CCNB1 and Ki67 staining. Lastly, we demonstrate the utility of the transcriptomic date to correctly classify disease or healthy states in the discovery and validation cohorts. Our data supplement recently published datasets providing insights into celiac pathogenesis using clinical pathology FFPE samples, and can stimulate new approaches to address this highly prevalent condition.Celiac disease is provoked by gluten exposure, but the complete pathogenic process in the duodenum and the loss of tolerance to gluten is not well understood. We aimed to define the core celiac transcriptomic signature and pathologic pathways in pre-treatment formalin-fixed paraffin-embedded (FFPE) duodenum biopsies used for clinical diagnosis. We use mRNAseq to define pre-treatment diagnostic duodenum gene expression in 54 pediatric celiac patients and non-celiac controls, and we validate our key findings in two independent cohorts of 67 adults and pediatric participants that used fresh frozen biopsies. We further define similar and divergent genes and pathways in 177 small bowel Crohn disease patients and controls. We observe a marked suppression of mature epithelial metabolic functions in celiac patients, overlapping substantially with the Crohn disease signature. A marked adaptive immune response was noted for the up-regulated signature including interferon response, alpha-beta, and gamma-delta T-cells that overlapped to some extent with the Crohn disease signature. However, we also identified a celiac disease specific signature linked to increased cell proliferation, nuclear division, and cell cycle activity that was localized primarily to the epithelia as noted by  It is characterized by a broad range of clinical presentations, a specific serum autoantibody response, and variable damage to the small-intestinal mucosa. Globally, the prevalence of celiac disease is increasing. Studies comparing serum stored from 1948–54 and 1974–89 to recent samples from the USA showed 4 and 2 fold increase over approximately 504 and 155 years respectively, and a similar increase was noted over 20 years in Finland6. In light of the increasing prevalence and improved recognition, more complete understanding of the underlining pathogenesis may elaborate on preventive strategies in high risk individuals7, and on ways to improve treatment strategies.Celiac disease is a systemic immune mediated enteropathy triggered by dietary gluten in genetically susceptible individuals8. To supplement recently published celiac disease mRNAseq transcriptomic studies10 done on fresh frozen biopsies, and to improve our understanding of celiac pathogenesis, we applied a standardized high throughput mRNA sequencing (RNAseq) approach on FFPE archived duodenum biopsies used for clinical diagnosis of active pre-treatment celiac disease and controls subjects (n = 54). Our cohort represent the largest celiac disease mucosal transcriptomic cohort to date12  tissue samples stored in pathology archives represent an invaluable biobank for clinical research, and its use for transcriptomics was previously tested with good results12 Table . We capt13 including positive IgA autoantibodies against tissue transglutaminase (anti-TTG) and villous blunting consistent with Marsh 3 on duodenal biopsy. Histopathologic assessment was completed by a single pathologist. To mimic real-life referrals, we included subjects with abdominal pain, poor growth, or anemia as non-celiac controls. The Sheba Local Research Ethics Committee granted ethic approval for the study and waived the need for patients’ written informed consent for using archived formalin-fixed paraffin-embedded (FFPE) material. All methods were performed in accordance with the relevant guidelines and regulations.Newly diagnosed celiac disease and age-matched controls (Ctl) subjects on a gluten-containing diet were included in the study Table . Celiac 14 and single-end 61 bp sequencing was performed15. Reads (mean of ~5.7 M per sample with 2.9 M Std. Deviation) were quantified by Kallisto v0.42.516, using Gencode v24 as the reference genome with 2.1 M pseudo aligned mean reads per sample, after excluding one sample due to poor coverage. Estimated counts were normalized to Reads per Million (RPM). 54 RNAseq samples were included and stratified into specific clinical sub-groups (21 Ctl and 33 celiac disease) and randomly assigned to gender and age matched discovery and validation cohorts with a 10:3 ratio between discovery and validation cohorts respectively. 48/54 (89%) were obtained and stored in the pathology core during 2017 and RNA was extracted within one year (mean of 251 days), and 6 (4 celiac and 2 controls) were obtained before 2017, and were processed within 4 years. We included 14,778 protein-coding mRNA genes with RPM above 3 in 20% of the samples in our downstream analysis.RNA was isolated from FFPE sections containing 4 pooled duodenal biopsies using the Qiagen AllPrep RNA/DNA FFPE Kit. Lexogen QuantSeq 3′ mRNA-Seq libraries17/ToppCluster18 and ClueGO19 platforms were used for functional annotation enrichment analyses and Cytoscape.v3.0.220 for visualization. Two recent celiac transcriptomics studies were used for validation; FASTQ files from Leonard et al.9 were processed similarly, and the processed differentially expressed genes from Bragde et al.10 were used for downstream analyses and comparison. R package random Forest21 version 4.6.14 with out of box (OOB) estimate of error rate, and the Support Vector Machine (SVM) in GeneSpring® software were used to build a classification model to differentiate celiac from controls using the discovery cohort. Those models were used to test the accuracy of the classification in the independent validation cohort.Differentially expressed genes were determined in GeneSpring® software using the discovery cohort (23 celiac and 15 controls) with fold change differences (FC) > = 1.5 and using the Benjamini–Hochberg false discovery rate correction  and not on the validation cohort due to samples numbers constrains. Unsupervised hierarchical clustering using Euclidean distance metric and Ward’s linkage rule was used to test for groups of duodenal biopsies with similar patterns of gene expression in both the discovery and validation cohorts. Principal Component Analysis (PCA) was performed to summarize variation in gene expression between patients in discovery and validation cohorts. ToppGene24 [GSE57945], we first confirmed that 92%  of the protein coding genes that passed the expression filtering criteria and were used for differential expression in the ileum in Crohn disease overlapped with the 14,778 protein-coding mRNA genes that passed expression filtering and were used for differential expression in the duodenum in the current study. We then used Venn diagrams that overlayed the core Crohn disease signature  with the core celiac disease signature  to test for similarities and differences, using only 1817/2160 of the Crohn disease differentially expressed genes that passed the current expression filtering criteria for downstream analyses.To compare between the celiac disease signature and our previous Crohn disease signatureSI and APOA1 mRNA expression was determined by SYBER Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions assays, after normalization to GAPDH. Relative mRNA levels were expressed as fold change (Rq). Primers used are in Table qPCR was performed on cDNA derived from FFPE extracted RNA as above. APOA4 , CCNB1 ,), SI , and MKI67 . APOA4, SI, and MKI67 were detected with UltraView and CCNB1 was detected with OptiView DAB Detection Kits . Sections were counterstained with Hematoxylin II . The slides were dehydrated in graded ethanol . Before cover-slipping, sections were cleared in Xylene and mount with Entellan.FFPE blocks were sectioned at 4μm and were processed by a fully automated protocol on a Benchmark Ultra staining module . Briefly, after sections were dewaxed and rehydrated, a CC1 Standard Benchmark Ultra pretreatment for antigen retrieval was selected for Duodenal mRNAseq data sets were deposited into GEO [GSE131705], and we used our previously published Crohn disease transcriptomics [GSE57945].14 that is designed for analyzing fragmented FFPE samples. Analyses of 3 FFPE and fresh paired biopsies obtained from the same endoscopic region showed correlation of ~0.8  and apolipoproteins (P < 5.07E-3), reduced vitamins metabolism and absorption (P < 4.29E-7), and lower oxidoreductase and NAD/P activities (P < 2.10E-6). Applying an independent ClueGO19 pipeline for functional annotation enrichment analyses is shown in Fig. SI) and APOA1 genes expression levels in celiac disease , and 85% (341/403) are within our core celiac signature , randomly assigned to 2:1 discovery and validation cohorts , alpha beta (P < 6.55E-55) and gamma delta (P < 3.34E-50) T cells, and interferon signaling (P < 6.71E-7). In addition, we noted a robust signature enrichment for mitotic cell cycle division (P < 2.4E-19), nuclear division (P < 7.05E-18), and in the key regulator of cell cycle CDK1 interactions (P < 6.18E-18). Many of those upregulated gene and pathways demonstrate substantial overlap with previous studies , a regulatory protein involved in mitosis, in celiac biopsies in comparison to controls. Furthermore, it demonstrated that the signal for induction of CCNB1 is noted substantially in the epithelial crypts  area under the curve (AUC) of 0.97 was obtained when using supervised learning Random Forests (RF) model and all 878 genes in the discovery cohort, and AUC of 1 in the validation cohort Fig. . The gen2B7 Fig. . After n24. Importantly, a substantial number of genes passed the expression filtering criteria in both studies (see methods). Using a Venn diagram, we show  is another inflammatory condition that involves the small intestine. We recently characterized the core signature of the inflamed Crohn disease ileumhow Fig.  that outIn contrast, a significantly smaller proportion [19%  regulate intestinal epithelial proliferation at the crypt base30, inducing or restricting intestinal epithelial proliferation and cell death32 depending on the circumstances. Differences between Crohn disease and celiac disease in this