers and facilitators to mail order use. Our focus groups and qualitative analysis showed that some diabetes patients may encounter important barriers to leveraging the opportunity to use mail order pharmacy, including difficulties using the ordering system, order accuracy, concerns about mail box security, longer efficacy . Our stuefficacy –39; addrPatients in our study cited numerous facilitators that encouraged opportunities to use the mail order pharmacy, including not having to wait in line in-person or having to travel to the pharmacy to refill their medications. Patients whose benefits included a mail order pharmacy incentive to receive an extra copayment’s worth of medication day’s supply for free when paying for two copayment’s worth (e.g. three for the price of two) and using mail order saw this as a facilitator as well. Since 90–100 day fills of diabetes medication are standard in these settings, most diabetes patients eligible for this incentive could likely benefit. This finding is consistent with that of a prior focus group study focused on understanding drug benefit decisions among adults ages 65 and older, which found that access to mail order pharmacy services was a valued attribute in prescription drug plans . PromotiIt is important to note that patients had a variety of perspectives on mail order pharmacy and themes occasionally contradicted one another. For example, some patients felt the mail order pharmacy system was unreliable and the delivery date unpredictable, whereas others felt the system was reliable and cited delivery notifications as facilitating its use. In another example, the need for in-person interactions with pharmacists and staff was cited as both a barrier and facilitator to mail order pharmacy use. Patients’ experience and understanding of mail order pharmacy services will not be universal, and health care systems should target outreach and communication to those who may be more in need of information. In addition, mail order pharmacy services should continue to offer the opportunity for one-on-one consultations with pharmacists to address medication questions and concerns, and allow patients to fill prescriptions at brick-and-mortar pharmacies in person if that is their personal preference .This study has limitations that should be noted. The focus group format and structure did not allow for understanding differences in results by patient characteristics such as age, gender, or socioeconomic status; prior studies suggest preference for mail order pharmacy vs. community pharmacy may vary by some of these characteristics , 42. HowThis study focused on diabetes patients; patients with other conditions may have different experiences with mail order pharmacy. However, other studies of mail order pharmacy use in patients with different chronic conditions have found similar themes. This study took place in two integrated delivery systems within the United States. Results might not be fully generalizable to other health care delivery systems, and the organizational roles and responsibilities for improving mail order pharmacy services may be different in other settings, particularly those that are less integrated or that don’t operate their own pharmacy services. Finally, we are unable to compare the effectiveness of the mail order pharmacy services examined in this study to those in other settings or countries, as these data are unavailable.Our study found that while patients with diabetes may benefit from mail order pharmacy use, they perceive numerous barriers and facilitators to using the service. These findings derived through this qualitative work, can be used to design interventions and quality improvement initiatives to increase mail order pharmacy use and promote improved medication adherence and outcomes in diabetes patients, across health care systems.Additional file 1:Focus Group Interview Questions. Questions for Mail Order and Non-Mail Order Pharmacy Users (DOC 37 kb)


TKTL1 gene suppression on basic metabolic parameters and on survival following oxygen restriction and ionizing radiation. TKTL1 was induced by hypoxia and by hypoxia-inducible factor-1α (HIF-1α). Knockdown of TKTL1 via shRNA increased the cells’ demand for glucose, decreased flux through the PPP and promoted cell death under hypoxic conditions. Following irradiation, suppression of TKTL1 expression resulted in elevated levels of reactive oxygen species (ROS) and reduced clonogenic survival. In summary, our results indicate a role of TKTL1 in the adaptation of tumor cells to oxygen deprivation and in the acquisition of radioresistance. Further studies are necessary to examine whether strategies that antagonize TKTL1 function will be able to restore the sensitivity of glioma cells towards irradiation and antiangiogenic therapies in the more complex in vivo environment.In several tumor entities, transketolase-like protein 1 (TKTL1) has been suggested to promote the nonoxidative part of the pentose phosphate pathway (PPP) and thereby to contribute to a malignant phenotype. However, its role in glioma biology has only been sparsely documented. In the present in vitro study using LNT-229 glioma cells, we analyzed the impact of  Such inconsistent findings might be due to either different methodological approaches or to context-specific regulation of transcription or translation in different subpopulations and environmental conditions. In particular, oxygen availability in tumors is known to fluctuate temporally and spatially [TKTL1 gene silencing with special regard to hypoxic conditions.Due to their high proliferation rate, malignant cells exhibit increased