Neuro-Oncology Overview

Neuro-Oncology concerns cancers of nervous system, including the brain and spinal cord. Brain tumors are often aggressive and life-threatening, presenting unique treatment challenges. These challenges include their localization, which restricts access for effective treatment delivery , high cellular heterogeneity, limited regenerative capacity of neuronal cells, as well as resistance to treatments and off-target neurotoxicity that is associated with therapeutics.

Neuro-oncology research can benefit from robust translational in vitro models to gain greater understanding of brain tumor progression in order to develop new therapeutic interventions. Live-cell analysis enables long-term measurements of brain tumor cell health and morphology using 2D and 3D models.

Key Advantages

Visualize and quantify cell health in 2D Neuroblastoma Model

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).

Model relevant solid brain Neuroblastoma and Glioblastoma tumors

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).

Single-Spheroid U87-MG Glioblastoma Validation

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).

Investigate Pharmacological Effects in Glioblastomas

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.


Gain New Insight into Invasive Potential with 96-well Analysis...

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).


Comparing invasiveness and compound effects on Glioblastoma types

Figure 6. Cell type-specific invasive capacity and pharmacology. Brightfield videos and time-course data of the invading cell area (outlined in blue) demonstrates the differential invasive capacity of glioblastoma cell types U87-MG and A172 (invading cell area ~8.5 x105 µM2  vs ~2 x105 µM2, respectively at 168h). U87-MG exhibited greater invasive potential over time and appeared more resistant to anti-metastatic compound treatment. PP242 was a strong inhibitor of A172 spheroid invasion (30 µM) but only appeared to partially inhibit U87-MG spheroids (~60% at 30 µM).

Ordering Information

Product

Qty.

Cat. No.

Incucyte® Spheroid Analysis Software Module

1

9600-0019

Incucyte® Nuclight Green Lentivirus (EF1α, puro)

1

4624

Incucyte® Nuclight Orange Lentivirus (EF1α, puro)

1

4771

Incucyte® Nuclight Red Lentivirus (EF1α, puro)

1

4625

Incucyte® Nuclight NIR Lentivirus (EF1α, puro)

1

4805

Incucyte®Cytolight Green Lentivirus (EF1α, puro)

1

4881

Incucyte® Cytolight Red Lentivirus (EF-1α, puro)

1

4882

Incucyte® Annexin V Green Reagent

1

4642

Incucyte® Annexin V Orange Reagent

4756

Incucyte® Annexin V Red Reagent

1

4641

Incucyte® Annexin V NIR Reagent

1

4768

Incucyte® Cytotox Green Reagent

1

4633

Incucyte® Cytotox Red Reagent

1

4632

Incucyte® Caspase-3/7 Green Apoptosis Reagent

1

4440

Incucyte® Caspase-3/7 Red Apoptosis Reagent

1

4704


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