Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis, and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin-4 (IL-4) is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs.[Keywords: Tumor-associated macrophage; tumor microenvironment; cysteine cathepsin; protease; interleukin-4; invasion] Supplemental material is available at http://www.genesdev.org.

We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.in-cell ͉ high-resolution ͉ liquid-state S tructural investigations of biomolecules are typically confined to artificial and isolated in vitro experimental setups. To study proteins in their native environment, i.e., within cells, recent attempts have aimed at the development of in vivo techniques for structural biology (1). X-ray crystallography and cryo-electron microscopy are intrinsically restricted from in vivo approaches because of their requirement for crystalline or vitrified specimens. NMR spectroscopy, the only other method for structural investigations at the atomic level, allows for the direct and selective observation of NMR-active nuclei within any NMR-inactive environment and can thus be used to structurally investigate labeled proteins inside living cells (2, 3). To date, all applications of in-cell NMR spectroscopy have been conducted in bacterial cells. Highresolution in vivo NMR experiments have been reported for the structural and functional characterization of cellular proteins (4-8), protein dynamics (9), and protein-protein interactions (10). Here, we have used intact Xenopus laevis oocytes to develop the first eukaryotic cellular system for intracellular NMR analyses of biomolecules at high resolution and at close-to-physiological levels of intracellular sample concentrations.Xenopus oocytes have long served as important laboratory tools in cellular and developmental biology (11-13). Their large size renders them suitable for microinjection and permits the precise cytoplasmic deposition of defined amounts of labeled, NMR-active proteins into an otherwise unlabeled, native cellular environment. This ensures quantitative sample delivery and a higher degree of reproducibility of in vivo measurements than is generally achievable by prokaryotic in-cell NMR methods, which overexpress labeled protein and conduct measurements within the same cells. Moreover, because labeled recombinant proteins are produced in Escherichia coli and conventionally purified before injection into oocytes, any background labeli...

Heparanase is an endo-␤-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and on the cell surface. Human proheparanase is produced as a latent protein of 543 amino acids whose activation involves excision of an internal linker segment (Ser 110 -Gln 157 ), yielding the active heterodimer composed of 8-and 50-kDa subunits. Applying cathepsin L knock-out tissues and cultured fibroblasts, as well as cathepsin L gene silencing and overexpression strategies, we demonstrate, for the first time, that removal of the linker peptide and conversion of proheparanase into its active 8 ؉ 50-kDa form is brought about predominantly by cathepsin L. Excision of a 10-amino acid peptide located at the C terminus of the linker segment between two functional cathepsin L cleavage sites (Y156Q and Y146Q) was critical for activation of proheparanase. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrates that the entire linker segment is susceptible to multiple endocleavages by cathepsin L, generating small peptides. Mass spectrometry demonstrated further that an active 8-kDa subunit can be generated by several alternative adjacent endocleavages, yielding the precise 8-kDa subunit and/or slightly elongated forms. Altogether, the mode of action presented here demonstrates that processing and activation of proheparanase can be brought about solely by cathepsin L. The critical involvement of cathepsin L in proheparanase processing and activation offers new strategies for inhibiting the prometastatic, proangiogenic, and proinflammatory activities of heparanase.

The Aurora family kinases are pivotal to the successful execution of cell division. Together they ensure the formation of a bipolar mitotic spindle, accurate segregation of chromosomes and the completion of cytokinesis. They are also attractive drug targets, being frequently deregulated in cancer and able to transform cells in vitro. In this review, we summarize current knowledge about the three family members, Aur-A, Aur-B and Aur-C. We then focus on Aur-A, its roles in mitotic progression, and its emerging roles in checkpoint control pathways. Aur-A activity can be controlled at several levels, including phosphorylation, ubiquitin-dependent proteolysis and interaction with both positive regulators, such as TPX2, and negative ones, like the tumor suppressor protein p53. In addition, work in Xenopus oocytes and early embryos has revealed a second role for Aur-A, directing the polyadenylation-dependent translation of specific mRNAs important for cell cycle progression. This function extends to post-mitotic neurons, and perhaps even to cycling somatic cells.