Continually emerging data indicate that sub-clinical, non-convulsive epileptiform activity is not only prevalent in Alzheimer’s disease (AD) but is detectable early in the course of the disease and predicts cognitive decline in both humans and animal models. Epileptiform activity and other electroencephalographic (EEG) measures may hold powerful, untapped potential to improve the translational validity of AD-related biomarkers in model animals ranging from mice, to rats, and non-human primates. In this review, we will focus on studies of epileptiform activity, EEG slowing, and theta-gamma coupling in preclinical models, with particular focus on its role in cognitive decline and relevance to AD. Here, each biomarker is described in the context of the contemporary literature and recent findings in AD relevant animal models are discussed. Alzheimer’s disease (AD) is the foremost cause of dementia NIA  and the It is now over 115 years since the first clinical report describing AD in 1906 , but theEarly detection and treatment at the very earliest stages of amyloid seeding may hold critical potential for the treatment of AD . Owing tEpileptiform activity is a broad term to describe sporadic discharges or trains of spiking activity resulting from highly synchronous oscillatory activity in the brain. The activity captured in a surface EEG is largely generated from reciprocal excitatory-inhibitory connections throughout thalamocortical circuits and is not necessarily equated to classical full body seizure type events; although severe events of this nature may result in such. In short, hyperactivity in layer VI pyramidal cells triggers an inhibitory response from GABAergic neurons within the thalamic reticular nucleus. During this inhibitory response, low-threshold calcium channels in thalamocortical neurons recover from initial inactivation. Consequently, as thalamocortical neurons recover from inhibitory post-synaptic currents generated by the thalamic reticular nucleus, this available pool of calcium channels trigger another burst of excitatory activity. This cycle produces reverberating oscillatory activity detectable as spike trains in EEG . PerturbCurrent theories hypothesize that progressive age-related decline of synaptic function in the earliest stages of AD alters firing properties of neurons within broad networks mediating genome, immune, and metabolic homeostasis, among other functions. Dysregulation within these networks feeds forward resulting in loss of function and cognitive decline as in AD . This unDespite broad acceptance of its occurrence, accurate coincidence of abnormal epileptiform activity in AD has proven difficult to estimate. Given that these neural perturbations are often subclinical and not presented with any overt motoric type of seizure events, awareness of seizure activity may be limited. Indeed, previous reviews have reported incidence ranging from 0.5 to 64% . HoweverRecent literature highlights the utility of EEG studies in animal models as demonstrating translational potential for AD . More spIn animal models, epileptiform activity is used broadly to refer to ectopic discharges that may present with  or without  motor features. Applications of EEG in animal models generally consists of the identification of spike wave discharges (SWD) or single spikes predominantly during NREM. SWDs are typically characterized by surface-negative spikes alternating with 7–10 Hz surface positive waves . Single Electroencephalographic slowing is a key biomarker in AD and AD related dementias (ADRD) . In the Hyperexcitability of hippocampal neurons has been used in animal models as a surrogate to hippocampal hyperactivation observed in AD patients during fMRI . In an fTheta-gamma coupling (TGC) is another electrophysiological biomarker with emerging relevance to AD . TGC refMuch of the preclinical AD research to date has utilized mouse models expressing FAD mutations, specifically amyloid precursor protein (APP) and presenilin 1 (PS1). Despite the causal mechanisms of these mutations in early-onset AD, FAD only comprises between 1 and 5.5% of all AD cases . MoreoveAmyloid precursor protein is a transmembrane protein with yet unknown endogenous functions, though it has been implicated in a broad array of roles such as synapse formation , plasticOne of the earliest APP mouse models is the Tg2576, overexpresses a mutant form of APP with the Swedish (APP KN670/671NL) mutation resulting in significant and early onset amyloid pathology. This is a double mutation directly adjacent to the β-cleavage site of APP, which facilitates β-secretase cleavage. Owing to its name, the Swedish mutation was first characterized in two Swedish families with a high degree of genealogical overlap. Affected members of these families experienced progressive memory loss consistent with AD . Late inNL–G–F, does not overexpress APP, but instead express fully humanized Aβ fragments from cleavage of Swedish (KM670/671NL), Arctic (E693G), and Beyreuther/Iberian (I716F) mutant APP  indicative of an anxiolytic-like phenotype, it may be difficult to distinguish between spatial memory deficits in an avoidance paradigm or an overall loss of inhibition/aversion to risk in these mice. Subsequent experiments showed that J20 mice do exhibit spatial memory deficits in the Morris Water Maze (MWM)  , althougnsistent . Howevernsistent . Spatialnsistent , neuron nsistent , and LTPnsistent  have beensistent . This pansistent . Overallnsistent . In a seutations , epileptderstood . Additioderstood . Reportsng water . Activatocedures . These pOver 70 mutations of the presenilin (PSEN) gene have been characterized , accountThe APP/PS1 mouse combines a transgene carrying both hAPP and PSEN mutations, though the specific mutations may vary per each strain. Mice expressing APP Swedish and PSEN L166P mutations are most common. These mice express the APP transgene at threefold higher levels compared to murine APP and human Aβ42 is expressed in higher concentrations over Aβ40. Amyloid deposition is detectable in 6 weeks old mice, beginning in the neocortex. Amyloid deposits in the hippocampus, striatum, thalamus and brainstem appear in young adult mice 3–5 months old, though tau inclusions have not been identified in this strain . DeficitAnother study comparing mice carrying a deletion of PSEN1 exon 9 (APP/PS1ΔE9) also reported a seizure phenotype exhibiting multiple forms of spontaneous epileptiform events. This is particularly interesting because indeed patients with the PSEN1 exon 9 mutation also demonstrate seizure activity . UtiliziInterestingly, a similar strain carrying the APP Arctic mutation (E693G) with the dE9 deletion have a comparatively mild seizure phenotype compared to the Swedish (APP KN670/671NL) mutation of APP. This mutation of APP also does not produce amyloidosis . This woTwo separate papers have recently described a startling phenomenon in APP/PS1ΔE9 mice regarding the use of selective serotonin reuptake inhibitors (SSRIs) and the induction of AD-related seizure activity. Treatment with fluoxetine  and paroThe 5xFAD mouse has rapidly gained widespread use in the study of AD. These mice express 5 human transgenes linked to familial AD: three APP mutations . AD-relevant pathology in these mice progresses aggressively. Amyloid plaques and gliosis have been characterized in as early as 2 months old mice . At 6 moThe ε4 allele of apolipoprotein E protein (APOE) is the strongest known risk factor for the development of non-familial LOAD. Three allelic variants of APOE are common in humans: ε2, ε3, and ε4. APOE is synthesized in the liver and the brain and mainly functions in the transport and homeostasis of cholesterol and other lipids throughout the body. With particular relevance to AD, APOE binds Aβ in the extracellular space and plays a role in clearing Aβ from the CNS through the blood–brain barrier (BBB). Of the three APOE isoforms, ApoE4 is poorest at binding Aβ and clearing it across the BBB; while APOE2 is the most effective. APOE2 is also known to be protective against AD. Thus, risk for LOAD is dose dependent on the APOE4 allele (APOE4/APOE4 > APOE3/APOE4 > APOE2/APOE4) . APOE4 cAn age-dependent seizure phenotype has been described in aged mice with a targeted replacement of the endogenous murine APOE allele with the humanized APOE4 allele (APOE4 TR). Female APOE4 TR mice were the first to develop a seizure phenotype of motoric jerks without arrest or clonus at six months of age. This same phenotype was present in males, albeit emerging later at 9 months. Cortical EEG reveled frequent spiking in the cortex of the APOE4 TR mice .Triggering receptor expressed on myeloid cells 2 (TREM2) is a frequent mutation related to AD. TREM2 is a transmembrane receptor involved in the immune and compliment systems. AD related mutations in TREM2 result in loss of function and are thought to mediate immunogenicity and inflammation that is enhanced in AD . Myoclonper se (The 3xTG mouse combines the APP Swedish mutation with the PSEN M146V mutation and the P301L mutation of Microtubule Associated Protein Tau (MAPT). The PSEN M146V mutation impairs γ-secretase cleavage of APP resulting in greater secretion of Aβ42 and increased Aβ42/Aβ40 ratio . Individper se . When coper se . Gliosisper se  and impaper se .Resting state functional magnetic imaging (re-fMRI) performed in 3xTg mice has revealed that, although tau immunoreactivity was present in 6–8 and 18–24 months old groups, the older cohort displays a higher degree of synchronous activity between multiple brain regions including hippocampus, auditory association cortex, temporal association cortex, and lateral entorhinal cortex. Thus, although development of tauopathy was not age-dependent in these mice, development of hypersynchronous activity in 3xTg is an age-dependent phenotype .In vivo anesthetized recordings in older 4–6 and 17–18 months 3xTg mice comparing evoked responses