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.

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.

A Core Outcome Set for inflammatory bowel diseases: development and recommendations for implementation in clinical practice through an international multi-stakeholder consensus process.

BACKGROUND AND AIMS: Standardising health outcome measurements supports delivery of care, enables data-driven learning systems, and secondary data use for research. As part of the Health Outcomes Observatory initiative and building on existing knowledge, a core outcome set (COS) for inflammatory bowel diseases (IBD) was defined through an international modified Delphi method. METHODS: Stakeholders rated 90 variables on a 9-point importance scale twice, allowing score modification based on feedback displayed per stakeholder group. Two consecutive consensus meetings were held to discuss results and formulate recommendations for measurement in clinical practice. Variables scoring 7 or higher by ≥80% of the participants, or based on consensus meeting agreement, were included in the final set. RESULTS: In total, 136 stakeholders (45 IBD patients (advocates), 74 healthcare professionals/researchers, 13 industry representatives and 4 regulators), from 20 different countries participated. The final set includes 18 case-mix variables, 3 biomarkers (haemoglobin to detect anaemia, C-reactive protein and faecal calprotectin to detect inflammation) for completeness and 28 outcomes (including 16 patient-reported outcomes (PROs) and 1 patient-reported experience). The PRO-2 and IBD-Control questionnaires were recommended to collect disease-specific PROs at every contact with an IBD practitioner, and the Subjective Health Experience model questionnaire, PROMIS Global Health and Self-Efficacy short form to collect generic PROs annually. CONCLUSIONS: A COS for IBD, including a recommendation for use in clinical practice, was defined. Implementation of this set will start in Vienna, Berlin, Barcelona, Leuven and Rotterdam, empowering patients to better manage their care. Additional centres will follow worldwide.

PD-1- CD45RA+ effector-memory CD8 T cells and CXCL10+ macrophages are associated with response to atezolizumab plus bevacizumab in advanced hepatocellular carcinoma.

The combination of atezolizumab plus bevacizumab (atezo/bev) has dramatically changed the treatment landscape of advanced HCC (aHCC), achieving durable responses in some patients. Using single-cell transcriptomics, we characterize the intra-tumoural and peripheral immune context of patients with aHCC treated with atezo/bev. Tumours from patients with durable responses are enriched for PDL1+ CXCL10+ macrophages and, based on cell-cell interaction analysis, express high levels of CXCL9/10/11 and are predicted to attract peripheral CXCR3+ CD8+ effector-memory T cells (CD8 TEM) into the tumour. Based on T cell receptor sharing and pseudotime trajectory analysis, we propose that CD8 TEM preferentially differentiate into clonally-expanded PD1- CD45RA+ effector-memory CD8+ T cells (CD8 TEMRA) with pronounced cytotoxicity. In contrast, in non-responders, CD8 TEM remain frozen in their effector-memory state. Finally, in responders, CD8 TEMRA display a high degree of T cell receptor sharing with blood, consistent with their patrolling activity. These findings may help understand the possible mechanisms underlying response to atezo/bev in aHCC.

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.

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.

The Dynamics of Nucleotide Variants in the Progression from Low-Intermediate Myeloma Precursor Conditions to Multiple Myeloma: Studying Serial Samples with a Targeted Sequencing Approach.

Multiple myeloma (MM), or Kahler's disease, is an incurable plasma cell (PC) cancer in the bone marrow (BM). This malignancy is preceded by one or more asymptomatic precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). The molecular mechanisms and exact cause of this progression are still not completely understood. In this study, the mutational profile underlying the progression from low-intermediate risk myeloma precursor conditions to MM was studied in serial BM smears. A custom capture-based sequencing platform was developed, including 81 myeloma-related genes. The clonal evolution of single nucleotide variants and short insertions and deletions was studied in serial BM smears from 21 progressed precursor patients with a median time of progression of six years. From the 21 patients, four patients had no variation in one of the 81 studied genes. Interestingly, in 16 of the 17 other patients, at least one variant present in MM was also detected in its precursor BM, even years before progression. Here, the variants were present in the pre-stage at a median of 62 months before progression to MM. Studying these paired BM samples contributes to the knowledge of the evolutionary genetic landscape and provides additional insight into the mutational behavior of mutant clones over time throughout progression.

Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases.

Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy specimen at a single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. This review provides an overview of the technical challenges that should be addressed when designing single-cell transcriptomics experiments that focus on glomerulopathies. The isolation of glomerular cells from core needle biopsy specimens for single-cell transcriptomics remains difficult and depends upon five major factors. First, core needle biopsies generate little tissue material, and several samples are required to identify glomerular cells. Second, both fresh and frozen tissue samples may yield glomerular cells, although every experimental pipeline has different (dis)advantages. Third, enrichment for glomerular cells in human tissue before single-cell analysis is challenging because no effective standardized pipelines are available. Fourth, the current warm cell-dissociation protocols may damage glomerular cells and induce transcriptional artifacts, which can be minimized by using cold dissociation techniques at the cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells; however, the efficacy of snRNA-seq on core needle biopsy specimens remains to be proven. The field of single-cell omics is rapidly evolving, and the integration of these techniques in multiomics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases.

A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer.

Immune-checkpoint blockade (ICB) combined with neoadjuvant chemotherapy improves pathological complete response in breast cancer. To understand why only a subset of tumors respond to ICB, patients with hormone receptor-positive or triple-negative breast cancer were treated with anti-PD1 before surgery. Paired pre- versus on-treatment biopsies from treatment-naive patients receiving anti-PD1 (n = 29) or patients receiving neoadjuvant chemotherapy before anti-PD1 (n = 11) were subjected to single-cell transcriptome, T cell receptor and proteome profiling. One-third of tumors contained PD1-expressing T cells, which clonally expanded upon anti-PD1 treatment, irrespective of tumor subtype. Expansion mainly involved CD8+ T cells with pronounced expression of cytotoxic-activity (PRF1, GZMB), immune-cell homing (CXCL13) and exhaustion markers (HAVCR2, LAG3), and CD4+ T cells characterized by expression of T-helper-1 (IFNG) and follicular-helper (BCL6, CXCR5) markers. In pre-treatment biopsies, the relative frequency of immunoregulatory dendritic cells (PD-L1+), specific macrophage phenotypes (CCR2+ or MMP9+) and cancer cells exhibiting major histocompatibility complex class I/II expression correlated positively with T cell expansion. Conversely, undifferentiated pre-effector/memory T cells (TCF7+, GZMK+) or inhibitory macrophages (CX3CR1+, C3+) were inversely correlated with T cell expansion. Collectively, our data identify various immunophenotypes and associated gene sets that are positively or negatively correlated with T cell expansion following anti-PD1 treatment. We shed light on the heterogeneity in treatment response to anti-PD1 in breast cancer.

Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities.

Multiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient's life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

Developing Organoids from Ovarian Cancer as Experimental and Preclinical Models.

Ovarian cancer (OC) represents the most dismal gynecological cancer. Pathobiology is poorly understood, mainly due to lack of appropriate study models. Organoids, defined as self-developing three-dimensional in vitro reconstructions of tissues, provide powerful tools to model human diseases. Here, we established organoid cultures from patient-derived OC, in particular from the most prevalent high-grade serous OC (HGSOC). Testing multiple culture medium components identified neuregulin-1 (NRG1) as key factor in maximizing OC organoid development and growth, although overall derivation efficiency remained moderate (36% for HGSOC patients, 44% for all patients together). Established organoid lines showed patient tumor-dependent morphology and disease characteristics, and recapitulated the parent tumor's marker expression and mutational landscape. Moreover, the organoids displayed tumor-specific sensitivity to clinical HGSOC chemotherapeutic drugs. Patient-derived OC organoids provide powerful tools for the study of the cancer's pathobiology (such as importance of the NRG1/ERBB pathway) as well as advanced preclinical tools for (personalized) drug screening and discovery.

Fungal Bezoars Mimicking an Enterovesica Fistula: A Unique Case Report.

