Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC

Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1 N), α-5 (hLM α-5 N) and β-3 (hLM β-3 N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1 N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function

Interplay Between LOX Enzymes and Integrins in the Tumor Microenvironment

Members of the lysyl oxidase (LOX) family are secreted copper-dependent amine oxidases that catalyze the covalent crosslinking of collagens and elastin in the extracellular matrix (ECM), an essential process for the structural integrity of all tissues. LOX enzymes can also remodel the tumor microenvironment and have been implicated in all stages of tumor initiation and progression of many cancer types. Changes in the ECM can influence several cancer cell phenotypes. Integrin adhesion complexes (IACs) physically connect cells with their microenvironment. This review article summarizes the main findings on the role of LOX proteins in modulating the tumor microenvironment, with a particular focus on how ECM changes are integrated by IACs to modulate cells behavior. Finally, we discuss how the development of selective LOX inhibitors may lead to novel and effective therapies in cancer treatment

Matritecture:Mapping the extracellular matrix architecture during health and disease

All cells in multicellular organisms are housed in the extracellular matrix (ECM), an acellular edifice built up by more than a thousand proteins and glycans. Cells engage in a reciprocal relationship with the ECM; they build, inhabit, maintain, and remodel the ECM, while, in turn, the ECM regulates their behavior. The homeostatic balance of cell-ECM interactions can be lost, due to ageing, irritants or diseases, which results in aberrant cell behavior. The ECM can suppress or promote disease progression, depending on the information relayed to cells. Instructions come in the form of biochemical (e.g., composition), biophysical (e.g., stiffness), and topographical (e.g., structure) cues. While advances have been made in many areas, we only have a very limited grasp of ECM topography. A detailed atlas deciphering the spatiotemporal arrangement of all ECM proteins is lacking. We feel that such an extracellular matrix architecture (matritecture) atlas should be a priority goal for ECM research. In this commentary, we will discuss the need to resolve the spatiotemporal matritecture to identify potential disease triggers and therapeutic targets and present strategies to address this goal. Such a detailed matritecture atlas will not only identify disease-specific ECM structures but may also guide future strategies to restructure disease-related ECM patterns reverting to a normal pattern

Laminin N-terminus α31 protein distribution in adult human tissues.

Laminin N-terminus α31 (LaNt α31) is a netrin-like protein derived from alternative splicing of the laminin α3 gene. Although LaNt α31 has been demonstrated to influence corneal and skin epithelial cell function, its expression has not been investigated beyond these tissues. In this study, we used immunohistochemistry to characterise the distribution of this protein in a wide-array of human tissue sections in comparison to laminin α3. The data revealed widespread LaNt α31 expression. In epithelial tissue, LaNt α31 was present in the basal layer of the epidermis, throughout the epithelium of the digestive tract, and in much of the epithelium of the reproductive system. LaNt α31 was also found throughout the vasculature of most tissues, with enrichment in reticular-like fibres in the extracellular matrix surrounding large vessels. A similar matrix pattern was observed around the terminal ducts in the breast and around the alveolar epithelium in the lung, where basement membrane staining was also evident. Specific enrichment of LaNt α31 was identified in sub-populations of cells of the kidney, liver, pancreas, and spleen, with variations in intensity between different cell types in the collecting ducts and glomeruli of the kidney. Intriguingly, LaNt α31 immunoreactivity was also evident in neurons of the central nervous system, in the cerebellum, cerebral cortex, and spinal cord. Together these findings suggest that LaNt α31 may be functionally relevant in a wider range of tissue contexts than previously anticipated, and the data provides a valuable basis for investigation into this interesting protein

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 Delta C solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease