Wilms tumour is the most common childhood renal cancer and is genetically heterogeneous. While several Wilms tumour predisposition genes have been identified, there is strong evidence that further predisposition genes are likely to exist. Our study aim was to identify new predisposition genes for Wilms tumour.In this exome sequencing study, we analysed lymphocyte DNA from 890 individuals with Wilms tumour, including 91 affected individuals from 49 familial Wilms tumour pedigrees. We used the protein-truncating variant prioritisation method to prioritise potential disease-associated genes for further assessment. We evaluated new predisposition genes in exome sequencing data that we generated in 334 individuals with 27 other childhood cancers and in exome data from The Cancer Genome Atlas obtained from 7632 individuals with 28 adult cancers.TRIM28, FBXW7, and NYNRIN. 21 of 33 individuals had a mutation in TRIM28; there was a strong parent-of-origin effect, with all ten inherited mutations being maternally transmitted (p=0·00098). We also found a strong association with the rare epithelial subtype of Wilms tumour, with 14 of 16 tumours being epithelial or epithelial predominant. There were no TRIM28 mutations in individuals with other childhood or adult cancers. We identified truncating FBXW7 mutations in four individuals with Wilms tumour and a de-novo non-synonymous FBXW7 mutation in a child with a rhabdoid tumour. Biallelic truncating mutations in NYNRIN were identified in three individuals with Wilms tumour, which is highly unlikely to have occurred by chance (p<0·0001). Finally, we identified two de-novo KDM3B mutations, supporting the role of KDM3B as a childhood cancer predisposition gene.We identified constitutional cancer-predisposing mutations in 33 individuals with childhood cancer. The three identified genes with the strongest signal in the protein-truncating variant prioritisation analyses were TRIM28, FBXW7, NYNRIN, and KDM3B—are involved in diverse biological processes and, together with the other 17 known Wilms tumour predisposition genes, account for about 10% of Wilms tumour cases. The overlap between these 21 constitutionally mutated predisposition genes and 20 genes somatically mutated in Wilms tumour is limited, consisting of only four genes. We recommend that all individuals with Wilms tumour should be offered genetic testing and particularly, those with epithelial Wilms tumour should be offered TRIM28 genetic testing. Only a third of the familial Wilms tumour clusters we analysed were attributable to known genes, indicating that further Wilms tumour predisposition factors await discovery.The four new Wilms tumour predisposition genes identified—Wellcome Trust. Wilms tumour is a childhood kidney tumour that affects one in 10 000 children. Its histology is similar to that of the developing kidney and is typically triphasic, with blastemal, stromal, and epithelial components.WT1, CTR9, and REST.PIK3CA-related overgrowth spectrum.5Wilms tumour is primarily a non-familial condition, with only about 2% of affected individuals having a relative with the tumour.Evidence before this studyWilms tumour is a rare childhood kidney tumour. We searched PubMed for papers in English with the terms “Wilms” AND “genetic” OR “mutation” OR “familial” OR “syndrome”, yielding 2801 papers that we reviewed to identify those relevant to genetic predisposition to Wilms tumour. This review identified 17 genes previously shown to predispose to Wilms tumour and showed that further Wilms tumour predisposition genes must exist, because many syndromic cases and familial clusters have not been explained.Added value of this studyTRIM28, FBXW7, KDM3B, and NYNRIN. We showed that FBXW7 and KDM3B are pleiotropic cancer predisposition genes, and that KDM3B and NYNRIN might also cause non-malignant phenotypes, particularly intellectual disability. Our study identified TRIM28 as a major Wilms tumour predisposition gene, making a similar contribution to familial and unselected Wilms tumour as those of constitutional WT1 and REST mutations. We also found an association between TRIM28 mutations and epithelial histology and a strong parent-of-origin-effect, because all inherited TRIM28 mutations were maternally transmitted. Functional enrichment analyses revealed remarkable diversity in the biological pathways affected by Wilms tumour predisposition genes. We also found limited overlap between the 21 constitutionally mutated Wilms tumour predisposition genes and 20 genes somatically mutated in Wilms tumour.To our knowledge, this study is the largest exome sequencing study to date of individuals with Wilms tumour, involving 890 individuals, including 91 individuals from 49 familial Wilms tumour pedigrees. We identified four new Wilms tumour predisposition genes, Implications of all the available evidenceThis study provides new insights into the causes of Wilms tumour and describes the overall landscape of Wilms tumour predisposition. Wilms tumour shows remarkable genetic heterogeneity and aetiological complexity, which have substantial clinical impact. Genetic testing should be made available to individuals with Wilms tumour, but will need to encompass both broad genetic testing, for example by exome sequencing, and testing for 11p15 epigenetic abnormalities. Moreover, our findings suggest that more Wilms tumour predisposition genes are likely to exist, which will have relevance for future research and clinical testing.Research over the past 25 years has led to tremendous advances in our knowledge of Wilms tumour predisposition. However, available evidence suggests that our knowledge is still incomplete and that further predisposition factors remain to be discovered. In particular, the cause of many familial clusters is still unknown.The Cancer Genome Atlas (TCGA) on May 13, 2014  Study. All children with a childhood solid tumour from the UK were eligible for participation in the FACT study; children with familial childhood cancer anywhere in the world were also eligible for participation in the FACT study. We analysed lymphocyte DNA from 1215 individuals with 28 different childhood tumours, of whom 1206 had one childhood tumour and nine individuals had two different childhood tumours . This coThe FACT study was approved by the London Multicentre Ethics Committee (05/MRE02/17), and its collaborators are listed in the TRIM28, FBXW7, NYNRIN, and KDM3B genes by use of Sanger sequencing in the probands and any available relatives, designing primers with BatchPrimer3. We used the QIAGEN Multiplex PCR kit  to prepare PCRs, and the resulting amplicons were bidirectionally sequenced with BigDye Terminator cycle sequencing kits  and an ABI 3730 sequencer . We analysed sequencing traces with Mutation Surveyor software and by visual inspection with Chromas, version 2.13. We validated the CDC73 mutation with the TruSight Cancer panel (Illumina). We did in-silico analyses of variant pathogenicity with Alamut Visual, version 2.9.0.We did exome sequencing in samples from all childhood cancer probands and 119 individuals from 49 familial Wilms tumour pedigrees who did not have Wilms tumour, by using 50 ng genomic DNA and the Nextera DNA sample preparation kit  or 1·5 μg genomic DNA and the TruSeq exome enrichment kit (Illumina). The captured libraries were amplified by PCR with the supplied paired-end PCR primers. Sequencing was done with HiSeq 2000 (Illumina) or HiSeq 2500 (Illumina). We used the OpEx v1.0 pipeline to do variant calling in childhood cancer, adult cancer (TGCA), and ICR1000 exome data.NYNRIN among 844 individuals with Wilms tumour from the 890 included in this study. We used the R function to calculate the probability of observing two individuals with two NYNRIN protein-truncating variants: analyse_inherited_enrichment from the R package recessiveStats with hgnc=”NYNRIN”, chrom=“14”, counts$biallelic_lof=2, counts$lof_func=9, and cohort_n=844.We used the methods described by Akawi and colleaguesTRIM28 mutations with known inheritance being maternally inherited, assuming the baseline probability of inheriting the variant from either parent was 0·5.We used a binomial test—dbinom function in R—to calculate the probability of all ten We did a functional enrichment analysis with use of g:Profiler (version r1665_e85_eg32).ACTB, BCOR, NONO) with at least three somatic mutations in the TARGET discovery series.COSMIC cancer gene census to establish which of the 21 Wilms tumour predisposition genes, and which of the 20 somatically mutated Wilms tumour driver genes, were also somatically mutated in other cancer types.For the somatic cancer driver comparisons, we used 20 genes reported to be somatically mutated in Wilms tumour. These included 17 established genes reported in more than one publication and three newly reported genes .TRIM28 mutations. We also did not find any TRIM28 protein-truncating variants in 334 individuals with 27 other childhood cancers or in 7632 individuals with adult cancers, suggesting that TRIM28 pathogenic mutations primarily predispose to Wilms tumour. Of note, the TRIM28 mutations in two families in our study (ID_0498 and ID_0506) were independently, and coincidentally, reported while we were preparing this manuscript.TRIM28 929G→A, leading to the protein change Gly310Asp . Pathology information was available for 16 tumours, of which 14 were epithelial or epithelial-predominant , one had been inherited from an unaffected father (in ID_0592), and two were of unknown provenance  and these data suggest that FBXW7 is a Wilms tumour predisposition gene. Two of the four individuals with truncating FBXW7 mutations have died , in a child with an extra-renal rhabdoid tumour (ID_7520), which we assumed to be pathogenic.We identified truncating ave died . AdditioNYNRIN in three children from two families . Additionally, the probability of finding two different families with the same phenotype and two truncating NYNRIN mutations by chance is 4·0 × 10−9. One of the affected children had an inguinal hernia and another had epilepsy, hypothyroidism, and intellectual disability  and a truncating mutation in a child with hepatoblastoma, hyperpigmentation and hypopigmentation, autism, and intellectual disability  and non-synonymous variation . Taken together, these data provide strong evidence that KDM3B is a childhood cancer predisposition gene.In addition to the agnostic protein-truncating variant prioritisation analyses, we reviewed the exome data in genes proposed as possible childhood cancer predisposition genes, identified through a ID_2086; . In 2018WT1, TRIM28, REST, and 11p15 epimutations and uniparental disomy that result in biallelic IGF2 expression—each account for about 2%.These new discoveries bring the number of constitutionally mutated genes confirmed as Wilms tumour predisposition genes to 21. We estimate that, together, these constitutional events contribute to about 10% of unselected Wilms tumour . Four coREST (five [8%] of 63), TRIM28 (five [8%] of 63), and WT1 (four [6%] of 63). CTR9 mutations were present in three families and H19 hypermethylation was found in two families. Biallelic BRCA2 mutations, biallelic NYNRIN mutations, and a CDC73 mutation were found in one family each. We identified the CDC73 mutation through this present study .We have investigated 65 families with two or more cases of Wilms tumour over the last 20 years, including the 49 familial Wilms tumour pedigrees in this study . In two nt study . CDC73 iWT1, IGF2, TP53, and DICER1—promoted Wilms tumour oncogenesis in both contexts, and all four were also somatically altered in other cancers. A further five constitutionally mutated genes—PIK3CA, FBXW7, ASXL1, BRCA2, and CDC73—were somatically mutated in other cancer types but have not been proven to be somatic drivers in Wilms tumour. The remaining 12 Wilms tumour predisposition genes are not known to be somatically mutated cancer drivers.Finally, we assessed the overlap between the 21 Wilms tumour predisposition genes and 20 somatically mutated Wilms tumour driver genes . Only foTRIM28, FBXW7, and KDM3B—and one autosomal recessive Wilms tumour predisposition gene, NYNRIN. Constitutional TRIM28 mutations join constitutional WT1 and REST mutations as a