Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem­cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell­induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non­random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

Expression and functional role of CRIPTO-1 in cutaneous melanoma.

BACKGROUND: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. METHODS: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. RESULTS: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. CONCLUSION: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

'Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma'.

Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Here, we show that PEDF expression is high in melanocytes, but it is lost during malignant progression of human melanoma. Using a high-throughput analysis of the data from microarray studies of molecular profiling of human melanoma, we found that PEDF expression is lost in highly invasive melanomas. In paired cell lines established from the same lesion but representing the high and low extremes of malignant potential, abundant PEDF expression was restricted to the poorly aggressive counterparts. We used RNA interference to directly address the functional consequences of PEDF silencing. PEDF knockdown in poorly aggressive melanoma cell lines augmented migration, invasion and vasculogenic mimicry, which translated into an increased in vivo metastatic potential. PEDF interference also significantly enhanced the migratory and invasive capability of normal melanocytes and moderately increased their proliferative potential. Our results show that loss of PEDF enables melanoma cells to acquire an invasive phenotype and, therefore, modulation of this multifunctional factor could be critical for the malignant progression of human melanoma.

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells.

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

The tumour suppressor gene maspin is differentially regulated in cytotrophoblasts during human placental development.

Identification of factors that play a role in regulating the highly invasive ability of human placental cells throughout gestation will contribute to a better understanding of this unique developmental process. The aims of this study were to determine whether the tumour suppressor gene maspin is present in the human placenta and plays a putative role in the regulation of cytotrophoblast invasion during placental development. The data showed that the expression of maspin mRNA was maximum in term placentae compared to the first and second trimester tissues, and absent in the HTR-SVneo (immortalized extravillous cytotrophoblast), JEG-3 and JAR (choriocarcinoma) cell lines. Maspin protein, detected by Western blot analysis, was twofold higher in the second trimester and 4.4-fold higher in the third trimester compared to the first trimester. Maspin immunohistochemical staining was localized in cytotrophoblasts with increased and more diffuse staining in the second and third trimesters. Corresponding to the period of maximum maspin expression, cytotrophoblasts isolated from term placentae had significantly lower invasive ability as compared to first and second trimester cytotrophoblasts (P< 0.03). Further, addition of recombinant maspin significantly decreased cytotrophoblast invasion in vitro by 40-50 per cent in all three trimesters of gestation. This study provides the first evidence of the temporal expression of maspin during human gestation and suggests a putative role for maspin in regulating the invasive activity of cytotrophoblasts at term. The down-regulation of maspin expression may be critical at the time of implantation and early placental development, whereas upregulation of maspin may serve as a signal for the end of cytotrophoblast invasion and gestation.