respect may be driven by the role of the gut microbiota that was already linked to Crohn disease pathogenic processes in several large cohorts33. The role of microbiota in celiac disease was linked to different metabolic patterns of gluten break down34 and was shown to be different in infants with an affected first degree relative35, but the overall microbial composition has not yet been fully defined in large human cohorts of celiac disease, and is still controversial36.We emphasize substantial similarities but also differences associated with Crohn disease and celiac disease pathogenesis. We demonstrate a large overlap of the repressed epithelial mature metabolic signatures in both. However, we noted a substantial divergence of the up-regulated epithelial and immune associated signatures. These differences include an intensified signature linked to innate granulocyte immune responses and extracellular matrix observed more specifically in Crohn disease Fig.  as opposCCNB1 and Ki67 immunohistochemistry staining. We used whole biopsies, composed of a mixture of cellular components, rather than single cell transcriptomics. Future studies using single cell preparations, prioritized by the current dataset, will be important for further cellular subset characterizations. However, there are also advantages in using whole biopsies in the clinical setting to capture the overall pathogenic process, and as a potential future diagnostic tool.Our study has several strengths, but also some limitations. Using FFPE clinically archived biopsies and novel analytic approaches we captured many of the previously reported pathways identified in recently published transcriptomics dataset that used research allocated fresh biopsies, supporting the robustness of our methodology and findings. In addition, we show that transcriptomic data of clinically archived samples was able to accurately classify disease or healthy states in both discovery and independent validation cohorts. We emphasize the robust signal of cell proliferation in the transcriptomic data and confirmed its specificity to epithelial crypts by 10 and in our validation dataset. We highlight important biologic differences between Crohn disease and celiac disease, two inflammatory conditions known to cause small intestine inflammation with a more intensified signature of innate granulocytes activation linked to Crohn disease and a more specific epithelial proliferative signature in celiac disease. Integrating this knowledge from transcriptomics datasets paves the way to more mechanistic studies that altogether will lead to new insights regarding pathogenesis of both diseases, and to future molecular-based prevention and therapies for those chronic conditions.In summary, our celiac disease transcriptomics cohort, based on clinically stored FFPE samples, is the largest to date, and was able to identify important molecular pathogenic signatures emphasizing a signal for epithelial proliferation over differentiation, coupled with increased adaptive immune signature. We validate those in recently published independent cohortsSupplementary informationDataset 1


Breast cancer is the leading cause of cancer related deaths in women worldwide and is classified into subtypes based on the cancer’s receptor status. Of these subtypes, those expressing the human epidermal growth factor receptor 2 (HER2) receptor were traditionally associated with poor prognosis. Several advances have been made in the treatment of HER2-positive breast cancer, yet issues of resistance and poor response to therapy remains prevalent. In this study we explored the impact of HER-family and homologous recombination deficiency SNPs on response to patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens. Using Cox regression analysis, we identified 6 SNPs that correlate with recurrence free survival in our patients and supported our findings using support vector machines. We also used reverse phase protein array analysis to examine the impact ERBB3 SNPs may have on both the PI3K/AKT and MAPK/ERK signaling pathways. Finally, using cell line models, we correlated SNP status with sensitivity to platinum based drugs and docetaxel. We found that patients on a TCH based regimen with the minor allele of the ERBB3 (rs2229046 and rs773123) and BARD1 (rs2070096) SNPs, were significantly more likely to relapse than those women who were not. Additionally, we observed that patients with these ERBB3 SNPs had shown elevated protein expression/phosphorylation of Src kinase, c-MET (Y1234/1235), GSK-3β (S9) and p27, indicating that these SNPs are associated with non-PI3K/AKT signaling. Finally, using cell line models, we demonstrate that the BARD1 SNP (rs2229571) is associated with greater sensitivity to both carboplatin and cisplatin. The BARD1 and ERBB3 SNPs can potentially be used to determine those patients that will have a worse response to TCH based treatment, an effect that may arise from the SNPs impact on altered cellular signaling. ERBB2) gene  and accounts for approximately 20% of all breast cancers. It is associated with a significantly worse survival [HER-2 positive breast cancer comprises cancers which exhibit the overexpression or amplification of the Erb-B2 Receptor Tyrosine Kinase 2 , ERBB2, ERBB3 or ERBB4 genes (HER-family genes) can be associated with either a worse relapse free survival (RFS) or worse overall survival (OS) rates in women with HER2-positive breast cancer who received adjuvant trastuzumab as part of their treatment regimen [Recent data has suggested that germline single nucleotide polymorphisms (SNPs) play a role in both sensitivity and resistance to targeted therapies ,8. In fa regimen ,10. Muta regimen .BRCA1/2 are the most common cause of HRD. However, any gene mutations which cause HRD can potentially result in a phenotype like that of BRCA1/2 mutated cancer. This phenomenon is called ‘BRCAness’ and is also characterized by HRD [Cancer cells with impairment in DNA repair mechanisms, such as homologous recombination deficiency (HRD), are sensitive to platinum based drugs, which directly damage a cell’s DNA. Mutations in the tumour suppressor genes d by HRD .Our current study aims to explore the effect on survival and functional impact of both HER-family and HRD related SNPs on response to TCH based treatment, versus patients who received a non-TCH based treatment.A total of 157 patients with operable primary BC were used in this study, which included 78 patient samples from the NCT01485926 phase II neo-adjuvant study of TCH/TCHL in women with early stage HER2-positive breast cancer. The remaining 79 patients came from the CTI-09-07 translational study, which enabled the collection of FFPE samples and the relevant clinicopathological data and treatment history. We selected an additional 32 samples from the CTI-09-07 translational study on which to perform high depth sequencing, as this discovery cohort had tumour blocks with sufficient tissue to perform NGS analysis. These studies were approved by the research ethics committees of all hospitals, were run by the All-Ireland Clinical Oncology Research Group  and included women who are confirmed as clinically HER2-positive by a 3+ HER2 immunohistochemistry score and or/ a FISH ratio of >2. Detailed clinical information is available in The patient data was analyzed anonymously, the study was approved by the ethics committee of all the individual hospitals in which the patients were treated, and approved the respective studies in accordance with the Declaration of Helsinki. CTI 09–07 is a translational study and enrolment on TCHL and CTI-0907 was possible. Central REC review for TCHL: University College Cork Clinical Research Ethics Committee, and Local REC reviews for 09–07: University College Cork Clinical Research Ethics Committee, Beaumont Hospital Ethics Committee, University Hospital Waterford Research Ethics Committee.In our initial SNP screen, we performed high depth sequencing on our discovery cohort, which contained 32 tumour formalin-fixed, paraffin-embedded (FFPE) samples, from patients with HER2-positive BC. Haematoxylin and eosin sections cut from the patient’s FFPE surgical blocks were analysed by a pathologist for tumour content and those with >50% tumour cellularity had a further 7*10μM sections cut, from which DNA was extracted using the Qiagen DDNA FFPE kit as outlined in a previous study [http://bio-bwa.sourceforge.net/) under default parameters. Duplicate reads were marked by Picard tools (http://broadinstitute.github.io/picard/) and local realignment and base recalibration were conducted with GATK [The reads from our discovery cohort were trimmed using Trimmomatic  and aligith GATK   was applied to confirm the allele calls of the 6 SNPs which are listed in http://cran.r-project.org/).Survival analysis was only performed using data from trastuzumab treated samples, with RFS as the survival endpoint. Survival curves are based on Kaplan-Meier estimates and the log-rank p-value is shown for difference in survival. The resultant p-values are adjusted for multiple testing using the Benjamini-Hochberg method . Cox reghttps://www.ncbi.nlm.nih.gov/geo/) in SRA format (accession number GSE48216). Samples were processed as per GATK [https://software.broadinstitute.org/gatk/best-practices/). Briefly, the SRA files were converted to FASTQ files using the SRA Toolkit (https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/), quality control was conducted using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), the adapter sequences were trimmed using BBmap (http://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbmap-guide/) and the FASTQ files were converted to SAM format using BWA [http://broadinstitute.github.io/picard/) and reordered before being passed on to GATK for base recalibration, using known sites as provided by the Broad Institute best practices guide. The files were then run through GATK Haplotype caller using discovery mode and recommended settings and the files were run through the GATK Variant Recalibrator, using the recommended settings for SNP discovery and known sites as provided by the Broad Institute best practices guide. Finally, the files were annotated using Oncotator [Exome sequencing data for 70 breast cancer cell lines treated with 90 therapeutic agents  were dowper GATK  best prasing BWA . Next, tncotator .50 values of the three groups were then compared using Tukey Honest Significant Difference and an ANOVA model. The GD50 values and the HER2 status of the cell line were as per the original authors [For statistical analysis, the locus status of the mutations in each HER2-positive cell line was used to divide the cell lines into three groups . The GD authors . This te authors .To perform pattern recognition analysis, a predictive model was built and tested using a Support Vector Machine (SVM). The features used were our 6 SNPs and the time to last follow-up (in months) to predict RFS with (1) or without (0) event and a radial-kernel SVM was built using the e1071 package in R . 