Magnetic resonance arthrography (MRA) requires intra-articular injection of gadolinium-based diluted paramagnetic contrast material. To our knowledge, gadobenate dimeglumine (Gd-BOPTA) has never been used for intra-articular applications. Our aim was to test in vitro different concentrations of Gd-BOPTA to be potentially used to perform MRA.Gd-BOPTA was diluted in saline (NaCl 0.9%) to achieve different concentrations . Six sets of five sterile pipes were prepared with 5 ml of each solution, five sets added with 0.5 ml of fresh synovial fluid. Two separate pipes were prepared with 5 ml of gadopentetate dimeglumine (Gd-DTPA) at 2 mmol/l, one pipe added with 0.5 ml of synovial fluid. Pipes were imaged using a T1-weighted sequence at 1.5 T. For each pipe, signal intensity (SI) in arbitrary units (au) was measured.P ≤ 0.002). Regarding pipes with synovial fluid, mean Gd-DTPA SI at 2 mmol/l was 1246 ± 27au. Compared with Gd-BOPTA, SI was not different at 0.5 mmol/l  while it was higher (P < 0.001) at all other concentrations (range + 13.3%[4 mmol/l] − + 28.3%[1 mmol/l]). Regarding pipes without synovial fluid, mean Gd-DTPA SI at 2 mmol/l was 1275 ± 56au. Compared with Gd-BOPTA, SI was lower at 0.5 mmol/l , while it was higher (P < 0.001) at all other concentrations (range + 6.1%[4 mmol/l] − + 19.6% [1 mmol/l]).SI reproducibility range was 86–99%. Mean Gd-BOPTA SI in pipes containing synovial fluid increased from 1236 ± 8au (0.5 mmol/l) up to 1610 ± 44au (1 mmol/l) and down to 1405 ± 33au (4 mmol/l). Mean Gd-BOPTA SI in pipes without synovial fluid increased from 1184 ± 29au (0.5 mmol/l) up to 1530 ± 38au (1 mmol/l), and down to 1347 ± 39au (4 mmol/l). SI of pipes without synovial fluid was lower than that of pipes with synovial fluid for both Gd-BOPTA and Gd-DTPA (In vitro, Gd-BOPTA at 1 mmol/ had a + 28% SI increase in comparison to Gd-DTPA 2 mmol/l. SI similar to Gd-DTPA can be obtained using one fourth concentration of Gd-BOPTA. Magnetic resonance imaging (MRI) has become the most important diagnostic tool for the assessment of joint disorders throughout the body, allowing for detecting abnormalities of the joint space, cartilage, fibrocartilages, tendons, ligaments, and synovial tissue . In this− 1 at 1.5 T and produce similar contrast enhancement when administered at the same dose [MRA requires intra-articular injection of gadolinium-based diluted paramagnetic contrast material , 4. Contame dose . Gadobename dose .To our knowledge, Gd-BOPTA has never been used for intra-articular applications.The purpose of our study was to test in vitro different concentrations of Gd-BOPTA to be potentially used for MRA.Six sets of six sterile pipes were prepared at different concentrations, 36 pipes overall Fig. a. For alTo simulate the environment of a joint, five sets of pipes prepared as reported above were added with 0.5 ml of fresh synovial fluid withdrawn from the knees of five patients presenting to our department for intra-articular injections  Fig. c. These All six sets of pipes were imaged independently using an axial T1-weighted sequence  at 1.5 T  using a 4-channel phased-array surface coil. The same sequence was repeated twice to test reproducibility. Fourteen slices per pipe were obtained.www.horosproject.info) placed approximately in the middle of the image. For each pipe, the mean and standard deviation (SD) of SI over the 14 regions of interest was calculated. To compare the SI of Gd-BOPTA and Gd-DTPA, data from all the pipes and all the measurements were averaged.For each image, signal intensity (SI) in arbitrary units (au) was measured by one observer using a 4-mm diameter region of interest  was used. P-value threshold was set at 0.003 for multiple comparison correction.Reproducibility between SI of each pipe was tested using the Bland-Altman method. Differences of SI between pipes at the same concentration of the two sets  and between pipes containing Gd-BOPTA at different concentrations and Gd-DTPA were assessed using the Mann-Whitney Figure The reproducibility of SI measurement on the same pipe ranged from 86%  to 99% . Full data is reported in Table P > 0.004).The mean SI of Gd-BOPTA in pipes containing synovial fluid increased from 1236 ± 8 au at 0.50 mmol/l  up to 1610 ± 44 au at 1.00 mmol/l  and down to 1405 ± 33 au at 4.00 mmol/l . SI within pipe sets was homogeneous, with no significant differences among them  up to 1530 ± 38 au at 1.00 mmol/l , and down to 1347 ± 39 au at 4.00 mmol/l .P ≤ 0.002).SI of pipes without synovial fluid was significantly lower than that of pipes with synovial fluid for both Gd-BOPTA and Gd-DTPA (Full data is reported in Table P = 0.587) while it was significantly higher at all other concentrations .Regarding pipes with synovial fluid, the mean SI of Gd-DTPA at 2 mmol/l was 1246 ± 27 au. In comparison with Gd-BOPTA, SI was not significantly different at 0.5 mmol/l  while it was significantly higher at all other concentrations . Full data is reported in Table Our in vitro experience demonstrated that synovial fluid slightly but significantly increased SI of Gd-BOPTA. In pipes containing synovial fluid, Gd-BOPTA at 1 mmol/l had a + 28% SI increase in comparison to Gd-DTPA 2 mmol/l.Gd-BOPTA has unique features that distinguish it from all other gadolinium-based contrast media. In particular, it has a dual route of elimination from the body that enables Gd-BOPTA to be used both as nonspecific agent and liver-specific agent . MoreoveRegarding MRA, the use of Gd-BOPTA has never been clearly reported before except for an animal study published in 2007 in which its effect on cartilage was tested . HoweverCompared to 2 mmol/l solution of Gd-DTPA, SI of Gd-BOPTA was higher at all concentrations except 0.5 mmol/l. The highest SI was achieved at 1 mmol/l, while a similar SI was obtained at 0.5 mmol/l in pipe sets added with synovial fluid. In clinical practice, these differences may be negligible, implying that also a low concentration of gadolinium-based solution can be considered acceptable. The main practical result of these findings is that, in a simulated joint environment, the same SI of pre-diluted Gd-DTPA syringes can be obtained with up one fourth concentration of Gd-BOPTA. However, in vivo testing of this speculation should be warranted.We acknowledge the limitations of an in vitro study in which joint environment has been reproduced only adding a small amount of synovial fluid to the contrast agent contained in sterile pipes. This implies that our data may be not directly applicable in vivo, as other factors  may be implicated in the interaction with Gd-BOPTA. Also, body temperature may somewhat affect behavior of contrast agents in vivo, while our experiment was conducted at room temperature. Moreover, we used the synovial fluid withdrawn from the knees of patients with a mean age of 68 years and mild degenerative osteoarthritis. It is known that synovial fluid of elderly subjects may contain less glycoproteins and glycosaminoglycans. Thus, in younger subjects, in whom MRA is more likely to be performed, results may be somewhat different. We can speculate that a higher content of glycoproteins may further improve SI, although at present we do not have data to confirm such hypothesis. Finally, we did not assess separately T1 and T2 effects of the contrast media and our results are only valid for one specific parameter set TR and TE. Also, relative signal changes with noise correction should also be measured to assess reliable relaxivity values R1 and R2. However, our study was primarily aimed to measure SI, that is the most useful parameter that may affect clinical practice.In conclusion, in vitro, Gd-BOPTA at 1 mmol/ had a + 28% SI increase in comparison to Gd-DTPA 2 mmol/l. A SI similar to Gd-DTPA can be obtained using one fourth concentration of Gd-BOPTA. Further in vivo studies are warranted to confirm clinical applicability and value of our results.


