TM4SF5-mediated Invasion Cell Behaviors in 3D Gel Systems Consisting of Collagen I and/or Matrigel

Abstract

Unlike 2 dimensional (2D) culture system, extracellular matrix (ECM)-surrounded 3 dimensional (3D) conditions differentially modulate cellular behaviors. Transmembrane 4 L six family member5 (TM4SF5), a branch of the tetraspanin superfamily, is a transmembrane glycoprotein. TM4SF5 is highly expressed in hepatocarcinoma. Previous studies showed that TM4SF5 is involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Here, we explored the TM4SF5-mediated behaviors of HCC cells embedded in 3D ECM gels, with regards to invasive properties. To growing cells in 3D ECM Gel, I used 3D Matrigel (basement membrane) and/or Collagen Type I gel systems. In 3D Matrigel contidion, TM4SF5-positive cells formed aggressively invasive foci depending on PI3K or JNKs activity, and actin cytoskeletal organization, whereas TM4SF5-null cells did not. No invasive foci formation of TM4SF5-null cells in 3D Matrigel were recovered by ROCK inhibition itself, or by adding collagen I into Matrigel and additional EGF treatment in collagen I plus Matrigel condition. This TM4SF5-mediated invasive foci formation was inhibited by suppression of either p27kip1, TM4SF5 or pharmacological inhibition of either JNK or AKT and also RhoA activation inhibited the invasive foci formation. However, ERK or RacI inhibition didn't affected this invasvie foci formation. Further, when cell spheroids were embedded in 3D collagen I gels, TM4SF5-positive spheroids showed more protrusive morphology, compared with TM4SF5-null spheroids. Also, TM4SF5-positive spheroids showed dramatic formation of invadopodia enriched with F-actin and cortactin at the tips of protrusive edges. Moreover, when TM4SF5-positive or TM4SF5-null cells were co-cultured in 3D Matrigel, even TM4SF5-null cells exhibited invasive foci formation surrounded by TM4SF5-positive cells. All these observations suggest that TM4SF5 can play crucial roles in regulation of invasive properties in 3D ECM environment.