Influence of the extracellular matrix on cellular behaviour – Development and application of a bioengineered 3D cell culture system for cancer research and high resolution microscopy

The aim of this thesis was the application and establishment of a semi-synthetic, bioengineered model system for ovarian cancer and a naturally based, optimised hydrogel system for non-invasive high resolution microscopy analysis of plasma membrane dynamics in a physiological relevant microenvironment. Providing ex vivo models for cancer research with physiologically relevant 3D conditions combined with controllable physical and chemical composition is an ongoing challenge in the field of tissue engineering and cell biology. In this study a semi-synthetic GelMA-based hydrogel system was successfully applied to the study of ovarian cancer cells. Thereby GelMA provided a biomaterial approach combining natural binding and cleavage sites with tuneable properties in an easy to handle and cost-effective hydrogel system. Using this model system, the unique metastatic pattern of ovarian cancer, found in patients with late stage disease, was replicated in vitro and in vivo. The polymer concentration (2.5-7% w/v) did directly impact the hydrogel stiffness (0.5± 0.2 kPa – 8.9 kPa ± 1.8 kPa) but had only minor impact on solute diffusion. Diffusion of FITC labelled 70 kDa dextran was in all tested hydrogels (2.5-7% w/v; 29.9±3.3 till 16.9 μm2/s) close to the diffusion coefficient measured in water (39.2 ± 2.8 μm2/s). In the stiffest tested hydrogel the diffusion coefficient was decreased by only 2.3 times. Spheroid formation, occurring in the tumour fluid (ascites) of ovarian cancer patients with late stage disease, combined with the highest metabolic and proliferation rates was reflected in medium stiff hydrogels (5%, 3.4 kPa). Inhibition of hydrogel degradation, with a MMP inhibitor, reduced spheroid growth and metabolic activity. Additional ECM components, Laminin-411 and hyaluronic acid increased spheroid growth, metabolic and proliferation activity significantly. Furthermore this hydrogel system allowed bio-molecular analysis of mRNA and protein levels. Ovarian cancer cells showed an increased mRNA level of integrin β1 compared to monolayer or other 3D cultures. Next to the ex vivo experiments it was shown that GelMA-based hydrogels can be successfully used as spheroid-based cancer cell carrier system for in vivo studies. Spheroid-seeded hydrogels were intraperioteneally implanted into female NOD/SCID mice, resulting in tumour development and metastasis, known from the clinical sequence of the disease. The developed tumours showed a response rate of 33% to the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. Combined treatment using both therapeutics resulted in 37.8% treatment response, while treatment with ATN-161 alone had no effect.Collagen-based hydrogel systems are widely used for investigations of cell-ECM crosstalk; however, they often lack reproducible, well characterised properties and are mostly not suitable for high resolution microscopy. A small volume collagen-based hydrogel system provided a 3D cell culture model utilised for high resolution microscopy approaches with optimised sample properties, such as sample immobilisation and minimised sample thickness. This study provided evidence of an alternative fluorescent labelling of collagen fibres after hydrogel polymerisation with E133 and Sirius Red. In particular E133 was a cost and timeeffective staining method, compatible with single photon CLSM and simultaneous cell-ECM imaging. Diffusion through collagen-based hydrogels was affected by surface properties of the diffusive particles rather than a size exclusive filtering. Carboxylated mircospheres (Ø 20 nm) did not diffuse through the hydrogel pores; they showed a high binding affinity towards collagen fibres. Also after 72 h, a gradient from the outer hydrogel parts to the centre was observable. This small volume collagen-based hydrogel system allowed culture of 4 different cell lines, showing high cell viability and being suitable for short and long term cultures. Compared to 2D cultures, adherent cell lines showed a different morphology in 3D, while non-adherent K562 cells did not show a morphological change. Studies on COS7 cells showed that next to morphology also cytoskeleton organisation was a function of the local microenvironment. In contrast, no differences in the intracellular organelle organisation between cells cultured in 2D or 3D was observed. Additionally this small volume collagenbased hydrogel system was suitable for single molecule microscopy measurements using the lipid like tracer molecule CellMask™Orange; a specific probe for plasma membrane labelling without biological function. This model system, combined with confined HILO excitation and a customised algorithm allowed single molecule microscopy also in a more complex, 3D microenvironment. Overall it was shown that mimicking a more natural, bioengineered microenvironment in a realistic experimental model can deepen the understanding of cancer cell behaviour and reaction to anti-cancer treatment. Moreover a 3D microenvironment does not exclude quantitative molecular assessment using single molecule microscopy, but therefore model system and analysis methods had to be adapted to this more complex situation

Small intestinal mucosa expression of putative chaperone fls485

<p>Abstract</p> <p>Background</p> <p>Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. <it>fls485 </it>coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze <it>fls48</it>5 expression in human small intestinal mucosa.</p> <p>Methods</p> <p><it>fls485 </it>expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several <it>in situ </it>techniques and usage of newly synthesized mouse monoclonal antibodies.</p> <p>Results</p> <p>fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c.</p> <p>Conclusions</p> <p>Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.</p

Influence of the extracellular matrix on cellular behaviour – Development and application of a bioengineered 3D cell culture system for cancer research and high resolution microscopy

