Modeling Solid Tumors with Scaffold-Free Models

Spheroids, or tumor cell aggregates, are more representative of in vivo conditions than cell monolayers, and tumor cells grown as spheroids exhibit several physiological traits including relevant morphology, increased cell survival, and a hypoxic core.

A growing body of evidence suggests that more relevant and translational observations can be made compared to 2D monolayer models, notably in the cancer biology and hepatotoxicity area. Though three-dimensional tumor cell culture has been shown to mimic the physiological cancer situation more closely than simple two-dimensional cell monolayers, most currently available three-dimensional techniques for generating and quantifying spheroids are time consuming, laborious, costly and/or lack reproducibility.  A simple and inexpensive model for solid tumors involves generating a single spheroid in a round bottom ULA plate.


Application

Introducing Incucyte® 3D Single Spheroid Assays

Effective analysis of 3D liquid-based multi-tumor spheroids can be challenging. Traditional plate reader assays lack multiple aspects of image-based analysis, including morphological information and ability to confirm data within images. Conventional imaging systems are inherently difficult to adapt to kinetic analyses of in vitro culture models due to various factors:

  • Incomplete data — Missed information between imaging intervals
  • Multiple uncontrolled environmental fluctuations — Repeated transportation from the incubator to the imaging system and lengthy 3D image acquisition protocols outside the incubator leading to temperature differentials and loss of control of oxygen and carbon dioxide conditions
  • Time-consuming development of optimal image acquisition parameters
  • Complex image processing requiring expert operators to generate quantitative information

Incucyte® 3D Single Tumor Spheroid Assays offer an integrated turnkey solution to automatically track and quantify tumor spheroid formation, growth and health in real time inside your tissue culture incubator.
 

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Video. Continuous monitoring of spheroid growth and cytotoxicity with the Incucyte® Live-Cell Analysis System and Incucyte® Cytotox Green Dye. Label-free SK-OV-3 human ovarian adenocarcinoma cells treated with and without 1µM Camptothecin, imaged in brightfield and green fluorescence over 10 days.

Key Advantages

Key Advantages of Incucyte® 3D Single Spheroid Assays

Derive more physiologically relevant information

Quantify label-free growth and investigate morphology of 3D single tumor spheroid cultures — inside your incubator.

Figure 1. Monitor spheroid size over time as they grow undisturbed inside your tissue culture incubator.  
Brightfield images show MDA-MB-231 breast cancer spheroids ± cytotoxic agent camptothecin (1 µM).  Vehicle treated spheroids increase in size while CMP treated spheroids remain compact.  Images taken automatically every 6h for quantification of brightfield area.

Figure 2. Reveal morphology with high quality HD phase and DF Brightfield images. High quality HD phase and corresponding DF Brightfield images of spheroids formed from A549 and MDA-MB-231 cells, 72-hours post seeding. Easily distinguish between loose aggregate and compact spheroid morphologies as exemplified here in A549 and MDA-MB-231 cells. Compaction of MDA-MB-231 aggregates into spheroids was achieved by the addition of 2.5% v/v Matrigel® post centrifugation. All images captured at 10x magnification.

Reveal cellular changes over time in mono- or co-culture

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


Figure 3. Establish cytotoxic vs cytostatic mechanism of action.   Compare brightfield and fluorescent readouts using Incucyte® Cytotox Green Dye. Images show green fluorescence within masked brightfield area of SK-OV-3 spheroids 10 days post-treatment.  Timecourse profiles of brightfield area show similar response to both drugs –spheroid growth is inhibited as drug concentrations increase.  Mean green intensity measured within brightfield boundary (bottom row) shows a differential response to cytotoxic (camptothecin, left) and cytostatic (cycloheximide, middle) agents.  In the presence of camptothecin cells die, yielding an increase in fluorescence intensity from the cytotoxicity reporter (Incucyte® Cytotox Green Dye); cycloheximide and vehicle treated spheroids show only a nominal amount of cell death as expected.

