br INTRODUCTION br Most cells
Most Elafibranor that are able to adhere, spread, and migrate on a two-dimensional extracellular matrix (ECM) can also adhere, change shape, and migrate when embedded in a biopolymer network of suitable adhesiveness, stiffness, and network porosity. However, when cells migrate through a three-dimensional (3-D) matrix, they must over-come not only the adhesion forces, as in a two-dimensional environment, but also the resisting forces imposed by the surrounding matrix (1,2). Resisting forces mainly arise from steric effects. This steric hindrance, in turn, depends on the matrix properties (pore size and fiber stiffness (1,3–6)) as well as cell properties (cell size and cell stiff-ness (4,7–11)). Studying cell-generated forces as the cells migrate through an ECM with varying degrees of steric hindrance is important for a mechanistic understanding of numerous physiological and pathophysiological cell func-tions in health and disease that involve cell adhesion, shape changes, and migration, such as tissue formation during
Submitted July 9, 2018, and accepted for publication February 1, 2019. *Correspondence: [email protected] Editor: Katharina Gaus.
2019 Biophysical Society.
embryogenesis, tumor metastasis formation, or the homing of immune cells.
To investigate cell migration under varying degrees of steric hindrance, previous studies have changed the protein concentration of a 3-D biopolymer network (1,3), the pore size (4,12), or the network fiber stiffness (3). These studies have consistently found a decreased cell migration or inva-sion with increasing steric hindrance of the matrix. What is unknown, however, is whether cells can partially compen-sate for this increase steric hindrance, either by an increased generation of traction forces or by changes in force polarity, which both have been previously shown to be essential for 3-D cell migration (13).
Although it is possible to measure cell-generated forces in a 3-D biopolymer network, it is problematic to compare measurements from gels with different protein concentra-tions and hence pore size and fiber stiffness because this can drastically change the nonlinear behavior of the matrix (14). Moreover, an altered matrix protein concentration inevitably leads to altered adhesive ligand density (1). An alternative way to modulate steric hindrance is to stiffen the biopolymer fibers with low doses of glutaraldehyde (3), but this, in turn, lowers the proteolytic degradability
Co´ndor et al.
of the matrix and may lead to changes in cell migration that are unrelated to effects of steric hindrance.
In this study, we follow an alternative approach: instead of changing the ECM properties, we alter the cell mechan-ical properties. To do so, we either increase the nuclear stiff-ness of breast cancer cells by overexpression of the nuclear protein lamin A (15) or we introduce into the cells polysty-rene beads with a diameter larger than the average pore size of the ECM. Although both interventions may also cause secondary cellular responses that are difficult to predict, we argue that the ability to measure cell-generated traction forces and migration behavior under identical matrix condi-tions compensates for the potential disadvantages.
We find that increasing the steric hindrance by stiffening the nuclear lamina causes a significant decrease in migration speed, which is partially compensated by an increase in directional persistence and force polarity. The traction force magnitude is equal to control cells.
Increasing the steric hindrance by loading the cells with polystyrene beads causes a small decrease in cell speed and directional persistence. To compensate, these cells in-crease their traction forces but not their force polarity. Taken together, our data demonstrate that breast cancer cells inde-pendently adapt their traction forces and force polarity to compensate for an increased steric hindrance imposed by the surrounding matrix.
MATERIALS AND METHODS
MDA-MB 231 cells (obtained from ATCC, Manassas, VA) are cultured in 75 cm2 cell culture flasks with low glucose (1 g/L) Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Cambridge, UK) supplemented with 10% fetal calf serum (Greiner, Kremsm€unster, Austria) and 1% peni-cillin and streptomycin at 37 C, 5% CO2, and 95% humidity. For lamin-A-transfected cells, 1 mg/mL puromycin is added to the medium. Cells are passaged every second day using 0.25% trypsin/EDTA.
MBA-MD 231 Lam-A lentiviral transduction and immunoblot analysis
For generating MDA-MB 231 cells expressing enhanced green fluorescent protein (eGFP)-lamin A, lentiviral transduction is used as described in (12). In brief, HEK293T cells are co-transfected with the vectors pMD2.G, psPAX2, and pLVX containing the coding sequence of lamin A N-termi-nally fused to eGFP using Lipofectamine LTX (Invitrogen, Carlsbad, CA). The cell culture supernatant is collected daily and replaced with fresh DMEM for the next 4 days. The collected medium containing assembled virus particles is pooled and filtered through 0.45-mm pores, supplemented with 8 mg/mL polyberene and added to MDA-MD 231 cells for 18 h. Start-ing from day 2 after lentiviral infection, cells are selected using 1 mg/mL puromycine. Previous studies demonstrated that after transduction, expres-sion of eGFP-lamin A is detectable in 95% of cells (12). Total average lamin A levels increased by 200% above endogenous lamin A levels (8,12,15), and cell stiffness after transfection increased by 47% above the stiffness of control cells (from 560 to 820 Pa), as estimated from the in-crease of transit time when the cells are flushed through 5-mm microcon-strictions (16).