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   Special Issue: Challenges and Opportunities in Breast Cancer

Epithelial-mesenchymal transition and stemness of breast cancer cells: Effect of viscoelasticity of the substrate to mimics microenvironment

Metastasis is one of the greatest challenges in cancer treatment today. Normal mammary epithelial cells are optimally supported by interaction with a soft matrix (microenvironment) with elastic modulus of about 800 Pa. However, after transformation, breast tissue becomes progressively stiffer and tumour cells become significantly more contractile and hyper-responsive to matrix elasticity. In addition, importantly, the cancer cells penetrate into blood vessel and enter the circulation during metastasis. The modulus of fluid such as blood or mucus has very low stiffness of around 50 Pa. For this reason, the critical association between cancer cell phenotype and the change of matrix rigidity with an order of magnitude smaller should be emphasizing. This review highlights the current understanding of epithelial-mesenchymal transition and cancer stem cells in metastasis, and identified importance for investigation on artificial extracellular matrix with different viscoelastic properties, which is required to mimics in vivo microenvironment. The substrate damping coefficient (tan) as potential physical parameter emerged the important linkage to cellular motility, cancer stemness, and epithelial-mesenchymal transition induction. Although further investigation is required to clarify the efficacy of environmental stimuli (tan) for tumors exhibiting stem cell-like properties, this review indicates that the cancer cells incubated on softer substrate might lead to express cancer stem cell biomarkers exhibiting high expression.
Keywords: breast cancer, metastasis, epithelial-mesenchymal transition, cancer stem cells, viscoelastic properties, microenvironment

ESMED Medical research Archives 11(10), (2023). doi.org/10.18103/mra. v11 i10.4580
  
    In the last 20 years, there has been a strong emphasis on the development of polymer nanocomposites, where at least one of the dimensions of the filler material is of the order of a nanometer. Polymer nanocomposites are fundamentally different from traditional filled polymers because of the immense internal interfacial area and the nanoscopic nature of the nanomaterials. The new multifunctional properties derived from the nano-structure of nanocomposites provide an opportunity to circumvent the traditional properties associated with traditional composites. Numerous examples can be found in the literature that show significant improvements in multifunctional properties of the nanocomposites and this new class materials now being introduced in structural applications, such as gas barrier film, flame retardant product, and other load-bearing applications. This review offers a comprehensive review on the basic concept, technology and application for polymer nanocomposites.

Okamoto, M. Polymer Nanocomposites. Eng 2023, 4, 457–479.
https://doi.org/10.3390/eng4010028

 
  Aim: This study aimed to examine the effect of polymer substrate surface topography and stiffness on the direct relationship between drug susceptibility and mesenchymal properties of cancer cells in both normal and hypoxic conditions.
Methods: The combination of both surface topographies (fiber alignments) and different stiffness of poly(L-lactic acid) (PLLA) substrates has been used to evaluate the effect of the surface topographies and stiffness of the substrate on the direct relation between anti-cancer drug (CDDP) sensitivity for MDA-MB-231cells and mesenchymal properties with induction of epithelial–mesenchymal transition (EMT).
Results: The CDDP treatment in hypoxia indicates weakened cell adhesion in MDA-MB-231 cells as well as a significant level of repression in E-cadherin (CDH1). The robust connection between the drug sensitivity and repression of epithelial cell marker of E-cadherin (CDH1) mediated by substrate surface topography had an implication in the anti-cancer drug resistance of MDA-MB-231cells.
Conclusion: PLLA substrates did not cause so significant change in induction and acquisition of the EMT, indicating the EMT has no effect on the drug susceptibility.

OBM Genetics (Link: http://www.lidsen.com/journals/genetics)
doi: 10.21926/obm.genet.2202155

 
 
   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 significantly upregulated with decreasing in coefficient of damping (tan). 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 tan-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 important linkage to cellular motility, cancer stemness, and EMT induction.