Paulownia trees are grown as a woody biofuel crop, and they yield large amounts of leafy biomass rich in nitrogen. However, there is limited information on the use of paulownia leaves as an animal feed resource. Hence, this study was conducted to assess the effects of paulownia leaf meal  on the growth performance, nutrient digestibility, blood biochemistry, and intestinal microbiota of growing rabbits. The chemical analyses of paulownia leaves indicated that most of the nutrients are similar to those in alfalfa hay. The in vivo results showed that the use of up to 15% paulownia leaf meal instead of alfalfa hay in the diets of the rabbits did not have any negative effects on their performance, nutrient digestibility, and blood constituents. In addition, a notable reduction in both cecal and feed pathogenic bacteria was observed when paulownia leaf meal was included in the diets.This experiment was conducted to study the effects of paulownia leaf meal (PLM) as a nontraditional feed on the growth, carcasses, digestibility, blood chemistry, and intestinal microbiota of growing rabbits. Sixty rabbits (5-weeks old) were randomly allotted to three dietary treatments containing three amounts of PLM . The results showed that PLM has a higher content of ether extract, organic matter, methionine, tyrosine, histidine, manganese, and zinc than alfalfa hay. Body weight gain decreased when 30% PLM was provided. The best feed conversion ratio was recorded in the rabbits fed 15% PLM. A notable increase in high-density lipoprotein levels with a significant decrease in low-density lipoprotein was noted in the rabbits fed the PLM diets. Total fungi and Enterobacteriaceae and total bacterial count in the feed were significantly reduced because of PLM. In the cecum, coliforms, Enterobacteriaceae species, and total bacterial count declined in the rabbits fed the PLM diets. Conclusively, up to 15% PLM can be used in rabbit diets without any deleterious effects on the performance, nutrient digestibility, and blood constituents. In addition, dietary inclusion of PLM has the potential to reduce cecal pathogenic bacteria in rabbits. In many countries, including Egypt, the animal feed industry is facing major challenges because of high prices of energy and protein sources such as maize and soybean meal, both of which are mainly used in animal feeds ,2. MoreoPaulownia trees are grown for their wood, which is used to make furniture, musical instruments, and flooring. Paulownia trees are produced in some countries, such as Bulgaria, and because of their good nutritional value, they can be used as an alternative feed ingredient. The leaves of paulownia could be used as an alternative feed ingredient for different animals because of their varied biochemical properties . AccordiHowever, to date, there is limited information on the optimal use of paulownia leaves as an animal feed resource to overcome the shortage of protein sources for livestock animals. In this study, paulownia leaf meal (PLM) was used as a novel feedstuff to replace alfalfa hay as a high-quality forage in the diet of growing rabbits. Thus, the objective of this study was to analyze the effects of PLM as a nontraditional feed on the growth, carcasses, nutrient digestibility, blood chemistry, and intestinal microbiota of rabbits.This experiment was performed at the Rabbit Research Farm, Animal Production Department, Agriculture College, Zagazig University, Egypt. All the protocols were performed according to the experimentation guidelines of the Ethics of Animal Use in Research Committee of Zagazig University .Paulownia tomentosa) leaves were harvested and collected in October 2016. They were collected from 100 six-month-old trees in an orchard located in Bani-Salama Village, Wadi El-Natroun in Beheira Governorate, Egypt (30° 26′N 30° 14′E). The orchard has an area of two acres containing 2500 trees. About 4 kg of fresh leaves was collected from each tree. The average moisture content was 80.22%. The paulownia leaves were transported to the lab of the Animal Production Department, Agriculture College, Zagazig University for further processing. The leaves were dried, ground, and stored in airtight bags until further analysis (proximate and amino acid profile) and use. The paulownia leaves were ground using a grinding machine designed at the Department of Agricultural Engineering, Faculty of Agriculture, Zagazig University for producing a nontraditional feed for growing rabbits to improve product quality. The highest values of machine productivity and overall machine efficiency were 435 kg/h and 90.60%, respectively. The optimum operating parameters of the milling machine were 1600 rpm drum speed by using a 450 kg/h feed rate of PLM.Paulownia  methods . All samThe mineral contents (method 984.27) were analyzed using the atomic absorption spectrophotometer in the Central Laboratory for Food and Feed analysis (CLFF), Agriculture Research Center, Ministry of Agriculture, Cairo, Egypt. Amino acid analyses of PLM and experimental diets were performed using an automated amino acid analyzer after hydrolyzing the samples of paulownia leaves and diets with 6 M HCl at 110 °C for 24 h, according to Ogunbusola et al. , and theSixty New Zealand White rabbits  were weighed and randomly allotted to three dietary treatments containing three amounts of paulownia powder  for 8 weeks. Each treatment involved 10 cages, where two animals were maintained in 1 cage. The rabbits were housed in the cages (30 cm width × 50 cm length × 40 cm height) and reared under the same managerial conditions, and they were fed to achieve their requirements, according to De Blas and Mateos .Feed intake (FI), body weight (BW), and body weight gain (BWG) as the FI-to-BWG ratio were measured according to the methods reported by Alagawany et al. . At weekAt week 13, four rabbits from each group were randomly selected and individually distributed into metabolic cages for seven days for the digestibility trial. The feces were collected daily from each rabbit and weighed, and a 50% subsample was obtained, oven-dried at 60 °C for 24 h, and stored in the lab for chemical analysis. Samples of the diets and feces were chemically analyzed . Total dThe following equation of Schieman et al.  was usedBlood samples were collected from the sacrificed rabbits (five rabbits/group) in sterile tubes. The samples were coagulated and centrifuged (4000 rpm for 10 min), and the sera were stored till further analysis. The serum samples were used to estimate the level of albumin (g/dl), total protein (g/dl), urea (mg/dl), creatinine (mg/dl), total cholesterol (mg/dl), triglyceride (mg/dl), low-density lipoprotein (LDL) (mg/dl), and high-density lipoprotein (HDL) by using kits and a spectrophotometer .Enterococcus, coliforms, and total fungi) were estimated using the methods of Xia et al. [2 in bottles and transferred immediately to the laboratory for microbiological investigation. The microbial counts  were determined according to the methods of Xia et al. [The dietary samples (25 g of each diet) of alfalfa hay, PLM, and their mixture (50/50) were subjected to microbiological analysis at the end of the experimental period. Microbial counts .All statistical analyses were performed using SAS . The datThe results obtained by Guoqiang and Jianqing  showed tThe mineral composition of PLM, alfalfa hay, and experimental diets is shown in Paulownia elongata leaves can be used as a new feed ingredient in diets for ruminants and other herbivorous agricultural species such as rabbits and horses. In the present study, the content of all nutrients was nearly similar in the experimental diets containing PLM and alfalfa hay. Paulownia species such as P. fortunei, P. tomentosa, and P. elongata are palatable and have a good chemical composition and nutritional value suitable for animal feed [When PLM is used in a complete rabbit diet, the nutrient composition of PLM is considered and mixed with appropriate feed ingredients to obtain a suitable diet formula that meets the nutritional requirements of rabbits. The nutrient composition of PLM, alfalfa hay, and experimental diets is shown in mal feed . The effects of dietary treatments on the performance of rabbits are illustrated in p = 0.027) decreased because of 30% paulownia when compared with 30% alfalfa hay. The lowest FI value (p = 0.003) was detected in the rabbits fed the diet containing 15% PLM. The best FCR value was observed in the rabbits fed 15% PLM during 5–9 weeks of age, when compared with 30% PLM (p = 0.001). The decrease in BWG (with 30% PLM) and FI (with 15% PLM) during the first 4 weeks of the experiment (5–9 weeks of age) might be due to the slight bitterness of the PLM possibly affecting palatability, some pharmacologically active agents, or the organic matter (OM) being simply less digestible. Leaves