relatively common contributor to Wilms tumour predisposition, accounting for about 8% of familial Wilms tumour and about 2% of unselected Wilms tumour.TRIM28 mutations and epithelial Wilms tumour, with most individuals with a TRIM28 mutation having Wilms tumour of predominantly epithelial histology. This suggests that TRIM28 mutations make a sizeable contribution to epithelial Wilms tumour, and we recommend that all children with this rare favourable subtype of Wilms tumour should be offered TRIM28 gene testing.To our knowledge, this is the largest exome sequencing study of individuals with Wilms tumour to date, including 890 affected individuals. Our analyses found three autosomal dominant Wilms tumour predisposition genes—TRIM28 mutations showed incomplete penetrance and a strong parent-of-origin effect, because all inherited mutations were maternally transmitted. TRIM28 is not imprinted, but it is located close to PEG3, which is imprinted and paternally expressed.PEG3 promotes growth but at the cellular level, it is a putative tumour suppressor.TRIM28 mutations is that somatic inactivation of the paternal wild-type TRIM28 allele, through mitotic recombination, also leads to loss of the paternally expressed PEG3, thus promoting tumourigenesis. There is precedence for this model in cancer predisposition: SDHD mutations predispose to phaeochromocytoma almost exclusively when inherited paternally.SDHD allele and maternally expressed growth inhibitory genes at the IGF1–H19 imprinting region has been proposed as the explanation for this pattern.TRIM28 is that the loss of heterozygosity in the tumour from ID_506_01 did not appear to include PEG3.TRIM28 mutations in Wilms tumour, confirmation of the parent-of-origin bias we observed should be possible in the near future. If this parent-of-origin effect is supported, DNA methylation analyses at the PEG3 imprinting control region and loss of heterozygosity and PEG3 expression analyses in tumours from individuals with TRIM28 mutations might help to provide a mechanistic explanation. From a clinical perspective, establishing if the penetrance of TRIM28 mutations is influenced by the parent-of-origin of the mutation is important, because this would have considerable impact on cancer risks and genetic counselling.Familial Wilms tumour pedigrees due to FBXW7 mutations predisposed to Wilms tumour and to other malignancies. Two of the five individuals carrying FBXW7 mutations had a malignancy other than Wilms tumour. ID_0811 developed osteosarcoma as an adult, after having Wilms tumour. ID_7520 had a rhabdoid tumour and did not have Wilms tumour. The assessment of additional individuals with rhabdoid tumour and de-novo FBXW7 mutations would be useful to further support the role of FBXW7 in rhabdoid tumour predisposition. Furthermore, a woman with Hodgkin lymphoma, adult Wilms tumour, early-onset breast cancer, and a constitutional FBXW7 deletion was reported in 2015,FBXW7 was reported in 2009.FBXW7 mutations might be at risk of multiple childhood and adult cancers and will require ongoing close monitoring. Notably, we believe that the in-frame FBXW7 variant reportedWT1 mutation.We provided evidence that constitutional KDM3B also appears to be a pleiotropic cancer predisposition gene. The four KDM3B pathogenic mutations reported to date have been associated with four different cancers: Wilms tumour and hepatoblastoma in our study, and acute myeloid leukaemia and Hodgkin lymphoma in the study by Diets and colleagues.FBXW7 and KDM3B in individuals with cancer will probably be required to establish the full spectrum of associated cancers, because of the rarity of truncating variants and the challenges in interpreting non-synonymous variation in these genes. There are indications that KDM3B and NYNRIN mutations might cause non-malignant phenotypes, particularly intellectual disability. More data on the contribution of these genes to non-malignant conditions will probably become available over the next decade through extensive exome and genome sequencing being done in children with developmental disorders.TRIM28 encodes a multidomain protein involved in the regulation of many cellular processes, including transcriptional repression, p53 degradation, pluripotency maintenance, autophagosome formation, epithelial-mesenchyme transition, and the DNA damage response.TRIM28 is highly expressed in many cancers, and its inactivation has not been previously associated with oncogenesis. This might explain why inactivating TRIM28 mutations seem to predispose to Wilms tumour alone. The mechanisms underlying this Wilms tumour predisposition are not known, but it is notable that TRIM28 is a major binding partner of AMER1, which is encoded by a gene that is frequently somatically mutated in Wilms tumour.The four genes we reported here have different functions, and it is unclear why or how they predispose to Wilms tumour. FBXW7 encodes the substrate recognition component of the E3-ubiquitin ligase SCF complex, which is responsible for recognising and binding phosphorylated substrates and regulating their turnover through proteosome degradation.FBXW7 is an established tumour suppressor gene and frequently mutated in many cancers, particularly endometrial and gastrointestinal cancers.FBXW7 is not a confirmed somatic driver in Wilms tumour because only one confirmed somatic FBXW7 point mutation has thus far been reported.PIK3CA. Constitutional mosaic PIK3CA mutations predispose to Wilms tumour, whereas somatic mutations at the same residue are common in many cancers but have not been reported in Wilms tumour.KDM3B encodes a histone H3 demethylase that specifically catalyses the demethylation of H3K9Me1 and H3K9Me2 residues, and is required for normal somatic growth in mice.KDM3B mutations have not been reported. Finally, there is very little known about the functions of NYNRIN, though NYN domains are thought to be involved in RNA processing and NYNRIN has been implicated in microRNA–mRNA regulation.The diverse functions of these four new Wilms tumour predisposition genes mirror the broad range of biological processes in which known Wilms tumour predisposition genes operate, as shown by our functional enrichment analysis . FunctioFWT1-linked families have not yet been discovered.Our analyses were designed to identify tumour suppressor genes in which constitutional truncating mutations predisposed to cancer, but were not designed to identify other mechanisms of cancer predisposition. For example, it is very possible that non-truncating coding variation might be contributing to familial and non-familial Wilms tumour, and future analyses to investigate this would be worthwhile. Non-coding, epigenetic, and mosaic abnormalities are all known to be relevant to Wilms tumour predisposition but were not investigated in our study. Notably, none of the known Wilms tumour predisposition genes are within the regions on chromosomes 2p24, 11q14, 5q14, 22q12, and Xp22 identified in a genome-wide association studyGenetic predisposition to Wilms tumour exhibits remarkable heterogeneity, and this is particularly noteworthy because childhood cancers are generally assumed to be aetiologically simpler than adult cancers. Furthermore, our study provides strong evidence that further genetic, genomic, or epigenetic Wilms tumour predisposition factors exist, because only a third of the familial Wilms tumour pedigrees we investigated have been explained. Any further familial Wilms tumour genes discovered will be highly likely to contribute also to non-familial Wilms tumour.Our study reveals new insights into the complexity, mechanisms, and clinical implications of Wilms tumour predisposition. Although our understanding of the genetic landscape of Wilms tumour predisposition is still far from complete, the available knowledge has considerable scientific and clinical use. Given the extensive heterogeneity and the absence of family history or additional clinical features in many individuals with a mutation, we believe routine genetic testing in all individuals with Wilms tumour would be scientifically and clinically valuable.


Background: Emerging evidence suggests that exposure to secondhand and thirdhand aerosol from electronic cigarettes may have serious health risks including respiratory and cardiovascular diseases. Social media data can help identify common locations referenced in vaping-related discussions and offer clues about where individuals vape. These insights can strengthen current tobacco regulations and prioritize new policies to improve public health. This study identified commonly referenced locations in vaping-related discussions on Twitter in 2018. Methods: Vaping-related posts to Twitter were obtained from 1 January 2018 to 31 December 2018. Rule-based classifiers categorized each Twitter post into 11 location-related categories  using a data dictionary of location-related keywords . Results: The most prevalent category was social venues (17.9%), followed by living spaces (16.7%), stores (15.9%), modes of transportation (15.5%), schools (14.9%), and workplaces (11.9%). Other categories pertained to: healthcare offices (2.0%), eateries (1.2%), correctional facilities (0.7%), and religious institutions (0.4%). Conclusion: This study suggests that locations related to socialization venues may be priority areas for future surveillance and enforcement of smoke-free air policies. Similarly, development and enforcement of similar policies at workplaces, schools and multi-unit housing may curb exposure to secondhand and thirdhand aerosol among the public. Smoke-free air policies are designed to protect non-smokers from involuntary smoke exposure, denormalize smoking among youth, and encourage cessation among those who smoke ,2,3,4,5.Vaping regulations vary across states and are inconsistent within states. For instance, some states prohibit vaping in public locations such as restaurants and bars but may not ban vaping in private locations such as multiunit housing . Other vTwitter is used by 24% of US adults , with 46% of users on the platform daily [Data Collectionhttps://twitter.com/) posts containing 26 vaping-related terms  drawn from previous research [Twitter .The analyses relied on public, anonymized data, adhered to the terms and conditions, terms of use, and privacy policies of Twitter. This study was performed under Institutional Review Board approval from the authors’ university. No Twitter posts were reported verbatim in this report to protect the privacy of the users. Python code and associated search keywords are publicly available on a repository  may not be captured in this study.social venues may be priority areas for future surveillance and enforcement of no-smoking policies. Similarly, development and enforcement of similar policies at workplaces, schools and multi-unit housing may curb exposure to secondhand and thirdhand aerosol among the public. As tobacco regulation continues to evolve with the growing use of electronic cigarettes, social media data can help inform policymakers about gaps in tobacco regulations by highlighting predominant locations referenced with vaping.This study identified common locations referenced in Twitter discussions related to vaping in 2018. Locations associated with