energy turnover; this characteristic is the basis for therapeutic strategies such as conventional chemo- and radiotherapy and some imaging techniques such as positron emission tomography. Tumorigenesis and tumor progression are associated with metabolic alterations and this re-programming of metabolic pathways has been found to profoundly impact cellular behavior, tumor macro- and microenvironment. Transketolase-like protein 1 (TKTL1) catalyzes the conversion of sedoheptulose 7-phosphate and hosphate , used fohosphate  and colo hypoxia . We have (TIGAR)  and publ (TIGAR)  and the patially , and hyppatially ,12. In oTKTL1 was upregulated under hypoxic conditions  and flow cytometry, we observed an increase in ROS following TKTL1 gene silencing in hypoxia. No impact of TKTL1 on intracellular ROS was detectable under normoxic conditions  staining . Additionally, we analyzed HCT-116 colon carcinoma cells and again applied two different shRNA sequences targeting TKTL1 B. In conS levels C and celS levels D specifiell line . Hypoxiaiagnosis E.TKTL1 expression was upregulated in hypoxia and depended on the presence of HIF-1α, (3) TKTL1 suppression was accompanied by a downregulation of PPP intermediate sedoheptulose 7-phosphate, by an elevated glucose turnover and higher lactate levels indicating accelerated glycolysis and by an increase in intracellular ROS under hypoxic conditions, and (4) TKTL1 knockdown facilitated hypoxia-induced cell death and lowered clonogenic survival following irradiation. Since the first description of TKTL1 by Coy et al. in 1996 , its rolTKTL1 by hypoxia has been described in T84 and Caco-2 cells, derived from a lung metastasis of human colon carcinoma and from a colon carcinoma, respectively [TKTL1 in nasopharyngeal carcinoma cell lines CNE2 and HONE1, Dong and Wang reported a drop in cell viability and in levels of NADPH and ribose 5-phosphate [TKTL1 suppression and cisplatin, they delineated additive cytotoxic effects. Further, TKTL1 knockdown inhibited human hepatoma HepG2 cell proliferation [TKTL1 inhibition on cell viability apparently depends on cell type-specific characteristics. Administering temozolomide, another DNA-damaging agent, and assessing cell density and clonogenic survival, we did not detect any difference between LNT-229-shTKTL1 and control cells (data not shown). Our findings do not basically contradict those of Dong and Wang as in our system TKTL1 knockdown per se was not associated with a loss of viability, and we noticed neither synergistic nor antagonistic effects of TKTL1 suppression on the activity of temozolomide. In contrast, oxythiamine, an antimetabolite and transketolase inhibitor, exhibited cytotoxicity in our LNT-229 cell line (data not shown). Xu et al. also used human HCT-116 colon carcinoma cell line and a similar shRNA-based technique, and found decreases in cell growth, glucose consumption and lactate production [TKTL1 was upregulated in a case of recurrent glioblastoma in comparison to the primary tumor is compatible with a role of TKTL1 in the process of tumor evolution in vivo, with tumor hypoxia, which is known to increase at recurrence [TKTL1 expression and the presence or absence of such conditions could account for the inconsistent reports on the significance of TKTL1 in tumor biology.An induction of ectively . Applyinhosphate . Combiniferation . The impoduction . As theyoduction . Anaerobcurrence , as the In tumors, hypoxia arises from an imbalance of rapidly proliferating cells and blood supply. Furthermore, some types of treatment, e.g., bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF)-A, induce hypoxia . HypoxiaHIF-1α and its control (Sima), kindly provided by T. Acker [2. For some experiments, serum- and glucose-free medium was supplemented with glucose to concentrations of 2 or 5 mM. After transfection using Attractene (Qiagen), cells stably expressing shRNA constructs targeting TKTL1 and scrambled control sequences, respectively, were selected by puromycin resistance (5 µg/mL). Hypoxia was generated by using Gas Pak pouches for anaerobic culture . For irradiation experiments, cells were exposed to single doses of 2 Gy photons using a linear accelerator  with 6 MeV/100 cm focus-surface distance and a dose rate of 4 Gy/min. 0 Gy-controls were kept in parallel at ambient temperature in the accelerator control room.Unless otherwise specified, reagents were purchased from Sigma-Aldrich  or Qiagen . Antibodies used were anti-glyceraldehyde-3-phosphate dehydrogenase  and anti-TKTL1 , shRNA sequences were TKTL1, TRCN0000055696 (GACAATCTTGTGGCAATCTTT) and TRCN0000415911 (TTCATCCCTAGTTCGGAAATT), and scrambled control 5′-GATCCCCACTACCGTTGT-TATAGGTCTTCAAGAGAGACCTATAACAACG-GTAGTTTTTTGGAAA-3′, siRNA sequences were TKTL1, antisense UAAAUAACCAUAGUUUCUGGU, sense ACCAGAAACUAUGGUUAUUUA , and antT. Acker , and HCTLysis buffer comprised 50 mM Tris-HCl, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 2 μg/mL aprotinin, 10 μg/mL leupeptin, 100 μg/mL phenylmethylsulfonyl fluoride, 50 mM NaF, 200 μM NaVO5 and phosphatase inhibitor cocktails I and II. Following estimation of protein content using a Bradford assay , 20 μg of total protein was separated under reducing conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on nitrocellulose . Membranes were blocked in Tris-buffered saline containing 5% skim milk and 0.1% Tween-20 and incubated with the appropriate primary (dilution 1:1000) and secondary (dilution 1:3000) antibodies. Immune complexes were detected by enhanced chemiluminescence .Total RNA was extracted using TRIzol™ (Invitrogen) and the RNeasy™ system (Qiagen), cDNA was synthesized using 