Barbora Dvorankova

Differentiation-Dependent Glycosylation of Cells in Squamous Cell Epithelia Detected by a Mammalian Lectin

The squamous stratified epithelia contain a proliferative (harboring mitotic activity) and a differentiating compartment. Due to the potential of protein-carbohydrate interactions to regulate cellular activities we introduced a mammalian lectin to cyto- and histochemical analysis. We answer the questions of whether and to what extent this new probe can pinpoint differentiation-dependent glycosylation changes in sections and in culture of keratinocytes. Material and Methods: Purification and labeling enabled monitoring of galectin-3 reactivity in frozen sections of human and pig epidermis and basal cell carcinomas as well as in culture of keratinocytes. The staining pattern of the lectin was correlated with the staining profile of other cell markers including desmosomal proteins, β1 integrin, and the proliferation marker Ki-67. The Dolichos biflorus agglutinin (DBA) sharing binding reactivity of galectin-3 to the A type histoblood group epitope was used for comparison. Results: Both lectins exhibit suprabasal binding. However, their profiles were not identical, substantiated by lack of coinhibition. Strong DBA reactivity was also observed in a limited number of basal layer cells, namely in cells without the expression of the proliferation marker Ki-67. Cultured mitotic epidermal cells have no reactivity for DBA. Presence of ligands for this plant lectin was connected with decreased positivity of nuclei for Ki-67 and the occurrence of ring-shaped nucleoli, micronucleoli or absence of nucleoli. Considering colocalization the pattern of galectin-3-binding sites coincided with the presence of desmosomal proteins such as desmoplakin-1 and desmoglein but not β1 integrin, a potential ligand. Interestingly, studied basal cell carcinomas expressed no binding sites for galectin-3, while a limited number of cells were DBA-reactive. Conclusion: The expression of galectin-3-binding sites and also DBA-reactive glycoligands correlates with an increased level of differentiation and/or cessation of proliferation in the examined squamous stratified epithelia. Further application of tissue lectins for characterizing ligand expression and its modulation is an important step to reveal functional relevance.

Defining the glycophenotype of squamous epithelia using plant and mammalian lectins. Differentiation-dependent expression of α2,6- and α2,3-linked N-acetylneuraminic acid in squamous epithelia and carcinomas, and its differential effect on binding of the endogenous lectins galectins-1 and -3

A thorough characterization of the properties of squamous epithelial cells is necessary in order to improve our understanding of the functional aspects of normal development and malignant aberrations. Up to now, studies have focused almost exclusively on monitoring distinct protein markers. With our growing awareness of the coding function of glycan chains of cellular glycoconjugates and their interaction with receptors (lectins) in situ, defining the glycophenotype of these cells has become an important issue. Whereas the commonly applied plant lectins are tools used to map the presence and localization of biochemically defined saccharide epitopes, the introduction of endogenous (mammalian) lectins to this analysis enables us to take the step from monitoring the presence of glycan to understanding the functional implications by revealing ligand properties of the detected epitope for tissue lectin. Thus, in this study we investigated a distinct aspect of glycosylation using plant and mammalian lectins, i.e. the linkage type of sialylation. We first mapped the expression profile of the type of sialylation (α2,3‐ or α2,6‐linked) by plant lectins. Based on the hypothesis that this factor regulates accessibility of ligands for endogenous lectins we introduced two labeled galectins to this study. Galectin‐3 (but not galectin‐1) binding was related to cell differentiation in normal adult and developing epithelia, cultured epidermal cells, and carcinomas derived from these epithelia. The presented data suggest that α2,6‐linked N‐acetyl‐D‐neuraminic acid moieties could serve to mask galectin‐3‐reactive glycoepitopes. As a consequence, monitoring of the linkage type of sialic acid in glycans by plant lectins therefore has implications for the extent of glycan reactivity with endogenous lectins, pointing to a potential function of changes in sialylation type beyond these cell and lectin systems.

Biocompatibility of HEMA copolymers designed for treatment of CNS diseases with polymer-encapsulated cells.

Surrounding the cells with a semipermeable polymeric membrane allows transplanting unmatched xenogeneic cells without a risk of their rejection. We prepared and tested several 2‐hydroxyethyl methacrylate (HEMA) copolymers with alkyl methacrylates or acrylates to find out which was the most valuable for cell encapsulation. On the basis of optimum physical properties and good results of cytotoxicity tests, HEMA‐EMA copolymer was chosen as a suitable candidate for encapsulation and immunoprotection of xenogeneic cells before their grafting into the central nervous system (CNS). To characterize the biocompatibility of p(HEMA‐co‐EMA) copolymer in the CNS, we implanted microcapsules made of this hydrogel into the brains of adult rats that were allowed to survive for 0.5, 1, 3, 6, and 9 months. Analysis of histological sections containing the implantation site was aimed at assessment of the cellular density at the implant‐brain interface and identification of cell types participating in a tissue reaction. Our results indicated that the tissue reaction that was observed was caused largely by the implantation procedure because HLA‐DR‐ and GSI‐B4‐positive macrophages/microglia infiltrated mainly the implantation channel. The number of these cells declined with time, which was true also for GFAP‐positive reactive astrocytes, as well as for foreign body giant cells. The amount of connective tissue components surrounding the implanted microcapsules increased only slightly. These findings indicated that p(HEMA‐co‐EMA) hydrogel was well tolerated after implantation in the brain.

