04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
Browse
30 results
Search Results
Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Transcriptional CDK inhibitors CYC065 and THZ1 induce apoptosis in glioma stem cells derived from recurrent GBM(2021) Juric, Viktorija; Düssmann, Heiko; Lamfers, Martine L. M.; Prehn, Jochen H. M.; Rehm, Markus; Murphy, Brona M.Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.Item Open Access Pharmacokinetic engineering of OX40-blocking anticalin proteins using monomeric plasma half-life extension domains(2021) Siegemund, Martin; Oak, Prajakta; Hansbauer, Eva-Maria; Allersdorfer, Andrea; Utschick, Karoline; Winter, Alexandra; Grasmüller, Christina; Galler, Gunther; Mayer, Jan-Peter; Weiche, Benjamin; Prassler, Josef; Kontermann, Roland E.; Rothe, ChristineAnticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (∼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t1/2 values of ∼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to ∼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs.Item Open Access Collective cell migration on collagen-I networks: the impact of matrix viscoelasticity(2022) Pajic-Lijakovic, Ivana; Milivojevic, Milan; Clark, Andrew G.Collective cell migration on extracellular matrix (ECM) networks is a key biological process involved in development, tissue homeostasis and diseases such as metastatic cancer. During invasion of epithelial cancers, cell clusters migrate through the surrounding stroma, which is comprised primarily of networks of collagen-I fibers. There is growing evidence that the rheological and topological properties of collagen networks can impact cell behavior and cell migration dynamics. During migration, cells exert mechanical forces on their substrate, resulting in an active remodeling of ECM networks that depends not only on the forces produced, but also on the molecular mechanisms that dictate network rheology. One aspect of collagen network rheology whose role is emerging as a crucial parameter in dictating cell behavior is network viscoelasticity. Dynamic reorganization of ECM networks can induce local changes in network organization and mechanics, which can further feed back on cell migration dynamics and cell-cell rearrangement. A number of studies, including many recent publications, have investigated the mechanisms underlying structural changes to collagen networks in response to mechanical force as well as the role of collagen rheology and topology in regulating cell behavior. In this mini-review, we explore the cause-consequence relationship between collagen network viscoelasticity and cell rearrangements at various spatiotemporal scales. We focus on structural alterations of collagen-I networks during collective cell migration and discuss the main rheological parameters, and in particular the role of viscoelasticity, which can contribute to local matrix stiffening during cell movement and can elicit changes in cell dynamics.Item Open Access Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins(2023) Steinlein, Sophia; Essmann, Frank; Ghilardi, Amanda Franceschini; Horn, Heike; Schüler, Julia; Hausser, Angelika; Sun, Lijun; Ott, German; Kalla, ClaudiaMalignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.Item Open Access Mechanosensory feedback loops during chronic inflammation(2023) Saha, Sarbari; Müller, Dafne; Clark, Andrew G.Epithelial tissues are crucial to maintaining healthy organization and compartmentalization in various organs and act as a first line of defense against infection in barrier organs such as the skin, lungs and intestine. Disruption or injury to these barriers can lead to infiltration of resident or foreign microbes, initiating local inflammation. One often overlooked aspect of this response is local changes in tissue mechanics during inflammation. In this mini-review, we summarize known molecular mechanisms linking disruption of epithelial barrier function to mechanical changes in epithelial tissues. We consider direct mechanisms, such as changes in the secretion of extracellular matrix (ECM)-modulating enzymes by immune cells as well as indirect mechanisms including local activation of fibroblasts. We discuss how these mechanical changes can modulate local immune cell activity and inflammation and perturb epithelial homeostasis, further dysregulating epithelial barrier function. We propose that this two-way relationship between loss of barrier function and altered tissue mechanics can lead to a positive feedback loop that further perpetuates inflammation. We discuss this cycle in the context of several chronic inflammatory diseases, including inflammatory bowel disease (IBD), liver disease and cancer, and we present the modulation of tissue mechanics as a new framework for combating chronic inflammation.Item Open Access A stiff extracellular matrix favors the mechanical cell competition that leads to extrusion of bacterially-infected epithelial cells(2022) Aparicio-Yuste, Raúl; Muenkel, Marie; Clark, Andrew G.; Gómez-Benito, María J.; Bastounis, Effie E.Cell competition refers to the mechanism whereby less fit cells (“losers”) are sensed and eliminated by more fit neighboring cells (“winners”) and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected “loser” cells by uninfected “winner” cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.Item Open Access Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport(2023) Lungu, Cristiana; Meyer, Florian; Hörning, Marcel; Steudle, Jasmin; Braun, Anja; Noll, Bettina; Benz, David; Fränkle, Felix; Schmid, Simone; Eisler, Stephan A.; Olayioye, Monilola A.The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase‐activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy‐based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP‐RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.Item Open Access Low-level endothelial TRAIL-receptor expression obstructs the CNS-delivery of angiopep-2 functionalised TRAIL-receptor agonists for the treatment of glioblastoma(2021) Krishna Moorthy, Nivetha; Seifert, Oliver; Eisler, Stephan; Weirich, Sara; Kontermann, Roland E.; Rehm, Markus; Fullstone, GavinGlioblastoma (GBM) is the most malignant and aggressive form of glioma and is associated with a poor survival rate. Latest generation Tumour Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding to death receptors. However, the blood–brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). We therefore investigated if antibody-based fusion proteins that combine hexavalent TRAIL and angiopep-2 (ANG2) moieties can be developed, with ANG2 promoting receptor-mediated transcytosis (RMT) across the BBB. We demonstrate that these fusion proteins retain the potent apoptosis induction of hexavalent TRAIL-receptor agonists. Importantly, blood–brain barrier cells instead remained highly resistant to this fusion protein. Binding studies indicated that ANG2 is active in these constructs but that TRAIL-ANG2 fusion proteins bind preferentially to BBB endothelial cells via the TRAIL moiety. Consequently, transport studies indicated that TRAIL-ANG2 fusion proteins can, in principle, be shuttled across BBB endothelial cells, but that low TRAIL receptor expression on BBB endothelial cells interferes with efficient transport. Our work therefore demonstrates that TRAIL-ANG2 fusion proteins remain highly potent in inducing apoptosis, but that therapeutic avenues will require combinatorial strategies, such as TRAIL-R masking, to achieve effective CNS transport.Item Open Access Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol(2021) Hellwig, Christian T.; Delgado, M. Eugenia; Skoko, Josip; Dyck, Lydia; Hanna, Carol; Wentges, Alexa; Langlais, Claudia; Hagenlocher, Cathrin; Mack, Alexandra; Dinsdale, David; Cain, Kelvin; MacFarlane, Marion; Rehm, MarkusCancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.
- «
- 1 (current)
- 2
- 3
- »