Nihon Reoroji Gakkaishi, 49, 3, 163-170, 2021
 
 

 To broaden the knowledge regarding regenerative medicine in natural rubber latex (NRL) nanoparticles, we have examined the chondrogenesis of human mesenchymal stem cells (hMSCs) with NRL nanoparticles in hypoxia. We have successfully fabricated the cartilage/NRL biocomposites via hMSCs spheroid under hypoxic condition, where the administration of NRL nanoparticles exhibits the suppression of the spheroid contraction due to the cellular proliferation of hMSCs. It has been emerged that the NRL nanoparticles acted as a main component, which provides surface heterogeneity of the spheroid, leading to the mechanically stable structure with higher modulus in comparison with the control as revealed by atomic force microscopy microindentation. In hypoxia the effective gene expression of in vitro chondrogenesis of hMSCs with administration of NRL was confirmed as revealed by the chondrogenic gene expression analysis as compared with that in normoxia.

Nanocomposites6,137-148,2020

 
   The presented research aims to verify whether physicochemical properties of lung fibroblasts, modified by substrate stiffness, can be used to discriminate between normal and fibrotic cells from idiopathic pulmonary fibrosis (IPF). The impact of polydimethylsiloxane (PDMS) substrate stiffness on the physicochemical properties of normal (LL24) and IPF-derived lung fibroblasts (LL97A) was examined in detail. The growth and elasticity of cells were assessed using fluorescence microscopy and atomic force microscopy working in force spectroscopy mode, respectively. The number of fibroblasts, as well as their shape and the arrangement, strongly depends on the mechanical properties of the substrate. Moreover, normal fibroblasts remain more rigid as compared to their fibrotic counterparts, which may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process. The chemical properties of normal and IPF-derived lung fibroblasts inspected using time-of-flight secondary ion mass spectrometry, and analyzed complexly with principal component analysis (PCA), show a significant difference in the distribution of cholesterol and phospholipids. Based on the observed distinctions between healthy and fibrotic cells, the mechanical properties of cells may serve as prospective diagnostic biomarkers enabling fast and reliable identification of idiopathic pulmonary fibrosis (IPF).

Materials Biomaterial Section (MDPI) 13, 4495-, 2020
 
   The induction of epithelial-mesenchymal transition (EMT) is believed to promote tumor cell motility and invasion. Using polymeric gel substrates with different viscoelasticity as microenvironment of cell culture substrates that influences cancer progression and metastatic potential, we have examined the effect of the viscoelasticity on the direct relation between cellular motility and mesenchymal properties with induction of EMT in MDA-MB-231 cells. The MDA-MB-231 cells cultured on gel substrates showed different cellular morphologies and these morphological differences were robustly correlated with cellular migration speed (S) and damping coefficient (tan) of gel substrates (not substrate stiffness). The linkage between the acquisition of the mesenchymal phenotype in the cells through the induction of EMT mediated by stiffer substrate and the promotion of the cellular motility were not observed. Understanding the biology of EMT with or without linkage to the cellular motility provides new approaches in development new therapeutic strategies.

Materials Today Chemistry 13, 8-17,2019
 
 
 

 Natural rubber latex (NRL) is mainly used around traditional industrial products, but currently their target application is continuously expanding into tissue engineering. Here, we have presented the chondrogenesis ability of NRL nanoparticles against human mesenchymal stem cells (hMSCs). The effective gene expression of in vitro chondrogenesis of hMSCs with administration of NRL was confirmed as revealed by the chondrogenic gene expression analysis. We have successfully for the first time fabricated the cartilage/NRL biocomposites via hMSC spheroid formation, where the administration of NRL nanoparticles exhibited the mechanically stable structure with hard surface structure in the spheroid.

Materials Today Chemistry, 12, 315-323, 2019
 
 




In the research we obtained porous scaffolds based on poly(L-lactide) and synthetic hydroxyapatite(HA) using thermal induced phase separation technique (TIPS) supported by salt leaching process(SL). The obtained series of composite foams differ in hydroxyapatite content(10,25,50,75,90 wt% of the HA in PLLA/HA systems). The infestigated scaffolds porosity ranged from 88 to 98%, density from 0.024 to 0.140 g/cm3. Water contact angle measurements indicated more hydrophilic scaffold surface with indicated more hydrophilic scaffold surface with increasing hydroxyapatite content in the composites. Thermogravimetric analysis revealed higher thermal stability of all composites comparing to neat PLLA, indicated presence of -3 wt% residual sodium chloride in the scaffolds. Differntial scanning calorimetry analysis showed nucleation effet of hydroxyapatite and higher crystallinity of PLLA in the composites as compared to the neat PLLA. The above tests were supplemented with compressive strength measurements, which showed an increase in the Young's modulus and compressive stresses values parallelly with the increase of the filler content. Proliferation tests of MC3T3-E1 mouse calvaria pre-osteoblast cells indicated directly proportional relationship between hydroxyapatite content and cells proliferation rate.

