Plants in soil are not solitary, hence continually interact with and obtain benefits from a community of microbes (“microbiome”). The meta-functional output from the microbiome results from complex interactions among the different community members with distinct taxonomic identities and metabolic capacities. Particularly, the bacterial communities of the root surface are spatially organized structures composed of root-attached biofilms and planktonic cells arranged in complex layers. With the distinct but coordinated roles among the different member cells, bacterial communities resemble properties of a multicellular organism. High throughput sequencing technologies have allowed rapid and large-scale analysis of taxonomic composition and metabolic capacities of bacterial communities. However, these methods are generally unable to reconstruct the assembly of these communities, or how the gene expression patterns in individual cells/species are coordinated within these communities. Single-cell transcriptomes of community members can identify how gene expression patterns vary among members of the community, including differences among different cells of the same species. This information can be used to classify cells based on functional gene expression patterns, and predict the spatial organization of the community. Here we discuss strategies for the isolation of single bacterial cells, mRNA enrichment, library construction, and analysis and interpretation of the resulting single-cell RNA-Seq datasets. Unraveling regulatory and metabolic processes at the single cell level is expected to yield an unprecedented discovery of mechanisms involved in bacterial recruitment, attachment, assembly, organization of the community, or in the specific interactions among the different members of these communities. Plants are holobionts and are associated with complex and very diverse microbiomes . The lonMost plant-associated microbial communities, for example root surface bacterial communities, are spatially organized structures composed of root-attached biofilms and planktonic cells arranged in complex layers . In addiMethods for isolation of bacterial communities from plant root surfaces and apoplastic compartments without plant tissue contamination are available . IsolatiEscherichia coli and Procholorococcus to develop a polymerase-based whole genome amplification method, polymerase cloning or “ploning”  can be used to detect and sort cells from a population based on their different chemical or physical characteristics. Cells in suspension are transported, one cell at a time, and pass through a laser beam. Scattered light is characteristic of individual cells based on their composition and/or physical properties and is used to gate cells into collection chambers . Typicalin situ-hybridization (FISH). Limitations of the traditional 16S rRNA FISH technology, for example the limited and variable amounts of rRNA, have been addressed by the development of liquid phase tyramide signal amplification FISH (TSA-FISH), which is compatible with flow sorting. Similarly, custom made μFACS systems have been developed that support faster throughput and less expensive applications with lower contamination risks due to their use of closed systems, and their higher sorting efficiency  with a cell size range of up to 50 μm, and the Fluidigm C1 platform  with a cell size range of 10–17 μm. Recently, single-cell printing methods were adapted for the encapsulation of single bacterial cells in droplets . These sAlternatively, it is possible to use custom-made drop-seq devices in combination with commercially available barcoded beads . It is iMany of the cell isolation methods have successfully been used to obtain genome sequences of individual bacterial cells, and can be adapted to obtain epigenomes as well. However, their adaptability for scRNA-seq is limited. Lack of polyadenylation in bacterial mRNAs, for example, requires methodologies to selectively enrich these molecules from the ∼10 times more abundant tRNA and rRNA molecules . The very low RNA content of bacterial cells is another challenge, and requires the amplification of RNA or cDNA molecules, while amplification biases are avoided (see section “Methods to amplify RNA or cDNA”). Finally, the highly complex bacterial cell wall poses a challenge since enzymatic and chemical approaches to disrupt the cell wall and membrane may not be compatible with the reagents for the subsequent steps in RNA-seq library construction.The need to prepare individually labeled sequencing libraries from individual cells precludes the use of typical affinity-based methods for enrichment of mRNAs or removal of rRNAs. In contrast, in-cell mRNA enrichment methods are suitable for this purpose.TM 5′-phosphate-dependent-5′- 3′exonuclease digests rRNAs and tRNAs, but not RNA molecules with a 5′-triphosphate, a 5′-cap, or a 5′-hydroxyl group. Consequently, TerminatorTM exonuclease can be used to selectively degrade rRNA and tRNA molecules with a 5′-phosphate structure, but not mRNA molecules. An exonuclease treatment at optimal concentrations enriched mRNA molecules from single cells of Burkholderia thailandensis and enables its potential use for next-generation sequencing  primer approach, random hexamers that match rRNA (or other non-target RNA) sequences are left out from the pool of random primers used for cDNA synthesis . This apB. thailandensis (DpnI), and the newly synthesized single-stranded cDNA (ss-cDNA) was circularized via 5′-end phosphorylation and intramolecular ligation. The circularized ss-cDNA was then randomly primed with RNA hexamers and subjected to multiple displacement amplifications using ϕ29 DNA polymerase. Thiophosphate-linked RNA random hexamers were used to reduce primer dimers and non-specific priming , a unique chimeric 5′-RNA/DNA-3′ primer is used for first strand cDNA synthesis. Second strand synthesis results in a double-stranded cDNA with a unique DNA/RNA heteroduplex at one end. RNase H–mediated degradation of RNA in this heteroduplex enables binding of another chimeric 5′-RNA/DNA-3′ primer which is then extended to displace the existing first strand. The process of chimeric DNA/RNA primer binding, DNA replication, strand displacement and repeated RNA cleavage leads to a rapid accumulation of amplified cDNA . SPIA wafication .A number of other methods typically used for the amplification of polyadenylated mRNA molecules from single cells of eukaryotic organisms  can alsoSplit pool ligation-based transcriptome sequencing, a recently developed alternative for scRNA-seq, labels the cellular origin of RNA through combinatorial indexing . This mein situ hybridization and PCR as well as the selective enrichment and amplification of bacterial mRNAs are very promising for the effective adaptation of SPLiT-seq for bacterial scRNA-seq . The successful application of methods for the permeabilization of bacteria for cRNA-seq .in situ localization of RNA through hybridization and PCR in a number of Gram-positive and Gram-negative bacteria (in situ reverse transcription (RT) and PCR with labeled nucleotides  can be polyadenylated at the 3′-end using a Poly(A) polymerase in the presence of ATP . The ligation plate wells would have dsDNA molecules with three distinct functional domains: a 5′-overhang that is complementary to the 5′-end on the cDNA molecule (originating from the RT primer), a unique well-specific barcode sequence, and the other 5′-overhang complementary to the 5′-overhang present on the DNA molecule that is ligated in the next ligation round  is a good starting point, as a reference resource. Following regular read mapping , normalide novo assembly of unmapped reads in each single cell to construct contigs using the assembly tool, Minia  and their expression values are determined by counting the number of reads that map to each TU. Machine learning algorithms such as SeqTU also enable accurate prediction and identification of TUs . A web sacterium . In 2019acterium . The preIn scRNA-seq of bacterial communities, clustering based on their expression profiles and the cluster can be evaluated in two different ways: (1) biological process-based, and (2) taxonomy-based. In the first approach, each cluster is evaluated for enriched biological processes compared to other clusters using Gene set enrichment analysis  and the Herbaspirillum seropedicae (20%), Azospirillum brasiliense (5%), Bacillus thuringiensis (10%), Rhizobium leguminosarum (15%), Flavobacterium frigidarium (8%), Actinokineospora diospyrosa (12%), Bradyrhizobium sp. (20%), and Methylibium sp. (10%) can be used  in a defined community. Ideally, all reads with the same barcode or those that came from a single cell should map to a single bacterial genome barring some highly conserved genes. Nevertheless, the results from these analyses can provide a benchmark to evaluate the accuracy with which scRNA-seq is able to distinguish transcripts from each individual bacterial species. Similar to the comparison of scRNA-seq data to bulk cell RNA-seq datasets from individual species , communiEvaluating the gene expression patterns in individual cells of plant-associated bacterial communities can provide transformative information not only about the gene expression levels and thereby function in individual members of different species but also about the spatial organization of bacterial communities in plant microbiomes. For example, cells with a higher expression of genes involved in exopolysaccharide synthesis are likely part of biofilms, while those expressing pili-encoding genes are likely attached to the plant surface. The estimated sequencing depth of 500 million reads per sample of 5000 cells  can be obtained from two sequencing runs  making this approach relatively inexpensive given the depth of information obtained. One of the major challenges is the complexity associated with multiple bacterial genomes present in the community, and the extent of genome sequence information available for each species. Nevertheless, since transcripts from each cell are tagged, general functional capacities expressed in individual bacterial cells can be determined. In fact, it becomes increasingly clear that the metabolic functions of bacterial communities are more important than their taxonomic composition .SS conceptualized the review topic. All authors wrote and edited the manuscript.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.


