Multi-Spheroid Assays to Reproduce Tumor Microenvironment

Traditional methods, like plate reader assays and conventional imaging systems, struggle with effective analysis of 3D scaffold-based multi-tumor spheroids,  due to limited morphological insight, lack of real-time data, and environmental disruptions during imaging.

Key Limitations:

• Incomplete data and missing information between imaging intervals
• Environmental fluctuations from moving cultures out of the incubator
• Time-intensive setup and image optimization
• Complex image analysis requiring expert users

Solution:

The Incucyte^® CX3 Live-Cell Analysis System, featuring spinning disk confocal imaging, empowers researchers to capture clear, kinetic 3D images of multi-spheroids with minimal disruption to culture conditions—enabling richer and more consistent analysis over time. Incucyte^® 3D Multi-Spheroid Assays further streamline and automate real-time monitoring of spheroid formation, growth, and viability directly within the incubator, using either embedded spheroids or those plated atop a Matrigel^® layer. For brightfield imaging, these assays remain fully compatible with Incucyte^® SX1, S3, and SX5 systems. Incucyte^® 3D Multi- Spheroid Assays provide a streamlined, automated solution for real-time 

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• Generate Reproducible, Quantitative Data: Lab-tested protocols, high quality images, and unbiased analysis deliver robust data suitable for pharmacological analysis.
• Derive More Physiologically Relevant Information: Quantify growth and investigate morphology of 3D multi-tumor spheroid cultures on a layer or embedded in Matrigel^® – inside your incubator.
• Reveal Cellular Changes Over Time: Investigate mechanisms of action with real-time multiplexed viability and toxicity measurements using non-perturbing reagents.
• Conduct Biologically Relevant Co-culture Assays: Incorporate additional cell types to recapitulate the tumor microenvironment and investigate cellular changes over time.

Lab-tested protocols, high quality images, and unbiased analysis deliver robust data suitable for pharmacological analysis.

Figure 1. Incucyte^®’s lab-tested multi-spheroid protocols reduce time spent troubleshooting 3D cell culture techniques and eliminate the need for trial-and-error approaches to obtain images suitable for quantitative analysis.

Quantify label-free growth and investigate morphology of 3D multi-tumor spheroid cultures on a layer or embedded in Matrigel^® – as they remain undisturbed, inside your incubator.

Figure 2 (videos). Monitor multi-spheroid growth and reveal differential spheroid morphologies over time. MCF-7 and MDA-MB-231 cells were seeded (1K cells/well) and allowed to form multi-spheroids (3 days) on a layer of Matrigel^® or embedded in Matrigel^® on a base. Time-lapse videos monitoring spheroid growth over time (0 – 7 days post formation) demonstrate morphological differences between round (MCF-7) and stellate (MDA-MB-231) multi-spheroids.

Figure 3. Quantify cell-type dependent growth profiles using real time analysis. MCF-7 and MDA-MB-231 cells were embedded in Matrigel^® (1K cells/well) on a base in flat bottom 96-well plates and treated with camptothecin (CMP) and Incucyte^® Annexin V Orange Dye to aid in visualizing cell death. Segmented (yellow outline) Brightfield and phase + orange fluorescent images combined with corresponding time course graphs illustrate cell type specific kinetic growth profiles and demonstrate the inhibitory effects of CMP on spheroid growth.

Investigate mechanisms of action with real-time multiplexed viability and toxicity measurements using non-perturbing reagents.

Figure 4. Establish cytotoxic mechanisms of action with kinetic, multiplexed growth and health measurements. MCF7-Green Nuclight cells (1K cells/well) were seeded in the presence of Incucyte^® Annexin V Orange Dye (1%) and spheroids  were allowed to form for 3 days. Spheroids were treated with a range of camptothecin (CMP) or Cisplatin (CIS) concentrations and imaged for an additional 7 days. (A) Brightfield (BF, top row), Incucyte^® Nuclight Green (green fluorescence, middle row), and Incucyte^® Annexin Orange Dye (orange fluorescence, bottom row) images are compared 6 days post-treatment. (B) A lack of growth (BF Area time-course) and increase in the Mean Orange Fluorescence Intensity was observed in both CMP and CIS treated spheroids. CMP and CIS EC50  from 4 days post treatment shows concentration-dependent loss of viability and increase in cell death. 

Figure 5. Perform robust, reproducible pharmacological analysis in physiologically relevant conditions. MCF-7-Nuclight red multi-spheroids were formed over 3 days prior to 7-day treatment with known cytotoxic compounds. Time-course plate views enable rapid visualization of treatment effects on both spheroid size (Total BF Area) and viability (red FLU Intensity within BF Boundary). Concentration response curves represent area under curve (AUC) analysis of the time-course data 0 - 7 days post-treatment. All compounds caused a concentration dependent inhibition of growth and viability with rank order of potency CMP > CHX > OXA.

Figure 6. Videos: Visualize cell culture and cellular mechanisms with live-cell confocal imaging on the Incucyte^® CX3. MDA-MB-231-Green Nuclight spheroids were treated with various compounds in the presence of Incucyte^® Annexin V Orange Dye (1%) to visualize their cytotoxic effect. A series of videos, captured using the Incucyte CX3 equipped with spinning disk confocal, illustrate the dynamic cellular responses to different drug compound treatments. The confocal imaging technique provides high-resolution insights into the morphological and phenotypic changes occurring within live cells, offering a comprehensive view of drug efficacy and cellular behavior over time.

Conduct Biologically Relevant Co-culture Assays 

Incorporate additional cell types to recapitulate the tumour microenvironment and investigate cellular changes over time.

Figure 7. Visualize and quantify the impact of stromal cells on tumor multi-spheroid morphology and assess immune cell-mediated toxicity within tumor multi-spheroids. (A) MDA-MB-231 cells were seeded in flat bottom 96-well plates on a bed of Matrigel^® in mono or co-culture with NHDFs (1:1 ratio, 1K cells/well for each) and multi-spheroids allowed to form (3 days). Incucyte^® DF-Brightfield (DF-BF) images compare mono and co-culture conditions over 3 days (6 days post cell seeding). Note the temporal impact of NHDFs on spheroid morphology. (B) BT474 cells stably expressing cytoplasmic restricted GFP were seeded (1K cells/well) on a base of Matrigel^® and allowed to form multi-spheroids (3 days) prior to the addition of freshly isolated PBMCs (E:T, 5:1) and Herceptin. Incucyte^® DF-BF and fluorescence images (7 days) compare the effect of Herceptin on spheroid proliferation in the absence (top panel) and presence (bottom panel) of PBMCs (Brightfield outline mask shown in yellow). Note the loss of fluorescence intensity in the presence of PBMCs. Time-course shows a Herceptin concentration-dependent decrease in green fluorescence, indicating a reduction in viable target cells. Concentration response curves to Herceptin show sensitivity differences between HER2-positive and HER2- negative multi-spheroids (BT-474 vs MCF7 respectively).