Related Experiment Video
Updated: Dec 21, 2025
Three-Dimensional 3D Tumor Spheroid Invasion Assay
Published on: May 1, 2015
Investigating TNS4 in the Colorectal Tumor Microenvironment Using 3D Spheroid Models of Invasion
Teresa P Raposo1,2, Susanti Susanti1,2,3, Mohammad Ilyas1,2
1Dr. T. P. Raposo, Dr. S. Susanti, Prof. M. Ilyas, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
Abstract:
TNS4 (Tensin 4 or Cten) is a putative oncogene in colorectal cancer (CRC) with a role in regulating cell adhesion, motility, invasion, and epithelial to mesenchymal transition (EMT). The objective is to study the role of TNS4 in CRC using more realistic models of the tumor microenvironment. CRC cells expressing TdTomato protein and shTNS4/shLUC hairpin oligos are grown in 3D spheroids with and without cancer-associated fibroblasts (CAFs). Adhesiveness to collagen I and CAFs is assessed in 2D and cell proliferation, volume, and invasion are assessed in 3D conditions. The role of TNS4 knockdown in gefitinib chemosensitivity and epidermal growth factor receptor (EGFR) and Ras protein levels are also tested. In general, TNS4 knockdown increases cell proliferation in cell lines producing compact spheroids. The addition of CAFs in spheroids supports CRC cell proliferation, whereas CAFs themselves do not proliferate, but increases ECM degradation. TNS4 knockdown reduces adhesiveness and 3D invasion and disrupts EGFR signaling which results in increased sensitivity to Gefitinib. In conclusion, in a 3D spheroid model, TNS4 inhibits cell proliferation and promotes cell invasion into the ECM, possibly by adhesion to the ECM and stromal cells. TNS4 knockdown enhances sensitivity to the EGFR inhibitor gefitinib and may be helpful for Kirsten ras oncogene homolog mutant CRC patients.
Insights
Tensin 4 (TNS4) promotes colorectal cancer (CRC) invasion but inhibiting it enhances gefitinib sensitivity. TNS4 knockdown may benefit patients with Kirsten ras oncogene homolog mutant CRC.
Area of Science:
- Oncology
- Cell Biology
- Cancer Research
Background:
- Tensin 4 (TNS4) is implicated as an oncogene in colorectal cancer (CRC), influencing cell adhesion, motility, invasion, and epithelial-to-mesenchymal transition (EMT).
- Realistic models of the tumor microenvironment are crucial for understanding TNS4's role in CRC progression.
Purpose of the Study:
- To investigate the function of TNS4 in colorectal cancer using advanced 3D spheroid models.
- To evaluate the impact of TNS4 knockdown on CRC cell behavior, including proliferation, invasion, and response to gefitinib.
Main Methods:
- Colorectal cancer cells were cultured in 3D spheroids with or without cancer-associated fibroblasts (CAFs), with TNS4 expression modulated using shRNA.
- Assessed cell adhesion to collagen I and CAFs, 3D spheroid volume, proliferation, invasion, and epidermal growth factor receptor (EGFR)/Ras signaling.
- Evaluated the effect of TNS4 knockdown on gefitinib sensitivity.
Main Results:
- TNS4 knockdown generally increased cell proliferation in compact spheroids but reduced adhesiveness and 3D invasion.
- Cancer-associated fibroblasts (CAFs) supported CRC cell proliferation and extracellular matrix (ECM) degradation but did not proliferate themselves.
- TNS4 knockdown disrupted EGFR signaling, leading to increased sensitivity to gefitinib.
Conclusions:
- In 3D models, TNS4 inhibits proliferation while promoting invasion, potentially via adhesion to ECM and stromal cells.
- TNS4 knockdown enhances gefitinib sensitivity, suggesting a potential therapeutic strategy for Kirsten ras oncogene homolog mutant CRC patients.
