Matrix stiffness modulates the activity of MMP-9 and TIMP-1 in hepatic stellate cells to perpetuate fibrosis

Liver fibrosis is characterised by a dense and highly cross-linked extracellular matrix (ECM) which promotes progression of diseases such as hepatocellular carcinoma. The fibrotic microenvironment is characterised by an increased stiffness, with rigidity associated with disease progression. External stiffness is known to promote hepatic stellate cell (HSC) activation through mechanotransduction, leading to increased secretion of ECM components. HSCs are key effector cells which maintain the composition of the ECM in health and disease, not only by regulating secretion of ECM proteins such as collagen, but also ECM-degrading enzymes called matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Uninhibited MMPs degrade ECM proteins to reduce external rigidity. Using fibronectin-coated polyacrylamide gels to alter substrate rigidity without altering ligand density, we show that fibrotic rigidities downregulate MMP-9 expression and secretion, and also upregulate secretion of TIMP-1, though not its expression. Using tissue immunofluorescence studies, we also report that the expression of MMP-9 is significantly decreased in activated HSCs in fibrotic tissues associated with hepatocellular carcinoma. This suggests the presence of a mechanical network that allows HSCs to maintain a fibrotic ECM, with external rigidity providing feedback which affects MMP-9 and TIMP-1 secretion, which may become dysregulated in fibrosis

Certifying forest-based ecosystem services: results from the world-first application of the FSC ecosystem service procedure in Italy

In recent years, several mechanisms have been put in practice to value forest-based ecosystem services in order to maintain or enhance their provision. Mechanisms range from more traditional - e.g. regulation of land use, taxes and subsidies - to more innovative market-based ones such as Payments for Ecosystem Services (PES). More recently, the use of voluntary third-party forest certification schemes based on standards and procedures specifically aimed at considering the provision of ecosystem services has gained interests. Certification schemes have already been established for specific ecosystem services, in particular focusing on carbon, in some cases including biodiversity conservation as a co-benefit. The Forest Stewardship Council\uae (FSC) is currently the only scheme promoting the certification of multiple ecosystem services by demonstrating the positive impacts of FSC-certified forest management practices on five regulating and cultural services. A dedicated procedure has been developed, that allows the assessment of the impact of forest management on carbon sequestration and storage, biodiversity conservation, watershed services, soil conservation and recreational services, by means of specific methodologies and indicators. We critically analyzed the world-first application of the FSC ecosystem services certification procedure with reference to the forest certification group Waldplus and Associazione Forestale di Pianura (Lowland Forest Association) in northern Italy. The case study includes 1,043 hectares of different forest types, from semi-natural productive forests to conservation forests, traditionally managed to deliver multiple benefits. Our objective is to give insights that could inform and improve future developments in the field of certification, promotion and marketing of ecosystem services

An expression signature of the angiogenic response in gastrointestinal neuroendocrine tumours: correlation with tumour phenotype and survival outcomes.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are heterogeneous with respect to biological behaviour and prognosis. As angiogenesis is a renowned pathogenic hallmark as well as a therapeutic target, we aimed to investigate the prognostic and clinico-pathological role of tissue markers of hypoxia and angiogenesis in GEP-NETs. METHODS: Tissue microarray (TMA) blocks were constructed with 86 tumours diagnosed from 1988 to 2010. Tissue microarray sections were immunostained for hypoxia inducible factor 1α (Hif-1α), vascular endothelial growth factor-A (VEGF-A), carbonic anhydrase IX (Ca-IX) and somatostatin receptors (SSTR) 1–5, Ki-67 and CD31. Biomarker expression was correlated with clinico-pathological variables and tested for survival prediction using Kaplan–Meier and Cox regression methods. RESULTS: Eighty-six consecutive cases were included: 51% male, median age 51 (range 16–82), 68% presenting with a pancreatic primary, 95% well differentiated, 51% metastatic. Higher grading (P=0.03), advanced stage (P<0.001), high Hif-1α and low SSTR-2 expression (P=0.03) predicted for shorter overall survival (OS) on univariate analyses. Stage, SSTR-2 and Hif-1α expression were confirmed as multivariate predictors of OS. Median OS for patients with SSTR-2+/Hif-1α-tumours was not reached after median follow up of 8.8 years, whereas SSTR-2-/Hif-1α+ GEP-NETs had a median survival of only 4.2 years (P=0.006). CONCLUSION: We have identified a coherent expression signature by immunohistochemistry that can be used for patient stratification and to optimise treatment decisions in GEP-NETs independently from stage and grading. Tumours with preserved SSTR-2 and low Hif-1α expression have an indolent phenotype and may be offered less aggressive management and less stringent follow up

Qualification of tumour mutational burden by targeted next-generation sequencing as a biomarker in hepatocellular carcinoma

Background & Aims: Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). Methods: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds ( 650.05, 650.1 and 650.2). Results: Eligible patients (n\ua0=\ua029) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P\ua0\ua0T transitions at CpG sites (median 60.3% vs 9.1%, P\ua0=.002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P\ua0=.041) and deamination counts (median 6393.5 vs 328.5, P\ua0=.041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. Conclusion: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC

A novel radiogenomics biomarker for predicting treatment response and pneumotoxicity from programmed cell death protein or ligand-1 inhibition immunotherapy in NSCLC

