Giant cell tumor of bone (GCTb) represents 5% of bone tumors, and although considered benign, 5% metastasize to the lung. The expression of proteins directly or indirectly associated with osteolysis and tumor growth was studied on 163 samples of GCTb. Of these, 33 patients developed lung metastasis during follow-up. The impact of tumor-host interaction on clinical aspects was evaluated with the aim of finding specific markers for new biological therapies, thus improving clinical management of GCTb. Protein expression was evaluated by immunohistochemical analysis on Tissue Microarray. The majority of GCTb samples from patients with metastatic disease were strongly positive to RANKL and its receptor RANK as well as to CAII and MMP-2 and to pro-survival proteins NFIB and c-Fos. Kaplan-Meier analysis indicated a significant difference in metastasis free survival curves based on protein staining. Interestingly, the statistical correlation established a strong association between all variables studied with a higher t coefficient for RANK/RANKL, RANK/NFIB, and RANKL/NFIB pairs. At multivariate analysis co-overexpression of NFIB, RANK and RANKL significantly increased the risk of metastasis with an odds ratio of 13.59 (95%CI 4.12-44.82; p < 0.0005). In conclusion, the interconnection between matrix remodeling and tumor cell activity may identify tumor-host endpoints for new biological treatments. Keywords: giant cell tumor; prognostic biomarkers; bone remodeling; metastasis GCTb is a locally aggressive tumor that occasionally metastasize to the lung. 1,2 It is characterized by a cellular network including multinuclear osteoclastlike giant cells, round cells, and spindle shaped mononuclear cells that represent a proliferative pattern. All cellular components interact with bone matrix mediating the release of soluble growth factors, cytokines, and transcription factors. 3 In a previous study, we selected a panel of proteins involved in cell cycle and apoptosis to identify highrisk patients and suggested the use of proteomic technique to identify new biomarkers associated with a more aggressive tumor behavior. 4 Our recent data demonstrated a significantly different expression of miR-136 between GCTb patients who progressed to lung metastases during follow-up and nonmetastatic patients, identifying the nuclear factor NFIB and the cytokine RANK as target genes involved in proliferation and osteolysis. 5 In GCTb, as well as in bone osteolytic tumors, massive bone resorption is triggered by the RANKL/RANK axis that stimulates osteoclast-dependent and -independent pathways 6 via activation of intracellular mediators such as TNF receptor associated factor (TRAF6), interleukins (IL), carbonic anhydrase II (CAII), metalloproteinases (MMPs), transcription factor c-Fos, and transforming growth factor (TGF-b). In the absence of RANKL inhibitors, a vicious cycle (bone destruction, tumor growth, and further bone destruction) supports tumor expansion. 7 The aim of the study was to find biological prognostic markers of GCTb tu...

Background: Little information is currently available concerning the relative contribution of cardiac parenchymal and stromal cells in the activation of the pro-inflammatory signal cascade, at the initial stages of diabetes. Similarly, the effects of early resveratrol (RSV) treatment on the negative impact of diabetes on the different myocardial cell compartments remain to be defined. Methods: In vitro challenge of neonatal cardiomyocytes and fibroblasts to high glucose and in vivo/ex vivo experiments on a rat model of Streptozotocin-induced diabetes were used to specifically address these issues. Results: In vitro data indicated that, besides cardiomyocytes, neonatal fibroblasts contribute to generating initial changes in the myocardial environment, in terms of pro-inflammatory cytokine expression. These findings were mostly confirmed at the myocardial tissue level in diabetic rats, after three weeks of hyperglycemia. Specifically, monocyte chemoattractant protein-1 and Fractalkine were up-regulated and initial abnormalities in cardiomyocyte contractility occurred. At later stages of diabetes, a selective enhancement of pro-inflammatory macrophage M1 phenotype and a parallel reduction of anti-inflammatory macrophage M2 phenotype were associated with a marked disorganization of cardiomyocyte ultrastructural properties. RSV treatment inhibited pro-inflammatory cytokine production, leading to a recovery of cardiomyocyte contractile efficiency and a reduced inflammatory cell recruitment. Conclusion: Early RSV administration could inhibit the pro-inflammatory diabetic milieu sustained by different cardiac cell types.

Osteosarcoma (OS) is the most frequent primary malignant tumour of bone and metastases occur in 30% of cases, the 5-year survival rate is 25–30%. Although pre- and post-operative chemotherapy has improved prognosis in osteosarcoma (OS), high toxicity and natural and acquired drug-resistance are the first cause of treatment failure. The identification of new predictive and therapeutic biomarkers may increase drug sensitivity and better control localized and metastatic disease. By the evidence that CXCR4 receptor by binding its ligand CXCL12 activates downstream critical endpoints for tumour malignancy, we first studied human OS progression correlating CXCR4 expression in OS biopsy with patient clinical data. By Real-time PCR and immunoistochemistry we found that high levels of CXCR4 gene and protein expression significantly correlated with OS progression, emphasizing the role of CXCR4/CXCL12 axis in tumour prognosis. This was supported by univariate analyses that showed a higher probability of local and/or systemic relapse in OS patients with a high CXCR4 gene expression and a significant increase of metastasis risk associated with an increasing score of CXCR4 protein staining intensity. Secondarily, to study the role of CXCR4 as a target for new therapeutic strategies, we evaluated the response of OS cells to the fully human CXCR4 antibody, MDX1338. In the study we also included AMD3100, the most studied CXCR4 antagonist. In CXCR4-positive OS cells cultured in CXCL12-rich BM-MCS-CM (bone marrow-derived mesenchymal stem conditioned medium), a decrease of cell proliferation up to 30%–40% of control was seen after drug exposure. However, an increase of apoptosis was seen in p53-positive U2OS and 143B after CXCR4 inhibitor incubation, while no changes were seen in treated SAOS-2 cells which also present a different labeling profile. The role of p53 in apoptotic response to CXCR4 inhibitors was confirmed by p53 silencing in U2OS cell line. Our data suggest that the response to anti-CXCR4 agents could be influenced by the genetic background and labeling profile which induces a different cross-talk between tumour cells and environment. The delay in cell cycle progression associated with increased apoptosis could sensitize p53-positive cells to conventional therapy and in vivo preclinical experiments are on going with the aim to suggest new combined target therapies in human OS.

Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression.