In addition, AZD5363 is shown to inhibit the proportion of proliferating tumour vascular endothelial cells in vivo, which may contribute to improved tumour control with adjuvant treatment. These new insights provide promise to improve outcomes with the addition of AZD5363 as an adjuvant therapy following radiotherapy.Radiotherapy is an important anti‐cancer treatment, but tumour recurrence remains a significant clinical problem. In an effort to improve outcomes further, targeted anti‐cancer drugs are being tested in combination with radiotherapy. Here, we have studied the effects of Akt inhibition with AZD5363. AZD5363 administered as an adjuvant after radiotherapy to FaDu and PE/CA PJ34 tumours leads to long‐term tumour control, which appears to be secondary to effects on the irradiated tumour microenvironment. AZD5363 reduces the downstream effectors VEGF and HIF‐1α, but has no effect on tumour vascularity or oxygenation, or on tumour control, when administered prior to radiotherapy. In contrast, AZD5363 given after radiotherapy is associated with marked reductions in tumour vascular density, a decrease in the influx of CD11b This has included establishing the optimal scheduling of AZD5363 and RT in order to maximise the efficacy of the combination.A number of potent inhibitors of Akt have recently entered clinical development. One such compound, AZD5363, is an orally bioavailable, adenosine triphosphate (ATP) competitive inhibitor of all three isoforms of Akt. AZD5363 is an attractive candidate as an adjuvant to RT as the drug has successfully completed phase I trial investigation with an acceptable safety and tolerability profile  and the subsequent impact of any alteration on tumour vascularity and oxygenation. In addition, we have considered the potential effect of AZD5363 on tumour vasculogenesis after RT and on vascular endothelial cell radiosensitivity and proliferation.These data collectively provide a strong case for the continued development of AZD5363 as a potential adjuvant to improve outcomes after RT. The study also provides potentially important proof of principle scheduling data to inform early phase clinical trial design.et al, in vitro. AZD5363 is a potent inhibitor of Akt and led to reductions in the levels of phosphorylated Akt substrate glycogen synthase kinase 3 beta (pGSK3β) and the phosphorylated downstream pathway S6 ribosomal protein (pS6) detectable by Western blot in all cell lines , in addition to a single 6 Gy dose of RT , when compared to the vehicle treatment group (median time 30 days vs. 17 days (P = 0.07) and 24 days vs. 17 days (P = 0.225), respectively; Fig P = 0.542 and P = 0.871, respectively; Fig P = 0.005; Fig P = 0.002; Fig P = 0.024; Fig P = 0.038). Administration of AZD5363 prior to RT did not significantly alter tumour control compared to RT alone , nor was an effect on tumour control demonstrated . Long‐term tumour control with no measurable tumour mass was achieved in 50% (4/8) of adjuvant‐treated mice. In the group of mice receiving the combination of continuous AZD5363 and RT, tumours began to regrow after a short delay following RT. However, these regrowing tumours showed signs of potential ulceration and these mice were culled for welfare reasons. In no other treatment group (including vehicle and RT control groups) was this effect observed.In order to confirm the validity of this observation, this experiment was undertaken in another head and neck human cancer xenograft model. The PE/CA PJ34 model was selected as AZD5363 had been shown to have direct cytotoxicity to this cell line, in contrast to FaDu cells, which we had already demonstrated are insensitive to AZD5363 monotherapy. Subcutaneous PE/CA PJ34 tumours were established in nude mice, and the mice were then randomised into neo‐adjuvant, continuous and adjuvant groups Fig A along wP = 0.032; The effect of AZD5363 on the Akt pathway in FaDu xenografts was evaluated. The levels of pAkt, pGSK3β and pS6 in lysates made from tumours harvested 2 h after oral drug dosing were measured by Western blot . Levels We hypothesised that the efficacy of AZD5363 in improving tumour control when given RT may result from effects on tumour vasculature. We initially carried out a limited exploratory study using FaDu tumours implanted into dorsal window chambers. Bright‐field imaging was used every 2–3 days, and 14 days after implant, a functional vascular supply was seen to have been established in these tumours. The tumours were then given a single dose of 6 Gy RT, after which mice received twice‐daily AZD5363 or vehicle. Imaging was continued every 2–3 days to allow measurement of total vessel length. Results suggested a marked reduction in total vessel length 7 days after RT in mice treated with adjuvant AZD5363 Fig A and B. 3, mice were treated with RT alone, or RT plus adjuvant or neo‐adjuvant AZD5363 as in previous experiments  and assessed histologically by staining for CD31 and pimonidazole. Combination treatments were assessed against the RT alone control. Analysis of CD31 showed the vessel density of the adjuvant‐treated tumours to be significantly reduced compared to the RT control  in fixed tumour tissue. The percentage of HIF‐1α+ viable tumour cells was significantly reduced in the tumours from mice treated with AZD5363, relative to vehicle‐treated mice  imaging to provide an assessment of tumour hypoxia and perfusion. There was no significant difference in MTV between the treatment groups. There was no difference in initial FAZA distribution between groups, as might occur if significant differences in perfusion existed. In addition, there was no difference in tumour hypoxia as measured by tumour FAZA uptake . Using BRDU uptake, we found a greater than 50% reduction in proliferation in HUVEC cells when treated with doses of between 2 and 3 μM AZD5363 for 96 h Fig A. Thus, et al, + cells of myeloid origin. We therefore investigated whether AZD5363 might reduce this CD11b+ population influx. Assessment of the number of CD11b+ cells present within the viable tumour portion of the sections  has previously been reported to be important in the recovery of tumour vasculature following RT, through a process termed vasculogenesis  and is schedule dependent. Importantly, AZD5363 delivered as an adjuvant after RT led to markedly improved tumour control. Half of the mice that received this combination schedule had no measurable tumour mass on day 100 of treatment. In contrast, the administration of AZD5363 before RT failed to improve tumour control and continuous drug dosing (both before and after RT), led to only modest improvements in tumour control. AZD5363 has no effect on the intrinsic radiosensitivity of tumour cells, as demonstrated in a wide range of human cancer cells In vitro experiments revealed that AZD5363 does not radiosensitise any of 15 different cell lines derived from tumour types commonly treated with RT in clinic. This was irrespective of the sensitivity of these cells to AZD5363 monotherapy, or the schedule of the drug relative to RT. Inhibitors of the Akt pathway have been shown to increase the radiosensitivity of human cancer cell lines  is reported to be required to allow a process termed “vascular normalisation” in which hypoxia is reduced . This is in contrast to some other reports with PI3K/Akt pathway inhibitors , inhibits vascular endothelial cell proliferation and reduces the recruitment of CD11bDetails of the original source and culture conditions of all cell lines used are contained in in vitro assays. For administration in vivo, AZD5363 was solubilised in a 10% DMSO 25% w/v Kleptose HPB (Roquette) buffer, and given by oral gavage.AZD5363 [(S)‐4‐amino‐N‐[1‐(4‐chlorophenyl)‐3‐hydroxypropyl]‐1‐piperidine‐4‐carboxamide] was prepared as a 10 mmol/l stock solution in DMSO (dimethyl sulphoxide). The final concentration of DMSO was < 0.1% in all et al, AZD5363‐treated tumour cells were lysed on ice with a complete cell extraction buffer, made by the addition of cOmplete™ EDTA‐free protease inhibitor cocktail (Sigma‐Aldrich) and PMSF (phenylmethanesulphonyl fluoride) to cell extraction buffer (Invitrogen). Snap‐frozen tumour portions were homogenised using a Fastprep™ homogeniser  in the same complete cell extraction buffer. Western blot analysis then proceeded as already published  and VEGF , all accet al, Tumour cells were treated with 0.1–10 μM AZD5363 for 96 h following which an MTT  assay was performed as previously published (Lunt Y=Bottom+(Top−Bottom)(1+10X−LogIC50)et al, Clonogenic assays were conducted in all tumour cell lines with 1 or 10 μM AZD5363, as previously published (Senra g for 5 min), and the cell pellet was resuspended in 300 μl hypotonic PI solution (50 