Summary
This protocol measures invasive outgrowth from preformed cancer spheroids embedded within LunaX ECM. The 96-well model surrounds each spheroid with a tuneable 3D matrix for studying stiffness-dependent invasion, treatment response and differences between cancer cell types.
What you’ll do
- Form compact cancer spheroids in an ultra-low-attachment plate.
- Embed individual spheroids in 30 µL of LunaX ECM precursor.
- Centre spheroids and photocrosslink the matrix to the selected stiffness.
- Capture daily images of invasion through the surrounding ECM.
- Quantify invasion area and invasion-front radius relative to day 0.
Protocol at a glance
- Model: Metastatic cancer cells such as MDA-MB-231, with adaptation possible for primary cells.
- Spheroid formation: 1,000–2,000 cells per well for 3 days.
- Embedding format: One spheroid in a 30 µL LunaX ECM volume.
- Stiffness range: 0.5–6 kPa using validated Crosslinker exposure settings.
- Readout: Brightfield imaging every 24 hours; invasion may begin within 1–3 days in softer matrices.
Expected outcome
Embedded spheroids should exhibit a time-dependent increase in invasion area. Softer matrices are expected to maintain a more compact morphology, while intermediate and stiffer conditions support collective protrusions, invasive spreading and increased single-cell dissemination under the reported model conditions.
Critical parameters
- Confirm compact spheroid formation and cell viability above 90% before embedding.
- Leave a small residual liquid volume during aspiration to avoid losing or drying spheroids.
- Fully submerge each spheroid in ECM and avoid air bubbles.
- Centrifuge at approximately 150 × g for 15 seconds to centralise spheroids before crosslinking.
- Record the Crosslinker program, irradiance and exposure duration for every condition.
