TY - JOUR A1 - Nanda, Indrajit A1 - Schröder, Sarah K. A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Weiskirchen, Ralf T1 - Rat hepatic stellate cell line CFSC-2G: genetic markers and short tandem repeat profile useful for cell line authentication JF - Cells N2 - Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - stress fibers KW - spectral karyotyping KW - rhodamine–phalloidin stain KW - next-generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288067 SN - 2073-4409 VL - 11 IS - 18 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Friedman, Scott L. A1 - Meurer, Steffen K. A1 - Schröder, Sarah K. A1 - Weiskirchen, Ralf T1 - Genetic characterization of rat hepatic stellate cell line HSC-T6 for in vitro cell line authentication JF - Cells N2 - Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - in vitro model KW - SKY analysis KW - phalloidin stain KW - next generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275178 SN - 2073-4409 VL - 11 IS - 11 ER - TY - JOUR A1 - Hauptstein, Julia A1 - Forster, Leonard A1 - Nadernezhad, Ali A1 - Horder, Hannes A1 - Stahlhut, Philipp A1 - Groll, Jürgen A1 - Blunk, Torsten A1 - Teßmar, Jörg T1 - Bioink Platform Utilizing Dual-Stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells JF - Macromolecular Bioscience N2 - 3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol–ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks. KW - hyaluronic acid KW - biofabrication KW - chondrogenic differentiation KW - dual-stage crosslinking KW - extracellular matrix Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257556 VL - 22 IS - 2 ER - TY - JOUR A1 - Ryma, Matthias A1 - Tylek, Tina A1 - Liebscher, Julia A1 - Blum, Carina A1 - Fernandez, Robin A1 - Böhm, Christoph A1 - Kastenmüller, Wolfgang A1 - Gasteiger, Georg A1 - Groll, Jürgen T1 - Translation of collagen ultrastructure to biomaterial fabrication for material-independent but highly efficient topographic immunomodulation JF - Advanced materials N2 - Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of “melt electrofibrillation” is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment. KW - biofabrication KW - extracellular matrix KW - immunomodulation KW - macrophages KW - melt electrofibrillation KW - melt electrowriting Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256381 VL - 33 IS - 33 ER - TY - JOUR A1 - Horder, Hannes A1 - Guaza Lasheras, Mar A1 - Grummel, Nadine A1 - Nadernezhad, Ali A1 - Herbig, Johannes A1 - Ergün, Süleyman A1 - Teßmar, Jörg A1 - Groll, Jürgen A1 - Fabry, Ben A1 - Bauer-Kreisel, Petra A1 - Blunk, Torsten T1 - Bioprinting and differentiation of adipose-derived stromal cell spheroids for a 3D breast cancer-adipose tissue model JF - Cells N2 - Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell–cell and cell–matrix interplay within the tumor-stroma microenvironment KW - adipose-derived stromal cells KW - adipose tissue KW - bioprinting KW - breast cancer model KW - extracellular matrix KW - hyaluronic acid KW - spheroids Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236496 VL - 10 IS - 4 ER - TY - JOUR A1 - Frischholz, Sebastian A1 - Berberich, Oliver A1 - Böck, Thomas A1 - Meffert, Rainer H. A1 - Blunk, Torsten T1 - Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs JF - Journal of Tissue Engineering and Regenerative Medicine N2 - When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques. KW - articular chondrocytes KW - cartilage KW - cell‐based therapy KW - extracellular matrix KW - IL‐1β KW - inflammation KW - osteoarthritis KW - resveratrol Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215471 VL - 14 IS - 7 SP - 897 EP - 908 ER - TY - JOUR A1 - Fecher, David A1 - Hofmann, Elisabeth A1 - Buck, Andreas A1 - Bundschuh, Ralph A1 - Nietzer, Sarah A1 - Dandekar, Gudrun A1 - Walles, Thorsten A1 - Walles, Heike A1 - Lückerath, Katharina A1 - Steinke, Maria T1 - Human Organotypic Lung Tumor Models: Suitable For Preclinical \(^{18}\)F-FDG PET-Imaging JF - PLoS ONE N2 - Development of predictable in vitro tumor models is a challenging task due to the enormous complexity of tumors in vivo. The closer the resemblance of these models to human tumor characteristics, the more suitable they are for drug-development and –testing. In the present study, we generated a complex 3D lung tumor test system based on acellular rat lungs. A decellularization protocol was established preserving the architecture, important ECM components and the basement membrane of the lung. Human lung tumor cells cultured on the scaffold formed cluster and exhibited an up-regulation of the carcinoma-associated marker mucin1 as well as a reduced proliferation rate compared to respective 2D culture. Additionally, employing functional imaging with 2-deoxy-2-[\(^{18}\)F]fluoro-D-glucose positron emission tomography (FDG-PET) these tumor cell cluster could be detected and tracked over time. This approach allowed monitoring of a targeted tyrosine kinase inhibitor treatment in the in vitro lung tumor model non-destructively. Surprisingly, FDG-PET assessment of single tumor cell cluster on the same scaffold exhibited differences in their response to therapy, indicating heterogeneity in the lung tumor model. In conclusion, our complex lung tumor test