br Introduction br Cancer ranks as the leading cause of
Cancer ranks as the leading cause of mortality in Japan. In 2017, around 30% of deaths have been attributed to cancer, making it a major health problem . Approximately 90% of deaths are caused by cancer invasion and metastasis. During cancer progression, cancer 1821-12-1 destroy the normal balance in the microenvironment via the induction of aberrant extracellular matrix (ECM) recon-struction. These disruption induced gene expression, cellular pro-liferation, and migration (invasion) to promote cancer malignancy .
The capability of metastatic cancers to shift motility modes is one of the main features of invasion. The tumor microenvironment is able to elicit epithelialemesenchymal transition (EMT), where an epigenetic program leads epithelial cells to lose their cellecell and celleECM interactions to undergo cytoskeleton reorganization and to gain morphological and functional characteristics of mesen-chymal cells.
* Corresponding author.
E-mail address: [email protected] (M. Okamoto).
Artificial nanofiber scaffolds can induce EMT for some breast and lung cancer cells [3e5]. We have previously investigated the combination of both surface topographies (fiber alignments) and different stiffness of the polymeric substrates to evaluate the effect on the cellular morphologies, proliferation, motility, and gene expression regarding EMT of two different types of breast cancer cells (MDA-MB-231 and MCF-7) . Considering these great efforts, designing scaffold properties has possibilities to control vasculari-zation, EMT, and inflammation phenomena. These phenomena are important for not only cancer progression but also tissue regener-ation [7e11].
It has been reported that the cancer cells prefer stiffer substrates (ranged from 1 to 50 kPa), and the cellular proliferation and motility were enhanced when they were cultured on a stiffer substrate [12e17]. The induction of EMT is believed to promote tumor cell motility and invasion. However, there is a lack of evi-dence regarding the correlation between cellular motility and acquisition of the mesenchymal phenotype through the induction of EMT mediated by stiffer substrates.
To solve this profound subject, artificial ECM with different viscoelastic properties is required which mimics the in vivo envi-ronment. The aim of this study was to examine the effect of
viscoelasticity of the substrate on the direct relation between cellular motility and mesenchymal properties with induction of EMT in hypoxia. Understanding the biology of EMT with or without linkage to cellular motility may provide new approaches in devel-opment of new therapeutic strategies.
2. Materials and methods
2.1. Materials and preparation of acrylamide copolymer gels
Acrylamide (AAm; Sigma-Aldrich), N-acryloyl-6-aminocaproic acid (ACA; Santa Cruz), N,N0-methylenebisacrylamide (BIS; Sigma-Aldrich), ammonium persulfate (APS; Sigma-Aldrich), and
N,N,N0,N0-tetramethylethylenediamine (TEMED; Sigma-Aldrich) were used without further purification. Millipore Milli-Q ultrapure (specific resistance: 18 MUcm, total organic carbon < 20 ppb, Merck Millipore Japan Co.) water obtained through a dialysis membrane was used in all experiments.
For the preparation of AAm-ACA copolymer (AC) gels, 562 mL of AAm solution (40 wt% in Milli-Q water), 360 mL of ACA solution (500 mM with 350 mM NaOH [Nacalai Tesque, Kyoto] in Milli-Q water), 15 mL of APS initiator solution (10 wt% in Milli-Q water), and 3 mL of TEMED catalyst were mixed at ambient temperature and polymerized by a free radical for ~ 1 h under nitrogen atmosphere. The viscoelastic properties of AC gels were controlled with varying amounts of cross-linker BIS solution (2 wt% in Milli-Q water) and Milli-Q water (Table S1).
After polymerization, the AC gels were fully hydrated (swelling) in phosphate buffer saline (PBS; Nacalai Tesque, Kyoto) and washed with 0.1 M of 2-(N-morpholino)ethanesulfonic acid buffer (MES; Dojindo) with 0.5 M NaCl (Sigma-Aldrich) at pH 6.1. The details were described in the previous article .
2.2. Immobilization of collagen on AC gels
For conjugation of type I collagen on the AC gel surface, the solution of 0.2 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodii-mide hydrochloride (EDAC; Tokyo Chemical Industry Co., Ltd. [TCI], Japan) and 0.5 M of N-hydroxysuccinimide (TCI) was prepared in MES buffer, and then, dehydration condensation reaction was performed for 30 min at ambient temperature. The gels modified with EDAC were washed with 40% cold methanol diluted with PBS and subsequently reacted with type I collagen (Cellmatrix I-C, Nitta Gelatin Inc.) overnight at 4 C in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES; Gibco, Life Technology)ebuffered saline solution (2 HBS: NaCl, 280 mM: Na2HPO4, 1.5 mM: HEPES, 50 mM) to adjust to pH 9.0. The AC gels were washed once with HEPES buffer at 4 C and then washed three times with PBS at room temperature. Then, AC gels modified with type I collagen were sterilized with germicidal UV light for 30 min. Finally, all AC gels were equilibrated in high-glucose Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Kyoto) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco®, Life Technologies) at 4 C overnight. Jiang et al.  reported that the resulting nano-structured collagen matrix was about 3 nm in thickness.