TNF-α and IFN-γ promote lymphocyte adhesion to endothelial junctional regions facilitating transendothelial migration.

Inflammatory conditions induce redistribution of junctional adhesion receptors toward the apical regions of endothelial cells promoting lymphocyte TEM. Much of the molecular structures of TEM have been revealed; however, the biophysical mechanisms underlying this process remain to be fully elucidated. Here, we used immunofluorescence microscopy and AFM to study endothelial distribution of adhesion molecules upon lymphocyte activation and transmigration. Our immunofluorescence results revealed redistribution of JAM-A and PECAM-1 but not ICAM-1 or VCAM-1 toward the apical junctional regions of HUVECs following a 6-h stimulation with TNF-α and IFN-γ. Consistently, our SCFS studies revealed that Jurkat cell adhesion to stimulated HUVEC monolayers was significantly greater in junctional regions. Enhanced adhesion was mediated mostly by JAM-A receptors. Further AFM adhesion mapping of the homophilic JAM-A/JAM-A interaction on the surfaces of HUVECs revealed a greater number of JAM-A receptors available for binding along junctional regions after TNF-α and IFN-γ stimulation. Our data reveal for the first time that adhesion "hot spots" of JAM-A receptors are involved in initiating lymphocyte TEM under inflammatory conditions.

Cancer cell recognition--mechanical phenotype.

The major characteristics of cancer metastasis is the ability of the primary tumor cells to migrate by way of the blood or lymph vessels and to form tumors at multiple, distant sites. There are evidences that cancer progression is characterized by disruption and/or reorganization of cytoskeleton (i.e. cellular scaffold). This is accompanied by various molecular alterations influencing the overall mechanical resistance of cells. Current approach in diagnosis focuses mainly on microbiological, immunological, and pathological aspects rather than on the biomechanics of diseases. The determination of mechanical properties of an individual living cell has became possible with the development of local measurement techniques, such as atomic force microscopy, magnetic or optical tweezers. The advantage of them lies in the capability to measure living cells at a single cell level and in liquid conditions, close to natural environment. Here, we present the studies on mechanical properties of single cells originating from various cancers. The results show that, independently of the cancer type (bladder, melanoma, prostate, breast and colon), single cells are characterized by the lower Young's modulus, denoting higher deformability of cancerous cells. However, the obtained Young's modulus values were dependent on various factors, like the properties of substrates used for cell growth, force loading rate, or indentation depth. Their influence on elastic properties of cells was considered. Based on these findings, the identification of cancerous cells based on their elastic properties was performed. These results proved the AFM capability in recognition of a single, mechanically altered cell, also in cases when morphological changes are not visible. The quantitative analysis of cell deformability carried out using normal (reference) and cancerous cells and, more precisely, their characterization (qualitative and quantitative) can have a significant impact on the development of methodological approaches toward precise identification of pathological cells and would allow for more effective detection of cancer-related changes.

Depth-sensing analysis of cytoskeleton organization based on AFM data.

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young's modulus, which is typically determined for indentation depths within the range 300-500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young's moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young's modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells.

The expression of N-cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N-cadherin expressed in various, but cancer-related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N-cadherin molecule, probed with the same antibody both on a surface of living non-malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N-cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non-malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N-cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non-malignant cells reveal less stable N-cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer-related changes in a structure of the binding site of the antibody, located at the extracellular domain of N-cadherin.