ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

Abstract

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet CellbedTM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using CellbedTM accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Application of “Tissueoid Cell Culture System” Using a Silicate Fiber Scaffold for Cancer Research

“…We evaluated the invasion depth of cancer cells in the vertical section of Cellbed, the results of which are shown in Figure 6. We had reported previously that cell projec- tions within HSC-3 and HSC-4 cells cultured in a Cellbed could be readily detected, in contrast to the results found using cells cultured in a 2D condition [8]. These cytological changes were also observed in this study, and we further show that HSC-3 and HSC-4 cells display similar cytological and histological characteristics regardless of the type of collagen (Fig.…”

“…Pathobiology 2020;87:291-301 DOI: 10.1159/000509133 the term "tissueoid cell culture system" to refer to our 3D cell culture system. The tissueoid cell culture system has several advantages over other 3D cell culture approaches, such as Matrigel coating and spheroid cultures for the following reasons [21,22]: (1) The procedures undertaken for the culture system are very simple; (2) as the scaffold resists heat and organic solvents such as xylene or alcohol, cultured cells can be directly used for various types of staining; (3) pure cultured cells can be used for various experiments [8]; and (4) we can coat ECM on the silicate fibers of Cellbed.…”

“…Cellbed is a high-purity silica glass fiber aggregate sheet which has ultrafine and continuous porosity ( Fig. 1a) [8]. Its lattice-like structure is thought to resemble the loose connective tissue in living organisms ( Fig.…”

“…Its lattice-like structure is thought to resemble the loose connective tissue in living organisms ( Fig. 1b) [8,9]. It is difficult to observe cells during culture as Cellbed is opaque in the culture medium; however, cells can be visualized using a mounting medium with a refractive index of 1.46, similar to that of silica fiber, after staining with various substances [8].…”

Expressions of ezrin, ERK, STAT3, and AKT in tongue cancer and association with tumor characteristics and patient survival

“…The inhibition of ERK signaling inhibits tumor growth in various types of cancer, including gastric cancer (Wu et al, 2013), renal cell carcinoma (Fang et al, 2013), and rhabdomyosarcoma (Renshaw et al, 2013). We have also revealed that the activation of ERK in tongue cancer cell lines cultured three-dimensionally using silica fiber is associated with the formation of invadopodia (Noi et al, 2018).…”