Most previous work on dynamic functional connectivity (dFC) has focused on analyzing temporal traits of functional connectivity (similar coupling patterns at different timepoints), dividing them into functional connectivity states and detecting their between-group differences. However, the coherent functional connectivity of brain activity among the temporal dynamics of functional connectivity remains unknown. In the study, we applied manifold learning of local linear embedding to explore the consistent coupling patterns (CCPs) that reflect functionally homogeneous regions underlying dFC throughout the entire scanning period. By embedding the whole-brain functional connectivity in a low-dimensional manifold space based on the Human Connectome Project (HCP) resting-state data, we identified ten stable patterns of functional coupling across regions that underpin the temporal evolution of dFC. Moreover, some of these CCPs exhibited significant neurophysiological meaning. Furthermore, we apply this method to HCP rsfMR and tfMRI data as well as sleep-deprivation data and found that the topological organization of these low-dimensional structures has high potential for predicting sleep-deprivation states (classification accuracy of 92.3%) and task types .In summary, this work provides a methodology for distilling coherent low-dimensional functional connectivity structures in complex brain dynamics that play an important role in performing tasks or characterizing specific states of the brain. The human brain is a hierarchically organized complex system that can be empirically parsed into functionally specialized units commonly referred to as functional brain networks. A large body of neuroimaging studies have made substantial progress in delineating this functional architecture mainly based on resting-state static functional connectivity (sFC). These stable spatiotemporal patterns of resting-state functional activity in a population closely resemble patterns of evoked task-based brain activity  and haveRecently, the use of the functional brain network has been extended from sFC measured based on the entire scan to its dynamic aspect  that assumes evident time-varying fluctuation of network connectivity during a period of unconstrained rest ,5,6 and In this paper, we ask whether there are intrinsic coupling patterns stably existing during the dynamics of functional connectivity, i.e., some functional connections can be grouped into different clusters based on the similarity of their correlation time courses. These clusters, if they exist, would reflect the functionally homogeneous regions that constitute the spatial components supporting dFC evolution. For example, a top-down parcellation strategy using time courses of instantaneous connectivity  has been confirmed to produce biologically valid functional subdivisions of the cortical cortex and subcortex . It has In particular, we used manifold learning of local linear embedding (LLE) to extract the consistent connectivity patterns (CCPs) underlying dFC. As a fully data-driven approach, manifold learning has been gradually applied to neuroimaging analysis with many advantages of its shared manifold nature, geometric intuition, and high-dimensional data visualization, including detecting activated voxels , reducinWe further demonstrated that these CCPs (the embedded dataset structures) have significant neurophysiological relevance by applying the proposed model to discriminate cognitive tasks from the resting state and to detect changes in brain activity induced by sleep deprivation. We assumed that different cognitive tasks or varying brain states could “activate” discrepant CCPs in dFC that have differentiated topological structures. The topological organization of these CCPs was quantitatively evaluated based on one core-quality score with each node (connection). We further demonstrated the potential of the CCPs’ topological organization for classifying the sleep-deprivation condition and different cognitive tasks with high accuracy, suggesting that these coherent low-dimensional functional coupling structures in complex brain dynamics play an important role in performing tasks or characterizing specific states of the brain.The primary dataset used in this work was selected from the Q3 release of the Human Connectome Project dataset (HCP). A second dataset (the sleep-deprivation dataset) was used to verify the effects of the proposed framework. These details are described below.https://www.humanconnectome.org).(1) HCP dataset: The first dataset consisted of 40 healthy subjects  were selected from the Q3 release of the HCP dataset with low mean head motion (<0.23 mm) during both resting-state sessions [(2) Sleep-deprivation (SD) dataset: The second dataset comes from the 36 h Acute Sleep Deprivation (ASD) study of 37 healthy, right-handed adult men , to remove non-neural spatiotemporal components from the data after the minimal preprocessing pipelines for HCP functional data which mainly includes spatial artifact and distortion removal, motion correction, within-subject cross-modal registration, and cross-subject registration to a standard space . In addiactivity . For tfMactivity .http://www.fil.ion.ucl.ac.uk/spm) was used for preprocessing rsfMRI ASD data. For each participant, the first 5 volumes of scan data were removed for magnetic saturation. The remaining 235 volumes were recalibrated by registering and re-slicing for head movements. Next, normalization to the EPI template in the Montreal Neurological Institute (MNI) space was conducted, and the resulting volumes were resampled to SPM12 software  is the number of the whole-brain functional connectivity pairs and D is the number of sliding windows. In this study, the slide step was selected as 5 TRs and the window size was 40 s, which were thought to achieve a balance between the ability to resolve the dynamics and quality of the covariance matrix estimation [A sliding-window correlation approach was applied to characterize dynamic functional connectivity (dFC) , which htimation .S. For an observation s was calculated as the average of A manifold learning approach of local linear embedding (LLE)  was usedS to the LLE algorithm as the input which includes three steps of transforming the matrix S in D-dimensional Euclidean space into the embedding matrix V in d-dimensional manifold space. Step 1: for the sample point k nearest neighbors W summed to one. Step 2: we obtained the optimal reconstruction linear coefficients W by resolving the minimum value of the cost function Equation  [d clusters were obtained. We displayed these clusters in the functional connectivity map and found the functionally consistent coupling patterns (CCPs). Thus, we defined these clusters with a new name “CCPs”. The d CCPs were therefore obtained.The low-dimensional parameterization of the dataset had a specific shape with extended branches, which was expected in the embedding structure with the LLE algorithm. The coordinates of the embedding spaces with the LLE algorithm are related to meaningful attributes: representative faces are shown next to the out-stretching points in the face image experiments and the intersection of two regions locates words with similar contexts in this continuous semantic space in the trial of the semantic associations of words . Therefocluster) . Then, wi-th CCP for one subject manifold structure model and j-th CCP for another subject manifold structure model are calculated using the following equation:i-th CCP from one subject model and the j-th CCP from another subject model during the scanning, and i-th CCP from the one subject model or the j-th CCP from another subject model.The identified CCPs were reranked based on their spatial overlap rate (SOR), which reflects the spatial correspondence between the CCPs identified from the K-means results of LLE embedding models, i.e., the two CCPs respectively obtained from the HCP dataset and AR group of the sleep models with the highest SOR were more likely to spatially correspond. Specifically, the SOR between the Spontaneous fluctuation in functional connectivity has been linked to changes in cognitive or vigilance states ,32, and V. Core-periphery detection was performed by using a nonlinear spectral (NSM) algorithm [n, with a higher rank denoting a more core connection. The core-periphery profile function A, which could be obtained via calculating the embedded representation V of the dFC dataset with the KNN algorithm and k, we regard x reveals a strong core-periphery structure if k. The value x varies per iteration of the NSM algorithm until Here, we quantified the manifold structure of CCPs (embedding dataset) by computing a core-quality value  for eachlgorithm  derived lgorithm ,35,36. Tolerance . Thus, tticipant D. This ci of matrix Herein, the spatial similarity between the core-quality value FC maps was calculated using eta2  as the fc for each subject as input features. To achieve good prediction, constructing the feature space for the classification model was important. The discriminative power of a feature can be quantized by the value of the two-sample t-test for the training dataset. A smaller value means a more discriminative power. For every training and testing loop, feature selection was conducted with the statistical results from the training dataset. Then, the prediction would be performed in the feature selection on the training dataset. Then, we used a linear support vector machine (SVM) classifier to validate the discriminative capacity of the CCPs topology for classifying cognitive task scans from the resting-state scans. For each of the seven classifiers, a leave-one-out cross-validation procedure was performed 100 times to estimate the generalization ability. Finally, the unbiased classification accuracy (ACC), sensitivity (SS) and specificity (SC) were computed based on the results of cross-validation [To exemplify the high potential of CCPs’ topological properties from providing specialized whole-brain configuration information at the single-participant level, we implemented the HCP task classification experiment E. First,lidation . To justt-test We further detected the changes in CCPs induced by sleep deprivation to link the CCP topological properties  into potential neurophysiological sources. We provided results of two experiments, including an individual prediction for sleep deprivation and a statistical comparison between sleep deprivation (SD) and resting-wake (AR) conditions E. In the testing E designend, 5th and 8th dimension in We found that local linear embedding of dFC based on the HCP resting-state dataset leads to a prominent manifold structure of a “specific shape with extended branches” , in whicWe also directly explored relations between the connectivity pairs of the dFC dataset and the linearly (PCA) embedded dataset. Using the Euclidean distance among the connectivity pairs with a K-means approach, the CCPs in a high-dimensional space were obtained . The higc and we rearranged the core-quality vector c into the core-quality matrix C using the ROI-based connectivity index in the brain template. Then, the rows and columns of the matrix C represent the ROI index in the brain template. After rearrangement using this rule, the elements in the matrix C quantify the importance of the functional connectivity in the whole scanning procession between ROIs. Consequently, the functional connectivity NSM value matrices for the resting state and 7 tasks were obtained and shown in More important connectivity in the core-quality value matrix was assigned a larger value within all the connectivity. The NSM algorithm assigned the node (connectivity) core-quality values with the core-quality value vector t values of the distinguished connections for each pair of brain states  with the NSM algorithm, we also assigned the core-periphery values to the connectivity pairs of the dFC dataset  and the topological organization of the linear structure (PCA dimension reduction) for the resting state and seven tasks. The SVM classifier results showed unbiased classification accuracy (ACC), sensitivity (SS) and specificity (SC) in shown in  could beApparent changes in the CCPs under ASD and AR conditions are shown in t-test t-test using the ASD and AD labels. The experimental results can be divided into two parts  prompted the finding of functionally consistent coupling patterns (CCPs) in the manifold space. This inspired the perspective in nonlinear couplings of the connectivity pairs. The high performance of supervised classification (SVM) certified the application potential of the nonlinear structure and our framework in fMRI data. There is no doubt that the use of supervised classification increases the practical applicability of our method and the embedding representation. Furthermore, the highly distinguishable connectivity pairs for the classifiers proved that the nonlinear structure we extracted has physiological significance.It is vital to understand the relationship between the neural basis of cognition and our study. Our results indicate that both the CCPs and their core-periphery structure exhibit significant task effects and ASD effects . This fiThis article presents a manifold learning of local linear embedding to distill the CCPs that reflect functionally homogeneous regions underlying the temporal evolution of dFC throughout the entire scanning period. Our results demonstrate these stable patterns of functional couplings across regions by uncovering the significant neurophysiological meaning underpinning the CCPs. The specific subcortex-cortical circuit is captured by the CCPs. Moreover, the topological organization of these low-dimensional structures exhibits high potential in predicting sleep-deprivation states (classification accuracy of 92.3%) and task types . Thus, these results suggest that our methodology for distilling coherent low-dimensional functional connectivity structures in complex brain dynamics is capable and competent.