μg/ml PI (Sigma‐Aldrich), 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X‐100) and left overnight at 4°C. 10,000 events were then analysed using a FACSCalibur cytometer (BD Biosciences), and the data examined using the software Summit version 4.3 (Dako).FaDu cells  were seeded in T‐25 flasks. The following day the flasks were treated with 1 μM AZD5363, and 2 h later, 4 Gy RT was delivered to the cells at a rate of 0.952 Gy/min (Faxitron). After 24 h, the contents of the flasks media were removed, centrifuged .Animal study protocols were approved by the Institutional Ethics Committee and the Home Office (project licenses 40/3212 and 70/7760) and designed in accordance with the Scientific Procedures Act (1986) and the 2010 guidelines for the welfare and use of animals in cancer research . To initiate tumour xenografts, 0.1 ml of cell suspension was implanted subcutaneously on the back of 8–12 weeks Female CD‐1® Nude Mice (Charles River) weighing 22–28 g. Mouse weights were monitored daily, and once palpable tumours were formed, the volumes were measured daily by the formula: tumour width × length × depth.Tumour cells in exponential phase were prepared to the desired concentration  alone, RT alone, vehicle alone and combination treatment groups. Mice receiving neo‐adjuvant treatment were dosed with AZD5363 for 7 days before RT, receiving the last dose of AZD5363 2 h before RT. The mice treated according to a continuous dosing schedule received AZD5363 for 7 days before, and continuously after, RT. The mice in the adjuvant group started treatment with AZD5363 from 2 h after RT and were then dosed twice daily until the end of the experiment. Mice were culled when the tumour volume exceeded 700 mm3, or if this end‐point was not reached, on day 100 after the beginning of treatment. Tumour control was considered to be lost if the tumour volume exceeded 700 mm3, and long‐term tumour control considered achieved if animals had no measurable tumour mass on day 100 after the beginning of treatment. MXR‐320/36 X‐ray apparatus  was used to deliver 6 Gy RT (2 Gy/min) to non‐anaesthetised, tumour‐bearing mice. The animals were restrained in jigs which used lead shielding to allow local RT to be delivered to the tumour, whilst protecting other tissues. Regular calibration was provided by the Medical Physics Department of the Christie Hospital . Where administered, pimonidazole  was given by intraperitoneal (i/p) injection 2 h before culling in all experiments.Mice bearing tumours measuring 100 mmet al, 7 FaDu cells). Mice received 6 Gy RT, delivered to the tumour, before commencing on 50 mg/kg AZD5363 or vehicle BD 2 h later. Repeat imaging was carried out on days 2, 4 and 7 after RT. Repeat imaging on vehicle/RT‐treated tumours was not possible beyond day 2 due to rapid outgrowth of the tumour window. Images at a 40× magnification were then inverted and the contrast enhanced using Photoshop Express (Adobe) and then analysed using AngioTool software /(injected activity/body weight), ml/g) were calculated for each tumour.After formalin fixation, xenograft tumour samples were paraffin‐embedded and freshly sectioned before staining. Sections were stained for CD31, pimonidazole, HIF‐1α, human Ki‐67 and CD11b , before ® Plus multi‐fluorophore (Cyanine 3 and Fluorescein) detection system kit (PerkinElmer) was used to amplify and visualise the multiple signals, as per manufacturer's instructions. After staining, samples were mounted using ProLong® Gold Antifade Mountant with DAPI  (Life Technologies). Stained sections were analysed using Tissue Studio.Fluorescent staining of formalin‐fixed paraffin‐embedded tumour sections for CD31 and mouse Ki‐67  was carrHUVEC cells  were seeded in a gelatin‐coated 96‐well plate and treated with doses of AZD5363 ranging from 0.1 to 10 μM for either 48 or 96 h. A colorimetric BrdU (bromodeoxyuridine) cell proliferation ELISA kit (Roche) was then used as per manufacturer's instructions. In a second experiment, HUVEC cells were plated then treated with AZD5363 for 2 h before, 2 h before and 96 h after, or for 96 h after a single 6 Gy dose of RT. The BrdU assay was then performed.in vivo, where time to a tumour volume of 700 mm3 reached was evaluated. Group MTVs were plotted graphically until the last day of possible comparison  and analysed on this day using unpaired t‐tests or one‐way ANOVA with Dunnett's test to adjust for multiple comparisons. Tumour volumes were log‐transformed to stabilise variance and remove size dependency; variance was unequal between groups as it increases with tumour volume. The Mann–Whitney test was used to assess the effects of AZD5363 on tumour levels of all markers, unless adjustment for multiplicity was required, in which case the Kruskal–Wallis test was used, followed by Dunn's multiple comparisons test. The effect of AZD5363 on the surviving fraction of cervical cell lines was tested using unpaired t‐tests. In order to analyse the effect of AZD5363 on cell cycle, with and without radiation, the Kruskal‐Wallis test was used, followed by Dunn’s multiple comparisons test. All statistical analyses were performed using GraphPad Prism 6 software .The effect of AZD5363 on PRAS40 phosphorylation, and of differing sequences of AZD5363 on the surviving fraction of FaDu cells was analysed using one‐way ANOVA with Dunnett's test to adjust for multiple comparisons. The Gehan–Breslow–Wilcoxon test was used to analyse the tumour control experiments in vitro studies with HUVEC cells. DMF performed the FAZA PET‐CT imaging and analysis. BRD and DM provided scientific expertise helpful to the design of experiments. All authors read and commented on the manuscript.EJS, KJW, IJS and TMI designed the study and the experiments and wrote the manuscript. EJS and BAT performed the majority of the experiments with the help of DM who carried out T.M. Illidge and I.J. Stratford received research grant support from AstraZeneca. B.R. Davies is an employee and shareholder of AstraZeneca.Radiotherapy is an important cancer treatment, delivered to approximately 50% of cancer patients. However, relapses following potentially curative radiotherapy treatment are common, causing significant mortality and morbidity. In an effort to improve clinical outcomes, there has been a growing interest in combining radiotherapy with drugs that target the molecular signalling pathways thought to be important in the response of tumours to radiotherapy. One such target is Akt, and in this study, we have looked at the effect of combining the potent Akt inhibitor AZD5363 with radiotherapy in mouse models of cancer.in vitro experimentation revealed that AZD5363 has no effect on the intrinsic radiosensitivity of tumour cells. This was irrespective of the dose of AZD5363, the extent of drug‐alone effect on tumour cell viability, or the sequence in which drug and radiation were delivered. Further in vivo investigation found the positive effect of AZD5363 on radiotherapy response is due to an impact on the irradiated tumour microenvironment. Whilst AZD5363 alone does not alter tumour vascularity or oxygenation as measured by histological and FAZA PET‐CT methods, it has dramatic effects on the response of tumour vasculature to radiotherapy. When given after radiotherapy, AZD5363 reduces tumour vascular density, the influx of pro‐vasculogenic myeloid cells and tumour regrowth. AZD5363 also inhibits vascular endothelial cell proliferation in vitro and in vivo, which may contribute to the schedule dependency of the tumour control effect.Our recent study has demonstrated that daily AZD5363 improves long‐term tumour control when commenced 2 h after a single dose of radiotherapy in two tumour models of head and neck cancer (FaDu and PE/CA PJ34). Interestingly, we also demonstrated that administration of AZD5363 up until 2 h before radiotherapy failed to improve tumour control and continuous drug dosing (both before and after radiotherapy) leads to only modest improvement. Whilst both models expressed pAkt, This study has demonstrated the potential of Akt inhibition to significantly improve long‐term tumour control following radiotherapy. The experiments performed in this work have also demonstrated the importance of establishing the correct sequence of radiotherapy and drug when using AZD5363 in patients receiving radiotherapy. AZD5363 is a promising clinical candidate providing the exciting possibility of early translation of this work into clinical trial.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.


In the Funding and Acknowledgements section, the grant number from the funder National Key Research and Development Program of China is listed incorrectly. The correct grant number is: 2017YFD0800102.


