Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl-/- and rank-/- pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl-/- mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.

Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1alpha. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)alpha, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1alpha and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1alpha expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1alpha in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1alpha construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1alpha. When exposed to hypoxia, infected cells failed to show HIF-1alpha and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1alpha in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin ␣ v ␤ 3 , has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of ␣ v ␤ 3 in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin ␣ v ␤ 3 on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. ␣ v ␤ 3 was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-␣ v ␤ 3 monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin ␣ v ␤ 3 , only L1 serves as a potential ligand for ␣ v ␤ 3 during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and ␣ v ␤ 3 antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin ␣ v ␤ 3 on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells. INTRODUCTIONThe process of tumor metastasis consists of a complex cascade of adhesive interactions between tumor cells and host tissues (Nicolson, 1988;Stetler-Stevenson et al., 1993;Orr et al., 2000). The endothelium of blood vessels constitutes a physical barrier to cells in the circulatory system and metastatic cells must penetrate the interendothelial junctions to invade the underlying tissue. Although much is known about the adhesive interactions during the invasion of the basement membrane, relatively little is known about the mechanism by which tumor cells pass through the endothelial junction. We have developed a novel in vitro coculture assay to investigate the molecular interactions and morphological changes during cancer cell extravasation (Sandig et al., 1997;Voura et al., 1998a). Our previous work has shown that although platelet-endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin are not required for melanoma transendothelial migration, heterotypic interactions with classic cadherins may play a role in this process (Sandig et al., 1997). However, inclusion of anti-cadherin antibodies in the coculture assay results in only a low level of inhibition of the transmigration process, implicating the involvement of other cell adhesion molecules in this process.In this report, we examine the role of ␣ v ␤ 3 during melanoma transendothelial migration in our in vitro assay system. The integrin ␣ v ␤ 3 was first identified as the "vitronectin receptor" but will adhere to a host of other extracellular matrix (ECM) proteins, including fibronectin, laminin, collagen, ...