[Show abstract][Hide abstract] ABSTRACT: 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 α-smooth muscle actin (α-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.
Full-text · Article · Jan 2016 · Journal of Neuropathology and Experimental Neurology
[Show abstract][Hide abstract] ABSTRACT: The Toll-like Receptor (TLR) family ensures prompt response towards pathogens, protecting the host against infections, and guarantees a realistic balance between protective and detrimental activities. Multiple regulating mechanisms characterize TLR activity that is not limited to innate and adaptive antimicrobial immune responses, as observed in the inflammatory (either infective, allergic or autoimmune) responses associated with tissue remodeling. Following the insult and the arise of inflammatory response, tissue remodeling takes place and might develop in fibrosis, depending on microenvironment as a result of imbalanced fibroblasts (FBs) and myofibroblasts (myoFBs) activation/survival. The process is driven by an epithelial-fibroblast-immune cell cross-talk. While the main FB function is the matrix metabolism for tissue homeostasis or repair, the myoFB differentiation represents a crucial step in attempting repair of injury. FBs/myoFBs provide more than structural support at site of injury, synthesizing and/or reacting to different cytokines, growth factors, neuromediators and soluble/lipid mediators. TLR-bearing FBs/myoFBs might contribute at the innate immune level, providing a second line of protection/defense as well as being a target/effector cell of tissue remodeling. TLRs might also interfere with acute inflammation as well as with established fibrosis, triggering structural/functional changes in agreement with the genetic background, the site of lesion, the entity of associated infection, the poor blood circulation or the pharmacological treatments, all together strictly influencing tissue repair/remodeling process. This review will focus on the recent findings on TLRs at launch and long-lasting tissue remodeling process, that strongly suggest TLRs as optional targets for future therapies. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Full-text · Article · Aug 2015 · Journal of Cellular Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Full-text · Article · Apr 2015 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Abstract
Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca2+-channels but they exhibited increased intracellular Ca2+ levels in response to ATP. These Ca2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle atrophy occurs during disuse and aging, or as a consequence of chronic diseases such as cancer and diabetes. It is characterized by progressive loss of muscle tissue due to hypotrophic changes, degeneration, and an inability of the regeneration machinery to replace damaged myofibers. Tumor necrosis factor (TNF) is a proinflammatory cytokine known to mediate muscle atrophy in many chronic diseases and to inhibit skeletal muscle regeneration. In this study, we investigated the role of Arg-vasopressin-(AVP-)dependent pathways in muscles in which atrophy was induced by local overexpression of TNF. AVP is a potent myogenesis-promoting factor and is able to enhance skeletal muscle regeneration by stimulating Ca(2+)/calmodulin-dependent kinase and calcineurin signaling. We performed morphological and molecular analyses and demonstrated that local over-expression of the AVP receptor V1a enhances regeneration of atrophic muscle. By upregulating the regeneration/differentiation markers, modulating the inflammatory response, and attenuating fibrogenesis, the stimulation of AVP-dependent pathways creates a favourable environment for efficient and sustained muscle regeneration and repair even in the presence of elevated levels of TNF. This study highlights a novel in vivo role for AVP-dependent pathways, which may represent an interesting strategy to counteract muscle decline in aging or in muscular pathologies.
