Investigating TNS4 in the Colorectal Tumor Microenvironment Using 3D Spheroid Models of Invasion

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.