3D Tumor Spheroid Invasion Assay in Collagen I Matrix for Quantifying Collective Invasion of U-87 MG Glioblastoma Cells

Conditions: vehicle (DMSO), GM6001 (10 µM, 25 µM), and a no-spheroid collagen-only blank. Each condition uses n = 6 individual spheroids per biological replicate (one spheroid per well), with N = 3 independent biological replicates. One uniform 5 x 10^3-cell spheroid per well is embedded. Invasion is imaged at 0, 24, 48, and 72 h at a fixed focal plane through the spheroid equator. Spheroid assignment to wells is randomized, and invaded-area measurements are made on coded images (blinded).

BSL-2 for human glioblastoma cells; use a class II biosafety cabinet. Collagen acid stocks and NaOH are corrosive — handle with gloves and eye protection, neutralize on ice. DMSO enhances skin penetration; avoid contact when co-handling inhibitors. Propidium iodide is a potential mutagen — handle and dispose as hazardous. Standard PPE (coat, nitrile gloves, goggles). Decontaminate media and surfaces with 10% bleach; segregate chemical waste.

Vehicle control: DMSO matched to the highest GM6001 carrier volume isolates drug effect from solvent. Positive invasion control: vehicle-treated spheroids define maximal invaded area. Negative/structural control: collagen-only wells (no spheroid) confirm the gel does not autofluoresce or show debris counted as cells. Viability control: live/dead staining ensures reduced invasion is not due to cell death. A matrix-concentration check (e.g., a 1.5 mg/mL vs 2.0 mg/mL pilot) controls for gel-stiffness effects on invasion mode.

Vehicle spheroids should show progressive radial outgrowth, with the invaded area increasing 2-4 fold over the initial core by 72 h and numerous detached leader cells. GM6001 should reduce invaded area in a concentration-dependent manner (e.g., 40-70% reduction at 25 µM) and lower leader-cell counts, while live/dead staining shows >90% viable core and front cells in all groups. Initial spheroid diameters should be uniform (400-500 µm, CV < 15%).

• U-87 MG cells (ATCC HTB-14), passage 4-15
• Round-bottom ultra-low-attachment 96-well plates (Corning) or 1.5% agarose-coated plates for spheroid formation
• EMEM with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate
• Rat-tail collagen type I, ~3-4 mg/mL stock, kept on ice
• 10x PBS, 0.5 M NaOH (for collagen neutralization), sterile water
• Final collagen working concentration 2.0 mg/mL, pH 7.2-7.4
• GM6001 (Ilomastat), 10 mM stock in DMSO; working 10 and 25 µM
• DMSO vehicle, matched (<0.25% final)
• Calcein-AM (2 µM) / propidium iodide (1 µg/mL) for live/dead
• Methylcellulose (0.24%) optional, to aid spheroid compaction
• Dulbecco's PBS, Trypsin-EDTA

To form size-controlled U-87 MG spheroids by liquid-overlay/hanging-drop, embed single spheroids in a 3D type I collagen matrix, and quantify the radial and area-based outgrowth of invading cells over 72 h by time-lapse phase imaging, with the goal of measuring how broad-spectrum MMP inhibition (GM6001/Ilomastat) alters the invasive area and the number of dispersed leader cells.

1. Seed 5 x 10^3 U-87 MG cells/well in 200 µL EMEM/10% FBS (with 0.24% methylcellulose) into round-bottom ULA 96-well plates; centrifuge 200 x g, 5 min; incubate 72 h to form a single compact spheroid (~400-500 µm).
2. On ice, neutralize collagen I to 2.0 mg/mL with 10x PBS and 0.5 M NaOH; keep on ice to prevent premature gelation. Verify pH 7.2-7.4 with a strip.
3. Add 50 µL neutralized collagen to each well of a flat-bottom plate and pre-gel 10 min at 37 C as a base layer.
4. Transfer one spheroid in minimal medium onto the base layer; overlay with 100 µL collagen containing vehicle or GM6001; gel 30-45 min at 37 C.
5. Add 100 µL EMEM/10% FBS with matching vehicle or GM6001 on top of the gel. Record t = 0.
6. Image each spheroid at the equatorial plane (4x or 10x phase) at 0, 24, 48, 72 h, keeping focus consistent.
7. At 72 h, stain live/dead (calcein-AM/PI, 30 min) and acquire fluorescence to confirm viability of core and invading cells.
8. Export images for blinded morphometric analysis.

Independent variable: GM6001 concentration (0, 10, 25 µM). Dependent variables: invaded area (total area minus initial core area), maximum radial invasion distance, and number of dispersed single (leader) cells beyond the front. Controlled variables: spheroid cell number/size, collagen concentration and pH, gel volume/thickness, embedding temperature/time, imaging plane and magnification, time points, passage number.

If U-87 MG collective invasion into collagen I requires matrix metalloproteinase-mediated proteolysis of the collagen network, then GM6001 (MMP inhibitor) will reduce the invaded area and the count of detached leader cells relative to DMSO vehicle, shifting invasion from a proteolytic, dispersed mode toward a more confined spheroid core, without reducing spheroid viability over 72 h.

Equatorial images are analyzed in Fiji: the spheroid core and the total invaded boundary are segmented (threshold + manual correction) to compute invaded area = total area - core area, normalized to the t = 0 core. Maximum radial distance is measured from the core centroid to the farthest invading cell. Dispersed leader cells are counted beyond a defined radius. Live/dead fraction is the calcein/PI ratio. All images are coded before measurement.

Loose or multiple spheroids per well: increase ULA centrifugation and add 0.24% methylcellulose; extend formation to 72 h. Collagen gels too fast or unevenly: keep everything ice-cold, neutralize just before use, and pre-chill pipette tips. Spheroid floats off the gel: include a pre-gelled base layer and embed gently. No invasion in any condition: collagen pH off (verify 7.2-7.4) or concentration too high/stiff — drop to 1.5-2.0 mg/mL. Out-of-focus invasion fronts in 3D: image the equatorial plane consistently or acquire a short z-stack and use a maximum-intensity projection of the relevant slices.

Invaded area at 72 h (per-biological-replicate means, N = 3) is compared by one-way ANOVA with Dunnett's test vs vehicle (alpha = 0.05). Time-course curves are analyzed by two-way ANOVA (time x treatment) with Sidak correction. Leader-cell counts, if non-normal, are compared by Kruskal-Wallis with Dunn's test. With 6 spheroids/condition x 3 biological replicates, the design has >0.8 power to detect a 40% reduction in invaded area at alpha 0.05 given spheroid-to-spheroid CV ~20%.