Isolation and identification of putative oral cancer stem cells (OSCCs) and changes associated with tumour progression

PhDOral squamous cell carcinomas (OSCC) appear to contain a sub-population of cells endowed with indefinite self-renewal capacity, usually referred to as cancer stem cells (CSCs). These cells are clonogenic and are responsible, in all probability, for tumour initiation and propagation. Cancer cell lines consistently show morphological patterns similar to normal keratinocytes (holoclones, meroclones and paraclones) reflecting a hierarchical organization associated with stem, amplifying and differentiated cells. In-vitro studies of OSCC cell lines have revealed further phenotypic differences in colony formation between the stem and non-stem fractions and have identified cell surface proteins such as CD44, E-Cadherin, ß-catenin and Vimentin which are differentially expressed in association with stemness. Recent studies suggest tumour progression to be associated with Epithelial Mesenchymal Transition (EMT), a process occurring in SCCs which confers invasiveness and motility to CSCs and also suggests a strong involvement of external factors such as those present within the tumour-host microenvironment. Analysis of these properties may provide insight into mechanisms of stem cell fate determination and provide mechanisms for therapeutic targeting of CSCs. Methods and Materials: Immunocytochemistry was used to analyze differential expression of cell surface and internal markers for their role in stemness and EMT, FACS was used to isolate and analyze sub-fractions of cells for clonal studies. Real time videos of cell lines were used to examine cell movement in colonies and cell acquisition of a fibroblastic phenotype. As a test of whether this process is actually EMT, cells were exposed to TGF-ß, a known inducer of EMT, to determine whether this enhanced the formation of the mesenchymal-looking cells at colony margins. Results and Conclusions: Video images showed cells at the margins of holoclones acquiring a motile phenotype. Immunohistochemical analysis revealed that certain stem-cell-related cell surface molecules were expressed at higher levels in holoclones than paraclones and at different levels within holoclones. Expression of CD44 was stronger in the center than at the edge of holoclones suggesting that this molecule plays a role in maintaining a stem-like state. Some images indicated nuclear translocation of CD44 and ß-catenin at colony margins. TGF-ß increased the incidence of mesenchymal-like cells at the edges of colonies and seemed to scatter and mobilize these cells with loss of the normal colony architecture. Further, TNF-α alsogreatly enhanced this effect by working synergistically with TGF-β suggestingthe involvement of external factors such as inflammatory cytokines and other cells present within the tumour-host environment,collectivelycontributing tothe generation of EMT cells with stem potential

Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

<p>Abstract</p> <p>Background</p> <p>Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia.</p> <p>Methods</p> <p>Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA.</p> <p>Results</p> <p>In both cancer biopsies and carcinoma cell lines a subset of CD44^highcells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44^highcarcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44^highcells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance.</p> <p>Conclusions</p> <p>These data indicate that both normal and malignant human epithelial cells with stem-like properties show greater resistance to apoptosis associated with extended G2 cell cycle phase, and that this property is not a consequence of neoplastic transformation. Targeting G2 checkpoint proteins releases these cells from the G2-block and makes them more prone to apoptosis, implying an opportunity for improved therapeutic approaches.</p

Enzyme treatment decreases the size of the CD44-positive population.

<p>Following isolation with trypsin, Accutase or enzyme-free (EF) buffer, CA1 cells were subject to FACS analysis either unstained, stained with an isotype control, or stained for CD44. Unstained and isotype-stained cells were unaffected by method of isolation but marked differences in the proportion of cells classified as staining positive for CD44 are seen.</p

Detection of CD44 variant isoform expression on the CD44^highESA^high cell sub-population.

