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Cancer-associated fibroblasts (CAFs) represent a crucial component of cancer microenvironment. CAFs significantly influence biological properties of various types of cancer in terms of local aggressiveness, recurrence, and metastatic behaviour. This chapter summarizes a simple protocol for isolation of normal fibroblasts and their cancer-associated counterparts from normal human skin and mucosa, respectively, as well as from samples of human tumours such as basal/squamous carcinoma, melanoma, and breast cancer, and employment of this procedure for other types of cancer is possible. Isolated fibroblasts can be expanded in vitro and employed for further analysis of, e.g., DNA, RNA, protein, etc.

Our review compares evolution of cancer in the human body to the origin of new species from a common ancestor organism with respect to the theory of Charles Darwin. Moreover, the functional role of the tumor microenvironment as a selective pressure actively participating in cancer progression is also demonstrated. Evolutionary aspects of tumor growth and invasion from the point of view of modern therapeutic challenges and opportunities in precision personalized medicine are also discussed., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

During the last decades, plant extracts containing phytoestrogens have increasingly been used as an alternative to oestradiol hormone replacement therapy. The aim of the present study was to compare the effects of genistein with those of different phytoestrogen-containing plant extracts (from red clover flowers and soybeans) on the proliferation and differentiation of NIH-3T3, HaCaT and MCF-7 cells. Our results showed poor correlations between direct anti/pro-oxidant effects and cytotoxicity of the tested samples. In contrast, genistein showed a direct correlation between significant pro-oxidative effects at cytotoxic concentrations and almost no pro-oxidative effects at non-cytotoxic concentrations. Moreover, the tested red clover extract and genistein induced keratin-8 (luminal and prognostic marker in breast cancer) expression only in MCF-7 cells, but this effect was not seen following treatment with the soybean extract. From this point of view, the effect of consumption of phytoestrogens in oestrogen-positive breast cancer remains to be elucidated. In conclusion, our study demonstrates that various phytoestrogen- containing plant extracts and genistein are able to specifically modulate antioxidant properties and differentiation of studied cells.

Melanoma represents a malignant disease with steadily increasing incidence. UV-irradiation is a recognized key factor in melanoma initiation. Therefore, the efficient prevention of UV tissue damage bears a critical potential for melanoma prevention. In this study, we tested the effect of UV irradiation of normal keratinocytes and their consequent interaction with normal and cancer-associated fibroblasts isolated from melanoma, respectively. Using this model of UV influenced microenvironment, we measured melanoma cell migration in 3-D collagen gels. These interactions were studied using DNA microarray technology, immunofluorescence staining, single cell electrophoresis assay, viability (dead/life) cell detection methods, and migration analysis. We observed that three 10 mJ/cm 2 fractions at equal intervals over 72 h applied on keratinocytes lead to a 50% increase (p < 0.05) in in vitro invasion of melanoma cells. The introduction cancer-associated fibroblasts to such model further significantly stimulated melanoma cells in vitro invasiveness to a higher extent than normal fibroblasts. A panel of candidate gene products responsible for facilitation of melanoma cells invasion was defined with emphasis on IL-6, IL-8, and CXCL-1. In conclusion, this study demonstrates a synergistic effect between cancer microenvironment and UV irradiation in melanoma invasiveness under in vitro condition.

The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease. For B‑Raf mutated melanomas, treatment with mutation‑specific drug inhibitors may be used. Unfortunately, tumors frequently acquire resistance to the treatment. Tumor microenvironment, namely cancer‑associated fibroblasts, largely influence this acquired resistance. In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B‑Raf V600E mutated). The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma‑associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B‑Raf inhibitor therapy, yet before clinical progression of the disease. Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates. MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma. Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression. A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)‑β1. Immunohistochemistry confirmed that melanoma cells potently produce TGF‑β1. Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale. Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels. The present results support recent experimental findings that tumor microenvironment is driving resistance to B‑Raf inhibition in patients with melanoma. Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

Melanoma represents a cancer with increasing incidence worldwide and limited curability of advanced stages of the disease. Similarly to other types of tumors, the microenvironment is an important factor that participates in the control of melanoma biological properties. This review summarizes data regarding the role of the microenvironment, namely fibroblasts, keratinocytes and infiltrating immune cells, on melanoma growth and spreading. The role of embryonic microenvironment on melanoma cell biological properties is also discussed. The potential of therapeutic targeting of the melanoma microenvironment is demonstrated.