Over the last few years, oncolytic virus therapy has been recognised as a promising approach in cancer treatment, due to the potential of these viruses to induce systemic anti-tumour immunity and selectively killing tumour cells. However, the effectiveness of these viruses depends significantly on their interactions with the host immune responses, both innate  and adaptive (e.g.,  The increase in the incidence of skin cancers, combined with the advances in understanding the molecular biological mechanisms involved in tumour progression and interactions between melanoma cells and immune cells, has led to the development of several immune strategies for the treatment of these cancers  cells; see Filley and Dey In this study, we focus on a highly metastatic cell line, B16F10 melanoma, which is also resistant against the To address this, in our paper we aim to investigate at a theoretical level , the interactions between the M1/M2 macrophages, the Mathematical and computational approaches have been widely used in the past to investigate the interactions between oncolytic viruses, tumour cells and immune cells (mainly cytotoxic T cells), with the overall aim of explaining existing observations or generating new testable hypotheses; see . The inH(t) that is usually a combination of Heaviside functions; see caption of Fig. H(t). The number of viral particles inside the tumour increases following the fast replication of these particles inside the tumour cells, causing the cells to burst open and release the particles. We denote by b the burst size, i.e., the number of viral particles released by one infected tumour cell. The half-life of these viral particles is The virus, described by Eq. , is injeentclass1pt{minimaM . As an example, experimental data in Dudley et al.  abo abo\docuTo investigate the dynamics of tumour–immune–virus system , we firsNevertheless, before we discuss these approaches, we need to emphasise that in mathematical and computational immunology, many researchers have used parameters already published in the literature: either measured experimentally following some particular experimental in vivo and/or in vitro studies, or parameters taken from other published mathematical and computational models  to 8000 PFU/cell, with an average of 2500 PFU/cell . For the oncolytic virus, we focus only on the wild-type VSV (and its impact on the B16F10 cells).We first fit model  with no n et al.  , which will be varied to reproduce different experimental and clinical approaches aimed at controlling tumour growth. In this context, we will discuss the effect of varying these parameters on the size of M1 and M2 populations, and how this correlates with tumour control/elimination. This investigation will address mainly question (I) from the Introduction. To address questions (II) and (III) we will combine numerical simulations for transient system dynamics with steady-state analysis of long-term dynamics.Before investigating the transient and long-term dynamics of model , we perfentclass1pt{minimar), the tumour carrying capacity (K), the activation rate of M1 cells  tumour sensitivity to the rate at which the In the following we use model  to invesWe start the investigation into the dynamics of model  by showiz et al. ]. In thiNext, we move away from the experimental studies in Fernandez et al. , Chen etChanges in virus-induced macrophages re-polarisation ratementclass2pt{minimChanges in virus-related parameters Next, we investigate the dynamics of model . To invOverall, Figs. Changes in anti-tumour immunity rates Finally, we investigate the effect of shifting the immune response from viral antigens to tumour antigens, for example, by injecting oncolytic viruses that carry TAAs Tumour-absent states Since the previous numerical results  thi thiTumousee also ppendix A(ii)Tumour-present virus-absent states Since the previous numerical simulations Tumour-prquations  that \doquations  + 1e), , Tumour-ase [see ]. In Figequality  is viola(iii)Tumour-present virus-present immune-present states To investigate the relation between anti-tumour and anti-viral immune responses, as well as the oncolytic activities of VSV particles, next we focus on the tumour-present virus-present and immune-present (quasi-) steady states  that was administered twice in 3 days, and innate and adaptive immune responses. We first parametrised the model without the oncolytic virus by fitting it to baseline experimental data in Chen et al. , which fn et al. ), and sin et al. , we expen et al. . Howevermentclass2pt{minimUsing the model derived in this study we then investigated the different tumour-immune-virus dynamics exhibited by model . Thus, tsis Fig.  showed tmentclass2pt{minimc) did not seem to have a big impact on small and large tumours, but might have some impact on medium-size tumours, i.e., tumours around the detection threshold (In the context of question (II) from the Introduction, we showed in Fig. Simplicity vs. complexity in mathematical immunology The complexity of the immune response, and in particular the tumour–immune interactions in the context of macrophages plasticity with multiple cell phenotypes, could lead us to think that incorporating more details into the mathematical models might shed light on the specific aspects of the immune responses that could control tumour growth. For example, one could think that understanding the detailed interactions between the adaptive and innate immunity, or the different aspects of innate immunity, or the saturated vs. linear interactions between tumour cells, immune cells and/or virus particles, might help us find the mechanisms that could control tumour growth. While these specific details could indeed help us investigate  their role on the overall dynamics of the model, the complexity of the new models that contain large numbers of equations/terms makes them very difficult to be investigated analytically , as the time series for the immune responses. To parametrise (relatively complex) mathematical models for tumour–immune interactions, one option is to use multiple data sets obtained in different experimental settings and to further investigate model sensitivity to the identified parameters. In this study we used two different data sets from Fernandez et al.  associated with system (The linear stability of the steady states mentclass2pt{minimmentclass2pt{minimAt the steady state  see Eq. ). Since Note that to calculate the tumour-free virus-free steady state Equation  to obtaiquations  and 1e)\documenttisfying ) to obtaAt the steady state The coefficient terms in this 6th-order polynomial are even more complicated if we consider the tumour-present virus-present immune-present states Not only that we cannot investigate the stability of the tumour-present virus-present immune-present steady stats, but even finding closed-form equations for these states is challenging. To obtain the implicit Eqs. –7) that that7) t, adding +1c) in  in \docuIf we now focus on the steady-state Eq. , and solEquating , 1b), \documentate Eqs.  and 1e)\documentmentclasspt{minimaTo obtain , we elimduces to .


Wettability, or preferential affinity of a fluid to a solid substrate in the presence of another fluid, plays a critical role in the statics and dynamics of fluid-fluid displacement in porous media. The complex confined geometry of porous media, however, makes upscaling of microscopic wettability to the macroscale a nontrivial task. Here, we elucidate the contribution of pore geometry in controlling the apparent wettability characteristics of a porous medium. Using direct numerical simulations of fluid-fluid displacement, we study the reversal of interface curvature in a single converging-diverging capillary, and demonstrate the co-existence of concave and convex interfaces in a porous medium—a phenomenon that we also observe in laboratory micromodel experiments. We show that under intermediate contact angles the sign of interface curvature is strongly influenced by the pore geometry. We capture the interplay between surface chemical properties and pore geometry in the form of a dimensionless quantity, the apparent wettability number, which predicts the conditions under which concave and convex interfaces co-exist. Our findings advance the fundamental understanding of wettability in confined geometries, with implications to macroscopic multiphase-flow processes in porous media, from fuel cells to enhanced oil recovery. Wettability is typically characterized by the static contact angle θ, measured between a smooth horizontal solid surface and the fluid-fluid interface2. Perfectly wetting systems (θ = 0) and partially wetting systems (θ > 0) exhibit fundamentally different behaviour in terms of liquid spreading3—in this study we are concerned exclusively with partially wetting systems. We adopt the convention that θ is measured through the defending phase (the fluid phase being displaced), and we will use the term wetting (to the defending phase) when θ < 90°, nonwetting (to the defending phase) when θ > 90°, and neutrally wetting for the particular case θ = 90°. From the assumption of local phase equilibrium, the relationship between θ and surface forces is described by Young’s law2:σ, σif and σdf are the interfacial energies of the fluid-fluid, invading fluid-solid, and, defending fluid-solid interfaces, respectively. According to Young’s law, the wetting case (θ < 90°) corresponds to σif > σdf, and the nonwetting case (θ > 90°) to σif < σdf. In the former case, the surface has higher affinity for the defending fluid, while in the latter case it has higher affinity for the invading fluid2.Wettability is the preferential affinity of a fluid with the solid surface in the presence of another immiscible fluid22. An important question, however, is how the effective wettability of a porous medium emerges from the surface wettability at the microscale (as determined by the contact angle θ). Early considerations23 suggest that contact angle alone provides an incomplete description, and that accounting for pore geometry is necessary to arrive at a more predictive description of the apparent wettability of a porous medium, which multiphase flow dynamics so critically depend on.Many previous studies have demonstrated the impact of wettability on different aspects of multiphase flow in porous media29, and a predictive theoretical framework linking the effect of surface wettability and pore geometry is missing. Here, we investigate the interplay of surface wettability and pore geometry in determining the apparent wetting characteristics of a porous medium. We do so by first performing direct numerical simulations of fluid-fluid displacement in a single channel of variable cross section, which allows us to assess and extend earlier descriptions of capillary pressure on converging-diverging capillaries23. We then study, via numerical simulations, the impact of pore geometry on the slow displacement of one fluid by another in a 2D porous medium under different surface wettability conditions, and demonstrate its profound control on macroscopic features of the displacement. We uncover the coexistence of convex and concave fluid-fluid interfaces at intermediate wettabilities—a feature that has remained absent from existing theories of multiphase flow in porous media—and provide experimental evidence of such convex-concave coexistence from direct imaging of immiscible fluid-fluid displacements in patterned microfluidic cells. Finally, we propose a simple relationship between the apparent wettability of the porous medium and the combined effect of surface wettability and pore geometry, which in turn can serve as a diagnostic tool for macroscale predictions.Despite the importance of apparent wettability on multiphase flow in porous media, to date only few recent studies have directly measured the local wetting characteristics in porous media30, and will not be repeated here. We simulate fluid-fluid displacement in two capillaries with square cross-section: one with a uniform cross section, and the other with a non-uniform (converging-diverging) geometry  simulations, where we solve the Navier-Stokes equations coupled with the volume of fluid method using a finite volume approach. The details of the numerical formulation are described elsewheretry Fig. .Figure 1β = 21.8°, and the length of the capillaries was kept constant at 2 × 10−3 m. The capillaries are initially filled with the defending fluid (viscosity μd = 1.0 × 10−3 Pa · s and density ρd = 1000 kg · m−3), which is displaced by the invading fluid  at a constant flow rate Q = 7.0 × 10−12 m3 · s−1. The interfacial tension between the fluids is σ = 70 × 10−3 N · m−1. The outlet of the capillaries is kept at atmospheric pressure in all simulations. The static contact angle θ, which corresponds to the angle between the defending fluid and the solid surface, is defined as an input parameter in the solver. The numerical simulations are performed at three different θ values: 45° (drainage), 90°  and 135° (imbibition). The simulations do not take into account contact angle hysteresis and dynamic contact angle effects. The relative importance of viscous to capillary forces is characterised