Such approach requires multiple hospital attendances and can be time and resource intensive. Recently, whole-body magnetic resonance imaging (WB-MRI) has been used in a single visit scanning session for several malignancies, including prostate cancer, with promising results, providing similar accuracy compared to the combined conventional imaging techniques. The LOCATE trial aims to investigate the application of WB-MRI for re-staging of patients with biochemical relapse (BCR) following external beam radiotherapy and brachytherapy in patients with prostate cancer.Accurate whole-body staging following biochemical relapse in prostate cancer is vital in determining the optimum disease management. Current imaging guidelines recommend various imaging platforms such as computed tomography (CT), Technetium 99 m  of WB-MRI for re-staging of BCR will be investigated against the enhanced reference standard on a per-patient basis. An economic analysis of WB-MRI compared to conventional imaging pathways will be performed to inform the cost-effectiveness of the WB-MRI imaging pathway. Additionally, an exploratory sub-study will be performed on blood samples and exosome-derived human epidermal growth factor receptor (HER) dimer measurements will be taken to investigate its significance in this cohort.The LOCATE trial will compare WB-MRI versus the conventional imaging pathway including its cost-effectiveness, therefore informing the most accurate and efficient imaging pathway.NCT02935816.LOCATE trial was registered on ClinicalTrial.gov on 18th of October 2016 with registration reference number  Prostate cancer (PCa) is the leading male cancer in the United Kingdom and a principal cause of cancer-related mortality with around 46,700 cases diagnosed in 2014  / DNA-RNA analysis. In total a maximum of 30 ml of blood will be taken:Up to 3 ethylenediaminetetraacetic acid (EDTA) containing tubes for peripheral blood mononuclear cells (PBMC) isolation and further FACS analysis, functional assays, and DNA/RNA isolation.Frozen aliquots-samples will be transported by courier service in dry ice-containing carrier boxes within 24 h after obtaining the blood sample. Samples will be processed at University College London (UCL) and King’s College London (KCL) laboratories:UCL: Exosome purification and analysis. Protein-DNA-RNA isolation and analysis.KCL: PBMC’s isolation, FACS analysis and functional assays.Data collection for the health economic analysis will be primarily concerned with cost data associated with the diagnosis and treatment of metastatic prostate cancer. Routinely collected administrative data from UCLH will be used to determine the relevant costs associated with the diagnostics test. To determine the consequence of detected metastatic disease on the treatment pathway, clinical experts will be interviewed, and patient notes will be reviewed noting changes to medication, radiotherapy, surgery, outpatient visits, inpatient visits and day cases, where possible. Data from existing trials such as STAMPEDE  and FOREThe study will be reported by two experienced radiologists, blinded to all other imaging investigations, initially independently and then in consensus using a locked sequential read paradigm (LSR). The images will be reviewed for the presence of nodal (N) and metastatic (M) disease and each radiologist will record the presence/absence of disease on the study-specific proforma.The initial WB-MRI read results will not be revealed to the clinical team. Where a significant additional/incidental finding is demonstrated this will be reported as per standard clinical practice and relayed to the clinical team.A 1–6 Likert scale , 25 willScoring will be conducted based on evaluation of imaging features of lymph nodes and bone metastases as indicated below in Table The study will be reported by the same pair of radiologists who reported the baseline scans. The findings on the follow-up study will be compared with the baseline WB-MRI with access to all conventional imaging. The presence of changes in size of lesions evident on the baseline WB-MRI and/or resolution of lesions evident on baseline WB-MRI will be recorded. Any discrepant findings will be reviewed, and the status of these lesions will be determined in a consensus panel review.Figure Patients will be divided into those with and without metastases evident on conventional imaging. For those with metastases  coupled with a PSA rise over the 12-month will classify the baseline WB-MRI as false negative, i.e. disease was present but not visualised. Whereas the absence of new lesions regardless of PSA status over the 12-month follow-up period will confer baseline WB-MRI true negative status.For positive findings on baseline WB-MRI discordant with conventional imaging, the baseline WB-MRI will be deemed true positive when a new lesion declared itself (on follow-up WB-MRI ± routine imaging) or the visualised lesion on baseline WB-MRI enlarged (on follow-up WB-MRI); and this was accompanied with a PSA rise within the 12-month follow-up period. Whereas, the baseline WB-MRI will be deemed false positive when the lesion evident at WB-MRI resolved or reduced in size at follow-up (in the absence of systemic therapy) regardless of PSA change. Lesions that resolved following systemic therapy  with corresponding decrease in PSA will be classified as true positive.Finally, patients with negative baseline conventional imaging with discordant lesion and PSA changes over the 12-month follow-up (i.e. lesion size/number increase without corresponding PSA increase) will be classified as unknown.The section below describes the thresholds for PSA and lesion size changes, and they will be interpreted in the context of treatment intervention and subsequent treatment response or progression.PSA progression defined as ≥25% and ≥ 2 ng/mL increase in PSA recorded at least 12 weeks from baseline or start of treatment and confirmed on a 2nd reading 3–4 weeks later as previously defined , 27.PSA response defined as ≥50% decline from baseline or start of treatment and confirmed on a 2nd reading 3–4 weeks later , 29.Nodal size change Increase of ≥2 mm and decrease of ≥3 mm in short axis diameter on contrast enhanced mDixon sequence will be considered true change (progression and response respectively) rather than a measurement variation, with a 95% confidence interval. .Bone lesion (disease progression) ≥ 25% increase in size (sum of short and long axes diameters) of lesions on CT, or MRI , 31.Bone lesion (treatment response) ≥ 50% decrease in size (sum of short and long axes diameters) of lesions on CT/ MRI or normalisation of signal intensity on MRI , 31.Soft tissue lesion size change (disease progression) At least a 20% increase in size of lesion taking as reference the baseline diameter of the lesion .Soft tissue lesion size change (treatment response) at least a 30% decrease in the size of lesion, taking as reference the baseline diameter or disappearance of the lesion .18F-choline PET-CT, CT-CAP and 99mTc Bone Scan) will be independently reported by two radiologists and nuclear medicine physicians, later reviewed in consensus to clarify any discrepant findings. The report will be transferred to a proforma and final nodal (N) and metastatic (M) stages will be derived base on the reported proforma.The conventional imaging .The blood sample will be used to investigate the significance of the Human Epidermal growth factor Receptor (HER) activated dimer in recurrent prostate cancer. Up-regulation of the HER1 (EGFR)/HER3 dimer was recently found, for the first time, to limit the efficacy of anti- epidermal growth factor receptor (EGFR) treatment in human breast cancer, as directly shown by imaging of residual disease; suggesting the potential of combined EGFR/HER3-targeted treatment . This moFor patients undergoing ADT treatment, retrospective quantitative analysis of WB-MRI’s ADC and FF signal intensities will be performed to derive heterogeneity indices across confirmed true positive metastatic sites. The heterogeneity indices will inform Bayesian models to predict high-risk patients with aggressive disease that progress on ADT within 12-months of treatment.The health economic analysis will compare the incremental costs and effects of WB-MRI for metastatic disease detection compared with conventional imaging