Quantification of forces involved in carbohydrate induced attachment of carcinoma cells

Abstract

Vital biological processes such as the adhesion of cells to their surroundings rely on short-term recognition processes, which are defined by a rapid assembly and disassembly of adhesion molecules. These processes were mostly attributed to interactions of membrane-bound proteins to complementary proteins (PPI) or carbohydrates (CPI), while direct interactions between carbohydrates (CCI) were underestimated due to their low homotypic binding affinity. However, the cellular plasma membrane offers an abundant supply of carbohydrate-containing molecules including glycosphingolipids (GSLs), which are exclusively displayed on the outer leaflet of mammalian cell membranes. GSLs are characterized by extremely high variability due to a large diversity of different carbohydrate head groups and were found to interact with high selectivity. In this thesis, two different methods based on atomic force microscopy (AFM), namely colloidal probe microscopy (CPM) as well as single-cell force spectroscopy (SCFS), were applied to assess the impact of CCIs between the GSLs GM3 and lyso-lactosylceramide (lyso-LacCer) on the initial adhesion of murine B16 melanoma cancer cells. To study the relation between CCI and tumor malignancy, B16-F1 and B16-F10 cells of the same origin but different malignancy were used. Surface-based analytical experiments including fluorescence and optical cell adhesion studies were applied to examine the distribution and organization of GM3 and lyso-LacCer in the cellular plasma membrane and in solid supported lipid membranes (SSLMs) and indicated the formation of GSL-enriched domains (GEMs). CPM employing a model membrane system allowed to examine the specific adhesion forces between GM3 and lyso-LacCer due to a full control over molecular composition in SSLMs. Our CPM results showed that strong maximum adhesion forces of approximately 100–400 pN exist between GM3 and lyso-LacCer at contact times of 0–60 s, which were comparable to adhesion forces found between other GSLs, CPIs and even PPIs. Since we assume similar amounts of binding partners in the contact area of the glass microsphere and the cells, maximum adhesion forces obtained by CPM can be compared to force values detected by SCFS. We found that CPM observed forces were quite similar to the maximum adhesion forces obtained by SCFS at contact times shorter than 5 s (∼100–500 pN) before high adhesion forces of 750 pN (B16-F1) and 1800 pN (B16-F10) were detected for contact times of 10–60 s. We propose that the CCIs between GM3 and lyso-LacCer have the potential to foster B16 cell attachment within the first seconds of adhesion. At longer contact times, associations between GSLs and adhesive molecules including proteins are presumably actively induced by cellular internal mechanisms leading to an enhanced adhesion caused by signaling processes. The stronger adhesion behavior of the invasive B16-F10 cells points at an enhanced recruitment of GM3 to the basal attachment site based possible on interactions with adhesive molecules leading to a more effective metabolism. In this thesis, I was able to show by using a combination of tailor-made model systems and live cell studies that CCIs play a significant role in early adhesion processes of the murine B16 melanoma cancer cells.