Integrative network analysis of miRNA-mRNA expression profiles during epileptogenesis in rats reveals therapeutic targets after emergence of first spontaneous seizure.

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.

ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2-BECN1 interaction.

Growing evidences indicate that dysfunction of autophagy contributes to the disease pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative disorders. The GGGGCC·GGCCCC repeat RNA expansion in chromosome 9 open reading frame 72 (C9orf72) is the most genetic cause of both ALS and FTD. According to the previous studies, GGGGCC·GGCCCC repeat undergoes the unconventional repeat-associated non-ATG translation, which produces dipeptide repeat (DPR) proteins. Although there is a growing understanding that C9orf72 DPRs have a strong ability to harm neurons and induce C9orf72-linked ALS/FTD, whether these DPRs can affect autophagy remains unclear. In the present study, we find that poly-GR and poly-PR, two arginine-containing DPRs which display the most cytotoxic properties according to the previous studies, strongly inhibit starvation-induced autophagy. Moreover, our data indicate that arginine-rich DPRs enhance the interaction between BCL2 and BECN1/Beclin 1 by inhibiting BCL2 phosphorylation, therefore they can impair autophagic clearance of neurodegenerative disease-associated protein aggregates under starvation condition in cells. Importantly, our study not only highlights the role of C9orf72 DPR in autophagy dysfunction, but also provides novel insight that pharmacological intervention of autophagy using SW063058, a small molecule compound that can disrupt the interaction between BECN1 and BCL2, may reduce C9orf72 DPR-induced neurotoxicity.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Spatial transcriptomic analysis reveals local effects of intratumoral fusobacterial infection on DNA damage and immune signaling in rectal cancer.

Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.

Meta-analysis of SATB2 immunohistochemical expression in colorectal cancer versus primary ovarian mucinous neoplasms.

BACKGROUND: Reliably distinguishing primary ovarian mucinous neoplasms (POMNs) from metastatic colorectal cancers (CRCs) is both challenging to the histopathologist and of great clinical importance. Special AT-rich sequence binding protein-2 (SATB2) has emerged as a useful diagnostic immunohistochemical marker of colorectal cancer. This meta-analysis compares SATB2 expression in POMNs and CRC. METHODS: A systematic literature search for relevant studies was conducted. Meta-analysis of SATB2 positivity was undertaken using a random effects model. RESULTS: Seven studies including 711 CRCs and 528 POMNs were included. SATB2 positivity was seen in 81 % (95 % CI: 72-88 %) of CRCs and 4 % (95 % CI: 1-11 %) of POMNs. Variation was seen in immunohistochemical methods used for SATB2 detection and threshold for positivity. CONCLUSION: SATB2 staining remains high in CRC and low in POMNs, supporting its use in differentiating these two pathologies with vastly differing prognosis and treatment.

Identification of unique rectal cancer-specific subtypes.

BACKGROUND: Existing colorectal cancer subtyping methods were generated without much consideration of potential differences in expression profiles between colon and rectal tissues. Moreover, locally advanced rectal cancers at resection often have received neoadjuvant chemoradiotherapy which likely has a significant impact on gene expression. METHODS: We collected mRNA expression profiles for rectal and colon cancer samples (n = 2121). We observed that (i) Consensus Molecular Subtyping (CMS) had a different prognosis in treatment-naïve rectal vs. colon cancers, and (ii) that neoadjuvant chemoradiotherapy exposure produced a strong shift in CMS subtypes in rectal cancers. We therefore clustered 182 untreated rectal cancers to find rectal cancer-specific subtypes (RSSs). RESULTS: We identified three robust subtypes. We observed that RSS1 had better, and RSS2 had worse disease-free survival. RSS1 showed high expression of MYC target genes and low activity of angiogenesis genes. RSS2 exhibited low regulatory T cell abundance, strong EMT and angiogenesis signalling, and high activation of TGF-ß, NF-κB, and TNF-α signalling. RSS3 was characterised by the deactivation of EGFR, MAPK and WNT pathways. CONCLUSIONS: We conclude that RSS subtyping allows for more accurate prognosis predictions in rectal cancers than CMS subtyping and provides new insight into targetable disease pathways within these subtypes.

Unlocking the secrets: exploring the influence of the aryl hydrocarbon receptor and microbiome on cancer development.