European Polymer Journal, 133, 313-320, 2019
 


 
Using polymeric fibers substrates with different mechanical features and surface topographies (fiber alignments) as microenvironment of cell culture substrates that influences cancer progression and metastatic potential, we have demonstrated systematically the effect of stiffness and topographies on two different types of breast cancer cells (MDA-MB-231 and MCF-7). The cells cultured on different substrates both normoxic and hypoxic conditions showed different morphologies and these morphological differences were correlated with cellular motility and gene expression. We discussed the effect of hypoxia on gene expression and cellular motility to understand the cell-cell and cell-substrate interactions. The progresses of microenvironment-mediated tumor progression in artificial extracellular matrix explored the design criteria to understand the cancer progression mechanism and metastatic potential.

Materials Today Chemistry
29-41, 11, 2019

 

 
  Aim: This study was aimed to assess multiple marker-loaded plasmid DNA-calcium phosphate (CaP) particles to allow a multiple tumor drug delivery for cancer therapy. Despite its widespread use as an in vitro plasmid DNA (pDNA)-loaded calcium phosphate (CaP) particles (pDNA-CaP) transfection, little attention has been given to the multiple deliver system. Methods: In this study, we optimized Ca:P ion ratios by varying ions concentration of simulated body fluid solutions to induce sufficient protein expression and examined the effect of the multiple marker-loaded pDNA-hydroxyapatite (HA) particles including three different markers of pDNA on the transfection to human embryonic kidney (HEK293) cells. Results: The existence of three different proteins inside the cells were produced after successful transfection of much higher multiple pDNAs when using the multiple plasmid-loaded (3-pDNA-HA) particles having 2.4 µm in size.
Conclusion: The 3-pDNA-HA-embedded gel substrate showed effective reverse transfection for HEK293 cells.


Journal of Unexplored Medical Data
doi.org/10.20517/2572-8180.2017.24 (2018)
 

 
   To broaden the knowledge of cytotoxicity of natural rubber latex (NRL) nanoparticles we for the first time examined the latex biocompatibility in vitro against mouse calvaria preosteoblast cells (MC3T3-E1) and human alveolar basal epithelial (A549) cells. For NRL nanoparticles, the half maximal inhibitory concentration (IC50) value for MC3T3-E1 cells is one order of magnitude higher in toxicity as compared to that of A549 cells (3.99 mg/mL for MC3T3-E1 and 0.33 mg/mL for A549 cells). Owing to fractionation of NRL nanoparticles by ultra-centrifuge, the effect of the non-rubber constituents on the cytotoxicity was clarified. The suppression on the proliferation for A549 cells incubated with NRL nanoparticles was demonstrated by the cell cycle distribution. The in vitro study on osteogenic differentiation and expressions of proteins and characteristic genes of MC3T3-E1 cells demonstrated the promising results of the NRL nanoparticles for application in bone tissue engineering.

e-Journal of Soft Materials
1-10, 12, 2017
 

 
   To broaden the knowledge of cytotoxicity of natural rubber latex (NRL) nanoparticles we for the first time examined the latex biocompatibility in vitro against a panel of cancer cells (A549, A2780, and MDA-MB-231). Owing to fractionation of NRL nanoparticles by ultra-centrifuge, the effect of the non-rubber constituents (intermediate of 5.8 wt.% and sediment of 0.2 wt.%) on the cytotoxicity was clarified. For intermediate constituent, the half maximal inhibitory concentration (IC50) values at 24 h was 1.05 mg/mL for A549 cells, which was one order of magnitude higher in toxicity as compared to that for A2780 (0.24 mg/mL) and MDA-MB-231 (0.36 mg/mL) cells. In addition, profound studies including cell cycle arrest abilities and apoptosis induction profiles against cancer cells were discussed in detail. It was found that the constituents exhibit some significant effect on the cell cycle arrest and trigger apoptosis for A2780 cells. This effective apoptosis induction profiles was more prominent in MDA-MB-231 cells incubated with NRL nanoparticles and sediment loading conditions. The percentage of apoptotic cells was ca. 6–8% of the total cells.

