Characterization of cancer stem cells (CSC) isolated from LI, a human glioblastoma (GBM) cell line

It is becoming increasingly clear that tumour development is due to a small fraction of mutated stem cells (CSC). Established cancer cell lines represent a good source of CSC with respect to tissue tumour samples because they do not contain any contaminating normal stem cells and it is easy to obtain large quantities of them. We isolated putative CSC via a non-adherent neurosphere (NS) assay from LI. Using a clonal assay, we selected from the primary NS two clones named F11 and D2. LI, F11 and D2 cells and these clones under differentiation were examined for expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox-2), markers of differentiation (βIII-Tubulin and GFAP) and Ca2+-channels, by immunocytochemistry, western blot analysis and confocal Ca2+ imaging. Both F11 and D2 clones expressed higher levels of stem cell markers with respect to LI cells. Markers of differentiation were expressed at high levels in both LI cells and clones. The expression of Nestin, Sox-2, and βIII-Tubulin was down-regulated in clones under differentiation, whereas Musashi-1 was increased. LI, F11 and D2 cells did not exhibit Ca2+ signals following KCl-induced membrane depolarization, thus suggesting that they do not express functional voltage-dependent Ca2+-channels. Nevertheless, transient increases in intracellular Ca2+ levels were observed after cell exposure to ATP in both F11 and D2 during differentiation. The ATP treatment did not affect cell proliferation. The increased expression of stem cell markers and their decrease in cells under differentiation demonstrate the stem characteristics of clones we selected. The findings regarding expression of differentiation markers and of Ca2+-channels require further investigation. After a deeper characterization, both F11 and D2 could represent a good model to improve the knowledge on CSC and to identify new therapeutic approaches in GBM

Effects of conditioned medium from human amniotic mesenchymal tissue cell cultures on prostate cancer cells

It has been recently demonstrated that human amniotic mesenchymal tissue cells (hAMTC) derived from term placenta inhibit lymphocyte proliferation and significantly reduce the growth of haemopoietic and non haemopoietic cancer cell lines (HeLa and Saos cells) in vitro (1). The aim of our study was to evaluate the effects of hAMTC-conditioned medium (CM) on two human prostate cancer cells lines: LNCaP, androgen responsive and well differentiated, and PC-3, androgen unresponsive and less differentiated. Cells were grown in their standard culture conditions in the absence or in the presence of various concentrations (0.001–50%) of hAMTC-CM or their own exhausted medium. Cell numbers were determined by using a haemocytometer, after three days. Moreover, E- and N-cadherin expression was evaluated in PC-3 cells cultured in medium with 0.01, 1 or 25% hAMTC-CM by Immunocytochemistry and Western blot analysis. Our findings indicate that hAMTC-CM reduces the growth of both PC-3 and LNCaP cells. The effect is more pronounced in PC-3 cells in which inhibition is about 25% vs control (p<0.001) at a very low concentration (0.001%) and reaches the maximum (about 55% vs control, p<0.001) with the highest concentration used (50%). In LNCaP cells only the highest concentration of hAMTC-CM (50%) inhibits cell proliferation (about 40% vs control, p<0.001). Interestingly, growth of LNCaP cells is reduced by their own exhausted medium, while proliferation of PC-3 cells is not affected by their spent medium. Both E- and N-cadherin expression have been detected at the membrane level in untreated PC-3 cells and the localization does not change in hAMTC-CM-treated cells. Preliminary data obtained by Western blot analysis seem to indicate an increase in both E- and N-cadherin levels. Our findings show that hAMTC-CM reduces prostate cancer cell proliferation in relationship to their androgen sensitivity and modifies the expression levels of adhesion molecules. Experiments are in progress to determine the mechanisms which underlie the observed effects and assess if hAMTC-CM can determine any variation in the differentiation status of prostate cancer cells

Relevance of tumour surrounding area in chemoresistance of glioblastoma (GBM)