Background/aim: Having previously initiated genome-wide expression profiling in head and neck squamous cell carcinoma (HNSCC) for regions of the tumor, the margin of surgical resecate (MSR) and normal mucosa (NM), we here proceed with respective analysis of cases after stratification according to the expression status of tenascin (Ten)., Materials and Methods: Tissue specimens of each anatomical site were analyzed by immunofluorescent detection of Ten, fibronectin (Fn) and galectin-1 (Gal-1) as well as by microarrays., Results: Histopathological examination demonstrated that Ten + Fn + Gal-1 + co-expression occurs more frequently in samples of HNSCC (55%) than in NM (9%; p<0.01). Contrary, the Ten - Fn + Gal-1 - (45%) and Ten - Fn - Gal-1 - (39%) status occurred with significantly (p<0.01) higher frequency than in HNSCC (3% and 4%, respectively). In MSRs, different immunophenotypes were distributed rather equally (Ten + Fn + Gal-1 + =24%; Ten - Fn + Gal-1 - =36%; Ten - Fn - Gal-1 - =33%), differing to the results in tumors (p<0.05). Absence/presence of Ten was used for stratification of patients into cohorts without a difference in prognosis, to comparatively examine gene-activity signatures. Microarray analysis revealed i) expression of several tumor progression-associated genes in Ten + HNSCC tumors and ii) a strong up-regulation of gene expression assigned to lipid metabolism in MSRs of Ten - tumors, while NM profiles remained similar., Conclusion: The presented data reveal marked and specific changes in tumors and MSR specimens of HNSCC without a separation based on prognosis., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The nonsyndromic cleft is one of the most frequent congenital defects in humans. Clinical data demonstrated improved and almost scarless neonatal healing of reparative surgery. Based on our previous results on crosstalk between neonatal fibroblasts and adult keratinocytes, the present study focused on characterization of fibroblasts prepared from cleft lip tissue samples of neonates and older children, and compared them with samples isolated from normal adult skin (face and breast) and scars. Although subtle variances in expression profiles of children and neonates were observed, the two groups differed significantly from adult cells. Compared with adult cells, differences were observed in nestin and smooth muscle actin (SMA) expression at the protein and transcript level. Furthermore, fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine, transforming growth factor-β1 (TGF-β1). Dysregulation of the TGF-β signalling pathway, including low expression of the TGF-β receptor II, may contribute to reducing scarring in neonates. Fibroblasts of facial origin also exhibited age independent differences from the cells prepared from the breast, reflecting the origin of the facial cells from neural crest-based ectomesenchyme.

Background: Knowledge of the phenotypic pattern of oral squamous epithelium is important in the histopathologic evaluation of lesions including cancer. The literature on normal epithelium is controversial as the phenotype has not been evaluated in samples from completely healthy tissue donors without a history of tobacco and alcohol exposure., Methods: In this study, we evaluated normal upper lip fornix and gingival mucosa from carefully selected young healthy donors without a history of smoking and alcohol exposure, and keratin types 8, 10, 14, and 17, filaggrin, and Ki67 were investigated in these donors. The results were compared with profile of epithelium from leukoplakia., Results: The results demonstrated that the phenotypic patterns of gingiva and upper lip fornix mucosa were different. Surprisingly, a high proportion of gingival samples exhibited keratin 8 and a suprabasal signal for keratin 14. These patterns were compared with that of human oral leukoplakia, and some phenotypic similarities were noted., Conclusions: These results demonstrated oral epithelium phenotypic plasticity based on functional requirements of the microenvironment, which can be used in diagnosis., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Incidence of malignant melanoma is increasing globally. While the initial stages of tumors can be easily treated by a simple surgery, the therapy of advanced stages is rather limited. Melanoma cells spread rapidly through the body of a patient to form multiple metastases. Consequently, the survival rate is poor. Therefore, emphasis in melanoma research is given on early diagnosis and development of novel and more potent therapeutic options. The malignant melanoma is arising from melanocytes, cells protecting mitotically active keratinocytes against damage caused by UV light irradiation. The melanocytes originate in the neural crest and consequently migrate to the epidermis. The relationship between the melanoma cells, the melanocytes, and neural crest stem cells manifests when the melanoma cells are implanted to an early embryo: they use similar migratory routes as the normal neural crest cells. Moreover, malignant potential of these melanoma cells is overdriven in this experimental model, probably due to microenvironmental reprogramming. This observation demonstrates the crucial role of the microenvironment in melanoma biology. Indeed, malignant tumors in general represent complex ecosystems, where multiple cell types influence the growth of genetically mutated cancer cells. This concept is directly applicable to the malignant melanoma. Our review article focuses on possible strategies to modify the intercellular crosstalk in melanoma that can be employed for therapeutic purposes.

Background/aim: Expression profiling was performed to delineate and characterize the impact of malignancy by comparing tissues from three sites of head and neck cancer of each patient, also determining interindividual variability., Materials and Methods: Genome-wide analysis was carried out covering the expression of 25,832 genes with quantification for each site of seven patients with tonsillar or oropharyngeal squamous cell carcinoma. Immunohistochemical analysis was performed for adhesion/growth-regulatory galectins, three pro-inflammatory chemo- and cytokines and keratins., Results: Up- and down-regulation was found for 281 (tumor vs. normal) and 276 genes (transition zone vs. normal), respectively. The profile of the transition zone had its own features, with similarity to the tumor. Galectins were affected in a network manner, with differential regulation and interindividual variability between patients, also true for keratins and the chemo- and cytokines., Conclusion: These results underline special features at each site of specimen origin as well as the importance of analyzing galectins as a network and of defining the expression status of the individual patient prior to reaching clinically relevant conclusions., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