5-fold The accuracy was chosen to represent the predictive power of the model, the Phi score was utilized to determine how strong of a relationship existed between features and the classes, and the AUC was chosen to assess how efficient the features are at separating the classes, where a value of 0.5 represents the machine just ‘guessing’ the classes. The Phi score is a metric with a value between -1 and 1, where -1 represents an inverse correlation between the features and the classes, 1 represents a strong positive correlation .ERBB2 and ERBB3 genes; the ERBB2 rs1136201 SNP, the ERBB3 rs2229046 and rs773123 SNPs, which were found in approximately 15%, less than 5%, and 6% of the 1000 genome UK population, respectively. Our analysis also included 3 SNPs related to HRD; RNF8 rs2284922 SNP, found in approximately 44% of the 1000 genome UK population and BARD1’s rs2070096 and rs2229571 SNPs, which occur in approximately 19% and 45% of the 1000 genome population, respectively. All 6 SNPs are exonic and occur in protein coding domains, where 3  are nonsynonymous variations, with rs2229046 being implicated in alternative splicing for HER3 [We focused on 6 SNPs identified in our high depth sequencing study of the exome of 28 genes  from DNAfor HER3 . The freIn our study, we assessed the impact of HER-family and HRD SNPs on the response of patients who received TCH-based treatment versus those who received other standard treatment regimens which did not include TCH.ERBB2 rs1136201 SNP and the ERBB3 SNPs rs2229046 and rs773123, were significantly more likely to relapse on a TCH based regimen  than those women who received a non-TCH based treatment (We found that women who were heterozygous for the reatment . After mBARD1 rs2070096 SNP were significantly more likely to relapse on a TCH-based treatment than those patients (p = 0.05) who received a non-TCH based treatment (p = 0.89) (-4). Conversely, we found that women who had the SNP for the RNF8, rs2284922, were significantly more likely to have a higher chance of relapse on non-TCH based treatment (p = 0.01), than those women who received TCH based treatment (p = 0.90)  , which m = 0.90) , Fig 2. Using machine learning, we demonstrate an increase in metric scores for support vector machine (SVM) model predicting RFS events in patients who underwent TCH based treatment versus those that received non-TCH based treatment . A rise ERBB3 rs2229046 SNP had a trend towards lower P27 expression  but had significantly higher expression of SRC kinase . These patients also had a trend towards higher phosphorylation of EGFR (Y1068) and Shc (Y317) . These results indicate that the ERBB3 rs2229046 SNP may be associated with tumours which preferentially signal through the EGFR/MAPK/SRC kinase pathway (Our results indicated that patients who had the  pathway .ERBB3 rs773123 SNP were significantly more likely to have lower expression of P27 (-3), whilst also having significantly lower phosphorylation of c-MET (Y1234/1235)  but significantly higher phosphorylation of GSK-3β (S9)  , it would be important to validate these findings in a larger dataset.We also found that patients who had the n of P27   . These rBARD1 rs2229571 were significantly more likely to be sensitive to a platinum-based drug relative to those cell lines that had either the reference allele or were heterozygous for the SNP  (Using available sequencing data from the Genomics of Drug Sensitivity in Cancer (GDSC) database  we deter = 0.02) . However = 0.02) .Despite the improvement in response rates of women with HER2-positive breast cancer to trastuzumab based treatments, a significant population of women have innate resistance to the trastuzumab based treatments such as the standard of care, TCH based therapy . This inPreviously we and others demonstrated that the minor allele of the ERBB–I655V SNP was associated with a worse RFS in women with HER2-positive breast cancer ,10. A liERBB3 SNPs, (rs2229046 and rs77123) and who received TCH based treatment are significantly more likely to have a worse RFS than patients who received alternate therapies. To support our hypothesis that this is a TCH dependent effect, we previously demonstrated that patients who had the minor allele of the ERBB3 SNP rs2229046 and who received trastuzumab as part of their therapeutic regimen did not have a significantly worse RFS [We have shown that patients who have the minor allele of the orse RFS .ERBB3 SNPs identified in our study represent both a synonymous (rs2229046) and non-synonymous (rs77123) SNP. SNPs which are synonymous variants were until recently believed to be silent, resulting in little to no impact on the ensuing protein. However, recent studies have shown that synonymous SNPs can play an important role in the functionality of the cancer cell and how a patient responds to targeted therapies [The herapies ,10,28,29ERBB3 SNPs rs77123 and rs2229046 SNPs not having a described role in cancer susceptibility [ERBB3 SNP rs2229046 have significantly elevated expression of Src kinase, and a nearly significant increase in EGFR Y1068 phosphorylation, whilst patients with the ERBB3 SNP (rs77123) have a significantly higher level of GSK-3β (s9) phosphorylation relative to those with the reference allele. HER2-positive breast cancer is generally classified as being associated with activation of the PI3K/AKT signalling pathway [ERBB3 rs2229046 and rs77123 SNPs could potentially indicate why these patients do not respond as well to TCH based regimens relative to the patients who receive non-TCH based treatment.Previous studies have shown that despite the tibility ,31, bothtibility . To date pathway ,33. TrasBARD1 SNP rs2070096 were significantly more likely to have a worse RFS than those patients who received a non-TCH based treatment. Our hypothesis that impediment of the HRD pathway would result in altered sensitivity to platinum based drugs given as part of the TCH backbone is supported by our result that patients who were homozygous for the minor allele of RNF8 rs2284992 had a worse RFS when they were given non-TCH based treatment, compared to those receiving TCH based treatment. The confounding effects of differing HRD related SNPs in relation to response to treatment would require further functional interrogation to elucidate why this effect occurs.Our analysis also identified that patients with the minor allele of the RNF8 rs2284922 as playing a role in cancer susceptibility or drug response [BARD1 SNPs  in this study have been associated with nephroblastoma and neuroblastoma [The role of RNF8 in DNA damage repair has been extensively studied, however there are no studies which associate response . Howeverblastoma –38, no s50 to platinum (cisplatin and carboplatin) and docetaxel based drugs. We demonstrated that those cell lines with the minor allele of the BARD1 SNP rs2229571 had greater sensitivity to both carboplatin and cisplatin. Interestingly, the BARD1 SNP rs2229571 had no significant effect on a patient’s response to TCH or non-TCH treatment. These results may indicate that whilst SNPs in HRD related genes do impact on a drug’s sensitivity in vitro, that their impact on a patient’s response clinically may be overcome using multiple therapies, which can have confounding effects on how a patient responds.To further examine the impact of HRD related SNPs on platinum response we analysed the SNP status of a panel of 11 HER2-positive cell lines and correlated this against the cell line GDBARD1 SNP results from patients in cell line models. We demonstrated that patients with the minor allele of the ERBB3 and BARD1 SNPs were more likely to have a worse RFS than those who received non-TCH based treatment. For the first time we also demonstrated using NGS analyses of cell line data that SNPs which occur in and BARD1 correlate with carboplatin or cisplatin sensitivity in vitro. Finally, we have shown that patients with the minor allele of the ERBB3 rs77123 and rs2229046 SNPs have elevated expression and phosphorylation of proteins in pathways not associated with PI3K/AKT signalling, likely indicating why these patients do worse to TCH based treatment.Our study does have limitations that must be taken into consideration, as these may lead to residual confounding biases. These issues include a small sample size and, in some cases, incomplete clinicopathological information. Future studies building from our work should test these variables to see if there are any residual effects on the results we have observed. One of the ways we addressed the small sample size in our study, was to perform rigorous statistical analysis to ensure that our results remain valid after correcting for multiple testing. We also used machine learning as another statistical tool to support that our patient response results are robust. Finally using the publicly available GDSC database we could determine the SNP status of cell line models which could be used to demonstrate the validity of our This study has demonstrated the potential of SNPs, both synonymous and non-synonymous to be important markers in tumour response to therapy. These hereditary changes should be taken into account when clinicians decide on a suitable course of treatment for women who will receive adjuvant treatment of their HER2-positive breast cancer.S1 File(CSV)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.


Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, activating transcription factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65–80% by 16 d) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knock-out-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemizygotes and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed phosphatase and tensin homolog (PTEN) line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons  and descending supraspinal neurons . We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes , and in probing the function of any number of genes as they relate to neuronal injury and regeneration. Understanding reactions to neurotrauma and overcoming obstacles to neural regeneration should benefit from the ability to genetically label or otherwise edit the genome of injured neurons. We sought to achieve this in mice by driving Cre recombinase expression under the control of activating transcription factor 3 (ATF3), which is robustly induced by axotomy in several populations of peripheral and central neurons. When crossed with reporter mice, recombination occurred only in injured neurons following sciatic nerve injury or spinal hemisection. Peripheral nerve injury (PNI)-induced neuronal phosphatase and tensin homolog (PTEN) excision also resulted in improved regeneration and more complete functional recovery. These results demonstrate the feasibility and utility of axotomy-induced recombination and represent a new tool for investigating genetic control of injury responses and regeneration.Advances in genome editing techniques have created opportunities to dissect out the function of specific genes and their contributions to health and disease. Among the most widely used of these tools is the Cre-Lox system, in which Cre expression can be restricted spatially, to a single tissue or population of cells through insertion under a specific promoter , or tempActivating transcription factor 3 (ATF3) is a transcription factor that belongs to the basic leucine zipper family . ATF3 isDamage to the PNS is a common outcome of motor vehicle accidents, penetrating trauma, and falls . The PNSvia viral Cre transfection induces a regenerative response in corticospinal neurons after a spinal cord injury (Phosphatase and tensin homolog (PTEN) is an inhibitor of the PI3K/AKT/mTOR cell growth and proliferation pathway . The losd injury , a findid injury . In the d injury . This alHere, we present a novel transgenic mouse model that allows for the selective genetic editing of injured neurons via the insertion of a Cre-ERT2 construct under the native ATF3 promoter. We confirm expression of the Cre-ERT2 construct in injured neurons after PNI using a fluorescent reporter line and we demonstrate that this model may also be used to effect recombination in select populations of axotomized CNS neurons. Finally, we show that neuronal injury-specific Cre-ERT2 expression can be used to functionally alter the regenerative capacity of neurons though excision of crucial PTEN exons, a proof-of-principle experiment that illustrates the utility and potential of this model in neurotrauma.All animal procedures were performed in accordance with the University of British Columbia and King’s College London animal care committees’ regulations. All mice were between two and four months of age and equally distributed between sexes.cre) strain used was generated and described by IMSR_JAX:007908; cre line was also crossed with conditional PTEN deletion line (PTENfl/fl) with LoxP sites flanking exon 5 of the PTEN gene  and ketoprofen  subcutaneously for prophylactic analgesia. Once anaesthetized with isoflurane , the sciatic nerve was exposed by blunt dissection and crushed for 15 s  with fine #5 forceps at the sciatic notch. For the brachial nerve crush the same anesthetic and analgesic protocols were used. The distal brachial plexus was exposed in the upper forelimb, the median radial, and ulnar nerves were crushed with fine forceps. Tamoxifen (Sigma) was dissolved in wheat germ oil  and injeSpinal hemisection was also conducted with the same anesthetic and analgesic protocols outlined above. A midline incision was made over the lower cervical/upper thoracic spinal cord, and the C8 and T1 laminae were removed. A 25-gauge needle was inserted dorso-ventrally at the midline between the C8 and T1 spinal segments to allow relatively atraumatic entry of one blade of a pair of microscissors. The cord was laterally transected with microscissors; after hemostasis was established, the muscle and skin were closed in layers with sutures. Mice were killed 7 d later.Atf3 and Pten alleles altered . Specifically, in the first set of experiments we compared ATF3+/+, ATF3+/cre, and ATF3cre/cre mice to determine similarities to previous knock-out models  on strong toe pinch was assessed in mice lightly anesthetized with 2% isoflurane. On loss of righting reflexes, each animal was removed from the induction chamber and placed prone on a table. To confirm light anesthesia, the base of a contralateral toe was pinched with curved serrated forceps. If there was no initial response, the other toes on the same foot were pinched in succession until either a response occurred, or there was an escape attempt. If a nocifensive response occurred, the ipsilateral toes were pinched starting with the first and ending with the fifth digit. As mice are prey species, they are prone to thanatosis (playing dead), and can suppress nocifensive withdrawal as they emerge from anesthesia. As such, a contralateral toe was pinched again following ipsilateral toe trials. An absence of a contralateral nocifensive response was almost always followed within 5 s by escape, and so the test was repeated. The test was also repeated if the mouse regained consciousness while the ipsilateral paw was being assessed. The presence or absence of a response to each ipsilateral toe pinch was recorded. If pinch to a particular toe elicited a response 2 d in a row, recovery was assigned to the first.For the grasping assay the mouse was suspended upside-down from a wire cage lid and the grasping ability of the hindpaw was assessed and scored. Scores were assigned using the following semi-quantitative metric: undirected paw placement = 0, directed paw placement = 1, occasional grasp = 2, consistent grasp = 3. Both tests were conducted every other day starting on the third postoperative day until the experimental endpoint (day 28).2O), and their sciatic nerves were exposed at mid-thigh. The nerves were bathed in paraffin oil and draped across a pair of silver wire hook electrodes (anode-cathode distance: 1 mm). EMG needle electrodes were placed subcutaneously over the lateral aspect of the hindpaw; one at the calcaneus, the other just proximal to the base of the 5th digit (over the abductor digiti minimi muscles). The nerve stimulated with 200 μs square wave current pulses using a stimulus generator  and stimulus isolator (A.M.P.I.). Signals were amplified using a Dual Bio-amp connected to a 16 channel Powerlab (ADInstruments). Signals were sampled at 40 kHz and filtered using LabChart7 software. After establishing appropriate electrode polarity, current pulses were delivered at increasing intensities until an EMG signal became apparent, and threshold current was recorded. The maximum EMG signal was then obtained, and the latency and amplitude of the first positive peaks were recorded.Four weeks following injury, mice were anesthetized with urethane . Once dissected, tissue was postfixed overnight in 4% paraformaldehyde, then overnight again in 20% sucrose (ThermoFisher) in 0.1 M phosphate buffer. The tissue was then frozen in Cryomatrix (ThermoFisher) and sectioned at 20 μm (DRG and sciatic nerve) or 50–100 μm . In some cases , whole s+-plus-tdtomato+ neuronal nuclei by the total number of ATF3+ neuronal nuclei. The 16-d recombination efficiency was calculated by dividing the number of tracer-positive neuron cell bodies by the total number of reporter-positive cell bodies. All image processing and quantification was done using ImageJ (Fiji version 2.0.0-rc-66/1.52b).All images were acquired with a Zeiss LSM 800 confocal microscope using Zen (Blue) software. Recombination efficiency 4 d after lesion was determined by dividing the number of ATF3+ axons and measuring the PTEN intensity of each axon.Both PTEN antibodies produced specific staining, but with high background, varying depending on tissue examined . After comparing both antibodies across all tissues, our analyses relied on the Cell Signaling 9188 antibody for the DRG images and the Cell Signaling 14642 for the ventral root images. PTEN immunoreactivity in the DRG was quantified by measuring the mean pixel intensity of the entire cell layer of the DRG. In the spinal cord, PTEN immunoreactivity was weak in motoneurons, but intense in motor axons on either side of the ventral root exit zones, and so we focused our attention there. PTEN immunoreactivity in ventral root axons was determined by selecting for tdtomatoTo determine the regeneration density and distance along the sciatic nerve one best 20-μm-thick section  was selected for each animal, which was then fully imaged (z-stack and tiled). The stack was orthogonally projected into a single image through its entire depth. The images were then processed to generate binary overlays. The average density of SCG10 immunopositive axons along the width of the nerve was measured and then averaged over 100-μm increments.t test (two groups). For toe pinch, we compared proportions of animals with five sensate digits  over time following injury using a log-rank (Mantel–Cox) test. The same test was used to compare proportions of animals which showed consistent grasping with the injured hindpaw. For EMG data, we compared ipsilateral and contralateral values  within groups using paired t tests. Because we used males and females (which differ in size), for between-group comparisons we used unpaired t tests on ipsilateral/contralateral ratios.All statistical analyses were conducted using Prism 7 (GraphPad). We used a one-way ANOVA followed by Tukey’s multiple comparison test to compare PTEN immunoreactivity among DRGs  or an un-paired two-tailed +/+) ipsilateral DRG neurons but did not label the same ipsilateral DRG neurons in the homozygous mutant mice  activity at the experimental endpoint. We found that for both the pinching and grasping assays that the ATF3cre/cre group had significantly diminished functional recovery  suggesting a gene dosage effect .To determine whether loss of the cre/cre group had