David Herrmann

Three-dimensional cancer models mimic cell–matrix interactions in the tumour microenvironment

Basic in vitro systems can be used to model and assess complex diseases, such as cancer. Recent advances in this field include the incorporation of multiple cell types and extracellular matrix proteins into three-dimensional (3D) models to recapitulate the structure, organization and functionality of live tissue in situ. Cells within such a 3D environment behave very differently from cells on two-dimensional (2D) substrates, as cell-matrix interactions trigger signalling pathways and cellular responses in 3D, which may not be observed in 2D. Thus, the use of 3D systems can be advantageous for the assessment of disease progression over 2D set-ups alone. Here, we highlight the current advantages and challenges of employing 3D systems in the study of cancer and provide an overview to guide the appropriate use of distinct models in cancer research.

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis

Fine-tuned manipulation of tumor tension and vasculature enhances response to chemotherapy and impairs metastatic spread in pancreatic cancer. ROCK-ing pancreatic cancer to the core Pancreatic cancer, one of the most deadly and difficult-to-treat tumor types in patients, usually has a dense stroma that can be difficult for drugs to penetrate. Stromal characteristics can also affect multiple other aspects of tumor biology, including metastatic spread, vascular supply, and immune response. Vennin et al. used Fasudil, a drug that inhibits a protein called ROCK and is already used for some conditions in people, to demonstrate the feasibility including short-term tumor stroma remodeling as part of cancer treatment. In genetically engineered and patient-derived mouse models of pancreatic cancer, priming with Fasudil disrupted the tumors’ extracellular matrix and improved the effectiveness of subsequent treatment with standard-of-care chemotherapy for this disease. The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or “priming,” using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

Without the protein phosphatase PTPN14, tumor cells secrete more prometastatic factors and send more growth-promoting receptors to the surface. Pez dispenses with metastasis Tumor cells have an increased amount of growth-promoting receptors on their surfaces, and they secrete factors that promote metastasis. Belle et al. found that the protein tyrosine phosphatase PTPN14 (also called Pez) suppressed the movement of prometastatic proteins toward the cell surface by binding and dephosphorylating the kinase PRKCD or the protein RIN1. Mice bearing breast cancer xenografts that lacked PTPN14 had larger tumors and a greater number of metastases. Breast cancers with decreased abundance of PTPN14 or increased abundance of RIN1 and PRKCD were more advanced. The findings explain how PTPN14 functions as a tumor suppressor. Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins.

Regulation of Tbx22 during facial and palatal development

Mutations in the gene encoding the T‐box transcription factor TBX22 cause X‐linked cleft palate and ankyloglossia in humans. Here we show that Tbx22 expression during facial and palatal development is regulated by FGF and BMP signaling. Our results demonstrate that FGF8 induces Tbx22 in the early face while BMP4 represses and thus restricts its expression. This regulation is conserved between chicken and mouse, although the Tbx22‐expression patterns differ considerably between these two species. We suggest that these species‐specific differences may result at least in part from differences in the spatiotemporal patterns of BMP activity, but we exclude a direct repression of Tbx22 by the BMP‐inducible transcriptional repressor MSX1. Together these findings help to integrate Tbx22 into the molecular network of factors regulating facial development. Developmental Dynamics 239:2860–2874, 2010.

Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

Summary E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.

ELF5 drives lung metastasis in luminal breast cancer through recruitment of Gr1+ CD11b+ myeloid-derived suppressor cells

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

The dynamics of Rho GTPase signaling and implications for targeting cancer and the tumor microenvironment

Numerous large scale genomics studies have demonstrated that cancer is a molecularly heterogeneous disease, characterized by acquired changes in the structure and DNA sequence of tumor genomes. More recently, the role of the equally complex tumor microenvironment in driving the aggressiveness of this disease is increasingly being realized. Tumor cells are surrounded by activated stroma, creating a dynamic environment that promotes cancer development, metastasis and chemoresistance. The Rho family of small GTPases plays an essential role in the regulation of cell shape, cytokinesis, cell adhesion, and cell motility. Importantly, these processes need to be considered in the context of a complex 3-dimensional (3D) environment, with reciprocal feedback and cross-talk taking place between the tumor cells and host environment. Here we discuss the role of molecular networks involving Rho GTPases in cancer, and the therapeutic implications of inhibiting Rho signaling in both cancer cells and the emerging concept of targeting the surrounding stroma.

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2−/− mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2—urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

Expression and regulation of ANTXR1 in the chick embryo.

Anthrax Toxin Receptor 1 (ANTXR1; also known as Tumor Endothelial Marker 8, TEM8) is one of several genes that was recently found to be up‐regulated in tumor‐associated endothelial cells. In vitro, the protein can link extracellular matrix components with the actin cytoskeleton to promote cell adhesion and cell spreading. Both, ANTXR1 and the closely related ANTXR2 can bind anthrax toxin and interact with lipoprotein receptor‐related protein 5 and 6, which also work as coreceptors in the WNT signaling pathway. Here, we report the cloning of chick ANTXR1 from a suppression subtractive hybridization screen for fibroblast growth factor (FGF) ‐inducible genes in chicken embryonic facial mesenchyme. We show that chicken ANTXR1 is dynamically expressed throughout embryogenesis, starting from Hamburger and Hamilton stage 10. Furthermore, we demonstrate that FGF signaling is sufficient, but not necessary, to induce ANTXR1 expression in chicken facial mesenchyme. Developmental Dynamics 239:680–687, 2010.

Monitoring the dynamics of Src activity in response to anti-invasive dasatinib treatment at a subcellular level using dual intravital imaging

Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting.