Anna Colabianchi

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression

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 &agr;-smooth muscle actin (&agr;-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.

Stearoyl-CoA desaturase 1 and paracrine diffusible signals have a major role in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts

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-β 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 cancer cell migration speed was markedly reduced or abolished by neutralising the above growth factors.Conclusion: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 strategies.

Stearoyl-CoA desaturase 1 and paracrine signal involvement in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts

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 this 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 assess 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 stimulatory effect of CAF-CM on tumor cell migration. SREBP1 inhibition impaired CAF-mediated up-regulation of SCD1 in poorly invasive but not in highly invasive tumor cells, in which SREBP1-independent mechanisms may account for the enhancement of SCD1 levels. 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 strategies.

Involvement of cancer stem cells in glioblastoma angiogenesis

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.

Breast cancer stroma influences the migratory behaviour of epithelial malignant cells: the role of secreted hepatocyte growth factor

In breast tumor, interactions occurring between malignant cells and stromal microenvironment 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 motility. 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 further promising therapeutic targets.

Role of neural cancer stem cells in angiogenesis

Growing evidence indicates the existence of small populations 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 vessel and lymphatic vasculature, which occurs during angiogenesis and lymphangiogenesis, plays a key role in the process of tumor growth. To this regards, an increasing number of studies showed that the employment of angiogenesis inhibitors might have significant therapeutic advantages. Fascinatingly, recent evidence demonstrated that CSCs play a role in angiogenesis, in particular in glioma, which, to date, represents a highly lethal tumor tough to treat. We demonstrated that CSCs isolated from both tumor (GCSCs) and peritumoral tissue (PCSCs) express a number of angiogenesis-related molecules, such as VEGF, HIF1a and HIF2a. In addition, VEGFR1 expression was found significantly reduced in PCSCs vs. GCSCs whereas VEGFR2 appeared 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 Boyden chamber assay. The results of these experiments indicated that ECs migration was stimulated in the presence of PCSCs but remained almost unaffected when endothelial cells were co-cultured with GCSCs. In conclusion, our data suggest that GCSCs and PCSCs contribute differently to tumor angiogenesis by activating distinct molecular mechanisms. PCSCs might, therefore, play a key role in the recruitment as well as activation of ECs in peritumoral tissue.

Epithelium-stroma reciprocal influence in breast cancer. Focus on plasma membrane features related to cell migratory/invasive ability

Interactions occurring between malignant cells and stromal microenvironment 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 carcinogenesis. 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 inhibition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could reasonably 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 previously described increase in tumor cell motility.

Angiogenic marker expression in cancer stem cells derived from glioblastoma and peritumor tissue

Glioblastoma (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with VEGF antibodies reduces GBM tumor growth. However, the clinical benefits are transient and invariably followed by tumor recurrence. Recently, it has been demonstrated that in 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+ cells and nestin+ and endoglin (CD105)+ vasculature, not only in the GBM but also in the tissue surrounding the tumor, suggesting the involvement of CSC endothelial differentiation also in peritumor tissue. In this study, with the aim of understanding CSCs involvement in angiogenesis, we investigated the expression of angiogenesis markers (VEGF, VEGFR1, VEGFR2, HIF1α and HIF2α) by immunocytochemistry, western blot or real time PCR analysis, 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 underlines 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 GBM local recurrence. The inhibition of multiple angiogenic pathways may be a new line of attack on this deadly cancer. We acknowledge Prof. Gigliola Sica for her effectiveness in coordinating this study supported by FIRB “Accordi di Programma” 2010.

Cancer stem cells from glioblastoma and peritumor tissue

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

Stemness features in peritumor tissue of glioblastoma

It has been recently shown that glioblastomas (GBMs) contain stem-like cells (CSCs) in both the tumor core and the tissue surrounding the tumor border. Nevertheless, these CSCs show diverse tumorigenic potential and distinct genetic anomalies. In agreement with these data, using immunohistochemical analysis, we found apparently normal cells expressing the stem cell marker nestin in GBM peritumor tissue at a distance of <1 cm and between 1 and 3.5 cm from the tumor edge. In addition, the same cell type revealed cytoplasmic immunoreactivity for GD3 ganglioside, which belongs to the predominant ganglioside species involved in the early developmental stages of the nervous system. In our experience, peritumor tissue harbored a florid microvascular proliferation with the same characteristics as those seen in GBM. Endothelial cells of microvessels expressed nestin, GD3 ganglioside and CD105, a membrane glycoprotein which is an accessory component of the transforming growth factor beta receptor complex. CD105 has been reported to distinguish between normal vessels and malignant neovascularisation. Endothelium of microvessels and hyperplastic microvessels was surrounded by pericytes, showing a positivity for alpha smooth muscle actin and for NG2 (neuro-glial proteoglycan 2), which is associated with neural progenitors. At present, pericytes are thought to be multipotent stem cells capable of migrating, rapidly adapting and differentiating along multiple lineages. Our findings indicate the presence of cells endowed with stemness features in both cellular and vascular compartments of GBM peritumor tissue. This might be a crucial event in the process of transformation of the areas surrounding the tumor.