The scientific objectives were organized in two work packages WP1 and WP2. In WP1 we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure and compare the interaction of cancer cells with high and low amount of mHsp70. We implemented and validated our approach by comparing the adhesion to unspecific substrates of two pairs of breast cancer cells and colon cancer cell lines that varied in their expression of mHsp70. We then explored their interaction with lining cells of the inner wall of blood and lymphatic vessels. The autologous colon cancer cell lines CX+ (mHsp70 positive) and CX- (mHsp70 negative) showed no differences in their adhesion to unspecific substrates, but signifficant differences in their interaction with the lining cells. MHsp70 positive CX+ cells adhere much less to blood vessel lining cells than mHsp70 negative CX- cells. Stronger adhesion indicates a higher tendency to remain at the primary site. Reduced adhesion presents and advantage dfor circulation through the blood stream and could contribute to the development of distant metastasis.
In WP2 we studied the insertion of mHsp70 into the plasma membrane by AFM and confocal fluorescence microscopy on the single protein level. We prepared model lipid membranes that mimicked the outside and insdide of the cell membrane in normal and cancer cells. Detailed investigations of a previously proposed anchoring mechanism that suggested a rare glycosphingolipid (Gb3) as interaction partner revealed shortcomings of this model. We then tested different lipid environments and conditions to induce insertion of Hsp70-1A into model lipid membranes and explored the influence of cholesterol, which is often enriched in cancer cells. We found that Hsp70-1A did not insert into the outer leaflet of the membrane under normal condition. Instead, we observed a concentration dependent in membranes that contained saturated phosphatidylserine (PS). Cancer cells are often enriched in saturated phospholipids, which protect them from oxidative damage and inhibit uptake of chemotherapeutics. PS is mostly present on the indide of the membrane but radiation treatment and chemotherapy can lead to the export of PS to the surface. Under normal condition the presence of PS on the surface triggers an immune response and the removal of the cell, but association with Hsp70-1A can mask PS. Thus the enrichment of cancer cells with saturated PS in combination with over-exessive production of Hsp70-1A and treatment with therapeutics and/or radiation may potentially result in the exhibition of mHsp70 in certain cancer cells and cause immunity.
The work performed in HspAdhesion a resulted in two book chapters and four journal manuscripts, of which one is already published. The project and its achievements were presented to the scientific community at 3 international conferences and to the general public.
