Neuro-Oncology Overview

Figure 1. mTOR inhibitor PP242 affects cell health in SH-SY5Y Neuroblastoma cell model. Mono-cultures of SH-SY5Y glioblastoma cells were seeded in 96-well plates (5,000 cells/well) and after 3 days were treated with the mTOR inhibitor PP242 (50 – 0.21 µM) in media containing Incucyte^®Annexin V NIR (0.5%; Sartorius). Phase and fluorescent images were captured in real-time using the Incucyte^® Live-Cell Analysis System. Representative images shown comparing PP242 treatment (16.7 µM) to vehicle at 72h post-treatment. Time-courses and drug-response curves show a concentration-dependent decrease in phase confluence and a corresponding increase in cell death (pIC50 4.8). Data is presented as Mean +/-SEM (3 replicates).

Figure 2. Morphological variation in growth rate and area of solid brain tumour 3D spheroids. Neuroblastoma (SH-SY5Y) and glioblastoma (U87-MG) cells were seeded independently in 96-well ULA round-bottomed plates (5,000 cells/well) and allowed to form single-spheroids (3 days). Spheroid formation and growth were monitored in the Incucyte for up to 10 days. Representative Brightfield images and segmentation masks used (blue outline) at 7 days post-formation are shown (A). Time-courses following formation show that spheroids varied in growth rate and area, with SH-SY5Y having a slightly greater area compared to U87s, 6.6 x105 µm² vs 5.0 x105 µm², respectively (B). Quantification of single-spheroid eccentricity shows U87 spheroids once formed are round, compact and maintain low eccentricity (0.33 on Day 1 vs 0.31 on Day 7), whereas SH-SY5Y spheroids have a higher eccentricity value and show some loss of compactness with proliferation over time (0.55 on Day 1 vs 0.78 on Day 7). Data presented as Mean +/- SEM, 12 replicates (C).

Figure 3. Validation and robustness of human glioblastoma U87 single-spheroid model. U87-MG cells stably expressing Incucyte^® Nuclight-Orange  were seeded into a 96-well ULA plate at a range of densities (1,000 – 7,500 cells/well) and formation was monitored over 3 days in the Incucyte^® (A). A high robustness of seeding and density-dependent difference in spheroid area was observed using orange fluorescence metrics (B). Data presented as Mean +/- SEM with  CV% values being shown. Representative Brightfield and Orange fluorescence images of a single-spheroid seeded following formation (7,500 cells/well, 3d) and the Orange segmentation mask used (Outline in Red) (C).

Figure 4. Differential cytostatic and cytotoxic effects of chemotherapeutic compounds. Glioblastoma (U87-MG) cells were seeded in 96-well ULA round-bottomed plates (5,000 cells/well) and allowed to form spheroids (3 days) with plates being monitored in the Incucyte for 10 days. Post-formation, spheroids were treated with DNA inhibitor Cisplatin (0.82 – 200 µM) or dual mTOR inhibitor PP242 (0.21 – 50 µM) in the presence of Incucyte Annexin V NIR (A). Time-course shows change in spheroid Brightfield area for top concentrations of Cisplatin (200 µM) and PP242 (50 µM) compared to vehicle (B). Time-course data and drug response curves suggest PP242 shows a strong cytostatic effect and is only apoptotic at higher concentrations, whereas Cisplatin shows higher levels of cytotoxicity (C). Data presented as Mean +/- SEM.

Figure 5. High-throughput investigation of compound effects on glioblastoma spheroid invasion. U87-MG cells were seeded in ULA round bottom 96-well plates (2,500 cells/well) and allowed to form spheroids (3 days). Spheroids were then treated with serial dilutions of anti-metastatic compounds and embedded in Matrigel (4.5 mg/mL) to induce invasion (up to 10 days). Incucyte microplate vessel views show effects of treatments on spheroid invasion (whole spheroid area; yellow outline mask) 3d post-treatment (A). Cytochalasin D (2.34 nM – 300 nM) and PP242 (0.01 µM – 30 µM) caused a concentration-dependent inhibition of U87-MG spheroid invasion, while little effect was observed by Blebbistatin (0.01 µM – 30 µM) (B).