in CA1 and DG of hippocampus following performant path stimulation revealed that short-term plasticity in response to brief high frequency stimulation is facilitated in both young and old 3xTg mice. Though responses to single or low frequency paired-pulses indicate that synapse integrity is nominal  present with increased hippocampal theta activity that is associated with increased tau phosphorylation. In these mice, this effect can be reversed by the potassium channel antagonist 4-aminopyradine . In vivo nominal . This hi nominal , audioge nominal , and cor nominal . This se nominal . Though  nominal .1–42 induced a selective upregulation of NaV 1.6 channels  leads to resting membrane depolarization and increased spike frequency. Interestingly, exposure of Tg2576 embryos to Aβchannels . In eachchannels , which iSeizure activity and the resulting cognitive deficits in J20 mice may be related to a deficient expression of calcium binding protein calbindin-D28k . CalbindA study in APP/PS1 mice compared induction of LTP in hippocampal slices of 1.5 and 8 months old mice following high frequency stimulation (HFS) of Schaffer collaterals. Small, but statistically non-significant differences in post HFS LTP were measured in 1.5 months old mice. At 8-months LTP was significantly diminished compared to younger mice. Smaller field excitatory post-synaptic potential (fEPSP) output in response to stimulation and increases in paired-pulse ratio at shorter inter-pulse intervals were also described. Treatment with the GABA agonist diazepam was sufficient to reduce paired-pulse ratios in 8 months old WT littermates, but did not produce a significant decrease in the APP/PS1 mice. Taken together, these studies indicate a loss of function in GABA signaling that produces dysfunction in hippocampal LTP , which mMultiple reports have characterized alterations in EEG power in 5xFAD mice. APOE4 mice were recently shown to have greater glucose metabolism in the entorhinal cortex measured by fMRI, increased activity of excitatory neurons in awake and freely moving mice, and longer duration extracellular field potentials with diminished LTP in hippocampal slices . These rRare gene variants of the disinterring and metalloproteinase domain-containing protein 10 (ADAM10) confer a low to moderate risk of AD. ADAM10 is an α-secretase that cleaves APP into non-toxic products preventing the production of Aβ fragments. Mutation or loss of function of ADAM10 will favor BACE cleavage and production of plaque forming Aβ fragments. Overexpression of ADAM10 in the hippocampus of C57BL/6J mice reduced markers of neuroinflammation  and protected against pilocarpine-induced seizures . On the Although not specific to AD, alpha-synuclein mutant mice used in the study of Dementia with Lewy Bodies (DLB) and other synucleinopathies may also be instructive to mechanisms related to seizure activity and hippocampal dysfunction. In DLB, inclusions of α-synuclein form when the presynaptic membrane is mislocalized and begins to aggregate . EEG sloThe AD-relevant neural activity has also been identified in some rat models as well. F344-AD rats express two transgenes: Swedish (APP KN670/671NL) and human presenilin PS1ΔE9. Urethane anesthetized and freely moving F344-AD rats exhibit diminished activity within alpha frequencies. Though, diminished activity was also observed in the theta frequency range, opposite of what is typically seen for EEG slowing. In both WT and F344-AD anesthetized rats, stimulation of the tegmental nucleus pontis oralis at 6 months of age strongly entrains a theta oscillation in the hippocampus. At 12 months, however, this evoked theta oscillation is diminished . TreatmeWhile the majority of EEG phenotypes in AD animal models have been reported in rodent species including in this present review, despite the utility of EEG in rodents for translational studies, it should not be dismissed that neurophysiological characteristics of rodents are largely different from those of humans. Thus, most pharmaceutical interventions that have been developed in rodents and reported as efficacious for improving cognitive impairment did not show any significant cognitive improvement in clinical trials . From thNL–F mice. Local field potentials were recorded in 5 months old homozygous male and female AppNL–F and five non-litter male and female C57BL/6 mice using tetrodes implanted and advanced through the entorhinal cortex. TGC was measured as the phase at the time of gamma oscillation maxima. Prominent gamma oscillations were characterized during theta states in both strains. Further the power of gamma oscillations did not differ between strains. However, though the number of gamma oscillations were not significantly affected in AppNL–F mice the temporal structure of gamma oscillations was impaired. Specifically, the peak of gamma oscillations observed in the falling phase to trough of theta was reduced in magnitude compared to C57BL/6. So, while epileptiform activity may be absent in AppNL–F mice, they are not spared in terms of EEG abnormalities (TGC is impaired in Appmalities .NL–G–F mice. Spike-gamma coupling refers to the generation of gamma oscillations generated by rhythmic synchronous activity of fast spiking interneurons. A loss of spike-gamma coupling could lead to impairments in theta-gamma coupling as temporal associations breakdown across oscillatory frequencies. Patch clamp recordings of fast spiking interneurons and pyramidal cells in 1, 2, 4, and 6 months old. AppNL–G–F mice revealed impairment of spike-gamma coherence as early as 2 months of age, preceding amyloid plaque formation. Gamma oscillations also become degraded with age in AppNL–G–F mice, which is correlated to Aβ1–42 concentration in the brain (NL–G–F mice may contribute to breakdown of theta-gamma interactions, though this remains to be demonstrated.Alterations in spike-gamma coupling have been observed in Apphe brain . This rein vitro prep combining patch and field recordings with optogenetics in hippocampal tissue from 30 to 39 day old mice, a number of circuit-level abnormalities were characterized. First, theta oscillations displayed less rhythmicity compared to non-transgenic mice. TG mice also exhibited reduced theta-gamma CFC. Therefore it is clear that these circuit level disruptions are detectable in very young J20 mice, preceding any signs of Aβ accumulation (J20 mice also exhibit breakdowns in cross frequency coupling. In an mulation . J20 micmulation . These r3xTg mice also exhibit deficient coordination between theta, slow gamma, and place cells in the hippocampus at 8–9 months of age. Mice were trained to run unidirectionally around a circular track to receive a reward that was always placed in the same location. Tetrodes aimed at the CA1 region of hippocampus recorded single units and continuous sampled local field potentials. Theta phase coupled increases in slow gamma power were diminished in 3xTG mice compared to WTs, suggesting that hippocampal slow gamma modulation is impaired in the 3xTg mouse . Though in vivo recordings in the hippocampus and mPFC of 9-month old F344-AD rats assessed phase-amplitude coupling, specifically the ability of low frequency theta oscillations to modulate high frequency gamma oscillations. This analysis showed a decrease in phase-amplitude CFC in both regions (Transgenic rats also exhibit impaired CFC. Anesthetized  regions . These rSubclinical seizure and epileptiform activity are potentially very powerful pre-diagnostic biomarkers of prodromal dementia and LOAD . Four diAside from clinical value, these EEG biomarkers have high potential to provide insights into the etiology and progression of AD in ways that go beyond amyloid and tau hypotheses that have failed to produce tenable, disease-modifying treatments for AD. In rodents, EEG and EMG can be recorded by wireless telemetric transmitters using skull surface electrodes that can be implanted with a relatively simple surgical preparation . This caThere is plenty of room for improvement in this arena. First, there is no animal model that fully recapitulates all aspects of AD; an important caveat that has been discussed extensively elsewhere see . AlthougSecond, understanding of the genetic mechanisms and environmental interactions contributing to LOAD are still poorly understood. Third, understanding of the timecourse of progressive degeneration and normal aging related changes in EEG in animal models has been limited. Finally, putative disease modifying drugs developed using these mice have all thus far failed to translate . If the NL–G–F/NL–G–F mice (Ideally, these EEG measures will begin to inform the onset of pathological changes in AD and predict cognitive decline, which has thus far eluded researchers to date. To date, EEG as an early biomarker has not yet fully realized its potential with respect to cognitive changes. Importantly, detection of spontaneous sub-clinical seizures may be difficult in a clinical setting where it may not be possible to record for extensive periods of time, which could limit its potential. Optimally, future investigations can begin incorporating EEG in animals performing cognitive tasks with discrete stimulus presentation and high temporal fidelity with recordings, such as the touchscreen batteries that are rapidly becoming increasingly commonplace in behavioral neuroscience . These aG–F mice  and methG–F mice . These mThe specificity of EEG signatures as biomarkers continues to remain under speculation across the field and requires more research. Measures of electroencephalographic hyperexcitability have been reported in several CNS disorders including in multiple sclerosis , autism Overall, pre-clinical AD research is in need of new approaches. Decades of research focused on behavioral testing in FAD mutant mouse models have not appreciably moved the needle on our ability to prevent degeneration or extend lifespan in patients. EEG assessment of epileptiform activity in diverse model systems including NHPs and new models genetically engineered with mutations relevant to LOAD risk has the potential to push this horizon in new ways and ultimately enable accelerating the pace of bringing effective therapeutics to patients.