1.5 μg of RNA and SuperScript VILO™ (Invitrogen). RNA extraction from a patient’s primary and recurrent glioblastoma after surgical resection was done with the patient’s written informed consent and with the approval of the institutional review board (UCT Frankfurt) and the ethics committee . For real-time PCR, ABsolute™ Blue qPCR SYBR-Green Fluorescein Mix  and an iQ5 real-time PCR detection system  were employed. Gene expression data were normalized to the internal control succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA) using the ddCT method and the iQ5 software . Primer sequences: SDHA forward 5′-TGGGAACAAGAGGGCATCTG-3′ and reverse 5′-CCACCACTGCATCAAATTCATG-3′, TKTL1 forward 5′-TAACACCATGACGCCTACTGC-3′ and reverse 5′-CATCCTAACAAGCTTTCGCTG-3′.R 24-well plates  and carefully overlaying cells with sterile paraffin oil.Cell-free supernatants were checked for glucose and lactate concentrations in a Hitachi 917 analyzer . Oxygen consumption was determined applying OxoDishCells were seeded, allowed to adhere overnight and then incubated for 8 h in serum-free medium containing 2 mM glucose. Further analysis was performed as described previously ,34,35.2DCFDA (Invitrogen) [Intracellular ROS were determined by flow cytometric analysis of Hitrogen) .Cell density was evaluated by crystal violet staining, resolubilizing the dye in 0.1 M sodium citrate and measuring the absorbance at 560 nm .Cell death was quantified by propidium iodide (PI) staining of adherent and non-adherent cells (1 µg/mL) and flow cytometry . Cytotoxicity was assayed by measuring the amount of released lactate dehydrogenase (LDH) with the Cytotoxicity Detection Kit . Both techniques, PI staining and LDH release, were based on the loss of membrane integrity . PI is aClonogenic survival was studied by seeding 500 cells in six-well plates, observing them for 7 days, staining them with crystal violet und counting colonies of more than 50 cells.p < 0.05 using the two-tailed Student’s t-test.Experiments were repeated at least three times with similar results. Results are depicted as mean + standard deviation (SD) and differences were considered significant if


Electroosmotic flow (EOF) of viscoelastic fluid with Linear Phan-Thien–Tanner (LPTT) constitutive model in a nanochannel connecting two reservoirs is numerically studied. For the first time, the influence of viscoelasticity on the EOF and the ionic conductance in the micro-nanofluidic interconnect system, with consideration of the electrical double layers (EDLs), is investigated. Regardless of the bulk salt concentration, significant enhancement of the flow rate is observed for viscoelastic fluid compared to the Newtonian fluid, due to the shear thinning effect. An increase in the ionic conductance of the nanochannel occurs for the viscoelastic fluid. The enhancement of the ionic conductance is significant under the overlapping EDLs condition.  In recent decades, micro/nanofluidics has received significant interest due to its promising applications in bioengineering and chemical engineering ,2,3,4,5.As is common in chemical and biomedical applications, solutions are often made from large molecules, such as polymer or DNA. These solutions exhibit non-linear rheological behavior that is distinctively different from the Newtonian fluid ,16, suchMost of the theoretical studies on EOF of viscoelastic fluid are in microscale, where the assumptions of small charge density and relatively thin EDL are reasonable. When the characteristic length of the channel is on nanoscale, the EDL thickness becomes comparable to the nanochannel height ,31, the cH, length cL, and width W connecting two reservoirs of height rH and length rL. A binary KCl electrolyte solution of bulk concentration V0 applied between the inlet (Anode) and outlet (Cathode). Assume that the nanochannel height is much smaller than its width and length , respectively; R, and T are the Faraday constant, gas constant, and the absolute temperature, respectively. In the above, cH as length scale, RT/F as electric potential scale, the bulk concentration Select the channel height At the symmetric axis, zero normal gradient is applied for all variables. At the Anode (or Cathode),On the nanochannel wall with a uniform surface charge density On the surfaces of reservoir , a symmetric boundary condition is imposed to account for the large size reservoirs. The initial conditions are set asWi [One of the most challenging problems for the numerical simulation of viscoelastic fluid flow is the high Weissenberg Number Problem (HWNP), i.e., the loss of numerical accuracy and stability at a relatively high Wi ,34,35. Lc, its matrix logarithm exists asc; and R is an orthogonal matrix composed of the eigenvectors of c.Due to the symmetric positive definite (SPD) property of conformation tensor The equation for the conformation tensor can be rewritten in terms of upferman . c can be obtained from Then, the conformation tensor To numerically solve the coupled set of Equations (7)–(8), (10)–(11), and (16) along with the boundary and initial conditions, a new solver is implemented in an open source software for CFD–OpenFOAM. QUICK, Gauss Linear, and MINMOD schemes are used to discretize the convection terms in Equations (8), (11), and (16), respectively. Pressure Implicit with Splitting of Operators (PISO) algorithm is used to solve Equation (8). Finer mesh is distributed near the charged wall and a mesh convergence study is conducted to ensure the accuracy of the following simulations. The developed solver has been shown to accurately simulate the viscoelastic fluid of EOF in a nanoslit . To furtwww.comsol.com) for