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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
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.
Zellmigration ist essentiell für die Invasion und Metastasierung maligner Tumore. Neben der Bewegung von Einzelzellen zeigen Tumore sowohl epithe¬lialen als auch mesenchymalen Ursprungs auch kollektive Migration und Invasion multizellulärer Zellverbände, die sich unter Beibehaltung von Zell-Zell-Adhäsionen koordiniert als Gruppe bewegen. Ziel der Arbeit war, primäre humane Melanomexplantate mittels organotypischer Kultur in 3D Kollagenmatrices einzusetzen, um mittels Zeit-raffermikroskopie und experimentellen Blockadestrategien die zellulären und molekularen Grundlagen kollektiver Migration darzustellen, insbesondere die Bedeutung von Zell-Matrix-Interaktionen und Integrinen. In 3D Explantatkulturen bildeten primäre Melanomexplantate reproduzierbar Invasionszonen und sich ablösende und kollektiv wandernde Zellcluster aus. Diese zeichneten sich durch eine ausgeprägte Polarität mit motiler Vorderfront mit zugartig reorientierten Kollagenfasern und nachgezogenem hinteren Teil der Gruppen aus, vergleichbar der Asymmetrie haptokinetisch migrierender Fibroblasten. β1 Integrine zeigten ein heterogenes Verteilungsmuster mit Fokalisierung an Zell-Matrix-Interaktionen vor allem an der Vorderfront und linearer Anordnung entlang der Zell-Zell-Grenzen. Adhäsionsblockierende anti- β1 Integrin-Antikörper bewirkten nahezu vollständige Hemmung der kollektiven Migration, mit Verlust der Zellgruppenpolarität und Migrationspersistenz. Nach Integrinblockade zerfielen Zellverbände infolge Loslösung von Einzelzellen, die sich mittels β1 Integrin-unabhängiger, amöboider Migration durch die Kollagenmatrix bewegten. Der Übergang von β1 Integrin-abhängiger, kollektiver Migration zu amöboider Einzelzellwanderung (kollektiv-amöboide Transition) ist ein Beispiel für die Plastizität von Tumorzellwanderung, die in Anpassung an das Milieu einen Wechsel der Migrationsstrategie erlaubt. Die Plastizität der Tumorzellmigration muss bei der Entwicklung therapeutischer Konzepte, die auf Hemmung von Tumorinvasion und -metastasierung abzielen, berücksichtigt werden.
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.
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.
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.
Articular cartilage is an exceptional connective tissue which by a network of fibrillar collagen and glycosaminoglycan (GAG) molecules allows both low- friction articulation and distribution of loads to the subchondral bone (Armiento et al., 2018, Ulrich-Vinther et al., 2003). Because of its very limited ability to self-repair, chondral defects following traumatic injury increase the risk for secondary osteoarthritis (OA) (Muthuri et al., 2011). Still, current OA treatments such as common nonsteroidal anti-inflammatory drugs (NSAIDs) and joint replacement primarily address end-stage symptoms (Tonge et al., 2014). As low-grade inflammation plays a pivotal role in the pathogenesis of OA (Robinson et al., 2016), there is a strong demand for novel therapeutic concepts, such as integrating application of anti-inflammatory agents into cartilage cell- based therapies in order to effectively treat OA affected joints in early disease stages. The polyphenolic phytoalexin resveratrol (RSV), found in the skin of red grapes, berries, and peanuts, has been shown to have effective anti-inflammatory properties (Shen et al., 2012). However, its long-term effects on 3D chondrocyte constructs cultured in an inflammatory environment with regard to tissue quality have remained unexplored so far. Therefore, in this study, pellets made from expanded porcine articular chondrocytes were cultured for 14 days with either the pro-inflammatory cytokine interleukin-1β (IL-1β) (1 - 10 ng/ml) or RSV (50 μM) alone, or a co-treatment with both agents. Constructs treated with chondrocyte medium only served as control. Treatment with IL-1β at 10 ng/ml resulted in a significantly smaller pellet size and reduced DNA content. However, RSV counteracted the IL-1β-induced decrease and significantly enhanced diameter and DNA content. Also, in terms of GAG deposition, treatment with IL-1β at 10 ng/ml resulted in a tremendous depletion of absolute GAG content and GAG/DNA. Again, RSV co-treatment counteracted the inflammatory stimulus and led to a partial recovery of GAG content. Histological analysis utilizing safranin-O staining confirmed these findings. Marked expression of the cartilage-degrading enzyme matrix metalloproteinase 13 (MMP13) was detected in IL-1β-treated pellets, but none upon RSV co- treatment. Moreover, co-treatment of IL-1β-challenged constructs with RSV significantly increased absolute collagen content. However, under non- inflammatory conditions, RSV induced gene expression and protein accumulation of collagen type X, a marker for undesirable hypertrophy. Taken together, in the present thesis, RSV was demonstrated to elicit marked beneficial effects on the extracellular matrix composition of 3D cartilaginous constructs in long-term inflammatory culture in vitro, but also induced hypertrophy under non-inflammatory conditions. Based on these findings, further experiments examining multiple concentrations of RSV under various inflammatory conditions appear desirable concerning potential therapeutic applicability in OA.
