Extensive experimental evidence shows that platelets support tumour metastasis. The activation of platelets and the coagulation system have a crucial role in the progression of cancer. Within the circulatory system, platelets guard tumour cells from immune elimination and promote their arrest at the endothelium, supporting the establishment of secondary lesions. These contributions of platelets to tumour cell survival and spread suggest platelets as a new avenue for therapy.

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p -Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2 + cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

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Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with beta1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, alpha3beta1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Brain metastases are diagnosed in 10 to 40% of all cancer patients, and the incidence is rising as patients live longer due to improved treatments for extracranial metastases.1,2 Brain lesions are most frequently associated with lung cancer, breast cancer, and melanoma. 1,2 Unfortunately, brain metastases are still very difficult to treat and the mechanisms underlying their establishment and progression are poorly understood. Thus, information in this direction and models for analysis are a prerequisite for the development of new, efficient therapies.The essential role of the tumor microenvironment in cancer progression has been well documented for extracranial malignancies, and recent findings indicate that the tumor microenvironment might be a suitable target in anticancer therapies, as well as a valuable biomarker for prognostic purposes.3-5 The brain provides a unique environment with paracrine growth factors that differ from most other organs. 6,7 The involvement of brain-resident cells including brain endothelial cells, microglia, and astrocytes in the pathology of primary and metastatic brain tumors is only partially understood. Brain endothelial cells are the first host cell type that circulating cancer cells encounter when they arrest within the brain microvasculature. In addition to posing the initial barrier for brain invasion, endothelial cells and their basement membrane seem to play important roles in supporting the growth of brain metastases as well as brain tumor stem cells. 8 -10

IntroductionDespite advances in clinical therapy, metastasis is still the leading cause of death in breast cancer patients (1). A clearer understanding of molecular mechanisms that drive metastasis will help to develop more effective therapies (2). Our present study focused on metabolism as an essential driver of tumor growth and metastasis, potentially common to all breast cancer types. Normal cells primarily use mitochondrial oxidative phosphorylation (OXPHOS) for energy production, whereas cancer cells depend on aerobic glycolysis (the Warburg effect) to generate energy and glycolytic intermediates for enhanced growth (3, 4). Tumor cells also generate high levels of reduced forms of NAD + , NADH, and NADPH as important cofactors and redox components (4, 5). These altered metabolic activities can be linked to mitochondrial dysfunction that inhibits OXPHOS, increases ROS, promotes uncontrolled growth, and causes DNA damage that further supports a metastatic phenotype (6, 7). Mitochondrial dysfunctions can be caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial proteins (6,8) that are essential for the respiratory chain/OXPHOS system. Due to the lack of protective histones and limited DNA repair (8), mtDNA mutations occur at high rates and were found in tumors including breast cancer (6,(9)(10)(11)(12)(13)(14), which suggests that defects in OXPHOS might contribute to tumorigenesis.By combining the nuclear genome of a recipient cell with the mitochondrial genome of a donor cell using cybrid technology, mitochondria from the triple-negative aggressive breast cancer cell lines MDA-MB-435 (15) and MDA-MB-231 facilitated tumor progression and metastasis in nonmetastatic tumor cells (7, 10). The donor cell lines harbor mtDNA mutations in tRNAs, in the

Expression of adhesion receptor integrin ␣v␤3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that ␣v␤3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. Expression of activated ␣v␤3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated ␣v␤3. When transferred, this factor also up-regulated ␣v␤3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated ␣v␤3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced ␣v␤3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin ␣v␤3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated ␣v␤3.M etastasis is the primary cause of death in breast cancer patients. Metastatic dissemination depends on tumor cell adhesion, migration, and invasion. These steps involve integrins, a family of transmembrane adhesion receptors, composed of noncovalently linked ␣ and ␤ subunits (1). Integrins are known to exist in distinct states of activation, and these determine integrin functionality and affinity for ligands (2). For example, integrin activation controls which ligands are recognized, whether an integrin can support cell arrest under dynamic flow conditions or only stationary adhesion, and whether cells can migrate on or toward specific substrates (3). The importance of integrin activation has long been appreciated in leukocytes and platelets, where it controls inflammatory responses and thrombus formation (4, 5). Recent findings indicate that other cell types, such as endothelial cells and tumor cells, can also regulate their interaction with extracellular matrix proteins by integrin activation (6-9). This regulation may help to control angiogenesis and tumor metastasis.In breast and ovarian cancer, as well as in melanoma and glioma, malignant progression is associated with expression of tumor cell integrin ␣v␤3 (10-14). We recently found that ␣v␤3 can exist in human breast cancer cells in an activated or a nonactivated functional state. Only the activated state supports breast cancer cell arrest du...

Brain metastases are among the most feared complications in breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cell growth within the brain tissue. To provide information in this direction, we established a human breast cancer cell model for brain metastasis based on circulating tumor cells from a breast cancer patient and variants of these cells derived from bone or brain lesions in immunodeficient mice. The brain-derived cells showed an increased potential for brain metastasis in vivo and exhibited a unique protein expression profile identified by large-scale proteomic analysis. This protein profile is consistent with either a selection of predisposed cells or bioenergetic adaptation of the tumor cells to the unique energy metabolism of the brain. Increased expression of enzymes involved in glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways suggests that the brain metastatic cells derive energy from glucose oxidation. The cells further showed enhanced activation of the pentose phosphate pathway and the glutathione system, which can minimize production of reactive oxygen species resulting from an enhanced oxidative metabolism. These changes promoted resistance of brain metastatic cells to drugs that affect the cellular redox balance. Importantly, the metabolic alterations are associated with strongly enhanced tumor cell survival and proliferation in the brain microenvironment. Thus, our data support the hypothesis that predisposition or adaptation of the tumor cell energy metabolism is a key element in breast cancer brain metastasis, and raise the possibility of targeting the functional differentiation in breast cancer brain lesions as a novel therapeutic strategy. [Cancer Res 2007;67(4):1472-86]