different bulk salt concentrations, C0 = 0.5, 5, and 50 mM, corresponding to C0 = 0.5 mM, as the thickness of EDL is larger than half of the channel height, i.e., the EDLs are overlapping, the velocity gradually increases from the wall and reaches maximum velocity at the center of the nanochannel. For high bulk salt concentration C0 = 50 mM with thin EDL, the velocity increases to its maximum value within a distance from the charged surface and remains at its maximum value. It is obvious that good agreement between our numerical results and the Comsol simulation is achieved for both cases of thin EDL and overlapping EDLs. Wi on the flow rate and ionic conductance. The viscosity ratio and the extensibility parameter for the LPTT viscoelastic fluid are set to β = 0.1 and ε = 0.25. The EOF of viscoelastic fluid in the same geometry is then solved by the validated solver to investigate the effects of Weissenberg number C0 = 0.5 mM, 5 mM, and 50 mM under different Weissenberg Wi, as shown in 0 = 0.5 mM, while it is the highest at C0 = 5 mM. The former is because for low bulk salt concentration C0 = 0.5 mM, the overall net charge density and thus the electric body force within the nanochannel is low. The highest flow rate at moderate bulk salt concentration C0 = 5 mM is due to the fact that the EDLs are slightly overlapped, so the net ionic concentration within the nanochannel is higher compared to those for both C0 = 0.5 mM and C0 = 50 mM. Under the same bulk salt concentrations, Wi within the investigated range, which is due to the shear thinning effect of viscoelastic fluid of the LPTT model. Besides this, the increase of flow rate is more obvious for Wi on the shear viscosity becomes less apparent with increasing Wi. At Wi =200, the flow rates are 4.95, 7.89, and 9.74 times of that for Newtonian fluid at C0 = 0.5 mM, 5 mM, and 50 mM, respectively. This indicates that the shear thinning effect is more obvious for the case with smaller EDL thickness. First of all, the volume flow rate across the channel is calculated at the middle of the channel . Wi = 200 at C0 = 0.5 mM, 5 mM and 50 mM, respectively. The diffusive component is not shown due to the fact that for all cases, the percentage of diffusive conductance is less than 0.5%, and thus its contribution is negligible. Besides this, to better compare the results of the Newtonian fluid and the viscoelastic fluid, the percentage of viscoelastic is shown with respect to the Newtonian fluid under the same C0. For viscoelastic of Wi = 200, an apparent increase in the convective ionic conductance is observed compared to that of the Newtonian fluid, while an obvious decrease of migrative component is seen for viscoelastic fluid. The increase of the convective ionic conductance is due to shear thinning effect, as it is proportional to the mainstream velocity. The decrease of migrative ionic conductance stems from the change in the distributions of the electric potential and the ionic concentration under the presence of viscoelasticity. The increase of the convective component exceeds the decrease of the migrative component, resulting in an overall increase of the ionic conductance. Besides this, at low bulk salt concentration, the ratio of the convective component to the migrative component is relatively large, thus the increase of convective component for viscoelastic fluid contributes significantly to the increase of the total ionic conductance. As the bulk salt concentration increases, the contribution of the convective component becomes smaller, thus the increase of the total ionic conductance for viscoelastic fluid becomes less significant. In Newtonian fluid, the migrative component dominates for all three salt concentrations, while in viscoelastic fluid the convective component dominates when the EDLs are overlapped. The variation of ionic conductance with Wi = 200 for the case of overlapped EDLs . Within the confined nanochannel, the value of otential . Numerical simulation on the EOF of viscoelastic fluid with an LPTT model in a nanochannel connecting two reservoirs is carried out with a new finite volume solver implemented in OpenFOAM. The implemented solver is validated by comparing the result of the current simulation with that from commercial finite element software Comsol for Newtonian fluid with the same geometry. For the first time, the condition of highly overlapped EDLs is taken into consideration for the EOF of viscoelastic fluid. Besides this, the surface charge density is much larger than the typical value used for the theoretical study in microscale, where the linearization is used to solve for the electric potential. Obvious increase in the volume flow rate is obtained for the viscoelastic fluid compared to Newtonian fluid due to the shear thinning effect of LPTT fluid. The enhancement is more significant under high bulk salt concentration where EDL is not overlapped. An enhancement in ionic conductance also occurs for viscoelastic fluid, and the enhancement becomes less significant as the bulk salt concentration increases. The increase of the ionic conductance arises from the increase of its convective component, which is directly proportional to the enhanced EOF velocity. In contrast to Newtonian fluid, where migrative ionic conductance always dominates over the convective and diffusive components, the convective current becomes dominant when the EDLs are overlapped for viscoelastic fluid. As the bulk salt concentration increases, the contribution of the convective ionic conductance decreases and the enhancement in the ionic conductance for the viscoelastic fluid becomes less significant.