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-small cell lung cancer (NSCLC) is commonly diagnosed at advanced stages limiting treatment options. Although, targeted therapy has become integral part of NSCLC treatment therapies often fail to improve patient’s prognosis. Based on previously published criteria for selecting drug combinations for overcoming resistances, NSCLC patient-derived xenograft (PDX) tumors were treated with a low dose combination of cabozantinib, afatinib, plerixafor and etoricoxib. All PDX tumors treated, including highly therapy-resistant adeno- and squamous cell carcinomas without targetable oncogenic mutations, were completely suppressed by this drug regimen, leading to an ORR of 81% and a CBR of 100%. The application and safety profile of this low dose therapy regimen was well manageable in the pre-clinical settings. Overall, this study provides evidence of a relationship between active paracrine signaling pathways of the Cellular Tumorigenic Network, which can be effectively targeted by a low-dose multimodal therapy to overcome therapy resistance and improve prognosis of NSCLC. The identification of novel therapeutic strategies for advanced non-small cell lung cancer (NSCLC) is essential as the outcome of most of these patients is poor. Here the authors provide a proof-of concept that specific low-dose drug combinations can disrupt intercellular crosstalk to overcome drug resistance in NSCLC patient-derived xenografts. In patients with NSCLC of squamous and non-squamous histology bearing a PD-L1 expression >50% an improved efficacy for a monotherapy with pembrolizumab compared to a platinum-based chemotherapy was observed and thus became the standard of care for these patients. For the treatment of NSCLC patients characterized by targetable and predictive oncogenic mutations, many different targeted therapies have already been approved or are under development, including drugs targeting Epidermal Growth Factor Receptor mutations (EGFR+), Anaplastic Lymphoma Kinase (ALK)/proto-oncogene tyrosine-protein kinase (ALK/ROS+) fusions, proto-oncogenes KRAS and B-Raf (BRAF+) mutations, respectively, as well as neurotrophic tyrosine kinase receptor (NTRK+) mutations4.Lung cancer is the leading cause of cancer‑related mortality worldwide. Most cases of non-small cell lung cancer (NSCLC) are diagnosed at advanced metastasized stages and therefore indicated for palliative treatment. These mainly non-resectable carcinomas are treated with platinum-based chemotherapy alone or in combination with immune-checkpoint inhibitors, which are the mainstay regimens in the absence of predictive, targetable oncogenic mutations and an expression of the immune-checkpoint inhibitor Programmed death ligand-1 (PD-L1) of <50%5. NSCLC patients without targetable oncogenic mutations and without PD-L1 expression are still a major challenge and there is a need for experimental generation of new, rational combination strategies with targeted therapies addressing factors of the Cellular Tumorigenic Network7, as apart from the tumor cells, non-neoplastic cells such as cancer-associated fibroblasts, endothelial cells, and immune cells contribute to tumor growth. Tumor cell signaling reprograms these cell types inducing processes such as angiogenesis, inhibition of apoptosis, immune evasion, and synthesis of soluble tumor microenvironment components. During the evolving tumor development, signaling networks within the tumor are reshaped further and programs such as hypoxia led to heterogeneity within specific tumor areas. This interdependence could be the initial cause of de novo and adaptive drug resistances in many cases10.The 5‑year survival rate of NSCLC patients is still below 25% and there is still a pressing medical need for the development of effective treatment optionsde novo and prevent adaptive drug resistances. Noteworthy, the direct suppression of cellular interdependency is most likely be achieved by targeting critical paracrine signaling axes of cell surface receptors and their respective ligands. These pathways are blocked at the receptor level while inhibiting multiple generic intracellularly connected downstream pathways involved in tumor development and treatment resistance such as the mitogen-activated protein kinase (MAPK), Janus kinase and the signal transducer and activator of transcription protein (JAK/STAT), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and protein kinase C (PKC) pathways Target expression or pathway activation proven by expression and/or mutational analysis(ii)Target should be part of a paracrine signaling pathway mediating intercellular interdependency within the Cellular Tumorigenic Network(iii)Drug combination should target non-overlapping intercellular signaling axes of the Cellular Tumorigenic Network(iv)Selected drugs should have shown proven activity in clinical studies(v)Manageable safety profile of drug combinations.In order to break the crosstalk within the tumorigenic network in late-stage NSCLC patients as described above, previously, we postulated five criteria for selecting a combinational targeted therapy approach15. Therefore, this effect is intended to be counter-regulated using afatinib for targeting the EGF-EGFR axis and plerixafor for targeting the SDF-1α—CXCR4 axis in the chosen therapy regimen. In addition, cabozantinib also targets the HGF-MET axis. The interruption of EGFR signaling by afatinib would lead to substitution by cancer-associated fibroblasts and tumor cells secreting HGF and SDF-1α17. SDF-1α secreting cells such as cancer-associated fibroblasts and tumor endothelial cells may substitute for interrupted EGFR signaling by paracrine signaling via CXCR418. Again, these anticipated resistances will be prevented by applying the inhibitors of the paracrine axes of HGF-MET and SDF-1α - CXCR4 through cabozantinib and plerixafor in this therapy regimen. A potential resistance against VEGFR inhibition with cabozantinib may also be initiated by the production of PGE2 via COX219. This effect is intended to be counter-regulated using etoricoxib in this therapy regimen , VEGFA, CXCR4, SDF-1α (CXCL12), MET, HGF, and COX2 were analyzed in this sample set and all genes were found to be expressed at different transcript levels.In order to assess the five criteria postulated for overcoming drug resistances 38 well characterized patient-derived, human NSCLC tumor xenograft models (PDX) from the EPO PDX panel were analyzed for mRNA expression of (i) paracrine signaling pathways mediating intercellular interdependency within the Cellular Tumorigenic Network that were (ii) non-overlapping and have been described (iii) as relevant for tumor proliferation previously and druggable by approved inhibitorsTP6320 was analyzed as control of the biological validity of this data set and correlated with the known tumor histology of these tumors . In contrast, the analysis of TP63 transcripts in adenocarcinomas indicated very low abundance or absence of detection in this NSCLC subpopulation. Therefore, TP63 could be used as a valid marker for squamous cell carcinoma histology within this data set. In conclusion, the high rates of sensitivity : ) and specificity  of TP63 expression demonstrates the high biological relevance of our RNA sequence NSCLC PDX data set.First, the specific marker for squamous cell carcinoma ors Fig. . Twenty-21 within the PDX panel.Utilization of ssGSVA in combination with a permutation-based test provided an indication for the importance of our selected nine genes within the tumorigenic network. The obtained high enrichment scores for all 38 NSCLC PDX models reveal an over-representation of the selected genes within a set of 19096 HGNC-annotated genes. The depicting chart of scaled enrichment scores distributes our 38 NSCLC PDX cohort into several segments representing the biological diversity and individual characteristics of each PDX model Fig. . The perIn order to evaluate a potential correlation between gene expression and treatment responses, we analyzed the expression data of the 38 NSCLC PDX models comprising 19096 HGNC-annotated genes and subsequently performed hierarchical clustering  identified in Cluster 1 exhibit the highest transcript expression for EGFR, BRAF, KRAS, MET, and RET  (Lu7406 and Lu7414) and tumors with at least stable disease (SD) or even better responses  for any chemo or targeted monotherapy tested previously. The existence of targetable oncogenic mutations was excluded by utilizing the Illumina amplicon cancer panel (TSACP), sequencing 212 regions in 48 cancer-related genes including RET Fig. . As an eTP63 expression analysis with the exception of false-negative detection for Lu7414 as discussed above  or squamous cell carcinoma , which was confirmed by the ove Fig. . All selN = 18) or monotherapy treatment (N = 12) group , α-SMA , and CD31 (blood vessels). No histological differences were observed between tumors of the treatment groups and tumors of the respective placebo groups Fig. . In addiEven though no differences between treatment and placebo groups in the three tumor models could be observed, a considerable membranous expression of PD-L1 was detected in all residual tumors of either group at different levels. This result again reflects the heterogeneous expression levels of PD-L1 in these models Fig.  