from a national health service (NHS) perspective and thus determine whether it is an efficient use of NHS resources. From an economic viewpoint, the cost-effectiveness of WB-MRI versus conventional imaging is determined by two main stages of the pathway: the treatment decision pathway and the treatment pathway (and its associated disease pathway). The former includes the time from presentation to treatment decision by the clinician and includes the imaging tests received; the latter includes the time period following the treatment decision and subsequent disease progression. In patients for whom the treatment decision with WB-MRI is the same as that with conventional staging, the subsequent disease pathways will be the same. Where the treatment decision with WB-MRI is different, the disease pathway will be different, yielding potentially different costs and health outcomes .The precise nature of the economic analysis will therefore depend on the degree of concordance between treatment decisions provoked by WB-MRI versus conventional imaging.There is concordance between the treatment decisions associated with WB-MRI and conventional imaging. In this case, the cost components included in the analysis will be conventional metastases imaging tests (as described above) and the costs of treating adverse events associated with conventional staging. The volume of resource use and unit costs will be taken from standard published sources, hospital administrative datasets and from trial data. Since the two algorithms yield the same treatment decisions, cost-effectiveness depends on the incremental cost only of WB-MRI versus conventional imaging.There is discordance between the treatment decisions associated with WB-MRI and standard imaging. In this case, cost-effectiveness depends on the incremental cost and effects of the treatment decision pathway and the subsequent disease pathway associated with WB-MRI versus conventional staging.The economic evaluation will also take into consideration the accuracy of WB-MRI versus conventional standard imaging techniques. Specifically, we will model the consequences, in both costs and effects, of true positives, false positives, true negatives and false negative outcomes. Thus, the economic study will evaluate both clinical and economic consequences of the novel imaging technique.Cost-effectiveness will be calculated as the mean cost difference between WB-MRI versus conventional imaging divided by the mean difference in outcomes  to give the incremental cost-effectiveness ratio (ICER). Also, a cost-consequence analysis will be performed by calculating the mean incremental costs per incremental gain (or loss) in clinical natural units, for example cost per metastases identified.Finally, to address uncertainty in our clinical and economic parameters, we will perform a comprehensive set of sensitivity analyses on our model estimates. Probabilistic sensitivity analysis using non-parametric bootstrapping methods will be performed. Outputs from this will inform a cost-effectiveness acceptability curve, which shows the probability that WB-MRI is cost-effective at different values of the NHS’ willingness to pay for an additional QALY. We will also subject the economic model to extensive one-way sensitivity analysis to identify key parameters that drive cost-effectiveness.At the time of writing, the LOCATE trial is the largest prospective multi-centre trial to compare WB-MRI with conventional imaging pathway specifically in patients with radio-recurrent prostate cancer.The LOCATE trial will be informative because it compares comprehensive imaging pathways and takes into consideration the cost-effectiveness of each pathway. This trial will therefore provide valuable information to guide potential implementation of new imaging platforms for prostate cancer management pathways.The results from the LOCATE trial would be relevant to the current clinical management pathway, specifically, should WB-MRI be advocated in the NHS in the setting of BCR.The LOCATE trial design includes important features: unlike most other trials, the initial finding on WB-MRI will be validated using 1 year follow up WB-MRI scans, corroborating the findings on initial scan in the context of patient management. In this way, we minimise the potential overcalling of disease positivity by WB-MRI that could lead to an overestimated sensitivity of WB-MRI compared to conventional imaging techniques.Additionally, and in order to define the most cost-effective imaging pathway for the health care system, we will perform an economic evaluation which includes a comprehensive set of sensitivity analyses to identify uncertainty in both clinical and economic estimates. Whilst recent trial designs incorporating WB-MRI are including health economic analysis –38, suchThe LOCATE study has some potential limitations. Firstly, we are using a combined biochemical/imaging follow-up to derive an enhanced reference standard against which the results of WB-MRI will be analysed. Although histopathological confirmation would have been preferable in cases where a discrepancy exists between WB-MRI and conventional imaging tests, such approach is technically challenging and ethically not feasible .Secondly, whilst the qualitative and metastatic staging findings can be generalised to other imaging platforms, such generalisability would be limited in quantitative biomarker imaging analyses (heterogeneity index). However, ongoing work is being carried out to improve standardisation of quantitative MRI imaging across different platforms that could ultimately aid in generalisation of such technique , 40.68Ga-PSMA PET-CT within a short time has led to significant advances in prostate cancer imaging both in primary and recurrent settings [68Ga PSMA PET-CT at our institution and therefore a head to head comparison was not possible. More importantly, there is significant cost difference between the two modalities and PSMA is routinely not available in many centres in the UK.Finally, it is important to note that settings , 42. HowAt the time of submission, the study is currently at the data collection stage and full trial data analysis is expected to begin shortly.


Impaired neural plasticity may be an important mechanism in the pathophysiology of major depressive disorder (MDD). Coupled with electromyography (EMG), repetitive transcranial magnetic stimulation (rTMS) is a useful tool to evaluate corticospinal excitability and cortical neuroplasticity in living humans. The goal of this study was to compare rTMS-induced cortical plasticity changes in patients with MDD and in healthy volunteers. In this single-blind controlled study, 11 drug-free patients with MDD and 11 matched healthy controls were analyzed. Cortical excitability, measured by the amplitude of motor evoked potentials (MEPs) evoked by single-pulse TMS, was assessed before and repeatedly after (for 30 min) participants received a single session of intermittent theta-burst stimulation (iTBS) and continuous TBS (cTBS). rTMS was applied over the left motor cortex using a neuronavigation system. Intensity was set at 80% of the active motor threshold (AMT). A large interindividual variability was observed after both iTBS and cTBS in the two groups. At the group level, we observed impaired iTBS-induced neuroplasticity in patients with MDD compared to that in controls. No differences were observed between the groups regarding cTBS-induced neuroplasticity. Our results suggest impaired long-term potentiation (LTP)-like mechanisms in MDD.Clinical Trial Registration: www.Clinicaltrials.gov, identifier #NCT02438163. Unipolar major depressive disorder (MDD) is a very frequently occurring disorder associated with high impairment of global functioning and significant societal economic burden  is a noninvasive brain stimulation method that can be used to evaluate some indexes of neural plasticity in living humans. Some authors have suggested that the modulation of motor corticospinal excitability measured by single pulse TMS following a repetitive TMS (rTMS) session may reflect neural plasticity . All patients provided written, informed consent. The trial was conducted in the Hospital Le Vinatier, University Department for treatment-resistant depression, University of Lyon, France. All patients were consecutively