of paulownia have been reported to be slightly bitter but palatable for some animal species such as sheep and cattle [Initially, at 5–9 weeks of age, BWG was significantly  statistically influenced by the PLM treatments  were not (eatments . An adeqp > 0.05) were observed in the nutrient digestibility, apart from NFE, because of the paulownia treatments. Inclusion of paulownia in the rabbit diets significantly reduced (p = 0.028) NFE digestibility. As a result, decreased TDN and DE were observed when the animals were fed 30% paulownia foliage. Conversely, the inclusion of 15% paulownia foliage, when compared with alfalfa hay, in the rabbit rations did not affect TDN and DE. Our results are consistent with those reported by Juniar et al. [Acacia saligna leaf meal did not affect nutrient digestibility and N balance. Tree leaves can be used as a supplement to enhance feed utilization and animal performance or to replace traditional feeds to achieve safe and economic production [p = 0.028), dry matter (p = 0.004), crude protein (p = 0.001), acid detergent fiber (p = 0.011), and NDF (p = 0.033); in addition, TDN (p = 0.045) and DCP (p = 0.041) were detected in the diets containing tree leaves [The effects of dietary inclusion of PLM on DM, CP, CF, OM, EE, and NFE digestibility and TDN, DCP, and DE values of growing rabbits are shown in r et al. , who posr et al.  observedoduction ,36. Rubaoduction  found thTo the best of our knowledge, the fibrous ingredients such as alfalfa hay and tree leaves used in the present study have not been examined previously, and, in this study, a high amount of this feed ingredient (up to 30%) was used. The digestibility of DM, OM, EE, CP, and CF can be assumed to be within the range of values reported in rabbits.p > 0.05) affected by the dietary treatments, except HDL and LDL. Our results were within the normal levels reported by Mader [Blood biochemical analysis is a defined way to evaluate the clinical and health status of animals and humans . The effby Mader . Similarby Mader . p = 0.037), but LDL was decreased (p = 0.042) in those that received 30% PLM. Our findings are partially consistent with those of Sun et al. [In contrast, Varlyakov et al.  found thd p = 0.0 in thosed p = 0.02 in thosn et al. . Our resRabbits have a special characteristic (cecotrophy) that helps them to use a high-roughage diet efficiently. The microflora in the cecum help in the digestion of the fibrous ingredients. p < 0.05). The populations of total fungi and Enterobacteriaceae and total bacterial count were decreased (p < 0.05) in all the paulownia groups when compared with the control. However, the coliform count increased in the 30% PLM group, followed by that in the control (0% PLM) and 15% PLM groups. In the cecum, inclusion of paulownia in the rabbit diets had a positive effect on the microbial populations (p ≤ 0.001) in the rabbits fed the paulownia diets. However, inclusion of paulownia in the rabbit diets did not affect (p = 0.131) cecal Clostridium populations. The inclusion of 15% and 30% paulownia delayed the growth of fungi and bacteria. The microbial populations in the digestive system of ruminants affect the food before it reaches the main place of absorption and digestion, but the microbial populations in the cecum of rabbits affect the undigested residues of feed. Tree leaves could be fed to rabbits separately as green fodder . As showulations . The popPseudomonas aeruginosa, Salmonella entericа, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans, and Paenibacillus alvei. The inhibitory impact of paulownia leaves is more evident against Gram-negative bacteria [The results indicated that the inclusion of paulownia in the rabbit diets resulted in a delay in spoilage emergence, which may be attributed to the antimicrobial effects of paulownia because of its biochemical composition . Flavonobacteria . Ibrahimbacteria  showed tOur study provides evidence for the suitability of using PLM as a feed resource for animals. Throughout the experiment period, the inclusion of 15% PLM in the rabbit diet did not adversely affect growth performance, blood biochemistry, carcass traits, nutrient digestibility, and nutritional values. However, the inclusion of 30% PLM significantly reduced the digestible energy and NFE digestibility; nonsignificantly decreased growth performance; and did not negatively affect blood measurements, carcass characteristics, and digestibility of most nutrients. Therefore, the current study recommends the inclusion of 15% PLM in rabbit diets instead of alfalfa hay to achieve the maximum benefit. In addition, the populations of fungi and Enterobacteriaceae and total bacterial count in the feed, and populations of coliforms and Enterobacteriaceae and total bacterial count in the cecum content, were decreased in the rabbits fed diets containing PLM.


Achieving the activation of drugs within cellular systems may provide targeted therapies. Here we construct a tumour-selective cascade activatable self-detained system (TCASS) and incorporate imaging probes and therapeutics. We show in different mouse models that the TCASS system accumulates in solid tumours. The molecules show enhanced accumulation in tumour regions via the effect of recognition induced self-assembly. Analysis of the molecular penetration in tumour tissue shows that in vivo self-assembly increases the penetration capability compared to typical soft or hard nanomaterials. Importantly, the in vivo self-assembled molecules exhibit a comparable clearance pathway to that of small molecules, which are excreted from organs of the reticuloendothelial system (liver and kidney), while are relatively slowly eliminated from tumour tissues. Finally, this system, combined with the NIR probe, shows high specificity and sensitivity for detecting bladder cancer in isolated intact patient bladders. The activation of drugs within cellular systems may provide targeted therapies for cancer. Here, the authors make a drug delivery system that is activated within the cell and exploits XIAP expression to cleave a linker region, resulting in the self-assembly of the system and drug release within cancer cells. Nanomaterial systems with targeting motifs and optimized particle sizes accumulate within the tumour and have enhanced molecular utilization efficiency5. Numerous strategies, such as developing small nano-sized superstructures6, enhancing long-term circulation4, and using physical tools, have been pursued to increase the accumulation and retention of nanoparticles8, decrease toxicity and optimize metabolism10. However, in the translation to clinical practice, the results remain unsatisfactory12. By taking advantage of small molecules and nanoscience, we expect in vivo self-assembly to outperform other strategies because (i) in addition to existing active/passive targeting processes, there is a new targeting mechanism which exhibit efficient accumulation and retention at desired sites; (ii) high penetration capability similar to that of small molecules in tumour region; and (iii) pharmacokinetics similar to that of small molecules except for reduced systemic toxicity in vivo. These advantages suggest that supramolecular systems may be promising for multifunctional nanomedicine.Traditional molecular chemotherapeutics and imaging agents exhibit relatively low bioavailability due to their fast metabolic clearance16 has been widely recognized as a promising tool for combating many life-threatening diseases19. Prominent examples in our previous work demonstrated some of the advantages of using peptide-based enzymatically induced assembly systems. For example, the aggregation/assembly induced retention (AIR) effect20 significantly enhanced bioactive molecule retention at disease sites, contributing to highly specific, sensitive and quantitative bioimaging21 and efficient tumour therapy22. Other groups have also confirmed that in vivo enzyme-triggered nanoassembly may facilitate endogenous enzyme imaging or induce cell death24. However, design of controllable building blocks with biofunctions and predicting the in vivo self-assembly behaviour still faces obstacles. Thus, de novo designed biocompatible small molecules with capability of self-assembly in living systems are imperative for in-depth biological studies. In this work, we attempt to construct a new tumour-selective cascade activatable self-detained system (TCASS) as a tumour nanomedicine, and reveal its mechanisms of efficient accumulation and retention at the tumour sites, and analyse the pharmacokinetics of the system.Currently, in vivo self-assembly strategy based on supramolecular chemistry28. Then the recognition process activates downstream caspase-3/7, which triggers the self-assembly process 31 and nanomaterials (5.2 ± 4.4% ID/g)32 reported in mouse models. Compared to typical