Importan1, the unmet medical need for many patients in this population remains high5.Despite some impactful advances in the treatment of NSCLC without targetable oncogenic mutations and without expression of PD-L1 resulting in an ORR of about 50%23.Our previously published hypothesis for the treatment of these NSCLC tumors by breaking the Cellular Tumorigenic Network has now been experimentally proven by the present study using PDX tumor models most closely resembling the clinical situation and being highly predictive for treatment responses in cancer patientsWe provide evidence that the expression of a respective set of target genes is important for NSCLC development, homeostasis and progression. Our ssGSVA obtained enrichment scores indicate the relevance of the underlying biological function of the selected pathways in NSCLC disease and treatment. Observing a relatively low statistically significant over-representation of the nine target genes for only 8 of 38 models by permutation-based test is even more confirmative highlighting the great range of individual NSCLC characteristics and phenotypes rather than pointing out a bias with our analysis.Furthermore, we showed that gene expression of a specific gene set of the tumorigenic network might be related to treatment resistance against chemo- or targeted monotherapies. A high expression of this nine gene set correlates mainly with disease progression in in vivo sensitivity testing with chemo- and targeted therapies.All tumors randomly selected from the NSCLC PDX biobank panel responded well to this low-dose combination therapy despite showing different expression levels of the target genes with simultaneous coverage of diverse clinical phenotypes. Our finding of a potential relationship between the expression of target genes within the tumorigenic network and the response to different anti-tumor therapies should be further expanded verifying our investigations by utilizing an even more extensive data set of well characterized NSCLC tumors.Nevertheless, to the best of our knowledge, for the first time, we show that the simultaneous and distinct targeting of paracrine signaling from different cell types of the Cellular Tumorigenic Network breaks the intercellular crosstalk and is highly effective in tumors that are resistant to monotherapies of chemotherapeutics and targeted drugs against EGFR and VEGFR. In all treated NSCLC tumors without a targetable oncogenic mutation randomly selected from a panel of 38 NSCLC tumors, growth was completely suppressed by the applied low dose combination regimen. Among them were two highly treatment-resistant tumors that did not respond to any tested therapy previously (Lu7406 and Lu7414). During the complete treatment duration, no tumor outgrowth was observed in either PDX model, indicating a strong therapy efficacy by preventing development of an adaptive resistance against this regimen. After treatment discontinuation growth suppression remained up to 18 days (Lu7860) with only slight increase in tumor growth. This indicates a long-lasting and sustained effect of the combination therapy on tumor biology.7 can be considered as a low dose treatment regimen. The two drugs with a more pronounced safety profile in this therapy regimen are cabozantinib and afatinib. Cabozantinib is approved for the treatment of renal cell carcinoma and hepatocellular carcinoma and is efficacious in different tumor models at dosages between 30–60 mg/kg once a day26. Therefore, the low dose drug combination used in our study only includes 50% or less of typically used cabozantinib concentrations in recently published preclinical studies. Afatinib is approved for the treatment of NSCLC either with activating EGFR mutation or in squamous cell carcinoma under or after progression on platinum-based chemotherapy and is efficacious in tumor models at dosages from 20 mg/kg or higher once a day27. In the combination therapy regimen used in this preclinical study, both drugs were used below these drug doses, each at 15 mg/kg and in addition to that paused after every 5 days of treatment. Plerixafor, approved for stem cell mobilization, and etoricoxib, approved for pain treatment and degenerative and auto-inflammatory diseases, were used at a commonly used concentration level in in vivo models29. The usage of this combination regimen showed an excellent safety profile and might lead to less pronounced side effects in patients. A database analysis for potential drug–drug interactions of this drug combination only revealed a potentially moderate clinically interaction between cabozantinib and afatinib that may result in a plasma level increase of afatinib when administered after exposure to a P-glycoprotein (P-gp) inhibitor. A monitoring of patients for the onset of adverse effects is recommended. Due to unavailability in this DDI database etoricoxib was replaced with celecoxib, another drug of the identical drug class of selective COX2 inhibitors30.The combination therapy regimen that was compiled on the five criteria published previouslyTogether, with its excellent efficacy, this combination would be superior to a prescribed medication of single cabozantinib or afatinib at higher drug dosages.Treatment with the combination therapy did not seem to change the histopathological composition of tumors when compared to placebo treatment regarding the extent of the tumor and the stroma compartment as well as the number of blood vessels. Since the studies have been performed in nude mice, it remains open whether this treatment regimen impacts the infiltration, composition, and effector phenotype of T cells. However, our studies also revealed that the expression of PD-L1, whose therapeutic targeting has essentially improved the prognoses of even metastasized NSCLC patients, was not affected by the combination therapy, offering the opportunity of combination therapies including immune-checkpoint inhibitors.31.Taken together the simultaneous inhibition of pathways forming the Cellular Tumorigenic Network in NSCLC tumors can overcome resistance mechanisms of targeted therapy drugs. All of the drugs used in this combination regimen are approved for different indications, and therefore their clinical profiles such as pharmacokinetics, pharmacodynamics, and toxicities are well known. They are readily available as study drugs for usage in a clinical trial. In order to identify NSCLC patient subgroups that may respond to this therapeutic concept, inclusion criteria should require the proof of expression of the respective target gene set. Collected tumor specimens can easily be used for mRNA expression analysis by specific RT‑real time PCR or by a customized gene expression array. The analysis of mRNA expression as a predictive biomarker is already in clinical developmentBased on our results we encourage the further validation of this combination therapy in additional cancer models and we recommend the setup of a clinical study in advanced stage NSCLC patients without a targetable oncogenic mutation combining the drugs cabozantinib, afatinib, plerixafor and etoricoxib in a low dose treatment regimen.RNA sequencing was performed for 38 NSCLC models, comprising 12 adenocarcinomas and 26 squamous cell carcinomas. One sample each from the established patient-derived xenograft models was analyzed.Total RNA was isolated from snap-frozen PDX tissue using InvitrogenTM TRIzolTM Reagent . 50 mg of tumor tissue was disrupted in 1.5 ml TRIzolTM using a gentleMACSTM dissociator and M tubes . RNA integrity was evaluated with Agilent Bioanalyzer 2100 and RNA 6000 Nano Kit . RNASeq libraries were prepared using illumina® TrueSeqTM Stranded mRNA Library Prep Kit. The 100-bp-PE-sequencings ran on an illumina® HigSeq 2500 device with a depth of 80–100 Mio reads (40–50 Mio cluster) .32 version 0.11.8. For classification of the xenograft-derived sequence reads (human/mouse read splitting) the tool xenome33 version 1.0.1 and human genome hg38 as graft reference as well as mouse genome mm10 as host reference were used. Mapping of human-specific reads against the homo sapiens reference hg38 was done using the STAR aligner34 version 2.6.1a. Quality of mapping was proven by QualiMap35 version 2.2.1 and quantification of transcripts was performed by eXpress36 version 1.5.1.The quality of reads was proven by the tool FastQC37 version 4.0.3 using packages provided by Bioconductor 3.738. Package DESeq239 was used to normalize gene level raw read count data by division with size factors or normalization factors, calculate a variance stabilizing transformation and transform the normalized read count data. The transformed data are on the log2 scale for large counts. Gene names are utilized in accordance with the approved annotations of the HUGO Gene Nomenclature Committee (HGNC)40. Further analyses were based on the resulting expression values of 19096 HGNC-annotated genes.The expression data analyses were performed in R41 (function heatmap.2) and RColorBrewer42 were used. The dendrogram next to the (magnified) heatmaps was based on the clustering  of all 19096 genes.For visualizing the expression data as heatmap and performing a hierarchical clustering of the PDX models, the packages gplotsTP63 as marker for squamous cell carcinoma histology, the expression value cut-off 12 was set for the given data sets. PDX models with TP63 expression values ≥ 12 were classified as squamous cell carcinoma. The obtained classification was compared to the histopathological classification. Sensitivity, specificity, and corresponding 95% confidence intervals (CI) were calculated using package epiR43.For the investigation of gene CXCL12, CXCR4, EGF, EGFR, HGF, KDR, MET, PTGS2, and VEGFA within all HGNC-annotated genes, a single-sample gene set variation analysis (ssGSVA) combined with a permutation-based test was performed utilizing RNASeq expression data of the 38 PDX models. Considering all 19096 HGNC-annotated genes (N), S ∈ N is the set of nine selected genes summarized above. In I = 100,000 iterations, datasets T were created for which each T consists of |S| genes sampled from N. ssGSVA (implemented in R package “GSVA”)44 was performed for S and T based on the expression data of