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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

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

Stromal fibroblasts have been reported to interfere with proliferation and invasion of cancer cells. Our objective was to elucidate, using a three-dimensional coculture system (nodules), the interaction between a human, estrogen receptor-positive, well differentiated breast cancer cell line (MCF-7) and fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in terms of cell growth and adhesion. The cell proliferating fraction of each cell type was evaluated by immunohistochemical detection of two cell cycle-associated nuclear proteins: Ki67 and proliferating cell nuclear antigen (PCNA). The expression of epithelial adhesion molecule E-cadherin was investigated in MCF-7 cells by confocal immunofluorescence microscopy. The MCF-7 cells/CAFs mixed nodules showed a significantly larger proportion (50%, p<0.001, Tukey’s multiple comparison test) of Ki67-positive tumor cells with respect to that observed in MCF-7cells/NFs (30.6%, p<0.001) or MCF-7 (34.8%, p<0.001) nodules. MCF-7 cells seemed to have no influence on the growth of both kinds of fibroblasts. Similar results were obtained with PCNA, even though a more heterogeneous and less intense nuclear staining was observed. Immunofluorescence analysis showed that in MCF-7 cells/NFs nodules E-cadherin expression level in tumor cells was about 2.5 fold higher than that observed in MCF-7 cultured alone and about 16 fold higher than that showed by MCF-7 cells cocultured with CAFs. These data support the hypothesis that the activated fibroblasts residing in the breast tumor stroma are able to transmit mitogenic signals to the epithelial cancer cells. Concerning cell adhesion, our results clearly demonstrate the ability of CAFs to negatively affect cell-cell contacts of epithelial cells by strongly reducing E-cadherin expression. The crosstalk of tumor cells with NFs seems to play a crucial role in preventing disruption of cell junctions, cell sha