nanomaterials (e.g. SiO2 nanoparticles and liposomes with diameters of approximately 100 nm), the TCASS exhibits remarkable penetration due to its monomeric molecular diffusion before self-assembly. Meanwhile, the organ competition behaviour of the TCASS is comparable to that of small molecules, which are rapidly excreted from the liver and kidneys. By integration of the chemodrug or contrast agent, the TCASS can be successfully employed in the process of drug delivery in mice or image-guided surgery in isolated patient intact bladders.Herein, we rationally design a TCASS system to optimize accumulation, penetration and organ competition. The modularized units of the system Fig.  consist ess Fig. . Hydroge34. The CD signals of molecule 4 showed a positive value at 199 nm and a negative value at 217 nm, which were considered to be β-sheet stacking self-assembly35 37. Furthermore, to study the blocking effect of Cy molecules in the peptide sequence, we synthesized two control molecules with high structural similarity to molecule 1, P1 (AVPIAQKDEVDKLVFFAECG) and 5 (AVPIAQKDEVDGC(Cy)KLVFFAEG) G) with recognition and self-assembled motifs, molecule 2 (MDEKAQKDEVDKLVFFAEC(Cy)G) with the self-assembled motif alone and molecule 3 (AVPIAQKDEVDEC(Cy)G) with the recognition motif alone  and 293T  for in vitro studies to evaluate the targeting mechanism. First, we examined the expression of XIAP in two cell lines by western blotting. As shown in Fig. 28. Then we found that the internalization of molecule 1 into cells was energy-dependent because the fluorescence intensity was decreased when the cells co-treated with energy inhibitor (oligomycin A)43  43 Fig. . As wide28, the fluorescence intensity of H460 cells treated with molecule 1 was significantly decreased compared with the untreated group. Additionally, down-regulating XIAP with P6 (ref. 49) in H460 cells and blocking the caspase-3 activity with z-VDK-FMK dramatically decreased the fluorescence intensity of molecule 2. All of the above results indicated that the activity of caspase-3 was the indispensable factor for molecule self-assembly. We speculate that the recognition of XIAP promotes the activity of caspase-3, which consequently cleaves molecule 1 and results in self-assembly. To study the supramolecular nanofibrils formation inside cells, we synthesized molecule 1 labelled 4-nitro-2,1,3-benzoxadiazole (NBD) (1-NBD)  and replaced the medium. After further incubation for 48 h, we still observed significant red fluorescence of molecule 1 in the cytoplasm by confocal microscopy. Under the same experimental protocol, only weak fluorescence was found in molecule 1-treated 293T cells Fig. . Statisttor Fig. . To obtator Fig. . To difftor Fig. . Similartor Fig. . Howevertor Fig. . Consequ5. Therefore, we speculated that the signal mainly came from the metabolite of the nanofibrils in tumour. These results implied that the molecular recognition and subsequent caspase-3 cleavage allowing self-assembly were both indispensable for tumour-specific targeting. To validate the generality of the TCASS in different tumours in vivo, besides the H460 lung xenografts mouse model, we carried out additional experiments in EJ bladder xenograft tumour model and evaluated the targeting, accumulation and retention  with xenografted XIAP-overexpressing H460 and EJ cells. Time-dependent fluorescence images of mice treated with molecules 1, 2 and 3 (14 mg/kg) were recorded at 1 h post injection and thereafter up to 5 days using a multispectral imaging system. Over time, during the clearance of the molecules, the stranded fluorescence signal in the tumour site of the mice treated with molecule 1 was clearly differentiable from the surrounding normal tissues as early as 4 h post injection. After 12 h, only the tumour continued to have a relatively strong fluorescence signal, which was stable and lasted up to 5 days Fig. . In contion Fig. . EJ cellion Fig. . The resght Fig. .Fig. 4Tht1/2β) values of molecules 1, 2 and 3 were 1.15 ± 0.10, 0.91 ± 0.23 and 1.06 ± 0.17 h, respectively  , the concentration of molecule 1 was six-fold and three-fold higher than that of 2 and 3, respectively  of molecule 1  were significantly longer than that of molecule 2  and 3 . Meanwhile, we noted that molecule 1 showed a similar distribution and clearance behaviour to molecules 2 and 3 at 2 h. Moreover, clearance of medicine (CL) of molecular 1 removal was significantly slower than that of molecules 2 and 3. These results suggested that molecule 1 exhibited small-molecule behaviour in major organs other than the tumour. The imaging of tissue lysates at different times showed that molecules 1, 2 and 3 followed a clearance pathway comparable to that of small molecules. The strong accumulation of molecule 1 observed in kidneys and liver were indicative of excretion from the organs of the RES, and the imaging of urine at different time points also indicated renal excretion -rich organs, we harvested the major organs of mice for ex vivo imaging at 48 h Fig. . Biodistely Fig. . Furtherely Fig. . After tely Fig. . We seleely Fig.  and 38 aely Fig.  formed bely Fig.  and lipoely Fig. . It was ion Fig.  and 43. ion Fig. –4132 rep2 nanoparticles and liposomes labelled with cyanine dye with diameters of approximately 100 nm, were used as controls  imaging system. Typical nanomaterials, such as SiOues Fig. . The quaour Fig. . This enels Fig. . It is i55 as a payload  and 3-DOX as a control molecule (positive only for recognition)  56 Fig.  and then 56 Fig. . Next, b 56 Fig. . Finallyays Fig. . In the ely Fig. . As we eely Fig. . The hisely Fig. . Althougely Fig. . These rely Fig. . Therefo57. This intravesical administration method minimized potential systemic toxicity of compounds including chemo-drugs, imaging agents, etc.57. Like 5-ALA was widely utilized for clinical intravesical instillation58, we used the same method to treat the EJ orthotopic bladder cancer mice model and the isolated intact bladder with molecule 1. Firstly, mice were sacrificed after instilled and incubated 100 uL molecule 1 in PBS for 1 h. Then, the bladder was removed and placed in a blackboard for ex vivo fluorescence imaging. As a result, the fluorescence signal was observed on the tumour site  on the 3, 7 and 15 days for blood collection. During our experiments, we did not notice any obvious signs of acute toxicity for molecule 1-injected mice within 15 days. To reveal any potential toxic effect of molecule 1 on the treated mice, we performed blood biochemistry and haematology analyses. As a result, the levels of major liver function markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and the albumin concentration, were all within reference normal ranges2 nanoparticles and liposomes with diameters of approximately 100 nm). Meanwhile, the organ competition behaviour of the TCASS was comparable to that of small molecules, which were rapidly excreted from the liver and kidneys, which linked directly to the reduced nanotoxicity. Furthermore, by integration of chemodrug , the TCASS has an enhanced therapeutic effect and negligible systemic toxicity. Importantly, the significantly prolonged median survival time further convinced the therapeutic outcome of this strategy. Finally, by integration of a contrast agent, our TCASS has a high specificity and sensitivity for detecting bladder cancer in an isolated human intact bladder, verifying its potential clinical translation. This study provided a de novo concept for tumour targeting that might be more efficient than active/passive targeting mechanisms.In summary, we presented a TCASS consisting of (i) a tumour-specific recognition motif, (ii) an enzymatically cleavable linker, (iii) a self-assembly motif and (iv) a functional molecule. We systematically studied the TCASS in different mouse models to optimize the tumour nanomedicine, including accumulation, penetration and organ competition. TCASS exhibited high-efficiency molecular accumulation and retention in tumour tissue. Moreover, this system enhanced tumour penetration compared with that of typical nanomaterials -1,1,3,3-tetramethyluronium hexafluorophosphate  was dissolved in 0.4 M 4-methylmorpholine (NMM)/DMF solution as a coupling reagent. The peptide was deprotected and cleaved from the resin by reacting with the mixture of trifluoroacetic acid , H2O , 1,2-ethanedithiol  and triisopropylsilane  for 2.5 h in ice bath. The peptide was precipitated into cold diethyl ether and dried under vacuum. The peptides were characterized with mass spectrometry (MALDI-TOF) and HPLC. The peptide was reacted with cyanine dye (Cy) (1.1 equiv) for 2 h in PBS. And then the final reaction solution was dialysed against deionized water to obtain a green solid with a 95–99% yield. The compounds were characterized with HPLC, mass spectrometry (MALDI-TOF) and nuclear magnetic