N resulting in  enrichment scores for each set in the corresponding PDX model. The p-value was calculated by dividing the number of occurrences E > F, where E represents the enrichment score of S and F the enrichment score of T, and the total number of iterations I.Investigation for statistically significant over-representation of the selected gene set including The high sequencing depth of the RNASeq we performed (80–100 Million reads), allowed a sensitive mutational analysis of the transcriptome including a detection of fusion events. The variant calling and annotation of mapped reads was performed using the GATK tools version 4.0.2.1 and the Ensemble Variant Effect Predictor (VEP). For the analysis of gene fusion events the STAR-fusion tool version 1.4.0 was used.45. For correlation studies only sequence variations, which passed the Illumina calling quality criteria and which occur with a frequency > 10% were considered.Total DNA from xenograft tissue samples was isolated using a commercially available column-based purification kit from QIAGEN (DNeasy Blood and Tissue Kit) according to manufacturer’s instructions. Purified DNA samples were analyzed with the Illumina TruSeq® Amplicon Cancer Panel (TSACP) which targets 212 amplicons from 48 tumor-related genes in a multiplexed reaction. All necessary reagents are supplied in the TSACP kit (cat. FC-130-1008) and the amplicon library preparation was performed according to the manufacturers protocol. Finally, next-generation sequencing was carried out on an Illumina MiSeq® Desktop Sequencer. Illumina MiSeq Reporter including Somatic Variant Caller 3.1.10.0 and Human GRCh37 hg19 (Feb 2009) as reference genome was used for sequence variant calling. The annotation and interpretation of detected variants was performed using the public IGV genome browser Mus musculus, Rj:NMRI-Foxn1nu/nu female nude recipient mice47. Once subcutaneous tumors became palpable, tumor size was measured twice weekly with a digital caliper. Individual tumor volumes were calculated by the formula V = (length × width2)/2 and related to the values at the first day of treatment . In addition, therapeutic efficacy was assessed by applying Response Evaluation Criteria In Solid Tumors (RECIST)21. According to these criteria, a reduction in tumor volume of at least 30% was defined as partial response (PR), an increase in tumor volume of at least 20% was defined as progressive disease (PD). Stable disease (SD) was defined as neither sufficient reduction to qualify for PR nor sufficient increase to qualify for PD. The Objective Response Rate (ORR) includes all tumors with Complete Response (CR) and PR and the Clinical Benefit Rate includes all tumors with CR, PR and SD expressed in percentage. The body weight of mice was determined on a regular basis and the change in body weight was taken as variable for tolerability. After 25–40 days, the mean tumor volume reached the indicated starting volume (150–200 mm3). Mice were randomly assigned to control and treatment groups (1–4 mice per group) and treatment was started on the day of randomization.For the establishment and characterization of PDX mouse models and lung PDX models, in particular, fresh tumor samples were cut into pieces of 3 × 3 mm and were subcutaneously transplanted into 6–8 weeks old The mice were treated using the following drug dosages and treatment schedules: cabozantinib 15 mg/kg, afatinib 15 mg/kg, etoricoxib 10 mg/kg orally and plerixafor 5 mg/kg intraperitoneally for 5 days on treatment and 2 days off treatment for four cycles in total  and a rabbit monoclonal anti-PD-L1 antibody . For staining of pan-cytokeratin, alpha-smooth muscle actin (α-SMA), and CD31, antigen retrieval was performed in a steamer for 20 min in citrate buffer pH 6.0. Blocking was carried out in PBS supplemented with 4% BSA for 1 h at room temperature (RT). Incubation with primary antibodies: mouse anti-human pan-Cytokeratin , rat anti-mouse CD31  and mouse anti-mouse α–SMA  was conducted 45 min at RT. Detection of primary antibodies (pan-Cytokeratin and α-SMA) was performed by using EnVision-mouse HRP  for 45 min at RT. Detection of anti-CD31 antibody was performed using a goat-anti-rat-HRP antibody . Visualization was done by using the AEC Substrate Kit  for 10–20 min. After final washing in PBS, sections were stained in Mayer’s Hemalaun  for 2 min. After washing in water for 10 min, cover slips were fixed with Kaiser’s glycerin gelatine . Specificity of all stainings has been ensured before by application of a respective isotype control revealing no or only weak staining. All evaluations were performed twice in a blinded manner.The number of independent biological replicates of PDX-tumor bearing mice used in every experiment is specified in the corresponding figure legend. Information on bioinformatics statistics and data processing are described in the specific methods section.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


V10:9183, 2020) established a novel non-invasive neurovascular coupling (NVC) assessment in newborns using dynamic wavelet transform coherence (WTC) analysis irrespective of different aEEG algorithms. As an extended study, the current paper examines whether the variability in processed EEG and amplitude-EEG (aEEG) outputs would impact the determination of NVC in newborns with encephalopathy. Concurrent processed EEG tracings and regional near infrared spectroscopy (NIRS)-based cerebral tissue oxygen saturation (SctO2) readings during a period of twenty hours in their first day of life were selected and processed in this study. After bandpass-filtered in 2–15 Hz, rectified, and down-sampled at 0.21 Hz, the processed EEG tracings along with NIRS-SctO2 (0.21 Hz) were used to perform WTC analysis, followed by comparison of WTC-metrics between SctO2-processed EEG coherence and SctO2-aEEG coherence using Bland–Altman statistics. Our results demonstrated high and significant correlation  between NVC assessments by SctO2-processed EEG and SctO2-aEEG coherence, confirming that band-passed, rectified, and down-sampled processed EEG, or aEEG, can be paired with NIRS-SctO2 to assess NVC in newborns with encephalopathy. Findings indicate the feasibility of a simpler approach to NVC in neonates by using directly processed EEG, instead of aEEG.There is a critical need for development of real time physiological biomarkers for birth asphyxia that constitutes a major global public health burden. Our recent study (Scientific Reports,  In 2017, we first established a new neurovascular wavelet bundle methodology which enables an unprecedented real time analysis of neurovascular coupling non-invasively at the bedside5. In a subsequent study in 2020, we have demonstrated the robustness of neurovascular coupling (NVC) based on wavelet transform coherence (WTC) analysis between various aEEG signal and SctO2 in newborns with encephalopathy irrespective of processing algorithms variability in aEEGs6.Asphyxia impairs fetal cerebral blood flow and is manifested postnatally by neonatal encephalopathy (NE) using the clinical Sarnat stages. A real clinical challenge has been the difficulty to clinically discern the encephalopathy severity within the short therapeutic window to guide decision making regarding the initiation of hypothermia. There is a critical need for development of real time physiological biomarkers for birth asphyxia that constitutes a major global public health burden. Brain function monitoring using amplitude integrated-EEG (aEEG) or EEG is recommended as a standard of care practice in neonatal hypoxic-ischemic encephalopathy (HIE)6. This approach may potentially expand or enable a conventional EEG system to simultaneously display continuous EEG (cEEG) and NVC by using processed EEG in real time. The impact of different processing methods of EEG with hybrid devices on the novel WTC-based NVC analysis is not known.Different EEG and aEEG devices are being used at the bedside in clinical practice and increasingly used in the neonatal intensive care unit (NICU) for infants with hypoxic-ischemic encephalopathy (HIE), which introduce possible heterogeneity of practice, complicate the analysis of results, and affect patient inclusions in future trials. The current paper demonstrates a simpler and more direct approach to measure NVC in neonates by using processed EEG, instead of amplitude-EEG (aEEG). The procedure to obtain processed EEG included three simple steps: (1) bandpass-filtering in 2–15 Hz, (2) rectification, and (3) down-sampling at 0.21 Hz to match with the sampling rate of SctO2Specifically, we now examine the rigor and consistency of the novel NVC methodology using processed EEG tracings directly from EEG hybrid or from AEEG machines to measure NVC assessment obtained from infants with asphyxia monitored in the first day of life, aiming to compare WTC-metrics between SctO2-processed EEG coherence and SctO2-aEEG coherence using Bland–Altman statistics, and evaluate agreement between two the NVC assessment tools.7 together with a three-step processed EEG (gray trace), both derived from the original EEG signal  between the NVC metrics determined by the two methods.Figure Key finding in the current study is that WTC-based NVC assessment can be performed by quantifying the NVC coherence using either directly processed EEG or aEEG machines ensuring the rigor of this analysis with variable devices in newborns with encephalopathy. This approach eliminates several unspecific data processing pipelines when using commercial devices and accommodates the large variability in practice as some centers use EEG, while others use amplitude EEG in the clinical protocols of monitoring HIE.9. Different aEEG and EEG devices are currently being used at the bedside in clinical practice and increasingly being used in NICU in the encephalopathic newborns. Such devices include new digital EEG systems that can display simultaneous cEEG and