The mechanisms responsible for resistance to damage in normal cells might determine chemoresistance in both tumor cells and cancer stem cells (CSC). Relapse due to chemoresistant residual disease is a major cause of death in GBM. Increasing body of evidence indicates that not only tumor area (TT), but also tissue surrounding the tumor border (pTT) of GBM contains tumor cells and CSC, which could contribute to the disease progression. Therefore, the need to have a deeper insight in this area through identification of the characteristics that confer chemoresistance. In this study, the expression of molecules involved in chemoresistance was investigated in samples derived from TT and from pTT at <1 cm from the tumor border, in 40 patients with GBM. The expression of O6-methylguanine-DNA methyltransferase (MGMT), a suicidal DNA repair protein; Breast Cancer Resistance Protein (BCRP1), a drug efflux transporter, and A2B5 (c-series gangliosides) has been determined by immunohistochemistry. The percentage of MGMT positive cells was higher (p<0.0001, paired Student’s t test) in pTT (median: 53.5, range: 0.6-92.4) with respect to TT (median: 3.3, range: 0.0-70.7). The same trend was observed in BCRP1 expression (p<0.02; pTT, median: 27.6, range: 1.0-95.6; TT, median: 10.1, range: 0.2-72.1). No difference was found between pTT and TT in A2B5 expression (p=0.69, pTT, median: 29.8, range: 0.0-98.4; TT, median: 26.0, range: 0.0-96.8). Patients were then divided into two groups according to presence (group A) or absence (group B) of tumor cells in pTT. The trend previous observed in MGMT expression was maintained in both groups, while only in group A a statistically significant difference in BCRP1 expression was observed. Our results confirm that the tissue surrounding GBM is not a normal tissue, and that it represents a frontline of tumor invasion, particularly for the presence of some molecules involved in chemoresistance, which could explain the disease recurrence after the conventional treatment of GBM. Experiments about the expression of above mentioned molecules in CSC from pTT and TT are in progress. Supported by FIRB, ACCORDI DI PROGRAMMA 201

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

Breast cancer-associated fibroblasts promote tumor cell migration: crucial role of Stearoyl CoA Desaturase1 and paracrine signalings

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

Breast cancer-associated fibroblasts promote tumor cell migration: crucial role of Stearoyl CoA Desaturase1 and paracrine signalings

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

Immunohistochemical Analysis of DNA Repair- and Drug-Efflux-Associated Molecules in Tumor and Peritumor Areas of Glioblastoma

Glioblastoma (GBM), the most commonly occurring primary tumor arising within the central nervous system, is characterized by high invasiveness and poor prognosis. In spite of the improvement in surgical techniques, along with the administration of chemo- and radiation therapy and the incessant investigation in search of prospective therapeutic targets, the local recurrence that frequently occurs within the peritumoral brain tissue makes GBM the most malignant and terminal type of astrocytoma. In the current study, we investigated both GBM and peritumoral tissues obtained from 55 hospitalized patients and the expression of three molecules involved in the onset of resistance/unresponsiveness to chemotherapy: O6-methylguanine methyltransferase (MGMT), breast cancer resistance protein (BCRP1), and A2B5. We propose that the expression of these molecules in the peritumoral tissue might be crucial to promoting the development of early tumorigenic events in the tissue surrounding GBM as well as responsible for the recurrence originating in this apparently normal area and, accordingly, for the resistance to treatment with the standard chemotherapeutic regimen. Notably, the inverse correlation found between MGMT expression in peritumoral tissue and patients' survival suggests a prognostic role for this protein

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-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 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

Degarelix activity on cell growth and cell migration of three human prostate cancer cell lines

The direct effects of Degarelix (10-11-10-5M), a third generation GnRH antagonist, on prolif-eration and cell migration of three human prostate cancer lines (LNCaP, androgen-sensitive; DU145 and PC3, androgen-independent) have been explored. Moreover, the capability of De-garelix of counteracting the mitogenic activity and the cell migration due to EGF has been evaluated in DU145 cells. Cell counts were performed with a hemocytometer and the “scratch test assay” was used to evaluate cell migration. Statistical significance of the data was assessed by Student’s t test. In both LNCaP and PC3 cells, the antagonist reduced the growth (~15%, p 60%, p60%, p<0.0001). EGF increased DU145 cell proliferation (~45%, p<0.01) and the addition of Degarelix reduced the stimulatory effect of the growth factor. Moreover, 50 ng/ml EGF increased (p<0.05), while 10-11M Degarelix inhibited (p<0.05) cell migration of DU145 cells. Finally, 10-11M Degarelix reduced (p<0.01) the cell migration induced by EGF. Our results clearly show that Degarelix has an inhibitory activity on proliferation of prostate cancer cell lines with different characteristics in terms of androgen sensitivity, differentiation and aggressiveness. This effect is observed at therapeutic concentrations of the GnRH antago-nist. In the androgen-independent DU145 cells, the lowest concentration of Degarelix(10-11M), which is ineffective on cell proliferation, reduces cell migration both in standard culture condition and in the presence of EGF. This phenomenon might be relevant in meta-static events