system features important characteristics of tumors and its microenvironment and allows monitoring of tumor growth and -metabolism in combination with functional imaging. In longitudinal studies, new therapeutic approaches and their long-term effects can be evaluated to adapt treatment regimes in future. KW - lung and intrathoracic tumors KW - trachea KW - adenocarcinoma of the lung KW - cancer treatment KW - secondary lung tumors KW - pulmonary imaging KW - extracellular matrix KW - collagens Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179678 VL - 11 IS - 8 ER - TY - JOUR A1 - Endres, Marcel A1 - Kneitz, Susanne A1 - Orth, Martin F. A1 - Perera, Ruwan K. A1 - Zernecke, Alma A1 - Butt, Elke T1 - Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1) JF - Oncotarget N2 - The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines. By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines. In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown. Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression. The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies. KW - LASP1 KW - c-Fos KW - extracellular matrix KW - AP-1 KW - matrix metalloproteinases KW - breast cancer Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176920 VL - 7 IS - 39 ER - TY - JOUR A1 - Henke, Erik A1 - Nandigama, Rajender A1 - Ergün, Süleyman T1 - Extracellular matrix in the tumor microenvironment and its impact on cancer therapy JF - Frontiers in Molecular Biosciences N2 - Solid tumors are complex organ-like structures that consist not only of tumor cells but also of vasculature, extracellular matrix (ECM), stromal, and immune cells. Often, this tumor microenvironment (TME) comprises the larger part of the overall tumor mass. Like the other components of the TME, the ECM in solid tumors differs significantly from that in normal organs. Intratumoral signaling, transport mechanisms, metabolisms, oxygenation, and immunogenicity are strongly affected if not controlled by the ECM. Exerting this regulatory control, the ECM does not only influence malignancy and growth of the tumor but also its response toward therapy. Understanding the particularities of the ECM in solid tumor is necessary to develop approaches to interfere with its negative effect. In this review, we will also highlight the current understanding of the physical, cellular, and molecular mechanisms by which the pathological tumor ECM affects the efficiency of radio-, chemo-, and immunotherapy. Finally, we will discuss the various strategies to target and modify the tumor ECM and how they could be utilized to improve response to therapy. KW - extracellular matrix KW - cancer therapy KW - drug transport KW - immunotherapy KW - chemotherapy (CH) KW - radiotherapy KW - tumor microenvironment KW - ECM Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-199341 SN - 2296-889X VL - 6 IS - 160 ER - TY - JOUR A1 - Han, Hong A1 - Kampik, Daniel A1 - Grehn, Franz A1 - Schlunck, Günther T1 - TGF-beta 2-Induced Invadosomes in Human Trabecular Meshwork Cells JF - PLoS ONE N2 - Primary open-angle glaucoma (POAG) is a leading cause of blindness due to chronic degeneration of retinal ganglion cells and their optic nerve axons. It is associated with disturbed regulation of intraocular pressure, elevated intraocular levels of TGF-β2, aberrant extracellular matrix (ECM) deposition and increased outflow resistance in the trabecular meshwork (TM). The mechanisms underlying these changes are not fully understood. Cell-matrix interactions have a decisive role in TM maintenance and it has been suggested that TGF-β-induced inhibition of matrix metalloproteases may drive aberrant ECM deposition in POAG. Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Here, we report on the effects of TGF-β2 on invadosomes in human trabecular meshwork cells. Human TM (HTM) cells were derived from donor tissue and pretreated with vehicle or TGF-β2 (2 ng/ml) for 3d. Invadosomes were studied in ECM degradation assays, protein expression and MMP-2 activity were assessed by western blot and zymography and ECM protein transcription was detected by RT-qPCR. HTM cells spontaneously formed podosomes and invadopodia as detected by colocalization of Grb2 or Nck1 to sites of gelatinolysis. Pretreatment with TGF-β2 enhanced invadosomal proteolysis and zymographic MMP-2 activity as well as MMP-2, TIMP-2 and PAI-1 levels in HTM cell culture supernatants. Rho-kinase inhibition by H1152 blocked the effects of TGF-β2. Concomitant transcription of fibronectin and collagens-1, -4 and -6 was increased by TGF-β2 and fibrillar fibronectin deposits were observed in areas of invadosomal ECM remodelling. In contrast to a current hypothesis, our data indicate that TGF-β2 induces an active ECM remodelling process in TM cells, characterized by concurrent increases in localized ECM digestion and ECM expression, rather than a mere buildup of material due to a lack of degradation. Invadosomal cell adhesion and signaling may thus have a role in POAG pathophysiology. KW - intraocular pressure KW - cell cultures KW - cell staining KW - proteolysis KW - glaucoma KW - extracellular matrix KW - actins KW - trabecular meshwork Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130074 VL - 8 IS - 8 ER -