An ageing population is a typical feature of many developed countries across the world. Analyzed from a biomedical and philosophical point of view, this phenomenon is also a potential risk factor for social sustainability of communities. The association between ageing and cancer seems to be more than apparent. Therefore, the further increase of epidemic-like incidence of malignant tumors in a population can be expected in the near future. Elderly people usually suffer from age-dependent diseases, and such polymorbidity can seriously affect the treatment of malignant tumors. Such an impending situation may be associated with multiple medical, social and economic issues. This article summarizes data about the possible molecular mechanism influencing rapid spreading of tumors in the elderly population. Reduction of the activity of DNA repair machinery is a likely genetic cause. Besides this, even epigenetic mechanisms can influence this process. In this context, the role of cancer stroma in controlling multiple biological properties of tumors is a prospective target for translational research with potential therapeutic outcomes., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: To assess the impact of Acanthamoeba keratitis (AK) and amniotic membrane transplantation (AMT) in corneal explants on presence of two multifunctional endogenous lectins, i.e. galectins-1 and -7., Methods: Ten corneal explants from AK patients (five with previous AMT and five controls without this treatment) and seven specimens of disease-free control cornea were processed by indirect fluorescent immunohistochemistry., Results: Immunostaining for both galectins was obtained in the epithelium, stroma and the endothelial layer of all controls, with the strongest positivity in the epithelium. Significantly decreased intensity for galectin-1 was recorded in the epithelium of corneal explants from patients with AK and AMT. The signal for galectin-7 was significantly decreased in the epithelium of AK patients and normalized after AMT., Conclusions: AMT has a marked impact on presence of the two galectins in opposite directions, encouraging complete profiling for this family of endogenous effectors.

Estrogen deprivation is considered responsible for many age-related processes, including poor wound healing. Guided by previous observations that estradiol accelerates re‑epithelialization through estrogen receptor (ER)‑β, in the present study, we examined whether selective ER agonists [4,4',4''-(4-propyl [1H] pyrazole-1,3,5-triyl)‑trisphenol (PPT), ER‑α agonist; 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), ER‑β agonist] affect the expression of basic proliferation and differentiation markers (Ki‑67, keratin‑10, ‑14 and ‑19, galectin‑1 and Sox‑2) of keratinocytes using HaCaT cells. In parallel, ovariectomized rats were treated daily with an ER modulator, and wound tissue was removed 21 days after wounding and routinely processed for basic histological analysis. Our results revealed that the HaCaT keratinocytes expressed both ER‑α and ‑β, and thus are well-suited for studying the effects of ER agonists on epidermal regeneration. The activation of ER‑α produced a protein expression pattern similar to that observed in the control culture, with a moderate expression of Ki‑67 being observed. However, the activation of ER‑β led to an increase in cell proliferation and keratin‑19 expression, as well as a decrease in galectin‑1 expression. Fittingly, in rat wounds treated with the ER‑β agonist (DPN), epidermal regeneration was accelerated. In the present study, we provide information on the mechanisms through which estrogens affect the expression patterns of selected markers, thus modulating keratinocyte proliferation and differentiation; in addition, we demonstrate that the pharmacological activation of ER-α and -β has a direct impact on wound healing.

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.

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.

Cancer-associated fibroblasts are bioactive elements influencing the biological properties of malignant tumors. Their origin from different cell types has been established, and the possibility of their formation by epithelial-to-mesenchymal transition from cancer cells is under debate. This study shows that human cancer cells grafted to nu/nu mice induced formation of tumor stroma with the presence of typical smooth muscle actin-containing cancer-associated fibroblasts. These cells seem to be of the host origin because they are not recognized by an antibody specific for human vimentin, as was also verified in vitro. These results suggest that cancer-associated stromal fibroblasts are not formed by epithelial-to-mesenchymal transition from cancer cells.

Detailed comparative analysis of at first sight not related process cascades is a means toward this aim: to trace common effector mechanisms and hereby eventually inspire innovative routes for therapeutic management. Following this concept, promotion of tumor progression by stroma, especially cancer-associated fibroblasts and smooth muscle actin-positive myofibroblasts, and beneficial activity of respective cells in wound healing have helped to delineate the involvement of endogenous lectins of the family of galectins. In addition to initiating conversion of fibroblasts to myofibroblasts, galectin-1 instructs the cells to produce a structurally complex extracellular matrix. This bioscaffold is useful for keratinocyte culture, also apparently operative in ameliorating wound healing. These functional aspects encourage to study in detail how lectin-(glycan) counterreceptor display is orchestrated. Such insights are assumed to have potential to contribute to rationally manipulate stem/precursor cells as resource in regenerative medicine.

Skin healing process is postnatally always associated with scarring of various extent. Based on the clinical experience of plastic surgeons, the healing after lip cleft reconstruction is surprisingly almost scar-less when it is carried out within a few first days after birth. This phenomenon is not seen in delayed cases. In order to decipher causative mechanism, we have isolated and studied principal cell populations, keratinocytes and fibroblast, from residual tissue samples after reconstructive operation (N=39) performed at various age (0-9 years). These cells play the pivotal role in the healing and that is why we focused on description of their phenotype and also functionality with respect to age. We have identified a population of remarkably small cells in explants from newborns (day 0-10). These small cells were strongly positive for markers of low differentiated keratinocytes, keratin-8 and -19, and moreover also for vimentin. In the explants cultures from older babies this population was missing. Fibroblasts from newborns and older patients differed namely in terms of nestin expression and also in the production of extracellular matrix components. We conclude that in vitro described properties of keratinocytes and fibroblasts in newborns could participate on the almost scar-less wound healing in earliest neonatal period.

