Colorectal carcinoma is the second most common type of cancer in both men and women in Sweden. Cancer of the colon and rectum are often considered together and their ten year survival rate is approximately 50 – 60 % depending on sex and location. Different histopathological characteristics of such cancers, including the complexity of growth, are of importance for prognosis. This thesis has compared different morphometric methods in order to achieve a quantitative and objective measurement of the invasive front of colon carcinoma. Since the growth pattern is dependent on the cell adhesiveness of different proteins we studied the distribution and localization of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin as well as screened the genes for mutations. We found a perturbed protein expression of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin in tumor sections compared to normal mucosa, but no relation to tumor volume or growth pattern could be seen. The tumor volume was found to be correlated to the growth pattern but not responsible to the perturbed protein expression. In the mutation screening we found a SNP in exon 13 the E-cadherin gene in the tumor, as well as in exon 2 of Claudin 1 and exon 4 of Claudin 7 in both tumor and normal mucosa. No correlation between mutations and growth pattern or tumor volume was found. In conclusion, this thesis shows that the computer image analysis with estimation of fractal dimension and number of free tumor cell clusters is superior to the semi quantitative visual grading of tumor invasive complexity. The aberrant expression of cell adhesion proteins in the tumor compared to normal mucosa as well as polymorphisms in the cell adhesion genes CLDN1 and CLDN7 in both tumor and normal mucosa can suggest that these aberrations are important in the tumorigenesis of colon carcinoma.

[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 298-310).

Previous studies have shown that the upregulation of CD151 and β1 is associated with poor prognosis in many cancers such as breast cancer. Studies have provided evidence that these proteins are associated with the adhesion and migration of tumor cells. In this study, a microfluidic flow chamber was utilized to determine how CD151 and β1 affected the firm and initial adhesion of metastatic breast cancer cells to a planar endothelial monolayer under shear stress. This system mimicked the adhesion of metastatic breast cancer cells to the endothelial cells of the circulatory system. CD151 and β1 increased the firm adhesion of metastatic breast cancer cells onto an endothelial monolayer when subjected to high shear stresses. CD151 and β1 increased the initial adhesion of metastatic breast cancer cells onto an endothelial monolayer. A transwell assay was utilized to determine how CD151 and β1 affected random migration through different matrixes and random transendothelial migration. CD151 and β1 decreased the random migration of metastatic breast cancer cells through matrices. Additionally, background information is provided related to the metastatic cascade, how it can be modeled with microfluidics, and how CD151 and β1 have been known to effect cancer and metastasis.

Polysialic acid is a unique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acid is an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma, strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST (ST8SiaIV) and STX (ST8SiaII), with STX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferase could lead to control of tumour dissemination and metastasis. The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferase inhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells (SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acid surface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acid surface expression and invasion. Of the potential polysialyltransferase inhibitors evaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p < 0.05) reduction in cell-surface polysialic acid re-expression and invasion in polysialic acid expressing cells. Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over other members of the wider sialyltransferase family (α-2,3- and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitro cell based assays to discovery more potent derivatives.

Title: Thin films of plasma polymers as stable supports for biomedical applications Author: Ivan Gordeev Institute: Charles University in Prague, Department of Macromolecular Physics Supervisor of the doctoral thesis: Doc. Ing. Andrey Shukurov, Ph.D, Charles University in Prague, Department of Macromolecular Physics. Abstract: Plasma polymers have been widely considered for use as bio-active coatings. In biomedicine, the surfaces that withstand accumulation of biofilms are of particular importance. This thesis is focused on development of new plasma-based methods for deposition of bio-resistant (non-fouling) plasma polymers. Poly(ethylene oxide) was the subject material. R.f. magnetron sputtering, plasma-assisted thermal vapour deposition and amplitude modulated atmospheric pressure surface dielectric barrier discharge were the methods adapted to fabricate thin films with tunable chemical composition, cross-link density and biological response. A new insight was gained into the processes of plasma polymerization as well as into composition/structure relationship and its effect on biological properties of resultant films. Keywords: plasma polymerization, PEO, 'non-fouling' properties, protein adsorption, cell adhesion