# Morphological Dynamics of Leukemia Cells on TiO\({}_{2}\) Nanoparticle Coatings Studied by AFM
## 3 Results and Discussion
### Time-Dependent Morphology of Leukemia Cells on TiO\({}_{2}\)-m Coatings  
Cells seeded on the TiO\({}_{2}\)-m coatings were characterized by AFM after different incubation times ranging from 10 to 60 min. Figure 2 evaluates the morphology of a representative cell after 10 min incubation. Within the first 10 min of contact with the coating, the cell membrane completely loses its original ellipsoidal shape (Figure 1a) and expands over the coating (Figure 2a,b), maximizing the contact area with the coating surface. This behavior is consistent with amoeboid locomotion on the TiO\({}_{2}\)-m coatings as previously observed [15]. In the absence of signals from extracellular matrix components and other cells, amoeboid locomotion is activated by chemotactic agents. Therefore, the spreading of the leukemia cell on the TiO\({}_{2}\)-m coating likely reflects the cell trying to find a gradient chemotactic signal and establish a direction of movement or anchor points [38]. Figure 2b shows that  
Figure 1: Representative AFM image of an acute lymphoblastic leukemia cell cultured on bare glass for 40 min. (**a**) Two-dimensional and (**b**) three-dimensional topography images, revealing pseudopods and microvilli structures. (**c**) Height profile taken along the dashed line in (**a**) showing the typical elliptical shape of the cell. (**d**) Three-dimensional representation of the zoomed area indicated by the dashed square in (**a**), revealing a corrugated membrane with pore-like structures. (**e**) Height distribution function calculated for the zoomed image shown in (**d**). The height \(z=0\) nm corresponds to the lowest height value within the zoomed region in (**d**). the expanded cell (highlighted with a green mask) adopts the topography of the coating, demonstrating the flexibility of the cell membrane to accommodate the rough topography of the coating surface, which exhibits numerous nanoparticle aggregates with heights up to about 1 \(\upmu\)m (Figure 2c). This results in a comparatively broad height distribution of the zoomed membrane region shown in Figure 2d, which now has become highly asymmetric and extends to heights well beyond 500 nm (Figure 2e). After 20 min of contact, the cell shown in Figure 3a,b can be seen to contract from all directions without a defined anchor point. This can be explained by the TiO\({}_{2}\)-m coating having a homogeneous chemical composition [15], so that the adhering cell could not detect any gradient in the chemotactic signals originating from the TiO\({}_{2}\)-m coating to define a migration direction during the initial expansion phase. Additionally, some TiO\({}_{2}\)-m nanoparticle aggregates up to about 2 \(\upmu\)m in height can be identified in the boundary region of the cell membrane (red arrows in Figure 3b,c). The cell membrane appears to be pinned by these large nanoparticle aggregates, which indicates that they are either surface features that hinder further membrane contraction or were dislodged from the coating and entrained in the previous contraction phase. However, since the performed detachment test showed only little nanoparticle release from the substrate over much longer time scales (see Supplementary Materials), we assume that the former is a more likely scenario. Regarding the morphology of the cell membrane, the pronounced topographic features seen before in the center of the cell (Figure 2d) have mostly disappeared during contraction (Figure 3d). This is also reflected in the height distribution function of the membrane in Figure 3e, which has become narrower again. Together with the observed increase in overall cell height from few hundred nanometers to about 1 \(\upmu\)m (Figure 3c), this further supports the above interpretation that the larger topographic features seen in Figure 2d were actually caused by the rough topography of the underlying coating surface that was closely followed by the highly spread and thus very thin cell. Figure 2: Representative AFM image of an acute lymphoblastic leukemia cell cultured on the TiO\({}_{2}\)-m coating for 10 min.