Polypropylene and PA66 are widely used in our daily life, but they cannot be welded by laser transmission welding (LTW) because of polar differences and poor compatibility. In this paper, grafting modification technology is used to improve the welding performance between polypropylene and PA66. Firstly, the strong reactive and polar maleic-anhydride (MAH) is grafted to polypropylene and infrared spectrometer is used to prove that MAH has been grafted to polypropylene. At the same time, the mechanical and thermal properties of the graft modified polypropylene (TGMPP) are tested. The results prove that the grafting modification has little influence on them. Also, the optical properties of TGMPP are measured. Then, the high welding strength between TGMPP and PA66 is found and the mechanism of the weldability is researched, which shows that there are two reasons for the high welding strength. By observing the micro morphology of the welding zone, one reason found is that the modification of polypropylene can improve the compatibility between polypropylene and PA66 and make them easy to diffuse mutually, which causes many locking structures formed in the welding region. The other reason is that there are chemical reactions between TGMPP and PA66 proved by the X-ray photoelectron spectrometer. However, there is a characteristic peak at 1775 cm−1 which cannot be found in the infrared spectroscopy of PP. Belonging to the peak of the MAH which is grafted to PP, the characteristic peak proves that the MAH has been grafted to the molecular chain of PP.Mixed with pure KBr powder, the trace purified graft modified polypropylene is ground to solid powder. Then, the solid power is pressed into a transparent sheet, which is put into the optical path of the infrared spectrometer and scanned from 690 cmTo do the tensile mechanical experiments which are used to explore the tensile mechanical properties of TGMPP, PP and TGMPP are made into L50 mm × W20 mm × H1 mm. The tensile speed is 2 mm/min and the working environment is room temperature when the tensile mechanical experiments are carried out. The tensile results are shown in Taking PP and TGMPP 10 mg, respectively, a dynamic heat flux differential scanning calorimeter is used to measure their melting peaks. The results are shown in In general, the grafting modification has a certain influence on the mechanical and thermal properties of PP. However, the original properties of PP change only a little.The optical properties of polymers have great influence on the welding quality, so research of the effect of grafting modification on the optical properties of PP is very important. The optical properties include transmissivity, reflectivity and absorptivity. For all of laser transmission welding, it is hoped that the laser energy can transmit to the interface of the upper materials and the lower materials to improve the utilization efficiency of laser energy and reduce the processing cost.RT) is defined as the ratio of the reflected energy and the total laser energy input.Due to the loss of the laser energy, the laser energy density will decrease. Some of the laser energy loss is due to the reflection of the upper surface and the lower surface of the upper materials and the reflection of the interface of different solid components  in the upper materials. The reflectivity (AT) is defined as the ratio of the absorbed energy and the total laser energy input. Then, the remaining laser energy passes through the upper materials to the interface of the upper materials and the lower materials. The transmissivity (TT) is defined as the ratio of this remaining laser energy and the total laser energy input. The reflectivity, absorptivity and transmissivity of one kind of material meet the following formula:When the laser penetrates the upper materials, some laser energy is absorbed by the impurities and the upper materials. Similar to the definition of reflectivity, the absorptivity .If the polymers are the upper materials, the laser reflection of the upper materials will transmit part of the laser energy back into the air, which results in a waste of energy and is bad for cost savings. Furthermore, if the laser reflectivity of the upper materials is too high, these upper materials cannot be welded with the lower materials by laser. So, the research of the reflectivity of TGMPP is very important. The reflectivity of PP at different wavelengths before and after grafting modification are shown in If the polymers are the upper materials, the high transmissivity of the upper materials will cause more laser energy to transmit to the welding zone while the low transmissivity of the upper materials will cause only a little laser energy to transmit to the welding zone, which is bad for cost savings. Furthermore, if the transmissivity of the upper materials is too low, it will prevent them from being welded with the lower materials under some extreme conditions. If the polymers are the lower materials and the transmissivity of the lower materials is high, most of the laser energy will pass through the lower materials to the air, which is also bad for the laser transmission welding. So, the research of the transmissivity of TGMPP is very important. The transmissivity of PP at different wavelengths before and after grafting modification are shown in If the polymers are the upper materials, the high absorptivity of the upper materials will cause only a little laser energy to transmit to the welding zone and a large amount of laser energy will be absorbed by the upper materials, which causes the high processing cost. If the polymers are the lower materials, the high absorptivity of the lower materials will cause a large amount of laser energy to be absorbed by the lower materials, which is good for the laser transmission welding. However, if the absorptivity of the lower materials is too low, the low absorptivity will lead to the low utilization of laser energy and prevent them from being welded with the upper materials under some extreme conditions. So, the effect of grafting modification on the absorptivity of PP needs to be researched. The absorptivity of PP at different wavelengths before and after grafting modification is shown in To do the next experiments, TGMPP, PP and PA66 are made into the same dimension of L50 mm × W20 mm × H1 mm. Prior to LTW, all samples are cleaned by ultrasonic cleaning machine and then placed in a drying oven for about 12 h. A Compact 130/140 semiconductor continuous laser made by Dilas is used for the experiments. The maximum power of the laser device is 130 W, the output wave-length is 980 ± 10 nm, the minimum beam diameter is 700–800 μm, cooling system is the built-in air cooling system and working temperature is 15–25 °C. The three axis table has a marble pedestal, table stroke is 300 mm × 300 mm × 200mm, repeat positioning accuracy is 0.01 mm and the scope of speed is 0–3000 mm/min.In the next experiments, TGMPP and PP are used as the upper materials while PA66 is used as the lower material. Also, the welding zone of PA66 is coated with clearweld as the absorbing layer. As shown in Passing through PP or TGMPP, the laser energy is absorbed by the clearweld absorbing layer at the interface of TGMPP or PP and PA66. The laser energy directly acts on PA66. Then, the upper materials (PP or TGMPP) are heated and melted by heat conduction. In the process of solidification, the melted PP or TGMPP and PA66 form the weld seam to connect the upper materials (PP or TGMPP) and the lower materials (PA66).The evaluations of the laser transmission welding quality are mainly (1) the welding strength; (2) the weld seam width and (3) the appearance of weld seam. In this paper, the welding strength is used to evaluate the quality of the welding.With tensile speed at 2 mm/min and working environment at room temperature, tensile tests finally break the samples (the welding of TGMPP and PA66) through loading tension at both ends of the samples to measure the limit stress of the samples, which can be used to evaluate the welding strength of the samples. The tensile schematic diagram is shown in F is the maximum pull-off force to make the welding failure (N); W is the width of the weld seam (mm) and L is the length of the weld seam (mm).The formula used  is shownUTM4104 microcomputer control electronic universal testing machine is used to do the tensile tests and a Keyence VHX-1000 ultra-depth electron microscope is used to measure the width of the weld seam.F). Through Keyence VHX-1000 ultra-depth electron microscope, the width of the weld seam can be obtained (W). The length of the weld seam is 20 mm (L), which is equal to the width of the original samples . According to Equation (2), the welding strength can be obtained (σ).Through UTM4104 microcomputer control electronic universal testing machine, the maximum pull-off force to make the welding failure can be obtained  and the lower materials (PA66). Comparing the micro morphology of the welding zone of TGMPP and PA66 with PP and PA66, TGMPP is researched to investigate how it improves the welding performance of PP and PA66.Before the observation of the micro morphology of the welding zone between TGMPP and PA66, welding TGMPP and PA66 by laser and putting the welding into formic acid need to be done first. Then, PA66 is dissolved by the formic acid, which makes the micro morphology of the welding zone easy to be observed.When PP and PA66 are welded by laser, the micro morphology of the weld mark on PP is shown in When TGMPP and PA66 are welded by laser, the micro morphology of the weld seam on TGMPP is shown in In order to investigate the effect of the bubbles caused by high laser power on the welding quality, welding TGMPP and PA66 by laser and putting the welding into formic acid need to be done first. Then, PA66 is dissolved by the formic acid, which makes the micro morphology of the welding zone easy to be observed.et al. studied joining of aluminum alloy to PET using friction lap welding. They also discovered that these bubbles generate high pressure pushing the fused polymers to the pits and micro voids on the surface of metal, which can supply more mechanical bonding and realize tight joining between metal and polymers [et al. also found that evenly distributed bubbles are beneficial to improving the welding strength [When the processing speed is 10 mm/s, the clamping force is 0.12 MPa and the laser power is 20 W, 35 W and 50 W, respectively, the micro morphologies of the weld zone on TGMPP are shown in polymers . Katayamstrength . Also, iTo prove that there are chemical reactions in the welding processes, X-ray photoelectron spectrometer (XPS) is used to detect the elements and the chemical bonds in the welding zone of TGMPP after TGMPP and PA66 are welded by laser. The XPS device used in the experiments is Escalab 250Xi manufactured by Thermo Fisher Scientific in Massachusetts, America, which uses the monochromatic Al target as X-ray source.Before observing the welding zone of TGMPP by XPS, welding TGMPP and PA66 by laser and then putting the welding into formic acid need to be done first. After the formic acid dissolves PA66, TGMPP needs to be washed by formic acid several times and at last by ethanol.After TGMPP is treated by formic acid and ethanol, the full element spectrum of the welding zone of TGMPP is obtained by XPS as shown in After TGMPP is treated by formic acid and ethanol, the high rate spectrum of the welding zone of TGMPP is obtained by XPS as shown in Containing unsaturated double bonds in the molecular structure, MAH is a kind of polar compound with various functional groups, which is easy to react with other polymers under the action of initiator. The strong active anhydride of MAH is easy to react with hydroxyl, carboxyl and amidogen. So, by being grafted to PP, MAH with strong reactive activity can reinforce the affinity and improve the welding performance between PP and PA66.et al. analyzed the fracture behavior at partially miscible polymer interfaces [et al. developed a kinetic model to describe the behavior of semicrystalline polymer interfaces [et al. investigated interfacial entanglements between glassy polymers [c, and the entanglement of the polymer chains occurs only when the molecular weight is more than the critical molecular weight Mc. The chemical reactions can make the molecular weight higher, namely cause the entanglement of the polymer chains to occur easily. Once the entanglement of the polymer chains occurs, the stress of one chain will spread to other molecular chains, which improves the welding strength. Furthermore, the chemical reactions form C–N bonds between the chains of TGMPP and PA66, which are also good for the improvement of the welding strength.Gorga terfaces . Lo et aterfaces . Horiuchpolymers . It can polymers ,20,21. F(1)Through grafting reactions, MAH with strong reactivity and polarity can be grafted to the side chain of PP to improve the welding performance of PP and PA66.(2)In general, the grafting modification has certain influence on the mechanical and thermal properties of PP. However, the original properties of PP change little.(3)The grafting modification has little influence on the optical properties of PP. The grafting modification causes the reflectivity and transmissivity of PP to decrease slightly and makes the absorptivity of PP increase slightly.(4)TGMPP and PA66 can be welded well. The micro morphology of the welding zone displays that because of the grafting modification, the affinity and the compatibility between PP and PA66 are improved and a large number of locking structures as similar to the rivet lock are formed, which is one of the reasons why the welding performance is so high.(5)Proved by the X-ray photoelectron spectroscopy (XPS), PA66 may react with the MAH of the side chain of TGMPP under the heating effect of laser, which is another reason for the high welding performance.(6)The evenly distributed bubbles caused by the thermal degradation are good for the high welding strength. However, when the laser power is too high, the serious thermal degradation causes many large bubbles to be formed in the welding zone, which account for a wide area in the welding zone and cause the poor welding strength.In this paper, using MAH to improve the welding performance of PP and PA66 by being grafted to the side chain of PP and the welding mechanism based on modification of PP for improving the laser transmission weldability to PA66 are researched. The following conclusions can be drawn from the experiments in this paper.