Gut microbiota regulates various aspects of human physiology by producing metabolites, metabolizing enzymes, and toxins. Many studies have linked microbiota with human health and altered microbiome configurations with the occurrence of several diseases, including cancer. Accumulating evidence suggests that the microbiome can influence the initiation and progression of several cancers. Moreover, some microbiotas of the gut and oral cavity have been reported to infect tumors, initiate metastasis, and promote the spread of cancer to distant organs, thereby influencing the clinical outcome of cancer patients. The gut microbiome has recently been reported to interact with environmental factors such as diet and exposure to environmental toxicants. Exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) induces a shift in the gut microbiome metabolic pathways, favoring a proinflammatory microenvironment. In addition, other studies have also correlated cancer incidence with exposure to PAHs. PAHs are known to induce organ carcinogenesis through activating a ligand-activated transcriptional factor termed the aryl hydrocarbon receptor (AhR), which metabolizes PAHs to highly reactive carcinogenic intermediates. However, the crosstalk between AhR and the microbiome in mediating carcinogenesis is poorly reviewed. This review aims to discuss the role of exposure to environmental pollutants and activation of AhR on microbiome-associated cancer progression and explore the underlying molecular mechanisms involved in cancer development.

Spatial Effects of Infiltrating T cells on Neighbouring Cancer Cells and Prognosis in Stage III CRC patients.

Colorectal cancer (CRC) is one of the most frequently occurring cancers, but prognostic biomarkers identifying patients at risk of recurrence are still lacking. In this study, we aimed to investigate in more detail the spatial relationship between intratumoural T cells, cancer cells, and cancer cell hallmarks, as prognostic biomarkers in stage III colorectal cancer patients. We conducted multiplexed imaging of 56 protein markers at single cell resolution on resected fixed tissue from stage III CRC patients who received adjuvant 5-fluorouracil-based chemotherapy. Images underwent segmentation for tumour, stroma and immune cells, and cancer cell 'state' protein marker expression was quantified at a cellular level. We developed a Python package for estimation of spatial proximity, nearest neighbour analysis focusing on cancer cell - T cell interactions at single-cell level. In our discovery cohort (MSK), we processed 462 core samples (total number of cells: 1,669,228) from 221 adjuvant 5FU-treated stage III patients. The validation cohort (HV) consisted of 272 samples (total number of cells: 853,398) from 98 stage III CRC patients. While there were trends for an association between percentage of cytotoxic T cells (across the whole cancer core), it did not reach significance (Discovery cohort: p = 0.07, Validation cohort: p = 0.19). We next utilized our region-based nearest neighbourhood approach to determine the spatial relationships between cytotoxic T cells, helper T cells and cancer cell clusters. In the both cohorts, we found that lower distance between cytotoxic T cells, T helper cells and cancer cells was significantly associated with increased disease-free survival. An unsupervised trained model that clustered patients based on the median distance between immune cells and cancer cells, as well as protein expression profiles, successfully classified patients into low-risk and high-risk groups (Discovery cohort: p = 0.01, Validation cohort: p = 0.003).

Direct neuronal reprogramming of NDUFS4 patient cells identifies the unfolded protein response as a novel general reprogramming hurdle.

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.

Integrated analysis of transcriptomic and proteomic alterations in mouse models of ALS/FTD identify early metabolic adaptions with similarities to mitochondrial dysfunction disorders.

OBJECTIVE: Sporadic and familial amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that results in loss of motor neurons and, in some patients, associates with frontotemporal dementia (FTD). Apart from the accumulation of proteinaceous deposits, emerging literature indicates that aberrant mitochondrial bioenergetics may contribute to the onset and progression of ALS/FTD. Here we sought to investigate the pathophysiological signatures of mitochondrial dysfunction associated with ALS/FTD. METHODS: By means of label-free mass spectrometry (MS) and mRNA sequencing (mRNA-seq), we report pre-symptomatic changes in the cortices of TDP-43 and FUS mutant mouse models. Using tissues from transgenic mouse models of mitochondrial diseases as a reference, we performed comparative analyses and extracted unique and common mitochondrial signatures that revealed neuroprotective compensatory mechanisms in response to early damage. RESULTS: In this regard, upregulation of both Acyl-CoA Synthetase Long-Chain Family Member 3 (ACSL3) and mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) were the most representative change in pre-symptomatic ALS/FTD tissues, suggesting that fatty acid beta-oxidation and mitochondrial protein translation are mechanisms of adaptation in response to ALS/FTD pathology. CONCLUSIONS: Together, our unbiased integrative analyses unveil novel molecular components that may influence mitochondrial homeostasis in the earliest phase of ALS.

