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WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals. Electronic supplementary material The online version of this article (10.1007/s10456-019-09688-8) contains supplementary material, which is available to authorized users.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

The topic of this work is the synthesis and properties of silanes with amino substituents and their corresponding silylium ions. In the first part of this thesis, the first aminosilylium ion was synthesized by hydride abstraction of (Me2N)3SiH. The dictionic ((Me2N)3Si+)2 could be fully characterized in solution and in the solid state. The compound can be described as the dimeric form of the silaguanidinium. Different factors that influence the dimerization process could be determined by suitable theoretical methods. In comparison to silylium ions with aliphatic substituents, the aminosilylium ion shows higher stability and compatibility with common organic solvents. Nevertheless, the dication is able to perform electrophilic, aromatic Friedel-Crafts silylation reactions with electron-rich, aromatic compounds or to initiate hydrodefluorination reactions. In the presence of acetonitrile, an insertion of the nitrile in one of the Si-N-bonds was achieved and the structure was characterized in the solid state. The synthesis of more sterically demanding, monomeric aminosilylium ions and the challenges around this are described in detail. In the second part of this thesis, the influence of aromatic triamido (NNN3−) and amidodiphenolato (ONO3−) substituents of the resulting silanes was investigated. The enforced geometrical strain resulted in a higher levels of Lewis acidity and thus dimerization of the silicon compounds. By installing substituents with different sterical and electronical properties around silicon, it was possible to influence the dimerization process on purpose. The species could be grouped in monomeric, structural-reversible and dimeric compounds. The high Lewis acidity resulting from the pre-organisation of the substituent was evident from the deviation from tetrahedral geometry and the planarization of the compounds in the solid state structures. Different factors which contribute to the dimerization process were determined by theoretical methods and their influence was explained in detail. For effective dimerization, the dispersion interactions of the substituents, low deformation energy at silicon, electrostatic attraction between silicon and nitrogen and a special σ-bond resonance phenomenon were identified as the main factors. The obtained results suggest unique stabilising effects and can be applied for more general conclusions about aggregation of amphiphilic species and Lewis acidity at silicon. The reactivity of the compounds towards different substrates was investigated with help of a model compound. In the third part, a silylium ylidene-like valence isomer will be synthesised in due course. With help of the redox-active substituent, a silylium ylidene compound was obtained via charge redistribution. The combination of the Lewis basic lone pair and the highly electrophilic silicon cation resulted in extraordinary reactivity. Although it was not possible to isolate the silylium ylidene compound, different adducts of the silylium ion with donor molecules could be obtained and its exceptionally high Lewis acidity could be proven by the Gutmann-Beckett method. By investigating secondary reaction products, the silylium ion showed a strong fluoride ion affinity in presence of SbF6− and a particular reactivity towards the weakly coordinating anion B(C6F5)4−.

3D-Zellkultur wird verwendet, um eine gewebeähnliche Umgebung, wie sie in-vivo zu finden ist, nachzuahmen. Ein weiterer Verwendungszweck ist die Untersuchung von molekularen und biochemischen Aspekten vieler physiologischer Funktionen unter in-vitro Bedingungen. Der „mammalian target of rapamycin (mTOR)-pathway” ist ein wichtiger Signalweg der Zellen, der essentielle Funktionen wie ribosomale Biosynthese, Zellteilung, Stoffwechsel und Zellgröße kontrolliert. Dieser wurde bisher ausführlich in Monolayerkulturen untersucht, jedoch nicht in Zellen in 3D Kultur. Da die Form und die Umgebung der Zellen ihr Verhalten und ihre Genexpression bestimmen können, wollten wir Unterschiede zwischen Fibroblasten aus 2D- und 3D-Kultur, in Bezug auf den mTOR-Signalweg, identifizieren. Weiters waren wir an ihrem Invasionspotenzial innerhalb der Kollagen-Gele interessiert. Nachdem wir die Kulturbedingungen für IMR-90 Fibroblasten in Kollagen-Gelen und ein Isolations-Protokoll etablierten, wurde der DNA-Gehalt und die Zellgröße von Zellen aus 2D- und 3D-Kultur mit Hilfe von FACS-Analyse bestimmt. „Spheroid invasion assays“ wurden unter Anwesenheit des spezifischen mTOR Inhibitors Rapamycin und siRNA vermittelter