processed EEG in real time. Currently those hybrid EEG/processed EEGs as in this investigation are best able to predict good and poor outcomes after hypoxic ischemic injury by providing optimal detection of seizures per the Standard American Clinical Neurophysiology Society recomendations11.Multimodal neuro-monitoring  to assess brain function and cerebral oxygenation delivery (NIRS) promises to provide estimates of neurovascular functions in new neuroprotective trials to target newborns with encephalopathy at higher risk who need added interventions. There is a long history of EEG as a tool for predicting outcomes after hypoxia ischemia and other neurologic conditions6. aEEG has traditionally been applied to surveil the brain development and screen cerebral pathologies by evaluating the background pattern and sleep-awake cycles in neonatal encephalopathy12. The digital calculation of aEEG was first described by Maynard et al.13. Many studies have been thereafter published using normal and abnormal amplitude margin ranges for preterm and term infants15. These output amplitude tracings are displayed in a semilogarithmic  and time-compressed (6 cm/h) format13, following some general procedure of aEEG data processing which allows clinicians to get an overall idea of the predominant background pattern and changes at the bedside.We have previously reported the rigor and robustness of the WTC analysis to quantify NVC enabling an unprecedented real time analysis of NVC non-invasively at the bedside using SctO2 and aEEG16. Caution is needed as differences in preprocessing can affect both the upper and the lower border of the tracing18. In contrast, the current study shown in Fig. The multitude of differences among aEEG algorithms have been discussed in our previous study; careful selection of the algorithm settings was recommended at the bedside, as these differences could potentially impact the clinical decision makings by the cliniciansTechnically, it is noted that amplitudes of raw EEG signal varied in a large range between ~ 10 and 60 μV within hours as shown in Fig. The results given in this study have two aspects. First, processed EEG can be used to quantify the WTC-based NVC in newborns since the results are highly and significantly correlated with those given by aEEG. Second, there exists a constant bias/offset in the NVC assessment by processed EEG with respect to that by aEEG. This offset can be possibly attributed to fewer processing steps in processed EEG, which resulted in a broader amplitude range of the resultant EEG output signal. On the other hand, aEEG after performing numerous processing steps resulted in a comparatively much narrower or smaller amplitude range as visualized in Fig. In conclusion, the key findings in this paper confirmed statistical consistency of WTC analysis to quantify NVC of neonates with HIE using either processed EEG or aEEG tracings. The advantage of using processed EEG in WTC analysis is the complexity reduction in post data processing when using a regular EEG device that can potentially reduce technical burden while keeping the scientific rigor for future translational applications.6) who had a minimum of twenty hours of simultaneous EEG and NIRS-SctO2 monitoring as a standard of care protocol. EEG signals were recorded at a sampling rate of 256 Hz from C3, C4, P3, P4, O1, O2, Cz, and Fz, which were placed on the infant’s scalp according to the 10–20 international system. Obtained electrical signals were then amplified and filtered within a frequency band of 0.1–100 Hz. An INVOS spatially resolved NIRS oximeter, consisting of a neonatal probe with a light emitting diode and two distant sensors, were placed on the infant’s forehead to record SctO2 at a speed of 0.2 Hz. Both EEG and NIRS-SctO2 were interfaced with a multi-device synchronization platform  for simultaneous recording of two modalities and then saved for off-line analysis using MATLAB . The time-series information from the cross cerebral electrode pair C3-C4 is used to perform dynamic wavelet coherence analysis with NIRS-SctO2 in all the neonates6. The study was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center and informed consent was obtained from parents of each newborn before enrollment.In this study, we selected a convenience sample of eight newborns with HIE  from Washington university, which is an open-source, MATLAB-compatible, clinically validated toolbox to quantify aEEGth order Butterworth filter. Second, the filtered signal was passed through a full wave rectification. Third, the peak-to-peak amplitudes of the 256-Hz rectified signal were measured and sampled at 4.78-s intervals between a common start and end point using cubic spline interpolation algorithm19 to match the dynamics of SctO2 signal (having a sampling rate of 0.21 Hz), which was needed to perform WTC analysis between them. The cubic spline interpolation algorithm allows resampling of the digitized EEG data with minimal distortion to the signal since the interpolation function does not reconstruct artificial spikes and rather assumes the function to be smooth20. The processing steps for the output traces derived using two methods are briefly summarized in Fig. The 256-Hz raw-EEG information from C3-C4 channel pair was downloaded from the multi-device synchronization platform . Three steps were performed for processed EEG. First, a 2–15 Hz bandpass filter was applied on the raw EEG signal using a 421 to perform WTC analysis between the spontaneous oscillations of SctO2 versus processed EEG and of SctO2 versus aEEG signals. WTC is a time–frequency domain analysis, which characterizes the squared cross-wavelet coherence, R2, against noise background, between two time series at multiple time scales and over time of two pre-specified variables. The two sets of variables selected for the evaluation of WTC-based NVC in this study are SctO2 → aEEG and SctO2 → processed EEG. Details of this method were previously published and can be found in refs. 5 and 6. The thresholding or cut-off criteria in coherence for “significant neurovascular coupling” were provided by the WTC analysis method21 that selects time-scale  regions having 95% confidence level after performing Monte Carlo simulations. Next, in the time-scale domain, NVC was assessed for each neonate by wavelet metric estimation of total pixel number of significant coherence within all selected regions for both aEEG and processed EEG. Then, the difference between the two types of total pixel numbers per neonate was calculated, followed by determination of the mean and standard deviation (SD) of the difference in NVC across the entire cohort. Statistical analysis to compare agreement between using aEEG and processed EEG tracings in WTC analysis was performed using a Bland Altman curve to detect statistically significant differences in the estimation of their total pixel counts.We used a MATLAB-based software packageAll methods were carried out in accordance with relevant guidelines and regulations of the institutional review board at the University of Texas Southwestern Medical Center.


FMR1) gene results in a “full mutation,” clinically Fragile X Syndrome (FXS), whereas 55 – 200 repeats result in a “premutation.” FMR1 premutation carriers (PMC) are at an increased risk for a range of psychiatric, neurocognitive, and physical conditions. Few studies have examined the variable expression of neuropsychiatric features in female PMCs, and whether heterogeneous presentation among female PMCs may reflect differential presentation of features in unique subgroups. In the current pilot study, we examined 41 female PMCs (ages 17–78 years) and 15 age-, sex-, and IQ-matched typically developing controls (TDC) across a battery of self-report, eye tracking, expressive language, neurocognitive, and resting state EEG measures to determine the feasibility of identifying discrete clusters. Secondly, we sought to identify the key features that distinguished these clusters of female PMCs. We found a three cluster solution using k-means clustering. Cluster 1 represented a psychiatric feature group (27% of our sample); cluster 2 represented a group with executive dysfunction and elevated high frequency neural oscillatory activity (32%); and cluster 3 represented a relatively unaffected group (41%). Our findings indicate the feasibility of using a data-driven approach to identify naturally occurring clusters in female PMCs using a multi-method assessment battery. CGG repeat count and its association with neuropsychiatric features differ across clusters. Together, our findings provide important insight into potential diverging pathophysiological mechanisms and risk factors for each female PMC cluster, which may ultimately help provide novel and individualized targets for treatment options.Over 200 Cytosine-guanine-guanine (CGG) trinucleotide repeats in the 5′ untranslated region of the Fragile X mental retardation 1 ( Fragile X mental retardation 1 (FMR1) gene over 200 and the consequent lack of protein product, Fragile X protein , results in a “full mutation,” clinically Fragile X Syndrome (FXS), whereas 55–200 repeats results in what is called a “premutation” (FMR1 premutation carriers (PMCs) have reduced production of FXP (Cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the 5′ untranslated region of utation” . FMR1 prn of FXP  and are n of FXP . Recentln of FXP  has beenn of FXP  and Fragn of FXP  as well n of FXP .Premutation status is approximately twice as likely to occur in females than males, highlighting the critical importance in better understanding neuropsychiatric features in female PMCs . PreviouTwo recent studies have applied clustering techniques to help identify potential subgroups of female PMCs and co-occurrence of clinical features. For example, n = 56; n = 81; However, a major limitation of these previous studies was their lack of biologically-linked measures