Satellite jitter (SJ) is an important error source that affects the geometric accuracy of high resolution satellite imagery. In this paper, the quantitative relationship between the jitter displacement (image displacement caused by SJ) and relative registration error obtained from parallax images is deduced to be theoretical in detail, and the jitter displacement estimation model is built to estimate the jitter displacement. Then, a simulation experiment is carried out to validate the feasibility of using the built jitter displacement estimation model to estimate the jitter displacement. Finally, experiments with real images in DengFeng (China) including multispectral images of Ziyuan-3 (ZY-3) satellite and high resolution (HR) images of Ziyuan1-02C (ZY1-02C) satellite are used to validate the effectiveness of utilizing the built jitter displacement estimation model to do the jitter estimation. High accuracy ground reference data are further used to evaluate the accuracy of the estimation. Experimental results show that the average estimated error for jitter displacement of ZY-3 is 2.96% and 0.11% in amplitude and frequency respectively, and the estimated error for jitter displacement of ZY1-02C is 8.46% and 0.35% in amplitude and frequency, respectively. With increased spatial resolution of satellite images, the influence of satellite jitter (SJ) on the imaging quality becomes more and more obvious, and seriously limits the application of satellite image products ,2,3,4,5.Much research on utilizing parallax images to estimate SJ has been performed under circumstances lacking high accuracy satellite attitude measuring instruments (star-tracker and gyro). Roques et al. proposed a method to identify micro-vibrations of satellite platforms in pitch, row and yaw—three directions—using stereo images . TeshimaIn this work, a jitter displacement (image displacement caused by SJ) estimation model was built according to the deduced quantitative relationship between the jitter displacement and relative registration error obtained from parallax images. Both simulation experiments and real image experiments were carried out to validate the feasibility and effectiveness of utilizing the built jitter displacement estimation model to do the jitter estimation.The principle of jitter displacement estimation is based on the design of satellite CCD sensors with parallax observation systems. In the parallax observation system, each linear sensor scans the same ground feature at different times and an object will be imaged at the same location in different images but at different times. However, some exterior orientation elements of the camera used in the observation system might have some small changes due to SJ during the observing interval time, and thus the same object might be imaged at slightly different locations in different images ,20. Thusith component, respectively. In order to simplify the derivation, it is assumed that the satellite platform contains only a single frequency jitter as follows:According to theory of Fourier transform, SJ can be converted into the attitude jitter component combined by several sinusoidal functions with different amplitudes, frequencies and phases , as showConsidering the angle of attitude fluctuation caused by SJ is small, the jitter displacement, According to the above-mentioned principle of jitter estimation using parallax images, relative registration error between parallax images with some imaging time interval can be denoted asSJ is time varying, so the relative registration error caused by SJ is also varying with the imaging time interval of parallax images. When Let Let As to Let Therefore, the quantitative relationship between the jitter displacement and relative registration error obtained from parallax images is determined and the jitter displacement estimation model is also established. After the relative registration error is obtained, components of jitter displacement including amplitude If SJ contains multiple frequencies, each component of the jitter displacement can be estimated from the corresponding component of the relative registration error .The jitter displacement estimation and validation are carried out based on dense matching between parallax images. Both parallax images and corresponding high accuracy ground reference data  or Digital Elevation Model (DEM)) are used to do the jitter displacement estimation and validation. The workflow is shown in (1) Jitter displacement estimationJitter displacement estimation is carried out based on the relative registration error between parallax images, where the relative registration error is computed by dense points matching. In matching, the correlation matching is used to get the initial values of corresponding points and the least squared matching is further used to obtain more precise positions of corresponding points. Then certain thresholds of coordinate differences for corresponding points are selected to filter out the mismatching points. In this paper, three times of root mean square error are chosen. After filtering mismatching points, coordinate differences of remaining corresponding points are fitted using sinusoidal function by the least squared method as the relative registration error curve. Finally, the estimated values of jitter displacement are estimated using the parameters of the fitted relative registration error curve including amplitude, frequency and phase, according to Equation (14).(2) Jitter displacement validationJitter displacement validation is carried out based on the registration error between parallax image  and reference DOM data, i.e., absolute registration error. Because of the high accuracy of the reference DOM data, it can be considered as a true reflection of the ground scene, i.e., ground truth. The registration error between parallax image and reference DOM data is thus the absolute registration error. It is also an exact measurement of the jitter displacement since there should be no registration error between parallax image and reference DOM data if there is no satellite jitter. Therefore, the absolute registration error can reflect the jitter displacement in some imaging time interval of the satellite and is used as a validation of jitter displacement in this paper.The absolute registration error between parallax image and reference DOM data is also computed by dense points matching. Parallax image should be rectified first using rational function coefficients (RPCs) and high accuracy DEM data. Then a dense points matching is conducted between the rectified parallax image and reference DOM data. Similarly, mismatching points in obtained corresponding points are also filtered out. Coordinate differences of remaining corresponding points are also fitted using sinusoidal function by the least squared method. Parameters of the fitted curve including frequency, amplitude and phase are considered as the reference values of jitter displacement and are used to evaluate the accuracy of the estimated values of jitter displacement purely based on parallax images.Both simulation experiments and real image experiments were carried out to validate the feasibility and effectiveness of utilizing the built jitter displacement estimation model to do the jitter estimation. Simulation experiments were carried out to analyze the change rule of jitter displacement at different imaging time intervals and to verify the built jitter displacement model. Real image experiments were conducted according to the workflow in t was a range from 0 to 2.5 s. Assuming (1)Whether the frequency of (2)Whether the relationship between amplitudes of (3)Whether the relationship between phases of Simulation experiments were used to verify whether the relationship between jitter displacement curves and registration error curves satisfied the built model given by Equation (14) if there was a jitter displacement with a single sin function. Simulation experiments were carried out with different The simulated Jitter displacements caused by SJ are in both cross-track and along-track directions. Because jitter displacements in along-track direction are covered up by errors caused by the terrain factor, jitter displacements in cross-track direction are usually more striking than those in the along-track direction . TherefoThe presented multispectral image of ZY-3 is a level 1A product which is generated by radiometric correction and sensor correction. As shown in The multispectral camera of ZY-3 satellite has four bands including blue (B1), green (B2), red (B3), and near-infrared (B4), and the corresponding CCD arrays are parallel to each other . The intThe detected and fitted relative registration error curves of different band combinations are shown in Reference data (DOM/DEM with high accuracy) are further utilized to obtain the absolute registration error curve as the reference values of jitter displacement in order to validate the accuracy of estimated values of jitter displacement. The detected and fitted results of absolute registration error curves obtained based on high accuracy ground reference data are shown in The presented HR image of ZY1-02C is a level 0 product including rational function coefficients. As shown in The process is similar to the estimation and validation using multispectral images. The detected and fitted relative registration error curve between CCD1 and CCD2 are shown in In this paper, the feasibility of jitter displacement estimation only using parallax images was analyzed in theory. Then the jitter displacement estimation model was built based on the quantitative relationship between the jitter displacement and the relative registration error obtained from parallax images. Finally, simulation experiments analyzed the change rule of jitter displacement estimated by registration error at different imaging time intervals and verified the feasibility of using the built jitter displacement model to estimate the jitter displacement. Real image experiments further evaluated the performance of the jitter displacement estimation based on the built model. In real image experiments, two typical images including multispectral images of ZY-3 satellite and HR images of ZY1-02C satellite were chosen as experimental data and high accuracy ground reference data were also used to evaluate the accuracy of the estimation. Experimental results indicated that the built jitter displacement estimation model can estimate the jitter displacement accurately and may be used to do the jitter detection for other sensors with parallax images.(1)The deduced quantitative relationship between the jitter displacement and relative registration error obtained from parallax images in the presented paper is more accurate than our previous work. In our previous works, the relationship has only been approximated. However, the present work deduced an accurate quantitative relationship between the jitter displacement and relative registration error obtained from parallax images without any approximation.(2)The deduced quantitative relationship between the jitter displacement and relative registration error obtained from parallax images in the presented paper is more robust than our previous work. The deduced quantitative relationship in our previous work is not an accurate expression, as its accuracy decreased when the imaging time interval increased. Obviously, such a relationship is not suitable to parallax images with large imaging time intervals such as those from the High Resolution camera of ZY1-02C. In the presented paper, the deduced quantitative relationship is accurate without any approximation. It is suitable to parallax images with any imaging time interval. Two typical images, the multispectral images of ZY-3 satellite  and the HR images of ZY1-02C satellite , were chosen as experimental data and validated the deduced relationship.(3)High accuracy ground reference data were used to evaluate the accuracy of the jitter displacement estimation in real image experiments in the presented paper. It is an objective method. In our previous work, the accuracy of the jitter displacement estimation in real image experiments wasn’t evaluated. Other references also didn’t evaluate the accuracy of the jitter displacement detection. Generally, they evaluated the images after jitter correction .This paper summarizes the work performed to improve upon our previous works ,19, and Future work must also determine whether the integration stage has a certain impact on the accuracy of the jitter displacement estimation. Whether the proposed jitter estimation approach is appropriate to estimate these high frequency satellite jitters also needs to be confirmed experimentally. More accurate fitting methods may also improve the performance of the jitter displacement estimation. These will be addressed in our future work.


