Non-adhesive Culture Research Articles

Quercetin Suppresses Drug-Resistant Spheres via the p38 MAPK–Hsp27 Apoptotic Pathway in Oral Cancer Cells

BackgroundTreatment failure in oral squamous cell carcinoma (OSCC) leading to local recurrence(s) and metastases is mainly due to drug resistance. Cancer stem cells (CSCs) are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive.MethodsA drug-resistant sphere (DRSP) model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo.ResultsOur data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis) and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27) via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu), suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC.ConclusionsThe p38 MAPK–Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis using Qu combined with Cis may be a treatment strategy to improve prognosis in patients with OSCC.

Dynamic Link between Histone H3 Acetylation and an Increase in the Functional Characteristics of Human ESC/iPSC-Derived Cardiomyocytes

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes in vitro, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA) recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.

Agarose Coated Culture Plate in Tumorsphere Culture of Cervical Cancer Cell Line HeLa: an Alternative to Non Adhesive Culture Plate

Cervical cancer recurs in 90% cases and linked to cancer stem cells that able to self-renew and responsible for recurrence, metastasis, and mortality of cancer. Isolation and identification of cancer stem cells using serum-free medium needs expensive growth factors and consume time. This study try to grow tumor sphere using culture plate coated with 1% agarose as an efficient and economical alternative to non-adhesive culture plate. HeLa cell line was grew in culture plate coated with 1% agarose and non-adhesive culture plate using similar medium and culture condition. Tumor spheres morphology was observed and the colonies were counted in 7 days followed by single cell assay. Tumor spheres then counted for CD133+, CD34+, and Sox2 expression using flowcytometry. Culture plate coated with 1% agarose can be used as an economic and efficient alternative to culture tumor sphere. Using culture plate coated with 1% agarose, the tumor spheres formed in 7 days with similar morphology to non-adhesive culture plate. Tumorsphere had three dimensional – sphere shape that tightly attached, colonized, and overlapped. The tumor sphere colony counts of two plates were statistically have no significant difference (p=0,667). Single cell assay of a tumor sphere shows that it can grow new tumor spheres with similar morphology. The tumor sphere from culture plate coated with 1% agarose express CD133+ and CD34+ as much as 8.78% ± 2.14 and Sox2 as much as 35.30% ± 23.82 whereas tumor sphere from non-adhesive culture plate express CD133+ and CD34+ as much as 62.36% ± 1.06 and Sox2 as much as 98.86% ± 0.56 (p = 0000).

Nonadhesive Culture System as a Model of Rapid Sphere Formation with Cancer Stem Cell Properties

BackgroundCancer stem cells (CSCs) play an important role in tumor initiation, progression, and metastasis and are responsible for high therapeutic failure rates. Identification and characterization of CSC are crucial for facilitating the monitoring, therapy, or prevention of cancer. Great efforts have been paid to develop a more effective methodology. Nevertheless, the ideal model for CSC research is still evolving. In this study, we created a nonadhesive culture system to enrich CSCs from human oral squamous cell carcinoma cell lines with sphere formation and to characterize their CSC properties further.MethodsA nonadhesive culture system was designed to generate spheres from the SAS and OECM-1 cell lines. A subsequent investigation of their CSC properties, including stemness, self-renewal, and chemo- and radioresistance in vitro, as well as tumor initiation capacity in vivo, was also performed.ResultsSpheres were formed cost-effectively and time-efficiently within 5 to 7 days. Moreover, we proved that these spheres expressed putative stem cell markers and exhibited chemoradiotherapeutic resistance, in addition to tumor-initiating and self-renewal capabilities.ConclusionsUsing this nonadhesive culture system, we successfully established a rapid and cost-effective model that exhibits the characteristics of CSCs and can be used in cancer research.

Neural crest-derived multipotent cells in the adult mouse iris stroma

The purpose of this study was to characterize neural crest-derived cells within the adult murine iris. The iris was isolated from P0-Cre/Floxed-EGFP transgenic (TG) mice. The isolated iris cells formed EGFP-positive spheres on non-adhesive culture plates. Immunostaining showed that these EGFP-positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere-forming ability were originally resided in the iris stroma (IS), in vivo. Real-time RT-PCR showed that the EGFP-positive spheres expressed significantly higher levels of the neural crest markers than EGFP-negative spheres and bone marrow-derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest-derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.