The aim of this thesis was the application and establishment of a semi-synthetic, bioengineered model system for ovarian cancer and a naturally based, optimised hydrogel system for non-invasive high resolution microscopy analysis of plasma membrane dynamics in a physiological relevant microenvironment. Providing ex vivo models for cancer research with physiologically relevant 3D conditions combined with controllable physical and chemical composition is an ongoing challenge in the field of tissue engineering and cell biology. In this study a semi-synthetic GelMA-based hydrogel system was successfully applied to the study of ovarian cancer cells. Thereby GelMA provided a biomaterial approach combining natural binding and cleavage sites with tuneable properties in an easy to handle and cost-effective hydrogel system. Using this model system, the unique metastatic pattern of ovarian cancer, found in patients with late stage disease, was replicated in vitro and in vivo. The polymer concentration (2.5-7% w/v) did directly impact the hydrogel stiffness (0.5± 0.2 kPa – 8.9 kPa ± 1.8 kPa) but had only minor impact on solute diffusion. Diffusion of FITC labelled 70 kDa dextran was in all tested hydrogels (2.5-7% w/v; 29.9±3.3 till 16.9 μm2/s) close to the diffusion coefficient measured in water (39.2 ± 2.8 μm2/s). In the stiffest tested hydrogel the diffusion coefficient was decreased by only 2.3 times. Spheroid formation, occurring in the tumour fluid (ascites) of ovarian cancer patients with late stage disease, combined with the highest metabolic and proliferation rates was reflected in medium stiff hydrogels (5%, 3.4 kPa). Inhibition of hydrogel degradation, with a MMP inhibitor, reduced spheroid growth and metabolic activity. Additional ECM components, Laminin-411 and hyaluronic acid increased spheroid growth, metabolic and proliferation activity significantly. Furthermore this hydrogel system allowed bio-molecular analysis of mRNA and protein levels. Ovarian cancer cells showed an increased mRNA level of integrin β1 compared to monolayer or other 3D cultures. Next to the ex vivo experiments it was shown that GelMA-based hydrogels can be successfully used as spheroid-based cancer cell carrier system for in vivo studies. Spheroid-seeded hydrogels were intraperioteneally implanted into female NOD/SCID mice, resulting in tumour development and metastasis, known from the clinical sequence of the disease. The developed tumours showed a response rate of 33% to the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. Combined treatment using both therapeutics resulted in 37.8% treatment response, while treatment with ATN-161 alone had no effect.Collagen-based hydrogel systems are widely used for investigations of cell-ECM crosstalk; however, they often lack reproducible, well characterised properties and are mostly not suitable for high resolution microscopy. A small volume collagen-based hydrogel system provided a 3D cell culture model utilised for high resolution microscopy approaches with optimised sample properties, such as sample immobilisation and minimised sample thickness. This study provided evidence of an alternative fluorescent labelling of collagen fibres after hydrogel polymerisation with E133 and Sirius Red. In particular E133 was a cost and timeeffective staining method, compatible with single photon CLSM and simultaneous cell-ECM imaging. Diffusion through collagen-based hydrogels was affected by surface properties of the diffusive particles rather than a size exclusive filtering. Carboxylated mircospheres (Ø 20 nm) did not diffuse through the hydrogel pores; they showed a high binding affinity towards collagen fibres. Also after 72 h, a gradient from the outer hydrogel parts to the centre was observable. This small volume collagen-based hydrogel system allowed culture of 4 different cell lines, showing high cell viability and being suitable for short and long term cultures. Compared to 2D cultures, adherent cell lines showed a different morphology in 3D, while non-adherent K562 cells did not show a morphological change. Studies on COS7 cells showed that next to morphology also cytoskeleton organisation was a function of the local microenvironment. In contrast, no differences in the intracellular organelle organisation between cells cultured in 2D or 3D was observed. Additionally this small volume collagenbased hydrogel system was suitable for single molecule microscopy measurements using the lipid like tracer molecule CellMask™Orange; a specific probe for plasma membrane labelling without biological function. This model system, combined with confined HILO excitation and a customised algorithm allowed single molecule microscopy also in a more complex, 3D microenvironment. Overall it was shown that mimicking a more natural, bioengineered microenvironment in a realistic experimental model can deepen the understanding of cancer cell behaviour and reaction to anti-cancer treatment. Moreover a 3D microenvironment does not exclude quantitative molecular assessment using single molecule microscopy, but therefore model system and analysis methods had to be adapted to this more complex situation

Wnt Lipidation and Modifiers in Intestinal Carcinogenesis and Cancer

The wingless (Wnt) signaling is suggested as a fundamental hierarchical pathway in regulation of proliferation and differentiation of cells. The Wnt ligands are small proteins of about 40 kDa essentially for regulation and initiation of the Wnt activity. They are secreted proteins requiring acylation for activity in the Wnt signaling cascade and for functional interactivity with transmembrane proteins. Dual lipidation is important for posttranslational activation of the overwhelming number of Wnt proteins and is probably involved in their spatial distribution. The intestinal mucosa, where Wnt signaling is essential in configuration and maintenance, is an established model to study Wnt proteins and their role in carcinogenesis and cancer. The intestinal crypt-villus/crypt-plateau axis, a cellular system with self-renewal, proliferation, and differentiation, is tightly coordinated by a Wnt gradient. In the review, some attention is given to Wnt3, Wnt3A, and Wnt2B as important members of the Wnt family to address the role of lipidation and modifiers of Wnt proteins in intestinal carcinogenesis. Wnt3 is an important player in establishing the Wnt gradient in intestinal crypts and is mainly produced by Paneth cells. Wnt2B is characterized as a mitochondrial protein and shuttles between mitochondria and the nucleus. Porcupine and ACSL5, a long-chain fatty acid activating enzyme, are introduced as modifiers of Wnts and as interesting strategy to targeting Wnt-driven carcinogenesis