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Any process of growth, decline and transformation can be understood from a spatial reading, unraveling the traces left on the ground and observing elements temporarily unable to adapt to the flow of time: wasteland. The concept of wasteland is an artificial ‘construction’ usually linked to the idea of (un)productivity. As society changes, the productive use of land changes through time. This chapter aims to reveal the cumulative alternation of western perception in land use value. The means to read this shift is the historical cartographic analysis on the Southside of Antwerp (Belgium). As constructions of reality, maps are loaded with intentions and interpretations of the culture in which they are produced. In particular, the historical mapping of wastelands generated by industrial and agricultural production focuses on understanding the evolution in interpreting fundamental issues such as time, processes and changes related to land ownership., Green Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care. Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., Environmental Technology and Design

Little is known about how the neighborhood social environment (e.g., safety, crime, traffic) impacts child physical activity. We examine the mechanism by which the neighborhood social environment is associated with child physical activity, moderated by individual-level cultural factors (e.g., language at home, immigrant generation) and mediated by neighborhood physical activity-related social norms (e.g., seeing walkers in the neighborhood). Data included 2749 non-Hispanic White and Hispanic children from the Healthy Communities Study. Multilevel regression was performed. The neighborhood social environment was not associated with physical activity in the full sample. However, Hispanic children speaking both English and Spanish and first- or second-generation Hispanic children engaged in more physical activity when the quality of the neighborhood social environment was higher (b = 1.60, p < 0.001 for Hispanic children speaking English and Spanish; b = 2.03, p < 0.01 for first-generation Hispanic children; b = 1.29, p < 0.01 for second-generation Hispanic children). Neighborhood physical activity-related social norms mediated the association between the neighborhood social environment and physical activity among Hispanic children speaking English and Spanish (b = 0.33, p < 0.001) and second-generation Hispanic children (b = 0.40, p < 0.001). Findings suggest heterogeneity in how neighborhood social environments impact physical activity by cultural factors. Health promotion programs may need to enhance neighborhood social environments to increase Hispanic children's physical activity.

BACKGROUND:Community initiatives to promote physical activity in children are common, but evidence supporting their effectiveness is limited. OBJECTIVES:The objective of this study is to examine the relationships between community programmes and policies and children's physical activity in a large and diverse sample of US communities. METHODS:Programmes and policies to promote children's physical activity were assessed in 130 communities by key informant interviews, and physical activity behaviours were measured by self-report and parental report in samples of children in each community (total n = 5138). Associations between composite indices of community programmes and policies and indicators of total and moderate-to-vigorous physical activity were examined without and with adjustment for demographic factors. RESULTS:An index reflecting the 6-year history of the number of behaviour change strategies used in community programmes and policies was positively associated with children's moderate-to-vigorous physical activity. This association was attenuated with adjustment for demographic factors. Effect modification analyses found that the association was positive among non-Hispanic children but was negative for Hispanic children. CONCLUSIONS:Community initiatives to promote physical activity in children were positively associated with children's physical activity in non-Hispanic children. Such initiatives were negatively associated with physical activity in Hispanic children, suggesting that future research should consider unique cultural factors when designing community initiatives to promote activity in this population sub-group.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Despite the acknowledged impact of the tumor stroma on breast cancer development and progression, the molecular basis of such effects remain partially unexplained. We previously reported that breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and an increase in cell membrane fluidity and migration speed in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. More recently, in order to better define the mechanisms responsible for the CAF-promoted tumor cell migration, we investigated the role of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, and demonstrated its CAF-triggered up-regulation as well as its crucial role in the migratory ability of the above tumor cells. Besides SCD1 induction, a CAF-promoted enhancement in the protein level and/or activity of the SCD1 transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), was observed. Moreover, the influence of stroma-derived signals in cancer cell migration speed was proved by cell tracking analysis in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor, where a marked reduction or abolishment of the fibroblast-triggered increase in cancer cell migration speed was observed. In the last part of the study, in order to verify if soluble CAF-derived factors stimulate breast cancer cell migration in a SCD1-dependent manner, tumor cells were exposed to CAF-conditioned medium (CM) and their migration evaluated by scratch assay in the presence of a small molecule inhibitor of SCD1. Moreover, to asses if the induction of SCD1 expression by CAFs might occur via SREBP1, the desaturase levels were also determined in SREBP1-inhibited tumor cells. These latest investigations indicate that SCD1 contributes to the promotion of breast cancer cell migration by CAF-derived soluble factors, since the desaturase inhibition completely suppressed the stim

