Biology subjects

Koch, A.

Publications and source records attributed to Koch, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Ovarian cancer-on-chip for patient-specific profiling of treatment responses to sequential chemotherapy and PD-L1 blockade

BackgroundOvarian cancer (OvCa) ranks as the most lethal gynecological malignancy in women worldwide. This complex disease, which can develop independently of a womans age, is characterized by late diagnosis, pronounced tumor heterogeneity, and an immunosuppressive tumor microenvironment (TME). Incremental diagnostic tools that could better inform clinicians on potential therapy resistance or subsets of patients that could benefit from new drug modalities represent a critical unmet need to improve patient care and potentially the identification of new biomarkers. ObjectiveThis study aimed to establish a reconfigurable patient-derived OvCa-on-chip platform for longitudinal functional profiling of tumor cell death, immune activation, and patient-specific responses to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. MethodsPatient-derived OvCa microtumors (PDM) were integrated with sequential integration of autologous tumor-infiltrating lymphocytes (TILs) into a perfusable microfluidic chip in the presence of different single and combination treatment regimens of chemotherapy and immune checkpoint inhibitors (ICIs). Treatment responses were assessed by longitudinal quantification of caspase-cleaved cytokeratin 18 (ccCK18) as marker of apoptotic epithelial tumor cell death, as well as cytokine/chemokine release in chip effluents, and multiplex flow cytometry-based characterization of autologous TIL subsets. ResultsThe perfusable OvCa-on-chip platform supported long-term culture of PDM while maintaining key structural and microenvironmental features of the primary tumor. Multidimensional analyses, including time-resolved assessment of tumor cell death, secretome profiling and correlative analysis of autologous TIL subsets revealed patient-specific tumor-immune response patterns and heterogenous sensitivity to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. Correlation analyses identified treatment-dependent associations between specific TIL phenotypes and functional tumor cell killing. PD-1-expressing CD4 TIL subsets correlated with enhanced tumor cell killing, whereas terminally exhausted CD8PD-1Tcf1- TILs negatively correlated with durvalumab responses. In contrast, tumor-reactive CD8CD39 TILs were associated with improved responses under sequential chemo-immunotherapy conditions. ConclusionCollectively, this OvCa-on-chip system represents a complex in vitro model (CIVM) that combines 3D tumor tissue with autologous immune cells in a microfluidic platform. Resembling a physiologically relevant human preclinical platform, it allows for the time-resolved functional assessment of patient-specific responsiveness to OvCa therapies, with direct implications for personalized treatment stratification.

cancer biology↗

bioRxiv2026

DLC1 loss drives multicellular streaming invasion by enforcing spatially coordinated Rho and β1 integrin signaling

Efficient cancer cell invasion requires coordinated control of actomyosin contractility, extracellular matrix (ECM) engagement and remodeling, yet how these processes are integrated in complex, three-dimensional (3D) environments remains unclear. Here, we identify the tumor suppressor and RhoGAP protein DLC1 as molecular brake on multicellular streaming invasion in collagen-rich ECM. Using CRISPRoff-engineered breast cancer spheroids, DLC1 reconstitution models and patient-derived organoids embedded in collagen gels, we show that DLC1 downregulation promotes an efficient multicellular streaming phenotype. This invasion program requires matrix metalloproteinase activity, {beta}1 integrin engagement and Rho-ROCK-dependent actomyosin contractility. Mechanistically, DLC1 downregulation stabilized the rear-polarization of RhoA activity and increased {beta}1 integrin abundance, plasma membrane localization and activation. Separation-of-function mutants revealed that DLC1 restrains invasion through a dual mechanism: its RhoGAP activity limited actomyosin-driven streaming, whereas its LD-like talin-binding motif controlled {beta}1 integrin enrichment at the plasma membrane. Together, our findings provide a molecular basis for the prominent role of DLC1 as a metastasis suppressor.