Background: The most demanding challenge in research on molecular aspects within the flow of biological information is posed by the complex carbohydrates (glycan part of cellular glycoconjugates). How the 'message' encoded in carbohydrate 'letters' is 'read' and 'translated' can only be unraveled by interdisciplinary efforts., Scope of Review: This review provides a didactic step-by-step survey of the concept of the sugar code and the way strategic combination of experimental approaches characterizes structure-function relationships, with resources for teaching., Major Conclusions: The unsurpassed coding capacity of glycans is an ideal platform for generating a broad range of molecular 'messages'. Structural and functional analyses of complex carbohydrates have been made possible by advances in chemical synthesis, rendering production of oligosaccharides, glycoclusters and neoglycoconjugates possible. This availability facilitates to test the glycans as ligands for natural sugar receptors (lectins). Their interaction is a means to turn sugar-encoded information into cellular effects. Glycan/lectin structures and their spatial modes of presentation underlie the exquisite specificity of the endogenous lectins in counterreceptor selection, that is, to home in on certain cellular glycoproteins or glycolipids., General Significance: Understanding how sugar-encoded 'messages' are 'read' and 'translated' by lectins provides insights into fundamental mechanisms of life, with potential for medical applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: Although the exact underlying mechanisms are still unknown, Plantago lanceolata L. (PL) water extracts are frequently used to stimulate wound healing and to drain abscesses. Therefore, in this experimental study the effect of PL water extract on skin wound healing was studied in Sprague-Dawley rats., Methods: Two excisional and one incisional skin wounds were performed on the back of each rat. Wounds were treated for three consecutive days with two different concentrations of the aqueous extract of PL. Rats were sacrificed 7, 14, and 21 days after surgery. Samples of wounds were processed for macroscopic (excisions - wound contraction measurement), biomechanical (incisions - wound tensile strength (TS) measurement) and histological examination (excisions)., Key Findings: It was shown that open wounds treated with PL extract contained myofibroblasts and demonstrated significantly higher contraction rates. Furthermore, significantly increased wound TSs were recorded in treated rats as a consequence of increased organization of extracellular matrix proteins, such as the collagen type 1., Conclusions: We demonstrated that PL aqueous extract improves skin wound healing in rats. However, further research need to be performed to find optimal therapeutic concentration, and exact underlying mechanism prior obtained results may be introduced into the clinical practice., (© 2014 Royal Pharmaceutical Society.)

Background/aim: Stromal cells in the tumor microenvironment are primarily considered as sources of promalignant factors. The objective of our study was to define the effect of extracellular matrix (ECM) produced by normal dermal or cancer-associated fibroblasts exposed to adhesion/growth-regulatory lectin galectin-1 on human umbilical vein endothelial cells (HUVECs)., Materials and Methods: Fibroblasts were cultured for 10 days with lectin, followed by removing cellular constituents after an osmotic shock. Freshly-isolated HUVECs were placed on the ECM. In parallel, HUVECs were seeded on untreated and gelatin-coated surfaces as controls. A positive control for growth of HUVECs culture using medium supplemented with vascular endothelial growth factor completed the test panel. Cells were kept in contact to the substratum for two days and then processed for immunocytochemistry., Results: HUVECs seeded on fibroblast-generated ECM presented a comparatively high degree of proliferation. Furthermore, contact to substratum produced by tumor-associated fibroblasts led to generation of a meshwork especially rich in fibronectin., Conclusion: Galectin-1 is apparently capable to trigger ECM production favorable for growth of HUVECs, prompting further work on characterizing structural features of the ECM and in situ correlation of lectin presence, ECM constitution and neoangiogenesis., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Cancer-associated fibroblasts (CAFs) play a role in the progression of malignant tumors. They are formed by conversion of fibroblasts to smooth muscle α-actin-positive (SMA-positive) myofibroblasts. Polyamines are known to change the arrangement of the actin cytoskeleton by binding to the anionic actin. We tested the effect of the synthetic polyamine BPA-C8 on the transition of human dermal fibroblasts to myofibroblasts induced either by TGF-β1 alone or by TGF-β1 together with adhesion/growth-regulatory galectin-1. Pre-existing CAFs, myofibroblasts from pancreatitis, and rat smooth muscle cells were also exposed to BPA-C8. BPA-C8 impaired myofibroblast formation from activated fibroblasts, but it had no effect on cells already expressing SMA. BPA-C8 also reduced the occurrence of an extracellular matrix around the activated fibroblasts. The reported data thus extend current insights into polyamine activity, adding interference with tumor progression to the tumor-promoting processes warranting study., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background Information: The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns., Results: We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation., Conclusions: The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies., (© 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Increasing evidence attributes tumour fates to a small population of cells (cancer stem cells) capable of surviving therapeutic interventions. Investigation of their characteristics, especially in cross-talk with other cell types of the tumour microenvironment, can pave the way to innovative therapeutic concepts. The central issue of this study was to evaluate the impact of stroma on tumour cells with stem cell-like features in a squamous cell carcinoma model (FaDu). Six different types of experimental conditions were tested using distinct compositions of the culture system, and both morphologic and molecular features of the tumour cells were analysed. In detail, FaDu cells alone were used as a control, compared to tumour cells from co-culture, with squamous cell cancer-derived stromal fibroblasts or normal skin human fibroblasts, both in the direct and indirect (insert) systems, adding analysis of side population cells of FaDu culture. Measurements were taken on days 2, 7 and 9 of culture and immediately after preparation in the case of the side population. A panel of antibodies against keratins 8, 10, 19, stem cell markers CD29, CD44, CD133, as well as biotinylated adhesion/growth-regulatory galectin 1 served as a toolbox for phenotypic characterization. Co-culture with fibroblasts prepared from tumour stroma and with dermal fibroblasts affected marker presentation, maintaining an undifferentiated stage phenotypically related to stem cells. Side-population cells showed close relationship to cancer stem cells in these characteristics. In conclusion, normal and tumour stromal fibroblasts are capable of shifting the marker expression profile of FaDu cells to a stem cell-like phenotypic pattern in co-culture.