resonance (H1 NMR).The peptides were synthesized by conventional solid phase peptide synthesis (SPPS). The 9-fluorenylmethoxycarbonyl (Fmoc) coupling chemistry on the Wang-resin was used. The Fmoc-protection groups were removed by piperidine/DMF (v/v = 1:5) solution. The Fmoc protected amino acids (10 equiv.) were dissolved in DMF.  and 293T cells (3111C0002000000112) were treated with molecule I-1 for 12 h, then fixed in 3% glutaraldehyde overnight at 4 °C, further washed triple with PBS, and fixed in buffer  for 1.5 h. Next, treated-H460 cells were rinsed three times in PBS. Subsequently, samples were dehydrated through increasing concentration of acetone solutions , then utilize 1:1 and 1:2 propylene oxide Epon 812 mixture for twice, respectively. Finally, the sample infiltrated with pure Epon 812 at 4 °C overnight, then solidified at 37 °C, 45 °C and 60 °C for 24 h, respectively. When the samples were accomplished, they were sectioned with a thickness of 70 nm, either transverse section or longitudinal section. Next, the sections were put onto copper grids; post-stained in 2% uranyl acetate in 70% ethanol for 0.5 h rinsed with HThe lysis buffer which contains 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor was used to re-suspend the pretreated cells. The estimation of the protein content was accomplished by a BCA kit (Applygen). Each sample (60 μg of protein) was subjected to SDS-PAGE and then transferred onto nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk, 0.1% (v/v) Tween-20 in 10 mM TBS, incubated overnight with primary antibodies at 4 °C on a shaker, incubated with an appropriate secondary antibody for 1 h at room temperature on a shaker and subsequently scanned on a Typhoon Trio Variable Mode Imager. Pretreated cells were re-suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor. The protein content was estimated using a BCA kit (Applygen). Each sample (60 μg of protein) was subjected to SDS-PAGE and then transferred onto nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk, 0.1% (v/v) Tween-20 in 10 mM TBS, incubated with primary antibodies overnight at 4 °C, incubated with an appropriate secondary antibody for 1 h at room temperature and subsequently scanned on a Typhoon Trio Variable Mode Imager. The uncropped and unprocessed scans are shown in Supplementary Figs. 18 and 33.n = 3). When the tumour grew to a single aspect of about 0.5 cm, we injected the molecules 1, 2 and 3 intravenously in the same dose of 14 mg/kg in PBS solution, respectively. Fluorescence images were measured by a Maestro II in vivo imaging system (CRi) at 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 h post injection. Further, tumours and other major organs  were collected for ex vivo fluorescence imaging at 48 h post injection. The organ biodistribution of the 1, 2 and 3 was examined using H460 xenografts mice. The mice were sacrificed at 2, 4, 8, 12, 24 or 48 h post injection. Finally, all the major organs and tumour were collected, weighed, and homogenized (0.3 mL of lysis buffer per 100 mg of tissues). The homogeneous tissue solutions were obtained for fluorescence imaging by IVIS Spectrum CT. Known concentrations of compound 1, 2 or 3 are used to make the standard curve (concentration from 2.8 to 25.2 μg/mL), the “Predicted R-Squared” value of 0.99. Then we utilized this standard curve to measure the molecule content in each tissue. In addition, EJ xenograft tumour mice model was built, as above described, to validate the generality of the strategy.Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Center for Nanoscience and Technology (NCNST), China. Female Balb/c nude mice  were purchased from Vital River Laboratory Animal Technology Co. Ltd. H460 cells were subcutaneously implanted into these mice on the right flank. Then the mice were randomly grouped (n = 3), liposome and SiO2 nanoparticles labelled by Cy  dissolved into PBS was intravenously injected through tail vein of mice. The control group was PBS at the same conditions. After injection, the mice were scanned with MOST (mode: MOST 128) at 12 h post injection individually, then the averaged PA signals of the tumour areas were obtained with the ViewMSOT software. Next, 3D reconstructions were generated from the PA images with Image J software. The continuous tomography was used with a space interval of 0.2 mm in a range of 2 cm. In order to quantify the PA signal intensity, the intratumoral distribution area was normalized with the tumour regions.All animal experiments were performed as previously described. H460 cells were also subcutaneously implanted on the right flank of xenografts mice. After the tumour had developed, Molecule 1  and i.v. administrated with PBS, DOX, 3-DOX and 1-DOX, under an identical DOX dose of 2.0 mg/kg. Treatment is administered once every other day for a total of five times. The body weight and the tumour volume were measured once every other day. Tumour volume (V) was calculated by the following equation: V = LW2 /2, where L was the longer and W was the shorter diameter (mm). Mice with a tumour volume of more than 2000 mm3 or a 20% weight loss are a humanitarian endpoint. Kaplan–Meimer survival curve was compared using the Cox Mantel log-rank test. Statistical analyses were performed using SPSS  version 22.0.All H460 xenografts mice were built as described above. When the tumour volume reached 50 mmn = 3) treated with molecule 1 was systematically investigated over 15 days. The mice were sacrificed after treatment with molecule 1 at a dose of 14 mg/kg on the 3rd, 7th and 15th day for blood collection. The blood biochemistry and haematology analyses were performed by Vital River Laboratory Animal Technology Co. Ltd. The histology evaluation was performed by Servicebio. All the toxicology evaluation was carried out in blind manner.The healthy female Balb/c nude mice  was infused and incubated in the bladder for 2 h. Finally, the mouse urethra was clamped to prevent tumour cells from overflowing along the urethra. The growth of the mice was observed daily to observe the presence or absence of gross haematuria as well as the growth of the bladder mass by palpation of the abdomen. After 3 weeks, a 1 cm incision is made through the skin and abdominopelvic wall, anddr then the bladder was exteriorized to confirm tumour growth.Mice were intraperitoneally anaesthetised with Pentobarbital sodium (40 mg/kg body weight) and fixed in the abdominal position. Then, the abdomen was gently squeezed to empty the bladder. Under sterile conditions, mice were catheterized with a modified IV catheter (24G) to slightly damage the inner mucosa of the bladder. Subsequently, 100 μL of EJ cells  urinary catheter with sterile saline, and 70 mL of molecule 1 in PBS  was instilled and incubated at 37 °C for 1 h. Then, the unbound molecule 1 was removed by irrigating with sterile saline three times for 5 min, the bladder was opened longitudinally on the anterior wall. Ex vivo fluorescent and white light imaging of the bladder cancer was performed using a near infrared imaging system. To explore the correlation between appearance of fluorescent signal and cancer lesions, standard pathological analysis of represented fluorescent spots were carried out after imaging.Data are mean ± standard deviation for in vitro and in vivo. Statistical significance was one-way ANOVA followed by post hoc Tukey’s test. Kaplan–Meimer survival curve was compared using the Cox Mantel log-rank test. Statistical analyses were performed using SPSS  version 22.0.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide and non-small-cell lung cancer (NSCLC) represents approximately 85% of all lung cancer patients . ImmunotTo evaluate subtype, frequency, phenotype and spatial distribution of TILs in patients with NSCLC, we isolated CD4+ and CD8+ TILs in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients [In light of these findings, we could anticipate that heterogeneity in the subtype, amount, and TCR repertoire of TILs in different tumour regions could be due to spatial difference in nature and abundance of tumour antigens within the same tumor , 6. FurtThese data were critical for immunotherapies for NSCLC. First, multiple biopsies were needed to obtain a comprehensive overview of TILs to an individual NSCLC. Second, compared with CD8+ TILs, the amount and TCR diversity of CD4+ TILs are generally higher. Third, adoptive cell therapy using TILs in tumour center could be more likely to achieve clinical efficacy in patients with NSCLC. Last, characteristics of TILs in tumour centers could more accurately predict response to immunotherapies, such as anti-PD1 immunotherapy.In summary, our continuous work would further profile characteristics of TILs in tumour center and identify tumour-specific TILs which could be applied to immunotherapy for patients with NSCLC.