Angiogenesis, a process of generation of new blood vessels from the pre-existing vasculature, has been demonstrated to be a basic prerequisite for sustainable growth and proliferation of tumour. Anti-angiogenic treatments show normalization of tumour vasculature and microenvironment at least transiently in both preclinical and clinical settings.In this study, we proposed a multi-scale mathematical model to simulate the dynamic changes of tumour microvasculature and microenvironment in response to anti-angiogenic drug endostatin (ES). We incorporated tumour growth, angiogenesis and vessel remodelling at tissue level, by coupling tumour cell phenotypes and endothelial cell behaviour in response to local chemical and haemodynamical microenvironment.Computational simulation results showed the tumour morphology and growth curves in general tumour progression and following different anti-angiogenic drug strategies. Furthermore, different anti-angiogenic drug strategies were designed to test the influence of ES on tumour growth and morphology. The largest reduction of tumour size was found when ES is injected at simulation time 100, which was concomitant with the emergence of angiogenesis phase.The proposed model not only can predict detailed information of chemicals distribution and vessel remodelling, but also has the potential to specific anti-angiogenic drugs by modifying certain functional modules. Angiogenesis is a physiological process of generation of new blood vessels required for normal body functioning, such as wound healing, endometrial growth during the menstrual cycle, tissue grafting, inflammation, and hypoxia. However, clinically and pathologically it has been demonstrated as a basic prerequisite for sustainable growth, proliferation and metastatic spread of solid tumours . CascadeGiven the prominent role of angiogenesis in cancer, many cancer therapies aim to block angiogenesis and thereby inhibit tumour growth. These approaches undertaken towards that goal include inhibition of growth factors required for ECs proliferation and survival, such as VEGF ; inhibitWhile the anti-angiogenic therapy have been approved for cancer treatment, it appears that the clinical application of anti-angiogenic therapy is more complex than originally thought. Since the anti-angiogenic drugs target tumour vasculatures without tumour cells, resistance to anti-angiogenic therapy caused by the pathological microenvironment in tumour is a prominent issue which may explains the variable results observed in the experiment and clinic using this approach . Unlike Mathematical modelling and numerical simulation offer powerful tools with which to study complex biological processes, such as tumour growth and angiogenesis. Billy et al.  developeBased on the continuous–discrete hybrid model proposed by Chaplain’s group for modelling angiogenesis , we starMulti-scale 3D tumour modelling is utilized to detail the mechanisms of tumour growth and angiogenesis. We have organized our model into three levels: tissue, intratumoral and cellular layers. The overall structure of this model is summarized in Fig. prol) and cell survival (θsurv) are introduced to describe the effects of oxygen field on the tumour cell actions. Each phenotype of tumour cell has a different coefficient of oxygen consumption rate and the production rate of VEGF and MDEs (Table The probabilistic hybrid model for tumour cell growth is based on the previous work . We assuEs Table  24].Tab.Tabprol)age/TTC. Tage is the tumour cell age, ranging from 1 to TTC and with an incremental 1 in each simulation time step. TTC is the tumour cell proliferation time . One of the two daughter cells will replace the parent cell and the other cell will move to a neighbouring element.If there is enough oxygen .When the local oxygen concentration at a tumour site is less than the cell survival threshold θsurv but lower than θprol and a space is available, a proliferating cell has a probability (50%) to become a migrating cell (MC), and will migrate to a neighbouring space which has the highest oxygen concentration in the neighbouring elements. It was also assumed that the migrating cells adjacent to the pre-existing vessel wall have higher probability of moving in the longitudinal direction  than the radial direction. The migration speeds of the two directions are the same. After a migrating cell completes its movement, the space it originally occupied will be released for other cells.When local oxygen level is higher than θe, A 3D hybrid discrete–continuum angiogenesis model was adopted to investigate the EC migration and proliferation through random motility, chemotaxis in response to VEGF distributions and haptotaxis in response to the local ECM density . EndotheVEGF will increase its radius R with the rate of 0.4 µm h−1 which will stop when the vessel radius reaches the maximum value of Rmax = 10 µm [p is increasing in a dilation vessel, and satisfiesp referred to the vessel permeability value in the normal tissue; p according to the experiments of vessel permeability value in a tumour microvessel.In our model, we consider vessel dilation as the first sign of a pre-existing vessel becoming an immature vessel. A vessel segment inside the tumour has a VEGF concentration larger than a threshold θ = 10 µm . At the p. In the simulation, vessel wall compliance is defined by the radius changing under the influence of intravascular and interstitial pressures and collapse pressure based on the empirical equation of Netti et al. [0 is the origin radius of the capillary; Pc is the collapse pressure; b is the compliance exponent; E is the compliance coefficient. The intravascular and interstitial pressures are calculated by fully coupled haemodynamic simulation, which will be detailed in “For a pre-existing vessel, once vessel dilation occurs, the vessel segment is treated as an immature vessel with increased Li et al. .3\documep will increase which causes higher Pi, and consequently vessel will be compressed. A compressed vessel, on the other hand will induce a higher flow resistance, lower flow which will then decrease the wall shear stress (WSS) level for the vessel. Vessel collapse will occur by either a significant reduced R or WSS criteria [Based on the above equations, when the vessel segment becomes immature, Lcriteria .f, Cm, Cv are the ECM, MDE and VEGF concentration, separately. The TCi,j and ECi,j terms represent a tumour cell and an endothelial cell located at a node position . Their values are either 1 if a cell is present or 0 if it is not. Dm is the MDE diffusion coefficient. Dv is VEGF diffusion coefficient. δ, μT, μE, λm, χ, ξ, ɛ, λv are positive constants.The tumour cell and endothelial cell behaviours are coupled by the changes of the chemicals in the extra-cellular matrix (ECM), such as oxygen, VEGF and matrix degradation enzymes (MDEs) . The traTo obtain a more realistic oxygen concentration field, the advection and diffusion of oxygen in the vessel network are introduced . The comThe haemodynamic model in this study is based on our previous work on the coupled modelling of intravascular blood flow with interstitial fluid flow , 22. Briv is the vascular flow rate without fluid leakage; Qt is the transvascular flow rate. v and Pi are the intravascular pressure and the interstitial pressure, respectively. Lp is the hydraulic permeability of the vessel wall. σT is the average osmotic reflection coefficient for plasma proteins; πv and πi are the colloid osmotic pressure of plasma and interstitial fluid, respectively.The main equations for blood flow calculation are as follows:v and interstitial flow Ui satisfiesThe velocity of intravascular U0 is the initial EC density. ɛmax is the maximum inhibiting effect of ES on ECs. CES50 is the ES concentration that induces 50% of the maximum inhibiting effect. CES is the concentration of ES, which satisfiesEndostatin (ES), identified in mice by O’Reilly et al.  in 1997,ES is the diffusion coefficient of ES. RES is the ES elimination rate in the plasma. UI,ex is the ES injection rate. Vp is the volume of the plasma. λES is the positive coefficient for ES decay of itself.DThe 3D model is defined on a 100 × 100 × 100 grid to cover a 1 mm × 1 mm × 1 mm volume, so the grid length corresponds approximately to the size of a tumour cell, i.e. 10 μm. Two separate lattices are constructed for tumour cells (TCs) and endothelial cells (ECs), with the constraint that each grid can contain only one single cell at each time point. We assume that there are two main parallel arterioles located at plane y = 100, which branch to form capillaries as initial pre-existing vasculature of the model Fig. .Fig. 2ThDirichlet boundary conditions of the chemicals are used in the simulation field. The initial condition of ECM density is set to be 1and other chemicals’ concentration  are 0. The chemicals’ concentrations are solved to steady state at each time step of the simulation with an inner iteration step of 5 s. The oxygen concentration has been normalized to be 0–1 in the "v through the two main parallel arterioles from plane x = 100 to x = 0 is set to be 3.5 mmHg as the driving force of blood in the network (or the boundary condition). The distribution of red blood cells (RBCs) at a microvascular bifurcation is calculated based on the blood rheology simulation .