Impact of elastic substrate on the dynamic heterogeneity of WC256 Walker carcinosarcoma cells

Abstract

Cellular heterogeneity is a phenomenon in which cell populations are composed of subpopulations that vary in their behavior. Heterogeneity is particularly pronounced in cancer cells and can affect the efficacy of oncological therapies. Previous studies have considered heterogeneity dynamics to be indicative of evolutionary changes within subpopulations; however, these studies do not consider the short-time morphological plasticity of cells. Physical properties of the microenvironment elasticity have also been poorly investigated within the context of cellular heterogeneity, despite its role in determining cellular behavior. This article demonstrates that cellular heterogeneity can be highly dynamic and dependent on the micromechanical properties of the substrate. During observation, migrating Walker carcinosarcoma WC256 cells were observed to belong to different subpopulations, in which their morphologies and migration strategies differed. Furthermore, the application of an elastic substrate (E = 40 kPa) modified three aspects of cellular heterogeneity: the occurrence of subpopulations, the occurrence of transitions between subpopulations, and cellular migration and morphology. These findings provide a new perspective in the analysis of cellular heterogeneity, whereby it may not be a static feature of cancer cell populations, instead varying over time. This helps further the understanding of cancer cell behavior, including their phenotype and migration strategy, which may help to improve cancer therapies by extending their suitability to investigate tumor heterogeneity.

Figure 1

Different subpopulations of WC256 adherent subline. (A) Mesenchymal (mesenchymal subpopulation), (B) polygonal (polygonal and bigonal subpopulation), (C) bigonal (polygonal and bigonal subpopulation), (D) pseudopodial (amoeboid subpopulation), (E) blebbing (amoeboid subpopulation). Magnification 10x , scalebar = 50 µm. Dynamic behavior of the same cells is presented in Supplementary Video S1.

Figure 2

(A) Subpopulations transitions observed in migrating cells of WC256 adherent subline. The main transition path marked with solid arrows starts from mesenchymal and goes through polygonal and bigonal to amoeboid subpopulation. Direct transition between mesenchymal and amoeboid subpopulation (marked with dashed arrows) was observed as well, although very rarely (3% of all observed cells). (B) Exemplary transition between mesenchymal and polygonal form. (C) Exemplary transition between amoeboid and polygonal form. Time scale in minutes:seconds, scalebar = 25 µm. Dynamic behaviors of the same cells are presented in Supplementary Video S3.

Figure 3

The influence of the mechanical properties of substrates on the dynamics of WC256 cells; (A) histograms of time spent in each subpopulation by WC256 cells; (B) histograms of number of transitions performed by dynamic cells, presented for two most common transitions (mesenchymal ↔ polygonal/bigonal; polygonal/bigonal ↔ amoeboid).

Figure 4

(A) The occurrence of different subpopulations in WC256 adherent subline plated on glass or elastic PA substrate. Each bar represents the share of frames of each subpopulation in the total number of registered frames; error bars represent the square root of counts. (B) Time evolution of total share of each subpopulation; error bars represent the square root of counts.

Figure 5

Box plots of velocities, turning angles, elongations and perimeter/area ratios of each WC256 subpopulation, cultured on glass or polyacrylamide (PA) substrate. Number of stars marks the statistical significance calculated by Mann–Whitney test (* for p < 0.05, ** for p < 0.01, *** for p < 0.001).

Figure 6

Selected quantitative parameters describing cell migration and morphology.