EPID images during VMAT treatments.The aim of this study was to develop a model that optimizes the fiducial marker locations in the prostate to increase detectability of the markers in the projected MATLAB . Three unique weighting factors were added to penalize the detectability score. The detectability scores of four different patterns containing 40 combinations of simulated fiducial marker locations were evaluated with 101 previously treated prostate treatment plans . The results were analyzed for each pattern group and each marker separation distance on the transverse plane.The fiducial marker tracking capability for each arc was evaluated through a proposed formula. The output of the formula, a detectability score, was calculated with the in‐house developed software written in The maximum detectability of the markers occurred when they were placed between 10 and 15 mm from the center of the prostate in the transverse plane and 6–13 mm in the superior–inferior direction. The detectability decreased when the markers were placed beyond 20 mm in both directions.MV imaging.The fiducial marker‐based detectability score can be used to predict the real‐time tracking capability. Suggestions for optimal insertion locations were given to improve prostate motion management using  However, there are two major fundamental challenges with MV‐based fiducial marker tracking techniques for VMAT deliveries: (a) frequently blocked radiation fields by the multileaf collimator (MLC) and (b) potential large time intervals between two detections.Thus, in this study, we aim to develop a model based on several simulated fiducial marker locations in the prostate to identify the optimal marker locations for MV EPID‐based tracking during VMAT treatments. In addition, we propose a detectability score to predict the detectability for MV‐based fiducial marker tracking techniques and recommend locations within the prostate to implant the fiducial markers to increase detectability of the prostate during treatments.2The first part of the study focused on developing a model based on several simulated fiducial marker locations. The origin of imaginary fiducial marker locations was positioned in the center of patient prostate, which was at the center of the MLC field. DICOM RT plan files from previously treated patients were used to generate the detectability score as described in the following sections.2.A3, was modeled as a 2 × 2 × 6 mm3 cuboid volume due to the scatter on the CT images. Typically, in our institute, four fiducial markers are inserted into the patient via an ultrasound‐guided needle and a grid template , a tool for pinpointing the prostate location in transverse plane to accurately deposit the marker into the designated spot. Throughout this manuscript, X‐Y defines the plane parallel to the template plane (transverse plane), from left to right (X) and posterior to anterior (Y) directions, accordingly. The inferior–superior direction is indicated by letter Z. In the X‐Y plane, the resolutions of the fiducial marker center locations were generated according to the resolution of grid template, which is 5 mm, while interval along the Z direction was determined by the size of the fiducial markers and the average‐sized prostate.The radiopaque fiducial marker used in our institution, having a size of 0.8 × 0.8 × 5 mm2.BTotal of 101 previously treated prostate VMAT cases, containing 202 arcs were used in this study. All plans were created using Varian Eclipse treatment planning system . The plans were chosen such that the target was limited to the prostate with corresponding PTV margin only.2.CThe projected fiducial marker locations that overlap at a particular EPID projection will reduce the detectability of the prostate at that EPID projection angle, therefore, the overlap ratio was introduced to take into account the reduction of prostate detectability due to overlap of projected fiducial marker locations on the EPID images. The overlapping ratio was defined as the ratio of number of control points having overlap of projected fiducial markers on EPID images to the total control points in an arc.2.D(Auij) is calculated for each fiducial marker i at control point j. The detectability score was calculated as the percentage of the summed unblocked area over the total area (A) for all fiducial markers at control points j.A particular fiducial marker is considered to be detectable if the area comprising a 3 mm radius around the corresponding fiducial marker center was unblocked by the MLC or the jaws. The unblocked area wd) was introduced to penalize the control points that had less than three unblocked fiducial markers detected . The wd weighting factor was set to 1 if three or more markers were visible on a projected EPID image for a particular control point. The factor is set to 2/3 for only two marker detections, 1/3 for only one marker detection, and zero if no markers were detected.The detectability of the prostate location on an EPID image is greatly affected by the number of fiducial