The Rho GTPase Rac1 is a multifunctional protein working through different effector pathways. The emerging physiological significance of glycanlectin recognition gives reason to testing the possibility for an influence of modulation of Rac1 expression on these molecular aspects. Using human colon adenocarcinoma (SW620) cells genetically engineered for its up- and down-regulation (Rac1+ and Rac1- cells) along with wild-type and mock-transfected control cells, the questions are addressed whether the presence of adhesion/growth-regulatory galectins and distinct aspects of cell surface glycosylation are affected. Proceeding from RT-PCR data to Western blotting after two-dimensional gel electrophoresis and flow cytofluorimetry with non-crossreactive antibodies against six members of this lectin family (i.e. galectins-1, -3, -4, -7, -8 and -9), a reduced extent of the presence of galectins-1, -7 and -9 was revealed in the case of Rac1 cells. Application of these six galectins as probes to determination of cell reactivity for human lectins yielded relative increases in surface labelling of Rac1- cells with galectins-1, -3 and -7. Examining distinct aspects of cell surface glycosylation with a panel of 14 plant/fungal lectins disclosed a decrease in α2,6-sialylation of N-glycans and an increase in PNA-reactive sites (i.e. non-sialylated core 1 O-glycans), two alterations known to favour reactivity for galectins-1 and -3. Thus, manipulation of Rac1 expression selectively affects the expression pattern within the galectin network at the level of proteins and distinct aspects of cell surface glycosylation.

With the increasing demand for noninvasive approaches in monitoring head and neck cancer, circulating nucleic acids have been shown to be a promising tool. We focused on the global transcriptome of serum samples of head and neck squamous cell carcinoma (HNSCC) patients in comparison with healthy individuals. We compared gene expression patterns of 36 samples. Twenty-four participants including 16 HNSCC patients (from 12 patients we obtained blood samples 1 year posttreatment) and 8 control subjects were recruited. The Illumina HumanWG-6 v3 Expression BeadChip was used to profile and identify the differences in serum mRNA transcriptomes. We found 159 genes to be significantly changed (Storey's P value <0.05) between normal and cancer serum specimens regardless of factors including p53 and B-cell lymphoma family members (Bcl-2, Bcl-XL). In contrast, there was no difference in gene expression between samples obtained before and after surgery in cancer patients. We suggest that microarray analysis of serum cRNA in patients with HNSCC should be suitable for refinement of early stage diagnosis of disease that can be important for development of new personalized strategies in diagnosis and treatment of tumours but is not suitable for monitoring further development of disease.

Malignant melanoma is a highly aggressive tumor with increasing incidence and high mortality. The importance of immunohistochemistry in diagnosis of the primary tumor and in early identification of metastases in lymphatic nodes is enormous; however melanoma phenotype is frequently variable and thus several markers must be employed simultaneously. The purposes of this study are to describe changes of phenotype of malignant melanoma in vitro and in vivo and to investigate whether changes of environmental factors mimicking natural conditions affect the phenotype of melanoma cells and can revert the typical in vitro loss of diagnostic markers. The influence of microenvironment was studied by means of immunocytochemistry on co-cultures of melanoma cells with melanoma-associated fibroblast and/or in conditioned media. The markers typical for melanoma (HMB45, Melan-A, Tyrosinase) were lost in malignant cells isolated from malignant effusion; however, tumor metastases shared identical phenotype with primary tumor (all markers positive). The melanoma cell lines also exerted reduced phenotype in vitro. The only constantly present diagnostic marker observed in our experiment was S100 protein and, in lesser extent, also Nestin. The phenotype loss was reverted under the influence of melanoma-associated fibroblast and/or both types of conditioned media. Loss of some markers of melanoma cell phenotype is not only of diagnostic significance, but it can presumably also contribute to biological behavior of melanoma. The presented study shows how the conditions of cultivation of melanoma cells can influence their phenotype. This observation can have some impact on considerations about the role of microenvironment in tumor biology.

In the present investigation a novel series of chalcone analogues were synthesized and evaluated for their anti-proliferative activity in human umbilical vein endothelial cells (HUVECs). Among 14 tested compounds, chalcone analogue (E)-3-(2'-methoxybenzylidene)-4-chromanone (KRP6) exhibited the most potent activity with IC50 19 μM. Moreover, HUVECs exhibited divergent, even opposing concentration-dependent responses to KRP6. This compound was the most potent inhibitor of cell proliferation and extracellular matrix formation (fibronectin and type IV collagen) at higher concentrations (20-50 μM). In contrast, KRP6 stimulated the compensatory increase in proliferative activity including extracellular matrix formation at low concentrations (1, 10 μM). KRP6 concentration-dependently modulated phosphorylation of Akt and mitogen-activated protein kinases such as extracellular signal-regulated kinase-1/-2 and p38 kinase, suggesting that these pathways play a role in the effect mediated by this compound. In addition, we found a selective effect on activated endothelial cells, in particular with resting endothelial cells. In conclusion, KRP6 is a potent modulator of selected steps of the angiogenic process in vitro. Accordingly, further in vivo research should be performed to facilitate its use in clinical practice.