recruited from March 2014 to October 2017.The study was approved by a local ethics committee (CPP Sud-Est 6), ANSM 2013-A00971-44 and registered in Fourteen patients with unipolar MDD according to DSM 5 and 14 matched healthy controls were enrolled in the study. Three patients with MDD were not included in the final analyzed sample. One patient with MDD was excluded because of an unexpected cerebral lesion discovered during the magnetic resonance imaging (MRI); one patient withdrew her consent; one was excluded because we were not able to obtain a 1 mV baseline MEP. It should be note that one patient only took part in the cTBS session because of strong nausea on the morning of the iTBS session, and setting up another day of investigation with the patient was not possible due to the introduction of antidepressant medication. In the healthy control group, three participants were not included in the final analyzed sample: two participants withdrew their consent, and one was excluded because she presented with an antecedent of a major depressive episode. Therefore, the final analyzed sample consisted of 11 healthy participants, 10 patients with MDD who received cTBS and 11 patients with MDD who received iTBS.Only right-handed patients (according to the Edinburgh Handedness Inventory) from both genders  with a Montgomery–Åsberg Depression Rating Scale (MADRS) score between 20 and 35 and free from any psychotropic drugs  were included. For patients with MDD under psychotropic drugs, the wash out period was at least of five half-life time of the concerned drugs. Exclusion criteria consisted of (i) melancholic features; (ii) presence of a neurological or psychiatric comorbidity, except for anxiety disorder; (iii) pregnancy; and (iv) contraindications for TMS.The group of healthy controls was composed of 11 right-handed individuals . The inclusion criteria consisted of the following: (i) no current psychiatric, neurologic or infectious disease with a potential effect on the brain; and (ii) free from any psychotropic drug. The exclusion criteria consisted of (i) pregnancy; and (ii) contraindications for TMS. Further characteristics of the participants are given in Participants were seated in a comfortable chair with both arms supported passively. Electromyographic (EMG) recordings from the right first-dorsal interosseus muscle (FDI) were taken using Ag/AgCl surface electrodes . Raw signals were amplified and digitized using a commercially available amplifier (Keypoint Portable System). All recordings were manually analyzed offline.TMS was applied over the left primary motor cortex (M1) using a posterior-anterior current direction through a standard figure-of-eight coil  connected to a MagPro-X100 stimulator. The coil was manually and tangentially placed with the handle pointing backwards at an angle of 45° to the midline. The stimulation site leading to large and stable MEPs was defined as the optimal coil position over the left M1. To ensure that the coil reliably remained over the same stimulation target throughout the entire experimental session , the coil was guided with an MRI-coupled neuronavigation system [SYNEIKA ONE (SYN1) version 1.5.1].To record MEPs at baseline and repeatedly after TBS, the TMS intensity was set to evoke MEPs of approximately 1 mV (S1mV) amplitude at baseline. We measured the peak to peak amplitude of 15 MEPs at baseline and 10 MEPs  to prevent diurnal influences on neurophysiologic measures  was undertaken with relative MEP value at the different time points as the dependent variable, group  as the between-subject factor, and time as the within-subject factor. Two RM-ANOVAs were conducted to analyze the effect of iTBS on the one hand and the effect of cTBS on the other hand.post hoc comparisons were performed to more specifically determine the changes in MEP amplitude. TBS-induced modulation of MEP size across the five time points  in both condition (iTBS and cTBS) were also investigated as the maximum peak amplitude at the individual level. Number of responders and non-responders after TBS according to Hamada et al. (p < 0.05.When appropriate (significant interactions in the RM-ANOVAs), a et al.  classifia et al. . SPSS 21There were no significant differences in age, gender, or AMT measures between groups at baseline. State and trait anxiety were significantly higher in patients with MDD than in healthy controls .p = 0.317). Before the cTBS session, the mean amplitude of 1 mV MEP in the MDD group was 1206.3 (385) vs. 975.4 (131) μV in the control group (p = 0.074).At baseline, there was no significant difference in the mean 1 mV MEP between the groups. Before the iTBS session, the mean amplitude of 1 mV MEP in the MDD group was 969.7 (SD 243) vs. 1055.7 (129) μV in the control group  = 2.504, p = 0.049).The RM-ANOVA revealed a significant group × time interaction when participants were exposed to iTBS . The difference was not significant at the other time points .The p = 0.009), whereas no modulation of MEP size was induced in patients with MDD (p = 0.339).Measured by the peak, the MEP size was significantly elevated by iTBS in the control group  = 0.986, p = 0.42).The RM-ANOVA revealed no significant group × time interaction when participants were exposed to cTBS .The aim of this study was to evaluate neural plasticity integrity in patients with MDD compared with that in healthy controls. Using TBS, we reported that iTBS-induced changes in neural plasticity were altered in patients with MDD. Whereas MEP size post iTBS was significantly increased in healthy controls, no effects of iTBS on MEP size were observed in patients with MDD. No effects of cTBS were observed in either group. Importantly, in addition to those effects, we observed a large interindividual variability in the effects of both iTBS and cTBS on MEP amplitude regardless of group.The effects of rTMS protocols have been proposed to relate to activity-dependent changes in synaptic neurotransmission, reflecting neural plasticity , the lack of a preliminary evaluation of the integrity of the cortico-spinal conductivity and the lack of a more accurate T2-MRI scan instead of only the T1-weight MRI  did not allow us to exclude that such comorbidities in our sample may have influenced current results.Further studies should investigate the close relationship between depressed mood and cognitive dysfunction since this aspect has crucial implications in determining changes of cortical excitability to TMS , a brain region critically involved in the pathophysiology of MDD (Concerto et al., Lastly, an important limitation of the current study was the relatively small sample size of included patients, which might hide significant differences between healthy subjects and patients with MDD at the group level. Nevertheless, our sample size is within the range of other TBS studies in patients with neuropsychiatric conditions (Eggers et al., Despite these limitations, the main strength of our study is that the included patients with MDD were drug free. Indeed, medication and especially psychopharmacological drugs are known to highly influence the cortical excitability parameters assessed by TMS (for a review see Paulus et al., In summary, iTBS-induced cerebral plasticity was altered in patients with MDD, whereas no effect of cTBS-induced cerebral plasticity was observed. These results suggested abnormal LTP-like plasticity mediated by glutamatergic neurotransmission in patients with MDD. These abnormalities should be considered an endophenotype biological marker of MDD. However, because of the small sample of the current study, results should be taken with caution and further studies are needed to explore this topic more thoroughly. Moreover, although MDD and the so called VD share some clinical similarities, VD may rely on distinct pathophysiological mechanisms (Concerto et al., philippe.vignaud@ch-le-vinatier.fr.The datasets for this manuscript are not publicly available because Data are available on reasonable request. Requests to access the datasets should be directed to PV, EP and JB contributed to the conception and design of the study. PV and CD organized the database and acquired data. PV and JB performed the statistical analysis. PV wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.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.


