Olivier De Wever

Role of tissue stroma in cancer cell invasion

Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Stromal changes at the invasion front include the appearance of myofibroblasts, cells sharing characteristics with fibroblasts and smooth muscle cells. The main precursors of myofibroblasts are fibroblasts. The transdifferentiation of fibroblasts into myofibroblasts is modulated by cancer cell‐derived cytokines, such as transforming growth factor‐β (TGF‐β). TGF‐β causes cancer progression through paracrine and autocrine effects. Paracrine effects of TGF‐β implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts. Autocrine effects of TGF‐β in cancer cells with a functional TGF‐β receptor complex may be caused by a convergence between TGF‐β signalling and β‐catenin or activating Ras mutations. Experimental and clinical observations indicate that myofibroblasts produce pro‐invasive signals. Such signals may also be implicated in cancer pain. N‐Cadherin and its soluble form act as invasion‐promoters. N‐Cadherin is expressed in invasive cancer cells and in host cells such as myofibroblasts, neurons, smooth muscle cells, and endothelial cells. N‐Cadherin‐dependent heterotypic contacts may promote matrix invasion, perineural invasion, muscular invasion, and transendothelial migration; the extracellular, the juxtamembrane and the β‐catenin binding domain of N‐cadherin are implicated in positive invasion signalling pathways. A better understanding of stromal contributions to cancer progression will likely increase our awareness of the importance of the combinatorial signals that support and promote growth, dedifferentiation, invasion, and ectopic survival and eventually result in the identification of new therapeutics targeting the stroma. Copyright

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

Stromal myofibroblasts are drivers of invasive cancer growth

Tissue integrity is maintained by the stroma in physiology. In cancer, however, tissue invasion is driven by the stroma. Myofibroblasts and cancer‐associated fibroblasts are important components of the tumor stroma. The origin of myofibroblasts remains controversial, although fibroblasts and bone marrow‐derived precursors are considered to be the main progenitor cells. Myofibroblast reactions also occur in fibrosis. Therefore, we wonder whether nontumorous myofibroblasts have different characteristics and different origins as compared to tumor‐associated myofibroblasts. The mutual interaction between cancer cells and myofibroblasts is dependent on multiple invasive growth‐promoting factors, through direct cell–cell contacts and paracrine signals. Since fibrosis is a major side effect of radiotherapy, we address the question how the main methods of cancer management, including chemotherapy, hormonotherapy and surgery affect myofibroblasts and by inference the surrogate endpoints invasion and metastasis.

Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)‐β stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF‐β‐upregulated extracellular matrix glycoprotein tenascin‐C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast‐stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)‐like repeats of TNC through EGF‐receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c‐Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA‐Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications.

Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

Background Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Methodology/Principal Findings Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearmans Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. Conclusions/Significance The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.

Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-β or wounding

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel™ when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-β (TGF-β) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-β. Functional N-cadherin activity is implicated in TGF-β stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-β1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-β stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Bone marrow stromal cell–derived exosomes as communicators in drug resistance in multiple myeloma cells

The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.