significantly longer peak latencies , and the ATF3+/cre group tested positive as a significant intermediary between groups  in ATF3-deficient mice. We then asked when differences in regeneration could be discerned between genotypes anatomically. To this end we assayed axonal regeneration along the sciatic nerve 2 and 3 d after crush by taking a single section (one that lacked sectioning artifacts like tears or folds) and imaged through its full 20-μm depth) for analysis. Two days after sciatic nerve crush, cumulative axon density was not yet different between ATF3e groups A,B. Thre+/cre line can excise floxed endogenous genes, we crossed it to a floxed Pten line, crushed the sciatic nerve, and measured PTEN expression 28 d after injury. Three sections each from five animals were used for analysis. High background with both PTEN antibodies rendered precise estimates of recombination efficiency difficult, but the antibody labeled small-diameter DRG neurons, as reported previously by +/cre PTENfl/fl mice (p = 0.0113) indicating that ATF3 driven Cre expression successfully excises the PTEN gene . PTEN immunoreactivity in ATF3+/cre PTENfl/fl mice was no different from background.To determine whether the ATF3TEN gene A,B. The +/cre and PTENfl/fl or PTEN+/+ alleles. Four control animals and three experimental animals were used (three sections from each). We found that PTEN expression was significantly reduced (p < 0.0001) in the axons that also expressed tdtomato in the PTENfl/fl group compared to controls  or sensorimotor (grasping) assays between the ATF3+/cre PTENfl/fl and control groups  was taken for analysis. Two days after sciatic nerve crush, axon density plotted as a function of distance was obviously increased in ATF3+/cre PTENfl/fl over that in ATF3+/cre PTEN+/+ mice, and the cumulative density at 2 mm was statistically greater  is highly transcribed once the neuron is axotomized, and there is a greater likelihood of ERT2 leak and subsequent recombination. Reduction of recombination by ICI 182780 (It is important to note that despite the Cre recombinase being anchored to a mutated estrogen receptor (ERT2) we have achieved significant recombination without the administration of tamoxifen. This can be attributed to the “leakiness” of the ERT2 construct where the ERT2 protein overwhelms its cytoplasmic anchor and translocates to the nucleus without tamoxifen (or its metabolites) binding. The high degree of tamoxifen-independent recombination we report is likely driven by massive upregulation of ATF3 in the PNS after injury: the I 182780  providesPten and significantly improve anatomic regeneration and functional recovery.We did not use tamoxifen to improve the recombination efficiency of the model, as it has been previously reported that tamoxifen administration, at the standard dose of 75 mg/kg, upregulates ATF3 without injury . NotionaPten deletion following axotomy in the sensory neurons that innervate the sciatic nerve. They excised Pten through a more traditional intraganglionic injection of an AAV Cre vector into the L4 and L5 DRG. Their study and ours agree on three key results: (1) PTEN immunohistochemistry preferentially labels small diameter sensory neurons, (2) PTEN deletion increased axonal regeneration along the sciatic nerve 3 d after axotomy, and (3) PTEN deletion after sciatic nerve crush did not significantly improve sensory functional recovery. The ATF3-CreERT2 line has several distinct advantages over more traditional Cre delivery models; the technical viral transfection setup is not necessary and Cre can be expressed in neuronal populations that may not be amenable to local injection. Moreover, the ATF3-ERT2 line allows for the selective editing of only injured neurons where a viral Cre transfection does not preclude the possibility of Cre expression in uninjured neurons.The potential utility of our model is strengthened by similarities to previous manipulations using different techniques. A study by in vitro uninjured nerve, this has indeed proven to be an issue in other cell types, such as muscle , we tested the validity of two reported ATF3-specific polyclonal antibodies: Novus (NBP 1-85816) and Santa Cruz (C-19). ATF3 signal was expectedly absent in the ATF3 null ipsilateral DRG after axotomy when using the Novus antibody. However, signal was still present in both the control and ATF3 null ipsilateral  DRG when tested with the Santa Cruz antibody suggesting that is not specific to ATF3, but likely another injury-dependent protein. This could call into question some of the conclusions made by the over 150 articles that have used this antibody. We then used the Novus ATF3 antibody to investigate ATF3 expression in the injured sciatic nerve. Despite the appearance of putative ATF3 upregulation, we were able to determine that this signal was restricted to white blood cells, non-nuclear, and in fact due to background fluorescence. s muscle . Histolos muscle . We thercre Ai14 reporter line. The absence of ATF3 immunoreactivity in these cells, along with the high numbers of RSCs that can be observed undergoing mitosis 4 d after injury show that their increase in density is due to cellular division rather than injury-induced ATF3-cre-mediated recombination.Tdtomato-positive RSCs increased in density following sciatic nerve injury. RSCs are a subtype of Schwann cell that are non-myelinating but still ensheathe small caliber axons to form Remak bundles . Gomez-Scre/cre null mutant also allowed us to test the effect of a loss of ATF3 function in PNS regeneration early after injury. While we found no significant differences between the groups in axonal regeneration along the sciatic nerve at the 2-d time point, we did find a statistically significant functional deficit of both ATF3-deficient groups in both our behavioral and EMG assays. Despite being a standard time point for this type of analysis, the lack of significantly different 2-d axonal regeneration along the sciatic nerve could be because the axons have not had sufficient time to differentially regenerate enough to produce a measurable difference. This supports previous work done by +/+ and ATF3cre/cre mice had emerged, along with evidence again for haploinsufficiency (the intermediate position of ATF3+/cre mice). To our knowledge, this is the first evidence that a loss of ATF3 mitigates functional recovery after injury.The ATF3Interestingly the existing literature suggests that ATF3 heterozygotes are haplosufficient  which is+/cre:X+/+ with ATF3+/cre:Xfl/fl , as we have done here substituting “X” with “PTEN.”ATF3 gene dosage effects  should not be an impediment to investigations of functions of other (floxed) genes or sequences provided the appropriate controls are used. There is simply a new baseline on which other genetic manipulations (using ATF3-Cre-driven excision of floxed gene “X”) can be evaluated. For this reason it is important that any investigation of the function of gene X compares ATF3ATF3 is not only upregulated after PNI but also after CNS trauma including spinal cord injury , albeit cre transgenic line. ATF3 is also expressed in injured neurons after spinal cord injury (The mammalian PNS is able to regenerate after injury, unlike the CNS where regeneration does not occur. This positions the PNS as an excellent model for study to better understand what is necessary for mammalian neuronal regeneration. The ability to edit the genes of injured neurons and further dissect what is necessary and/or sufficient to produce this regeneration is of obvious value, and is now possible with the ATF3d injury , traumatd injury , and iscd injury , making


Tlr2−/−), knockout mice lacking Toll-like receptor 4 (Tlr4−/−), and wildtype (WT) control mice, and evaluated endpoint histology at 2 and 16 weeks after implantation. Tlr4−/− mice exhibited significantly lower BBB permeability at acute and chronic time points, but also demonstrated significantly lower neuronal survival at the chronic time point. Inhibition of the Toll-like receptor 2 (TLR2) pathway had no significant effect compared to control animals. Additionally, when investigating the maturation of the neuroinflammatory response from 2 to 16 weeks, transgenic knockout mice exhibited similar histological trends to WT controls, except that knockout mice did not exhibit changes in microglia and macrophage activation over time. Together, our results indicate that complete genetic removal of Toll-like receptor 4 (TLR4) was detrimental to the integration of intracortical neural probes, while inhibition of TLR2 had no impact within the tests performed in this study. Therefore, approaches focusing on incomplete or acute inhibition of TLR4 may still improve intracortical microelectrode integration and long term recording performance.We have recently demonstrated that partial inhibition of the cluster of differentiation 14 (CD14) innate immunity co-receptor pathway improves the long-term performance of intracortical microelectrodes better than complete inhibition. We hypothesized that partial activation of the CD14 pathway was critical to a neuroprotective response to the injury associated with initial and sustained device implantation. Therefore, here we investigated the role of two innate immunity receptors that closely interact with CD14 in inflammatory activation. We implanted silicon planar non-recording neural probes into knockout mice lacking Toll-like receptor 2 ( Brain machine interfaces are a growing area of interest for basic research, rehabilitation, and commercial applications Winkler, . IntracoThe correlation between the neuroinflammatory response to intracortical microelectrodes and recording performance remains a commonly debated topic for over a decade  at the microelectrode-tissue interface , knockout mice lacking TLR4 (Tlr4−/−), and WT control mice, and evaluated endpoint histology at 2 and 16 weeks after implantation. Gaining a more detailed understanding of innate immunity receptors associated with the CD14 pathway should further our understanding of CD14 mediated neuroinflammation to intracortical microelectrodes.In the current study, we will focus on the complementary receptors associated with CD14 activation, Toll-like receptors 2 and 4 (TLR2 and TLR4). TLR4 is an innate immunity receptor closely associated with CD14 that is involved in the recognition of PAMPs and DAMPs to promote inflammation , Tlr4−/− mice , and WT mice  were acquired from the Jackson laboratory and bred in-house. Both male and female mice were used as to not bias the results based on sex. Mice were handled according to the approved Case Western Reserve University IACUC protocol and the NIH Guide for Care and Use of Laboratory Animals.Strains of mice were verified prior to surgery by extracting DNA from tail snips, running PCR, and running gel electrophoresis. Genotyping protocols were performed as suggested by the mouse vendor (Jackson Laboratories), following similar protocols described in previous studies within the lab  using methods adapted from Ravikumar et al. ,b and PoMouse skulls were exposed by a midline incision on the scalp and retraction of the skin using tissue spreaders. Craniotomies were carried out using a 3 mm biopsy punch (PSS select) lateral to midline, and between lambda and bregma, to minimize heat associated with drilling  subcutaneously twice on the first day after surgery. Mice were administered meloxicam  once or buprenorphine (0.1 mg/kg) twice on the first day after surgery and as needed thereafter. Mice were monitored daily for 5 days after the operation for signs of pain and distress and then weekly thereafter.