markers detected on that image. A detection‐number weighting factor (wo) was incorporated into the detectability score to take into account the overlapped markers on the projected EPID images. This factor is different from the aforementioned overlapping ratio in two aspects: (a) it was applied only for the unblocked markers while the overlapping ratio was calculated for the open field, and (b) for each control point, the total number of overlapped markers are counted and treated differently. The wo weighting factor was set to 1 if there is no overlap between all four fiducial markers, wo was set to 0.5 when two markers overlap, and 1/3 for all other cases.In addition, an overlap weighting factor (wts). wts was given for the entire arc. Each segment of consecutive control points that had no detection was first counted. The threshold of 15 control points was given to sift out the segments that were not deemed as large time interval. One minus the half of summed control points of all remaining segments over the total number of control points was the final wts value.The fiducial markers that were blocked by the MLC or jaws for consecutive control points were additionally penalized in the detectability score with a weighting factor (D) was calculated as:i is the fiducial marker number, j is control point number, n corresponds to the total number of control points for that arc, wd is the weighting factor of detectable number of markers, wo is the overlapping weighting factor, wts is the time span weighing factor, Auij is the unblocked area for ith fiducial marker at control point j, and A is the total area of the four fiducial markers.Thus, the detectability score  value was calculated for each arc of all the prostate plans. The calculation method was based on the equation described by McNiven.3As shown in Fig. The overall detectability scores are shown in Fig. Figure Figure 4As seen from Fig. Figures Meanwhile, placing the fiducial markers closer to the CAX means that the changes limited to the very peripheral region of the prostate would not be detected, especially the rotational changes of the prostate. It is a balance between detecting most of the markers in the projected EPID images to determine the position of the prostate during the entire treatment and accurately determining the rotation of the prostate during treatment. The optimum placement of the markers should be toward the higher limit of the proposed range to maximize the detectability of the markers and to accurately monitor rotational changes in the prostate.In certain clinical environment, one marker is considered sufficient enough to pinpoint the target location at a time. Then, the detection‐number weighting factor would be assigned to 1 for cases that at least one valid detection is observed and 0 for no detection. To take these situations into account, a recalculation of the detectability score with the new detection weighting factor was presented here in Fig. The detectability score would tend to favor the ones scattered more on the X‐Y plane. This is reasonable. Majorly, the MLC openings are around the central region. The MLC will irradiate occasionally around the peripheral area, which, with no detection number penalty, gives a boost to detectability score in the original scores. Compared to the original results, the more peripheral the markers get, the higher the chance will be blocked. Therefore, with the original detect number weighting factor, the patterns with more scattered markers got lower values. Whether to choose the original format and simple detection‐number weighting format is purely depended on how it is going to be implemented in the clinical environment. However, the final results remain the same.The relationship between MCS and detectability indicated that lower complexity of the treatment field would improve the overall performance of the marker tracking technique via MV imager. Future work can be done to maintain the same treatment plan quality while decreasing the complexity of the MLC movements.D designates the interval between each transverse plane 6–13 mm is recommended for ΔD. The color from dark blue to white (transparent) indicated the degree of preference from highly recommended to not‐recommended marker placement. Dark blue indicates the recommended fiducial marker location bands/spots. The rate gradually decreased as the position moves away from the central dark blue region.Figures 5Tracking the movement of the prostate is crucial during treatment to ensure the intended dose is given to the target while minimizing the dose to surrounding healthy organs. From our results, the optimal locations to place the fiducial markers are determined to be 10–15 mm from the center of the prostate in the transverse plane and 6–13 mm in the superior–inferior direction. These fiducial marker locations will enable the best detectability of the prostate to provide better real‐time tracking with the MV imaging technique.The authors have no other relevant conflicts of interest to disclose.