Cancer Biology↗

bioRxiv2026

Integrated risk assessment reveals low biological activity of RNA-based crop protection formulations across plant and non-target systems

RNA-based crop protection products are considered promising alternatives to conventional pesticides due to their high target specificity and selectivity profile. While current risk assessments mainly focus on sequence-dependent off-target effects, potential sequence-independent effects of RNA molecules and formulation components remain poorly understood. Here, we assessed the potential of naked and formulated RNA molecules to induce unintended biological responses in plants and non-target organisms using complementary plant stress and cellular toxicity assays. Single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), circular RNA (circRNA), and chitosan-based RNA formulations were evaluated for their ability to induce early stress responses in plants and adverse effects in insect cells. Cytosolic Ca{superscript 2} signaling and reactive oxygen species (ROS) production were monitored in barley and Arabidopsis, while impedance-based phenotypic profiling (ECIS) was used to assess cellular responses in Spodoptera frugiperda (Sf21) cells. Neither naked nor formulated RNA preparations triggered pronounced Ca{superscript 2} influxes or ROS bursts, indicating an absence of substantial activation of early plant defense signaling. Likewise, RNA formulations caused only minor effects on insect cell attachment and spreading as a measure for cell viability. Observable responses occurred primarily at elevated concentrations of RNA formulations and were formulation-dependent rather than RNA-dependent. Overall, the investigated RNA molecules exhibited low biological activity across the tested non-target systems. Our results highlight the importance of evaluating RNA active ingredients and delivery matrices separately and demonstrate the value of integrated screening approaches for the early hazard assessment of RNA-based crop protection products.

Plant Biology↗

bioRxiv2026

A luciferase-based assay for assessing IRES-mediated translation in Wheat Germ Extract

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1457b00org.highwire.dtl.DTLVardef@8e7405org.highwire.dtl.DTLVardef@6303eforg.highwire.dtl.DTLVardef@974d71_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

molecular biology↗

bioRxiv2026

Efficiency of RNAi based gene silencing in fungi - a review and meta-analysis

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

plant biology↗

bioRxiv2025

Generating Synthetic MR Perfusion Maps from DWI and FLAIR in Acute Ischemic Stroke using Deep Learning

BackgroundMagnetic resonance imaging (MRI) is critical for acute stroke triage, but time-consuming, and often requires contrast injection for perfusion imaging. This study aimed to synthesize T-map perfusion maps from routinely available, non-contrast DWI and FLAIR using deep generative models. We hypothesized that relevant perfusion information could be inferred from these modalities to streamline imaging and reduce reliance on dynamic susceptibility contrast perfusion. MethodsAcute MRI data from 355 patients with anterior circulation stroke, including dynamic susceptibility contrast perfusion, were retrospectively collected from two European centers (Heidelberg: 2010-2018; Bordeaux: 2021-2022). Six versions of a denoising diffusion probabilistic model (DDPM) and a GAN architecture were trained to generate synthetic T-max perfusion maps from DWI, FLAIR, and infarct core mask as inputs. Performance was assessed by comparing synthetic and ground truth T-max maps using image similarity metrics. Regions with T-max >6s were compared using Dice coefficients, and mismatch volume distributions were analyzed. An ablation study quantified the contribution of each input. ResultsThe best performance was achieved by a DDPM with a 2.5D architecture using DWI, FLAIR, infarct core mask, and a perfusion-weighted loss function. It produced synthetic perfusion T-max maps with high similarity to ground truth under 110 seconds. The model showed strong spatial overlap for T-max >6s regions in internal validation (average Dice = 0.82, SD = 0.08), and external validation average (Dice 0.59, SD = 0.13), respectively. Synthetic maps closely matched ground-truth mismatch distributions, capturing key perfusion patterns. The infarct core mask played a critical role in model performance, alongside DWI and FLAIR inputs. ConclusionsWe propose a non-invasive, scalable framework to generate synthetic T-max perfusion maps from non-contrast MRI. This approach could expand access to perfusion data in acute stroke, shorten imaging protocols, and accelerate treatment decisions by eliminating the need for contrast-enhanced acquisition. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/684079v2_ufig1.gif" ALT="Figure 1"> View larger version (94K): org.highwire.dtl.DTLVardef@164235forg.highwire.dtl.DTLVardef@14e5489org.highwire.dtl.DTLVardef@190214eorg.highwire.dtl.DTLVardef@17a9e3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