Materials Today Chemistry
63-71, 5, 2017
 

 
   Natural rubber latex (NRL) is mainly used around traditional industrial products, but currently their target application is continuously expanding into tissue engineering. To broaden our knowledge of application in tissue engineering of NRL, we have focused on the surface modification of NRL nanoparticles through the biomineralization of hydroxyapatite (HA) using simulated body fluid in order to create a better cytocompatibility with controlled cell adhesion and mineralization properties in osteogenic culture to determine the effect on bone outcomes. Using MC3T3-E1 mouse osteoblastic-like cells incubated with NRL nanoparticles coated with HA layer, we have examined the osteogenic differentiation and expressions of multiple proteins and characteristic genes of mature osteoblast. We have successfully prepared the biocomposites composed of NRL and bone tissue.

Nanocomposites
76-83, 3, 2017
 

 
   The interpretation of the local microenvironment of extracellular matrix for malignant tumor cells is in intimate relation with metastatic spread of cancer cells involving the associated issues of cellular proliferation and drug responsiveness. This study was aimed to assess the combination of both surface topographies (fiber alignments) and different stiffness of the polymeric substrates (PLLA and PCL) and collagen substrates (coat and gel) to elucidate the effect of the cellular morphology on cellular proliferation and drug sensitivities of two different types of breast cancer cells (MDA-MB-231 and MCF-7). The morphological spreading parameter of (Nuclear/Cytoplasm) induced by the anthropogenic substrates has correlated intimately with the cellular proliferation and the drug sensitivity (IC50) of cancer cells. This study demonstrated the promising results of the parameter for the evaluation of cancer cell malignancy.

Journal of Functional Biomaterials
8, 18, 2017

 

 
   The complexation of allophane nanoparticles with cisplatin, cis-dichlorodiammineplatinum (II) (CDDP) to deliver platinum prodrug into cancer cells was investigated. Using human lung carcinoma (A549) cells, profound studies including cellular uptake (endocytosis) analysis of allophane nanoparticle, cell cycle arrest abilities and apoptosis induction profiles were discussed in detail. As well as the features against A549 cells to emerge a promising strategy to enhance their anti-cancer activity and to mitigate side-effects were discussed.

Applied Clay Science
422-429, 143, 2017
 

 
   Progresses of microenvironment-mediated tumor progression in artificial extracellular matrix explore the design criteria to understand the cancer progression mechanism and metastatic potential. This study was aimed to assess the combination of both surface topographies (fiber alignments) and different stiffness of the polymeric substrates (PLLA and PCL) to evaluate the effect on the cellular morphologies, proliferation, motility and gene expression regarding epithelial to mesenchymal transition (EMT) of two different types of breast cancer cells (MDA-MB-231 and MCF-7). The cellular morphologies (roundness and nuclear elongation factor), E-cadherin and vimentin expression, and cellular motility in terms of cellular migration speed, persistent time and diffusivity were discussed comprehensively. We have demonstrated that the microenvironment of cell culture substrates influences cancer progression and metastatic potential.

Journal of Materials Chemistry B
2588-2600, 5, 2017
 

 
 
 Clay minerals are mainly used around traditional cosmetics and industrial products, but currently their target application is continuously expanding into pharmaceutical industry and tissue engineering. To broaden the knowledge of in vitro cytotoxicity of allophane nanoparticles against human cancer cells, the cytotoxicity of both natural and synthetic allophane nanoparticles for cultured human alveolar basal epithelial (A549) cells was examined. For both natural and synthetic allophones, the A549 cell viability was maintained at > 70% for concentration up to 3160 mg/mL, implying higher biocompatibility of allophane nanoparticles as compared with that of hectorite nanoparticles. The cell adhesion kinetics coupled with cytotoxic characteristics against A549 cells was analyzed using quartz crystal microbalance (QCM) technique to distinguish the dynamic cell adhesion signatures.