Effect of cancer-associated fibroblasts on the migration of glioma cells in vitro

Cancer-associated fibroblasts (CAFs) significantly influence biological properties of many tumors. The role of these mesenchymal cells is also anticipated in human gliomas. To evaluate the putative role of CAFs in glioblastoma, we tested the effect of CAF conditioned media on the proliferation and chemotaxis of glioma cells. The proliferation of glioma cells was stimulated to similar extent by both the normal fibroblasts (NFs) and CAF-conditioned media. Nevertheless, CAF-conditioned media enhanced the chemotactic migration of glioma cells significantly more potently than the media from normal fibroblasts. In order to determine whether CAF-like cells are present in human glioblastomas, immunofluorescence staining was performed on tissue samples from 20 patients using markers typical for CAFs. This analysis revealed regular presence of mesenchymal cells expressing characteristic CAF markers α-smooth muscle actin and TE-7 in human glioblastomas. These observations indicate the potential role of CAF-like cells in glioblastoma biology.

Decrease of Nuclear Reactivity to Growth‐regulatory Galectin‐1 in Senescent Human Keratinocytes and Detection of Non‐uniform Staining Profile Alterations upon Prolonged Culture for Galectin‐1 and ‐3

Multipotent stem cells (source for interfollicular epidermis, hairs and sebaceous glands) are localized in the bulge region of the outer root sheath of hair follicles, while stem cells giving rise to interfollicular epidermis reside in its basal. Using the multifunctional lectin galectin‐1 as a marker to localize accessible binding sites in situ as a step to figure out galectin functionality in stem cells, we studied hair follicle‐derived keratinocytes. Specific nuclear binding of galectin‐1 associated with expression of ΔNp63α, a potential marker of epidermal stem cells, was detected. Binding of chimera‐type galectin‐3 to a nuclear site was not found in parallel assays. During the process of ageing in culture when cells acquire properties of senescence, disappearance of the nuclear signal for galectin‐1 binding was accompanied by a similar decrease of nuclear ΔNp63α expression and increased binding of galectin‐3 to the cell membrane, namely in regions of intercellular contacts. Expression of cytokeratin 10, a marker of the terminal differentiation was seen only in a small fraction of the cell population. These data extend the evidence for nuclear sites with galectin‐1 reactivity in squamous epithelial cells, the expression of which is modulated upon senescence. Moreover, the results document the divergence of galectin‐1 and ‐3 on the level of ligand selection in this cell type, underscoring the importance of the technical aspect to employ tissue lectins as probe and to perform a fingerprinting with several markers of the galectin family in parallel.

Cancer Microenvironment: What Can We Learn from the Stem Cell Niche.

Epidermal stem cells (ESCs) are crucial for maintenance and self- renewal of skin epithelium and also for regular hair cycling. Their role in wound healing is also indispensable. ESCs reside in a defined outer root sheath portion of hair follicle—also known as the bulge region. ECS are also found between basal cells of the interfollicular epidermis or mucous membranes. The non-epithelial elements such as mesenchymal stem cell-like elements of dermis or surrounding adipose tissue can also contribute to this niche formation. Cancer stem cells (CSCs) participate in formation of common epithelial malignant diseases such as basal cell or squamous cell carcinoma. In this review article, we focus on the role of cancer microenvironment with emphasis on the effect of cancer-associated fibroblasts (CAFs). This model reflects various biological aspects of interaction between cancer cell and CAFs with multiple parallels to interaction of normal epidermal stem cells and their niche. The complexity of intercellular interactions within tumor stroma is depicted on example of malignant melanoma, where keratinocytes also contribute the microenvironmental landscape during early phase of tumor progression. Interactions seen in normal bulge region can therefore be an important source of information for proper understanding to melanoma. The therapeutic consequences of targeting of microenvironment in anticancer therapy and for improved wound healing are included to article.