In planta gene expression via exogenous mRNAs has a wide range of potential biotechnological applications from genome editing to plant protection and to circumventing tissue culture. Yet, regarding mRNA delivery into intact plant cells, the purity, stability and translational efficiency of exogenous mRNAs appear as obstacles for effective exogenous mRNA application. Our study investigates the potential of exogenous mRNAs in mediating protein synthesis in plant cells. We applied unformulated, unmodified green fluorescent protein (GFP)-coding mRNA to monocot (Triticum aestivum, Hordeum vulgare, Zea mays) crops and dicot model plant Arabidopsis thaliana and observed GFP signals in all species, confirming the successful uptake and translation of mRNA into protein. This approach opens possibilities for broader, GM-free applications in plant science.

plant biology↗

bioRxiv2024

Double-stranded DNA reduces dsRNA degradation in the saliva and significantly enhanced RNAi-mediated gene silencing in Halyomorpha halys

The invasive pest Halyomorpha halys (Hemiptera: Pentatomidae) poses a significant threat to agriculture, necessitating effective control methods beyond chemical pesticides. Our research explores RNA interference (RNAi) as a targeted gene silencing approach for H. halys population management. However, the variable efficacy of RNAi across insect orders, particularly in hemipteran insects like H. halys, poses challenges. Ex vivo degradation assays revealed rapid degradation of double-stranded RNA (dsRNA) in H. halys (Hh) saliva and extracts of salivary glands across several growth stages, attributed to the high expression of the DNA/RNA non-specific nuclease (HhNSE). A key discovery from our research was that double-stranded DNA (dsDNA) can act as a protective agent, increasing the stability of dsRNA in saliva probably by competitive inhibition of HhNSE. Based on the well-established lethality of silencing the gene encoding the heavy chain of clathrin (HhCHC) in insects, this gene was chosen as a target to test the functionality of our dsDNA-based formulation for enhancing dsRNA-mediated gene silencing. Our in-vivo experiments showed increased HhCHC silencing after 72 hours of feeding initiation with a mixture of dsRNA-CHC and dsDNA, as opposed to dsRNA alone. This discovery indicates potential for enhancing the efficiency of orally delivered dsRNA through formulations based on dsDNA. Although the injection of dsRNA-CHC resulted in near-total mortality, the dsDNA formulation did not significantly enhance mortality rates when fed together with dsRNA-CHC. This emphasises the necessity for further investigation into additional factors beyond nuclease activity, such as the understanding of dsRNA uptake and release mechanisms within the gut epithelial cells of H. halys. Nevertheless, our study opens avenues for developing cost-effective formulations to enhance RNAi efficacy in H. halys and perhaps other insects where nucleases hinder dsRNA delivery, representing a promising solution for sustainable pest control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/617619v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1517e13org.highwire.dtl.DTLVardef@12c4977org.highwire.dtl.DTLVardef@8b243borg.highwire.dtl.DTLVardef@f79eca_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Acute exposure to caffeine improves navigation in an invasive ant

Invasive alien species are a major and growing problem, devastating ecosystems and costing billions of euros in damage and control efforts. Argentine ants, Linepithema humile, are particularly concerning, with control efforts often falling short likely due to a lack of sufficient bait consumption. Using neuroactives to manipulate ant navigation and learning could increase recruitment and consumption, ultimately leading to more efficient control strategies. Caffeine is naturally occurring, cheap, and has been found to cause motivational and cognitive improvements in bees. Here, we subject L. humile to a wide range of caffeine concentrations and a complex but ecologically relevant task: an open landscape foraging experiment. Without caffeine, we find no effect of consecutive foraging visits on the time the ants take to reach a reward, suggesting a failure to learn the rewards location. However, low (25ppm) to intermediate (250ppm) concentrations of caffeine lead to a decrease of up to 38% in the time taken to find the reward during each consecutive visit, implying that caffeine boosts learning. Interestingly, such improvements are lost at high (2000ppm) doses. In contrast, caffeine appears to have no impact on the ants homing behaviour, as the time required to reach the nest was similar across treatments. The effect of caffeine is thus not only dose-dependent, but also differentially targets neurologically distinct navigational mechanisms. Adding moderate levels of caffeine to baits could be a simple way to improve ants ability to learn its location, potentially leading to increased recruitment to, and consumption of, the toxicant.