−1 ketamine, 1 mg ml−1 xylazine solution, administered subcutaneously. Additional ketamine-xylazine solution was administered as needed. Following perfusions, the mouse heads were removed and post-fixed in 4% paraformaldehyde dissolved in 1xPBS overnight. Liquid dentin skull caps were carefully removed up from the skulls to remove implanted electrodes and minimize damage to the tissue. Brains were gently extracted from skulls and transferred to a series of sucrose solutions with concentrations of 10%, 20%, and two rounds of 30% in 1xPBS for cryoprotection. Upon equilibration , brains were advanced to the next solution in the series and stored at 4°C. Next, brains were frozen in blocks of Optical Cutting Temperature gel over dry ice and moved to a −80°C freezer. Finally, brains were cryostat sectioned into 16 μm horizontal slices and directly mounted to slides at a ~ −20 to −25°C, and stored at −80°C until removed for immunohistochemical staining.Mice were transcardially perfused 2 or 16 weeks after probe implantation using protocols adapted from Ravikumar et al. . Prior tTwo cohorts of animals were implanted with neural probes to assess both the acute and chronic neuroinflammatory response to implanted intracortical microelectrodes. For the purposes of this study, four histological markers were investigated. First, since neurons are the source of electrical signals recorded by intracortical microelectrodes, sections of cortical tissue were stained with an antibody directed against NeuN, a nuclear protein specific to neurons . Similarly, examination of the accumulation of inflammatory activated microglia and macrophages via CD68 revealed no significant differences between either of the knockout conditions and WT mice . Additionally, examination of the chronic glial scar as a function of GFAP expression also indicated no significant differences were observed between either of the knockout conditions and WT mice . Tlr2−/− mice did not exhibit any differences in IgG expression from WT controls.Plots of percentage of background neuronal density  or normalized fluorescence intensity  with respect to distance from probe hole for e Figure  revealede Figure . Unlike e Figure . SpecifiTlr2−/−, Tlr4−/−, and WT mice revealed a trend of increasing neuronal survival with distance from the probe at the 16 week time point . Examination of the accumulation of inflammatory activated microglia and macrophages via CD68 expression indicated that Tlr2−/−, Tlr4−/−, and WT mice all exhibit a trend of decreasing CD68 expression with distance from the probe hole at the chronic 16 week time point . Similarly, examination of the chronic glial scar as a function of GFAP expression also indicated no significant differences between either of the knockout conditions and WT mice  .An additional cohort of animals was implanted with identical non-functional probes for 16 weeks, to assess the chronic neuroinflammatory response  Figure . Unlike ) Figure . SpecifiTlr2−/−      Figure . Additio) Figure . In cont) Figure . Similar) Figure .Tlr2−/− mice will identify potential effects of TLR2 removal on the standard progression of chronic neuroinflammatory mechanisms in response to implanted neural probes. Contrary to the trend in WT mice, examination of neuronal density in Tlr2−/− mice exhibited significantly higher neuronal density at 16 weeks than 2 weeks after probe implantation in the distance interval 250–300 μm from the probe hole, *p < 0.05 ;   .Examining the time course of immunohistochemical markers in ; Figure . While t) Figure , which l) Figure . Astrocy5 Figure      Figure . Similar) Figure .This study sought to interpret the roles of TLR2 and TLR4 in the neuroinflammatory response to implanted intracortical microelectrodes. Overall, this study reveals that full removal of TLR4 results in reduced blood-brain barrier permeability at an acute (2 weeks) and chronic time points (16 weeks), and reduced neuronal survival at chronic time points, compared to WT animals. Further, microglia/macrophage activation and blood-brain barrier permeability significantly decreased from acute to chronic time points, whereas astrocytic encapsulation significantly increased from acute to chronic time points in WT mice. Mice lacking TLR2 or TLR4 exhibited similar trends in decreasing blood-brain barrier permeability and increasing astrocytic encapsulation, but significant changes in microglia/macrophage activation close to the electrode-tissue interface from 2 to 16 weeks post-implantation. The findings presented here introduce more questions regarding the role of innate immunity receptors in the neuroinflammatory response to intracortical microelectrodes.The first major finding of this study indicated that knocking out TLR4 resulted in decreased blood-brain barrier permeability around implanted intracortical microelectrode at both acute and chronic time points. The blood-brain barrier is a network of endothelial cells with tight junctions that protects parenchymal brain tissue from neurotoxic molecules and infiltrating inflammatory cells. Damage to the blood-brain barrier following intracortical microelectrode implantation has been linked to poor recording performance . Differences in CD68 expression may be limited to the first layer of cells around the neural probe hole. The decrease in CD68 expression in WT mice over time may indicate a diminishing importance of TLR2 and TLR4 in the chronic neuroinflammatory response to intracortical microelectrodes over time, or that lacking either TLR2 or TLR4 retards the rate of wound healing / scar progression. For example, TLR4 deficient mice exhibited delayed skin wound closure paired with decreased Il-1β and IL-6 production   but not chronic (2–16 weeks) time range, with no differences in neuronal survival, microglia/macrophage activation, astrocytic encapsulation, or blood-brain barrier permeability at 16 weeks after implantation , without any differences in 16 week endpoint histology. Inhibition via a small molecule antagonist is less complete as full removal of a receptor via knockout. Residual TLR4 signaling from incomplete inhibition may protect neurons from the processes detrimental to neuronal survival in Further studies by Bedell et al. investigated the effects of knocking out CD14 in specific cell populations on intracortical microelectrode recording performance (Bedell et al., Tlr2−/− mice did not exhibit any significant differences in endpoint histology, changes in blood-brain barrier permeability and neuronal survival in Tlr4−/− mice would suggest a role of TLR4 in the neuroinflammatory response to intracortical microelectrodes. Differences in the time course of microglia/macrophage activation observed in Tlr2−/− mice suggest a subtler role in the neuroinflammatory response to intracortical microelectrodes. Further, we proposed TLR2 and TLR4 signaling as a mechanism for the activation of microglia and macrophages and subsequent release of pro-inflammatory cytokines, ROS, and RNS in response to an implanted intracortical microelectrode. Here, we did not observe significant changes in microglia/macrophage activation via expression of CD68 in ether Tlr2−/− or Tlr4−/− mice at acute or chronic time points. However, the paradoxical decrease in blood-brain barrier permeability and decrease in neuronal survival observed in Tlr4−/− mice may be affected by decreased or increased release of pro-inflammatory factors. As stated earlier, differences in expression of CD68 may not be sensitive enough to detect functional differences in cytokine, ROS, and NOS release. Alternatively, activation of TLR4 on other infiltrating myeloid cells not expressing CD68, endothelial cells, or neurons may be responsible for the observed histological changes (Ravikumar et al., Initially, we hypothesized that TLR2 and TLR4 play a role in the neuroinflammatory response to implanted intracortical microelectrodes. Although Building off of the previous studies of Hermann et al. and Bedell et al. innate immunity signaling pathways appear to play a role in the neuroinflammatory response against intracortical microelectrodes (Bedell et al., Complete removal of TLR2 via genetic knockout did not result in meaningful changes in the expression of the markers stained for in this study. Thus, TLR2 does not likely play a significant role in the foreign body response to intracortical microelectrodes. Complete removal of TLR4 via genetic knockout results in reduced blood brain barrier permeability in response to implanted neural probes at acute and chronic time points, as well as reduced neuronal survival around implanted neural probes. Benefits of fully removing TLR4 are overshadowed by detrimental effects on neuronal survival. Inhibition of TLR4 without complete removal of the pathway or for intermittent time courses may still be worth investigating as an intervention for improving intracortical microelectrode integration.JH, SMS, RM, and JRC contributed substantially to the conception or design of the work, analysis, and interpretation of data for the work, drafting, and revising the manuscript for important intellectual content, approved the final version to be published, and agreed to be accountable for all aspects of the work. SL, AS, CW, VS, JC, SmS, ShS, WT, and GP aided in the collection and analysis of histological data. JH, SL, AS, CW, VS, JC, SmS, ShS, WT, GP, SMS, RM, and JRC approved the final version to be published and agreed to be accountable for all aspects of the work.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.