Non-adhesive organ culture of human biliary epithelium with stroma

Objective. Explanted tissue has been shown to keep adult human cells in organ culture with a preserved morphology for at least one month as spheres in a non-adhesive organ culture. In the present study, we explored whether also human biliary epithelium can be grown in this manner, because the result may be of interest in studies of hepato-biliary-pancreatic carciogenesis. Material and methods. Small tissue samples were obtained from the gallbladder wall of patients who had been operated upon with cholecystectomy. Fragments of about 300 µm in diameter from each patient were cultured and investigated with light microscopy at the time of explantation and after 5, 10, 20, 30 and 40 days of culture. Scanning and transmission electron microscopy were performed to demonstrate the ultrastructure. Incubation of cultured fragments with the vital dyes revealed a viable epithelium. Results. At the time of explantation, all the tissue fragments had a rough appearance with an uneven, torn periphery, while during the first few days of culture they became rounder with a smooth-looking surface covering the entire circumference. This spheroid morphology persisted for the remainder of the culture period. The core of the fragments harboured connective tissue with vascular elements, fibroblasts and leucocytes. Immunostaining for cytokeratin 7, 19 and 20 revealed a strong positive staining of the epithelium. Conclusions. These results show that biliary epithelium can be grown in vitro in a non-adhesive organ culture with their stroma.

Monocytes secrete interleukin-6 when co-cultured in vitro with benign or malignant autologous fragment spheroids from squamous cell carcinoma patients.

Biopsies from tumour and benign mucosa were removed from patients with head and neck squamous-cell carcinoma (HNSCC), chopped into cubes and transferred to a nonadhesive culture system where in vitro fragment (F)-spheroids were established. The F-spheroids stabilized within 14 days of culture in vitro with epithelial cells and fibroblasts on the surface. F-spheroids were co-cultured with freshly isolated autologous monocytes. The monocytes of 10 of 11 patients secreted interleukin (IL)-6 at a level similar to that of the average monocyte endotoxin-stimulated response. Secreted IL-1beta or tumour necrosis factor-alpha (TNF-alpha) levels greater than 0.1 times the endotoxin-stimulated secretion were determined in one and two of the 11 co-culture experiments, respectively. This different monocyte response to F-spheroids compared with endotoxin stimulation was also present at the mRNA expression level. HNSCC monocytes secreted no IL-6 after co-culture with autologous fibroblasts. When monocytes and F-spheroids were cultured separated by a semipermeable membrane, the IL-6 supernatant level was only approximately 25% of that observed during co-culture with direct contact. F-spheroids secreted only trace amounts of IL-6. In conclusion, monocytes of HNSCC patients generally secrete IL-6, but not IL-1beta or TNF-alpha, after stimulation with epithelial-associated components of F-spheroids upon direct contact and in part by a soluble substance.

Tissue transglutaminase is not increased during apoptosis of HT-1080 human fibrosarcoma cells

Tissue transglutaminase (tTGase), a cytosolic enzyme which catalyzes the covalent cross-linking of proteins, is thought to be involved in the apoptosis. Here, we tested whether tTGase is involved during HT-1080 fibrosarcoma cell apoptosis induced by the YIGSR (Tyr-Ile-Gly-Ser-Arg) peptide. This sequence is derived from the laminin alpha1 chain, and its potency is increased by the formation of a 16mer polymerization using a lysine tree structure. Cells were treated with several different concentrations of Ac-Y 16 for 16 hours, and apoptosis was increased in dose-dependent manner. When assayed by incorporation of [14C] putrescine into succinylated casein, total transglutaminase activity was decreased in parallel with the change in the number of attached cells. Western blot analysis using polyclonal antibody against tTGase showed that the tTGase protein level had not been significantly changed when equal amounts of the protein were applied. To confirm this result, we induced apoptosis of these cells by coating the tissue culture plates with non-adhesive poly-hydroxyethyl methacrylate (HEMA). Western blot analysis showed that the tTGase protein level did not change during this process of apoptosis. Although it has been suggested that tTGase is involved in the process of apoptosis of various cells in vitro and in vivo, our data demonstrate that tTGase is not involved in the process of apoptosis of HT-1080 human fibrosarcoma cell induced by either Ac-Y 16 or a non-adhesive culture surface.

Induction of collagen synthesis in response to adhesion and TGF beta is dependent on the actin-containing cytoskeleton.

Adhesive interactions between cells and the extracellular matrix (ECM) are thought to influence gene expression. Previously, we showed that the regulation of α1(I) collagen expression is anchorage-dependent in Swiss 3T3 fibroblasts (Dhawan and Farmer, 1990). Non-adhesive culture conditions established by suspending cells in methylcellulose containing media result in a suppression of procollagen synthesis. Replating these suspended cells onto a tissue culture dish surface rapidly activates procollagen synthesis, and serum factors are not required for this response. The changes in type I procollagen synthesis reflect the levels of α1(I) collagen mRNA. Induction of procollagen synthesis during replating is the result of regulation at transcriptional and post-transcriptional sites (Dhawan et al., 1991). Collagen mRNAs are destabilized in suspended cells and restabilized when adhesive contacts are restored. Transcription of the α1(I) collagen gene is suppressed in suspended cells and reactivated by 18 hours after reattachment. In addition, the 5’ flanking region of the rat α1(I) collagen gene was found to contain sequences that conferred adhesion-responsiveness to a CAT reporter gene.