Biological properties of titanium implants covered with hydroxyapatite and zirconia layers by pulsed laser : In vitro study

The biological and physical properties of dental implants coated by the sandwich technique with a thin layer of hydroxyapatite and an interlayer of zirconia were evaluated. The implant samples were covered by pulsed laser deposition. The aim of our study is to evaluate the cytotoxicity and the surface characteristics of the titanium targets modified with zirconia and hydroxyapatite. The titanium substrates were analyzed physically by x-ray diffraction and scanning electron microscopy. We used a direct test of cytotoxicity to compare the prepared samples with other reference materials. No changes in the morphology or the proliferation rate of the cells used were found in the presence of the modified titanium targets. The adhesion, proliferation, and fibronectin expressions of human fibroblasts were also evaluated on the surface of the modified titanium targets. The results show that the modified titanium samples are at least as attractive as the tissue grade polystyrene in promoting fibroblasts’ adhesion a...

Postmitotic basal cells in squamous cell epithelia are identified with Dolichos biflorus agglutinin – functional consequencesNote

Dolichos biflorus agglutinin (DBA) is a plant lectin specifically recognizing α‐N‐acetylgalactosamine. Controversial reports regarding the binding of DBA to the epidermis have been published. Using a double labeling procedure at the single‐cell level, we studied the expression of DBA‐reactive binding sites in conjunction with markers of cell proliferation and differentiation in normal human epidermis, cornea, and malignant tumors as well as in cultured keratinocytes. The results characterize the cells recognized by DBA as postmitotic early differentiating cells, identifiable by their lack of expression of the proliferation marker (Ki‐67). The Golgi complex of a limited number of cultured keratinocytes was recognized by DBA and some of these cells show the accumulation of β1 integrin chain in the Golgi complex. This process seems to be important for the migration of postmitotic cells from the basal to the suprabasal layers.

Biological and physical properties of pulsed-laser-deposited zirconia/hydroxyapatite on titanium : In vitro study

Coating dental implants with hydroxyapatite (HA) may give certain advantages such as active encouragement of new bone growth, a lower rejection rate, and an improved long-term prosthesis fixation. This study examined the mechanical and biological properties of titanium alloy implant cores with an interlayer of zirconia and a coating of HA created using pulsed Laser deposition (PLD). The thickness of the zirconia layer was 50–100 nm, and the HA layer was ∼600 nm. The crystallinity, morphology, wettability, and Ca/P ratio of the HA layer were investigated by electron microscopy, X-ray diffraction, goniometric measurement of contact angle, and wavelength dispersive X-ray analysis. The physical tests indicated adequate mechanical properties and a satisfactory adhesion to a titanium core modified with zirconia and HA. Cell proliferation and metabolic activity of human embryonal lung fibroblasts were determined using counting of harvested cells and providing an MTT assay. it was demonstrated that none of the samples were cytotoxic and their surfaces promoted cell colonization. PLD was found to be a promising method of applying coatings to a metal core for dental implants, and the in vitro biological tests suggest that the crystalline HA coating can improve the biological properties of titanium covered with zirconia.

Abstract LB-A23: Galectin-8 favors growth of endothelial cells and induces production of extracellular matrix by cancer associated fibroblasts

Galectins (Gal) belong to a family of endogenous lectins specifically recognizing sugar motives and playing an important role in the biology of tissue repair and cancer. It has been shown that the tandem-repeat lectin Gal-8 plays an essential role in tumor angiogenesis by improving endothelial cell migration and capillary tube formation. Interestingly by a more prominent way than the proangiogenic growth factor VEGF. Thus, new question arises, i.e. does Gal-8 improves endothelial cell proliferation? To provide answer to that question the MTT and BrDu proliferation assays using HUVEC (human umbilical vein endothelial cells) as a model were used. We found that Gal-8 induces cell proliferation in combination with VEGF, but Gal-8 alone does not exhibit any significant effect. Furthermore, we studied whether human Gal-8 induces production of a bioactive extracellular matrix (ECM) scaffold by cancer associated fibroblasts (CAF). In parallel, the effect of Gal-8 on ECM production by human dermal fibroblasts (HDF) was studied as well. Adding Gal-8 to the culture medium led the CAF to generate a very fine structured 3D meshwork especially rich in fibronectin. Contrary, the ECM production by HDF was not that apparent. Since ECM-cell interactions play an important role in the ability of tumors to spread and form metastasis further research focused on roles of the tandem-repeat-type Gal-8 in other steps of tumorigenesis is warranted by present in vitro study. This study was supported in part by the Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA-1/0404/15 and VEGA-1/0048/15) and the Agency for Science and Research under the contract no. APVV-0408-12 and APVV-14-0731. Citation Format: Peter Gal, Lenka Varinska, Martin Kello, Jan Mojzis, Barbora Dvorankova, Karel Smetana Jr.. Galectin-8 favors growth of endothelial cells and induces production of extracellular matrix by cancer associated fibroblasts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A23.