While a number of alternative radionuclide-based blood pool imaging agents have been proposed, none have yet to achieve widespread clinical use. Here, we present both in vitro and small animal in vivo imaging evidence that the high physiological expression of the glucose transporter GLUT1 on human erythrocytes allows uptake of the widely available radiotracer 2-deoxy-2-(18F)fluoro-D-glucose (FDG), at a rate and magnitude sufficient for clinical blood pool positron emission tomographic (PET) imaging. This imaging technique is likely to be amenable to rapid clinical translation, as it can be achieved using a simple FDG labeling protocol, requires a relatively small volume of phlebotomized blood, and can be completed within a relatively short time period. As modern PET scanners typically have much greater count detection sensitivities than that of commonly used clinical gamma scintigraphic cameras, FDG-labeled human erythrocyte PET imaging may not only have significant advantages over 99mTc-labeled erythrocyte imaging, but may have other novel blood pool imaging applications.99m-Technetium-labeled ( The current prevalent clinical indications for 99mTc-labeled erythrocyte imaging are the detection of drug-induced cardiomyopathy in cancer patients undergoing potentially cardiotoxic chemotherapy, and anatomic localization of sites of occult lower intestinal bleeding in patients .Unless otherwise specified, all chemicals were cell culture grade and obtained from Sigma Aldrich, St. Louis MO. Packed human erythrocytes collected in standard anticoagulant citrate dextrose (ACD) solution (either ≤ 24 hours post phlebotomy or ~ 5 day post phlebotomy) were obtained from Zen-Bio, Inc. 370–740 Megabecquerel (MBq) (1 ml) United States Pharmacopeia (USP) grade 2-deoxy-2- rats [Charles River] were collected and labeled with FDG using the previously described FDG labeling protocol. 250 μl of washed FDG-labeled rat erythrocytes were then incubated with 375 μl of rat plasma at 37° C for 30, 60, and 90 minutes. The samples were then centrifuged 1000g for 10 minutes, and counted with the Atomlab 500 dose calibrator. When accounting for additional time passage during the centrifugation step, sample activity measurements were made approximately 46 minutes, 76 minutes and 106 minutes after start of plasma incubation.5 rat erythrocytes in 40 μl 1X distilled phosphate buffered saline pH 7.4 were incubated in 10 μl Annexin V-PE [BD Biosciences], 80 μM calcein AM dye, and 40 μl 1X Annexin V Binding Buffer [BD Biosciences] to a final volume of 100 μl at 37° C for 30 minutes in the dark. As control groups, unperturbed rat erythrocytes or unperturbed rat erythrocytes then treated with 20 mM CaCl2 were stained with both calcein AM and Annexin V-PE. Rat erythrocytes were then evaluated with flow cytometric analysis at the Moffitt Cancer Center (MCC) flow cytometry core facility.To evaluate the degree of mechanical erythrocyte membrane damage induced by the centrifugation and washing steps in the cell labeling process, fresh rat venous blood samples were subjected to the previously described FDG erythrocyte cell labeling protocol, but with substitution of 1X EDTA buffer solution for FDG. The erythrocytes were then incubated with acetoxymethyl ester of calcein , a fluorescent marker of cell membrane integrity and viability . Erythroscid gamma (NSG) immunodeficient mice were obtained from The Jackson Laboratory. Mice were placed in specialized housing for immunodeficient mice in the MCC vivarium. Approval for all animal experimentation was first obtained from the University of South Florida (USF) Institutional Animal Care and Use Committee (IACUC). All animal experimentation was performed in accordance to federal regulations and USF IACUC principles and procedures. Animal imaging was performed in the MCC Small Animal Imaging Laboratory with an Inveon PET/CT  preclinical scanner. Mice were either injected with FDG-labeled human erythrocytes (4 mice) or were injected with intravenous FDG (4 mice) prior to PET imaging. Mice were first fasted the night before microPET imaging and underwent phlebotomy of ≈ 200–250 μl whole blood via retro-orbital venous plexus puncture immediately prior to microPET imaging. Blood glucose from the phlebotomized blood was measured. A tail vein microcatheter was placed in each mouse under inhalational isoflurane anesthesia. Mice were maintained under inhalational anesthesia and immobilized on the microPET platform before injection of 500 μl of FDG-labeled erythrocyte suspension through the catheter. The PET portion of the system has 64 detector blocks with a total ield of view (FOV) of 12.7 cm and a spatial resolution of 1.4 mm. Raw PET data were acquired for 10 minutes in list-mode data format, followed by the CT attenuation correction scan. Imaging was started immediately after tail-vein injection of -FDG infused erythrocytes. The ECG signal in each mouse was detected with three flat electrodes placed on two front limbs and one hind limb of each animal (ground lead on a rear leg). The signals detected by these electrodes were recorded during the 10 minute time period by BioVet [m2m Imaging] physiological monitoring and heating system. The threshold for TTL cardiac gating signals was set in a rising mode of R-wave peak. The PET list-mode data was reconstructed using 3D-OSEM iterative algorithm with four iterations and eight subsets, with a final image volume of 256x256x256 voxels. Voxel effective dimensions were 1.4x1.4x1.4 mm. For each animal there were three data sets: standard 3-dimensional (3D) PET reconstruction, resulting in a motion—time average 3D PET image; dynamic 3D PET reconstruction with 30 frames; and the phase-based 4-dimensional  PET cardiac reconstruction, with four cardiac gate binning. In all cases, CT attenuation correction was applied to the PET images.4–6 week old male splenectomized NOD PET images of the mice were analyzed using Inveon Workstation Software . Vendor software-supplied Patlak compartment plot option was selected . For 3D All mice in the protocol are evaluated daily in the vivarium to assure well being where their behavior was observed for any signs of distress or illness. A few mice experienced transient distress during microPET image acquisition as perceived by intra-procedural tachypnea and tachycardia, during which inhalational anesthesia was carefully monitored and continuously titrated to reduce any perceived discomfort. These mice were also carefully monitored immediately after PET imaging, and noted to have quickly recovered from previous tachypnea and did not display signs of obvious discomfort, such as labored breathing or impaired movements. Immediately after imaging, mice were placed under a heating lamp and monitored for signs of distress for 1 hour. Breathing was monitored visually and recovery from inhalational anesthesia was assessed by response to sternal rubbing. After completion of the study, mice were euthanatized with an overdose of inhalational anesthesia, and death was verified by cessation of cardiovascular and respiratory movements during prolonged observation of at least 10 minutes, in accordance with USF IACUC protocol.The FDG radiolabeled erythrocytes were assumed to distribute uniformly throughout the blood pool within a few minutes of injection (“well-mixed” assumption). Dosimetry calculations were made under two scenarios. In the first scenario, it is assumed that there is no appreciable efflux of erythrocyte internalized FDG into the plasma during imaging and beyond. In essence, there is no assumed excretion and the entire injected dose is therefore considered to undergo physical decay within the model. It was also assumed there is no significant variation in blood pool specific activity related to decay while in transit to various organ sites. These assumptions result in a static biodistribution of radiotracer throughout the body, with the organ fraction of the injected dose being equal to the organ blood volume. In the second scenario, dosimetry values were calculated under conditions of poor FDG labeling of human erythrocytes, leading to a 25% urinary excretion of released/free FDG in vivo. The values for the organ blood volumes were obtained from ICRP Publication 89 .Human organ absorbed doses were estimated using the FDA approved OLINDA/EXM 1.1  dosimetry software which contains several idealized human and tumor phantoms spanning both genders and various age groups ,36. DoseThe Christy-Eckerman phantom (and therefore OLINDA/EXM 1.1) assumes the self-dose to the hollow organs  arises from radioactive material within the lumen  rather than the viscus wall itself as would be expected with a blood pool agent. The assumption results in an erroneous calculation of the electron self-dose that can be corrected .2 = 0.997.We examined FDG uptake efficiency of human erythrocytes either 1 day or 5 days after phlebotomy. The percent of FDG uptake by human erythrocytes collected ≤ 24 hours prior to FDG labeling was significantly higher than that of erythrocytes collected ≈ 5 days prior to FDG labeling . The meaFDG uptake by human erythrocytes was then measured over a time period of up to 2 hours. 