using the capillary number vi is the injection velocity of the invading fluid31. All simulations are performed at Ca = 10−7, a value for which capillary forces dominate viscous forces. Gravity forces do not play a role in the simulations since the fluid densities are identical. The visualization and post-processing of the numerical results are performed using ParaView32.The non-uniform capillary converges and diverges at an angle of orientation θ is shown in Fig. pc as a function of the local capillary radius r(radius of the capillary at the contact line) are presented in Fig. pc, represents the pressure difference across the interface, pc ≡ pi − pd, where pi is the pressure of the invading fluid and pd is the pressure of the defending fluid, and is given by the Laplace equationκ is the curvature of the interface, computed as the median value of the curvature distribution from the diffuse interface produced by the numerical solution30.The movement of the interface in the uniform and converging-diverging capillary for different values of the contact angle θ = 135° or 45° the sign of pc does not change as the interface traverses the converging-diverging capillary with β = 21.8°, and the convexity of the interface is the same in the uniform and non-uniform capillaries. For θ = 45°, the invading fluid is the non-wetting fluid (convex interface and positive pc) and the displacement process is regarded as drainage. For θ = 135°, the invading fluid is the wetting fluid (concave interface and negative pc) and the displacement process is regarded as imbibition. In contrast, for θ = 90°, there is a difference in the morphology of the interface in the uniform and converging-diverging capillaries. In the uniform capillary, the interface is flat, and pc = 0. In the converging-diverging capillary, however, the interface flips from concave to convex  surface wettability and pore geometry on the wettability characteristics of porous media. Figure θ in Young’s equation [Eq. (θ ~ 90°), however, the orientation angle β exerts a strong control over the apparent wettability: the invading fluid imbibes in the converging section (pc < 0) and drains defending fluid in the diverging section of the capillary (pc > 0).Understanding the sign of mentclass2pt{minimpc with static contact angle and the geometric characteristics of the pore. Following Rabbani et al.30 (Eq. (14)), and replacing cos θ by cos(θ ± β), one obtains the analytical expression describing the capillary pressure in the converging (+) and diverging (−) sections of the capillary:h is the corner angle of the capillary cross-section, P is the perimeter of the capillary cross-section, and G = C/P2 is the so-called shape factor, where C is the cross-sectional area of the capillary.To gain more insight into the phenomenon of curvature reversal in the converging-diverging capillary, we have obtained an analytical model relating the capillary pressure θ and β that controls the sign of interface curvature κ, as suggested in previous studies33. Indeed, the equation cos(θ ± β) = π/2 determines the boundaries of the phase diagram in θ–β space  function ‘bwdist’ on the binary image to compute the distance between each pixel in the image and the nearest boundary. Subsequently, the MATLAB function ‘bwmorph’ was used to retain only the center pixel of each pore. The fluid properties and boundary conditions employed in the simulation are similar to those used for the simulations in the single capillary, except for the changes to μd = 10−1 Pa · s and Ca = 10−5. The static contact angle θ imposed at the grain boundaries is uniform throughout the micromodel. Figure θ ranging from 45° to 135°.We simulate immiscible fluid-fluid displacements in a 2D porous medium or micromodel to investigate the macroscopic consequences of geometrically induced appparent wetting. The design of the 2D micromodel  and imbibition (concave interface) are taking place simultaneously, even in neighboring pores  at θ = 45°. The co-existence of concave and convex interfaces is observed for θ ranging from approximately 60° to 120°.Figure θ = 90°), where the invading fluid advances via drainage (convex interface) in the converging section of the pore throat, and imbibes (concave interface) in the diverging section of the pore throat . The trigonometric identity apparent wettability numberIn Eq. , the sigθ over β in controlling the interface shape. When θ alone—this is the case for strongly wetting (θ ≈ 0) or strongly nonwetting conditions (θ ≈ 180°). In contrast, when β—this occurs for neutrally wetting conditions (θ ≈ 90°). Combinations of θ and β such that |W| ~ 1 correspond to scenarios for which the interface is flat at either the converging or the diverging section of the pore.This quantity describes the relative importance of W in terms of characteristic quantities describing the pore-space geometry in porous media. Assimilating the transition from a pore body (radius rb) to a pore throat (radius rt) as a converging channel, and assuming that the length of the channel is equal to the pore-body radius, we have tan β = (rb − rt)/rb. Futhermore, assuming a relatively uniform, densely packed porous medium, we take the typical pore body as the space between four grains, so that the number of pore bodies np ~ N and the number of pore throats nt ~ 2N, where N is the number of solid particles (grains). These assumptions lead to a relationship between rt and rb, ϕ and A are porosity and total area of the porous medium (per unit depth), respectively. Substituting the mathematical expressions for tan β and rt in Eq. , from the regions where apparent wetting corresponds to strict drainage (W > 1) or strict imbibition (W < −1) conditions , the apparent wettability number W predicts that the co-existence of concave and convex interfaces to occur for θ between 48° and 132°. This prediction is consistent with our direct numerical simulation results, which shows the co-existence of concave and convex interfaces for θ between 60° and 120° . We show that while apparent wettability is a function of θ alone under strong wetting conditions , pore geometry plays a crucial role in governing the apparent wettability under intermediate-wet conditions, which leads to co-existence of concave and convex interfaces. In the case of a converging-diverging capillary tube, the critical angles delineating the regime of apparent mixed wetting have been determined theoretically and shown in in Fig. β. For porous media, the critical transition contact angles have been quantified via numerical simulations (Figs Consistent with past studies, our results elucidate the interplay between pore geometry and contact angle ons Figs  and 4. IW that estimates the range of θ values where pore geometry exerts a fundamental influence on the wettability of porous media and, likely, on the macroscopic displacement processes it mediates, such as enhanced oil recovery41 and geologic carbon sequestration43.To capture the coupled effect of pore geometry and contact angle on apparent wettability, we derive the apparent wettability number Supplementary information


In the original article, we neglected to include the funder Grant # 2018/00163-0 São Paulo Research Foundation (FAPESP), to for all authors.The FUNDING is: “This research was funded by grant #2013/14957-5, grant #2013/09265-7 and grant #2018/00163-0 São Paulo Research Foundation (FAPESP) by the Coordination for the Improvement of Higher Education Personnel (CAPES) together with the Post-Doctoral National Program (PNPD), the Programa de Apoio ao Desenvolvimento Científico (PADC) of School of Pharmaceutical Sciences/UNESP, Brazil and the authors thank UNESP for the data of the dissertation Freitas, .”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Here we established a repeated dose “safficacy” test assay for the simultaneous generation of safety and efficacy data. Therefore, a commercially available multi-organ chip platform connecting two organ culture compartments was adapted for the microfluidic co-culture of human H292 lung cancer microtissues and human full-thickness skin equivalents. Repeated dose treatment of the anti-EGFR-antibody cetuximab showed an increased pro-apoptotic related gene expression in the tumour microtissues. Simultaneously, proliferative keratinocytes in the basal layer of the skin microtissues were eliminated, demonstrating crucial inhibitory effects on the physiological skin cell turnover. Furthermore, antibody exposure modulated the release of CXCL8 and CXCL10, reflecting the pattern changes seen in antibody-treated patients. The combination of a metastatic tumour environment with a miniaturized healthy organotypic human skin equivalent make this “safficacy” assay an ideal tool for evaluation of the therapeutic index of EGFR inhibitors and other promising oncology candidates.Antibody therapies targeting the epithelial growth factor receptor (EGFR) are being increasingly applied in cancer therapy. However, increased tumour containment correlates proportionally with the severity of well-known adverse events in skin. The prediction of the latter is not currently possible in conventional  This receptor is frequently overexpressed in many tumours, such as carcinomas and glioblastoma3, and serves as a prime target for tumour therapies. Inhibitors of EGFR activation, using monoclonal antibodies or small-molecule inhibitors, have been established successfully and are increasingly being used in first or later line cancer therapy4. However, its inhibition induces adverse responses primarily in skin and intestine – the organs with the highest physiological cell turnover. Receptor-mediated (EGFR) adverse cutaneous effects result in dose reduction or discontinuation of treatment, limiting the effectiveness of tumour therapy5.The human epidermal growth factor receptor (EGFR), a member of the erbB family of receptors, is a transmembrane receptor tyrosine kinase expressed in epithelial, mesenchymal and neuronal tissues. It is a key regulator of organ homeostasis mediating physiological cell turnover – proliferation and differentiation – in adultsin vivo animal studies. Standard in vitro assays are limited to the evaluation of the anti-EGFR response in either human tumour models or skin equivalents, each in a separate conventional static tissue culture. This restricts cross-talk and perfusion-based pharmacokinetic studies with relevant dosing background for further quantitative in vitro to in vivo extrapolation. Consequently, preclinical development of novel effective EGFR-inhibitors would require a reliable holistic human assay platform generating both target-mediated efficacy and safety data based on the homeostasis of a patient’s skin and tumour tissue co-culture.The mechanisms underlying the correlation between the intensity of the adverse effects in skin and the efficacy of tumour treatment are still poorly understood due to the lack of suitable assay formats. Human skin biology differs substantially from that of most laboratory animal species, which hampers a simultaneous evaluation of anti-tumour efficacy and adverse cutaneous effects by 7. A first drug-testing assay based on the co-culture of a rat lung type II epithelial cell line together with either H4IIE rat hepatocytes or the human HepG2/C3A hepatocyte cell line was first described in 20048. Since then, the number of cell types used in such MPS-based suspension or monolayer co-cultures has increased immensely9. Furthermore, the degree of complexity of the organ equivalents used in MPS platforms has improved from suspension and monolayer culture to three-dimensional (3D) tissue culture, emulating their human counterparts more precisely. The first successful examples were single organ equivalents for lung alveoli10 and various skin equivalents11. The latter have been improved into a robust commercially available multi-organ chip (MOC) platform, where human skin biopsies can be co-cultured homeostatically over four weeks with a human 3D spheroid liver model12. Subsequently, microfluidic channels of the MOC co-cultures can be endothelialized13. A roadmap towards full integration of MPS into industrial use with regulatory acceptance within the next few decades has been outlined recently by representatives of leading academic groups, MPS technology providers, large industries and regulatory bodies from major countries14. Intensive industrial adoption of such MPS-based assays fitting the different purposes within the drug development cycle is ongoing15.Co-cultures of human microtissues in microphysiological systems  emulating organ cross-talk and mechanisms of disease progression are used increasingly for detailed temporal studies of pharmacological effects of drugsWe established a novel human tumour–skin co-culture assay to evaluate anti-EGFR antibody effects on both tumour and human skin tissue, the latter being the site of target-mediated adverse effects in patients. We investigated the impact of repeated cetuximab (trade name: Erbitux®) exposure on the systemic behaviour of the co-culture and individual tissue responses. We present here an MPS-based co-culture assay that has the potential to provide a platform for evaluation of the therapeutic window of drug candidates.16. Activation of EGFR-tyrosine kinases is a key for lung cancer progression17. The EGFR-targeting therapies, such as monoclonal antibodies, specific tyrosine kinase inhibitors or a combination of both, are a promising approach in the pipeline18. Innate or acquired resistance of distant metastatic tumour foci to EGFR-targeted therapies after primary tumour resection and severe adverse effects in skin are major challenges for therapeutic success19. Preclinical in vitro assays for the evaluation of EGFR-targeting lung cancer therapies lack the ability to investigate efficacy simultaneously on distant tumour metastasis and adverse skin reaction at their interplay. Prime requirements for such an assay tool are stable long-term homeostasis of a human metastatic lung tumour model and a human skin equivalent, and low fluid flow forces in and microscopic access to the tumour microtissue compartment.Lung cancer is the most common cause of cancer death in the United States2 at the air–liquid interface. This corresponds to 1/30,000 of the average skin surface of a human adult. However, the skin could also be replaced by any other organ equivalent, produced in, for example, 24-well Millicell® standing inserts, such as intestine or lung equivalents, depending on the toxicity target to be analysed. The tumour microtissue compartment is designed to mount a minimum of 15 homogeneous 3D tumour spheroids and support their adherence-induced metastatic outgrowth within the compartment over five days of co-culture. The HMOC has been designed to circulate 150–180 μl fluid in the channel system of each circuit to match a surrogate blood volume, which is 30,000-fold smaller than the original blood volume of an adult human. Additionally, the HMOC consists of an artificial media reservoir of 600 μl above the tumour microtissue culture compartment. Such a reservoir obviously does not exist in vivo. However, in the HMOC, it is necessary to provide sufficient nutrition to the tissues for daily feeding.We designed a new microphysiological HMOC accommodating two identical microfluidic circuits, each interconnecting a 96- with a 24-well sized culture compartment. Henceforth, any tissue model cultured on standard 24-well based cell culture inserts can be easily transferred and cultured for the desired culture time in this new MOC platform. In this study, a co-cultivation of tumour and skin microtissue was established. Figure 2 in the venous circulation and 4.0–30.0 dyn/cm2 in the arterial circulation20. The wall shear stress in the microfluidic channels reaches up to 2.7 dyn/cm² and, therefore, lies in the physiological range of the venous circulation. Former studies identified mechanical stress as an important regulator of apoptosis and autophagy in circulating tumour cells23. By comparison, a tumour cell in metastatic growth after extravasation from the circulation would experience much lower forces. Therefore, the system was adjusted to low shear stresses in the tumour compartment. The mean wall shear stress at the surface of the tumour spheroids is of the order of 10−4 dyn/cm2 36.We selected the human NCI-H292 cell line for the establishment of the tumour microtissue in the HMOC assay because of five essential features: its epithelial lung tissue origin, metastatic track record, capability to undergo epithelial to mesenchymal transformation, compliance with existing 3D spheroid formation protocol and availability of a standardized monolayer efficacy test for anti-EGFR antibodies. The cell line has been derived from a cervical lymph node metastasis of a high-grade pulmonary mucoepidermoid carcinoma from a 32-year-old Negro femaleoid Fig. . No apopoid Fig. . In addioid Fig. . Fifteen35; and 2) cetuximab half-life time is around five days.A robust five-day skin-tumour co-culture regimen has been established and qualified by five series of experiments involving 69 HMOC co-culture circuits. The assay longevity was limited to five days for two reasons: 1) Robust 3D H292 cell spheroid cultures have been reported in static ultra-low attachment plate culture for that time frame38. Thus, the monolayer assay proved the pleomorphic nature of the H292 cells generating distinct subpopulations of cells under well-defined culture conditions. Furthermore, it proved the suitability of the H292 cell culture lot used for the experiments.Prior to starting the HMOC assay, a validated standardized 2D efficacy assay for H292 cell treatment with cetuximab was performed to demonstrate the H292 cell line sensitivity to the drug for the cell batch used Fig. . Our dat39. It has been shown to bind to NSCLC and colorectal cancer from patients, and to H292 tumour cells38. It also triggers antibody-dependent cell-mediated cytotoxicity in vivo40. The antibody has been authorized for the treatment of a number of cancer indications since 2003. Exposure to cetuximab antibody was customized from a treatment cycle of cancer patients to fit the HMOC assay. Doses have been extrapolated from human exposure regimens established for the use of cetuximab in head and neck cancer41. At day 1, these patients receive a single intravenous dose of 400 mg/m2 of skin surface. Considering that the skin surface of each circuit in the HMOC has a surface of 0.6 cm2, this would correspond to a systemic application of 24 µg cetuximab per circuit. Patients then, from day 7 onwards, receive radiotherapy accompanied by weekly infusions of 250 mg/m2 cetuximab, corresponding to 15 µg per circuit of an HMOC. However, due to the lack of the other organ equivalents, the total biomass in the HMOC is lower than in vivo. Therefore, we have reduced the first exposure to 10 µg at day one. The further application regimen has been set to a daily treatment of 5 µg per circuit combined with a 50% medium exchange  of the tumour vessels43. We calculated antibody residence times by integrating 1/v (v: magnitude of velocity) over streamlines in a contact zone of 50 µm around the tumour microtissues, which corresponds to about two diameters of the H292 cells. This contact zone distance is much bigger than the molecule size of an antibody (~10 nm) but provides the first rough insight into antibody–tumour interaction potential at reasonable computational modelling costs. This model revealed an increase of the median residence time of the antibody molecules in proximity to the dispersed metastatic tumour microtissues from 4.5 min at day 0 to 8 min on day 3 and 11 min on day 5. This increase of the residence time is due to the rapidly changing architecture of the metastases, which then provides an increased exposure surface to the convective fluid flow on day 5 compared to day 0 and mimics the situation in patients when metastases are extending rapidly in size.The distribution of antibodies in patient’s tumour metastases depends on tumoural pathophysiology and antibody characteristics, such as affinity and molecular weightThe LDH served as a marker for cell death in the co-culture. Antibody-treated co-cultures showed a significantly higher systemic release of LDH 24 h after the first cetuximab application, indicating a toxic antibody-dependent effect Fig. . Afterwa44. This can lead to proliferation inhibition and apoptosis induction in tumour cells45. We, therefore, checked for genes related to apoptosis induction. The tumour suppressor gene p53 plays a crucial role in tumour initiation and progression. As a transcription factor, it can regulate downstream target genes such as BAX, a pro-apoptotic protein, belonging to the Bcl-2 family. Gene expression analysis of tumour microtissues revealed an increase of p53 and a significant upregulation of BAX within the antibody exposure group in comparison to the control group  decreased EGFR receptor expression and density together with a higher intra-tumoural basal EGFR phosphorylation decreases antibody sensitivity35. It remains unclear whether the dead proportion of tumour cells in the control and antibody exposure group are related to antibody-specific death or to intra-tumoural death due to oxygen limitation.Cetuximab binds to the extracellular domain of EGFR, which induces receptor internalization, degradation of the receptor leading to the downregulation of EGFR at the cell surface. Therefore, autophosphorylation and activation of the receptor is prevented and further downstream signal transduction pathways are inhibitedoup Fig. . This daoup Fig. . These foup Fig.  within toup Fig. . Togetheoup Fig.  at the snce Fig. . There ain vitro assays considering these aspects of modulation of antibody effects in patients are the next logical step towards improved antibody efficacy testing.Based on these factors, our tumour spheroid-based microfluidic culture approach enables a robust reproducible creation of a heterogeneous tumour tissue architecture with zones of proliferation, dormancy and cell death creating distinct areas of mucoepidermoid and fibroblastoid histology. Validation of such 48. Early human cellular assays for these outcomes are lacking. Using the HMOC co-culture model, several key side effects on the cetuximab-exposed skin microtissues could be detected at a very early stage. The haematoxylin and eosin staining revealed severe damage to the arrangement of the epidermal stratum basale, seen evidently by the lacking columnar keratinocytes in the lowest layer of the epidermis  was instead shown to be increased due to anti-EGFR treatment54. In this study, CXCL8 remained at a low level in the control group and the antibody exposure group in a first phase (day 2 and 3). It then increased in both groups (day 4 and 5), with a lower release (non-significant) in the antibody exposure group  with elevated levels in the antibody exposure group  with a laminar inflow condition corresponding to a given flow rate and a zero-pressure outlet condition. The medium was modelled using the properties of water at 37 °C. The position and size of the mounted tumour spheroids were approximated from microscopic images to set final microfluidic flow conditions guaranteeing low shear stress for the tumour microtissues. Finally, antibody residence times in tumour microtissue areas were calculated by integrating 1/v (v: magnitude of velocity) over streamlines in a contact zone within 50 µm around the tumour microtissues.The commercially available microphysiological MOC platformids Fig. . CAD sof64 at frequencies of 0.1 to 0.9 Hz, pressures of 200 to 800 mbar and a constant vacuum of -500 mbar. The red blood cells were prepared by centrifugation of human blood at 3,000 g for 10 min, and subsequent resuspension in phosphate-buffered saline (PBS) at a haematocrit of 2.5%. The average flow rate was determined with a high-speed CMOS camera  coupled to an inverted microscope . Magnification was set to 2.5×, which resulted in a resolution of 0.23 px/µm with an acquisition rate of 1,470 fps. The displacement of the red blood cells in recordings of 13.6 s was computed by the open source toolbox “PIVlab”65. Only velocity vectors in the central portion of the channels were considered. Details of the underlying equations have been discussed elsewhere13. For the rheological analyses we utilised three chips with a setup identical to the biological experiments and assessed the flow at three imaging points A, B and C d C Fig. . All mea2, 50 µg/ml ascorbic acid  and 10 ng/ml keratinocyte growth factor . After a resting phase of 24 h at 37 °C and 5% CO2, the 24-well Millicell® standing inserts containing the skin microtissues were transferred as a whole into the 24-well culture compartment of the HMOC. Additional skin tissues were maintained unchanged under static culture conditions for morphological evaluation.Reconstructed human full-thickness skin microtissues were obtained from the Fraunhofer Institute IGB . Human foreskin biopsies for isolation of primary human keratinocytes and dermal fibroblasts were obtained, with informed consent from legal representatives and ethics approval , after routine circumcisions in compliance with the relevant guidelines and regulations. Skin microtissues were shipped on day nine of the air–liquid culture. Upon arrival, skin tissues were transferred into 1 ml of E3 medium composed of EpiLife™ medium  supplemented with human keratinocyte growth supplement, including 0.2 ng/ml human epidermal growth factor , 1.4 mM CaCl2. Compact tumour spheroids formed within three days. Fifteen spheroid microtissues were then transferred to the tumour culture compartment of the HMOC. Additional tumour microtissues were embedded in Tissue-Tek® O.C.T.