Figure 4. Continuously monitor spheroid growth and cell health in Incucyte® Live-Cell Analysis System. SK-OV-3 human ovarian carcinoma cells stably expressing nuclear restricted fluorescent protein (Incucyte® Nuclight Red Lentivirus). A time-dependent increase in fluorescence (measured within the spheroid area defined by the brightfield mask) is inhibited by the cytotoxic drug camptothecin (1 µM).

Figure 5 (Video). Quantify antibody-dependent cell-mediated cytotoxicity (ADCC) in a 3D cell culture model. Trastuzumab (Herceptin®) induced immune cell killing of SK-OV-3 ovarian cancer cells shown in a spheroid model. Incucyte® Nuclight Red HER2-positive SKOV-3 spheroids were seeded with PBMCs and treated with Herceptin (mAb targeting HER2 receptors). Herceptin induced inhibition of SKOV-3 spheroid growth.

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Generate reproducible, quantitative data

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

Figure 6. Incucyte® lab-tested and validated Single Tumor Spheroid Protocol is easy to follow. Reduce time spent troubleshooting 3D cell culture techniques and eliminate the need for a trial-and-error approach to obtain images suitable for quantitative analysis.

Figure 7. Spheroid growth assay shows robustness and reproducibility. Incucyte® VesselView shows masked brightfield area of three spheroid types (lung carcinoma, fibrosarcoma, ovarian carcinoma) at four cell densities. The brightfield area plot indicates that the recommended seeding density (2500 cells/well) for each of these cell types yields a robust timecourse.

Figure 8. Perform robust pharmacological analysis in physiologically relevant conditions. Effect of camptothecin (CMP), cisplatin (CIS) and oxaliplatin (OXA) on growth of SKOV-3 cells in a spheroid assay performed inside a tissue culture incubator and without labels. SKOV-3 cells were plated at a density of 5,000 cells per well and spheroid allowed to form (72-hours). Cells were then treated with serial compound dilutions and kinetics of spheroid growth were obtained. Microplate Graph shows the individual well Largest Brightfield (BF) Area (µm2) over time. Concentration response curves represent the Largest BF Area (µm2) at 204-hours post-treatment. Data were collected over 240-hour period at 6-hour intervals. Each data point represents mean ±SEM, n=8.
 

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Unlock your productivity

Automatically acquire, analyze and graph thousands of images from up to six 96/384-well plates in parallel and get to answers faster.

Figure 9. Guided interface is easy to use for even first-time users.  Automated image acquisition and analysis tools provide a ‘set up and walk away’ experience. View images remotely to monitor experimental progress and analyze in real time for rapid decision-making.

Ordering Information

Ordering Information

Product

Qty.

Cat. No.

Incucyte® Spheroid Analysis Software Module

1

9600-0019

Incucyte® Cytotox Green Dye

1

4633

Incucyte® Cytotox Red Dye

1

4632

Incucyte® Annexin V Red Dye

1

4641

Incucyte® Annexin V Green Dye

1

4642

Incucyte® Nuclight Green Lentivirus (EF-1a Promoter, Puro selection) 

1

4624

Incucyte® Nuclight Red Lentivirus (EF-1a Promoter, Puro selection)

1

4625

Incucyte® Cytolight Green Lentivirus (EF-1a Promoter, Puro selection) 

1

4481

Incucyte® Cytolight Red Lentivirus (EF-1a Promoter, Puro selection) 

1

4482


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Resources

Literature and Documentation

Application Note

Validation and Pharmacological Utility of Real-time, Live-cell Assays for Single 3D Spheroids

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Protocol

Incucyte® Immune Cell Killing of Tumor Spheroids Assay

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Protocol

Incucyte® Single Spheroid Assay

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Protocol

Incucyte® Spheroid Assay

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Application Note

Tumor Spheroid Brightfield Analysis

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MIPTEC 2017 Poster

Non-invasive real-time analysis of growth, shrinkage and cell health in living 3D spheroids

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AACR Poster

A 3D Culture Model for Screening of Cancer Therapeutics

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