Stemness and Epithelial-Mesenchymal Transition of Breast Cancer Cells Incubated on Viscoelastic Gel Substrates

Nihon Reoroji Gakkaishi Vol.49, No.3, 163~170 (Journal of the Society of Rheology, Japan) ©2021 The Society of Rheology, Japan Article DOI: 10.1678/rheology.49.163 Stemness and Epithelial-Mesenchymal Transition of Breast Cancer Cells Incubated on Viscoelastic Gel Substrates † Ryohei OHTA and Masami OKAMOTO Department of Advanced Science and Technology, Graduate School of Engineering, Toyota Technological Institute, 2-12-1 Hisakata, Tempaku, Nagoya 468-8511, Japan (Received : January 26, 2021) Acrylamide copolymer-based gel substrates with different viscoelasticity were employed to evaluate the viscoelasticity effect on the direct relation among cancer stemness, cellular motility and mesenchymal properties with induction of epithelial-mesenchymal transition (EMT) of human breast adenocarcinoma (MCF-7) cells in both normoxia and hypoxia. Cellular migration speed (S) of MCF-7 cells was signifi cantly upregulated with decreasing in coeffi cient of damping (tanδ). The softer gel substrate produced a large amount of surface molecule of cancer stem cells (CSC) marker CD44. In contrast, for the stem cell biomarker CD133 expression, their tanδ-dependent manner was not contributed by EMT phenomenon and was an independent from acquisition of the EMT. The substrate damping as potential physical parameter emerged the im- portant linkage to cellular motility, cancer stemness, and EMT induction. Key Words: Viscoelastic gel substrates / Epithelial-mesenchymal transition / Cancer stem cells 1. INTRODUCTION leads epithelial cells to lose their cell-cell and cell-extracellu- Breast cancer is one of the most common malignancies lar matrix (ECM) interactions to undergo cytoskeleton reor- in women and is the higher incidence and mortality rates ganization and to gain morphological and functional charac- among all female malignant tumors in Japan1-3). The average teristics of mesenchymal cells. The tumor microenvironment 5-year overall survival rate of breast cancer is reported to be can elicit EMT. The capability of metastatic cancers to shift around 55 % due to the poor outcome of the therapy for met- motility modes by EMT is one of the main features of inva- astatic disease in the resistance to radiation and chemothera- sion. The understanding the interaction between microenvi- py4). The breast cancer therapies having poor prognosis ronment and cancer cells is also critical subject to progress in prompt us to study an effective cancer therapy. That is, break- the cancer treatment7,8). throughs in effective cancer therapies are imperative. Most proliferative cancer cells are killed by chemora- A recently proposed hypothesis suggests that cancer diotherapy. However, similar to sarcomas, CSCs undergoing stem cells (CSCs) and epithelial-mesenchymal transition EMT, which present mesenchymal features, are resistant to (EMT) play a pivotal role in cancer metastasis, recurrence anti-cancer therapies. Thus, CSCs survive and cause cancer and drug resistance5). CSCs were fi rst proposed by Virchow recurrence. Recent studies showed that inducible factors of and Conheim; a subpopulation of cancer cells resembles the EMT not only induce EMT, but also enhance CSC features of same traits as embryonic cells such as the ability to prolifer- cancer cells9). These fi ndings suggest that EMT is closely re- ate, and cancer is derived from the activation of dormant cells lated to cancer progression. of the same tissue. A few CSCs do self-renew and produce a Targeting the population of CSCs and EMT may result large number of heterogeneous and highly proliferative can- in breakthrough in cancer treatments. The connection be- cer cells to form primary tumors6). tween CSCs and EMT has attracted considerable attention. EMT causes morphological and functional changes in The induction of EMT is believed to promote CSC features. epithelial cells, resulting in the acquisition of mesenchymal However, there is a lack of more detailed analysis on the role cell-like features by cancer cells, where an epigenic program of EMT in modulating the stemness of breast cancer cells. To solve this profound subject, artifi cial ECM with dif- † Corresponding author. ferent viscoelastic properties is required which mimics in E-mail : [email protected] (M. Okamoto) Tel: +81-52-809-1861 vivo microenvironment. This study was aimed at examining 163 Nihon Reoroji Gakkaishi Vol.49 2021 the effect of viscoelasticity of the substrate on the direct rela- comparison, type-I collagen coated plates (TCP-coat) were tion among cancer stemness, cellular motility and mesenchy- prepared by adding Cellmatrix I-C solution onto 24-well tis- mal properties with induction of EMT in both normoxia and sue culture plates (TCP) and heated at 37 ºC for 2 h. Then hypoxia10). added solution was removed. The details of preparation and characterization were described in our previous paper12). 