Cell melanoma cells, having altered invasive characteristics, were

Celladhesion depends on multiple and even multivariate individual molecularconnections, where the individual players are difficult to identify. Due to thehigh force-resolution provided by the AFM besides maximal adhesion forces,individual de-adhesion events can be identified and compared accurately, whichare the hallmark of the established connection 10,11. On single-cell forcespectroscopy based mechanical examination the interaction of three differenttypes of melanoma cells, having altered invasive characteristics, wereperformed against brain endothelial cells. Our results show that nanomechanicalproperties can be associated to higher metastatic potential and invasive characteristicsmay rely on stronger adhesive properties mediated by altered tether formationdynamics. Arrestof melanoma cells on the inner surface of the brain blood vessels is a crucialbut not a sufficient step in the process of brain metastasis formation.

Nevertheless,those blood-travelling melanoma cells which show higher adhesiveness to thebrain endothelium might have higher chance to successfully colonize the brain.The mechanism of melanoma cell arrest and establishment of firm contact tobrain endothelial cells is still only partially described and understood. Herewe show a comparison of the dynamics of the first short term contact of threetypes of melanoma cells (WM35, A2058 and A375) with brain endothelial cells.

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The WM35 is a cutaneous, non-metastatic cell line, while A2058 and A375 arehighly metastatic cell lines 26. The difference in the metastatic potentialbetween the A2058 cells and A375 cells has not been clearly established so far.Both cell lines are VCAM-1 negative 27, however, Rolland et al. found thattransmigration of A2058 cells across bovine brain capillary endothelial cells,was twice as effective compared to A375 28. Both A2058 and A375 cells showsimilar adhesive properties to brain endothelial cells with similar junction damagingpotential in static models 29,30. Pogoda et al.

denotes A375 cells as highlyinvasive 31 and compares its elasticity to WM35 emphasizing that the formerhas lower elastic modulus (which correlates well with our results). The abovementioned studies deal with the total transmigration process of the melanomacells across the endothelium, which includes but is not restricted to initialaffinity dynamics of firm adhesion establishment. Furthermore, these studieswere conducted on static conditions.Intercellularadhesion dynamics depends on multiple factors, amongst which we can findapparent whole cell elasticity, visco-elastic properties, surface chargedensity, surface linked adhesion molecule distribution and glycocalyx thicknessas well.

The parameters investigated by us include relative elasticity, maximaladhesion force, size and location of de-adhesive rupture events. Referring tothe first short term contact to brain endothelial cells our data suggests thefollowing order from lower to higher metastatic potential – WM35, A2058 andA375 – based on apparent affinity to brain endothelial cells.Unfortunately,no proper model exists to obtain elastic or plastic properties when two cellsare pushed against each other. Therefore, in order to compare the elasticproperties of the studied cell types in situ. Similarly to the plasticity index23 we have used the relative elasticity, or elastic index, as a dimensionlesscomparing factor, which consists of the ratio between the remanent and totalwork needed to obtain the pre-set load (for details see section of Materialsand methods). In our case the perfect elasticity would be 1 while totalplasticity 0. As presented in Fig.

3 the obtained relative elasticity valuesare similar both in the case when melanoma cells were pushed against the Petri dishsurface and against the endothelial layer. This indicates that the calculatedrelative elasticity is predominantly the property of the melanoma cells and theendothelial cells have low contribution. The highest relative elasticity valueswere shown by WM35 cells, followed by A2058 and A375 cells respectively. Theseresults are in line with the findings of Lekka et al., which demonstrate ahigher Young’s modulus for WM35 cells compared to A375 cells 32.

Furthermore,Xu et al. has reported the same relation between Young’s Modulus and metastaticpotential in case of ovarian cancer cells probed with a spherical indenter33. In our case Fig. 4 depicts similar relation in case when the studiedmelanoma cells were pushed to a Petri dish surface: WM35 has the highestapparent Young’s modulus succeeded by A2058 and A375 cells respectively.

Thisrelation vanishes when the same cells were pushed to endothelial cells. Thesupporting connection between apparent Young’s modulus and invasiveness is animportant control, which in case of melanoma-endothelial interaction might behindered by several factors. In our opinion the calculated apparent Young’s modulus(Fig.