Background: Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. Methods: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1- inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-b or basic fibroblast growth factor. Results: A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in c

It is widely accepted that glioblastoma (GBM) develops from cancer stem cells (CSCs), a subset of stem-like cells displaying high resistance to treatment. In fact, despite aggressive therapy, 90% of patients relapse within 2 cm from tumor edge. Our recent findings showed the existence of a CSC type, residing in GBM peritumor tissue (PCSCs), that bears distinct characteristics from CSCs of the tumor mass (GCSCs). It should be considered the possibility that, after surgical resection, PCSCs might represent a reservoir of cells able to recapitulate the tumor. In this setting, characterization of PCSCs appears to be crucial in order to identify novel effective therapeutic targets. Thus, our aim was to investigate GCSCs and PCSCs role in angiogenesis, a key event in both GBM and peritumor tissue, whose vasculature shows features similar to those found in the tumor mass. In particular, we analyzed, by immunocytochemistry (ICC), Western blotting or real-time PCR, the expression of molecules involved in hypoxia and angiogenesis, such as HIF1α, HIF2α, and VEGF along with its receptors (VEGFR1, VEGFR2). ICC has highlighted the presence and the specific localization of these molecules in both GCSCs and PCSCs. The two cell populations showed comparable levels of VEGF. The transcript of VEGFR1 was in general expressed at higher levels in GCSCs than in PCSCs, while VEGFR2 mRNA and protein did not show a unique trend of expression. The expression of VEGF and its receptors in both GCSCs and PCSCs suggests that, besides well-known paracrine loops, autocrine signalings are also involved in tumor angiogenesis. Moreover, the expression of angiogenesis markers in PCSCs suggests these cells to have a direct role in peritumor tissue new vessel formation. In this regard, PCSCs should be considered a promising therapeutic target to counteract the angiogenesis-supported tumor progression. Supported by FIRB Accordi di Programma 2010

The key role played by the stroma in breast cancer development and progression has been widely recognized. Recently, we reported that the cross-talk between epithelial and stromal cells affects structural and functional features correlated with the invasive phenotype of breast cancer cells by co-culturing mammary cancer cells with different metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs) [1]. This study was designed to deepen the knowledge of the role played notably by CAFs in promoting breast tumor cell migratory skill through the analysis of the expression/activity of downstream potential target molecules and of the contribution of paracrine signalings. Thus, we investigated the influence of fibroblasts on the expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the level and activity of its transcription factor, SREBP1, in breast cancer cells. The ability of CAFs to promote a 2-3 fold increase in SCD1 mRNA and protein expression as well as an induction of SREBP1 DNA binding activity has been shown in the two cancer cell lines. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration. To clarify the possible role of tumor-stroma paracrine interaction in the previously reported improvement of cancer cell migratory ability, cocultures were set up in presence of neutralizing antibodies against hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor. Cell tracking analysis demonstrated that the CAF-mediated increase in tumor cell migration speed was reduced or abolished by neutralizing the above soluble factors. These results provide new insights in understanding the role of CAFs in promoting tumor cell invasiveness and may help to devise new targeted therapeutic approaches. References [1] Angelucci et al. (2012), PLoS

