Qiacube machine

We used the NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs) to assemble gene inserts, synthesized (Integrated DNA Technologies) or amplified from previous plasmids33 in the lab, into restriction digested in-house cloning vectors. We designed the insert sequences for expression in human cells using a codon optimization tool (Integrated DNA Technologies). To generate mutant plasmids, we performed Gibson assembly of synthesized mutant genes or used the Q5 Site-Directed Mutagenesis Kit (New England BioLabs). For transfection, we purified the plasmids manually using the QIAprep Spin Miniprep Kit (Qiagen) or by the Qiacube machine (Qiagen). To verify their sequences before use, we sent the plasmids to Primordium or Laragen.

For the 3D‐printed scaffold (3DPS) synthesis, alginate of 8% (wt/vol %; Protanal LF 10/60; FMC) and 5% hydroxyapatite (wt/vol %; Sigma‐Aldrich) were mixed in water (Synergy Elix 15; Merck) using an Ultra‐Thurrax T50 digital dispenser (IKA), equipped with an S 25N‐25G dispensing tool, at 5000 rpm overnight and printed in four layers (⌀20 × 2 mm; grid distance 1.5 mm, 90°) using a bioplotter (Envisiontech) equipped with 400 µm extrusion needles. Each layer was cross‐linked in 0.1 M CaCl₂ (VWR) and ready prints were stored in 0.1 M CaCl₂ (VWR) at 4°C for up to 2 weeks.
For cell culture, 3DPS were washed in cell culture media, placed in 24 wells‐plates and seeded with 3 × 10⁵ MCF7 cells in supplemented DMEM. After 24 h, 3DPS were moved to a six well‐plates with fresh media, and this process was repeated every 4 days to a total culturing time of 21 days. After that, 3DPS were treated with 5‐FU and DOX at the 10X concentration as the PDS treatments. For RNA extraction, 3DPS were washed twice in cell media, lysed in 700 µl QIAzol (Qiagen) and homogenized for 2 × 2.5 min at 25 Hz. Automated isolation of total RNA from lysates was performed in a QIAcube machine (Qiagen) using a RNeasy Micro Kit (Qiagen) with QIAzol extraction directives. Reverse transcription and qPCR were performed as explained above.

Cells in PDS and cells grown in adherent conditions were harvested in 350 µl of RTL buffer (Qiagen) and stored at −80°C. PDSs were homogenized using a stainless steel bead in TissueLyzer II (Qiagen) for 2 × 5 min at 25 Hrz, and centrifuged at full speed for 3 min. RNA extraction was performed using the RNeasy Micro Kit including DNase treatment in a QIAcube machine (all from Qiagen). RNA concentration was measured by NanoDrop (Thermo Fisher Scientific).