Applied Clay Science
485-492, 135, 2017

 

 
     Poly-L-lactide (PLLA)/natural hydroxyapatite (n-HA) composite scaffolds with different morphology and porosity were prepared using thermally induced phase separation (TIPS) technique. The morphological features of the scaffolds were observed by field emission scanning electron microscope (FE-SEM). The solvent concentration had much effect on the porosity and pore morphology. The double-stranded deoxyribonucleic acid (ds-DNA) adsorption behavior was investigating with the aim of their applications in gene therapy. The propensity of the scaffolds and n-HA particles to adsorb ds-DNA was assessed by batch experiments at pH 4. The aggregated size of the ds-DNA molecules and agglomeration after adsorption were characterized by dynamic light scattering (DLS) and Fourier transform infrared (FTIR) spectroscopic imaging.
The adsorption data were fitted into the Freundlich equation and the adsorption parameters were assessed. Although the adsorption capacity of the scaffolds was lower as compared to n-HA particles, it was sufficient for ds-DNA adsorption. The in vitro cell culture test was conducted on the scaffolds with human mesenchymal stem cells (hMSCs). The cells on PLLA/n-HA/ds-DNA scaffold showed more significant increases than neat PLLA and PLLA/n-HA scaffolds. 

Polymer

73-81, 56, 2015
 

 
   Clay mineral surfaces have been important for the prebiotic organization and protection of nucleic acids. The morphological observation to provide insight into the adsorption structure and characteristics of single-stranded DNA (ss-DNA) by natural allophane particles was presented. The molecular orbital (MO) computer simulation has been used to probe the interaction of ss-DNA and/or adenosine 5’-monophosphate and allophane with active sites. Our simulations predicted the strand undergoes some extent of the elongation, which induces the alteration of the conformation of the phosphate backbone, base-base distance and excluded volume correlation among bases. This work demonstrates the ss-DNA adsorption by the allophane particles with novel insights into the morphological features and detailed molecular level information. The overall results support a general adsorption mechanism for the ss-DNA/allophane complexation

Applied Clay Science
591-597, 101, 2014

 

 
   To understand the enzymatic degradation behavior of crosslinked polylactide (PLA), the preparation and enzymatic degradation of both thermoplastic (linear) and crosslinked PLAs having pore structure with different dimension were carried out. The porous structures of the linear PLA samples were of micro and nanoporous range prepared by batch foaming at supercritical CO2 and compared with the porous structures of crosslinked PLA (Lait-X) created by salt leaching method. The surface and cross-sectional morphologies of the porous structures were investigated by using scanning electron microscopy. The morphological analysis of Lait-X porous showed a rapid loss of physical features with 120 h of exposure to the proteinase-K enzymatic degradation at 37 °C. Due to higher affinity for water led to enhanced enzymatic activity as compared to the linear PLA porous structures in the micro and nanoporous range.

International Journal of Molecular Science
9793-9808, 15, 2014
 

 
   Allophane nanoparticles were synthesized through a hydrothermal treatment of precursor with Si/Al molar ratio of 0.75 at 100°C for 48 h and characterized by using field emission scanning electron microscopy, pore-size distribution based on the Cranston-Inkley method, 29Si and 27Al magic angle spinning nuclear magnetic resonance, X-ray diffractometry, and energy dispersive X-ray spectroscopy. It is demonstrated that the synthetic allophane nanoparticle leads to a novel allophane-Pt nanocomposite with the metal particle size of ~ 2 nm and narrow size distribution. The synthetic allophane particles promote the reduction of K2PtCl4 to Pt0 and act as a support substratum. The molecular orbital computer simulation was performed to provide insight into the structure and stability of Pt nanoparticles during complexation by the functional (OH)Al(OH2) groups on the wall perforations of the synthetic allophane.

Applied Clay Science
191-196, 95, 2014