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Despite increasing knowledge of the GBM biomolecular characterization and breakthrough in treatment, the prognosis in the great majority of patients is poor as almost all tumors recur in peritumor tissue. This site of alterations seems to harbor CSCs that show some differences when compared to CSCs derived from GBM. By means of immunocytochemistry, Western blotting and RT-PCR, we have compared CSCs derived from GBM (GCSCs) to those isolated from peritumor tissue (PCSCs) as to the expression of molecules linked to stemness (Nestin, Musashi-1, SOX2), proliferation and apoptosis (pERK1/2 and pJNKs), migration/invasion, angiogenesis, and hypoxia (reelin, GD3, NG2, VEGF, VEGFR1/2, HIF1α, and HIF2α). Furthermore, comparison of growth rate, frequency of tumor initiating cells (TICs), tumorigenicity in Scid/bg mice, and ultrastructure between the two CSC populations was performed. Finally, we analyzed the effect of different chemotherapeutic agents (temozolomide, etoposide, irinotecan and carboplatin) on proliferation of both GCSCs and PCSCs. All the molecules analyzed were expressed in the two populations of CSCs. Among the investigated markers, nestin, pJNKs, Musashi-1, NG2 and VEGFR1 were found to be significantly increased in GCSCs if compared with PCSCs. Moreover, GCSCs grew at a higher rate and showed a greater frequency of TICs. Animal survival time revealed that mice receiving GCSCs died significantly earlier than mice inoculated with PCSCs. Only PCSCs ultrastructure showed fully organized desmosomes and gap junctions. All the chemotherapeutic agents used inhibited proliferation of both GCSCs and PCSCs, although with different efficiency. Moreover, each drug had similar effect on the GCSCs and PCSCs derived from the same patient. These findings support the need for a complete characterization of GCSCs and PCSCs to optimi

Glioblastoma (GBM) is characterized by extensive angiogenesis, mostly triggered by tumor hypoxia. We previously reported that in GBM and peritumor tissue microvessels show a similar morphology and endothelial cells express CD105, the best marker for newly-formed tumor vessels. In this study, to better understand the role played by angiogenesis in GBM progression, the expression of HIF-1α and -2α, VEGF and its receptors (VEGFR-1 and -2) in GBM and in peritumor tissue was investigated. Tissue samples were obtained from fifty patients. In paraffin-embedded specimens, derived from enhanced lesions (first area) of GBM and white matter at a distance ≤1 mm from the tumor edge (second area), the expression of HIF-1α and -2α, VEGF, VEGFR-1 and -2 was evaluated by immunohistochemistry. Immunoreactivity for all markers was detected not only in the tumor, but also in the peritumor tissue and it was present in neoplastic cells, in endothelium and in apparently normal glial cells. HIF-1α and -2α expression was mainly confined in the nuclei. VEGF, localized in the cytoplasm, showed diffused expression with intense staining in the first area. VEGFR-1 and -2 immunopositivity was dominantly observed at the membrane level. A significant difference in the expression of VEGF and VEGFR-2 between GBM and peritumor tissue was observed. The high heterogeneity in the expression of the other molecules might explain the absence of significant differences between the first and second area. No correlation was observed between the analyzed molecules and survival time. Our study demonstrates that HIF-1α, HIF-2α, VEGF and its receptors are present in peritumor areas, probably reflecting the reduced oxygen availability. Since the response to anti-VEGF therapy is transient and the majority of patients relapse, our findings may lead to consider the molecules expressed in the peritumor tissue as a target for new treatments counteracting angiogenesis. Supported by FIRB Accordi di Programma 2010

Background: Despite the recognized contribution of the stroma to breast cancer development and progression, the effective therapeutic targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and increase in cell membrane fluidity and migration in both well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. Materials and Methods: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. Migration was evaluated by wound healing assay in SCD1-inhibited (by small interfering RNA, siRNA, or pharmacologically) tumor cells. To assess the role of stromal-derived signals in cancer cell migration speed, cell tracking analysis was performed in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor. Results: A 2-3 fold increase in SCD1 mRNA and protein expression as well as an induction of SREBP1 DNA binding activity has been induced, particularly by CAFs, in the two cancer cell lines. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralizing the above growth factors. Conclusions: These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic stra