2. MATERIALS AND METHODS 2.3 Cell culture 2.1 Preparation of acrylamide copolymer gels Human breast adenocarcinoma cell line, MCF-7 (ATCC) Acrylamide (AAm, Sigma-Aldrich), N-acryloyl-6- were cultured in high glucose Dulbecco’s modified Eagle’s aminocaproicacid (ACA, Santa Cruz), N,N’- medium (DMEM) (Nacalai Tesque, Japan) supplemented Methylenebisacrylamide (BIS, Sigma-Aldrich), ammonium with 10 % (v/v) FBS, 100 unit/mL penicillin (Nacalai Tesque, persulfate (APS, Sigma-Aldrich), and N,N,N’,N’- Kyoto), and 100 μg/mL streptomycin (Nacalai Tesque, tetramethylethylendiamine (TEMED, Sigma-Aldrich), were Kyoto), grown at 37 ºC under 5 % CO2 atmosphere and 95 % used without further purification. Millipore Milli Q ultrapure relative humidity (normoxia) or hypoxic condition (94 % N2, (specific resistance: 18 MΩcm, total organic carbon 5 % CO2, and 1 % O2) at 37 ºC. Cells were grown to 70–80 % (TOC) < 20 ppb, Merck Millipore Japan Co.) water through confluence at normal culture condition. Cells were digested dialysis membrane was used in all experiment. with trypsin and diluted to 5 × 103 cells cm-2 before being After polymerization, the AAm-ACA copolymer (AC) seeded onto the gel substrates. gels were fully hydrated (swelling) in phosphate-buffer saline (PBS, Nacalai Tesque, Kyoto) and washed with 0.1 M of 2.4 Real time polymerase chain reaction (RT- 2-(N-morpholino) ethanesulfonic acid buffer (MES, Dojindo) PCR) with 0.5 M NaCl (Sigma-Aldrich) at pH 6.1. The details of The total RNA was extracted from the cells cultured for preparation11) and rheological characterization12) were de- three days using Fast GeneTM RNA Premium Kit (Nippon ge- scribed in previous papers. netics). Then the RNA was subjected to reverse transcription using a Transcriptor Universal cDNA Master (Roche) follow- 2.2 Immobilization of collagen on AC gels ing the manufacturer’s instructions. The reaction solutions For conjugation of type-I collagen on the AC gel sur- included 5 µL of KAPA SYBR® FAST qPCR Master Mix face, the solution of 0.2 M of 1-ethyl-3-(3-dimethyl amino- (KAPA BIOSYSTEMS), 200 nM forward and reverse prede- propyl) carbodiimide hydrochloride (EDAC, Tokyo Chemical signed primers13), and 1/10 of cDNA template in a 10 µL vol- Industry Co., Ltd., Japan (TCI)) and 0.5 M of ume. The resulting cDNA yield was then subjected to RT-PCR N-hydroxysuccinimide (NHS, TCI) was prepared in MES using Light Cycler® 96 system (Roche). The results were an- buffer and then dehydration condensation reaction was per- alyzed with Light Cycler® 96 software (Roche). The cDNA formed for 30 min at ambient temperature. The gels modified samples were analyzed for expression of E-cadherin (CDH1), with EDAC were washed with cold 40 % methanol diluted N-cadherin (CDH2), transforming growth factor beta with PBS and subsequently reacted with type-I collagen (TGF-β), hypoxia-inducible factor 1α (HIF-1α) and vimen- (Cellmatrix I-C, Nitta Gelatin, Inc.) for overnight at 4 ºC in tin, relative to the glyceraldehyde-3-phosphatase dehydroge- HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-sulfon- nase (GAPDH) as an internal standard for sample normaliza- ic acid, Gibco, Life Technology) buffered saline solution tion. In addition, the cDNA samples of day 7 were analyzed (2 × HBS: NaCl, 280 mM: Na2HPO4, 1.5 mM: HEPES, for expression of snail family zinc finger 2 (SNAI2) and zinc 50 mM) to adjust at pH 9.0. The AC gels were washed once finger E-box binding homeobox 1 (ZEB1). The details of with HEPES buffer at 4 ºC and then washed three times with characterization were described in our previous paper12). PBS at room temperature. Then AC gels modified with type-I collagen were sterilized with germicidal UV light for 30 min. 2.5 Cellular migration Finally, all AC gels were equilibrated in high glucose MCF-7 cells were seeded on the gel substrates and cul- Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai tured under normoxic or hypoxic conditions. Both cells were Tesque, Kyoto) supplemented with 10 % (v/v) FBS at 4 ºC seeded at the density of 5 × 103 cells cm-2 for 3 days, pre-cul- for overnight. Jiang et al. reported the resulting nanostruc- tured for 24 h to attach the cells to the substrates. An environ- tured collagen matrix was about 3 nm in thickness12). For ment of 37 ºC temperature, normoxic or hypoxic condition 164 OHTA • OKAMOTO : Stemness and Epithelial-Mesenchymal Transition of Breast Cancer Cells Incubated on Viscoelastic Gel Substrates was applied during the time-lapse measurement. The cells 2.7 Statistics were stained with 0.1 µg mL–1 of Hoechst 33342 for 20 min All data presented are expressed as the mean and stan- prior to time-lapse experiment. The blue fluorescent images dard deviations (±SD). Statistical analysis was performed of the tracers were obtained every 15 min for 16 hours by using one-way analysis of variance with Tukey-Kramer’ EVOS FL Auto fluorescent microscope (Life Technologies). post-hoc testing using Excel 2013 with Statcel4 v.2.0 To track the cell movement, the position of 20 individual cen- Software (OMS Publishing Co., Tokyo, Japan), and signifi- troids [x(t), y(t)] of cell nucleus were obtained by a Mosaic cance was considered at a probability of p < 0.05.

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