Alginate–Matrigel hydrogel preparation

Sodium alginate of an average molecular weight of 138 kDa (FMC Biopolymer) was used to prepare alginate–Matrigel interpenetrating networks of different stiffnesses, as described previously⁷⁷. Briefly, the day before the experiment, alginate was reconstituted in DMEM/F12 (Gibco, 31331093). From this step forward, all the solutions were kept on ice. Next, an alginate–Matrigel mixture was prepared into intermediate concentrations of 1.25% and 5.4 mg ml^–1, respectively (volumes must be adjusted based on the Matrigel batch concentration).

For each hydrogel, two Luer-Lok syringes were prepared (on ice). One syringe was filled with the alginate–Matrigel intermediate mixture, with sufficient volumes for a final concentration of 1% alginate and 4.4 mg ml^–1 Matrigel. The second syringe containing the medium, spheroids (formed of MCF10A β4 or MCF10A β4R1281W) and calcium sulphate (final concentrations, 24.5 mM (stiff gels) and 8.9 mM (soft gels)). Next, both syringes were attached with a female–female Luer-Lok connector, taking care not to introduce bubbles or air into the mixture. The contents of the two syringes were rapidly mixed and the alginate gel was immediately deposited on top of a sterile plate and transferred to the incubator.