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Abstract Background The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. Methods We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. Results ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. Conclusion In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.

Abstract Background Mutations in the gene DISC1 are associated with increased risk for schizophrenia, bipolar disorder and major depression. The study of mutated DISC1 represents a well-known and comprehensively characterized approach to understand neuropsychiatric disease mechanisms. However, previous studies have mainly used animal models or rather heterogeneous populations of iPSC-derived neurons, generated by undirected differentiation, to study the effects of DISC1 disruption. Since major hypotheses to explain neurodevelopmental, psychiatric disorders rely on altered neuronal connectivity observed in patients, an ideal iPSC-based model requires accurate representation of the structure and complexity of neuronal circuitries. In this study, we made use of an isogenic cell line with a mutation in DISC1 to study neuronal synaptic phenotypes in a culture system comprising a defined ratio of NGN2 and ASCL1/DLX2 (AD2)-transduced neurons, enriched for glutamatergic and GABAergic neurons, respectively, to mimic properties of the cortical microcircuitry. Results In heterozygous DISC1 mutant neurons, we replicated the expected phenotypes including altered neural progenitor proliferation as well as neurite outgrowth, deregulated DISC1-associated signaling pathways, and reduced synaptic densities in cultures composed of glutamatergic neurons. Cultures comprising a defined ratio of NGN2 and AD2 neurons then revealed considerably increased GABAergic synapse densities, which have not been observed in any iPSC-derived model so far. Increased inhibitory synapse densities could be associated with an increased efficiency of GABAergic differentiation, which we observed in AD2-transduced cultures of mutant neurons. Additionally, we found increased neuronal activity in GABAergic neurons through calcium imaging while the activity pattern of glutamatergic neurons remained unchanged. Conclusions In conclusion, our results demonstrate phenotypic differences in a co-culture comprising a defined ratio of DISC1 mutant NGN2 and AD2 neurons, as compared to culture models comprising only one neuronal cell type. Altered synapse numbers and neuronal activity imply that DISC1 impacts the excitatory/inhibitory balance in NGN2/AD2 co-cultures, mainly through increased GABAergic input.

Abstract Fibroblast growth factor receptor (FGFR)−2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.

In the context of multimodal treatments for abdominal cancer, including procedures such as cytoreductive surgery and intraperitoneal chemotherapy, recurrence rates remain high, and long-term survival benefits are uncertain due to post-operative complications. Notably, treatment-limiting side effects often arise from an uncontrolled activation of the immune system, particularly peritoneally localized macrophages, leading to massive cytokine secretion and phenotype changes. Exploring alternatives, an increasing number of studies investigated the potential of plasma-activated liquids (PAL) for adjuvant peritoneal cancer treatment, aiming to mitigate side effects, preserve healthy tissue, and reduce cytotoxicity towards non-cancer cells. To assess the non-toxicity of PAL, we isolated primary human macrophages from the peritoneum and subjected them to PAL exposure. Employing an extensive methodological spectrum, including flow cytometry, Raman microspectroscopy, and DigiWest protein analysis, we observed a pronounced resistance of macrophages towards PAL. This resistance was characterized by an upregulation of proliferation and anti-oxidative pathways, countering PAL-derived oxidative stress-induced cell death. The observed cellular effects of PAL treatment on human tissue-resident peritoneal macrophages unveil a potential avenue for PAL-derived immunomodulatory effects within the human peritoneal cavity. Our findings contribute to understanding the intricate interplay between PAL and macrophages, shedding light on the promising prospects for PAL in the adjuvant treatment of peritoneal cancer.

Summary: Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-β inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research.

Abstract Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β catenin stabilization by inhibitory phosphorylation of GSK3β, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCα activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.

Serology is an important way to monitor SARS-CoV-2 infection in the population and support vaccine development. Here the authors develop a multiplex immunoassay including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses with high specificity and sensitivity.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL’s growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.

Abstract Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet β‐cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen‐1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic β‐cells via the αvβ3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen‐1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen‐1 in ischemic conditions.

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders.

Abstract Background Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers. Methods Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest. Results PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p

Non-invasive physical plasma (NIPP) is a novel therapeutic tool, currently being evaluated for the treatment of cancer and precancerous lesions in gynecology and other disciplines. Additionally, patients with cervical intraepithelial neoplasia (CIN) may benefit from NIPP treatment due to its non-invasive, side-effect-free, and tissue-sparing character. However, the molecular impact of in vivo NIPP treatment needs to be further investigated. For this purpose, usually only very small tissue biopsies are available after NIPP treatment. Here, we adapted DigiWest technology, a high-throughput bead-based Western blot, for the analysis of formalin-fixed paraffin-embedded (FFPE) cervical punch biopsies with a minimal sample amount. We investigated the molecular effects of NIPP treatment directly after (0 h) and 24 h after in vivo application. Results were compared to in vitro NIPP-treated human malignant cervical cells. NIPP effects were primarily based on an inhibitory impact on the cell cycle and cell growth factors. DigiWest technology was suitable for detailed protein profiling of small, primary FFPE biopsies.

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.

Floor-plate-derived extracellular signaling molecules, including canonical axon guidance cues of the Netrin family, control neuronal circuit organization. Despite the importance of the floor plate as an essential signaling center in the developing vertebrate central nervous system, no systematic approach to identify binding partners for floor-plate-expressed cell-surface and secreted proteins has been carried out. Here, we used a high-throughput assay to discover extracellular protein-protein interactions, which likely take place in the zebrafish floor-plate microenvironment. The assembled floor-plate network contains 47 interactions including the hitherto-not-reported interaction between Netrin-1 and Draxin. We further characterized this interaction, narrowed down the binding interface, and demonstrated that Draxin competes with Netrin receptors for binding to Netrin-1. Our results suggest that Draxin functions as an extracellular Netrin signaling modulator in vertebrates. A reciprocal gradient of Draxin might shape or sharpen the active Netrin gradient, thereby critically modulating its effect.

To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients.The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines.After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p