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  • br MATERIALS AND METHODS br

    2022-08-06


    MATERIALS AND METHODS
    Cell culture
    MDA-MB 231 Metformin (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). 
    Preparation of 3D collagen hydrogels
    To Metformin prepare 1.2 mg/mL collagen type I hydrogel, we mix 1.2 mL of rat tail collagen (collagen R, 2 mg/mL; Matrix Bioscience, Mo¨rlenbach, Ger-many), 1.2 mL bovine skin collagen (collagen G, 4 mg/mL; Matrix Biosci-ence), 270 mL NaHCO3 (23 mg/mL), 270 mL 10 DMEM (Biochrom), and 43 mL NaOH (1 M) to adjust the pH to 10. The solution is then diluted with 3 mL of a mixture of one volume part NaHCO3 (23 mg/mL), one part 10 DMEM, and eight parts distilled H2O. All ingredients are kept on ice during the preparation process. 2 mL of the final collagen solution is pipetted in a 35-mm petri dish and polymerized in a tissue culture incubator at 37 C, 95% relative humidity, and 5% CO2 for 1 h. After polymerization, 2 mL of complete cell culture medium is added to prevent dehydration of collagen gels (17).
    Collagen gel mechanical properties
    To quantify the traction-force-induced deformations of the biopolymer network during cell migration, we implement the finite element approach described in (14). Finite elements are randomly filled with one-dimensional collagen fibers that buckle under compressive strains according to a buck-ling strain scale d0 and display a constant stiffness K0 during extension up to a linear strain range LS, beyond which the stiffness increases exponentially with a strain scale dS. For a 1.2 mg/mL collagen gel, these four material pa-rameters are K0 ¼ 1645 Pa, d0 ¼ 0.00032, LS ¼ 0.0075, and dS ¼ 0.033, as estimated from the stress versus strain relationship measured with a cone-plate rheometer and from the vertical gel contraction under uniaxial stretch (14) (Fig. S9).
    3-D cell migration assays
    To study the invasiveness of individual cells in 3-D collagen gels, 30,000 cells are mixed with 2.5 mL 1.2 mg/mL collagen solution and incubated for 4 h to ensure that cells have attained their typical elongated shape within the collagen gel. Subsequently, we image z-stacks with a z-distance of 10 mm, using a 10 objective with numerical aperture 0.30, a time interval of Dt ¼ 5 min between subsequent stacks, and a total duration of 24 h. We automatically track the x/y-position of all cells in the image series based on their characteristic intensity profile in the minimal and maximal intensity projections of the z-stacks using a custom Python script. All three cell conditions (MDA-control, MDA-lamA, and MDA-beads) are imaged in parallel.