Rat tail collagen

To fluorescently label collagen, rat tail collagen (Advanced Biomatrix) was precipitated using 1 M NaCl. The collagen was redissolved in 2 M HCl and neutralized at a 4:1 volume of neutralization buffer (0.5 M NaCl and 0.1 M NaHCO₃, pH 8.2). Alexa Fluor 488 5-TFP was added to the neutralized collagen and reacted for 1.5 h at room temperature on a shaker. The reaction was stopped by adding a 1:1 volume of stop buffer (1 mM glacial acetic acid and 1.5 M NaCl). Dialysis was performed on tagged collagen using the 10 K MWCO Slide-A-Lyzer Casettes with 0.02 N acetic acid. Dialysis was performed overnight, with the acetic acid exchanged three times. The labeled collagen was extracted from the dialysis cassette using a 22G needle, snap frozen in liquid nitrogen, and stored at −80 °C.
A quantity of 5 mg/ml collagen I gels were made with a 1:5 fluorescent collagen I incorporation. The collagen was neutralized with NaOH and polymerized for 1 h at 37 °C. Cells were seeded onto the gels at a concentration of 500 000 cells/cm² overnight. Medium was changed to serum free medium, and spheroids were collected from gels after 72 h. Spheroids were imaged and analyzed for fluorescent collagen incorporation.

Human U-87 and U-251 glioblastoma cells were obtained from ATCC (Manassas, VA, USA); Dulbecco’s modified Eagle medium (DMEM), from Mediatech (Manassas, VA, USA); fetal bovine serum (FBS) and penicillin–streptomycin, from Invitrogen (Carlsbad, CA, USA); poly-L-lysine solution (0.01%), from Sigma Aldrich (Saint Louis, MO, USA); sodium pyruvate, MEM non-essential amino acids, and GlutaMax, from Life Technologies (Carlsbad, CA, USA); and trypsin, from CellGro (Manassas, VA, USA). A WST cell proliferation assay kit was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). High-viscosity alginic acid sodium salt from brown algae (alginate) was purchased from Sigma Aldrich (Saint Louis, MO, USA); sterile alginate covalently coupled with GRGDSP, from FMC BioPolymer (Philadelphia, PA, USA); rat-tail collagen, from Advanced BioMatrix (San Diego, CA, USA); and agarose (low melting), from Thermo Fisher Scientific (Waltham, MA, USA), used as received. The chemical inhibitor of the RhoA-ROCK pathway (Y-27632) was purchased from Selleck Chemicals (Houston, TX, USA), and that of the Rac pathway (NSC23766), from EMD Biosciences (La Jolla, CA, USA). Integrin inhibitor RGD was purchased from Selleck Chemicals (Houston, TX, USA); GRGDSP, from Sigma Aldrich (Saint Louis, MO, USA); and cilengitide, from MedChem Express (Monmouth Junction, NJ, USA).