It is widely accepted that glioblastoma (GBM) develops from cancer stem cells (CSCs), a subset of stem-like cells displaying high resistance to treatment. Our recent findings showed the existence of a CSC type, residing in GBM peritumor tissue (PCSCs), that bears distinct characteristics from CSCs of the tumor mass (GCSCs) and that, after surgical resection, might represent a reservoir of cells able to recapitulate the tumor. In this setting, characterization of PCSCs appears to be crucial in order to identify novel effective therapeutic targets. Thus, our aim was to investigate GCSCs and PCSCs role in angiogenesis, a key event in both GBM and peritumor tissue, whose vasculature shows features similar to those found in the tumor mass. In particular, we analyzed, by immunocytochemistry (ICC), Western blotting or real-time PCR, the expression of molecules involved in hypoxia and angiogenesis, such as HIF1α, HIF2α, and VEGF along with its receptors (VEGFR1, VEGFR2). ICC has highlighted the presence and the specific localization of these molecules in both GCSCs and PCSCs. The two cell populations showed comparable levels of VEGF. The transcript of VEGFR1 was in general expressed at higher levels in GCSCs than in PCSCs, while VEGFR2 mRNA and protein did not show a unique trend of expression. The presence of VEGF and its receptors in both GCSCs and PCSCs suggests that, besides well-known paracrine loops, autocrine signalings are also involved in tumor angiogenesis. Moreover, the expression of angiogenesis markers in PCSCs suggests these cells to have a direct role in peritumor tissue new vessel formation. In this regard, PCSCs should be considered a promising therapeutic target to counteract the angiogenesis-supported tumor progression. Supported by FIRB Accordi di Programma 2010

Growing evidence indicates the existence of small population of cells endowed with distinctive self-renewal capacity, tumorigenesis and resistance to conventional treatments, defined as cancer stem cells (CSCs) or tumor initiating cells. In addition, it is widely appreciated that the growth of new blood vessels and lymphatic vasculature, which occurs during angiogenesis and limphoangiogenesis, plays a key role in the process of the tumor growth. To this regards, an increasing number of studies showed that the employment of angiogenesis inhibitors might have significant therapeutic advantages. Fashinatingly, recent evidence demonstrated that CSCs play a role in angiogenesis, in particular in glioma, which, to date, represents a highly letal tumor tough to treat. We demonstrated that CSCs isoleted from both tumor (GCSCs) and peritumoral tissue (PCSCs) express a number of angiogenesis-related molecules, such as VEGF, HIF1alpha and HIF2alpha. In addition, VEGFR1 expression was found significantly reduced in PCSCs vs GCSCs whereas VGFR2 appeard to be largely heterogeneous in both stem cell types. With the aim to investigate the aptitude of CSCs derived neurospheres to regulate the angiogenesis process we performed in vitro migration analysis by means of boiden chamber assay. The results of this experiments indicate that ECs migration was stimulated in the presence of PCSCs but remained almost unaffected when endothelial cells where co-coltured with GCSCs. In conclusion, our data suggest that GCSCs and PCSCs contribute differently to tumor angiogenesis by activating distinct molecular mechanisms. PCSCs might therefore a key role in the recruiment as well as activation of ECs in peritumoral tissue.

In breast tumor, interactions occurring between malignant cells and stromal mi-croenvironment heavily influence various aspects of cancer biology. We recently reported that this cross-talk affects structural and functional features correlated with the invasive phenotype of breast cancer cells by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts, particularly those isolated from breast tumor stroma (cancer-associated fibroblasts, CAFs). In the present study, we aimed to elucidate some aspects of the epithelium-stroma interaction, related to two linked CAF-triggered effects we previously described: a) increase in cancer cell plasma membrane fluidity; b) increase in speed and directness of cancer cell migration. Thus, we tested the effect of fibroblast/breast cancer cell co-culturing on the expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating the fatty acid membrane composition whose up-regulation has been reported in different cancer cells, generally leading to loosely packed membranes. Western blot analysis revealed a dramatic CAF-promoted increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells. Quantitative real-time PCR demonstrated similar variations in the SCD1 transcript levels. With the aim of clarifying the possible role of soluble factors in the CAF-induced increase in speed and directness of cancer cell migration, we performed co-culture assays in presence of a neutralizing antibody against hepatocyte growth factor (HGF) which is released by CAFs and notoriously affects cancer cell motili-ty. We demonstrated that speed and directness increases were strongly reduced or completely abolished by the addition of the anti-HGF neutralizing antibody to the culture medium. These results provide new insights in understanding the role of CAFs, the main host stromal component of breast cancer, in promoting the tumor cell invasive attitude and may help in defining furthe

