Investigating TNS4 in the Colorectal Tumour 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). Our objective was to study TNS4 role in CRC using more realistic models of the tumour microenvironment. CRC cells expressing TdTomato protein and shTNS4/shLUC hairpin oligos were grown in 3D spheroids with and without cancer-associated fibroblasts (CAFs). Adhesiveness to collagen I and CAFs was assessed in 2D and cell proliferation, volume and invasion were assessed in 3D conditions. The role of TNS4 knockdown in Gefitinib chemosensitivity and EGFR and Ras protein levels were also tested. In general, TNS4 knockdown increased cell proliferation in cell lines producing compact spheroids. The addition of CAFs in spheroids supported CRC cells proliferation, whereas CAFs themselves did not proliferate, but increased ECM degradation. TNS4 knockdown reduced adhesiveness and 3D invasion and disrupted EGFR signalling which resulted 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 KRAS mutant CRC patients.