™ Compound  and subsequently snap-frozen for morphological evaluation of preformed tumour spheroids.The human lung tumour cell line NCI-H292 , used in standard two-dimensional (2D) monolayer efficacy testing, were grown in RPMI1640 medium supplemented with 10% foetal calf serum, 50 µg/ml gentamycin sulphate and 0.25 µg/ml Amphotericin B . Standard 384-well ultra-low attachment spheroid microplates (Corning) were used for the formation of H292 lung tumour spheroids. Briefly, 50 µl of medium containing 10,000 H292 cells was pipetted into each well of the microplates. The microplates were centrifuged at 300 g for 1 min at room temperature and then incubated on a 3D rotator  at 37 °C and 5% CO2). Afterwards, the supernatant was aspirated and 130 µl of lysis buffer, consisting of Isopropanol  and 3% sodium dodecyl sulphate , was applied and incubated for an additional 15 min. Absorbance was measured using a microplate reader  at 570 nm. Mean absorbance of the control group was set to 100% of viability for subsequent calculations.A static monolayer proliferation assay was performed for the evaluation of cetuximab-dependent cytotoxicity of H292 cells. Thus, 5000 H292 lung tumour cells were seeded per well in a 96-well plate using 100 µl of supplemented RPMI1640 medium. The next day, a concentration series of cetuximab was applied originating from a stock solution of 15.5 mg/ml . Cells were incubated for additional 96 h followed by a standard MTT -2,5-diphenyltetrazolium bromide) viability assay. Briefly, 20 µl of MTT reagent  was added to each well and incubated for 2 h at 37 °C  for the evaluation of tumour microtissue remodelling.PLUS . Measurement was performed according to the manufacturer’s instructions, with minor modifications: 12.5 µl of reaction mix was added to 12.5 µl of prediluted sample into 384-well non-treated microplates (Corning) and incubated for 30 min at room temperature. Photometric measurement took place using a microplate reader at 490 and 680 nm. Calculations were conducted via MARS Data Analysis Software and further analysed using GraphPad Prism® 7 software.Furthermore, tissue viability was monitored daily by the measurement of lactate dehydrogenase (LDH) released in the supernatant using the Cytotoxicity Detection KitMedium was collected from the cell cultures at the time points indicated and stored at −80 °C. Samples were thawed and centrifuged to remove any debris on the day of analysis. Chemokines were quantified in the supernatants using the V-PLEX Chemokine Panel 1 Human Kit, on the QuickPlex Meso SQ 120 , according to the kit manuals.Skin and tumour microtissues from each culture condition were embedded in Tissue-Tek® O.C.T.™ Compound and subsequently snap frozen for end-point analysis. Tissue blocks were sectioned with a Leica CM1950 Cryostat  at a thickness of 8 µm. Standard haematoxylin and eosin staining was used to analyse the sections histologically. A combined TUNEL (TdT-mediated dUTP-digoxigenin nick end labeling)/Ki67 immunofluorescence staining was performed to detect proliferating and apoptotic cells. Thus, representative cryostat central sections of the microtissues were stained using the Apo Direct Apoptosis Detection Kit (Thermo Fischer Scientific Inc.), according to the manufacturer’s instructions. Subsequently, slides were blocked with 10% (v/v) goat serum in PBS for 20 min and primary mouse anti-human Ki67 antibody (Thermo Fischer Scientific Inc.) was applied overnight, washed with PBS and then developed by goat anti-mouse CF594  for 45 min. DAPI (Roche) was added for nuclei staining. Further single immunofluorescence staining on skin microtissue was performed using mouse anti-human Collagen IV antibody (Sigma–Aldrich) combined with goat anti-mouse CF594 for visualisation of the basement membrane on skin sections. Tumour microtissue sections were single stained with rabbit anti-human Vimentin (Thermo Fisher Scientific Inc.) or mouse anti-human E-Cadherin antibody with the appropriate secondary antibody, goat anti-rabbit CF594 or goat anti-mouse CF594 (Biotium). A Keyence BZ-9000 Microscope with BZ-II-Viewer software was used for microscopic imaging. Images were merged using the BZ-II-Analyser software. DAPI and Ki67 positively stained cells were counted manually using this software to determine the proportion of proliferative cells.Real-time qPCR endpoint analysis was performed to evaluate tumour and skin microtissue gene transcription at an mRNA level. Thus, microtissues from both culture conditions were collected for RNA isolation using the NucleoSpin RNA Kit . cDNA was synthesized by reverse transcription of 100 ng total RNA using the TaqMan® Reverse Transcription Kit (Thermo Fisher Scientific Inc.). Quantitative PCR was performed using the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific Inc.) and the SensiFAST SYBR Lo-ROX Kit , according to the manufacturer’s instructions. The real-time qPCR primers were as follows: tumour protein 53 (p53) forward 5′-TATGAGCCGCCTGAGGTTGG-3′ and reverse 5′-GGCACAAACACGCACCTCAA-3′ Bcl-2-associated X protein (BAX) forward 5′-CAACCACCCTGGTCTTGGAT-3′ and reverse 5′-CTGACGGCAACTTCAACTGG-3′, tumour necrosis factor alpha (TNFα) forward 5′-CTCGAACCCCGAGTGACAAG-3′ and reverse 5′-ATGGTGTGGGTGAGGAGCAC-3′, Keratin 1 (KRT1) forward 5′-GCTGGCAGACATGGGGATAG-3′ and reverse 5′-CATCCTTGAGGGCATTCTCG-3′, Glyceraldehyde 3-phosphate dehydrogenase 5′-TGTTGCCATCAATGACCCCTT-3′ and reverse 5′-CTCCACGACGTACTCAGCG-3′ and TATA-binding protein (TBP) forward 5′-CCTTGTGCTCACCCACCAAC-3′ reverse 5′-TCGTCTTCCTGAATCCCTTTAGAATAG-3′.Differences among the control group and the antibody exposure group were analysed using unpaired two-tailed Student’s t test and a p value < 0.05 was considered statistically significant.


The effect of TLR5 down‐regulation was detected with qPCR and Western blot. 125I‐anti‐TLR5 mAb and control isotype 125I‐IgG were prepared and injected to TLR5+/− 4T1‐bearing mice models, respectively. Whole‐body phosphor‐autoradiography, fluorescence imaging and biodistribution were performed. Furthermore, ex vivo tumour TLR5 expression was proved through immunohistochemistry staining. We found that 125I‐anti‐TLR5 mAb could bind to TLR5+ 4T1 with high affinity and specificity. Whole‐body phosphor‐autoradiography after 125I‐anti‐TLR5 mAb injection showed TLR5+ 4T1 tumour images in 24 hours, more clearly in 48 hours. Radioactivities in tumour tissues were positively related with TLR5 expression. Biodistribution assay showed that 125I‐anti‐TLR5 mAb was mainly metabolized through the liver and kidney, and 125I‐anti‐TLR5 mAb was much more accumulated in TLR5+ 4T1 tumour than TLR5− 4T1. In vivo fluorescence imaging successfully showed tumour tissues clearly both in TLR5+ and TLR5− 4T1 mice compared with lentivirus untreated 4T1 tumour. Immunohistochemistry staining showed that TLR5 expression in tumours was indeed down‐regulated in TLR5− 4T1 mice. Our results indicated that 125I‐antiTLR5 mAb was an ideal agent for non‐invasive imaging of TLR5+ tumours; TLR5 may be as a novel molecular target for TNBC non‐invasive diagnosis.Triple‐negative breast cancer (TNBC) is a highly aggressive tumour that lacks marker for targeted diagnosis. Recently, it was reported that toll‐like receptor 5 (TLR5) was associated with some kind of tumours, especially in TNBC, but whether it could be used as a non‐invasive monitoring target is not fully understood. Here, we established TLR5 Searching the better molecular target for tumour diagnosis and therapy is critical for clinic.Nuclear medicine molecular imaging is a kind of non‐invasive whole‐body scanning modality based on metabolism and function abnormal, more suitable for the detection and quantification of tumour target molecular, compared with traditional imaging CT (computed tomography) and MRI (magnetic resonance imaging) in diagnosis and tumour staging., such as monocyte/macrophage, vascular endothelial cells,, so we choose TNBC cell line to investigate whether it could be a target molecule of non‐invasively diagnosis for TNBC.Recently, many reports showed that innate molecules were participated in tumour developments and prognosis, such as CCAT2 18F‐labelled aptamers of human epidermal growth factor receptor 2 (Her2/ErbB2),125I/131I‐labelled anti ICAM‐189Zr‐Transferrin.For breast cancer molecular imaging, there were some reports about target molecules with labelled radioisotope, for example 22.12. The lentivirus‐shRNA for TLR5 knock‐down and negative control virus was purchased from GenePharma.The murine triple‐negative breast cancer (TNBC) cell line 4T12.25/well) overnight, then discarded the supernatant, replaced with virus dilution and then incubated at 37°C in a humidified incubator with 5% CO2 for 24 hours. Finally, replaced the virus dilution with fresh medium and continued to incubate at 37°C in a humidified incubator with 5% CO2 for another 48 hours. Then, we calculated the transfected efficiency. Puromycin was used to filter out successfully transfected cells. If the transfection is successful, the cells will express green fluorescent protein (both of negative control virus and lentivirus‐shRNA TLR5).4T1 cells were plated in 24‐well plates  according to the instructions of manufacturer and then determined RNA concentration. cDNA first strand was synthesized from RNA using TransScript First‐Strand cDNA Synthesis SuperMix (TRANSGEN BIOTECH). Finally, mRNA was measured using TransStart Tip Green Qpcr SuperMix (TRANSGEN BIOTECH). TLR5 quantitative primers: 5′‐GCAGGATCATGGCATGTCAAC‐3′(forward) and 5′‐ATCTGGGTGAGGTTACAGCCT‐3′(reverse).For Western blot (WB), cells were harvested at about 70% confluency. Total protein concentration was measured using a protein assay kit (Beyotime Biotechnology). Twenty micro gram of total protein was loaded into Bio‐Rad gel (Bio‐Rad Laboratories), along with Chameleon Duo ladder protein marker (SMOBIO). Gel electrophoresis was performed at 80 mV for 30 minutes and then up to 100 V for 60 minutes. All proteins were then transferred to a nitrocellulose membrane at 200 mA for 100 minutes. The membrane was blocked with skim milk (5%) blocking buffer for 2 hours at room temperature and further incubated with a rabbit antimouse TLR5 mAb  and rabbit antimouse GAPDH mAb  overnight at 4°C. Next, the membrane was washed three times with TBS with Tween 20 (TBS‐T) and incubated with anti‐rabbit secondary antibodies  for 1 hours at room temperature. The washed membrane was scanned and quantitatively analysed by Tanon 4200 imaging system.2.4+/− 4T1 cells (3 × 103/well) were respectively plated in 96‐well plates overnight, and then microplate reader was used to measure the absorbance at 450 nm at 0, 6, 24 and 48 hours.CCK‐8 assay was used to evaluate its proliferation. Furthermore, flow cytometry was used to detect apoptosis. For CCK‐8, TLR5+/− 4T1 cells were respectively digested with 0.25% trypsin (no EDTA and no phenol red) and then centrifuged to collect the cells. The cells were washed twice with cold PBS, suspended in 400 μL of 1× Annexin V binding solution at a concentration of approximately 1 × 106 cells/mL, added 5 μL of Annexin V‐YF647A and 10 μL of PI staining solution, mixed gently and incubated at 4°C for 15 minutes in the dark and filtered with a 200‐mesh filter and immediately detected by flow cytometry.For apoptosis, the TLR52.5125I, the mixture was incubated for 20 minutes at room temperature, and then the reaction was stopped by adding 150 μL of 0.05 M pH 7.4 PB and incubated for another 10 minutes at room temperature.125I‐anti‐TLR5 mAb and 125I‐IgG was determined in PBS and serum.Briefly, 10 μg of anti‐TLR5 mAb/IgG was added to 100 μL of 0.05 M phosphate buffered , followed by the addition of 11.1 MBq (300 μCi) of Na2.6+/− 4T1 cells were seeded in a 24‐well plate at 5 × 105 cells per well, respectively. 