The dorsal  mesial frontal cortex contains the supplementary motor area (SMA) and the pre-supplementary motor area (pre-SMA), which play an important role in action and cognition. Evidence from cytoarchitectonic, stimulation, and functional studies suggests structural and functional divergence between the two subregions. However, a microstructural map of these areas obtained in a representative sample of brains in a stereotaxic reference space is still lacking. In the present study we show that the dorsal mesial frontal motor cortex comprises two microstructurally different brain regions: area SMA and area pre-SMA. Area-specific cytoarchitectonic patterns were studied in serial histological sections stained for cell bodies of ten human postmortem brains. Borders of the two cortical areas were identified using image analysis and statistical features. The 3D reconstructed areas were transferred to a common reference space, and probabilistic maps were calculated by superimposing the individual maps. A coordinate-based meta-analysis of functional imaging data was subsequently performed using the two probabilistic maps as microstructurally defined seed regions. It revealed that areas SMA and pre-SMA were strongly co-activated with areas in precentral, supramarginal and superior frontal gyri, Rolandic operculum, thalamus, putamen and cerebellum. Both areas were related to motor functions, but area pre-SMA was involved in more complex processes such as learning, cognitive processes and perception. The here described subsequent analyses led to converging evidence supporting the microstructural, and functional segregation of areas SMA and pre-SMA, and maps will be made available to the scientific community to further elucidate the microstructural substrates of motor and cognitive control.The online version of this article (10.1007/s00429-018-1738-6) contains supplementary material, which is available to authorized users. The human mesial frontal motor area has for a long time been thought of as being one entity. Campbell  identifiThe question if and how the mesial frontal cortex in the superior frontal gyrus is parcellated into functional units or modules has been a matter of interest in the past decades  or horizontal plane  on a large-scale microtome . Each 15th section was mounted on a glass slide and stained for cell bodies using modified silver staining  to the inner contour line (the border between layer VI and the white matter), which were manually traced on each high-resolution scanned image. A set of ten features  was extracted from each GLI profile and used to quantify the laminar pattern of the cortical ribbon at that position . To avoid interferences caused by tangential sectioning, ROIs were collected at sites, where the cortex appeared to be cut perpendicular to the surface.To quantitatively detect the cortical layer(s) responsible for the cytoarchitectonic differences between areas SMA and pre-SMA, the mean GLI profiles of each area were divided into ten bins with a 10%-interval: bin 1 (0–10%), bin 2 (10–20%), …, bin 10 (90–100%). The mean GLI profile of each area in each brain was calculated from six samples (three samples per hemisphere and brain). In each sample, representative blocks of 15 GLI profiles were extracted, and mean GLI profiles produced. The mean GLI profiles of area SMA were then compared with those of area pre-SMA using pairwise permutation comparisons  .The percentage of area volume and the total brain volume were calculated to enable comparison of differently sized brains and to analyze individual variability in the size of SMA and pre-SMA. Furthermore, a shrinkage factor was defined for each postmortem brain as the ratio between the fresh volume and the histological volume to compensate for shrinkage during the histological processing . Then, the mapped areas were three-dimensionally (3D) reconstructed using the MR image sets of the ten corresponding postmortem brains. The individual brains and the delineations of the cortical areas were spatially normalized to the T1-weighted single-subject template of the Montreal Neurological Institute (MNI)  of the individual probabilistic maps were produced  .The seed regions were defined by the former created MPMs of areas SMA and pre-SMA. Only those experiments were included, that reported stereotaxic coordinates from normal individual mapping studies in healthy humans using fMRI or PET. Accordingly, 1398 functional imaging data of pre-SMA  and 597 functional imaging data of SMA  were included. The analysis set a threshold at a familywise error (FWE) corrected cluster-level , and the absence of Betz cells in layer V. Area pre-SMA differed essentially from area SMA by its dark layer V, which was well demarcated from layers III and VI. In SMA the boundary between layers III and V was not clearly visible. Layer III pyramids were clearly smaller in pre-SMA than in SMA Table ; Fig. 3.Areas SMA and pre-SMA were restricted to the medial surface of the hemisphere in each brain. Although their dorsal borders were often found close to the dorsal convexity of the hemisphere, they never encroached onto its lateral surface . The ventral borders of areas SMA and pre-SMA was found in the region of the cingulate sulcus. For both areas, the border was found on the dorsal bank of the sulcus, though that of pre-SMA was closer to the free surface of the hemisphere. There were no gyri or sulci reliably associated with the border between areas SMA and pre-SMA . However, the border between areas SMA and pre-SMA was closely associated with the vertical plane through the anterior commissure, i.e., the VCA line.Areas SMA and pre-SMA were limited dorsally by the dorsal premotor cortex (PMd), i.e., by the portion of BA6 found on the lateral surface of the superior frontal gyrus. SMA mainly abuts the caudal part of PMd (PMdc), and pre-SMA its rostral part (PMdr). Ventrally, areas SMA and pre-SMA were followed by cingulate areas 24d and 24c, respectively  in layer Vb. Unlike BA4, SMA showed no giant pyramidal cells in layer Vb, although some large pyramidal cells were found in its caudal part. Most importantly, SMA showed an increased cellular density in the lower part of layer III .Both areas were relatively poorly laminated, but had a conspicuous layer IIIc, which was denser and contained larger pyramids in PMdc than in SMA. Furthermore, layer VI of PMdc was more pronounced than that of SMA .Both areas were agranular, but differed considerably in layers III and V. Area SMA, presented a prominent layer IIIc with medium size pyramids, whereas in area 24d cells in the lower part of layer III were considerably smaller. Moreover, whereas layer V is inconspicuous in SMA, a dark layer V with big or even giant pyramidal cells and well demarcated layer VI were easily identified in area 24d .Both pre-SMA and PMdr presented a relatively prominent layer V and a diffuse border between layers II and III. However, layer V neurons were larger and layer VI was more demarcated in PMdr than that in pre-SMA .Both areas were characterized by a clear lamination and a relatively prominent layer V. However, layer V of 24c presented a higher packing density and larger cells than that of pre-SMA. Furthermore, the border between layers V and VI was sharper in 24c .The border between these two areas was clearly identifiable, because layer IV was not visible in cell-body-stained sections of pre-SMA, while in BA8 this layer is clearly recognizable .The average total volume proportions of left and right SMA, and left and right pre-SMA in relation to the whole brain were 1.3%, 1.27%, 1.51%, and 1.47%, respectively. No gender and hemispheric differences were found ; the border between areas SMA and pre-SMA was closely associated with the vertical plane through the anterior commissure (y = 0). The mean center of gravity of area SMA was caudal to the anterior commissure and that of the pre-SMA rostral to the anterior commissure in the anatomical MNI space  and some paradigms . Apart from that, area SMA was significantly related with domains such as interoception , and somesthetic perception. Area pre-SMA was correlated with domains such as action , cognition (in particular with the specific music domain), and the visual perception of motion. Moreover, the area pre-SMA was associated with tasks like sequence recall/learning, saccades and music comprehension. The contrast analysis of behavioral domains revealed that area SMA was more associated with action execution than area pre-SMA Fig. . These rIn the present study, we applied a novel approach for 3D quantitative and statistically testable mapping to identify and differentiate areas SMA (SMA proper) and pre-SMA on the mesial surface of the hemisphere rostral to the primary motor cortex. Specifically, in stereotactic space the pre-SMA was localized rostral and the SMA caudal to the vertical plane through the anterior commissure. Furthermore, the different cytoarchitectonic patterns of the two areas were used to investigate if the two regions were part of different functional networks within the brain as evident from functional brain imaging. For that purpose, a meta-analysis of resting state functional imaging data was performed to demonstrate differential connectivity patterns. Furthermore, a meta-analysis of functional activation data classified according to different behavioural categories was employed to explore to what extent areas SMA and pre-SMA belonged to different functional networks. The combined findings were expected to provide evidence for a functional differentiation of human areas SMA and pre-SMA from the microscopical cytoarchitectonic to the macroscopical functional imaging level.Agranular area SMA was characterized by a poor lamination, and densely packed large size pyramidal cells in the lower part of layer III. Agranular area pre-SMA presented a more differentiated laminar pattern and a more pronounced layer V. These findings are in accordance with previous cytoarchitectonic studies , which roughly corresponded to the position of lower layer III. This suggests that the most prominent differences between areas SMA and pre-SMA were differences of cell packing density in layer IIIc.Y = 0), which is in good agreement with earlier cytoarchitectonic data provided by Zilles et al. , dorsomedial prefrontal cortex (dmPFC), and PMd which also were characterized by multimodal mapping similarly as in this study. The functionally defined region within the aMCC