Full-text · Article · May 2014 · BioMed Research International
[Show abstract][Hide abstract] ABSTRACT: Reelin is a large secreted extracellular matrix glycoprotein which contributes to positioning, migration and laminar organization of several central nervous system structures during neurodevelopment. Recent studies reported the expression of Ree-lin and its intracellular adapter protein DAB1 in neuroblastoma, where it appears to be involved in cell motility and invasiveness. Interestingly, our data obtained by immunolocalization analysis show the expression of Reelin in both tumor and peritu-moral area of glioblastoma (GBM). It is known that many solid tumors may originate from cancer stem cells (CSC) which are usually resistant to common therapies and might be involved in tumor progression. Therefore, we evaluated the expression of Reelin in CSC neurospheres isolated from both tumor (GCSC) and peritumoral area (PCSC) of GBM. Immunocytochemistry analysis showed the expression of Reelin in both cell types, suggesting that this protein may contribute to neurosphere tridimen-sional organization and possibly to cell migration during tumor progression. This is the first evidence of Reelin expression in human GBM which might indicate a piv-otal role of this protein in the regulation of tumor development. Our data may open potential avenues for GBM treatment by targeting Reelin signaling activity. References  Biamonte et al. (2009) Interactions between neuroactive steroids and Reelin haploinsufficiency in Purkinje cell survival. Neurobiol Dis 36(1): 103-15.  Becker et al. (2012) Reelin signalling in neuroblastoma: migratory switch in metastatic stages. Int J Oncol 41(2): 681-9.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The therapeutic use of stem cells and tissue engineering techniques are emerging in urology. Here, stem cell types, their differentiating potential and fundamental characteristics are illustrated. The cancer stem cell hypothesis is reported with reference to the role played by stem cells in the origin, development and progression of neoplastic lesions. In addition, recent reports of results obtained with stem cells alone or seeded in scaffolds to overcome problems of damaged urinary tract tissue are summarized. Among others, the application of these biotechnologies in urinary bladder, and urethra are delineated. Nevertheless, apart from the ethical concerns raised from the use of embryonic stem cells, a lot of questions need to be solved concerning the biology of stem cells before their widespread use in clinical trials. Further investigation is also required in tissue engineering utilizing animal models.
[Show abstract][Hide abstract] ABSTRACT: High cell-surface GnRH receptor (GnRH-R) levels have been shown to have a major influence on the extent of GnRH agonist-mediated tumor growth inhibition. The ability of the GnRH agonist leuprorelin acetate (LA) to induce a post-transcriptional upregulation of GnRH-R at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells has been previously demonstrated by Western blotting. Here we performed single molecule force spectroscopy by using Atomic Force Microscopy (AFM), which has proven to be a powerful tool allowing for investigation of living cell surface biological features, such as the so far unclear GnRH agonist/receptor interaction. Thus, in the hormone-insensitive PC-3 cells, we characterized the strength of the LA-receptor binding, and the amount and distribution of the functional receptor molecules on the cell surface. The effect of a long and continuous treatment (up to 30 days) with the agonist (10(-11) and 10(-6) M) on the same parameters was also investigated. A GnRH-R increase was observed, reaching the maximum (∼80%) after 30 days of treatment with the highest dose of LA (10(-6) M). The analogue-induced increase in GnRH-R was also demonstrated by Western blotting. In addition, two different receptor bound strengths were detected by AFM, which suggests the existence of two GnRH-R classes. A homogeneous distribution of the unbinding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the membrane of these living cells may warrant the maintenance of the response to LA also in androgen-unresponsive PCa. Moreover, the determination of ligand/receptor bond strength could shed light on the poorly understood event of LA/GnRH-R interaction and/or address structural/chemical agonist optimizations.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Objective:
Treatment of glioblastoma recurrence can have a palliative aim, after considering risks and potential benefits. The aim of this study is to verify the impact of surgery and of palliative adjuvant treatments on survival after recurrence.
From January 2002 to June 2008, we treated 76 consecutive patients with recurrent glioblastoma. Treatment was: 1-surgery alone--17 patients; 2-adjuvant-therapy alone--24 patients; 3-surgery and adjuvant therapy--16 patients; no treatment--19 patients. The impact on median overall-survival (OS-time between recurrence and death/last follow-up) of age, Karnofsky performance scale (KPS), resection extent and adjuvant treatment scheme (Temozolomide alone vs low-dose fractionated radiotherapy vs others) was determined. Survival curves were obtained through the Kaplan-Meier method. Cox proportional-hazards was used for multivariate analyses. Significance was set at p<0.05.
Median OS was 7 months. At univariate analysis, patients with a KPS≥70 had a longer OS (9 months vs 5 months--p<0.0001). OS was 6 months for patients treated with surgery alone, 5 months for patients that received no treatment, 8 months for patients treated with chemotherapy alone, 14 months for patients treated with surgery and adjuvant therapy--p=0.01. Patients with a KPS<70 were significantly at risk for death - HR 2.8 - p=0.001. Subgroup analysis showed no significant differences between patients receiving gross total or partial tumor resection and among patients receiving different adjuvant therapy schemes. Major surgical morbidity at tumor recurrence occurred in 16 out of 33 patients (48%).
It is fundamental, before deciding to operate patients for recurrence, to carefully consider the impact of surgical morbidity on outcome.
No preview · Article · Sep 2012 · Clinical neurology and neurosurgery