animal behavior and cognition↗

bioRxiv2023

Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

The non-clinical assessment of CAR-T cells demands innovative models that are capable of predicting safety and efficacy in the clinical setting. Here, we present a novel solid tumor-on-chip model that allows CAR-T cell perfusion and integrates the vasculature and tumor lesions to recapitulate key events of CAR-T cell performance including extravasation, tumor infiltration and cytokine release. We assessed CAR-T cells targeting the ROR1 antigen against tumor aggregates that were derived from a breast cancer cell line and primary breast cancer organoids. The data show the temporal kinetic of ROR1 CAR-T cell migration and expansion, lytic activity and cytokine production over the course of 8 days, and reveal a correlation between anti-tumor efficacy and ROR1 antigen density on tumor cells. CAR-modified T cells extravasated faster, infiltrated tumor lesions stronger, persisted longer and in higher numbers than non-CAR modified T cells. Intriguingly, we detected cytokine release levels and kinetics typically observed in patients who developed cytokine release syndrome, and administered dasatinib as a pharmacologic OFF switch to control this inflammatory response. The data illustrate the ability of this tumor-on-chip platform to assess parameters associated withherapeutic outcome and the potential to aid in patient stratification and monitoring of CAR-T cell therapy.

bioengineering↗

bioRxiv2023

NEMO reshapes the protein aggregate interface and promotes aggrephagy by co-condensation with p62

NEMO is a ubiquitin-binding protein which regulates canonical NF-{kappa}B pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identified an NF-{kappa}B-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including -synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at -synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

cell biology↗

bioRxiv2022

Optimistic ants: Positive cognitive judgement bias but no emotional contagion in the ant Lasius niger

Understanding the emotional states of animals is key for informing their ethical treatment, but very little attention has been directed towards the emotional lives of invertebrates. As emotions influence information processing, one way to assess emotional states is to look for an individuals cognitive bias, i.e., their tendency to make optimistic or pessimistic judgements. Here we developed a free-running judgment bias task for the ant Lasius niger, and applied the judgement bias to assess ants reactions towards positive and negative stimuli. After an initial learning phase in which individuals were trained to associate two odour stimuli with positive or negative reinforcement, their reaction towards ambiguous stimuli, i.e., a mixture between both odours, was assessed. We also explored our study species capacity to socially transmit emotional states ( emotional contagion) by investigating whether social information could elicit emotional responses. We find L. niger to be optimistic, showing a baseline positive judgement bias, with 65-68% of ants preferring an ambiguous 1:1 mix of positive and negative cues over no cues. Providing an unexpected food reward prior to the judgement bias task increases positive judgement bias (c. 75% positive). There was a non- significant tendency towards a negative judgement bias after experiencing a mild electric shock (c. 75% negative). Neither positive nor negative social information (trail and alarm pheromones, respectively) affected the ants judgement biases, thus providing no indication for emotional contagion. The development of a powerful, simple, and ecologically relevant cognitive judgement task, deployable in the lab and in the field, opens the door to systematic comparative studies of the evolutionary and ecological causes of judgement bias.

animal behavior and cognition↗

bioRxiv2022

IFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

cancer biology↗

bioRxiv2022

Protein profiling of breast carcinomas reveals expression of immune-suppressive factors and signatures relevant to patient outcome

BackgroundIn cancerous tissue, a complex interplay of tumour cells with different cell types from the tumour microenvironment is causing modulations in signalling processes. By directly assessing expression of a multitude of proteins and protein variants, extensive information on signalling pathways, their activation status and the effect of the immunological landscape can be obtained providing viable information for treatment response. MethodsProtein extracted from archived breast cancer tissue from patients without adjuvant therapy was subjected to high-throughput Western blotting using the DigiWest technology. Expression of 150 proteins and protein variants covering cell cycle control, apoptosis, Jak/Stat, MAPK-, Pi3K/Akt-, Wnt-, and, autophagic signalling as well as general tumour markers was monitored in a cohort of 84 patient samples. The degree of immune cell infiltration was investigated and set against treatment outcome by integrating patient specific follow-up data. ResultsCharacterization of the tumour microenvironment by monitoring CD8, CD11c, CD16 and CD68 expression revealed a strong correlation of event-free patient survival with immune cell infiltration. Furthermore, the presence of tumour infiltrating lymphocytes was linked to a pronounced activation of the Jak/Stat signalling pathway and apoptotic processes. Elevated phosphorylation of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, pS112) in non-immune infiltrated tumour tissue suggests a novel immune evasion mechanism in breast cancer characterized by increased PPAR{gamma} activation. ConclusionMultiplexed immune cell marker assessment and protein profiling of tumour tissue provides functional signalling data facilitating breast cancer patient stratification.

