17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) mediated concentration-dependent inhibition of tumor spheroid invasion into Matrigel. (a, b) Day 4 U-87 MG spheroids were embedded into Matrigel for three-dimensional invasion. 17-AAG was used over a range of concentrations and control spheroids were treated with vehicle. Automated image analysis was carried out at intervals from t0 to 72 h on a Celigo cytometer using the Colony Counting Confluence application scanning 1/16 field of view/well. Invasion was normalized to t0. (a) A representative example of three separate experiments is shown. Values are means ± SD; n = 8. Student t test with Welch's correction was performed relative to 72 h values: vehicle control vs 0.1 to 2.5 μM P < 0.001. (b) Representative images of U-87 MG spheroid invasion obtained on a Celigo cytometer show the reproducibly sized spheroids at t0 and the typical segmentation around the invading cells with a clear inhibition by the 17-AAG at 24 h (also seen at concentrations below the concentration inhibiting cell viability by 50% (GI50) for this cell line (see Table 2)). Images of one field of view/well were cropped and magnified (20 ×) to allow visualization of the invasion. Scale bar: 500 μm.