Metastatic clear cell renal cell carcinomas (mRCC) over-express the vascular endothelial growth factor (VEGF). Hence, the anti-VEGF antibody bevacizumab/Avastin (BVZ) combined with interferon alpha (IFN) was approved for the treatment of mRCC. However, approval was lost in July 2016 due to the absence of sustained efficacy. We previously showed that BVZ accelerates tumor growth in experimental models of mRCC in mice, results in part explained by down-regulation of the phospho tyrosine phosphatase receptor kappa (PTPRκ) in tumor cells. The epidermal growth factor receptor (EGFR) is a direct target of PTPRκ. Its down-regulation leads to constitutive activation of EGFR, an observation which prompted us to test the effect of the EGFR inhibitor erlotinib/Tarceva (ERLO) in addition to BVZ/IFN. The influence of the long non-coding RNA, EGFR-AS1, on ERLO efficacy was also addressed.Methods: The effect of BVZ/IFN/ERLO was tested on the growth of experimental tumors in nude mice. The presence of germline mutation in the EGFR was evaluated on cell lines and primary RCC cells. In vitro translation and transfections of expression vectors coding the wild-type or the EGFR mutated gene in HEK-293 cells were used to test the role of EGFR mutation of the ERLO efficacy. Correlation between EGFR/EGFR-AS1 expression and survival was analyzed with an online available data base (TCGA).Results: Tumor growth was strongly reduced by the triple combination BVZ/IFN/ERLO and linked to reduced levels of pro-angiogenic/pro-inflammatory cytokines of the ELR+CXCL family and to subsequent inhibition of vascularization, a decreased number of lymphatic vessels and polarization of macrophages towards the M1 phenotype. Cells isolated from surgical resection of human tumors presented a range of sensitivity to ERLO depending on the presence of a newly detected mutation in the EGFR and to the presence of EGFR-AS1.Conclusions: Our results point-out that the BVZ/IFN/ERLO combination deserves testing for the treatment of mRCC that have a specific mutation in the EGFR. In vitro, the EGFR inhibitor erlotinib/Tarceva (ERLO), which is approved for the treatment of lung cancers harboring specific mutations in EGFR, strongly inhibited proliferation of cells derived from BVZ-resistant tumors Before the development of anti-angiogenic therapies (AAT), the outcome of mRCC was poor. The first treatment approved for mRCC was the humanized monoclonal antibody bevacizumab/Avastin (BVZ) in combination with the standard treatment interferon alpha (IFN), the only treatment that showed a modest efficacy The Ethics departments of the University hospital, the Cancer Centre (Centre Antoine Lacassagne), Nice, France and the Princess Grace Hospital of Monaco approved this study and participants provided their written informed consent. Cells were isolated from tumors as previously described Quantitative PCR (qPCR) experiments were performed after cell passage 11. One microgram of total RNA was used for reverse transcription, using the QuantiTect Reverse Transcription kit , with blend of oligo(dT) and random primers to prime first-strand synthesis. For real-time PCR, we used the master mix plus for SYBR assay . The PCR conditions were 10 minutes at 95°C followed by 40 cycles 15 seconds at 95°C, 1 minute at 60°C. The sequences of the different couples of oligo-nucleotides are detailed in The following antibodies were used for immuno-blotting: anti-phospho ERK 1,2 and anti-tubulin , anti-phospho S6 Kinase, total anti-EGFR/HER1 and anti-pEGFR/HER1  and anti ERKs .Tumor sections were handled as described previously Frozen tumor tissues were homogenized using a Precellys tissue homogenizer  in cell extraction buffer . The intra-tumor hemoglobin content, CXCL cytokines, VEGF and VEGFC were measured as previously described Five million 786-O or A498 cells were injected subcutaneously into the flank of 5-week-old nude (nu/nu) female mice (Janvier). The tumor volume was determined with a caliper (v ¼ L _ l2 _ 0.5). When the tumor reached 100 mm3, mice were treated twice a week with control or ERLO (50 mg/kg) or BVZ  plus IFN (9MIU) plus or minus ERLO (50 mg/kg).This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Our experiments were approved by our internal ethic committee.The assay was performed as already described t-test or Wilcoxon test for quantitative data.Statistical analyses were two-sided and were performed using R-2.12.2 for Windows. Statistical comparisons were performed using the Student Figure ). Therefore, ERLO is cytostatic rather than cytotoxic and the cytostatic effect was stronger for 786-O cells. ERLO induced dose-dependent inhibition of EGF production by 786-O cells whereas this was not modified in A498 cells . Therefore, the more potent effect of ERLO on cell proliferation observed for 786-O cells may be explained by inhibition of an EGF/EGFR autocrine pathway. Consistent with this, the phosphorylated/active form of EGFR (pEGFR) was dose-dependently inhibited by ERLO in 786-O cells. In A498 cells, the EGFR levels were higher compared to 786-O cells and ERLO had no incidence on pEGFR, which remained low whether or not ERLO was present, as compared to basal levels in 786-O cells . We observed a decrease in the activity of the ERK/MAP kinase proliferation pathway for both cell lines. However, the ERK activity was lower and was more strongly inhibited by ERLO in 786-O cells. The AKT activity (pAKT) was high and was inhibited by ERLO in 786-O cells but almost undetectable in A498 cells. This result may explain the differential effect exerted by ERLO on proliferation for the two independent cell lines .Activation of the EGFR pathway in response to BVZ was demonstrated previously in experimental mRCC in mice Figure ). The opposite result was observed for CXCL8 . These results suggest that ERLO may indirectly inhibit angiogenesis through decreased production of pro-angiogenic factors by tumor cells.Gefitinib, another EGFR inhibitor used to treat lung cancers Figure ). These results highlight the differences in response to AAT and EGFR pathway-targeting treatments, which probably reflects tumor heterogeneity Considering that activation of the EGFR pathway is one of the causes of relapse when on anti-angiogenic treatment with BVZ Figure  and ) but was not different for A498 tumors . However, these treatments increased the number of vessels (CD31 positive) lined with αSMA-positive cells, a pattern of vessel normalization . The triple combination decreased the number of blood vessels but also increased coverage with αSMA labelled cells for 786-O and A498 tumors . The amount of tumor hemoglobin was significantly decreased for only the triple combination suggesting that the treatment reduced tumor perfusion and/or hemorrhagic vessels . As previously reported, BVZ stimulated the development of a lymphatic network in 786-O tumors Figure  and ). ERLO stimulated the development of lymphatics for both tumor model systems. However, the triple combination strongly reduced the BVZ/IFN- or ERLO-dependent development of the lymphatic network for both model systems  and the basal level of lymphatics for the A498 tumors. These results suggest that the triple combination inhibited tumor growth partly by inhibiting the formation of blood and lymphatic networks.We showed previously that BVZ alone stimulated experimental tumor growth. This unexpected result correlated with tumor vessel normalization and the development of a lymphatic network shown in the literature to be involved in tumor cell dissemination Table 1 summarizes the modifications to the mRNA analyzed by qPCR or proteins analyzed by ELISA. First, gene expression differed for the two cell lines highlighting the importance of the tumor genetic background. However, some genes were consistently modified by the different treatments in both cell lines. PTPRκ mRNA levels were decreased by BVZ Figure . The expression of angiogenic factors redundant for VEGF was suspected to promote BVZ resistance To understand the better efficacy of BVZ/IFN/ERLO, we investigated the genes involved in the adaptation of cancer cells (proliferation genes) and cells of the tumor environment  to a given treatment. Figure ).The different treatments generated a wide range of profiles of tumor growth. Therefore, we hypothesized that due to the selection pressure exerted by the different drugs, tumor cells acquired specific genotypic/phenotypic profiles. Thus, we analyzed their proliferation after amplification and selection from the tumors, as previously described Figure ), which reflected their strong level of EGF production (Table 1). The proliferation rates of A498 cells extracted from the different tumors were higher than that of 786-O cells (200 %) whereas they were lower for parental cells . However, they were similar whatever the treatment . In these cells, the intra-tumor levels of human/mouse EGF and EGFR varied according to the treatment (Table 1). We showed previously that exposure to BVZ sensitized resistant cells to ERLO because of PTPRκ down-regulation Table 1). Cells from triple-treated tumors were still sensitive to ERLO whatever the model. This persistent response to ERLO was linked to increased expression of EGF in both model systems . Hence, the chronic inhibition of the EGF/EGFR proliferation pathway is consistent with the in vivo efficacy of the triple combination.However, cells from BVZ/IFN/ERLO 786-O treated-tumors proliferated three times more than those from control tumors . However, the decreased level of PTPRκ in cells from the triple-treated tumors resulted in a modest increase in basal pEGFR levels for both systems. ERLO inhibited pEGFR in the different cells for both cellular models except for A498 cells from the triple-treated tumors. This result reflects an alternative mechanism of EGFR activation probably through the increased expression of EGF by cells of the microenvironment (Table 1). Inhibition of the EGFR activity correlated with inhibition of ERK and preferentially with the AKT activity . However, the persistence of ERK and AKT activity independently of the EGFR activity reflects activation of alternative proliferation pathways independent of the EGF/EGFR pathway after chronic exposure to treatments.We then analyzed the level and activity of EGFR and the sensitivity to ERLO of signaling pathways involved in cell proliferation (ERK/MAP Kinase and PI3Kinase/AKT). Total EGFR levels were increased following treatment of the 786-O model system and were slightly decreased in the A498 model. Basal levels of the phosphorylated/active form of EGFR (pEGFR) decreased in 786-O and A498 cells after BVZ/IFN treatment. This result is consistent with increased levels of PTPRκ  for ERLO and for sunitinib, is reported in Table 2 for our reference 786-O and A498 cell lines and the already described primary cells ). EGFR mRNA levels in 786-O cells are 50% and 25% percent those of RCC4 and A498 cells respectively. However, EGFR protein levels in 786-O cells are 20% and 6.6% percent those of RCC4 and A498 cells respectively. Of note ERLO did not influence the EGFR level . This discrepancy for 786-O cells may be related to the high levels of a long non-coding EGFR antisense mRNA (EGFR-AS1) already described as a marker of poor prognosis in RCC ). The relationship between EGFR/EGFR-AS1 levels and tumor aggressiveness was evaluated by using the online available data of the TCGA. EGFR is overexpressed