Determining the rate of breast cancer (BC) growth in vivo, which can predict prognosis, has remained elusive despite its relevance for treatment, screening recommendations and medicolegal practice. We developed a model that predicts the rate of in vivo tumour growth using a unique study cohort of BC patients who had two serial mammograms wherein the tumour, visible in the diagnostic mammogram, was missed in the first screen.SM-INVIGOR) index was developed using tumour volumes from two serial mammograms and time interval between measurements. We then developed a machine learning-based surrogate model called Surr-INVIGOR using routinely assessed biomarkers to predict in vivo rate of tumour growth and extend the utility of this approach to a larger patient population. Surr-INVIGOR was validated using an independent cohort.A serial mammography-derived in vivo growth rate (SM-INVIGOR stratified discovery cohort patients into fast-growing versus slow-growing tumour subgroups, wherein patients with fast-growing tumours experienced poorer BC-specific survival. Our clinically relevant Surr-INVIGOR stratified tumours in the discovery cohort and was concordant with SM-INVIGOR. In the validation cohort, Surr-INVIGOR uncovered significant survival differences between patients with fast-growing and slow-growing tumours.Surr-INVIGOR model predicts in vivo BC growth rate during the pre-diagnostic stage and offers several useful applications.Our  In addition, BC growth rate evaluated using tumour size from mammograms may predict tumour response to chemotherapy, and may help in determining the likely time of tumour initiation and previous tumour size in medicolegal cases.7 BC growth rate is also associated with prognostic variables, such as lymph node status, stage and vascular invasion;8 however, the prognostic and predictive value of BC growth rate has not been harnessed in routine practice due to the inherent difficulty in its assessment in the short intervals between diagnosis and treatment.Breast cancer (BC) is a heterogeneous disease with tumours exhibiting variable morphology, molecular profiles, behaviour and response to therapy. Mounting evidence demonstrates that BC shows variable rates of growth, which has important clinical and medicolegal implications.12 The complexity of the processes controlling BC growth and the interaction with the tumour microenvironment make assessment and prediction of BC growth rate a challenging task. Therefore, serial imaging of BC at different time points is considered as the best model available for assessing the in vivo growth rate and for determining associations between potential intrinsic growth rate determinants and BC behaviour, including response to therapy.Although the growth rate of BC in vivo is strictly regulated, it appears to be dependent on the balance between several variables including growth fraction (the tumour cells that are proliferating and leading directly to the addition of new tumour cells), the rate of tumour cell loss by apoptosis and/or necrosis, tumour cells’ doubling-time/kinetics, and the surrounding microenvironment including angiogenesis, blood supply and host immune response to the proliferating tumour cells.This study utilises a discovery cohort comprising clinically and molecularly well-characterised data from BC patients who underwent serial mammography. It is a unique and rare cohort because the second mammogram illuminated that the tumour was indeed “missed” during the first mammogram. We find that this one-of-a-kind cohort can be interrogated to (a) identify predictors of BC in vivo growth rate, (b) evaluate the impact of BC growth rate on disease outcome and (c) develop a surrogate model that robustly predicts pre-diagnosis in vivo growth rate for patients who would normally not have tumour volume data from two serial mammograms. In contrast to a matched first-presentation-only BC patients’ cohort, BC growth rate in this study is determined by the changes in tumour volume between sequential mammograms, wherein the first mammogram “mistakenly” reported the case as normal/benign, and the cancer was identified in the screening mammogram on a retrospective review subsequent to the second (diagnostic) mammogram Fig. .Fig. 1ScThe study cohort comprises 114 BC patients aged between 50 and 70 years who were presented at the Nottingham City Hospital from 1988 to 2008 with BC, and for whom review of the previous screening mammogram showed a previously undetected tumour at the same affected site. This may have been due to either a false-negative screening outcome or due to minimal visible signs of malignancy on the previous mammogram. Mammographic abnormalities included measurable soft tissue abnormality  on screening and diagnostic films. On retrospective review of the previous mammogram after the disease diagnosis, two radiologists (blinded to each other’s observations) confirmed the “missed” cancer. We selected patients in whom a soft tissue abnormality was detected (upon retrospective review of prior screening mammograms) at the site of the subsequent cancer. Due to a misdiagnosed mammogram, this cohort uniquely comes with an earlier screening measurement with a visible tumour. Clinicopathological data including age, histological tumour type, primary tumour size, lymph node status, histological grade, Nottingham Prognostic Index (NPI), vascular invasion and patients’ outcome data were obtained. BC-specific survival (BCSS) was defined as the time interval (in months) between the primary surgeries and death from BC. The mean survival time of this cohort of patients was 120 months. Clinicopathological variables were available for 92 cases, and the BCSS was available in 90 cases; thus, we restricted our study to these cases Fig. .13 For tumour growth rates, we tested exponential growth,15 the Gompertz model,16 and power law growth with the exponent set to both the classic value of 2/318 and 1/219 as shown in Table The two measurements in the screening and diagnostic mammograms were assumed as tumour diameter and tumour height, which were then used to calculate tumour volumes at the time of screening and diagnosis. The greater mammogram dimension was assumed as height corresponding to the diameter of the semi-major axis, and the other dimension was regarded as diameter of the semi-minor axis. For tumour volume calculation, we considered the aforementioned dimensions as volume inputs for a cylinder, sphere and an oblate spheroid.19 were tested to find the optimal combination that was prognostic. Growth rate indices that combined tumour volume  and individual growth functions  or Gompertz growth) were compared on the basis of their prognostic ability. Growth rates were used either as a continuous variable or through a fast/slow growth cut-off determined through optimising the log-rank statistic.21 Both forms of all growth rates were analysed univariately in a Cox proportional hazard regression model using 10-year breast cancer-specific survival (BCSS), and corresponding model fits were ranked with the Akaike Information Criterion (AIC).22 The best-fitting growth rate index was chosen via the lowest relative AIC and was used in subsequent analyses. The data related to changes in volume of the lesion between the time of screening and at diagnosis, as well as the time between screening and diagnosis, were used to estimate the Serial Mammography-derived In-vivoGrowth Rate (SM-INVIGOR)  Fig. . To contOR) Fig. .SignalStain®Cleaved Caspase-3 (Asp175) IHC Detection Kit #8120, Cell Signaling Technology) was used following the manufacturer’s instructions. Other markers were stained using our protocols as previously published.24For each patient, a representative formalin-fixed paraffin wax-embedded (FFPE) tumour block of the resected tumour was obtained from the Nottingham breast tumour bank Fig. . Full-fa26 Ki67 and cleaved caspase-3 were assessed and scored as previously described.24 BC molecular subtypes were defined based on their IHC expression profile into (a) luminal (ER + and/or PR + /HER2-), (b) HER2 + (HER2-positive), (c) triple-negative  and (d) basal-like breast cancer (BLBC: TN + CK5/6 + ).24 A total of 92 cases were informative for IHC biomarkers, and these comprised the study cohort in the subsequent analyses including molecular markers  blinded to each other’s scores and clinicopathological data. Cases with discordant results were further reviewed by both observers to achieve scoring consensus. For each marker, the percent and intensity of staining were assessed, and H-scores were generated. For ER, PR and HER2, cut-offs according to published guidelines were used.ers Fig. .SM-INVIGOR to predict fast or slow in vivo growth rate for cases where only a single (diagnostic) mammogram is available  with optimised hyperparameters28 were then tested. The machine-learning algorithm and feature set that resulted in the maximum fivefold cross-validated accuracy (mean of 100 iterations) were chosen. For each trained machine-learning model (combination of biomarkers), hyperparameters were fit through Bayesian optimisation28 over 180 iterations  was tested in an independent, well-characterised large validation cohort of 1241 BC patients using Kaplan–Meier survival analysis .Most patients in the study cohort showed features associated with good prognosis, including lower grade and negative (65%) or early positive pN1; 26%) lymph nodes. Age at the time of diagnosis ranged from 50 to 73 years . There was a predominance of the luminal A subtype with 85% positive for ER, while HER2 overexpression was identified in only 6% of the patients. Ki67 staining ranged from 0 to 96%, with a mean expression of 19%  stratified the tumours into slow-growing and fast-growing subgroups and produced a minimum cross-validated AIC of 152.621 (Table 3 (mean of 2742 ± 7619 mm3). This contrasted with tumour volumes at diagnosis, which ranged from 61 to 61,562 mm3 (mean = 5573 ± 8768 mm3). The mean time difference between date of first screening and that of second diagnostic screening was 18 months . Tumour growth rate differed considerably from patient to patient, ranging from 0 to 0.53 mm3/day (mean = 0.08 ± 0.13 mm3).Since fast in vivo growth prior to diagnosis is a sign of aggressive disease and could lead to poor outcomes, we reasoned that the growth rate model of choice would be the one that is most prognostic. Thus, we evaluated various combinations of growth rate functions and assumptions regarding the tumour’s three-dimensional shape. The best-fitting model of tumour volume and growth rate was obtained using the assumption that the study cohort comprises spherical tumours growing at a power law (α = 0.5) rate; this growth rate function (21 Table . Using tSM-INVIGOR used a cut-off of 0.045 mm3/day to stratify tumours into slow-growing (n = 53) and fast-growing (n = 37) subgroups. Faster SM-INVIGOR significantly associated with clinicopathological factors normally associated with poorer prognoses, such as larger histological tumour size (p = 0.0023), high grade (Grade 3) (p = 0.0186), more mitotic divisions (p = 0.0134), apparent vascular invasion (p = 0.0139) and a poor NPI (p = 0.011)  compared with other subtypes (p < 0.05). Among the proliferation/apoptosis-related biomarkers that were immunohistochemically assessed (Table p = 0.0003) between the fast-growing (24%) versus slow-growing (11%) tumour subgroups. Furthermore, patients with higher tumour growth rate showed significantly poorer survival (BCSS = 71.7%) relative to the slow-growing tumours (BCSS = 91.9%) as shown in Kaplan–Meier’s survival graph  upon controlling for common clinicopathological variables, including grade, age and ER status. In fact, SM-INVIGOR was the only variable significantly associated with BCSS in our multivariable analysis  Fig. . SM-INVIed Table , only Kiaph Fig. . SM-INVIsis Fig. .Fig. 2PrSM-INVIGOR is limited in its utility to derive in vivo tumour growth rate for most BC patients in routine clinical practice. Therefore, to extend the benefits of having growth rate data (or estimates) to a much larger group of patients lacking a second mammogram, we developed a machine learning-based surrogate growth rate model for SM-INVIGOR and called it Surr-INVIGOR (described in Supplementary Data). Surr-INVIGOR non-linearly combines multiple clinicopathological variables and immunohistochemical biomarkers to predict in vivo growth rate. First, we evaluated the ability of individual clinicopathological variables to serve as potential surrogate features and discriminate between the fast-growing and slow-growing tumour subgroups of our study cohort , mitotic score , tumour size (p = 0.003619), NPI (p = 0.004163) and grade (p = 0.021128) differed significantly between the fast-growing and slow-growing tumours. The seven variables  with p-value < 0.2 were then tested in multiple machine learning-based classification algorithms via sequential selection  were used in a K-nearest neighbour algorithm or KNN (accuracy or concordance with the classification yielded by SM-INVIGOR = 0.706). The ensemble also yielded a 70% accurate classifier, but required four additional features; the more parsimonious KNN was thus selected for use in Surr-INVIGOR. Fitting an optimal regression model to predict the growth rate continuously resulted in a poor R2, peaking at 0.22, as shown in Fig. Surr-INVIGOR model was a clinically relevant, superior choice compared with regression-based models.Unlike the patients in our unique discovery cohort, most begin therapy at an initial cancer diagnosis, and are therefore unlikely to have two serial mammograms with two tumour volume measurements. Because of this difference, ion Fig. . The maxSurr-INVIGOR in an independent BC case series (n = 1241) from Nottingham University Hospital, UK. Patient age at the time of diagnosis ranged from 21–71 years . Most patients showed features associated with good prognosis, including negative lymphovascular invasion (55.3%), and negative (61%) or showed 1–3 positive (30%) lymph nodes. Patient follow-up time ranged from 1 to 120 months . The clinicopathological features of patients are summarised in Table We then evaluated the prognostic ability of ries n = 241 from Surr-INVIGOR model, using the same input parameters on this naive validation cohort resulted in significant BCSS stratification. Patients in the fast growth rate group  had a significantly lower survival than patients in the slow growth rate group  alongside grade as shown in Fig. The clinicopathological variables that discriminated between slow-growing and fast-growing tumours are depicted in Fig. 3/day) for the slow-growing subgroup, we can estimate the date after which the tumour was definitely present within the patients in the slow-growing tumour subgroup. Using these assumptions, we determined that the average tumour age at diagnosis of slow-growing tumours was 4.7 years  power law (α = 0.5) growth rate  available from a pair of serial mammograms to derive their in vivo growth rates (SM-INVIGOR). We explored the potential association of a larger number of molecular biomarkers with their in vivo BC growth rate, reaffirmed that fast tumour growth rate has a profound impact on prognosis, developed and validated a surrogate model (Surr-INVIGOR) that can predict a gross scale (fast versus slow) in vivo growth rate accurately in routine practice, and its medicolegal consequences.Although several studies have investigated variables associated with pre-diagnosis in vivo BC growth rate, only clinicopathological variables and a few molecular biomarkers have been studied in this context, and the available tumour dimensions were limited due to the measurement of the tumour’s long-axis only.34 Even though some authors quote high sensitivity (>90%) for diagnostic mammography, such results are not universal.35 Among many factors, age appears to be one of the important factors underlying false-negative reporting, because the high-radiographic density of breast in young women makes detection difficult.6 Mammograms are generally capable of detecting tumours as small as 2 mm in diameter, which equates to a tumour of ~107 cells and about 23 tumour doublings.36 In our study cohort, however, patients with tumours ranging from 4 to 55 mm received false-negative diagnoses in their screening mammograms showing the imperfection associated with this technology and inherent human limitations associated with reading radiology films. Whether the spread of a tumour is due to delays in diagnosis and initiation of treatment, or due to the inherently more aggressive nature of the tumour cells themselves  is another highly controversial matter. Natural fears that the delay in diagnosis has reduced their chances of survival or of avoiding the life-sapping effects of chemotherapy, or the feeling that cosmetic outcomes which would have been better had the tumour been detected earlier, are frequent causes of patients seeking legal redress. The importance of breast imaging in BC diagnosis and the use of mammography in screening has thus pushed breast radiologists into the frontline for medicolegal actions.37 Cancers missed at screening but followed by a positive diagnostic mammogram are not common, yet false-negative mammography is amongst the principal reasons for delayed diagnosis of BC.34 Only few population-screening programmes have reported the data on this group of cancers, which makes our study cohort uniquely valuable. This cohort allowed us to develop a model to predict pre-diagnostic in vivo tumour growth rate, and provide insights into the potential prognostic consequences of delays in BC diagnosis.The success of breast screening lies in the timely detection of cancer on mammography. False-negative mammography is among the principal reasons for delayed diagnosis of BC.SM-INVIGOR varies considerably, and is consistent with findings by Weedon-Fekjaer et al.5 who reported that the time BC takes to grow from 10 mm to 20 mm in diameter varied from less than 1.2 months to more than 6.3 years. Our current study also reinforced previous findings that higher grade and larger tumours with high proliferative activity are likely to have faster SM-INVIGOR, and that faster pre-diagnosis growth rate predicted shorter survival.39 We also found that the status of lymphovascular invasion (LVI) correlated with growth rate; with highly proliferative and fast-growing tumours more likely to develop when there is increased provision of nutrients to the tumour cells from the leaky invaded blood vessels. Our results indicated that increasing SM-INVIGOR increases the risk of mortality of the disease. However, SM-INVIGOR cannot be included as a prognostic variable in routine clinical practice because of difficulty in evaluating it in the short interval between diagnosis and treatment.Our study has yielded several key insights into features and the prognostic significance of the rate of tumour growth in its early stages. In our study, we found that Surr-INVIGOR to predict the pre-diagnosis in vivo BC growth rate after testing multiple clinicopathological and molecular variables  using diverse machine-learning algorithms. The optimal algorithm, a KNN which used Ki67, MI and size, stratified both the study and validation cohorts into two subgroups with very distinct outcomes. Surr-INVIGOR further allowed routine clinical parameters to be used in patients with slow-growing tumours to determine tumour size at various time points before the diagnosis of the tumour. For fast-growing tumours, immediate surgery is often recommended, as delays may result in upgrading of clinical T stage. Surr-INVIGOR may thus have a potential use in medicolegal cases, and may be used to guide screening and perhaps even follow-up intervals in selected groups of BC patients.Therefore, we developed 41 the results from our validation cohort showed a significant correlation between BC molecular subtypes and pre-diagnosis tumour growth rate, wherein a higher growth rate was observed in triple-negative/basal-like BC patients. Previous studies have indicated that faster-growing tumours lead to poorer survival.45 Our results compellingly demonstrated that high pre-diagnosis in vivo BC growth rate increases the risk of mortality from the disease regardless of potential clinicopathological cofounders. Some previous studies did not find such statistically significant associations,4 which might be because in those studies, the tumour volume was calculated using only one dimension—a method that can introduce considerable inaccuracy into growth rate calculations. In this study, we utilised a combination of power law growth rate and spherical volume, both of which were significant in a previous study using two-dimensional breast mammogram data,19 and showed the most significant prognostic relevance in our data.Consistent with previous studies,30 This can be explained by the unique nature of these slow-growing early-stage tumours in this study. By contrast, aggressive tumours are likely to present without prior mammographic abnormality.46 In line with these results, Kalager et al.47 have reported that BCs presenting as interval cancers were slightly larger than symptomatic BC, but there was no difference between the two groups regarding lymph node status or patient outcome. Moreover, our results indicated that the impact of SM-INVIGOR on disease stage and development of LVI is limited. However, this study holds a few limitations: due to the unique nature of the study cohort and the lack of similar missed cancer cohorts, the SM-INVIGOR growth index could not be readily validated. In addition, this is a retrospective, single centre study and adjuvant treatment regimens were not factored in our analyses. Validation of the model in diverse cohorts is necessary before it can be applied for the prediction of in vivo growth rate and determination of the likely tumour initiation date and previous tumour size in clinico-legal cases. If validated in further studies, the model developed herein could potentially guide treatment selection, as it prognostically distinguishes fast-growing tumours from slow-growing ones. For example, for fast-growing tumours, immediate treatment in the form of primary systemic therapy (rather than surgery) may be required. Moreover, HER2 is known to be related to rapid growth of tumours, and might be a good marker to add to the Surr-INVIGOR; however, our study cohort was overwhelmingly HER2 negative and thus its impact within a prognostic model could not be properly measured. Further analysis may be required in a diverse cohort.Review of previous mammography is carried out as a routine practice at Nottingham Hospital, and cases that show an abnormality at the same site as the diagnosed tumours are considered as cancers potentially missed in the prior screening. Some of these tumours are only detectable in retrospect with knowledge of the diagnostic mammograms, and if all such subtle areas were recalled for further assessment, this would likely increase the false-positive rate beyond what is regarded as acceptable in the NHS breast screening programme. The impact of such delay in the diagnosis on the presentation and outcome of these tumours compared with matched population of women who presented for the first time as symptomatic or with screen-detected BC remains to be defined. Most tumours included in our study (similar to other studies looking at screen-detected tumours) by their very nature, were small, slow-growing luminal tumours and infrequently expressed basal markers or HER2 with similar nodal status.In conclusion, this study has demonstrated that multiple factors control BC growth; when considered together Ki67, mitotic index and tumour size produce a robust prediction model of pre-diagnostic growth rate and can be used to classify BCs as slow growing or fast growing. The impact of missing subtle cancers in screening mammography seems to depend on whether the tumour was slow growing or fast growing prior to diagnosis, as fast-growing tumours were associated with poorer outcomes and perhaps reflected more aggressive tumour biology. Independent validation of these findings in multiple and more diverse cohorts is warranted.Supplementary files