<p>FACS analysis of CD44 variant isoform expression on cells of the CA1 (A), Met1 (B) and Met2 (C) cell lines isolated using enzyme-free buffer showing the CD44 variants on the y-axis and ESA expression on the x-axis. For all cell lines, similar total levels of CD44 are detected on the CD44^highESA^low and CD44^highESA^high cells but lower levels of the v3, v5, v6 and v9 isoforms are detected on the CD44^highESA^low cells. (D) FACS analysis of CD44 (y-axis) and ESA (x-axis) expression on CA1 (top), Met1 (middle) and Met2 (bottom) cells treated with either trypsin (left) or enzyme-free buffer (right). Loss of detection of CD44 variant isoforms after trypsin treatment results in a large increase in the fraction of motile CSCs within the 5% of cells showing the highest CD44 expression. Representative plots. (E) Quantification of the experiments depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057314#pone-0057314-g004" target="_blank">figure 4D</a>, showing the percentage of EMT CSCs within the 5% CD44^high population after treatment with trypsin or enzyme-free buffer.</p

Trypsin treatment prevents cell surface detection of CD44 variant isoforms.

<p>Histograms showing expression, assessed by FACS analyses of CA1 cells, of CD44 (“epitope 1” antibody, top left panel) and of CD44 variants after treatment with trypsin (green) or enzyme-free buffer (blue). The isotype control is in red.</p

Differential staining for CD44 on cells isolated from fresh tumour samples using different methods.

<p>Panels A–D illustrate the procedure used to identify staining patterns. Cell smears were prepared and co-stained with DAPI (A) with a pan-keratin antibody (B) and with the CD44 “epitope 1” antibody (C). These panels show images manipulated by adjusting capture thresholds in PhotoShop to identify cells with set levels of staining above a background level. For each field, this level was held constant to generate the three images, one for each fluorochrome, containing only the stained cells. These images were then combined, slightly out of lateral register, to allow identification of cells with absent or low levels staining for CD44 or keratin (blue nuclei only), staining for CD44 only (CD44+), keratin only (K+), or both CD44 and keratin (CD44+K+). Treatment with trypsin+collagenase or collagenase alone consistently reduced the fraction of cells that was positive for CD44 (E) and for both CD44 and keratin (F). Displaying the 5 assayed tumours individually (G) demonstrates the striking variation in CD44 staining between tumours, and that this variation is reduced following enzymatic treatment. Statistical analysis was conducted using a Mann-Whitney test.</p

CD44 expression in EMT and non-EMT CSCs.

<p>(A) FACS sorting of CA1, Met1 and Met2 cells by expression of CD44 (using the “epitope 1” antibody that detects all forms of CD44) and ESA. The CD44^highESA^low and CD44^highESA^high sub-populations are gated and show higher levels of staining of CD44^highESA^low cells for CD44. (B) QPCR analysis of CD44 variant gene expression in CD44^highESA^low cells relative to the CD44^highESA^high cells for the CA1, Met1 and Met2 lines shows marked differences in the isoform expression patterns.</p

Quality attributes of dehydrated pear (Pyrus communis L.) as affected by conventional and novel blanching techniques

ABSTRACTA study was conducted to assess the impact of different blanching methods and blanching time on the quality of dried pear slices. Pears are only available seasonally, making the production of high-quality dried slices a valuable commodity. The pears were prepared and sliced before being blanched using three different methods: traditional blanching with hot water, blanching with steam, and microwave blanching for varying lengths of time. After blanching, the slices were cooled and then placed on filter papers to dry. The dried slices were then evaluated for chemical properties, such as sugars, total soluble solids, titratable acidity, ascorbic acid, Brix/acid ratio, ash content, and moisture content. Sensory properties such as color, taste, texture, and overall acceptability were also evaluated using a 9-point hedonic scale. The data generated were analyzed using a two-way analysis of variance (ANOVA) to determine the significant differences between variables. The results showed that the blanching method used had a non-significant effect (p < .05) on the chemical properties of the pear slices, such as total sugars (reducing and non-reducing), total soluble solids, titratable acidity, ascorbic acid, Brix/acid ratio, ash content, and moisture content. However, there was no significant effect on the sensory properties of color, texture, taste, and overall acceptability. In conclusion, blanching is an effective technique for preventing microbial growth and chemical and enzymatic reactions that can lead to the deterioration of dried pear slices. It also helps to remove moisture without adversely affecting the physicochemical and sensory quality attributes of the final product