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The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin αvβ3 in brain metastatic growth and recruitment of blood vessels. Although αvβ3 is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the αvβ3 activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell αvβ3 supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated αvβ3, VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated αvβ3 depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell αvβ3 exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis. [ABSTRACT FROM AUTHOR]

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy. [ABSTRACT FROM AUTHOR]

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors, Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives). [ABSTRACT FROM AUTHOR]

Selective antitumor chemotherapy can be achieved by using antibody-drug conjugates that recognize surface proteins upregulated in cancer cells. One such receptor is integrin α3β1, which is overexpressed on malignant melanoma, prostate carcinoma, and glioma cells. We previously identified a human single-chain Fv antibody (scFv), denoted Pan10, specific for integrin α3β1 that is internalized by human pancreatic cancer cells. Herein, we describe the chemical introduction of reactive thiol groups onto Pan10, the specific conjugation of the modified scFv to maleimide-derivatized analogs of the potent cytotoxic agent duocarmycin SA, and the properties of the resultant conjugates. Our findings provide evidence that Pan10-drug conjugates maintain the internalizing capacity of the parent scFv and are cytotoxic at nanomolar concentrations. Our Pan10-drug conjugates may be promising candidates for targeted chemotherapy of malignant diseases associated with overexpression of integrin α3β1. [Copyright &y& Elsevier]

Overexpression of the cell-surface glycosphingolipid G[SUBM3] is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G[SUBM3] epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G[SUBM3]-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3AS, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K[SUBd]) ranging from 4.2 × 10[SUP-7] to 2.1× 1[SUP-5] M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G[SUBM3] panning reagent can be used to isolate a fully human scFv that is highly specific for native G[SUBM3] on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer. [ABSTRACT FROM AUTHOR]

Despite advances for the treatment of cancer, the prognosis for patients suffering from malignant brain tumors remains dismal. High-grade neoplasms, such as gliomas, are highly invasive and spawn widely disseminated microsatellites that have limited the efficacy of surgical and adjunctive therapies. The cancer stem cell hypothesis suggests that conventional chemotherapeutic treatments kill differentiated and differentiating cells which often form the bulk of the tumor. One major concern is that the cells which give rise to the tumor, the cancer stem cells, remain untouched and may be responsible for a relapse of the disease. Therefore, an adjunctive therapy to current cancer treatment is critical for the survivability of patients suffering from brain tumors. We have successfully engineered tumor-tropic neural stem cells to deliver antineoplastic gene products directly to the tumor-producing cells. This potential therapeutic strategy may safely eradicate tumor-producing cells in the brain while minimizing damage to normal, healthy cells.

Selective antitumor chemotherapy can be achieved by using antibody-drug conjugates that recognize surface proteins upregulated in cancer cells. One such receptor is integrin alpha3beta1, which is overexpressed on malignant melanoma, prostate carcinoma, and glioma cells. We previously identified a human single-chain Fv antibody (scFv), denoted Pan10, specific for integrin alpha3beta1 that is internalized by human pancreatic cancer cells. Herein, we describe the chemical introduction of reactive thiol groups onto Pan10, the specific conjugation of the modified scFv to maleimide-derivatized analogs of the potent cytotoxic agent duocarmycin SA, and the properties of the resultant conjugates. Our findings provide evidence that Pan10-drug conjugates maintain the internalizing capacity of the parent scFv and are cytotoxic at nanomolar concentrations. Our Pan10-drug conjugates may be promising candidates for targeted chemotherapy of malignant diseases associated with overexpression of integrin alpha3beta1.

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin alpha v beta 3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin alpha v beta 3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either alpha v beta 3, alpha IIb beta 3 or no beta 3 integrin, we found that only alpha v beta 3 strongly supported metastasis. Inhibition of tumor cell alpha v beta 3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow alpha v beta 3, but not alpha IIb beta 3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing alpha v beta 3, but not those expressing alpha IIb beta 3 or no beta 3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between alpha v beta 3 expression and protease processing. Furthermore, alpha v beta 3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin alpha v beta 3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin alpha IIb beta 3 cannot substitute for alpha v beta 3 in this respect.