in RCC from non- metastatic (M0) and metastatic (M1) patients as compared to healthy tissue. Surprisingly, EGFR levels decreased in tumors from metastatic patients (compared M0 to M1) . Over-expression of EGFR was indicative of a longer overall survival (OS) for M0 patients (p = 0.00209) whereas an inversion of this trend was observed for M1 patients although it did not reach statistical significance . In M1 patients, overexpression of EGFR was correlated to a shorter progression-free survival  and a trend was observed for a shorter disease-free survival  . EGFR-AS1 is also overexpressed in RCC from M0 and M1 patients as compared to healthy tissues. No statistically significant difference was observed between M0 or M1 tumors . High EGFR-AS1 levels were correlated with a shorter OS in M1 patients (p = 0.0468) and a trend was observed in M0 patients although non-significant (p = 0.121) . However, high EGFR-AS1 levels were associated with a longer DFS in M0 patients (p = 0.0145) and a shorter PFS in M1 patients (p = 0.0434) . Hence, in M0 patients a mirror image of the role of EGFR and EGFR-AS1 on OS and DFS was observed with an unexpected beneficial role of EGFR on OS. However, EGFR and EGFR-AS1 were systematically associated with shorter OS and DFS in M1 patients. These results are consistent with the pejorative role of EGFR and the relevance of its inhibition in metastatic patients.We showed previously that treatment response to AAT, especially to the current reference treatment sunitinib, was equivalent in metastatic patients and in primary cells derived from the patients' surgically removed tumor EGFR gene. The different mutations/deletions determining ERLO sensitivity in lung cancers were not detected in RCC cells Figure ). The corresponding amino acid is located within the kinase domain (Q 787). This specific mutation modifies a frequently used codon for Q to a rare codon ). In addition to the differences in mRNA levels, this result may explain the difference in the total amounts of EGFR detected in the different cell lines and their sensitivity to ERLO (Supplementary https://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=1050171). Sensitivity to ERLO was tested in the different primary cells. The IC50 for ERLO was the lowest for cells with the heterozygous mutation and the highest for the cells with the homozygous mutation and intermediate for wild-type cells . The differences in ERLO sensitivity were confirmed using another specific EGFR inhibitor: AZD3759 . Considering these results, we investigated whether the G2618A mutation could be responsible for the discrepancy between the mRNA and protein levels observed in the different cell lines. We hypothesized that a higher efficiency of translation of mRNA carrying the A mutation occurs. To functionally test this hypothesis, we performed an in vitro transcription and translation assay using an EGFR construct for both the wild-type and the mutated allele. The wild-type construct was translated less efficiently than the mutant . To confirm these results, HEK293 cells expressing very low EGFR levels were transfected with expression vectors coding for the wild-type or the mutated EGFR. A luciferase construct was co-transfected as a control for transfection efficiency as already described Figure ). These results strongly suggest that patients carrying an homozygous wild-type genotype express the highest levels of EGFR.EGFR levels and its activity varied from tumor to tumor, a situation that may explain the general failure to ERLO in clinical trials. In lung cancers, for which ERLO is routinely used, EGFR protein levels and activity, that are crucial for ERLO efficacy, are never assessed before ERLO treatment. Moreover, ERLO is efficient for lung tumors but only if EGFR has mutations in the kinase domain The presence of high amounts of EGFR in mRCC cells suggested that EGFR inhibitors may have a potent therapeutic effect. A phase II clinical trial with the EGFR pharmacological inhibitor To gain insight into the related molecular mechanisms, we scrutinized the different pathways that were involved in relapse on treatment with BVZ in our previous study Our current study was based on the BVZ-mediated decrease of PTPRκ, down-regulator of EGFR activity. However, BVZ/IFN increased PTPRκ levels. Hence, IFN indirectly decrease the activity of EGFR and other tyrosine kinase receptors that are PTPRκ targets . EGFR is not only expressed by tumor cells but also by endothelial cells and the EGF/EGFR pathway participates in processes of tumor vascularization The decrease of CSF1R amounts with the triple treatment argues strongly for a reduction in tumor growth since the CSF1/CSF1R pathway exerts an autocrine proliferation loop in RCC and CSF1R is indicative of poor prognosis Triple treatment also played a prominent role on the polarization of macrophages that can alternate between pro-inflammatory (M1) and pro-tumorigenic (M2) phenotypes The prognostic score we generated for the different combinations, was not indicative of the ideal treatment and differed for the two tumor models. These results suggest that these treatments may be efficient but need to be used with caution depending on specific genetic characteristics. Cells isolated from tumors exposed to triple treatment showed a higher ability to proliferate in only one model. However, the cells were still sensitive to ERLO in both models. This result suggests that ERLO must be maintained to prevent acceleration of tumor growth.The increase in PDL1, which participates in evasion of immune surveillance in vivo and in vitro results (better efficacy of ERLO for the A498 model in vivo and better efficacy of ERLO for the 786-O model in vitro). This discrepancy may be explained by the ability of the different tumor cells to shape the microenvironment. As illustrated in Table EGFR inhibitors are currently used for the treatment of lung cancers, but treatment is efficient only if the receptor has specific mutations in the kinase domain Another possibility is the difference in perfusion (measurement of hemoglobin levels) of the 786-O versus the A498 model. Strikingly, the hemoglobin in A498 tumors is twice that of 786-O tumors. Therefore, ERLO may have a better access to tumor cells in the A498 model.Finally, the EGFR genotype status is unknown in nude mice and may mitigate the relative efficacy of ERLO. Nevertheless, these experiments aimed at demonstrating the relevance of adding EGFR inhibitors to the previously approved BVZ/IFN treatment. Since the mutation appears as germinal, we can estimate that the triple combination would be more efficient for patients with a heterozygous genotype.The analysis of genome sequences in cancer revealed that silent mutations can control the speed of mRNA translation, mRNA folding, pre-mRNA splicing, and through translational pausing, the folding of proteins In conclusion, EGFR is a relevant therapeutic target for mRCC in combination with anti-angiogenic treatment but only in the presence of a relevant mutation, different to those described in lung cancer. Association of first-generation immunotherapy with IFN should be revisited because of the associated debilitating side effects and new associations with immune checkpoint inhibitors may have a strong therapeutic impact.Supplementary figures and tables.Click here for additional data file.


CABS-dock standalone is a multiplatform Python package for protein–peptide docking with backbone flexibility. The main feature of the CABS-dock method is its ability to simulate significant backbone flexibility of the entire protein–peptide system in a reasonable computational time. In the default mode, the package runs a simulation of fully flexible peptide searching for a binding site on the surface of a flexible protein receptor. The flexibility level of the molecules may be defined by the user. Furthermore, the CABS-dock standalone application provides users with full control over the docking simulation from the initial setup to the analysis of results. The standalone version is an upgrade of the original web server implementation—it introduces a number of customizable options, provides support for large-sized systems and offers a framework for deeper analysis of docking results.https://bitbucket.org/lcbio/cabsdock.CABS-dock standalone is distributed under the MIT licence, which is free for academic and non-profit users. It is implemented in Python and Fortran. The CABS-dock standalone source code, wiki with documentation and examples of use and installation instructions for Linux, macOS and Windows are available in the CABS-dock standalone repository at  Molecular docking of peptides to proteins is often a difficult modelling task. One of the main challenges is modelling conformational flexibility of a protein–peptide system .In 2015, we released a web server implementation of the CABS-dock method for flexible protein–peptide docking , while the protein size is primarily limited by the computer memory size (accessible sizes are in the range of thousands of residues). CABS-dock standalone allows customization of every modelling step and provides a number of newly introduced options and tools. New features include tools for generating initial peptide conformations, optional user-defined distance restraints that drive the conformational search towards preferred conformations, settings of conformational flexibility, tools for docking more than one peptide and modification of simulation parameters (among others: temperature and number of Monte-Carlo procedure steps). The package also serves as a comprehensive framework for analysis of simulation results, including calculation of root-mean squared deviation to the reference complex, custom clustering or residue–residue contact frequency maps using user-defined cut-off. The only required input for CABS-dock is the receptor protein structure and the amino acid sequence of the peptide. The receptor may be provided either as a local PDB file or a PDB code.https://bitbucket.org/lcbio/cabsdock.All the options, examples of use and installation instructions are described in the CABS-dock standalone repository available at The CABS-dock standalone application extends the capabilities of the original web server and introduces new features that provide a framework for customizable protein–peptide docking simulations and their analysis. CABS-dock standalone can be particularly useful for simulating large-scale conformational changes of protein–peptide systems and it is well suited for restraint-driven docking. The customizable distance restraints allow the user to impose weak or strong biases towards the desired type of conformations. Thanks to its customizability, modularity and implementation in Python 2.7, CABS-dock standalone (or its modules) may be readily incorporated into existing information-driven docking pipelines  Grant [MAESTRO2014/14/A/ST6/00088].Conflict of Interest: none declared.