Fibroblast-derived matrix models desmoplastic properties and forms a prognostic signature in cancer progression.

The desmoplastic reaction observed in many cancers is a hallmark of disease progression and prognosis, particularly in breast and pancreatic cancer. Stromal-derived extracellular matrix (ECM) is significantly altered in desmoplasia, and as such plays a critical role in driving cancer progression. Using fibroblast-derived matrices (FDMs), we show that cancer cells have increased growth on cancer associated FDMs, when compared to FDMs derived from non-malignant tissue (normal) fibroblasts. We assess the changes in ECM characteristics from normal to cancer-associated stroma at the primary tumor site. Compositional, structural, and mechanical analyses reveal significant differences, with an increase in abundance of core ECM proteins, coupled with an increase in stiffness and density in cancer-associated FDMs. From compositional changes of FDM, we derived a 36-ECM protein signature, which we show matches in large part with the changes in pancreatic ductal adenocarcinoma (PDAC) tumor and metastases progression. Additionally, this signature also matches at the transcriptomic level in multiple cancer types in patients, prognostic of their survival. Together, our results show relevance of FDMs for cancer modelling and identification of desmoplastic ECM components for further mechanistic studies.

Modeling Metastatic Colonization in a Decellularized Organ Scaffold-Based Perfusion Bioreactor.

Metastatic cancer spread is responsible for most cancer-related deaths. To colonize a new organ, invading cells adapt to, and remodel, the local extracellular matrix (ECM), a network of proteins and proteoglycans underpinning all tissues, and a critical regulator of homeostasis and disease. However, there is a major lack in tools to study cancer cell behavior within native 3D ECM. Here, an in-house designed bioreactor, where mouse organ ECM scaffolds are perfused and populated with cells that are challenged to colonize it, is presented. Using a specialized bioreactor chamber, it is possible to monitor cell behavior microscopically (e.g., proliferation, migration) within the organ scaffold. Cancer cells in this system recapitulate cell signaling observed in vivo and remodel complex native ECM. Moreover, the bioreactors are compatible with co-culturing cell types of different genetic origin comprising the normal and tumor microenvironment. This degree of experimental flexibility in an organ-specific and 3D context, opens new possibilities to study cell-cell and cell-ECM interplay and to model diseases in a controllable organ-specific system ex vivo.

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.

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.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Author Correction: Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Framing cancer progression: influence of the organ- and tumour-specific matrisome.

The extracellular matrix (ECM) plays a crucial role in regulating organ homeostasis. It provides mechanical and biochemical cues directing cellular behaviour and, therefore, has control over the progression of diseases such as cancer. Recent efforts have greatly enhanced our knowledge of the protein composition of the ECM and its regulators, the so-called matrisome, in healthy and cancerous tissues; yet, an overview of the common signatures and organ-specific ECM in cancer is missing. Here, we address this by taking a detailed approach to review why cancer grows in certain organs, and focus on the influence of the matrisome at primary and metastatic tumour sites. Our in-depth and comprehensive review of the current literature and general understanding identifies important commonalities and distinctions, providing insight into the biology of metastasis, which could pave the way to improve future diagnostics and therapies.

Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.

AMPK negatively regulates tensin-dependent integrin activity.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a ß1-integrin inhibitor in fibroblasts. Loss of AMPK promotes ß1-integrin activity, the formation of centrally located active ß1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases ß1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind ß1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.