250 μl of 1 day old human erythrocytes were incubated with 37–74 MBq FDG at 37° C at 30 minutes, 1 hour, and 2 hours. The 30 minute:2 hour FDG incorporation ratio = 0.92 ± 0.02. The 1 hr:2 hr FDG incorporation ratio = 0.99 ± 0.02 . , original incubation solution, and subsequent 3 washes was measured and the relative % of total FDG were as follows: Washed erythrocyte fraction (500 μl) = 59% ± 1%; Original supernatant  = 36% ± 2%; 1 1% ± 1% . , and had a much larger cellular fraction expressing high levels of Annexin V-PE binding (≈ 94–98%)  group , the sam 94–98%)   or intravenous FDG (370–740 MBq) would be 3.89–7.77 mSv and 7–14 mSv, respectively, if the SNMMI Guidelines for administered doses are followed.The effective dose to the patient accounts for the relative susceptibility of the various organs to radiation damage and is estimated to be 3.90E-02 mSv per administered MBq using the tissue weighting factors outlined in ICRP Publication 60  and usedPer the dose limits outlined in 21 CFR 361.1 for both single dose administration and annual limits, the dose limiting organ is the heart wall . During 99mTc-labeled compounds remain the dominant radionuclide-specific clinical blood pool imaging agents. In particular, 99mTc-labeled erythrocytes are commonly used to non-invasively search for sites of occult lower intestinal bleeding and to measure cardiac contractility (left ventricular ejection fraction) in patients at risk for chemotherapy-induced cardiotoxicity. Given the inherent advantages of PET over gamma camera imaging, development of 18-fluorine-based blood pool imaging agents for these and other clinical applications remains an area of active research.Currently, SPECT blood pool imaging has also been described in the medical literature, primarily in the setting of radionuclide ventriculography with 99m-Tc-labeled erythrocytes. While results comparable to that obtained with planar-based radionuclide ventriculography have been described, its use in the clinical setting is likely limited, as other imaging tests including myocardial perfusion SPECT can yield similar information . MagnetiHere, we take advantage of the high physiologic glucose uptake rate of FDG by human erythrocytes to show that human erythrocytes can be labeled with a sufficient amount of FDG for in vivo imaging of the vasculature of immunodeficient NSG mice with microPET/CT imaging. Rebinning of ECG-gated microPET images allowed visualization of the cardiac cycle in the mouse heart.While erythrocytes collected ≤ 24 hours from the time of phlebotomy retained the ability to internalize significant amounts of FDG, we observed a negligible rate of internal FDG uptake when human erythrocytes are collected and used ≈ 5 days after phlebotomy, suggesting a significant drop in FDG uptake by glucose transporters and/or intracellular FDG metabolism in these somewhat older cells.Importantly, FDG erythrocyte labeling was accomplished using a straightforward incubation and wash procedure that can be completed in a relatively short period of time  and with minimal equipment. We demonstrate that the repeated cell centrifugation and wash steps in the FDG labeling protocol resulted in minimal cell membrane damage, at least as assessed by flow cytometric analysis with the cell viability/eryptosis markers calcein AM and Annexin V-PE. In addition, the simple incubation/wash buffer used in the protocol is composed of physiologic concentrations of a few salts, and a subclinical (≤1%) dose of the FDA approved chelating compound EDTA. These buffer constituents should thus not represent a significant impediment to clinical translation . Our resOne potential limitation to this technique is that there is a slow gradual release of intracellular FDG from erythrocytes over time. We have found that the relative FDG leakage fraction is approximate 11% at 46 minutes and 19% at 76 minutes. The relevance of the magnitude of FDG leakage would likely depend on the particular clinical application, but it would be reasonable to assume that the typical time course for clinical imaging using this technique would be up to 1 hour, with a resulting estimated leakage fraction of approximately 14%.Recently, Matsusaka et al demonstrated that adult rat erythrocytes were able to incorporate FDG in vitro and could be used to image the body vasculature of rats using a microPET scanner. Our results are similar to those of the Matsusaka group, as they showed an estimated FDG-rat erythrocyte labeling efficiency of approximately 70% with their labeling procedure, and a low fraction (≈ 5–10%) of released FDG from erythrocytes after a 30 minute incubation period .99mTc-labeled, heat-damaged autologous human erythrocytes [Given the lack of clinically significant adverse events associated with transfusion of either autologous erythrocytes or intravenous FDG, it is hypothesized that intravenous administration of FDG-labeled autologous human erythrocytes is unlikely to induce serious adverse clinical events. We have shown that repeated centrifugation and wash steps did not result in significant erythrocyte membrane damage. In addition, we did not detect a significant amount of urinary FDG excretion via PET imaging in these mice to suggest intravascular release of large amounts of intracellular FDG or byproducts from erythrocyte hemolysis or gross membrane leakage. It is also hypothesized that in the clinical setting, any mechanically damaged erythrocytes would be asymptomatically sequestered primarily in the spleen and the reticuloendothelial system, as this is the rationale for clinical imaging of patients with suspected/occult splenic tissue after injection of hrocytes .Mice imaged with FDG-labeled human erythrocytes were previously splenectomized over concerns of potential xenogeneic-specific splenic sequestration of human erythrocytes. As a result, human erythrocytesequestration in the spleen of NSG mice would be difficult to accurately characterize. It is possible that similar imaging results can be obtained in mice with intact spleens; however, this remains to be further investigated by our group. As adult mouse erythrocytes have been found to have minimal membrane expression of GLUT1 transporters, mouse blood pool imaging using FDG-labeled mouse erythrocytes was not pursued to address this potential issue .We found that all mice infused with human erythrocytes survived after imaging; however, some of the mice subjected to repeat injections of human erythrocytes experienced tachycardia and respiratory distress during and immediately after imaging that resolved 24 hours later. We speculate that this could be related to transient pulmonary/systemic edema from the relatively rapid injection of a large volume (≈500 microliters) of FDG-labeled erythrocyte suspension, despite prior retro-orbital plexus phlebotomy. It could also be related to hematopoietic engraftment of residual peripheral blood mononuclear cells contained in the first injection of human erythrocytes. Subsequent injection of human erythrocytes from a different human donor might have induced a type of allogeneic blood transfusion reaction in mice, although this remains somewhat speculative. This possibility could potentially be obviated in the future by using erythrocytes from a single human blood donor when repeated immunodeficient mice imaging is to be performed.In addition, we present calculated human organ dosimetry data for FDG-labeled erythrocyte PET imaging using the OLINDA v.1 dosimetry software, with comparison made to organ doses from either 99m-Tc-labeled erythrocyte imaging or from intravenous FDG PET imaging. These idealized estimates were calculated using conditions where there is assumed to be either 100% tracer decay in the body with no FDG excretion, or under conditions where there is poor FDG labeling of human erythrocytes with resulting 25% urinary excretion of free/released FDG. As we have found that approximately 5% of the FDG in the labeled erythrocyte preparations is excreted into the mouse bladder over the imaging course of our in vivo experiments, we postulate that the calculated organ dosimetry range under these two theoretical conditions would provide reasonable guidance for translation to initial human clinical trials. Mouse organ dosimetry data was not collected, as the use of a splenectomized mouse model was expected to result in skewing of the relative blood volume distribution within the remaining mouse organs, and thus diminish the accuracy of any resulting organ dosimetry results for this blood pool imaging technique.Our calculations show that the organ limiting dose from FDG-labeled erythrocyte imaging would be the myocardial wall; however, it should be noted that the calculated dose is only mildly higher than the dose received from intravenous FDG. As there are several organs where there is ≈ 2-3X higher organ dose compared to that from intravenous FDG , patient exposure to 185–370 MBq of FDG-labeled erythrocytes would be predicted to have an overall dosimetry level somewhat comparable to that received from amounts of intravenous FDG used in clinical practice (370–740 MBq).99mTc-labeled erythrocyte imaging, when used for the detection of either occult intestinal bleeding or chemotherapy-related cardiotoxicity. We speculate that this technique may have inherent advantages over the cerebrovascular perfusion SPECT imaging agents HMPAO and ECD for similar reasons. We also speculate that the relatively long radioactive half-life of 18F-FDG may allow for real-time detection of subtle changes in blood flow in the brain as well as other organs under varying conditions/stimuli that may not be discernable with clinical magnetic resonance imaging (MRI) platforms, due to the significantly higher sensitivity of PET over MRI [We show that human erythrocytes can rapidly incorporate sufficient amounts of FDG to obtain in vivo images of the mouse vasculature using microPET/CT. We believe this imaging technique can be readily translated to the clinical setting, given the simplicity, high labeling efficiency, and relatively short turn-around time of the labeling protocol, as well as the use of a simple incubation/wash solution composed of a few physiologic salts and a subtherapeutic dose of a clinically used compound (EDTA). As modern medical PET/CT scanners generally possess count detection sensitivities much higher than that of medical gamma camera scintigraphy, FDG-erythrocyte PET imaging may have significant advantages over over MRI .S1 FigUnperturbed sample 1.(TIF)Click here for additional data file.S2 FigUnperturbed sample 2.(TIF)Click here for additional data file.S3 FigUnperturbed sample 3.(TIF)Click here for additional data file.S4 Fig2-treated sample 1.CaCl(TIF)Click here for additional data file.S5 Fig2-treated sample 2.CaCl(TIF)Click here for additional data file.S6 Fig2-treated sample 3.CaCl(TIF)Click here for additional data file.S7 FigMock FDG-labeled sample 1.(TIF)Click here for additional data file.S8 FigMock FDG-labeled sample 2.(TIF)Click here for additional data file.S9 FigMock FDG-labeled sample 3.(TIF)Click here for additional data file.S1 FileRelative fraction of intracellular release of FDG from rat erythrocytes over time.(XLSX)Click here for additional data file.S2 FileCalculated organ-specific absorbed dose per unit activity administered (mGy/MBq).(XLSX)Click here for additional data file.