−1 day−1 after different time point of tumour growth . All simulations were performed on an Intel Xeon 3.2 GHz CPU, 24 GB Memory desktop. The parameter values of baseline model are listed in Table Initially, 20 proliferating cells are placed in the central computational area (see the tumour morphology in Fig. Figure The numbers of tumour cells with different phenotypes at different time step are presented as curves in Fig. inj = 100. However, only 30 and 21% of reduction can achieved respectively if injection time of ES is earlier or later than simulation time 100.In order to study the influence of anti-angiogenic drug ES on tumour growth and morphology, we tested five drug strategies with different drug concentration and different injection time. Compared to base case Fig. a, a low i is significantly higher than that of surrounding tissue and drops dramatically in the tumour margin (Fig. The model predicts the chemical and haemodynamical substances distribution including oxygen, VEGF, interstitial pressure and vessel radius, as shown in Fig. gin Fig. c, which gin Fig. d, arrow.p and maximum inhibiting effect of ES ɛmax. The variation ranges of tested parameters are ±1 and ±10% from their default values listed in Table max.In the current model, the main factors that directly influence ECs distribution are the chemotaxis of VEGF, the haptotaxis of ECM, the vessel remodelling in response to local haemodynamical microenvironment and the inhibition effect due to ES treatment. The sensitivity of tumour growth to the important parameters are analyzed, including chemotaxis coefficient entclass1pt{minimaIn this paper, we proposed a multi-scale coupled modelling system to investigate the dynamic process of tumour growth and angiogenesis in response to the changes in chemical and haemodynamical microenvironment caused by anti-angiogenic therapy. At tissue level, we considered tumour growth, tumour angiogenesis and vessel remodelling. Tumour growth and neo-vessel growth are coupled by the chemical microenvironment including oxygen, VEGF, ECM and MDE, which are described by continuous partial differential equations. At the same time, haemodynamic calculation is carried out by coupling intravascular blood flow and interstitial fluid flow. According to the microenvironmental information, detailed and comprehensive mechanisms of tumour cell phenotype and endothelial cell proliferation and migration are generated at cellular level, which in turn influence the modelling at intratumoral and tissue levels. In addition, we extended the model to study the dynamic changes of tumour growth in response to anti-angiogenic therapy. Anti-angiogenic drug ES is assumed to target immature vessels and may cause endothelial cell apoptosis.Simulation results revealed multiple characteristics of tumour progression, including tumour expansion, morphology changes and angiogenesis. The growth curves of cell population with different cell phenotypes are consistent with the pathophysiological knowledge. Furthermore, different anti-angiogenic drug strategies are designed to test the influence of ES on tumour growth and morphology. The largest reduction of tumour size is found when ES is injected at simulation time 100, which is concomitant with the emergence of angiogenesis phase. With pharmacokinetics information of specific anti-angiogenic drugs, the proposed model can be adopted for cancer drug discovery and testing.There are a few major limitations of the present work. On calculating the tumour mechanical environment, only interstitial fluid static pressure was included. Mechanical stress caused by the rapid proliferation of tumour cells was not simulated in the model. This solid mechanical stress may influence tumour cell behaviour. In addition, although most of the simulation parameters were set based on published experimental data, some of them cannot be found, such as tumour migration speed.A multi-scale coupled modelling system to investigate the dynamic process of tumour growth and angiogenesis in response to the changes in chemical and haemodynamical microenvironment during the anti-angiogenic therapy, is established. The simulation results demonstrate the inhibition of tumour growth and progression due to the reduction of neo-vasculature in response to anti-angiogenic therapy. Different anti-angiogenic drug strategies can be designed based on the proposed mathematical model, to test the influence of anti-angiogenic drug on tumour growth and morphology, with pharmacokinetics information of specific drugs


Cymbomonas tetramitiformis—a marine prasinophyte—is one of only a few green algae that still retain an ancestral particulate-feeding mechanism while harvesting energy through photosynthesis. The genome of the alga is estimated to be 850 Mb–1.2 Gb in size—the bulk of which is filled with repetitive sequences—and is annotated with 37,366 protein-coding gene models. A number of unusual metabolic pathways (for the Chloroplastida) are predicted for C. tetramitiformis, including pathways for Lipid-A and peptidoglycan metabolism. Comparative analyses of the predicted peptides of C. tetramitiformis to sets of other eukaryotes revealed that nonphagocytes are depleted in a number of genes, a proportion of which have known function in feeding. In addition, our analysis suggests that obligatory phagotrophy is associated with the loss of genes that function in biosynthesis of small molecules . Further, C. tetramitiformis and at least one other phago-mixotrophic alga are thus unique, compared with obligatory heterotrophs and nonphagocytes, in that both feeding and small molecule synthesis-related genes are retained in their genomes. These results suggest that early, ancestral host eukaryotes that gave rise to phototrophs had the capacity to assimilate building block molecules from inorganic substances . The loss of biosynthesis genes, thus, may at least partially explain the apparent lack of instances of permanent incorporation of photosynthetic endosymbionts in later-divergent, auxotrophic eukaryotic lineages, such as metazoans and ciliates. Chloroplastida (or Viridiplantae) is one of the major eukaryotic lineages, comprising green algae and land plants . The groCymbomonas tetramitiformis was definitively confirmed to engulf bacteria by transmission electron microscopy , the amoebozoans rmophila . In thisC. tetramitiformis sp. (PLY262) was obtained from the Plymouth Algal Culture Collection. The original strain was cocultured with a heterotrophic stramenopile of uncertain taxonomic identity; a clonal algal culture was established by single-cell isolation techniques. This reisolated culture strain was used for genome and transcriptome sequencing. The culture was maintained in f/2-Si medium . The cells were permeabilized in 0.03% Triton X-100 and 100 µg/ml bovine serum albumin (BSA), buffered in phosphate buffered saline (PBS) for 5 min at RT. Alexa Fluor 488-phalloidin (Invitrogen) was then added to the cell solution according to the manufacturer’s recommendation. Phalloidin staining proceeded for 20 min at RT. The cells were resuspended in 50 mM glycine in PBS to quench autofluorescence from unreacted aldehydes. The cells were washed three times with PBS and the final pellet was resuspended in filter-sterilized seawater. In all relevant steps, cells were pelleted by centrifugation at 500 × g for 90 s. Microscopic imaging was done using a Zeiss Axiovert 100 inverted microscope  equipped with an Olympus DP73 digital camera .Exponentially growing −1) using a PureLink Genomic DNA kit . For RNA extraction, 50 ml of culture was mixed with an equal volume of RNAlater solution (Life Technologies) prior to being pelleted by centrifugation at 1,000 × g for 5 min. Total RNA was isolated using the TRIzol Plus RNA Purification System (Life Technologies). Quantitative and qualitative assessments of purified nucleic acids were conducted by Qubit assays (Life Technologies) and agarose gel electrophoresis, respectively.Total DNA of the alga was obtained from 50 ml of culture in a midexponential phase . The suspended solution that contained nuclei was filtered through 30-µm nylon mesh, and incubated at 37 °C for 30 min (for staining and RNA digestion) before flow cytometric analysis alongside with standard nuclei. The mean nuclear DNA content, measured in picograms, was based on 1,000 scanned nuclei.DNA content analysis of C. tetramitiformis genome. Gene model inputs to MAKER proceeded from an annotation pipeline that was adapted from a previous study (C. tetramitiformis. The transcriptome was assembled in Trinity (C. tetramitiformis to update the gene models.The MAKER  (V 2.28)us study . Evidenc Trinity  (V Trini Trinity  (V 2006- Trinity  (V 2.7).e-value cutoff of 1e−5, using the peptide sequence output from MAKER as query sequences and the curated UniProt-SProt peptides as the database. This first step assigned potential homologs to 60.6%  of the C. tetramitiformis peptides. 2) Because statistically significant similarity does not necessarily mean equivalent function  A DELTA-BLAST  homologyfunction , for eacfunction  of equatot-SProt  fasta fi_uniprot  that wasC. tetramitiformis genome assembly as well as select genomes that were downloaded from publicly available sources  (V 2.4) , 2009 wa sources  online.  