125I‐antiTLR5 mAb in PBS solution  was added into the plates, then incubated at room temperature for 2 hours and discarded the supernatant, and cells were washed twice with iced 1 × PBS , then harvested and determined radioactivities with a gamma counter. Non‐specific binding was evaluated by the presence of non‐labelled anti‐TLR5 mAb  in the same wells. The binding results including maximum binding ability (Bmax) and dissociation constant (Kd) were obtained through GraphPad Prism software. For competitive binding, TLR5+ 4T1 cells were seeded in a 24‐well plate at 5 × 105 cells per well. 0.1‐1000 nmol/L anti‐TLR5 mAb and 10 nmol/L 125I‐anti‐TLR5 mAb were used. The mixture was incubated at 37°C for 45 minutes and finally discarded the supernatant, and cells were washed twice with iced 1 × PBS containing 0.1% BSA, then harvested and the radioactivity determined with a gamma counter.TLR52.7+ and TLR5− 4T1 cell lines were induced in 5‐week‐old male nude mice. TLR5+ 4T1 Tumour cells (2 × 106) suspended in 200 μL PBS  were subcutaneously injected into their lower right flank. TLR5− 4T1 Tumour cells (2 × 106) suspended in 200 μL PBS were subcutaneously injected into their lower left flank. Mice were monitored every other day. The animals were used for phosphor‐autoradiography imaging, fluorescence imaging and biodistribution once the tumour had reached about 10 mm in diameter.All animal studies were conducted in accordance with protocols approved by the Animal Care and Use Committee of Shandong University, China. For tumour‐bearing mice models, subcutaneous tumours of TLR52.8125I‐antiTLR5 mAb (0.38μg) or 125I‐IgG (0.38μg) through tail vein. For blocking group, unlabelled anti‐TLR5 mAb (100 μg) was injected 30 minutes before 125I‐antiTLR5 mAb injection. Phosphor‐autoradiography scanning was conducted at 24, 48 and 72 hours after injection. 0.6% sodium pentobarbital was used for anaesthesia. Anaesthetized mice were placed on the storage phosphor screen plate (back to the plate). After 20 minutes in the dark, the plate was immediately covered with an opaque plastic sheet and then transferred to the Cyclone Plus scanner (PerkinElmer Life Sciences). Semi‐quantitative analysis was performed by manually drawing rectangular regions of interest (n = 5) within the target area at each time‐point. Digital light units (DLU)/mm2 were obtained using OptiQuant™ image analysis software 5.0 (PerkinElmer Life Sciences). In addition, we stripped the tumour separately and performed the imaging.Three days before injection of radiotracers, 4% potassium iodide was added to the drinking water to block the thyroid gland uptake of iodine. Tumour‐bearing mice (n = 5 per group) were injected for each with 0.37 MBq of 2.9− and TLR5+ 4T1 cells were induced described as above. TLR5+ 4T1 on lower right flank and TLR5− 4T1 on lower left flank and the 4T1 cells without lentivirus transfected on back. When tumour reached 10 mm in diameter, anaesthesia was performed, then surface skin of the tumour was peeled, and the mouse was placed on the imaging plate and photographed (Belly down).4T1 cells transfected with lentivirus‐TLR5 knock‐down and negative control lentivirus express green fluorescent protein (GFP) after integrated into 4T1 cells. Subcutaneous tumours of the TLR52.10125I‐anti‐TLR5 mAb or 125I‐IgG (0.37 MBq) was injected into model mouse (n = 5 per group), respectively, and mice were killed and dissected at 24, 48 and 72 hours. The tumours, blood and major tissues/organs  were harvested and weighed. Samples and primed standards were measured with gamma counter. Tissue radioactivity is expressed as the per cent injected dose per gram (%ID/g). The target‐to‐non‐target ratio was defined as the tumour‐to‐opposite‐muscle radioactivity (T/NT) ratio.Three days before injection of radiotracers, 4% potassium iodide was added to the drinking water to block the thyroid gland uptake of iodine. 2.11Tumour‐bearing mice (n = 5) were executed at 72 hours after whole‐body phosphor‐autoradiography finished. Tumours were harvested for immunohistochemical staining with rabbit polyclonal TLR5 antibody  and DAB chromogen . Immunohistochemistry was performed with SP‐9002 Histostain™ Plus kits (ZSGB‐BIO) according to the manufacturer's protocols. The slides were visualized at a magnification of 200× and 400×. Corresponding positive areas of slides were analysed （five fields per slides） by the Image‐Pro Plus software version 4.5.0.29 (Media Cybernetics).2.12t test was used by GraphPad Prism version 5 software . Significant difference was indicated by *P < .05, ***P < .01. SAS version 9 (SAS Institute Inc) and was used for statistical analyses.The data are presented as the means ± standard deviation from three independent experiments. Student's 33.1− 4T1 cells compared with negative virus transfected 4T1 cells (P < .05) and also lower than no virus transfected 4T1 cells (P < .05. data not shown).Lentivirus transfection efficiency is shown in Figure 3.2− 4T1 cells was apparently higher than TLR5+ 4T1 cells which indicated that TLR5 may down‐regulate the 4T1 cell proliferation .CCK‐8 assay showed that the proliferation ability of TLR53.3+ 4T1 and TLR5− 4T1 cells. For TLR5+4T1 vs TLR5− 4T1, a Scatchard plot and computer curve fitting of the saturation data revealed a Kd value of 6.069 vs 6.463 nmol/L and a Bmax value of 124 vs 56.22 cpm/104 cells. The results suggested that even total TLR5 expression decreased in TLR5− 4T1 cells, and the affinity between 125I‐anti‐TLR5 mAb and TLR5 was not changed apparently in two groups. Labelling rate was 95.90 ± 0.60% for 125I‐anti‐TLR5 mAb and 92.62 ± 0.34% for 125I‐IgG. The radiochemical purity of 125I‐antiTLR5 mAb and 125I‐IgG were both greater than 95%, respectively. Radiochemical purity of 125I‐anti‐TLR5 and 125I‐IgG were more than 90% up to 72 hours, relatively stable in serum and NS, and no significant difference between them was detected. Competitive binding analysis with excess unlabelled anti‐TLR5 mAb (>500‐fold) could almost completely block the binding of 125I‐anti‐TLR5 mAb (<5%), while only about 3% non‐specific binding observed for 125 I‐IgG /mm2 of tumour area reached 121 042 ± 5587 cpm for TLR5+ 4T1 tumour, whereas only 34 245 ± 2747 cpm for TLR5− 4T1 tumour. Block group  had a lower %ID/g, which was in agreement with the imaging data. As shown in Figure 125I‐antiTLR5 mAb exhibited higher targeting efficiency in TLR5+ 4T1 tumours, compared with TLR5− 4T1 tumours. The uptake of 125I‐antiTLR5 mAb in TLR5+ 4T1 tumours at 24, 48 and 72 hours post‐injection was 7.725 ± 0.7525, 4.9225 ± 0.36 and 2.5775 ± 0.1825 (%ID/g), T/NT ratio of 6.481 ± 0.6023, 8.413 ± 0.5270 and 7.152 ± 1.040, respectively, while the uptake of 125I‐antiTLR5 mAb in TLR5− 4T1 tumours was 2.8225 ± 0.1975, 1.4325 ± 0.1125 and 0.845 ± 0.0475 (%ID/g), with T/NT ratio of 2.353 ± 0.091, 2.489 ± 0.1541 and 2.308 ± 0.1631. The T/NT ratio of 125I‐antiTLR5 mAb group in TLR5− 4T1 tumours was significantly lower than in TLR5+ 4T1 tumours group, P < .05 .Ex vivo biodistribution studies were performed to validate the imaging results and to further quantify the 3.6− and TLR5+ 4T1 tumours, representative microscopy images at 200× and 400× magnification from the HE staining (left) and immunohistochemistry staining for TLR5 (right) are shown in Figure + is 69.75 ± 5.25%, much higher than that in TLR5− tumours (21.75 ± 3.15%), n = 5, ***P < .01.For TLR54The promising era of cancer diagnosis is greatly developed through the targeted molecules finding.Recently, it was reported that TLR5 was associated with tumour growth and metastasis,− 4T1) with lentivirus‐shRNA TLR5 transfection, which is stable technique in down‐regulation a definite gene expression. Our results proved that with this treatment, TLR5 expression was indeed down‐regulated both on mRNA and protein level. Since TNBC has no targeting molecules reported until now, we prepared a radio‐iodine 125 labelled anti‐TLR5 tracer which showed higher labelled rate and high affinity to TLR5 on 4T1 cell in vitro, and relatively stable in serum and NS.Here, firstly we proved that TNBC cell line 4T1 expressed TLR5 both on mRNA and protein level; then, we constructed a TLR5 known down 4T1 cell line  tumours than in 4T1 (TLR5−) tumours at all checking time‐points both ex vivo distribution and in vivo whole‐body phosphor‐autoradiography. In block group with unlabelled anti‐TLR5 antibody pre‐treatment, 125I‐antiTLR5 mAb failed to target TLR5 in tumour‐bearing mice, no tumour radio image could be obtained, which revealed the specificity of the 125I‐antiTLR5 mAb imaging. 125I‐IgG is isotype control to show non‐specific imaging of Fc fragment. 125I‐IgG failed to target TLR5 accurately in tumour‐bearing mice, exhibiting non‐specific retention in tumours. Using fluorescent reporter gene markers to detect the growth, migration labelled cells in living organisms is applied in many kinds of diseases in vivo.Further, we investigated that whether TLR5 could be used in targeting molecule imaging for TNBC in vivo. 125I‐antiTLR5 mAb make it a promising radiotracer for TNBC imaging in vivo.These data suggest that TLR5 should be considered as an in vivo biomarker of TNBC. It could be applied for non‐invasive molecular imaging to monitor tumour development and metastasis, and evaluate therapy response, even as a target for therapy. Low uptake of tracer in main organs such as the heart and high uptake in tumours apparently increased image contrast. These tumour cell‐specific and favourable non‐target clearance features of 125I‐antiTLR5 mAb may provide a method for visualizing TLR5 expression in vivo. Our data suggested that 125I‐antiTLR5 mAb could be used in these TNBC tumour models, which is more clinically relevant.Targeted radioimmunoimaging with monoclonal antibody has been considered a successful imaging for many kinds of cancer.However, there are some limitations for this study: The Molecular 125 weight of monoclonal antibodies is large and it metabolizes slowly in the human body. So it isn't well suitable for clinical application. To improve clinical application, the optimized choice is to reduce extended circulation through selecting antibody fragment or adapter or other small size molecules specifically binding to TLR5, and also we need further study for TLR5 targeting probe with different radioisotope labelled suitable for clinical application, such as iodine 131 or positron nuclide F‐18. Another question is the molecular mechanism underlying regulation of TLR5 in different histological subtype of breast cancer. So, further in vitro and in vivo investigations about TLR5 in breast cancer are required.125I‐antiTLR5 mAb was successfully prepared; in vitro and in vivo experiments results revealed that TLR5 expressed on the surface of 4T1 cells was a specific imaging target of 125I‐antiTLR5 mAb; TLR5 is a new reporter for triple‐negative breast cancer non‐invasive imaging. Our results supply a new strategy for TLR5‐positive tumour monitoring.In conclusion, TLR5 expression on TNBC cell line 4T1 stable was knocked down successfully. The authors confirm that there are no conflicts of interest.Guihua Hou designed research; Dai Shi conducted research; Dai Shi analysed date; Guihua Hou, Dai Shi, Weiwei Liu and Shanshan Zhao wrote the paper; and all members had primary responsibility for final content. All the authors read and approved the final manuscript.