In addition, the influence of these anticoagulants on vascularisation was examined using the chorioallantoic membrane assay (CAM) model and compared to the outcome of treatment with Bevacizumab. Using this model the influence of pharmacological concentrations of the anticoagulant on the growth, invasion and vascularisation of tumours derived from WM-266-4 and AsPC-1 cells was also measured in vivo. Tinzaparin and Daltepain reduced tumour formation and invasion by the cell lines in vitro, but with dissimilar potencies. In addition, treatment of CAM with LMWH reduced the local vascular density beyond that achievable with Bevacizumab, particularly suppressing the formation of larger-diameter blood vessels. In contrast, treatment with DOAC was largely ineffective. Treatment of CAM-implanted tumours with LMWH also reduced tumour vascularisation, while treatment of tumours with Apixaban reduced tumour growth in vivo. In conclusion, LMWH and DOAC appear to have anti-cancer properties that are exerted through different mechanisms.The bidirectional association between coagulation and cancer has been established. However, anticoagulant therapies have been reported to have beneficial outcomes by influencing the vascularisation of the tumours. In this study the influence of a set of anticoagulants on tumour formation, invasion and vascularisation was examined. WM-266-4 melanoma and AsPC-1 pancreatic cancer cell lines were treated with LMWH , and DOAC (Apixaban and Rivaroxaban) and the rate of tumour formation, growth and invasion were measured  Favourable biological effects of LMWH in the treatment of cancer patients appear to extend beyond the treatment of thrombosis7 although these have not definitively translated into clinical gains9. These beneficial influences include the inhibition of chemotaxis11, tumour growth12, metastasis14 and the inhibition of angiogenesis19, and are not mutually exclusive. In contrast, other studies do not point to an anti-proliferative property for LMWH21. Furthermore, although similarities exist between the different preparation of LMWH22, there are differences in physicochemical properties and biological actions of these agents which may explain some of the variability of results in clinical studies24. LMWH is an anti-coagulant which acts by inhibiting factor Xa (fXa) in an antithrombin-dependant manner. However, preparations of LMWH differ in size and charge22 which in turn differentially bind a large variety of proteins within the blood stream25 resulting in variable responses by the cells. One such outcome of LMWH includes the release of tissue factor pathway inhibitor (TFPI) from endothelial cells26. The release of TFPI in turn inhibits the components of the extrinsic pathway of coagulation26 as well as constraining angiogenesis by blocking the phosphorylation of VEGF-receptor 227. Furthermore, non-anticoagulant heparins such as S-NACH have been shown to possess anti-oncogenic properties28 which further suggests that the beneficial effects of LMWH may be distinct from the anticoagulant function25.Thrombotic complications are among the leading causes of morbidity and mortality in cancer patients2. The anticoagulant function of agents such as Apixaban and Rivaroxaban is derived from the inhibition of coagulation factor Xa, and is therefore similar to that of a number of LMWH. However, these agents do not require the function of antithrombin III, which also possesses independent anti-angiogenic properties29.The recent development of clinically relevant direct anti-fXa inhibitors provides an opportunity to apportion elements of anti-cancer influence of LMWH to particular mechanisms with greater accuracy. Recently, the use of direct oral anticoagulants (DOAC) have become an option in the treatment of cancer-associated venous thromboembolism31. Recently, a number of studies have used this procedure to examine and compare the influence of preparations of LMWH as anti-angiogenic agents36. In this study, we have used the CAM model in chick embryo to explore the ability of selected anticoagulants to influence tumour vascularisation, using tumours prepared from MW-266-4 melanoma and AsPC-1 pancreatic cancer cell lines. The CAM were supplemented with the LMWH; Tinzaparin and Dalteparin, and also with direct oral anti-coagulants; Apixaban and Rivaroxaban, and were compared to the effects of the VEGF-receptor blocker; Bevacizumab, in vivo. In addition, the direct influence of these anticoagulants on tumour formation, growth and invasion was examined by preparing spheroid tumours which were then implanted into the invasion matrix in vitro.The chorioallantoic membrane (CAM) based model is an established model to explore the anti-angiogenic potential of biological agents and to examine the properties of tumoursTinzaparin solution  and Dalteparin solution  were diluted in sterile phosphate buffered saline (PBS) as required. Apixaban and Rivaroxaban were obtained as pure compounds from, Bistrol-Myers Squibb  and Bayer  respectively. These were then dissolved gradually in dimethyl sulfoxide  and then diluted to 4 mg/ml in PBS. Controls were also prepared by diluting DMSO. Bevacizumab solution  was diluted in sterile PBS as required.38.WM-266-4 melanoma cell line and AsPC-1 pancreatic cancer cell lines  were cultured in RPMI-1640 supplemented with 10% (v/v) foetal calf serum (FCS). Cell numbers were determined by crystal violet staining as previously described and calculated from a standard curve4) in non-adherent Nunclon Sphera 96 wells plates  in 50 µl of media. The cells were maintained at 37 °C, 5% CO2 for 4 days to permit the formation of tight tumour spheroids. Invasion matrices were