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.
Ziel der vorliegenden tierexperimentellen Studie war es, Unterschiede im Einheilverhalten der Werkstoffe Titan und VA-Stahl (316L) anhand der Matrixproteine Kollagen Typ I (C1), Kollagen Typ III (C3) und Fibronektin im implantatumgebenden Interface zu untersuchen und darzustellen. Hierzu wurden die Einheilkapseln der Implantate nach subkutaner, intramuskulärer und intraossärer Implantation nach den Bewertungskriterien Kapselqualität, Kapseldicke und Verteilungsmuster der Matrixproteine mittels konventioneller Mikroskopie und Konfokaler Laserscanning Mikroskopie (CLSM) analysiert. Nach subkutaner Implantation zeigten beide Werkstoffe in Übereinstimmung mit den Ergebnissen von SHANNON et al. (1997) vermehrt locker angeordnete, teils parallel orientierte Kollagenfasern mit erhöhtem Zellaufkommen an Fibroblasten und Makrophagen. Nach intramuskulärer Implantation jedoch fanden sich vorwiegend parallel angeordnete, teils dicht gepackte Kollagenfasern mit nur mäßig erhöhtem Zellaufkommen. Intramuskulär eingebrachte Implantate heilten in dünneren Kapseln ein, als subkutan eingebrachte Implantate. Es ergab sich keine Korrelation zu den ermittelten Kapselqualitäten. Dies erstaunt umso mehr, da unter der fortwährenden funktionellen Beanspruchung der intramuskulären Implantate im Bereich der Bauchmuskulatur gegenüber der statischeren Platzierung im subkutanen Rückenfett eine erhöhte Zell- und Matrixreaktion erwartet worden war. Im Lokalisationsvergleich zeigte sich intramuskulär für beide Werkstoffe ein erhöhtes Aufkommen an Fibronektin. Dies könnte auf die erhöhte Stoffwechselaktivität und funktionelle Belastung im Muskelgewebe zurückgeführt werden (ROSENGREN et al. 1994). Nach intraossärer Implantation konnten dünnere Kallusformationen für VA-Stahl gegenüber Titan in allen Proteinfluoreszenzen nachgewiesen werden. Die Qualität der Kallusformation und die histologische Kallusstruktur glichen sich mit zunehmender Implantationsdauer der regulären Knochenstruktur an. Die semiquantitativ beurteilte Verteilung der Matrixproteine mittels CLSM zeigte bei deutlichen Standardabweichungen für beide Werkstoffe erhöhte Fluoreszenz-Intensitäten nur in den implantatnahen Kapselanteilen. In den mittleren und den implantatfernen Kapselabschnitten waren für beide Werkstoffe inkonstant höhere Fluoreszenzwerte gegenüber den Vergleichskollektiven messbar. Der intraossäre Materialvergleich ergab implantatnahe und implantatferne Fluoreszenzmaxima für alle Matrixproteine, die mit zunehmender Implantationsdauer abfielen. Reproduzierbare, materialspezifische Unterschiede waren in Analogie zu BERGER-GORBET et al. (1996) nicht zu finden. In den mittleren Kallusabschnitten konnten reproduzierbare Fluoreszenzunterschiede nur bei Detektion von Kollagen Typ I (C1) in allen Zeitintervallen gesehen werden. Im Vergleich zur Literatur konnte die von VIROLAINEN et al. (1997) beschriebene biphasische Proteinanhäufung, wie auch ein wechselndes Proteinaufkommen (LINDHOLM et al. 1996) nach intraossärer Implantation nicht nachvollzogen werden. Ergänzende Beobachtungen der hier vorgestellten Studie verdeutlichen, dass die lokale, intraossäre Anreicherung von Matrixproteinen, unabhängig von Implantatinsertion oder gar Werkstoffeigenschaften, nach jeglicher Traumatisierung von Knochengewebe den knöchernen Reparationsprozess begleitet. Unter dem Aspekt der Restitutio ad Integrum von Knochenwunden können diese Beobachtungen auf das implantatnahe und das implantatferne Restitutionszentrum übertragen werden. Die Aktivität dieser Restitutionszentren hält bis zum Abschluss der knöchernen Remodellierung über 12 Wochen hinaus an. Dies deckt sich mit Aussagen von STEFLIK et al. (1998), wonach der periimplantäre Knochenumbau langfristig dynamisch bestehen bleibt. Um der zunehmenden Verbreitung nicht nur dentaler Implantate gerecht zu werden, muss auch zukünftig ein besseres Verständnis der Komunikationswege zwischen Implantaten und Biosystemen gewonnen werden. Dies bedeutet für die Herstellung und Weiterentwicklung von Implantaten, dass nicht nur die Werkstoff- und Oberflächenauswahl wichtig ist, sondern auch die funktionell erforderliche Oberflächenstrukturierung auf die gewünschte Wechselwirkung mit Bestandteilen der EZM und den Zellen angepasst sein sollte (THULL 2005). Die CLSM kann hierbei aufgrund der Möglichkeit der 3-dimensionalen in-situ-Darstellung des Implantatinterface biologisch-strukturelle und molekularbiologisch-immunologische Fragestellungen beantworten.
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.