Extracellular matrix players in metastatic niches

Nature 481 7379, 85–89 (2012); published online December072011Metastatic niches support the survival and fitness of disseminated tumour cells (DTCs) in otherwise inhospitable tissue environments. The components of metastatic niches have remained a matter of conjecture, but recent reports, including one in a current issue of Nature, point at the extracellular matrix (ECM) proteins periostin and tenascin C (TNC) as key metastatic niche molecules. By enhancing Wnt and Notch signalling in cancer cells, these proteins provide physical as well as signalling support for metastasis-initiating cells. These findings underscore the importance of the ECM environment in cancer and provide potential drug targets against metastasis.In many cancers, tumour cells start spreading through the body long before the primary tumour is detected and removed (Pantel et al, 2009). Although cancer cells may enter the circulation and egress into distant tissues by the millions, only a few of these cells manage to form overt metastases. This rate is far too low to be explained solely by a scarcity of metastasis-initiating cells, but it rather suggests that the tissue environment in the target sites is generally inhospitable to DTCs. Some DTCs do thrive nonetheless, and form metastases, implying that these metastasis-initiating cells found exceptional spots that provided support to resist the new environment and remain fit for eventual outgrowth. A context that provides DTCs with this kind of support is referred to as a ‘metastatic niche'', by analogy to the niches that support stem cells in healthy tissues.In recent years, much attention has been devoted to stromal cells that rally to tumours and secrete enzymes, growth factors and angiogenic cytokines for tumour growth and metastasis (Joyce and Pollard, 2009). Another important source of regulatory signals in normal tissues and tumours is the ECM (Hynes, 2009). Owing to the complex composition and interactions of the ECM components, and the rarity of oncogenic ECM mutations in cancer, the specific roles of these components in metastasis have remained elusive. However, several reports have recently revealed that the ECM proteins periostin and TNC play key roles as metastasis niche components for tumour-initiating cells that invade the lungs (Figure 1A; Malanchi et al, 2011; Oskarsson et al, 2011; O''Connell et al, 2011).In the most recent of these reports, Malanchi et al (2011) show that the ECM protein periostin, is expressed in the end buds of mammary glands. The authors also detect periostin expression in myofibroblasts of mouse mammary tumours and their metastases in the lungs and demonstrate a role for periostin in metastasis initiation by means of periostin null mice. These mice can develop mammary tumours driven by a polyoma virus middle T antigen (PyMT) transgene. However, the ability of these tumours to metastasize to the lungs is significantly diminished compared with PyMT-driven tumours in wild-type mice. The in vitro growth of tumour cell populations in suspension oncospheres (an assay that enriches for tumour-initiating cells) could be blocked by anti-periostin antibodies. The authors show that stromal fibroblasts increase periostin production in response to TGF-β3, and periostin acts by presenting Wnt to the cancer cells leading to enhanced colonization of the lungs (Figure 1B). Moreover, they demonstrate that the only cancer cells able to benefit from periostin, respond to Wnt, and initiate metastasis are contained within a subpopulation defined by Thy-1 and CD24 markers. This population comprises ∼3% of the PyMT mammary tumour cells, and has been shown to represent cells enriched with tumour-initiating capacity in mouse models (Cho et al, 2008). Based on this, Malanchi et al propose that the role of periostin in progression of lung metastasis is to concentrate Wnt ligands in the metastatic niche for the stimulation of stem-like metastasis-initiating cells. These findings provide an exciting example of the role of the ECM in metastasis outgrowth.Open in a separate windowFigure 1(A) The ECM components periostin and TNC in the metastatic niche help activate developmental pathways for the viability of metastasis-initiating cells in the lungs. (B) In the pulmonary parenchyma, TGF-β3 stimulates myofibroblasts to produce periostin, which binds stromal Wnt factors for presentation to stem-like metastasis-initiating cells (Malanchi et al, 2011). Myofibroblasts and the cancer cells themselves also produce TNC, which promotes the intracellular functioning of the Wnt and Notch pathways (Oskarsson et al, 2011). A direct biochemical connection between these functions is likely, as periostin binds TNC and anchors it to ECM components including fibronectin and type I collagen (Kii et al, 2010).These new findings have striking parallels with recent findings on the role of TNC in breast cancer metastasis to the lungs (Oskarsson et al, 2011). TNC forms radial hexamers (hexabrachions) and interacts with various membrane receptors and ECM proteins. TNC is present in stem cell niches and tumour invasive fronts, and its expression in breast tumours is clinically associated with lung metastasis. TNC is expressed not only in cancer-associated fibroblasts but also in breast cancer cells. Stem-like human breast cancer cells expressing TNC showed a superior ability to form lung metastases when implanted as orthotopic tumours in mice (Oskarsson et al, 2011). TNC was found to support the survival and fitness of metastasis-initiating cells by enhancing their responsiveness to Wnt and Notch (Figure 1B). This effect was mediated by TNC-dependent signalling to components of the Notch pathway (Musashi) and the Wnt pathway (LGR5). Although cancer cell-derived TNC provides an advantage in metastasis initiation, stromal TNC is important too. Indeed, TNC-deficient mice implanted with mammary cancer cells show resistance to the formation of lung metastases, suggesting a significant role for stromal TNC, which is produced by S100A4+ fibroblasts (O''Connell et al, 2011).The functional similarities between periostin and TNC as ECM components of the metastatic niche may not be coincidental. Earlier biochemical studies have shown that these two proteins bind tightly, with periostin additionally binding type I collagen and fibronectin, and thereby anchoring TNC to these general ECM components (Kii et al, 2010) (Figure 1B). The recent findings suggest that the collaboration between periostin and TNC in the metastasis niche and, more generally, in stem cell niches, may extend beyond building a proper ECM architecture. Periostin may gather Wnt for stem cells while TNC may enhance the ability of these cells to respond to Wnt and Notch (Figure 1B). Thus, periostin and TNC may represent two sides of the same metastasis niche coin.The role of these molecules in promoting metastasis initiation raises several interesting questions. Why are stem-like cancer cells the only population that can respond to Wnt ligand presented by periostin? Are these cells uniquely capable of ‘reading'' periostin–TNC ECM units? And, what is the source of the TGF-β3 that induces periostin expression in myofibroblasts in the first place? Cancer cells and various stromal components can produce TGF-β, but the recent finding that cancer cell-associated platelets can act as carry-on source of TGF-β provides additional clues (Labelle et al, 2011).The new roles of periostin and TNC as ECM components of the metastatic niche, and other recent studies, in turn underscore the importance of developmental and cell survival pathways in metastasis. The key roles of the Wnt, Notch and PI3K pathways in metastatic progression is increasingly evident, as is the nature of the molecules that metastasis-initiating cells resort to in order to maximize the activity of these pathways in difficult microenvironments (Chen et al, 2011; Malanchi et al, 2011; Oskarsson et al, 2011). This wave of newly identified molecular components of the metastatic niche provides exciting opportunities to develop novel therapies to target the survival and viability of DTCs, to complement and eventually replace adjuvant chemotherapy in the oncology clinic. This may be particularly relevant in cancer-like breast cancer where DTCs must survive in latency for long periods while they await a chance for outgrowth (Pantel et al, 2009). Targeting the signalling provided by the metastatic niche could reduce the probability of a relapse.     

Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.     

Challenges and Solutions in the Development of Genomic Biomarker Panels: A Systematic Phased Approach

In the post-genome era, high throughput gene expression profiling has been successfully used to develop genomic biomarker panels (GBP) that can be integrated into clinical decision making. The development of GBPs in the context of personalized medicine is a scientifically challenging and resource-intense process. It needs to be accomplished in a systematic phased approach to address biological variation related to a clinical phenotype (e.g. disease etiology, gender, etc.) and minimize technical variation (noise). Here we present the methodological aspects of GBP development based on the experience of the Cardiac Allograft Rejection Gene Expression Observation (CARGO) study, a study that lead to the development of a molecular classifier for rejection screening in heart transplant patients.     

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.     

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

Background  

Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.     

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.     

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.     

Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor

The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Suspension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and microbial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/m3. day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were performed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detachment forces. The higher biomass content is associated with a lower biomass substrate loading rate and therefore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the observed biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuberances), denser and thinner compact biofilms were obtained when the biomass surface production rate decreased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance between biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low detachment forces, the biofilm becomes highly heterogeneous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. Copyright 1998 John Wiley & Sons, Inc.     

Stent implantation in acute myocardial infarction using a heparin-coated stent: a pilot study as a preamble to a randomized trial comparing balloon angioplasty and stenting

Preliminary experience with primary stenting in myocardial infarction has suggested a greater benefit in clinical outcome than has been obtained with direct balloon angioplasty. However, subacute thrombosis (SAT) remains a limitation for this new mode of therapy. In the BENESTENT II Pilot and main trials, the incidence of SAT with the heparin-coated Palmaz-Schatz stent was only 0.15%. Therefore, as a preamble to a large randomized trial, the feasibility and safety of the use of the Heparin-Coated Palmaz-Schatz trade mark Stent in Acute Myocardial Infarction (AMI) was tested in 101 patients enrolled between April and September 1996 in 18 clinical centres. In 101 stent-eligible AMI patients, as dictated by protocol, a heparin-coated stent was implanted. The primary objectives were to determine the in-hospital incidence of major adverse cardiac events (MACE: death, MI, target lesion revascularization) and bleeding complications, while the secondary objectives were the procedural success rate and the MACE, the restenosis and reocclusion rates at 6.5 months. Stent implantation (n 3 129 stents) was successful in 97 patients of the 101 who were included in this trial. During their hospital stay, two patients died and no patient experienced re-infarction, ischaemia prompting re-PTCA or CABG. Four patients suffered a bleeding complication, three major and one minor, of whom three required surgical repair. At 210 days follow-up, 81% of the patients were event free. At 6.5 months restenosis was documented in 18% of the 88 patients who underwent follow-up angiography, including three total occlusions. The results, both with respect to QCA and the occurrence of MACE, compare favourably with studies using elective stenting in both stable and unstable angina patients. As a result of this pilot study, a large randomized trial comparing direct balloon angioplasty with direct stenting in 900 patients with AMI was initiated in December 1996.