knock-downs von Raptor und Rictor, den Komponenten zweier unterschiedlicher mTOR Multiproteinkomplexe, durchgeführt. Immunfluoreszenz-Analysen wurden mit Antikörpern gegen mTOR Komponenten, phospho Histone H3 und cleaved caspase-3 durchgeführt. Im Unterschied zu 2D fanden wir in Fibroblasten aus 3D- Kultur eine massive Reduktion der Zellgröße, geringere Zellteilung und Veränderungen in der Granularität. Veränderte Zellgröße und Zellzyklusverteilung weisen auf Änderungen des mTOR-Signalweges hin, Immunfluoreszenzanalysen zeigten jedoch keine signifikanten Unterschiede in der Menge oder der Aktivität der beteiligten Proteine. Einige Komponenten des mTOR-Signalweges waren in Bezug auf ihre Lokalisation in den Fibroblasten verändert, z. B. pAKT (S473), welches eine vesikulärere Verteilung zeigte und das Totalprotein AKT, das innerhalb der Kerne in 3D Kultur hochreguliert war. Weiters zeigten in Kollagen-Gelen kultivierte Zellen reduzierte Invasion nach Behandlung mit Rapamycin und Transfektion mit Raptor und Rictor siRNA. Die invasiven Eigenschaften von IMR-90 Zellen wurden nach Behandlung mit Rictor siRNA hochgradig reduziert, während die Transfektion mit Raptor siRNA eine mittelgradige Wirkung zeigte. Immunfluoreszenz-Analyse zeigte eine Induktion von Apoptose in Rapamycin behandelten Sphäroiden durch einen erhöhten Anteil von früh (cleaved capase-3+) und spät apoptotischen (cleaved caspase-3+/kondensierten Kernen+) Fibroblasten. Zusammenfassend stellten wir Veränderungen in Fibroblasten aus 3D-Kultur verglichen mit 2D-Kultur fest. Weiters fanden wir Einflüsse des mTOR-Signalweges auf die invasiven Eigenschaften von IMR-90 Fibroblasten, allerdings werden noch weitere quantitative Auswertungen des mTOR-Signalweges (zB durch Western-Blot-Analyse) benötigt, um die molekularen Mechanismus, die zu reduzierter Zellgröße und Zellzyklus Verteilung, sowie zu einem Rückgang der invasiven Kapazität durch mTOR-Hemmung führen, zu bestimmen., 3D cell culture is supposed to mimick a tissue-like environment found in-vivo and it is used to study molecular and biochemical aspects of many physiologic functions under in-vitro conditions. A key regulatory pathway of important cellular functions such as ribosomal biosynthesis, proliferation, metabolism and cell size is the mammalian target of rapamycin (mTOR) pathway. It was previously extensively studied using monolayer culture, however mTOR signalling has not been studied very well in cells cultivated in 3D. Since the environment and the shape of cells can determine its behaviour and gene expression, we wanted to identify differences between 2D and 3D culture in respect to mTOR signalling. Furthermore we were interested in the invasion potential of fibroblasts in collagen gels under these experimental settings. After establishing culture conditions for IMR-90 lung fibroblasts in collagen gels and a harvesting protocol, cells from 2D and 3D culture were subjected to FACS analysis and DNA content and cell size was determined. Spheroid invasion assays were performed in the presence of the specific mTOR inhibitor rapamycin and siRNA mediated knockdowns of raptor- and rictor, two components of different mTOR multiprotein complexes. Immunofluorescence analyses were done with antibodies for mTOR pathway components, α-phospho Histone H3 and α-cleaved caspase-3. We found a massive cell size reduction, less proliferation and changes in the granularity in fibroblasts cultivated in 3D as compared to cells in 2D. Altered cell size and cell cycle distribution indicate changes in the mTOR pathway, however immunofluorescence analyses did not show significant differences in the amount or activity of the proteins. Some mTOR pathway components were altered in respect to localisation, e.g. pAKT (S473) displayed a more vesicular like distribution and total AKT protein was upregulated within the nuclei in fibroblasts cultivated in 3D. Furthermore cells cultured in collagen gels displayed reduced invasion after treatment with rapamycin and transfection with raptor and rictor siRNA. The invasive properties of the IMR-90 cells were profoundly reduced upon rictor knockdown, whereas raptor knockdown displayed an intermediate effect. Immunofluorescence analysis showed induction of apoptosis in rapamycin treated spheroids as demonstrated by an increase in the proportion of early apoptotic (cleaved capase-3+) and late apoptotic (cleaved caspase+/condensed nuclei+) fibroblasts. In summary we found profound alterations in fibroblasts cultivated in 3D as compared to 2D and an influence of mTOR signalling in the invasive properties of IMR-90 fibroblasts, however, further quantitative evaluation of the mTOR pathway (e.g. by Western blot analysis) is needed to determine the molecular events leading to reduced cell cycle progression and size as well as to a decrease in invasive capacity upon mTOR inhibition.