Growing evidence indicates the existence of small population of cells endowed with distinctive self-renewal capacity, tumorigenesis and resistance to conventional treatments, defined as cancer stem cells (CSCs) or tumor initiating cells. In addition, it is widely appreciated that the growth of new blood vessels and lymphatic vasculature, which occurs during angiogenesis and limphoangiogenesis, plays a key role in the process of the tumor growth. To this regards, an increasing number of studies showed that the employment of angiogenesis inhibitors might have significant therapeutic advantages. Fashinatingly, recent evidence demonstrated that CSCs play a role in angiogenesis, in particular in glioma, which, to date, represents a highly letal tumor tough to treat. We demonstrated that CSCs isoleted from both tumor (GCSCs) and peritumoral tissue (PCSCs) express a number of angiogenesis-related molecules, such as VEGF, HIF1alpha and HIF2alpha. In addition, VEGFR1 expression was found significantly reduced in PCSCs vs GCSCs whereas VGFR2 appeard to be largely heterogeneous in both stem cell types. With the aim to investigate the aptitude of CSCs derived neurospheres to regulate the angiogenesis process we performed in vitro migration analysis by means of boiden chamber assay. The results of this experiments indicate that ECs migration was stimulated in the presence of PCSCs but remained almost unaffected when endothelial cells where co-coltured with GCSCs. In conclusion, our data suggest that GCSCs and PCSCs contribute differently to tumor angiogenesis by activating distinct molecular mechanisms. PCSCs might therefore a key role in the recruiment as well as activation of ECs in peritumoral tissue.

The gene expression pattern of glioblastoma (GBM) is well documented but the expression profile of brain adjacent to tumor is not yet analysed. This may help to understand the oncogenic pathway of GBM development. We have established the genome-wide expression profiles of samples isolated from GBM tumor mass, white matter adjacent to tumor (apparently free of tumor cells), and white matter controls by using the Affymetrix HG-U133 arrays. Array-CGH (aCGH) was also performed to detect genomic alterations. Among genes dysregulated in peritumoral white matter, 15 were over-expressed, while 42 were down-regulated when compared to white matter controls. A similar expression profile was detected in GBM cells. Growth, proliferation and cell motility/adhesion-associated genes were up-regulated while genes involved in neurogenesis were down-regulated. Furthermore, several tumor suppressor genes along with the KLRC1 (a member of natural killer receptor) were also down-regulated in the peritumoral brain tissue. Several mosaic genomic lesions were detected by aCGH, mostly in tumor samples and several GBM-associated mosaic genomic lesions were also present in the peritumoral brain tissue, with a similar mosaicism pattern. Our data could be explained by a dilution of genes expressed from tumor cells infiltrating the peritumour tissue. Alternatively, these findings could be substained by a relevant amount of "apparently normal" cells presenting a gene profile compatible with a precancerous state or even "quiescent" cancer cells. Otherwise, the recurrent tumor may arise from both infiltrating tumor cells and from an interaction and recruitment of apparently normal cells in the peritumor tissue by infiltrating tumor cells.