sources , genewis sources , HMMER . If an enzyme was present in C. tetramitiformis based on the functional genome annotation criteria discussed in “Functional Genome Annotation” section, it was considered present in the corresponding MetaCyc pathways. Presence/absence of pathways was determined by comparing pathway scores in C. tetramitiformis with those of three other green algae, Chlamydomonas reinhardtii, O. tauri, and Micromonas pusilla. False positives in the presence/absence list were eliminated by manually investigating each peptide in the respective organisms by BLASTP analysis.MetaCyc  is a free-value cutoff of 10−5. Up to the top 1,000 DELTABLAST hits were retained for subsequent phylogenetic analysis. Sequences were aligned in MAFFT (V 7.205)  with an V 7.205) . AlignmeV 7.205) . MaximumV 7.205)  on the CV 7.205)  using thV 7.205) . BootstrThe algorithm Bowtie2  V 2.2.3 V 0.1.1 (V 1.0).RepeatMasker  (V 4.0.5) was used to identify conserved repeats using the 20140131 version of the Repbase  (GIRI) rC. tetramitiformis genome against the full complement of proteins in a group of organisms known to be phagocytotic: The amoebozoan D. discoideum, the parasitic amoebozoan E. histolytica, the fruit fly Drosophila melanogaster, the mouse Mus musculus, the apusomonad Thecamonas trahens, the ciliate T. thermophila, and the phago-phototrophic chlorarachniophyte Bigelowiella natans; and a group of organisms where an active form of phagocytosis has not been observed: The plants Arabidopsis thaliana  Saccharomyces cerevisiae, and the chytrid fungus Batrachochytrium dendrobatidis (see C. tetramitiformis genome if BLASTP hits to a C. tetramitiformis peptide matched the following two criteria: 1) An e value of 1e−4 or less   algorith but see  and Oryzdatabase ) and 2) Mu. musculus, Dr. melanogaster, and D. discoideum was obtained from E. histolytica from T. thermophila from A list of proteins observed in purified phagosomes for C. tetramitiformis were inferred in the following procedures. First, sets of proteins shared between C. tetramitiformis and a phagocyte or C. tetramitiformis and a nonphagocyte were obtained by parsing BLAST results with custom scripts in the R programming environment  for that organism; if there was no match that met those criteria, it received a score of 0 (absent) for that organism  the number of genes in the enrichment set, 2) the proportion of those genes found within the known phagosome, and 3) the distance between the phagosome proportion scores among phagocytes and nonphagocytes for a given grouping strategy.C. tetramitiformis peptides share homology with known phagosome components. This number was used to weight sets of peptides such that a set of peptides derived from an optimal grouping strategy should include at least 4,305 peptides. Having too many or too few peptides in a set would incur a penalty. A weighted proportion score (wps) was thus calculated that included a penalty for distance from 4,305 peptides:The scoring scheme was derived from several observations. First, for each group , the proportion of peptides with homology to known phagosome components versus the stringency of the comparison (absence criteria)  online s penalty  online w penalty  online.  penalty  online aC. tetramitiformis peptides that are found in six or seven of seven phagocyte genomes but are absent from three or more nonphagocyte genomes    term enrogy (GO) . The ref P value . Organis P value  using th P value . Heat maC. tetramitiformis by flow cytometry estimated the nuclear DNA content  at 1.73 Gb ± 10.8 Mb—in other words, a genome size of 850 Mb. On the other hand, K-mer spectrum analysis of the sequencing reads of the alga suggested a genome size of 1.2 Gb. The unique (nonrepetitive) portion of the genome was estimated to be 270 Mb, by calculating the area under the curve around the peak K-mer coverage. The algal nuclear genome was assembled using the AllPaths-LG algorithm , RepeatModeler (Smit and Hubley 2008–2010) identified 9,044 long terminal repeat (LTR) retrotranspoable elements totaling 5.2 Mb and 1.88% of the assembled genome . AdditioC. tetramitiformis genome assembly, in comparison to genomes of select eukaryotes , it was predicted that 19 pathways that are present in C. tetramitiformis are absent from the three other green algae   . Cymbomoplastida .C. tetramitiformis were inferred through a comparative genomics approach. When the entire complement of predicted C. tetramitiformis peptides was compared with those of other phagocytes and nonphagocytes, 399 C. tetramitiformis peptides emerged that were present in C. tetramitiformis and at least six  other phagocyte genomes, but absent in three or more nonphagocyte genomes , 702 C. tetramitiformis peptides that are enriched among nonphagocyte sets were identified (N] or N − 1 phagocytes and absent in at least three nonphagocytes) were identified by a weighted scoring scheme that gauges both the sensitivity and inclusiveness of peptides in the set as described in Materials and Methods.Peptides related to phagocytosis in  genomes  online. entified  online. C. tetramitiformis peptides enriched among phagocytes or nonphagocytes were analyzed by function. The 399 C. tetramitiformis peptides associated with phagocyte genomes revealed, as predicted, significant enrichment of several functional categories strongly associated with feeding by phagocytosis. These include macropinocytosis, receptor-mediated endocytosis, actin cytoskeleton organization, cGMP metabolic process, cell adhesion, and cell projection organization , functional enrichment was, predictably, not strongly associated with phagosome components, but was rather enriched in metabolic and in particular biosynthetic processes. The top enriched biological processes included biosynthesis of amino acids  and intermediate compounds  (An interesting pattern emerged when the sets of nization  online. nthesis)  online.C. tetramitiformis nuclei and by estimation based on sequencing reads is between 850 Mb and 1.2 Gb. This is among the largest green algal genomes sequenced to date  (O. tauri and M. pusilla (<10% repeats)  , which may be suggestive of active rearrangement and expansion of the mixotrophic green algal genome  , and mucrepeats) , but on repeats)  and the s (>65%) . Note thcoverage . Althougl genome .C. tetramitiformis assembly was annotated with 37,366 protein models, 18,698 (50%) of which are grouped into 8,354 ortholog groups by orthoMCL analysis ; however, Ch. reinhardtii has a comparable number of orthologous groups  to that of C. tetramitiformis  and GT51 (MurG) and a glucoside hydrolase family protein (GH103 in this case)—that were suggested to be key indicators of the presence of PG metabolism according to M. pusilla and the bryophyte Physcomitrella patens, encode the same set of three PG-related proteins. Phylogenetic analyses of individual protein data sets  is a major cell-wall component of bacteria, including cyanobacteria from which plastids evolved . Of plasynthesis . Rather ynthesis . Furtherata sets  online sve GH103  online, proteins  online. proteins  online. C. tetramitiformis contains KDO and lipid A synthesis pathways similar to those described in the plant A. thaliana, in which the protein activities of the pathways were seen largely to localize to the mitochondria , the C. tetramitiformis glucoamylase may be involved in degradation of foods in the algal phagosome.monomers . This engionella   phagocytes but are absent in three or more nonphagocytes.Inference of phagocytosis-associated genes in rganisms . Our appmponents . Becausemponents , our appC. tetramitiformis peptides were found to be enriched among phagocytes when compared with nonphagocytes. GO , their compulsory feeding is mostly likely a derived status as their close photosynthetic relatives are not obligatory phagotrophs (Our analysis shows that two phago-mixotophic algae—the green alga etazoans . This isetazoans  (which cgotrophs . An integotrophs  unless agotrophs . An irongotrophs , led to gotrophs —despite gotrophs . PerhapsSupplementary DataClick here for additional data file.


Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery. Drugs that sensitize tumour cells to ionizing radiation are prized because they can overcome resistance to radiotherapy. Here, the authors show that anti-tubulin drugs conjugated to cetuximab or trastuzumab can radiosensitize EGFR- or HER2-expressing tumors by increasing DNA damage and cell death due to ionizing radiation. Concurrent chemotherapy and radiotherapy form the basis of curative organ sparing therapy for patients with locally advanced cancers1245678To be clinically useful, radiosensitizing chemotherapies must improve the therapeutic index, that is, the level of tumour cell sensitization must be greater than surrounding normal tissue101113141516171819212223242627293031We hypothesized that therapeutic antibodies to ErbB receptors could direct delivery of highly potent anti-tubulin drugs in a receptor-restricted manner to selectively radiosensitize tumours. To test this hypothesis in tumour model systems, we initially synthesized two ADC in which the anti-tubulin drug monomethyl auristatin E was conjugated to cetuximab or trastuzumab . C-MMAE and T-MMAE bound and restricted MMAE activity and toxicity to EGFR and HER2 expressing tumour cells, respectively. Importantly while free MMAE radiosensitized indiscriminately, antibody conjugation resulted in targeted MMAE radiosensitization to EGFR or HER2 expressing tumours. To delineate the translational potential of these findings, we extended our studies to the clinically approved anti-tubulin ADC, ado-trastuzumab emtansine (T-DM1). We found that T-DM1 radiosensitized HER2 expressing tumours specifically resulting in significantly increased tumour xenograft control. On the basis of these findings, we propose antibody drug conjugate based chemo-radiotherapy paradigms designed to focus on antibody directed delivery of highly potent radiosensitizing chemotherapies as an alternative to receptor signal inhibition.2/M. Morphologically, CAL-27 cells treated with either free MMAE or C-MMAE appeared identical with cells rounding up, indicative of G2/M  and trastuzumab (T-MMAE) and labelled them with Cy5 for tracking . Cetuxim of G2/M . OE19 anll cycle . In OE19ll lines .Key advantages driving the clinical development of MMAE are its potency and its ability to be tumour targeted through antibody conjugation271250 of mertansine was fairly consistent across cell lines ∼10 nM (50 of T-DM1 was <1 nM but was >100 nM in HER2- cells(HCT116 and CAL-27). From a translation point of view, T-DM1 was a more potent cytotoxic drug in HER2+ OE19 cells compared with clinically used radiosensitizers that are either cytotoxic (paclitaxel and cisplatin) or targeted to HER2 (trastuzumab and lapatinib) or EGFR (erlotinib)  radiosensitized by increasing IR induced DNA double-strand breaks in both HER2+ and HER2− cell lines  as they accumulated in the G2/M phase of the cell cycle , maytansinoids  are another potent class of anti-tubulins29s ∼10 nM . In contlotinib) . Howeverlotinib) . Functioll cycle . While Tll lines . Importall cycle .in vivo ADC visualization, tracking and serves as a surrogate for tumour drug delivery following intravenous injection in mice. Mice bearing OE19 tumours were intravenously injected with Cy5-labelled C-MMAE or T-MMAE . Since an average of four molecules of MMAE were conjugated to each antibody molecule, a dose of 2 nmol of free MMAE was injected to maintain MMAE dose equivalence to C-MMAE. Because C-MMAE persisted in murine xenografts for up to 72 h  were equally efficacious in slowing tumour growth compared with control or trastuzumab by day 14  delivered once (day 0). On the basis of our above results with our Cy5-labelled ADC, ADC localized within tumours for up to 72 h. Therefore, we delivered 2.5 Gy of IR on days 1, 2 and 3 after T-DM1 injection. In both OE19 and NCI N87 HER2+ tumour xenografts, this regimen resulted in significantly prolonged tumour regression  Combinatorial therapy attacks cancer cells by multiple mechanisms decreasing the risk of tumour resistance emerging. 