D2429

The following cell lines were used in the study: SW480, SW620, DLD-1, HT29, HCT116, LoVo (all human colon cancer), MC38 (mouse colon cancer), OE33 (human esophageal cancer), and HeLa (human cervical cancer). All the cell lines were purchased from American Type Culture Collection (ATCC), except for MC38 and OE33 which were purchased from Kerafast and Sigma, respectively. Early passage SW480, SW620, DLD-1, HT29, HCT116, and OE33 cell lines were cultured in Roswell Park Memorial Institute (RPMI-1640) media (R8758; Sigma). Early passage MC38, and HeLa cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media (D2429; Sigma). All media were supplemented with 10% fetal bovine serum (FBS, 16000-044; Thermo Fisher), and 100 IU/ml penicillin/streptomycin (15140-122; Thermo Fisher). All cells were cultured under sterile conditions and maintained in a 37 °C incubator with 5% CO₂, regularly tested for mycoplasma and genetically authenticated by CRUK Cancer Centre Genomics Facility, Leeds, UK. Cell lines were passaged upon reaching ∼80–90% confluency and their morphology was regularly checked to ensure the absence of cross contamination between cell lines or mycoplasma contamination. To determine cell number, the automated cell counter NucleoCounter NC-100 (Chemometec) or manual hemocytometers were used.

HeLa cells and NIH3T3 Fibroblasts were maintained in high glucose DMEM (SIGMA D-2429, non-pH buffered), supplemented with 10% Fetal Bovine Serum, 1% Glutamine and 1% Penicillin/Streptomycin. HeLa H2B-mCherry cells were a gift from Daniel Gerlich and showed the nucleus in red fluorescence, were kept as above but Puromycin was added to the culture medium every other day. Medium was changed every other day and cells were passaged once a week. For the particle movement in the presence of tumorous cell conditioned, acidic medium, 2 × 10⁴ HeLa cells (tumoural) or Fibroblasts (control) were seeded to cover one half of a petri dish with 35mm diameter. The experiments were conducted within 2 days. For experiments with the conditioned medium only, cells were seeded into a 25 cm² cell culture bottle and allowed to acidify the medium. The medium was aspirated 4–5 days later and used for experiments.

Collagen networks were polymerized without fluorescence labels following a procedure established by Mickel et al.16 (link) In brief, acid-soluble rat-tail tendon collagen (type I, Collagen R, 354236, BD Biosciences, NJ, USA) and bovine-dermis collagen (type I, Collagen G, 354231, BD Biosciences, NJ, USA) were mixed at relative concentrations of 1:2. The mixture was then diluted to a total collagen concentration of 2.4 mg ml⁻¹ by adding equal parts of 10 × DMEM (D2429, Sigma Aldrich, MO, USA) and 0.27 M NaHCO₃. To induce gel polymerization, the pH of the solution was raised to pH 10 using 1 M NaOH. All components were kept on ice during mixing. The mixture was then quickly pipetted into a preassembled sample chamber consisting of a glass coverslip attached by vacuum grease to a metal washer (Supplementary Fig. 9), and left to polymerize for >45 min at 37 °C in a 5% CO₂ atmosphere. Polymerizing the network inside the sample chamber ensures its attachment to the coverslip, which is a prerequisite for a mechanically stable assay. Networks were between 500 μm and 1 mm thick. After polymerization, the gel was gently rinsed with 1 ml of 1 × PBS. Care was taken to never let the network dry out.