The gene expression pattern of glioblastoma (GBM) is well documented but the expression profile of brain adjacent to tumor is not yet analysed. This may help to understand the oncogenic pathway of GBM development. We have established the genome-wide expression profiles of samples isolated from GBM tumor mass, white matter adjacent to tumor (apparently free of tumor cells), and white matter controls by using the Affymetrix HG-U133 arrays. Array-CGH (aCGH) was also performed to detect genomic alterations. Among genes dysregulated in peritumoral white matter, 15 were over-expressed, while 42 were down-regulated when compared to white matter controls. A similar expression profile was detected in GBM cells. Growth, proliferation and cell motility/adhesion-associated genes were up-regulated while genes involved in neurogenesis were down-regulated. Furthermore, several tumor suppressor genes along with the (a member of natural killer receptor) were also down-regulated in the peritumoral brain tissue. Several mosaic genomic lesions were detected by aCGH, mostly in tumor samples and several GBM-associated mosaic genomic lesions were also present in the peritumoral brain tissue, with a similar mosaicism pattern. Our data could be explained by a dilution of genes expressed from tumor cells infiltrating the peritumour tissue. Alternatively, these findings could be substained by a relevant amount of "apparently normal" cells presenting a gene profile compatible with a precancerous state or even "quiescent" cancer cells. Otherwise, the recurrent tumor may arise from both infiltrating tumor cells and from an interaction and recruitment of apparently normal cells in the peritumor tissue by infiltrating tumor cells.

Glioblastoma (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with VEGF antibody reduces GBM tumor growth. However, the clinical benefits are transient and invariably followed by tumor recurrence. Recently, it has been demonstrated that in glioblastoma (GBM), cancer stem cells (CSCs) are responsible for either tumorigenesis or neoangiogenesis. In particular, a fraction of CSCs is represented by a population of endothelial progenitors. In our previous studies, we showed the presence of nestin+ cell and nestin+ and endoglin (CD105) + vasculature, not only in the GBM but also in the tissue surrounding the tumor, suggesting the involvement of CSC in endothelial differentiation also in peritumor tissue. In this study, with the aim to understand CSCs involvement in angiogenesis, we investigated the expression of angiogenesis markers (VEGF, VEGFR1, VEGFR2, HIF1α and HIF2α) by immunocytochemistry, western blot or RT- PCR analyisis, in CSCs derived from GBM (GCSC) and peritumor tissue (PCSC) of 4 patients. Both GCSCs and PCSCs were immunopositive for HIF1α and HIF2α, VEGF and its receptors VEGFR1 and VEGFR2, which underline the presence of autocrine and paracrine growth factor loops. Immunoblotting showed that VEGF is expressed at higher levels in GCSCs than PCSCs, with the exception of a patient who showed an up-regulation in PCSCs. Moreover, real time PCR showed that both VEGFR1 and VEGFR2 were expressed at low level in the two types of CSCs with a down-regulation of VEGFR1 in PCSCs. These results indicate the presence of all angiogenic markers in PCSCs suggesting their involvement in the GBM local recurrence. The inhibition of multiple angiogenic pathways may be a new line of attack on this deadly cancer.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of "cadherin switching", another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration veloci

It has been reported that distinct cancer stem cell-like (CSC) populations can be isolated from different regions of the same human glioblastoma (GBM), namely from the tumor core or from peritumor tissue. Although originating from common ancestor cells, these populations can exhibit different characteristics in terms of growth properties, genetic anomalies, and their tumor initiating ability. In particular, CSCs deriving from the GBM core (GCSCs) induced tumors in immunodeficient mice with an efficiency of 95% while CSCs isolated from peritumor tissue (PCSCs) have much less, if any, tumorigenic potential. In this study, we aimed at gaining a deeper insight into the features of both GCSC and PCSC populations. By means of immunocytochemical and Western blot analysis, we investigated the expression of stem cell markers such as nestin, Sox2, Musashi-1, CD133, and also NG2 and GD3 (associated with invasion and angiogenesis), as well as markers linked to proliferation (i.e., pJNK). Immunocytochemistry showed that all these molecules are expressed in both GCSCs and PCSCs. Furthermore, Western blot analysis on nestin, Musashi-1, and pJNK proteins, revealed a higher expression in GCSCs than in PCSCs. The Sox2 protein was equally expressed in GCSCs and PCSCs, probably because Sox2 is critical for the maintenance of broader stem cell potential. Yet, GCSCs that grew as neurospheres showed a higher growth rate than PCSCs, which were generally found to grow as adherent cells or floating neurospheres. TEM showed differences between GCSCs and PCSCs in terms of cytoskeletal component expression and presence of cell-cell junctions. Taken together, our data confirm that, at least in some instances, GCSCs can be more aggressive than PCSCs which are likely to begin their malignant transformation in a microenvironment that may be influenced by various factors deriving from the main tumor mass. Supported by FIRB “Accordi di Programma” 2010