2) Instead of higher individual doses of a single agent to achieve effective tumour kill, combinatorial therapy allows for dose reduction of each individual modality thereby decreasing the toxicities inherently associated with each therapy. 3) concurrent delivery of full dose chemotherapy and radiotherapy allow for attacking not only known local disease but also potential micrometastases. (4) Ideally, chemotherapies used in conjunction with radiotherapy will be highly potent alone, synergize with IR, and be spatially targeted to tumours and not normal tissue. (5) The precise timing with which IR can be delivered is valuable for defining the temporal window(s) when radiosensitization should be maximal. Auristatin and maytansinoid based ADC fulfill these criteria. Currently, there are only two indications for which ADC has shown clinical value, CD30 lymphomas and HER2 metastatic breast cancer30 Instead  concurre43−1 hydrocortisone. OE19 and NCI N87 cells were cultured in RPMI supplemented with 10% FBS. CALU3 cells were cultured in MEM supplemented with 10% FBS. On receipt, each cell line was expanded, cryopreserved as low passage stocks and routinely tested for mycoplasma. Cisplatin (Enzo Biosciences), paclitaxel (Sigma), MMAE (Concortis) and mertansine (Abcam) were reconstituted in DMSO. Clinical-grade erlotinib, lapatinib, cetuximab, trastuzumab, ado-trastuzumab emtansine (T-DM1) were obtained from UCSD Moores Cancer Center pharmacy.All cell lines used in these studies are summarized in −1) of cetuximab  or trastuzumab  was treated with sodium bicine buffer  and sodium diethylenetriaminepentaacetic acid . Following reduction with four equivalents of tris(carboxyethyl)phosphine (TCEP) at 37 °C for 2 h, the solution was added to four equivalents of maleimidocaproyl-valine-citrulline-PABA-MMAE (MC-VC-MMAE)−1 cm−1 (cetuximab) or 225,000 M−1 cm−1 (trastuzumab) at 280 nm and 12,500 M−1 cm−1 and 250,000 M−1 cm−1 at 280 and 650 nm, respectively, for Cy5. Hydrophobic interaction chromatography (HIC) of a reaction sample after labelling with MMAE revealed nine peaks corresponding to antibody modified with 0–8 MMAE derivatives if up to four disulfides are reduced by TCEP per antibody46A solution  and analysed by FACS using FloJo software.Cells were plated in 96-well plates and exposed to a range of concentrations of MMAE, mertansine, cisplatin or paclitaxel, cetuximab, cetuximab-MMAE, trastuzumab-MAME or T-DM1 for 72 h. Alamar Blue (resazurin) was added to the cells and allowed to incubate for 2–4 h at 37 °C. Plates were analysed using a plate reader with fluorescence measured at 560 nm.Cells were treated with MMAE, mertansine, cetuximab or cetuximab-MMAE for 24 h and then irradiated with 2 Gy. Following IR, cells were counted, re-plated at varying cell numbers in drug-free media. 10–14 days after initial seeding formed colonies were methanol fixed, stained with crystal violet and counted. Surviving fraction at 2 Gy (SF2) was calculated as the fraction of cells surviving 2 Gy compared with non-irradiated cells.Cells were treated with indicated times doses of MMAE, mertansine, cetuximab, cetuximab-MMAE, trastuzumab, trastuzumab-MMAE or T-DM1 overnight, and then irradiated with 6 Gy. Cells were collected 15 min post IR, suspended in agarose gel and lysed per assay directions (Trevigen). Samples underwent electrophoresis under neutral conditions and were then stained with Sybr Green. Comet tails were counted in multiple fields (>60 cells per sample) and analysed using CometScore (TriTek Corp). Comet tail length was normalized to vehicle-treated, non-irradiated cells.Cells were collected and lysed in RIPA buffer  with protease and phosphatase inhibitors . Lysate protein was quantitated by BCA technique (Pierce). Twenty micrograms of lysate underwent electrophoresis using 4–12% Bis-Tris gels (Life Technologies), transferred to PVDF membranes (iBlot) and incubated with indicated primary antibodies HER2, GAPDH  and EGFR  at dilutions of 1:1,000, 1:3,000 and 1:3,000, respectively. Blots were developed by ECL (Pierce). Uncropped blots are shown in relevant Tissue sections were cut from blocks of formalin-fixed paraffin embedded xenografts. Four micron thick tissue sections were stained with antibodies to phospho-histone H3  and cleaved caspase-3  and used at dilutions of 1:300 and 1:900, respectively. Slides were stained on a Ventana Discovery Ultra . Antigen retrieval was performed using CC1 for 24–40 min at 95 °C. The primary antibodies were incubated on the sections for 1 h at 37 °C. Primary antibodies were visualized used DAB as a chromagen using the UltraMap system  followed by hematoxylin as a counterstain. Slides were rinsed, dehydrated through alcohol and xylene and coverslipped.Immunostained slides were scanned using an Axio Scan.Z1 . Axio Scan uses the software Zen2 (Zeiss) for automatic thresholding and tissue detection. The entire tissue section encompassing the xenograft was scanned at 40 ×  using the default stitching parameters to combine the individual tiles into a single image. Whole-slide images were imported into Definiens Software for quantitative analysis . Using Definiens Tissue, tumour regions of interest (ROI) were chosen per slide based on the pH3 immunostain. The software uses the color contrast of the DAB and hematoxylin counterstain to determine the basic ROI. Training was done to exclude non-tumour tissue including necrotic regions and mouse stroma. The ROI defined for the pH3 stain was also used to analyse the adjacent section stained for ClC3. For analysis of pH3 (a nuclear stain), the total number of nuclei and the total number of nuclei showing DAB staining (pH3 positive cells) were recorded within each ROI. The percent of positive nuclei was then calculated for each sample. For analysis of ClC3 (a cytoplasmic stain), mean DAB chromagen intensity was measured and calculated within the ROI for each sample.6 CAL-27, SQ9-G, SCC-35, SCC-61, A549, HCT-116, OE19 or LN229 tumour cells in a 1:1 Matrigel (BD) and PBS solution. After tumours grew to >100 mm3 they were injected with 0.5 nmol of Cy-labelled cetuximab-MMAE, trastuzumab-MMAE  as described above. For imaging, mice were anaesthetized (1:1 mixture of 100 mg ml−1 of ketamine and 5 mg ml−1 of midazolam). Animals were imaged using a Maestro Small Animal Imager (CRI) with excitation filter of 620/22 and 645 nm long-pass emission filter with dichroic filter tuned to 670 nm. Imaging was done both with skin on and after skin removal to decrease autofluorescence and scattering. For blood clearance studies, athymic nu/nu mice were anaesthetized with isoflurane and dosed with 5 nmol of the antibody conjugates (∼36.4 trastuzumab and ∼38.6 mg kg−1 T-MMAE). At various time points after injection, the tail was pricked and a small volume (5–10 μl) of blood was collected in a heparinized hematocrit tube. Fluorescent images were taken using the Maestro, with the filters mentioned above, and the integrated fluorescent intensity was measured using Image J.All animal work was done in compliance with the University of California San Diego Institutional Animal Use and Care Committee. Six-to-eight-week-old female athymic nu/nu mice purchased from the University of California San Diego Animal Care Program breeding colony were injected subcutaneously into the bilateral upper thighs with 5 × 102. Mice were randomized into groups once the average tumour volume reached >100 mm3. Mice were assigned to indicated groups in ‘Results'. MMAE, cetuximab, trastuzumab, C-MMAE, T-MMAE or T-DM1 was intravenously injected in 50 μl. For irradiated mice, the tumour-bearing hindlimbs were focally irradiated while the remainder of the mouse was shielded from IR with custom designed lead blocking >95% of the dose as verified by dosimeters placed on the mouse. Free MMAE was injected on an equimolar basis to C-MMAE in final volume of 3% DMSO. Drug doses and IR fractionation are as indicated in ‘Results'. To prevent unnecessary morbidity, mice were killed if tumour length exceeded 15 mm per protocol. Nonlinear regression least squares fit was used to calculate tumour volume doubling times. Tumour doubling times were extrapolated for T-DM1+IR groups where tumour doubling was been achieved. To minimize the number of mice used, tumours were grown in the bilateral flanks. For CAL-27 tumour xenografts in experiment CAL-27, OE19, NCI N87 and HCT116 tumour xenografts were established and tumour growth was measured with digital calipers. Tumour volume was measured blindly to treatment group and calculated using the formula as ½ × length × widthg, 10 min), then the supernatants were collected and diluted two-fold by addition of 2% acetic acid in acetonitrile, then centrifuged again . MMAE concentration was determined by LC-MS/MS with Luna-2 C18 column and Agilent Trap XCT mass spectrometer and extracted fragment ion currents at 686.4 and 506.4 were integrated and combined to improve sensitivity.Tumours were excised, weighed and homogenized in 10 volumes of PBS with a point sonicator (Fisher Scientific) using an amplitude range of 5–15% for a maximum of 20 s while on ice. The homogenates were centrifuged . Statistical analysis for all tumour xenograft data are presented in Unpaired two-sided All relevant data are available from the authors.How to cite this article: Adams, S. R. et al. Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize. Nat. Commun.7, 13019 doi: 10.1038/ncomms13019 (2016).Supplementary Figures 1-11 and Supplementary Tables 1-9.