Summary
This protocol quantifies invasive outgrowth from preformed cancer spheroids seeded onto LunaX ECM hydrogels. The 96-well assay supports comparisons of matrix stiffness, treatment effects and intrinsic invasive potential using daily brightfield imaging and image-based quantification.
What you’ll do
- Generate compact spheroids in a 96-well ultra-low-attachment plate.
- Prepare cell-free LunaX ECM surfaces across a defined stiffness range.
- Transfer one spheroid to the centre of each crosslinked hydrogel.
- Image surface invasion every 24 hours under consistent acquisition settings.
- Quantify spheroid core area, total outgrowth area and invasion-front radius in ImageJ/Fiji.
Protocol at a glance
- Model: Metastatic cancer cells such as MDA-MB-231; primary cancer cells may also be adapted.
- Spheroid formation: 1,000–2,000 cells per well, aggregated undisturbed for 3 days.
- ECM format: 60 µL cell-free LunaX ECM per flat-bottom 96-well.
- Stiffness range: 0.5–6 kPa using validated LunaX Crosslinker settings.
- Assay window: Daily imaging, typically for 10–14 days.
Expected outcome
Surface-seeded spheroids should show progressive, stiffness-dependent outgrowth. Under the reported conditions, softer matrices support compact morphology with limited radial migration, intermediate matrices support collective protrusive outgrowth, and stiffer matrices produce more pronounced spreading and single-cell dissemination.
Critical parameters
- Confirm cell viability is above 90% before spheroid formation.
- Use wide-bore or cut tips and slow pipetting to avoid disrupting spheroids during transfer.
- Allow spheroids to settle for 3–5 minutes before adding medium slowly against the well wall.
- Keep gels hydrated and use identical imaging settings at every timepoint.
- Exclude displaced, damaged or off-centre spheroids from analysis.
