Characterization of the mechanical properties of cancer cells in 3D matrices in response to collagen concentration and cytoskeletal inhibitors

Abstract

Cellular processes, such as cell migration, adhesion, and proliferation depend on the interaction between the intracellular environment and the extracellular matrix (ECM). While many studies have explored the role of the microenvironment on cell behavior, the influence of 3D matrix mechanics on intracellular activity is not completely understood. To characterize the relationship between the mechanical components of the microenvironment and intracellular behavior, we use particle-tracking microrheology of metastatic breast cancer cells embedded in 3D collagen gels to quantify the intracellular activity from which the molecular motor activity and stiffness can be determined. Our results show that increasing collagen concentration of the 3D environments leads to an increase in intracellular stiffness and motor activity. Furthermore, our studies demonstrate that intracellular fluctuations depend on collagen concentration, even in the presence of a number of frontline chemotherapeutic and anti-MMP drugs, indicating that ECM concentration is an important and indispensable parameter to consider in drug screening.

Figure 1. Schematic of microrheology procedure

a) Cells are embedded into varying collagen concentrations for 24 hours and incubated with MitoTracker for 1 hour. Image acquisition performed on cells with confocal microscope. b) Imaris software used to trace the displacement vs. time curve for each tracer and the mean-squared displacements (MSDs) are calculated

Figure 2. Microrheological characterization of MDA-MB-231 cells in 3D collagen

a) MSD curves of MDA-MB-231 cells in 1–4 mg/mL collagen. The initial MSD at t = 50 ms decreases with increasing collagen density. b) Beta curves of MDA-MB-231 cells in 1–4 mg/mL collagen. c) Comparison of MSDs at 50 ms with varying collagen densities (1–4 mg/mL) d) Comparison of β’s at t = 1 s with varying collagen densities. Error bars are s.e.m. * indicates p<0.05. Color code of the curves and bar graph are the same as in Fig. 2a and Fig 2c.

Figure 3. Microrheology of MDA-MB-231 cells with different drug treatments

a) MSD curves of MDA-MB-231 cells treated with GM6001 in 1–4 mg/mL collagen. The initial MSD at t = 50 ms decreases with increasing collagen density. b) MSD curves of MDA-MB-231 cells treated with Y27632 in 1–4 mg/mL collagen. c) MSD curves of MDA-MB-231 cells treated with Paclitaxel in 1–4 mg/mL collagen. d) Beta curves of MDA-MB-231 cells treated with GM6001 in 1–4 mg/mL collagen. The beta increases with increasing collagen density. e) Beta curves of MDA-MB-231 cells treated with Y27632 in 1–4 mg/mL collagen. f) Beta curves of MDA-MB-231 cells treated with Paclitaxel in 1–4 mg/mL collagen. g) Comparison of MSDs at 50 ms with and different drug treatments (GM6001, Y27632, Paclitaxel) b) Comparison of β’s at t = 1 s with varying drug treatments. Error bars are s.e.m. * indicates p<0.05. Color code of the curves is the same as in Fig. 3a.

Figure 4. Relationship between collagen concentration and the fundamental parameters of the intracellular environment

a) A is the power generated by the molecular motors b) B is the shear modulus coefficient

Figure 5. Image analysis of untreated, GM6001, Y27632, and Paclitaxel treated cells in 2 mg/mL collagen

a) Cell area b) Perimeter c) Aspect ratio, and d) Circularity analysis of untreated cells (n = 21), GM6001 treated cells (n = 22), Y27632 treated cells (n = 22), and Paclitaxel treated cells (n = 23) with phalloidin stain. Error bars are s.e.m. * indicates p<0.05.

Figure 6. Images of untreated, GM6001, Y27632, and Paclitaxel treated cells in 2 mg/mL collagen

a–h) Bright field and fluorescence images of (a,b) untreated cells (c,d) GM6001 treated cells (e,f) Y27632 treated cells and (g,h) Paclitaxel treated cells with phalloidin stain. Green represents actin. The scale bar is 20 µm.