prepared by adding 20 µl of Cultrex Spheroid Invasion matrix  in 12-well ibidi µ-chambers  and supplemented with the appropriate media (100 µl) containing a range of concentrations of Tinzaparin or Dalteparin (0–5 IU/ml), Apixaban (0–1 µg/ml) or Rivaroxaban (0–0.6 µg/ml). The tumours were gently transferred to individual invasion assay wells and allowed to set for 1 h at 37 °C. The spheroid tumours were examined daily for up to 72 h by white-light microscopy and photographed using multiple exposures. The multiple images from each sample were then collated and stitched into a composite figure. The observable area of the spheroid and the invasion of the cells into the matrix were analysed using ImageJ software.Tumour spheroids were prepared by seeding out WM-266-4 or AsPC-1 cells (2 × 104) in non-adherent Nunclon Sphera 96 wells plates in media (100 µl) containing a range of concentrations of Tinzaparin or Dalteparin (0–5 IU/ml), Apixaban (0–1 µg/ml) or Rivaroxaban (0–0.6 µg/ml). The cells were maintained at 37 °C, 5% CO2 for 48 h to permit the formation of the spheroid tumours. The spheroid tumours were examined daily for up to 72 h by white-light microscopy and photographed. The relative tumour intactness was then assessed from the size of the formed tumours as well as the number of detectable tumour particles. However, this assay was not usable as a means of measuring the tumour cell proliferation or tumour spreading.Tumour spheroids were prepared by seeding out WM-266-4 or AsPC-1 cells  were soaked with a range of concentrations of the test reagents or with controls. Test reagents, Tinzaparin (0–5 IU/ml), Dalteparin (0–5 IU/ml) and the VEGF-receptor blocker Bevacizumab (0–12.5 µg/ml), were tested alongside the PBS control. FXa inhibitors Apixaban (0–1 µg/ml) and Rivaroxaban (0–0.6 µg/ml) were examined along with a DMSO-treated control. The pads were then placed on the top of the CAM  were prepared and examined as follows. Fertilized white Leghorn chicken eggs were provided by Henry Stewart & Co. Eggs  and were stored at 12–18 °C for up to 5 days before use. The eggs were placed in an incubator at 70% relative humidity and 37 °C. On the fifth day, a window of approximately 4 cm6) within Matrigel matrix  containing Tinzaparin (5 IU/ml), and Apixaban (0–1 µg/ml) as well as the respective vehicle controls. The Matrigel containing the tumours were permitted to set and then placed on top of the CAM and the orientation of the recorded. The size of the tumour was recorded on each day by white-light microscopy as above. The CAM tests were inspected daily for up to 3 days after which the tumours and surrounding tissue were excised and fixed in 4% (v/v) paraformaldehyde. In order to determine tumour vascularisation, the tumours and the underlying membranes were separated. The underlying membranes were washed three times with PBST (PBS containing 0.5% (v/v) Tween 20), and developed using a FITC-conjugated anti-PECAM-1 antibody  diluted 1:400 (v/v) in PBST. The samples were then washed a further three times with shaking (10 min each) and images were acquired using a Ziess Axio Vert.A1 inverted fluorescence microscope with . The extent of vascularisation was determined by ImageJ program as above.Tumour were prepared by resuspending WM-266-4 or AsPC-1 cells  and the calculated means values shown. The number of experiments was as stated in each figure legend ± the calculated standard error of the mean. The significance values were calculated by paired t-test, or the one-way ANOVA  procedure with Tukey’s honesty significance test.Spheroid tumours were prepared by culturing WM-266-4 or AsPC-1 cells in non-adherent plates which were then transferred to the invasion matrix containing the test reagents. The growth and invasion of the tumours was then determined by measuring the size of the main spheroid and the reach of the invading cells. The rate of invasion through the matrix was dissimilar between the two cell lines with WM-266-4 demonstrating a faster rate. Consequently, the data presented depicts the days of measurement most appropriate for each cell line. Incubation of WM-266-4 spheroids with Tinzaparin (5 IU/ml) or Dalteparin (5 IU/ml) significantly reduced tumour invasiveness within the matrix (40% and 30% reduction respectively) by the third day  were placed on the CAM for 3 days and recorded daily Fig. . Images in vivo. WM-266-4 and AsPC-1 tumours were implanted on the CAM and the tumours were permitted to grow and vascularise over 3 days. The tumours were then excised and the underlying membrane was probed using an FITC-conjugated rabbit polyclonal anti-PECAM-1 antibody. The membranes were examined by fluorescence  but the mechanism was not investigated further, in this study.Finally, the examination of either the CAM-implanted tumours, or the tumour growth The anticoagulant function of LMWH arises from its ability to provide a surface for the approximation of the coagulation factor Xa (fXa) and antithrombin III. However, LMWH is also capable of interfering with other mechanisms due to the ability to interact with a large range of proteins within the bloodstream and on the surface of the cells. Among these are a number of receptors involved in cell adhesion, cell migration and signals that control vascularisation and angiogenesis. In conclusion, this study has highlighted the dissimilar outcomes of treatment of cancer cells with therapeutic concentrations of Tinzaparin, Dalteparin, Apixaban and Rivaroxaban on tumour growth, invasion and vascularisation. We have established some of the non-anticoagulant functions of LMWH and elucidated the mode action of these therapeutic agents.Supplementary information