The emerging insights into the physiological significance of endogenous lectins prompted us to characterize the effect of monosubstitution with poly(ethylene glycol) (PEG; 5 kDa) on a human lectin. As role model, we used a member of the galectin family, that is, galectin-2, the Cys57Met (single-site) mutant and its monoPEGylated derivative. The activities of these three proteins were comparatively studied by biochemical, cell biological, and histochemical methods, using surface-immobilized glycoproteins, different types of cells presenting gangliosides or (glyco)proteins as counterreceptors in vitro and tissue sections. PEGylation led to decreases in affinity/signal intensity with context dependence. The introduction of the mutation, too, can influence reactivity. Assays on haemagglutination and inhibition of cell proliferation underscored that mutational engineering and substitution can (but must not necessarily) affect this protein's activity. Serum clearance in rats was markedly retarded by PEGylation. Overall, the bulky substitution, spatially comparable to N-glycans, can markedly reduce binding of the galectin to physiological binding sites.

Introduction: One route of translating the information encoded in the glycan chains of cellular glycoconjugates into physiological effects is via receptor (lectin) binding. A family of endogenous lectins, sharing folding, a distinct sequence signature and affinity for β-galactosides (thus termed galectins), does so effectively in a context-dependent manner., Areas Covered: An overview is given on the multifunctional nature of galectins, with emphasis on galectin-1. The broad range of functions includes vital processes such as adhesion via glycan bridging, glycoconjugate transport or triggering signaling relevant, for example, for growth regulation. Besides distinct glycoconjugates, this lectin can also interact with certain proteins so that it can target counterreceptors at all sites of location, that is, in the cytoplasm and/or nucleus, at both sides of the membrane or extracellularly. Approaches to strategically exploit galectin activities with therapeutic intentions are outlined., Expert Opinion: The wide versatility of sugar coding and the multifunctionality of galectin-1 explain why considering to turn the protein into a therapeutic target is an ambitious aim. Natural pathways shaped by physiologic master regulators (e.g., the tumor suppressor p16(INK4a)) are suggested to teach inspiring lessons as to how the lectin might be recruited to clinical service.

Galectins are potent effectors of cell adhesion and growth regulation. Their expression as comples network necessitates systematic study of each member of this family. Toward this aim, we here focus on the tandem-repeat-type galectin-9. Its presence is monitored in normal squamous epithelium of the head and neck, the surgical margin, and four types of squamous cell carcinoma. Lectin presence was detected in cells of the basal layer of the epithelium. All galectin-9-negative epithelia showed aberrant positivity for keratins 14 and 19. The surgical margin presented either a normal pattern of galectin-9 and keratin presence or a mosaic-like presence/absence of galectin-9 and aberrant expression of both keratins 14 and 19. All studied specimens of squamous cell carcinoma were negative for galectin-9. When biotinylated galectin-9, or its N-terminal domain, was tested, no significant tissue reactivity for both probes was observed. Neuraminidase treatment generated reactivity to the N-domain. In conclusion, galectin-9 is expressed in the majority of samples of normal epithelium, along with regular presence of keratins 14 or 19. This lectin can represent a potential marker of normality in the cases of the studied squamous cell epithelia., (© 2012 John Wiley & Sons A/S.)

It has been shown that fibroblasts within the stroma of malignant tumours can affect the tumour's biological character, influencing such properties as local aggressiveness and metastasis potential. This influence is asserted via paracrine secretion of multiple cell factors, including chemokines. This study demonstrates that both normal keratinocytes and cancer cells can stimulate the secretion of chemokines IL-8 and CXCL-1 from normal dermal fibroblasts and stromal fibroblasts from squamous cell carcinoma. The effect of epithelia on normal fibroblasts leads to a transient secretory change, in contrast to stromal fibroblasts which generate a more prolonged one. This observation demonstrates that stimulated expression of both IL-8 and CXCL-1 is not specific to cancer, supporting the hypothesis that similar mechanisms exist between wound healing and oncogenesis. It also shows that stromal fibroblasts isolated from a tumour have significantly different features from normal fibroblasts.

Data about the possible correlation between reduction of the regeneration capacity in the course of phylogeny and formation of malignant tumours have been summarized from invertebrates to mammals. The evolutionarily increasing complexity of body building plane and expectancy of longevity in the course of phylogeny seems to be grossly negatively correlated with diminished regeneration capacity, but positively with increased occurrence of malignant tumours. A certain evolution-based switch-off mechanism reducing the extent of regeneration in developmentally complicated and long-living animals such as mammals and birds can be hypothesized and benefits of loss of this ability are discussed. This high incidence of malignancies seems to be related, in addition to other factors, to prolonged and cumulative exposure to cancerogenic stimuli in the course of lifetime. Longevity, supported by the progress and availability of medical care to the population, has been unveiling this phenomenon during recent decades. From this point of view, ageing represents the main risk for cancer acquisition. The probable role of microenvironment in all the discussed phenomena such as healing/regeneration, inflammation, and cancer is discussed and targeting of microenvironment is consequently predicted as a possible therapeutic target where controlled manipulation may represent a new approach to the treatment of cancer patients.