to define clusters . A bottoForty-one females with PMC status between 17 and 78 years and 15 healthy TDC participants age-, IQ-, and sex matched to PMC participants were included in the study . Only feFMR1 polymerase chain reaction (PCR) with quantification of allele-specific CGG repeat length via review of medical records or through new blood collection. Blood samples were obtained from PMC participants only. Testing for FMR1 gene CGG expansion and gene methylation was conducted at Rush University at the laboratory of Dr. Elizabeth Berry-Kravis. Six PMC participants did not complete blood draws as part of the current study. We did not include their CGG repeat count in our analysis as PCR results were not from the same laboratory.Premutation status was confirmed by The EEG data was collected with 128 lead channels referenced to Cz using EGI NetAmp400  with hydrocel nets. Consistent with our prior studies , 5-min oAll participants completed via the Beck Depression Inventory, Second Edition BDI-II;  to measuParticipants completed the computerized Kiddie Test of Attentional Performance (KiTAP). KiTAP examines executive functioning over multiple domains, including processing speed, cognitive flexibility, and behavioral response inhibition. Raw scores were used for statistical analysis. Though KiTAP was originally designed and normalized on children, it has been validated for use in adults with full mutation FXS , and rawEye tracking data were collected using a Tobii  T120 infrared binocular eye tracker with sampling at a rate of 120 Hz. Participants completed one emotional face paradigm and one social interest paradigm. Data collection and analysis methods are described in detail elsewhere . BrieflyThe Expressive Language Sampling Task (ELS) was completed by all participants . This taFirst, we conducted separate univariate ANOVAs for each variable with the between subjects’ factor group to examine whether female PMCs differed from matched controls. Age and IQ were evaluated as covariates, but neither significantly altered results and thus were not included in final analyses.K-means cluster analysis identifies non-hierarchical, non-overlapping clusters with the lowest within-cluster variance and the highest between-cluster variance. In order to identify the optimal number of clusters, we used the dendrogram with all possible variables and identified a three cluster solution. The optimal k was verified with the elbow point of a least-squares fit line plotted across the cluster validity index. Because k-means clustering algorithm produces round clusters, it is critical to standardize data to improve good quality clusters and improve the accuracy of the clustering algorithm (z-score) based on means and standard deviations of our TDC sample prior to cluster analysis (n = 45), we performed a data reduction step that was both statistically- and clinically-guided. First, we examined all variables that demonstrated significant or marginal findings (p’s < 0.10 and/or η2’s > 0.05) from between-group ANOVAs (m = 8 variables selected). When more than one variable from a specific KiTAP subtest met this criteria, we removed the variables with the lower η2 (m = 6). Next, we added whole brain resting state relative power in frequency bands known to be relevant to FMR1 . We also included the proportion of time looking at happy eyes as most likely to differentiate between subgroups based on previous reports [(m = 12)]. Last, clinical expert opinion reviewed variables and identified additional variables of ASI total believed to be relevant to the female PMC phenotype . A total of 12 variables were used for k-means cluster analysis analysis . The CGGGiven the large age range of our analyses (17–78 years), we also completed all analyses on a smaller subset of adults 18–55 years. However, findings were nearly identical to those performed with the larger data set, thus we report only findings from the larger complete sample. Due to descriptive nature of these group comparisons and the relatively small sample size for this pilot study, corrections for multiple comparisons were not performed.F = 5.90, p = 0.019) and increased errors during a flexibility task  on the KiTAP than TDC. During ELS, female PMCs had significantly less lexical diversity than TDC , suggesting the size of female PMCs’ expressive vocabularies is smaller than those of TDC. However, groups were similar on other ELS variables (p’s > 0.137). No significant group differences were found across eye tracking variables or in resting relative power for any frequency band . Cluster 2 is primarily defined by impaired cognitive processing in the form of executive dysfunction as well as elevated high frequency resting gamma power . Cluster 3 represents a relatively unaffected group with minimal abnormalities across neuropsychiatric features . Power band comparisons by clusters are available in We found a three cluster solution with maximum distance between the different clusters confirmed by F-tests . ClusterF’s < 0.39, p’s > 0.67; X2’s < 1.7, p’s > 0.42). Talkativeness during ELS differed between clusters , with female PMCs in Cluster 2 being significantly more talkative than those in Cluster 3 . CGG repeat count also differed based on cluster membership . Female PMCs in Cluster 1 had higher CGG repeat counts compared to those in Cluster 2  and Cluster 3 . However, Cluster 2 and 3 did not differ on repeat counts (p = 0.97).To further examine our defined PMC clusters, we compared clusters on variables not included in the original cluster analysis. There was no difference in IQ or age between clusters . Specifically, Cluster 1 had more individuals in the High CGG repeat category (67%) compared to Cluster 2 (17%) and Cluster 3 (21%). PMCs considered to have mid-range CGG repeat count composed 50% of Cluster 2 and Cluster 3 each, but only 22% of Cluster 1.We also examined difference between clusters when using categorized repeat count based on previous papers and as recommended by the American College of Medical Genetics .Among female PMCs in Cluster 1, we found higher CGG repeat count was significantly related to fewer correct trials during the KiTAP Distractor subtask , but higher gamma1  and gamma2  power. Lower theta also was associated with greater proportion of time viewing social scenes during the dynamic eye tracking task , but higher gamma also was associated with greater proportion of looking at calm eyes during static face task  in Cluster 2.In female PMCs classified as Cluster 2, higher CGG repeat count was associated with lower theta power  but reduced gamma1  and reduced gamma2  were associated with greater proportion of time looking at eyes across emotions. Elevated theta also was related to greater number of errors during KiTAP Flexibility in Cluster 3 .Last, among female PMCs in Cluster 3, elevated theta ; Cluster 2 represented a group with executive dysfunction and elevated high frequency (gamma) neural oscillatory activity (32%); and Cluster 3 represented a relatively unaffected group (41%). These represent three clinically-meaningful clusters with good clinical face-validity  and consPsychiatric features and executive dysfunction have been well-documented in female PMCs; however, the majority of previous studies have examined these features independently  and to be reflected in elevated gamma band power  did not differ in EEG relative band power distribution. In contrast, Cluster 3 (the “unaffected” subgroup) demonstrated increased theta power and reduced alpha power in comparison with TDC. Female PMCs in Cluster 3 also showed the lower gamma power compared to TDC and other PMC clusters. These findings suggest a potential compensatory mechanism in female PMCs in Cluster 3 to dampen high frequency gamma activity, which could ultimately lead to their relatively spared neuropsychiatric phenotype. Future studies with larger female PMC samples are needed to replicate and further explore these electrophysiological profiles.Female PMCs in Cluster 1 were noteworthy for the presence of elevated psychiatric symptoms and high CGG repeat counts, although these specific features did not interrelate with each other. In contrast, we found higher CGG repeat count and greater elevations in high frequency power was associated with more severe distractibility in this subgroup. Though we generally observed psychiatric features and executive dysfunction to be the predominant phenotypes of distinct subgroups, vulnerability to specific executive function deficits in Cluster 1 may result from underlying biology. This notion also could help account, in part, for our finding of a relatively preserved electrophysiological profile in female PMCs in Cluster1 since executive dysfunction was largely absent in this subgroup. Instead, environmental factors, including the stress associated with being a (grand)parent to or sibling of a child with FXS, may be important sources of emotional distress in this subgroup.lowest percentage of females in the high CGG repeat category. It is possible that executive dysfunction may be present in Cluster 2 regardless of degree of electrophysiological “deficit” or CGG repeat count, such that other biological and/or environmental determinants determine the degree of executive dysfunction. A recent study documented CGG repeat count and stress independently contributed to executive dysfunction in female PMCs , percent methylation, non-AUG (RAN) translation, heritable factors not related to the FMR1 gene, and environmental factors including increased stress in family members of individuals with FXS all are thought to play a role in the heterogeneous clinical presentation of female PMCs . Yet, it is notable that psychiatric medication use was consistent across the three clusters. This suggests that some female PMCs may have been effectively treated and demonstrate reduced or subthreshold psychiatric symptoms, which could have affected cluster