Fungal colonization or infection of the urinary tract system is relatively common in patients with diabetes or a compromised immune system. However, fungal intravesical bezoars are extremely rare. We present a unique case with multiple, gas-holding fungals bezoars and emphysematous cystitis caused by Candida tropicalis.

Gene and Mirna Regulatory Networks During Different Stages of Crohn's Disease.

BACKGROUND AND AIMS: Early treatment of Crohn's disease [CD] is required in order to optimize patient outcomes. To this end, we need to gain a better understanding of the molecular changes at the onset of CD. METHODS: As a model for the earliest mucosal CD lesions, we study post-operative recurrent CD [Rutgeerts score ≥ i2b]. We are the first to analyse gene and microRNA [miRNA] expression profiles in ileal biopsies from these patients, and compare them with those of newly diagnosed [≤18 months] and late-stage [>10 years after diagnosis] CD patients. RESULTS: Except for one gene [WNT5A], there are no differential genes in CD patients without post-operative recurrence [i0], showing that previous disease did not influence gene expression in the neoterminal ileum, and that this model can be used to study early mucosal CD lesions. Gene expression and co-expression network dysregulation is more pronounced in newly diagnosed and late-stage CD than in post-operative recurrent CD, with most important modules associated with [a]granulocyte adhesion/diapedesis, and cholesterol biosynthesis. In contrast, we found a role for snoRNAs/miRNAs in recurrent CD, highlighting the potential importance of regulatory RNAs in early disease stages. Immunohistochemistry confirmed the expression of key dysregulated genes in damaged/regenerating epithelium and immune cells in recurrent CD. CONCLUSIONS: Aside from regulatory RNAs, there are no clear gene signatures separating post-operative recurrent, newly diagnosed, and late-stage CD. The relative contribution of dysregulated genes and networks differs, and suggests that surgery may reset the disease at the mucosal site, and therefore post-operative recurrent CD might be a good model a good model to study to study early mucosal CD lesions.

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Difference in Pathomechanism Between Crohn's Disease and Ulcerative Colitis Revealed by Colon Transcriptome.

BACKGROUND: We aim to identify the differences in colonic mucosal transcriptome between Crohn's disease (CD) and ulcerative colitis (UC) for a better understanding of the molecular pathology. METHODS: Differentially expressed genes (DEG) in the colonic mucosa of CD and UC were identified with a global gene expression microarray dataset generated from the colon biopsies of CD and UC patients and normal controls. The DEGs were then processed to identify altered pathways and modularized DEGs and pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis with an independent cohort of samples was performed to validate the microarray data. RESULTS: At the pathway level, virus infection and autoimmune pathways were upregulated in CD but not in UC when compared with controls. Some of the relevant DEGs (such as TAP1 and TAP2) were elevated in both CD and UC, with CD exhibiting more pronounced elevations. Gene expression levels in viral infection pathways were correlated with those of autoimmune pathways. In contrast, pattern recognition-mediated innate immune pathways (TLR4 and TLR2) were significantly elevated in UC but not in CD. Similar results were observed with an independent cohort by qRT-PCR. CONCLUSIONS: Our data support the hypothesis that viral infection induced autoimmunity may represent a pathomechanism for IBD, especially CD. However, pattern recognition-mediated innate immunity targeting microbiome may play a more important role in UC compared with CD. Our findings identified different intervention targets for CD and UC, which may lead to more effective treatments for IBD patients.

Selective Suprascapular and Axillary Nerve Block Versus Interscalene Plexus Block for Pain Control After Arthroscopic Shoulder Surgery: A Noninferiority Randomized Parallel-Controlled Clinical Trial.

