AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication

Abstract

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190^BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 10⁵ cells ml^-1) and are poised to grow at low cell density (LD, <1 × 10⁴ cells ml^-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Figure 1

A scheme of the AFM probe deforming a cell loosely attached to the bottom of the culture dish. An elastic cell body is surrounded by the pericellular brush layer.

Figure 2

Growth and proliferation profile of the ALL3 cells at different starting cell densities. ALL3 cells were grown in ALL3 medium as described in ‘Methods’. Comparative growth of the ALL3 cells at starting cell density of a) LD of 5,000 and 10,000 cells/ml and b) 200,000 and 300,000 cells/ml. Y-axis represents total number of viable (V) and non-viable (NV) cells on different days as determined using the trypan-blue exclusion method. Note: In both cases, a) and b) Y-axis are of different scale. Images of LD (c) and HD (d) cells on Day 2 that were stained with the MIB-1 Ki67 antibody.

Figure 3

An example of processing of the AFM curve recorded on the surface of the ALL3 cell through the brush model. (a, c and d) fitting of the different regions of raw Z-d curve with the eq.(1) (points represent the raw AFM Z-d curve, solid (red) lines show region of data used for fitting and solid (dashed) (blue) lines are the calculated Hertz curve). (b) Forces of interaction between the AFM probe and the brush layer obtained by processing raw Z-d curve for the d=85-100 nm (here open circles correspond to the force-separation data, solid line is the model fitting eq.(3)).

Figure 4

Results for biomechanical properties of ALL3 cells at low and high starting cell densities. The elastic modulus (a,b), equilibrium pericellular brush length (c,d), and grafting density (e,f).

Figure 5

Comparison of the effective brush size (N*L parameters) for (a) low and (b) high density ALL3 cells.