membership. Though four female PMCs at the time of testing were taking certain psychiatric medications may interfere with electrophysiological recordings, excluding these participant’s data from our analyses did not substantively change findings.Specific measures, including KiTAP and ELS, previously have not been used in PMC samples, thus additional caution was taken with data interpretation. PMC group performance was relatively similar to controls, consistent with numerous studies using other measures of executive function and language, suggesting its use in the current study is appropriate. Last, we also did not obtain blood from TDC, and thus it is possible that a control participant may have had CGG repeat expansion in the “gray zone” or the premutation range.Consistent with prior reports, our overall sample of female PMCs has relatively preserved psychiatric, cognitive, social, and electrophysiological functioning compared to matched controls. However, given the X-linked nature of the disorder and variability in CGG repeat length, Fragile X protein levels, degree of mRNA toxicity, and environmental factors, variable neuropsychiatric profiles are both well-documented and expected in female PMC even if group comparisons are null. Our pilot study using a wide array of quantitative assessments to subgroup clusters demonstrated that we were able to identify three clusters of female participants with PMC status, each demonstrating a unique profile of neuropsychiatric features—a psychiatric feature group, and an executive dysfunction and elevated high frequency resting state power group, and an unaffected group. Together, our findings implicate the feasibility of using multimodal data to identify subgroups of female PMCs that are clinically-meaningful and face-valid, with the promise of providing important insight into potential diverging pathophysiological mechanisms and risk factors for each cluster. Future studies with larger samples are warranted to replicate cluster findings and expand on molecular correlates in order to improve our understanding of illness mechanisms that in the longer term may provide novel and individualized targets for treatment options.NIMH Data Archive.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Cincinnati Children’s Hospital Medical Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.LS analyzed and interpreted data and wrote full draft of the manuscript. KD and ES assisted with data interpretation and manuscript. RL, LE, and EP analyzed and interpreted resting EEG findings. JS and CE participated in the conceptualization of the study and edited the manuscript. All authors contributed to the article 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Significance: Morphological collagen signatures are important for tissue function, particularly in the tumor microenvironment. A single algorithmic framework with quantitative, multiscale morphological collagen feature extraction may further the use of collagen signatures in understanding fundamental tumor progression.Aim: A modification of the 2D wavelet transform modulus maxima (WTMM) anisotropy method was applied to both digitally simulated collagen fibers and second-harmonic-generation imaged collagen fibers of mouse skin to calculate a multiscale anisotropy factor to detect collagen fiber organization.Approach: The modified 2D WTMM anisotropy method was initially validated on synthetic calibration images to establish the robustness and sensitivity of the multiscale fiber organization tool. Upon validation, the algorithm was applied to collagen fiber organization in normal wild-type skin, melanoma stimulated skin, and integrin Results: Normal wild-type skin collagen fibers have an increased anisotropy factor at all sizes scales. Interestingly, the multiscale anisotropy differences highlight important dissimilarities between collagen fiber organization in normal wild-type skin, melanoma stimulated, and integrin Conclusions: This objective computational collagen fiber organization algorithm is sensitive to collagen fiber organization across multiple scales for effective exploration of collagen morphological alterations associated with melanoma and the lack of  All collagen molecules are triple helical molecules characterized by a common tripeptide motif (Gly-X-Y) where the aminoacid glycine (Gly) resides in every third position. Collagen molecules are stabilized via hydrogen binding and are further stabilized into both fibril and fiber structures via covalent bounds.,––,,,,,,––The role of collagen in the ECM is complex and multifaceted. Collagen is both a mechanical scaffold and an acellular regulator of cellular processes. The mechanical properties of collagen are tissue specific; however, desmoplasia, or increased collagen content and altered collagen alignment in tumor microenvironments, has been shown to be tumorigenic. There have been several publications highlighting the importance of SHG microscopy mapping both collagen features and collagen stiffness to tumor initiation, promotion, and metastasis in several epithelial cancers.,,––,,,––,,,––SHG microscopy and computational image analysis are widely used to characterize tissue and disease-specific tissue remodeling. Unbiased, robust quantitative image analysis routines are essential tools. Currently, the quantitative analysis of collagen fiber features is broadly categorized into two types: (1) machine-learning/AI-based features for tissue classification and (2) physical collagen features. Machine-learning/AI-based feature extraction can be extremely powerful and has clearly identified relevant features for accurate classification in both 2D and 3D;In this paper, an adaptation of the 2D WTMM anisotropy is described and validated through a rigorous calibration on thousands of synthetic images of simulated fibers from a wide range of random alignments. After validation, the method is applied to characterize the multiscale morphological features of collagen fibers acquired using SHG microscopy from normal wild-type skin, skin stimulated with a melanoma tumor, and skin from integrin 22.1B16F10 melanoma cells were obtained from ATCC  and cultured in DMEM medium with 10% FBS, 1% Pen-strep, and 1% sodium pyruvate.2.2To generate integrin α10 knockout mice , acting much like a “mathematical microscope,” is used to quantify image intensity fluctuations. Use of the continuous WT allows us to investigate a continuous range of size scales. The WT of an image is the gradient vector (strength and direction of largest intensity variation) of the image smoothed by dilated versions of a Gaussian (smoothing) filter. Specifically, consider the wavelets,–,At a given scale An image having an anisotropic signature means that the intensity variation in the image will differ according to the direction considered. These images can be easily characterized from the directional information provided, at all size scales ctor see . A flat 2.6To obtain quantitative information from the angle pdfs Therefore, a theoretically isotropic surface will have a pdf of 2.7,Batches of synthetic images were created using a Matlab script previously described in Ref. 2.8To test the robustness of the method and its capacity to properly evaluate the strength of the anisotropy in noisy conditions, increasing levels of white noise were added to a subset of synthetic images. In 2.9,FT-based approaches have been used by several research groups to characterize morphological properties of collagen fibers.33.1Groups of 100 synthetic images each containing 50 simulated fibers with diameters of 2, 4, 6, and Computed , did not3.2To further investigate the sensitivity of the 2D WTMM anisotropy method to discriminating between synthetic images of simulated fibers with different ranges of directional angles, 19 groups of different ranges of angles were analyzed, as described in Sec. 3.3As shown in 3.4Cohorts of images from the mouse skin SHG images listed in 3.4.1At an μm scale , the aniμm scale , the dif3.4.2At all scales considered, the normal skin collagen fibers are more aligned . Clearly, it would be impossible to observe the drastically different behaviors at different size scales that are presented in this study. For example, if a single-scale analysis had been performed on these data at an μm scale , then itWe hypothesize that the superiority of the 2D WTMM anisotropy method is due to the wavelet transform’s ability to analyze both space and scale , compared with Fourier techniques being limited only to scale (frequencies). There are drawbacks that come with this additional analytical power associated with the WTMM method, namely algorithmic complexity and higher computation time, versus the simpler and faster FT.4.2in vivo. To this end, previous evidence suggests that collagen fiber spacing, density, and orientation play important roles in regulating tissue morphology, cell shape, and integrin-mediated signaling that collectively helps control cell behavior.,,–,,–,,Given the sensitivity of the 2D WTMM method to detecting specific differences in collagen fiber orientation over multiple size scales, this important technique allows for a more precise analysis of subtle changes in collagen organization that may play functional roles in governing cellular behavior –,,Integrins including in vivo may not only provide a better understanding of how integrins contribute to changes in collagen structure but also how biophysical changes in collagen organization in vivo regulate cellular behavior. Taken together, our studies, in conjunction with existing insight into how collagen orientation contributes to physiological and pathological processes, may ultimately not only help identify new therapeutic strategies for the treatment of diseases controlled by ECM remodeling but also help optimize the use of engineered collagen containing tissue scaffolds for multiple applications.The exact mechanism by which integrin