cancer biology↗

bioRxiv2021

A platform of patient-derived microtumors identifies treatment response and therapeutic vulnerabilities of ovarian cancer

BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses. MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format. ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment.

cancer biology↗

bioRxiv2021

Patient-derived endometrial organoids from MRKH patients: Insight in disease causing pathways

The uterus is responsible for the nourishment and mechanical protection of the developing embryo and fetus and is an essential part in mammalian reproduction. The Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. Although heavily studied, the cause of the disease is still enigmatic. Current research in the field of MRKH mainly focusses on DNA-sequencing efforts and, so far, failed to decipher the nature and heterogeneity of the disease, thereby holding back scientific and clinical progress. Here, we developed long-term expandable organoid cultures from endometrium found in uterine rudiment horns of MRKH patients. Phenotypically, they share great similarity with healthy control organoids and are surprisingly fully hormone responsive. Transcriptome analyses, however, identified an array of dysregulated genes that point at potentially disease-causing pathways altered during the development of the female reproductive tract. We consider the endometrial organoid cultures to be a powerful research tool that promise to enable an array of studies into the pathogenic origins of MRKH syndrome and possible treatment opportunities to improve patient quality of life.

developmental biology↗

bioRxiv2021

Localization of TRP channels in healthy oral mucosa from human donors

The oral cavity is exposed to a remarkable range of noxious and innocuous conditions, including temperature fluctuations, mechanical forces, inflammation and environmental and endogenous chemicals. How such changes in the oral environment are sensed by oral cells and tissues is not completely understood. Transient receptor potential (TRP) ion channels are a diverse family of molecular receptors that are activated by chemicals, temperature changes, and tissue damage. In non-neuronal cells, TRP channels play roles in inflammation, as well as tissue development and maintenance. In somatosensory neurons, TRP channels mediate nociception, thermosensation and chemosensation. To assess whether TRP channels might be involved in environmental sensing in the human oral cavity, we investigated the distribution of TRP channels in human tongue and hard palate. Oral biopsies were collected from volunteers and underwent fluorescent immunohistochemistry followed by confocal imaging. We analyzed immunoreactivity of TRP channels in human oral epithelia including TRPV3, TRPV4, TRPV1, TRPM8, and TRPA1. TRPV3 and TRPV4 were expressed in epithelial cells with inverse expression patterns where they are likely to contribute to epithelial development and integrity. TRPA1 immunoreactivity was found in fibroblasts, subsets immune cells, and neurons, consistent with known roles of TRPA1 in sensory transduction, as well as in response to damage and inflammation. TRPM8 immunoreactivity was found in lamina propria cells and some neuronal subpopulations including some neurons within the end bulbs of Krause, consistent with a role in thermal sensation. TRPV1 immunoreactivity was identified in intraepithelial nerve fibers, in some end bulbs of Krause, and in epithelial cells, consistent with roles in nociception and thermosensation. Immunoreactivity of TRPM8 and TRPV1 in end bulbs of Krause suggest that these structures contain a variety of neuronal afferents, including those that mediate nociception, thermosensation and mechanotransduction. Collectively, these studies support the role of TRP channels in oral environmental surveillance and response.

neuroscience↗

bioRxiv2021

Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing. Significance StatementFusarium graminearum is one of the most devastating fungal pathogens in cereals, while understanding the mechanisms of fungal pathogenesis is a prerequisite for developing efficient and environmentally friendly crop protection strategies. We show exploratory data suggesting that fungal small RNAs play a critical role in Fusarium virulence by suppressing plant immunity.

microbiology↗