Tumor stroma is an active part influencing the biological properties of malignancies via molecular cross-talk. Cancer-associated fibroblasts play a significant role in this interaction. These cells frequently express smooth muscle actin and can be classified as myofibroblasts. The adhesion/growth-regulatory lectin galectin-1 is an effector for their generation. In our study, we set the presence of smooth muscle actin-positive cancer-associated fibroblasts in relation to this endogenous lectin and an in vivo competitor (galectin-3). In squamous cell carcinomas of head and neck, upregulation of galectin-1 presence was highly significantly correlated to presence of smooth muscle actin-positive cancer-associated fibroblasts in the tumor (p = 4 × 10(-8)). To pinpoint further correlations on the molecular level, we applied microarray analyses to the transcription profiles of the corresponding tumors. Significant correlations of several transcripts were detected with the protein level of galectin-1 in the cancer-associated fibroblasts. These activated genes (MAP3K2, TRIM23, PTPLAD1, FUSIP1, SLC25A40 and SPIN1) are related to known squamous-cell-carcinoma poor-prognosis factors, NF-κB upregulation and splicing downregulation. These results provide new insights into the significance of presence of myofibroblasts in squamous cell carcinoma., (Copyright © 2012 UICC.)

Background Information: Considering an analogy between wound healing and tumour progression, we studied chemokine and cytokine transcription and expression in normal fibroblasts by co-culture and in situ., Results: Whole-genome transcriptome profiling revealed strong upregulation for the interleukin (IL)-6, IL-8 and the chemokine CXCL-1 in in vitro co-cultures of normal fibroblasts with either normal or malignant epithelial cells compared to fibroblast cultures. The same ILs/chemokines were distinctly upregulated in clinical samples of squamous cell carcinoma when compared with paired normal mucosae. Analysis of culture supernatants showed that during the course of co-culture of the fibroblasts with the epithelial cells, IL-6, IL-8 and CXCL-1 were secreted to the culture medium. Experiments with addition of any of the proteins to the culture medium supported the notion that these ILs/chemokines strongly contributed to maintenance of a low-differentiation phenotype of epithelial cells, evaluated by the detection of keratin-8. Simultaneous addition of all factors increased the extent of the effect. These studies were extended by experiments with epithelial cells, either cultured in medium conditioned by preceding use for malignant keratinocytes without and in the presence of normal or cancer-associated fibroblasts or medium containing antibodies against IL-6, IL-8 and CXCL-1., Conclusions: Our results indicate an analogy between wound healing and tumour growth, support the importance of epithelial-mesenchymal interaction in this model system and establish a potential bio-inspired anticancer therapy., (Copyright © 2012 Wiley-Liss, Inc.)

It is widely recognized that stromal fibroblasts significantly influence biological properties of multiple tumors including breast cancer. However, these epithelial-mesenchymal interactions seem to be essential in tumor biology and it is not fully clear whether this interaction is tumor type-specific or has a more general non-specific character. To elucidate this question, we tested the effect of cancer-associated fibroblasts (CAFs) isolated from different types of tumors (breast cancer skin metastasis, cutaneous basal cell carcinoma and melanoma, squamous cell carcinoma arising from oral cavity mucous membrane) on the EM-G3 breast cancer cell line. The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by CAFs prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. The CAFs also elevated the number of cells with positivity for both keratins 8 and 14 that are similar to ductal originated precursor cells. The expression of proliferation marker Ki67 was not influenced by any of the tested fibroblasts. In conclusion, our data indicate that CAFs are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type-unspecific mechanism. Finally, a clear functional difference between normal and CAFs was demonstrated.

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.

Tracheotomy may be associated with numerous acute and chronic complications including extensive formation of granulation tissue. The emerging functional versatility of the adhesion/growth-regulatory galectins prompted us to perform a histochemical study of wound healing using rat trachea as model. By using non-cross-reactive antibodies and the labelled tissue lectins we addressed the issue of the presence and regulation of galectin reactivity during trachea wound healing. Beside localization of high-molecular-weight keratin, wide-spectrum cytokeratin, keratins 10 and 14, α-smooth muscle actin, vimentin, fibronectin, and Sox-2, galectins -1, -2, and -3 and their reactivity profiles were measured in frozen sections of wounded and control trachea specimens 7, 14, and 28 days after trauma. A clear trend for decreased galectin-1 presence and increased reactivity for galectin-1 was revealed from day 7 to day 28. Sox-2-positive cells were present after seven days and found in the wound bed. Interestingly, several similarities were observed in comparison to skin wound healing including regulation of galectin-1 parameters.

Galectins are a family of carbohydrate-binding proteins that modulate inflammation and immunity. This functional versatility prompted us to perform a histochemical study of their occurrence during wound healing using rat skin as an in vivo model. Wound healing is a dynamic process that exhibits three basic phases: inflammation, proliferation, and maturation. In this study antibodies against keratins-10 and -14, wide-spectrum cytokeratin, vimentin, and fibronectin, and non-cross-reactive antibodies to galectins-1, -2, and -3 were applied to frozen sections of skin specimens two days (inflammatory phase), seven days (proliferation phase), and twenty-one days (maturation phase) after wounding. The presence of binding sites for galectins-1, -2, -3, and -7 as a measure for assessing changes in reactivity was determined using labeled proteins as probes. Our study detected a series of alterations in galectin parameters during the different phases of wound healing. Presence of galectin-1, for example, increased during the early phase of healing, whereas galectin-3 rapidly decreased in newly formed granulation tissue. In addition, nuclear reactivity of epidermal cells for galectin-2 occurred seven days post-trauma. The dynamic regulation of galectins during re-epithelialization intimates a role of these proteins in skin wound healing, most notably for galectin-1 increasing during the early phases and galectin-3 then slightly increasing during later phases of healing. Such changes may identify a potential target for the development of novel drugs to aid in wound repair and patients' care.

