Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.
Henderson N, Markwick LJ, Elshaw SR, Freyer AM, Knox AJ, Johnson SR. Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration. Am J Physiol Lung Cell Mol Physiol 292: L1030-L1038, 2007. First published December 22, 2006; doi:10.1152/ajplung.00317.2006.-Increased proinflammatory mediators and ECM deposition are key features of the airways in asthma. Matrix metalloproteinases (MMPs) are produced by airway smooth muscle (ASM) cells and have multiple roles in inflammation and tissue remodeling. We hypothesized that components of the asthmatic airway would stimulate MMP production and activation by ASM and contribute to airway remodeling. We measured human ASM-derived MMP mRNA, protein, and activity by real-time RT-PCR, zymography, Western blotting, and MMP activity assay. Collagen I and thrombin caused a synergistic increase in MMP-2 protein and total MMP activity but paradoxically decreased MMP-2 mRNA. Additionally, collagen I activated MMP-2 in culture supernatants independent of the cell surface. Together, collagen I and thrombin strongly enhanced MMP-14 mRNA and protein but had no effect individually, suggesting increased MMP-14, the activating protease for MMP-2, may be partially responsible for MMP-2 activation. Furthermore, collagen I reduced tissue inhibitor of metalloproteinase-2 protein (TIMP-2). We examined the role of MMPs in functions of ASM related to airway remodeling and found migration and proliferation were MMP dependent, whereas adhesion and apoptosis were not. Ilomastat inhibited migration by 25%, which was also inhibited by TIMPs 1-4 and increased by the MMP-2 activator thrombin. These in vitro findings suggest that the environment within the airways of patients with asthma enhances MMP-2 and -14 protein and activity by a complex interaction of transcriptional and posttranscriptional mechanisms, which may contribute to ASM migration. matrix metalloproteinases; asthma PATIENTS WITH ASTHMA DEVELOP structural airway changes termed remodeling. Starting in infancy, remodeling is categorized by airway smooth muscle (ASM) hypertrophy, hyperplasia, increased subepithelial myofibroblasts, altered ECM deposition, infiltration of ASM by mast cells, increased mucosal vascularity, epithelial shedding, metaplasia, and mucus gland hyperplasia (1,22,43). Remodeling results in bronchial hyperresponsiveness and airflow obstruction and, therefore, increased symptoms and use of asthma medication and health care resources (1, 34). The ASM cell is emerging as a key cell in airway remodeling being increased in both size and number in the airways of patients with asthma (4). Moreover, ASM cells produce ECM components (10), pro-and anti-inflammatory mediators, and growth factors of relevance to airway remodeling, including vascular endothelial growth factor (30), interleukin-8 (52), eotaxin (41), and prostaglandin E 2 (39). Thus the ASM cell is an important modulator of inflammation, inflammatory cell influx, and angiogenesis in the asthmatic ...
1 Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells. 2 MMPs and TIMPs were examined using quantitative real-time RT-PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity. 3 The most abundant MMPs were pro-MMP-2, pro-MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4. 4 In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling.
