Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Abstract

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma. J. Vis. Exp. (2020)

The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Protocol

1. Generation of uniform size tumor spheroids

NOTE: Stem-like cells are cultured in neurobasal medium complemented with B27 supplement, heparin, Fibroblast growth factor 2 (FGF-2), penicillin, and streptomycin. These cells spontaneously form spheroids in culture.

1. Wash the tumor cells with 5 mL phosphate-buffered saline (PBS) and incubate the cells with 0.5-1 mL dissociation enzyme for 5 minutes at 37 °C.
2. Wash with 4-4.5 mL PBS and add 10 mL of the complete growth medium (complete neurobasal medium, cNBM).
3. Count the cells using an automatic counting technique with trypan blue and a cell counting chamber slide.
4. To generate 100 spheroids with 10⁴ cells per spheroid (according to preferred size), mix 10⁶ cells in 8 mL of NBM with 2 mL of 2% methylcellulose.
5. Transfer the suspension to a sterile system container and dispense 100 µL/well with a multichannel pipette into a 96 well round bottom plate.
6. Incubate the plate at 37 °C, 5% CO₂ and 95% humidity. Equal sized spheroids will form and can be used after 3-4 days.

2. Three-dimensional experiments

1. Invasion
   1. Preparation
      1. Prepare the collagen matrix in a tube on ice with type I collagen at 1 mg/mL final concentration, 1x PBS, 0.023xV_collagen, 1 M sodium hydroxide, and sterile H₂O. Incubate the solution on ice for 30 min.
      2. Collect spheroids from the round bottom well plate in 500 µL tubes and wash 2x with 200 µL 1x PBS.
      3. Pipette the spheroids carefully into 100 µL of the collagen matrix and insert in the center of a well in normal 96 well plates.
      4. Incubate the collagen gel for 30 min at 37 °C and then add cNBM on top of the gel. Inhibitors or activators (e.g., hydrogen chloride as shown in Figure 1B, 1C) can be added to the medium at this step.
   2. Image acquisition and analysis
      1. Take pictures sequentially with a video microscope in brightfield mode 24 h after collagen inclusion.
      2. Use Fiji to analyze pictures either manually or in a semiautomated manner. To do so manually, draw around the core and the total area of the spheroid with the freehand selection tool and measure the invasive area of each spheroid by subtract total area with core area. To analyze the images in a semiautomated manner, use a macro to determine the invasive area.

Results

Figure 1: Glioblastoma P3 spheroid in proliferation, invasion, or migration assays. (A) Proliferation assay. Left panel: Representative pictures with DMSO as control or with 20 µM of rotenone (respiratory chain...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well round-bottom plate	Falcon	08-772-212
Accutase	Gibco	A11105-01	Stored at 4 °C, sphere dissociation enzyme
B27	Gibco	12587	Stored at -20 °C, defrost before use
Basic Fibroblast Growth Factor	Peprotech	100-18B	Stored at -20 °C, defrost before use
DPBS 10X	Pan Biotech	P04-53-500	Stored at 4 °C
Methylcellulose	Sigma	M0512	Diluted in NBM for a 2% final concentration
NBM	Gibco	21103-049	Stored at 4 °C
Neurobasal medium	Gibco	21103049	Stored at 4 °C
Penicillin - Streptomycin	Gibco	15140-122	Stored at 4 °C
Type I Collagen	Corning	354236	Stored at 4 °C