VPZ2 and VPZ3. Through spectral titration, VPZ3 was found to yield the desired threshold-limited signal. VPZ3 was suitable for in vitro bioimaging of viscosity under one-photon or two-photon excitation. VPZ3 is potentially useful in many downstream applications. Future work includes fine-tune of the threshold to allow tailored limit for fluorescence turn-on to better meet the need of different applications. Besides the implications in the real-world applications, the design concept could also be translated to design of alternative substrates.Viscosity of body fluid is an established biomarker of pathological conditions. Abnormality of cellular viscosity occurs when cells are challenged with external stresses. Small molecule probes to assess the viscosity are sought after for both disease diagnostics and basic studies. Fluorescence based probes are particular attractive due to their potentials for convenient and high spatiotemporal resolution microscopic monitoring of biological samples. The dyes with a floppy push-pull backbone or dyes with a rotatable substituent exhibits a viscosity responsive fluorescence enhancement and therefore viable viscosity probes. The scaffold of the existing viscosity probes contains typically one such floppy site. Therefore, they typically linearly respond to log(viscosity). We argue that minor viscosity fluctuation could potentially be physiological as the biological system is dynamic. We wish to develop a type of conceptually-new, threshold-limited viscosity probes, to complement the existing probes. Such probes do not exhibit a fluorescence enhancement when challenged with minor and presumably physiological enhancement of viscosity. When the viscosity is higher than a certain threshold, their fluorescence turns on. We hypothesize that a dye with two far-apart floppy sites could potentially yield such a threshold-limited signal and designed  Viscosity is a biophysical parameter of homeostasis Tsien, . By alteP1  for microscopic viscosity.Herein, we report the synthesis, spectral titrations and proof-of-concept bioimaging of threshold-limited fluorescent probe  was condensed with ethylene glycol in the presence of p-toluene-sulfonic acid to the corresponding 1,3-dioxolane derivative (2) in an 85% yield. Then 2 was treated with nBuLi at −78°C to generate the nucleophilic intermediate phenyllithium reagent in situ, which was quenched with DMF to yield the benzaldehyde derivative (3) in good yield. Through aldol condensation of 3 and 1,4-cyclohexanedione monoacetal under basic conditions, 4 was obtained in an 95% yield. (2-Phenoxyphenyl) lithium was added into a solution of 4 in THF. The resulting carbinol product was isolated and treated with methylsulfonic acid without further purification to afford 5 through a cascade of reactions. The crystal structure of the compound 5 was obtained and the diphenylether moiety and the bottom dinaphthylmethanone unit are perpendicular. Replacement of fluorine atoms of 5 with methoxide gave 6 in an 35% yield. Then the two methoxys groups of 6 was demethylated by BBr3 to get 7, the hydroxyls of which were converted to triflate by treatment of triflic anhydride to obtain the key intermediate 8. The viscosity probes (VPZ2 and VPZ3) were furnished by the Suzuki–Miyaura cross coupling reactions with 9 and 10, respectively. The NMR and the HRMS spectra of all new compounds are provided in the SI , tetramethylrhodamine (25 nm), BODIPY (20 nm), Cy5 (20 nm), Cy7 (23 nm), which is desirable for imaging-based applications. We tested the viscosity related spectral responses  with the increase of viscosity. Very interestingly, the fluorescence intensity of VPZ3 did not immediately increase when the solvent viscosity increased (VPZ3 enhanced linearly with respect to Log(viscosity). This is in good agreement with the design rational of the VPZ series of probes. The fluorescence life-time decay of VPZ3 in different solvent mixtures of H2O and glycerol was acquired  test (The short absorption wavelength of (δ) test  since thVPZ3 was determined by MTT. In short, HepG2 cells were incubated with VPZ3 with different concentration for 24 h. The cell viability remained 85% at up to 25 μM  at 37 and 25°C for 30 min separately. The ptimages of the cells were collected under two-photon excitation. As we known, lower temperature means higher viscosity for the cells. The fluorescence intensity of cells incubated at 25°C was higher than those at 37°C significantly  can cause cell death, the micro-viscosity of the cells will change greatly during the apoptosis process. HepG2 cells were incubated with etoposide. The two-photon fluorescence images were collected at different times during the apoptosis process. As shown in VPZ3 could be used to monitor the viscosity changes during the apoptosis process.To further demonstrate the potentials of VPZ) for viscosity monitoring based on a symmetric D-A-D framework. Among the three VPZ probes, VPZ3 showed a great “off-on” fluorescence response (ca. 126-fold) with increasing viscosity in the glycerol-water system. VPZ3 shows low cytotoxicity and could be used to monitor the real-time viscosity change during the cell apoptosis process. We expect VPZ3 to find applications in basic biological studies related cell apoptosis and disease diagnostics.In summary, we have developed a series of new fluorescence probes (All datasets generated for this study are included in the manuscript and/or the ZL prepared the compound synthesis. KX and SQ involved in the synthesis and carried spectral titrations. LH carried out biological studies. YY and XM conceived the project. Everyone contributed to manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To review the imaging characteristics of Morel-Lavallée lesions with both ultrasound and magnetic resonance imaging (MRI).We retrospectively analyzed 31 patients (mean age = 46 years), diagnosed with a Morel-Lavallée lesion, on ultrasound (n = 15) or MRI (n = 16). On ultrasound the echogenicity, internal septations, hyperechoic fat globules, compressibility and Doppler signal were evaluated. On MRI, T1- and T2-signal intensity, capsule presence, internal septations, enhancement, mass-effect and fluid-fluid levels were assessed. The MR images were classified according to the classification of Mellado and Bencardino.Most of the lesions were situated peritrochanteric, around the knee or the lower leg. The majority of the lesions had a heterogeneous hypoechoic appearance with septations and intralesional fat globules. On MRI, most of the collections were hypointense on T1-weighted images and hyperintense on T2-weighted images. Half of the collections were encapsulated, and most collections demonstrated septations. The collections were classified as seroma (n = 10), subacute hematoma (n = 2) and chronic organizing hematoma (n = 5).Ultrasound is the imaging method of choice to diagnose Morel-Lavallée lesions. MRI can be of use in selected cases . Characteristic imaging features include a fusiform fluid collection between the subcutaneous fat and the underlying fascia with internal septations and fat globules. On MRI, six types of ML lesion can be differentiated, with the seroma, the subacute hematoma, and the chronic organizing hematoma being the most frequently observed lesions. In 1863, the French surgeon Victor Auguste François Morel-Lavallée originally described posttraumatic fluid collections dissecting the subcutaneous fat, resulting from soft tissue injury to the proximal thigh 34. These35Severe trauma to the pelvis or thigh, such as motor vehicle accidents or low-velocity crush injuries, may result in a ML lesion . This le98The mechanism of injury is a sudden shearing force that occurs tangential to the fascial planes, causing the somewhat mobile dermis and subcutaneous fat to abruptly move relative to the firmer underlying fascia Figure  12]. Th. Th12]. 3The peritrochanteric area is particularly sensitive to this injury due to the increased mobility of the soft tissues in this region, the superficially located bone, a strong underlying fascia lata attaching to the iliotibial band, and a rich vascular plexus piercing the fascia lata .Several forms of subcutaneous fluid collections may result, depending on the severity of the injury, ranging from seroma to hematoma. After some time, the blood is reabsorbed and replaced by serosanguineous fluid . This he2This serosanguineous collection may either spontaneously resolve or undergo a secondary inflammatory reaction with subsequent fibrous capsule formation 4791112134791112In the literature, six types of ML lesions have been described based on the chronicity, tissue composition and lesion appearance on magnetic resonance imaging (MRI) . Mellado2The goal of our study was to retrospectively analyse patients with ML lesions on both ultrasound and MRI. We classified the lesions according to the above mentioned classification, and we subsequently briefly reviewed the imaging features of ML lesions.Morel-Lavallée and closed degloving injury were inserted in the search engine of the PACS system; the imaging reports and associated MR-images were analysed and 31 patients were included in the study. Fifteen patients had ultrasound examinations available and 16 patients were evaluated with MRI. The location and morphology were analysed for all patients on both imaging modalities:From 2005 to 2016, 31 patients [mean age = 46 years (range 13–85)] were included in this retrospective cohort study. The search terms location: greater trochanter/hip, proximal thigh, suprapatellar region of the knee, abdominal wall, periscapular, scalp, arm, hand, lumbosacral region.morphology: fusiform, oval, laminar.The ultrasound examinations (both report and images) were specifically evaluated for the following characteristics: echogenicity, internal septations, hyperechoic fat globules, compressibility and Doppler signal.The following characteristics were evaluated on the MR-images: T1- and T2-SI, presence of a (pseudo) capsule, septations or nodules within the collection, internal enhancement (if intravenous contrast was administered), mass-effect and fluid-fluid levels.The patients with MR-images were classified according to the classification of Mellado and Bencardino, who proposed an MRI classification of ML lesions corresponding to their histopathological stadium . Six cattype I ML lesion is a seroma which exhibits fluid-like signal characteristics on MRI . Other locations were the anterolateral compartment of the lower leg (7/31 = 23%), greater trochanter/hip (8/31 = 26%), gluteal (1/31 = 3%), ankle (2/31 = 6%), upper arm (1/31 = 3%) and lumbosacral region (2/31 = 6%). The predominant morphology was fusiform (26/31 = 84%), and oval collections were only seen to a minor extent (5/31 = 16%). Only 3 of the injuries extended in different compartments and demonstrated underlying muscle contusion.The majority of the collections demonstrated a heterogeneous hypoechoic ultrasound appearance with intralesional septations 13/15 = 87%) and/or hyperechoic fat globules (10/15 = 67%)  and all cases were hyperintense on T2-WI. More than half of the collections were encapsulated (11/16 = 68%) and demonstrated internal septations (9/16 = 56%). Fat globules were present in fifty percent of the cases (8/16). None of the collections showed enhancement (note that intravenous gadolinium contrast was only administered in three patients). Three collections revealed mass-effect on surrounding structures and fluid-fluid levels were present in one patient. The collections were classified according to Mellado and Bencardino as seroma  . Characteristic imaging features include a fusiform fluid collection with septations and fat globules between the subcutaneous tissues and the underlying fascia.On MRI, six types of ML lesion can be differentiated, with the seroma, the subacute hematoma, and the chronic organizing hematoma being the most frequently observed lesions. Knowledge of the common locations, the specific location between the subcutaneous tissue and the fascia, the imaging signs, and knowledge of the classification system may all be helpful in the differential diagnosis with other fluid-filled collections in the soft tissues.