Performance of TMRM and Mitotrackers in mitochondrial morphofunctional analysis of primary human skin fibroblasts.

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes ("probes") like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMH2Xros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ "flickering" was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized.

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells.

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise.

Ketone body ß-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics.

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, ß-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca2+-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

Complexation of histone deacetylase inhibitor belinostat to Cu(II) prevents premature metabolic inactivation in vitro and demonstrates potent anti-cancer activity in vitro and ex vivo in colon cancer.

PURPOSE: The histone deacetylase inhibitor (HDACi), belinostat, has had limited therapeutic impact in solid tumors, such as colon cancer, due to its poor metabolic stability. Here we evaluated a novel belinostat prodrug, copper-bis-belinostat (Cubisbel), in vitro and ex vivo, designed to overcome the pharmacokinetic challenges of belinostat. METHODS: The in vitro metabolism of each HDACi was evaluated in human liver microsomes (HLMs) using mass spectrometry. Next, the effect of belinostat and Cubisbel on cell growth, HDAC activity, apoptosis and cell cycle was assessed in three colon cancer cell lines. Gene expression alterations induced by both HDACis were determined using RNA-Seq, followed by in silico analysis to identify master regulators (MRs) of differentially expressed genes (DEGs). The effect of both HDACis on the viability of colon cancer patient-derived tumor organoids (PDTOs) was also examined. RESULTS: Belinostat and Cubisbel significantly reduced colon cancer cell growth mediated through HDAC inhibition and apoptosis induction. Interestingly, the in vitro half-life of Cubisbel was significantly longer than belinostat. Belinostat and its Cu derivative commonly dysregulated numerous signalling and metabolic pathways while genes downregulated by Cubisbel were potentially controlled by VEGFA, ERBB2 and DUSP2 MRs. Treatment of colon cancer PDTOs with the HDACis resulted in a significant reduction in cell viability and downregulation of stem cell and proliferation markers. CONCLUSIONS: Complexation of belinostat to Cu(II) does not alter the HDAC activity of belinostat, but instead significantly enhances its metabolic stability in vitro and targets anti-cancer pathways by perturbing key MRs in colon cancer. Complexation of HDACis to a metal ion might improve the efficacy of clinically used HDACis in patients with colon cancer.

Chlorpromazine affects autophagy in association with altered Rag GTPase-mTORC1-TFEB signaling.

Autophagy is a critical protein and organelle quality control system, which regulates cellular homeostasis and survival. Growing pieces of evidence suggest that autophagic dysfunction is strongly associated with many human diseases, including neurological diseases and cancer. Among various autophagic regulators, microphthalmia (MiT)/TFE transcription factors, including transcription factor EB (TFEB), have been shown to act as the master regulators of autophagosome and lysosome biogenesis in both physiological and pathological conditions. According to the previous studies, chlorpromazine (CPZ), an FDA-approved antipsychotic drug, affects autophagy in diverse cell lines, but the underlying mechanism remains elusive. In our present study, we find that CPZ treatment induces TFEB nuclear translocation through Rag GTPases, the upstream regulators of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Meanwhile, CPZ treatment also blocks autophagosome-lysosome fusion. Notably, we find a significant accumulation of immature autophagosome vesicles in CPZ-treated cells, which may impede cellular homeostasis due to the dysfunction of the autophagy-lysosome pathway. Interestingly and importantly, our data suggest that the expression of the active form of Rag GTPase heterodimers helps in reducing the accumulation of autophagosomes in CPZ-treated cells, further suggesting a major contribution of the Rag GTPase-mTORC1-TFEB signaling axis in CPZ-induced autophagic impairment.

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers.

BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

Corrigendum: Mucinous adenocarcinoma of the rectum: a whole genome sequencing study.

[This corrects the article DOI: 10.3389/fonc.2020.01682.].

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes.

Activation of the ATP-gated P2X7 receptor (P2X7R), implicated in numerous diseases of the brain, can trigger diverse responses such as the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. However, despite the known species-specific differences in its pharmacological properties, to date, most functional studies on P2X7R responses have been analyzed in cells from rodents or immortalised cell lines. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 ß-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining. P2X7R protein expression was also confirmed by Western blot. Importantly, stimulation with the potent non-selective P2X7R agonist 2',3'-O-(benzoyl-4-benzoyl)-adenosine 5'- triphosphate (BzATP) or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the central nervous system (CNS).

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.