4) is less sensitive to the characteristics of the probing melanoma cell,while value of relative elasticity (Fig. 3) is more suitable for propercomparison in our case. Cellelasticity is mainly determined by cytoskeletal structures and low elasticitymay reflect disorganization of the cytoskeletal characteristic to aggressivecancer cells 34. Besides mechanical properties of a cell, physical aspects ofcell-cell interaction may influence cellular behaviour, too. The forcenecessary to move apart two cells (adhesion force) reflects well the strengthof the connection. Maximal adhesion force is a well-established parameter tocharacterize adhesion properties of biological samples, ranging from individualmolecules to living cells 10. Fig.

5 shows the comparison of the calculatedmaximal adhesion forces between the three studied cell types contacting a bare Petridish and the endothelial layer. In case of the Petri dish, no differences canbe found between the investigated cells suggesting a nonspecific interactionbetween the plastic surface of the Petri dish and the cells. In contrast, whenmelanoma cells were pushed against the endothelium a clear difference can beobserved.

WM35 cells show the lowest adhesion force, higher values can beobserved in the case of A2058 cells, whereas the A375 cells show the highestadhesion forces from brain endothelial cells. This indicated that the moreaggressive melanoma cell types adhere stronger to the cerebral endotheliumpossibly leading to an enhanced transmigratory and metastasis forming capacity.Interestingly, – although independent of cell type – adhesion forces are higherin case of Petri dish – melanoma cell contacts compared to melanoma cell –endothelial cell contact. An explanation of this observation could be thespecially treated, cell culture grade plastic surface to which cultured cellscan easily adhere.Theprocess of de-adhesion is not continuous; it can be decomposed into a series ofde-adhesion events. Intercellular adhesion is largely determined by specificcell-cell adhesion molecules and non-specific interactions of the glycocalyx.When pulling apart two adhering cells these interactions have to be released.

These bond ruptures can occur close to the contact point if the adhesionmolecules are well anchored to the underlying cytoskeleton. In case of non-anchoredadhesion molecules during the release these are either ripped out from themembrane or form a tether (membrane nanotube), depending on the membrane properties35. Dynamics of these tethers highly depends on the physical parameters(pulling speed, temperature) as reported in case of rolling neutrophils 36and adhesion of the monocytic cell line THP-1 to a surface coated with ICAM-137.

In our case membrane nanotube formation can also be observed during therelease of the contact.Comparingthe number of the observed rupture events (Fig. 6), which is directly relatedto active contact points, the same relation can be observed as for the maximaladhesion force. This implies the presence of a surface size related activebinding process, which has higher weighting in case of more inelastic cells(see Fig. 3). Size distribution of the active de-adhesive events is depicted inFig.

8 and Fig. 9 for melanoma – Petri surface and melanoma – endothelialcontact respectively. The most abundant values are around 30 pN, presenting aslight downshift with the cell type for both cases. According to literature,this value is associated with de-coupling of membrane bound adhesion moleculesin case of membrane tether ruptures 38, although it is very close to theadhesion forces reported in case of E-cadherin fragments 39. Additionally,the occurrence place of these de-adhesion events is an important characteristicof membrane dynamics, since in many cases they appear at several micrometredistances from the contact point. Since physical parameters (pulling speed,contact time, temperature) were not altered through the experiments, allmelanoma cell types were subjected to the same set of external parameters.

Hence, differences in release dynamics are associated to alterations inmembrane or cytoskeletal network properties. Based on our data, we can concludethat the shift towards higher distances of occurrence places of detectedde-adhesion events (Figs. 10 and 11) might indicate that the role of tetherbased adhesive properties of invading melanoma cells cannot be neglected in themetastasis formation process. Tether formation and dynamics might contributeconsiderably to site selection of melanoma cells ending in successful arrest onthe surface of brain endothelial layer. Although it is not an easy task toquantify the weighting of tether based adhesive contribution within the fulldetachment force, it might grant metastatic melanoma cells one step forward tosuccessful colonization.

Finally,as a conclusion we can say that we have used successfully the AFM based singlecell spectroscopy for comparison and analysis of adhesion force dynamicsbetween a confluent brain endothelial layer and three different type ofmelanoma cells presenting different invasive characteristics. Apparentmechanical properties such as elasticity, maximal adhesion force, number, sizeand distance of individual rupture events showed altered values pointingtowards cell type dependent aspects. Our results underline the importance ofmechanical details in case of intercellular interactions. Nevertheless, itsuggests that in adequate circumstances elastic and adhesive characterizationsmight be used as biomarkers

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