Abstract
Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.
Highlights
Metastatic cancer cells must communicate in a bidirectional manner with the extracellular environment, including the extracellular matrix (ECM), cytokines, growth factors, and neighboring cells [1]
We found that Transmembrane 4 L six family member 5 (TM4SF5) expression in 2 dimensional or 3 dimensional (3D) gelembedded cells promoted the formation of invasive foci, invadopodia, and an endothelial-like network via the intracellular signaling activity of TM4SF5 and extracellular environmental cues
Compared with SNU449Cp cells, SNU449Tp cells embedded in 3D Matrigel for 24 h showed increased mesenchymal markers, loss of epithelial markers, increased protein kinase B (Akt) activity, and increased cytosolic p27Kip1 level, which are downstream of TM4SF5 [12] (Supplementary Figure 1A and Figure 1F)
Summary
Metastatic cancer cells must communicate in a bidirectional manner with the extracellular environment, including the extracellular matrix (ECM), cytokines, growth factors, and neighboring cells [1]. Depending on the extracellular cues, cancer cells may adopt different migratory or invasive morphologies to navigate flexible or rigid extracellular contexts [3]. Migration and invasion depend on the dynamic regulation of cell adhesion properties via efficient formation of invadopodia, which www.impactjournals.com/oncotarget are cellular structures where cell-ECM adhesions occur and ECM proteases localize to resolve ECMs [4, 5]. Cells cultured in vitro on flat, two-dimensional (2D) substrates can differ considerably in their morphologies and cell adhesion properties from those grown in threedimensional (3D) in vivo-like environments surrounded with ECMs [6, 7]. Embedding tumor cells in 3D Matrigel or collagen type I gels can allow dynamic behaviors that replicate the behavior of metastatic tumor cells traveling through the ECM-enriched stromal area [8]. Cell cultures in 3D ECM gel may allow exploration of the mechanisms of cancer metastasis and screening for antimetastatic reagents [9]
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