Oestrogen deprivation is one of the major factors responsible for many age-related processes, including poor wound healing in women. Previously, it has been shown that oestrogens have a modulatory effect in different wound-healing models. Therefore, in this study, the effect of selective oestrogen receptor (ER) agonists (PPT - ER-α agonist, DPN - ER-β agonist) on excisional and incisional wound-healing models was compared in ovariectomised rats in vivo as well as on human dermal fibroblasts (HDF) and human umbilical endothelial cells (HUVEC) in vitro. In the in vivo study, 4 months after either ovariectomy or sham ovariectomy, Sprague-Dawley rats were randomly divided into four groups and subjected to two incisional and excisional wounds: (i) control - sham operated, vehicle-treated; (ii) ovariectomised, vehicle-treated; (iii) ovariectomised, PPT treated; (iv) ovariectomised, DPN treated. In the in vitro study, HDFs and HUVECs were used. After treatment with ER agonists, cells were processed for immunocytochemistry and gelatin zymography. Our study shows that stimulation of ER-α leads to the differentiation of fibroblasts into myofibroblasts both in vivo and in vitro. On the other hand, the formation of extracellular matrix was more prominent, and wound tensile strength (TS) was increased when ER-β was stimulated. In contrast, stimulation of ER-α led to a more prominent increase in the expression of MMP-2 and decrease in wound TS. New information is presented in this investigation concerning oestrogen replacement therapy (ERT) in different wound-healing models. This study demonstrates that the ERT should be both wound and receptor-type specific., (© 2011 John Wiley & Sons A/S.)

Background Information: Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer-associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT-PCR (quantitative real-time PCR) and immunohistochemistry., Results: We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin-like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential., Conclusions: Our observations indicate the pro-stem cell activity of cancer-associated fibroblasts and underline the role of epithelial-mesenchymal interaction in tumour biology.

Nuclear galectins participate in splicing of pre-mRNA. In this study we detected galectins-1, -2, -3 and -7 and their glycoligands in three types of cells: fibroblasts, cancer epithelial cells and melanoma cells. The results demonstrated that the nuclear expression of distinct types of galectins and their ligands in interphasic nuclei is dependent on the cell type. The extensive binding of labelled galectins-1 and -2 to mitotic cells (around chromosomes, in mitotic spindle and in bridge connecting both daughter cells) suggests their role during the cell division.

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum., (Copyright © 2011 S. Karger AG, Basel.)

Mesenchymal-epithelial interaction is important in the morphogenesis of squamous epithelia and their appendages, and in the control of the hair cycle postnatally. This review summarizes data regarding the interaction between stromal fibroblasts and tumor cells, with an emphasis on tumors originating from squamous epithelium. Tumor stromal fibroblasts as important element of the cancer stem cell niche are able to participate in the control of the biological properties of tumors. We propose these stromal cells and their products as novel targets for anticancer therapy.

Epithelial-mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.

Objective: It is an open question whether multifunctional galectin-3 can be a serum marker in inflammatory bowel disease., Methods: Western blots and commercial ELISA detected and quantitated the lectin immunocytochemistry using double labeling localized it in tissue sections., Results: Serum concentrations were significantly increased in specimen of patients with active and remission-stage ulcerative colitis and Crohn's disease, associated with emerging positivity of CD14(+) cells., Conclusion: Enhanced concentration of galectin-3 in serum reflects presence of disease and points to its involvement in the pathogenesis.

Background: Benign and malignant fibrous histiocytoma present with a considerable difference concerning cellular organization in their vicinity., Objective: Normally appearing epithelium covers the malignant form in contrast to hyperplastic epidermis for benign tumors. It is an open question as to whether the tumor-associated fibroblasts are capable to affect phenotypic features of normal keratinocytes, prompting this comparative analysis., Methods: Fibroblasts were isolated from benign and malignant fibrous histiocytomas, respectively, and also from normal dermis. The resulting cell populations were thoroughly characterized immunocytochemically using a large panel of antibodies. The three fibroblast preparations were cocultured with normal interfollicular keratinocytes. Their phenotype was characterized for distinct properties including differentiation and proliferation., Results: Fibroblasts prepared from both tumor types were phenotypically practically identical with normal dermal fibroblasts. Their activities on keratinocytes were different. Cells prepared from benign fibrous histiocytoma were capable to effect strong expression of keratin 19 and production of a galectin-1-rich extracellular matrix. Fibroblasts isolated from malignant fibrous histiocytoma led to a phenotype very similar to that when keratinocytes were cocultured with normal dermal fibroblasts., Conclusion: Fibroblasts prepared from benign fibrous histiocytoma were biologically active on keratinocytes in a particular manner. Our results on fibroblast activity are suggested to be relevant for morphologic differences observed in vivo between normal epidermis and epidermis adjacent to the studied tumor types.

The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology.