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Cell Biology JoVE (Open Access) Citable · DOI

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

DOI: 10.3791/59567-v
What you'll learn
  • ✓ Generate multicellular tumor spheroids for 3D invasion assays
  • ✓ Prepare human leiomyoma-based extracellular matrix for spheroid culture
  • ✓ Segment and analyze cell invasion using Ilastik and Fiji ImageJ
Protocol

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Difficulty
advanced
Total time
~5–7 days (spheroid generation 2–3 days, invasion assay 2–3 days, image analysis 1 day)
Model organism
Human tumor-derived cells, human leiomyoma fibroblasts
Biosafety
BSL-1

Steps

1
Generate multicellular tumor spheroids

Culture tumor cells under conditions that promote spheroid formation. Establish 3D spheroid structures prior to invasion assay setup.

▶ 01:10
2
Perform three-dimensional spheroid invasion assay

Embed tumor spheroids in human leiomyoma-based matrix and culture to permit cell invasion into the biologically relevant extracellular matrix over several days.

▶ 02:53
3
Segment cell invasion images with Ilastik

Train and apply machine-learning image segmentation software to distinguish invading cells from background and matrix in microscopy images.

▶ 04:39
4
Quantify invasion area using Fiji ImageJ

Measure segmented invasion area and extract quantitative metrics for statistical analysis of cell invasion extent.

▶ 06:59
5
Interpret representative cell movement results

Review typical invasion patterns and cell behavior observed in the 3D matrix model to validate assay performance.

▶ 07:36
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