br Stiffening and aligned extracellular matrices are observe
Stiffening and aligned extracellular matrices are observed in the vicinity of tumors. Collagen fibers near the tumor tend to be aligned, and cancer 13095-48-2 reorganize collagen fibers to be aligned. This collagen alignment contributes the tumor progression [12,13]. It has been reported that the cancer cells prefer stiffer substrates, and the proliferation and motility were enhanced when they were cultured on stiffer substrate [14e18]. Thus, the celleECM interac-tion should be elucidated to understand the mechanism of cancer progression. To solve this profound subject, artificial ECM is required, which mimics in vivo environment.
Artificial nanofiber scaffolds can induce epitheliale mesenchymal transition (EMT) for some breast and lung cancer cells [19e21]. In our previous article , we have 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). Artificial nanofiber scaf-folds can induce EMT for some breast and lung cancer cells [20e22]. Considering these great efforts, designing scaffolds properties has possibilities to control vascularization, EMT, and inflammation phenomena. These phenomena are important not only for cancer progression but also for tissue regeneration [23e27]. However, the celleECM interactions using nanofiber scaffolds in hypoxia have not been elucidated comprehensively in the literature. Hypoxia or low oxygen concentration levels are believed to enhance the tumor cell activity (more aggressive) and gene expression profiles .
This study was aimed at examining the effects of hypoxia on gene expression and cellular motility to understand the cellecell and celleECM interactions, which are overlaid on the top of fea-tures of the tumor microenvironment, such as hypoxia, low pH, and nutrient deprivation.
2. Materials and methods
2.1. Materials and electrospinning
Poly(L-lactic acid) (PLLA) with a D content of 0.8% (Mw ¼ 102 kDa, Mw/Mn ¼ 2.71 ) and poly(ε-caprolactone) (PCL) (Mw ¼ 80 kDa, Mw/Mn < 2) were used as previously described . All other reagents (dichloromethane, dimethylformamide, chloro-form, methanol, and 1,4-dioxane) were purchased from Nacalai Tesque, Kyoto. Millipore Milli-Q ultrapure (specific resistance: 18 MUcm, total organic carbon < 20 ppb, Merck Millipore Japan Co.) water through dialysis membrane was used in all experiments. Electrospinning was conducted according to our published litera-ture .
Briefly, for the preparation of aligned nanofibers, the electro-spinning was carried out using 19-gauge blunt needle (SANSYO Co., Ltd) mounted on a digital syringe pump (SPS-2, ASONE Co.) at 16 kV (HST-30K033P-100, Izumi Electric Co.) and a flow rate of 3.0 mL/h. The rotating collector covered with aluminum foil was used and maintained at a constant distance of 10 cm from the needle. Random nanofibers were collected using grounded collector of the aluminum sheet (15 15 cm2) at a constant distance of 10 cm from the needle under an operated flow rate of 2.0 mL/h. Both aligned and random nanofibers were placed on the slide glass of an 8-well chamber slide (Watson Co., Ltd). All substrates were dried over-night in vacuum at room temperature to remove the residual sol-vents. Then, they were sterilized with germicidal UV light for 30 min and further sterilized with 30% ethanol. Finally, all sub-strates were coated by 2% gelatin (Sigma-Aldrich) to enhance cell adhesion. In this study, spin-coated substrates were also used as a control of electrospun fiber substrates. The details were described in our previous article .
Human breast adenocarcinoma cell line, MDA-MB-231 (ATCC) and MCF-7 (ATCC), was cultured in high-glucose Dulbecco's modified Eagle's medium (Nacalai Tesque, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco®, Life Technologies), 100 unit/mL of penicillin (Nacalai Tesque), and 100 mg/mL of
streptomycin (Nacalai Tesque), grown at 37 C under 5% CO2 at-mosphere and 95% relative humidity (normoxia) or hypoxic con-dition (94% N2, 5% CO2, and 1% O2) at 37 C. Cells were grown to 70e80% confluence at normal culture condition before being seeded onto the fiber substrates.
2.3. Immunofluorescence staining
MDA-MB-231 and MCF-7 cells were seeded at the density of 1.0 104 cells cm¡2 on the spin-coated flat substrates (designated as Fe), random fibers (designated as R-), and aligned fibers (designated as A-) substrates. All the substrates were coated with gelatin before seeding and cultured under normoxic or hypoxic conditions for a period of 3 days and 7 days. The cells were fixed with 4% of paraformaldehyde for 15 min at room temperature. The cells were then washed with phosphate-buffered saline (PBS, Nacalai Tesque) and permeabilized with 0.1% of Triton X (Nacalai Tesque) for 6 min. The fixed cells were washed twice with PBS and blocked with 2% of bovine serum albumin (BSA, Wako) in PBS for