It has been reported that distinct cancer stem cell-like (CSC) populations can be isolated from different regions of the same human glioblastoma (GBM), namely from the tumor core or from peritumor tissue. Although originating from common ancestor cells, these populations can exhibit different characteristics in terms of growth properties, genetic anomalies, and their tumor initiating ability. In particular, CSCs deriving from the GBM core (GCSCs) induced tumors in immunodeficient mice with an efficiency of 95% while CSCs isolated from peritumor tissue (PCSCs) have much less, if any, tumorigenic potential. In this study, we aimed at gaining a deeper insight into the features of both GCSC and PCSC populations. By means of immunocytochemical and Western blot analysis, we investigated the expression of stem cell markers such as nestin, Sox2, Musashi-1, CD133, and also NG2 and GD3 (associated with invasion and angiogenesis), as well as markers linked to proliferation (i.e., pJNK). Immunocytochemistry showed that all these molecules are expressed in both GCSCs and PCSCs. Furthermore, Western blot analysis on nestin, Musashi-1, and pJNK proteins, revealed a higher expression in GCSCs than in PCSCs. The Sox2 protein was equally expressed in GCSCs and PCSCs, probably because Sox2 is critical for the maintenance of broader stem cell potential. Yet, GCSCs that grew as neurospheres showed a higher growth rate than PCSCs, which were generally found to grow as adherent cells or floating neurospheres. TEM showed differences between GCSCs and PCSCs in terms of cytoskeletal component expression and presence of cell-cell junctions. Taken together, our data confirm that, at least in some instances, GCSCs can be more aggressive than PCSCs which are likely to begin their malignant transformation in a microenvironment that may be influenced by various factors deriving from the main tumor mass. Supported by FIRB “Accordi di Programma” 2010

It has been reported that distinct cancer stem cell-like (CSC) populations can be isolated from different regions of the same human glioblastoma (GBM), namely from the tumor core or from peritumor tissue. Although originating from common ancestor cells, these populations can exhibit different characteristics in terms of growth properties, genetic anomalies, and their tumor initiating ability. In particular, CSCs deriving from the GBM core (GCSCs) induced tumors in immunodeficent mice with an efficiency of 95% while CSCs isolated from peritumor tissue (PCSCs) have much less, if any, tumorigenic potential. In this study, we aimed at gaining a deeper insight into the features of both GCSC and PCSC populations. By means of immunocytochemical and Western blot analysis, we investigated the expression of stem cell markers such as nestin, Sox2, Musashi-1, CD133, and also NG2 and GD3 (associated with invasion and angiogenesis), as well as markers linked to proliferation (i.e., pJNK). Immunocytochemistry showed that all these molecules are expressed in both GCSCs and PCSCs. Furthermore, Western blot analysis on nestin, Musashi-1, and pJNK proteins, revealed a higher expression in GCSCs than in PCSCs. The Sox2 protein was equally expressed in GCSCs and PCSCs, probably because Sox2 is critical for the maintenance of broader stem cell potential. Yet, GCSCs that grew as neurospheres showed a higher growth rate than PCSCs, which were generally found to grow as adherent cells or floating neurospheres. TEM showed differences between GCSCs and PCSCs in terms of cytoskeletal component expression and presence of cell-cell junctions. Taken together, our data confirm that, at least in some instances, GCSCs can be more aggressive than PCSCs which are likely to begin their malignant transformation in a microenvironment that may be influenced by various factors deriving from the main tumor mass. Supported by FIRB “Accordi di Programma” 2010