BACKGROUND AND OBJECTIVES: This randomized trial aimed to assess if a combined suprascapular-axillary nerve block (SSB) is noninferior (margin = 1.3 on a 0- to 10-point scale) to interscalene block (ISB) in treating pain after arthroscopic shoulder surgery. Secondary end points included opioid consumption, dyspnea, discomfort associated with muscle weakness, and patient satisfaction. METHODS: One hundred patients undergoing arthroscopic shoulder surgery were randomized to receive ultrasound-guided ISB (n = 50) or SSB (n = 50). Pain intensity at rest, dyspnea, and discomfort were recorded upon arrival in the recovery room, discharge to the ward, and at 4, 8, and 24 hours after surgery. Piritramide consumption was recorded for the first 24 hours. Patient satisfaction was assessed on the second postoperative day. RESULTS: During the first 4 hours after surgery, the difference in mean pain score between SSB and ISB was higher than 2.5 (±0.8). The difference gradually decreased to 1.1 (±1.0) at 8 hours before resulting in noninferiority during the night and at 24 hours. Piritramide consumption was significantly higher in the SSB group in the first 8 hours. The incidence of dyspnea and discomfort was higher after ISB. Treatment satisfaction was similar in both groups. CONCLUSIONS: Suprascapular-axillary nerve block is inferior to ISB in terms of analgesia and opioid requirement in the immediate period after arthroscopic shoulder surgery but is associated with a lower incidence of dyspnea and discomfort. The difference in pain and opioid consumption gradually decreases as the blocks wear off in order to reach similar pain scores during the first postoperative night and at 24 hours. CLINICAL TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov, identifier NCT02415088.

Prognostic Biomarkers in the Progression From MGUS to Multiple Myeloma: A Systematic Review.

Multiple myeloma (MM), characterized by malignant plasma cells in the bone marrow, is consistently preceded by asymptomatic premalignant stage monoclonal gammopathy of undetermined significance (MGUS). These MGUS patients have an annual risk of 1% to progress to MM. Clinical, imaging, and genomic (genetic and epigenetic) factors were identified, whose presence increased the risk of progression from MGUS to MM. In this systematic review we summarize the currently identified clinical, imaging, and genomic biomarkers suggested to increase the progression risk or shown to be differentially expressed/present between both cohorts of patients. Despite the wide range of proposed markers, there are still no reliable biomarkers to individually predict which MGUS patient will progress to MM and which will not. Research on biomarkers in the progression from MGUS to MM will give more insight in the unknown pathogenesis of this hematological malignancy. This would improve research by elucidating new pathways and potential therapeutic targets as well as clinical management by closer follow-up and earlier treatment of high-risk MGUS patients.

Biopsy-derived Intestinal Epithelial Cell Cultures for Pathway-based Stratification of Patients With Inflammatory Bowel Disease.

BACKGROUND: Endoplasmic reticulum [ER] stress was shown to be pivotal in the pathogenesis of inflammatory bowel disease. Despite progress in inflammatory bowel disease [IBD] drug development, not more than one-third of patients achieve steroid-free remission and mucosal healing with current therapies. Furthermore, patient stratification tools for therapy selection are lacking. We aimed to identify and quantify epithelial ER stress in a patient-specific manner in an attempt towards personalised therapy. METHODS: A biopsy-derived intestinal epithelial cell culture system was developed and characterised. ER stress was induced by thapsigargin and quantified with a BiP enzyme-linked immunosorbent assay [ELISA] of cell lysates from 35 patients with known genotypes, who were grouped based on the number of IBD-associated ER stress and autophagy risk alleles. RESULTS: The epithelial character of the cells was confirmed by E-cadherin, ZO-1, and MUC2 staining and CK-18, CK-20, and LGR5 gene expression. Patients with three risk alleles had higher median epithelial BiP-induction [vs untreated] levels compared with patients with one or two risk alleles [p = 0.026 and 0.043, respectively]. When autophagy risk alleles were included and patients were stratified in genetic risk quartiles, patients in Q2, Q3, and Q4 had significantly higher ER stress [BiP] when compared with Q1 [p = 0.034, 0.040, and 0.034, respectively]. CONCLUSIONS: We developed and validated an ex vivo intestinal epithelial cell culture system and showed that patients with more ER stress and autophagy risk alleles have augmented epithelial ER stress responses. We thus presented a personalised approach whereby patient-specific defects can be identified, which in turn could help in selecting tailored therapies.