Background/aims-Posterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The 1 family of integrins, v 3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement in uveal melanoma we analysed the expression of the 1 integrins, v 3, MMP-2, and MMP-9 in 10 primary posterior uveal melanomas, and correlated expression with invasive potential in vitro. Comparable studies were undertaken on cultures of melanocytes. Methods-Expression of integrins was studied by immunohistochemistry, secretion of MMP-2 and MMP-9 by zymography, and the invasive potential was assessed using a transwell model. Results-MMP-2 was secreted by all uveal melanomas and seven of 10 secreted MMP-9. Among uveal melanoma, invasion levels of 4-25% were observed and the major integrins expressed were 1 1, 2 1, 3 1, 5 1, and av 3. Melanocytes did not express 1 1, 4 1, and 6 1. Conclusion-The laminin binding 6 1 integrin was not expressed by either melanocytes or tumours with spindle morphology, which are considered to have a better prognosis. It is possible that expression of the 6 1 integrin may prove useful as a prognostic indicator. (Br J Ophthalmol 2001;85:732-738) Posterior uveal melanoma is the most common primary intraocular tumour in adults.1 Despite new methods of treating the primary melanoma, the survival rate has remained unchanged because of the problem of detection and treatment of metastases.2 Unlike cutaneous melanoma, which metastasises via lymphatic or haematogenous routes to various sites, uveal melanoma mainly disseminates haematogenously and preferentially establishes secondary disease in the liver.3 At least 30% of patients will develop metastases within 10 years of successful treatment of the primary tumour, 4 but at initial presentation, only 1-2% of patients show evidence of hepatic involvement.5 Detection of liver micrometastases is diYcult with currently available clinical tests and it is thought that many patients have subclinical metastases at diagnosis.5 Once metastatic disease has been detected, survival is usually less than 1 year.2 6 Traditional prognostic indicators are based on assessment of the diameter and histological appearance of the tumour 7 8 but it remains diYcult to determine which patients at initial presentation are at high risk of developing metastases.Metastasis is a "multistep" process and tumour cells are required to invade through extracellular matrices (ECMs) at various times during the process. It is proposed that a number of cells detach from the primary tumour and attach via adhesion molecules to components of the ECM or vascular endothelium to be invaded.9 ECM proteins are then degraded by proteolytic enzymes released from the primary tumour or the surrounding stroma.
Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n = 79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P = 0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (Po 0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P = 0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P = 0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.
Posterior uveal melanoma is the most common intraocular malignancy in adults. Metastasis occurs in approximately 40% of all cases and spread is primarily to the liver. Once secondary hepatic disease has developed the prognosis is poor. Metastasis involves a series of adhesion and de-adhesion events, coupled with regulated tissue degradation to facilitate tumour cell invasion and spread to both local and distant sites. These processes are assisted by the expression of integrins and degradative enzymes by both tumour and host cells. Using a series of 10 uveal melanomas, we investigated the expression of a panel of integrins, degradative enzymes and their inhibitors that have been shown to be associated with metastasis. In addition, we undertook to establish if there might be differential expression in response to growth under artificial conditions. All the tumours expressed matrix metalloproteinases (MMP)-2 and-9, tissue inhibitor of metalloproteases (TIMP)-2, urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI)-1 and PAI-2. Differences in the expression of the integrins alpha1beta1, alpha2beta1 and alpha6beta1 were observed; in particular, these differences appeared to relate to expression as a consequence of growth in culture. In summary, uveal melanoma cells express both degradative enzymes and their respective inhibitors, which are important in metastasis. It would appear that differential expression of integrins is present, probably as a response to in vitro stimulation.
Aims To establish if invasive and noninvasive uveal melanomas have differences in expression of adhesion molecules, and whether their adhesive interactions with the extracellular matrix (ECM) and endothelium vary. Methods Cells from an invasive and noninvasive uveal melanoma cell line and hepatic and dermal microvascular endothelial cells were assessed by flow cytometry for adhesion molecule expression. Tumour cell adhesion to ECM substrates (collagens I and IV, fibronectin, laminin, and vitronectin) and endothelial cells was also investigated using a commercially available assay or a fluorescencebased in vitro assay, respectively. The significance of results comparing cell lines was determined using a Student's t-test, whereby P-values of less than 0.05 were taken as significant. Results a1-and a4-integrins were not expressed by noninvasive cells, but were detected on invasive cells. The invasive cell line also expressed higher levels of other integrins than the noninvasive line. Correspondingly, invasive cells adhered in higher numbers to ECM substrates and endothelial cells, and for the latter, the difference was highly significant (Po0.001). No preference in adhesion of invasive cells for the hepatic endothelium was observed. Conclusions Successful attachment to and migration through the ECM, basement membrane, and endothelium are vital processes involved in malignant progression. Differential expression of a1-and a4-integrins by invasive and noninvasive cells infers a role for these receptors in invasion, while the ability of invasive cells to adhere more efficiently to the endothelium suggests that this is a critical factor in uveal melanoma invasion.
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