INTRODUCTION: Patient selection for checkpoint inhibitor immunotherapy is currently guided by programmed death-ligand 1 (PD-L1) expression obtained from immunohistochemical staining of tumor tissue samples. This approach is susceptible to limitations resulting from the dynamic and heterogeneous nature of cancer cells and the invasiveness of the tissue sampling procedure. To address these challenges, we developed a novel computed tomography (CT) radiomic-based signature for predicting disease response in patients with NSCLC undergoing programmed cell death protein 1 (PD-1) or PD-L1 checkpoint inhibitor immunotherapy. METHODS: This retrospective study comprises a total of 194 patients with suitable CT scans out of 340. Using the radiomic features computed from segmented tumors on a discovery set of 85 contrast-enhanced chest CTs of patients diagnosed with having NSCLC and their CD274 count, RNA expression of the protein-encoding gene for PD-L1, as the response vector, we developed a composite radiomic signature, lung cancer immunotherapy-radiomics prediction vector (LCI-RPV). This was validated in two independent testing cohorts of 66 and 43 patients with NSCLC treated with PD-1 or PD-L1 inhibition immunotherapy, respectively. RESULTS: LCI-RPV predicted PD-L1 positivity in both NSCLC testing cohorts (area under the curve [AUC] = 0.70, 95% confidence interval [CI]: 0.57-0.84 and AUC = 0.70, 95% CI: 0.46-0.94). In one cohort, it also demonstrated good prediction of cases with high PD-L1 expression exceeding key treatment thresholds (>50%: AUC = 0.72, 95% CI: 0.59-0.85 and >90%: AUC = 0.66, 95% CI: 0.45-0.88), the tumor's objective response to treatment at 3 months (AUC = 0.68, 95% CI: 0.52-0.85), and pneumonitis occurrence (AUC = 0.64, 95% CI: 0.48-0.80). LCI-RPV achieved statistically significant stratification of the patients into a high- and low-risk survival group (hazard ratio = 2.26, 95% CI: 1.21-4.24, p = 0.011 and hazard ratio = 2.45, 95% CI: 1.07-5.65, p = 0.035). CONCLUSIONS: A CT radiomics-based signature developed from response vector CD274 can aid in evaluating patients' suitability for PD-1 or PD-L1 checkpoint inhibitor immunotherapy in NSCLC

Neuronal chemotaxis by optically manipulated liposomes

We probe chemotaxis of single neurons, induced by signalling molecules which were optically delivered from liposomes in the neighbourhood of the cells. We implemented an optical tweezers setup combined with a micro-dissection system on an inverted microscope platform. Molecules of Netrin-1 protein were encapsulated into micron-sized liposomes and manipulated to micrometric distances from a specific growth cone of a hippocampal neuron by the IR optical tweezers. The molecules were then released broken the liposomes with UV laser pulses. Chemotaxis induced by the delivered molecules was confirmed by the migration of the growth cone toward the liposome position. Since the delivery can be manipulated with high temporal and spatial resolution and the number of molecules released can be controlled quite precisely by tuning the liposome size and the solution concentration, this technique opens new opportunities to investigate the effect of physiological active compounds as Netrin-1 to neuronal signalling and guidance, which represents an important issue in neurobiology

Matrix stiffness modulates the activity of MMP-9 and TIMP-1 in hepatic stellate cells to perpetuate fibrosis

Liver fibrosis is characterised by a dense and highly cross-linked extracellular matrix (ECM) which promotes progression of diseases such as hepatocellular carcinoma. The fibrotic microenvironment is characterised by an increased stiffness, with rigidity associated with disease progression. External stiffness is known to promote hepatic stellate cell (HSC) activation through mechanotransduction, leading to increased secretion of ECM components. HSCs are key effector cells which maintain the composition of the ECM in health and disease, not only by regulating secretion of ECM proteins such as collagen, but also ECM-degrading enzymes called matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Uninhibited MMPs degrade ECM proteins to reduce external rigidity. Using fibronectin-coated polyacrylamide gels to alter substrate rigidity without altering ligand density, we show that fibrotic rigidities downregulate MMP-9 expression and secretion, and also upregulate secretion of TIMP-1, though not its expression. Using tissue immunofluorescence studies, we also report that the expression of MMP-9 is significantly decreased in activated HSCs in fibrotic tissues associated with hepatocellular carcinoma. This suggests the presence of a mechanical network that allows HSCs to maintain a fibrotic ECM, with external rigidity providing feedback which affects MMP-9 and TIMP-1 secretion, which may become dysregulated in fibrosis

Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interesting is to investigate the morphology and function of the central nervous system (CNS) cultured using such scaffolds. Here, we used an elastomer, polydimethylsiloxane (PDMS), to produce lattice-type scaffolds from a photolithography defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist and downward mounted on a rotating stage at angle of 45\ub0. After exposure for three or more times keeping the same exposure plan but rotated by the same angle, the photoresist was developed to produce a 3D porous template. Afterward, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. Culture test has been done using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenously cell infiltration and 3D attachment. As expected, a much higher cell number was found in 3D PDMS lattices than in 2D culture. We also found a higher neuron to astrocyte ratio and a higher degree of cell ramification in 3D culture compared to 2D culture, due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies

An inflammation based score can optimize the selection of patients with advanced cancer considered for early phase clinical trials.

Background: Adequate organ function and good performance status (PS) are common eligibility criteria for phase I trials. As inflammation is pathogenic and prognostic in cancer we investigated the prognostic performance of inflammation-based indices including the neutrophil (NLR) and platelet to lymphocyte ratio (PLR). Methods: We studied inflammatory scores in 118 unselected referrals. NLR normalization was recalculated at disease reassessment. Each variable was assessed for progression-free (PFS) and overall survival (OS) on uni- and multivariate analyses and tested for 90 days survival (90DS) prediction using receiving operator curves (ROC). Results: We included 118 patients with median OS 4.4 months, 23% PS>1. LDH 65450 and NLR 655 were multivariate predictors of OS (p<0.001). NLR normalization predicted for longer OS (p<0.001) and PFS (p<0.05). PS and NLR ranked as most accurate predictors of both 90DS with area under ROC values of 0.66 and 0.64, and OS with c-score of 0.69 and 0.60. The combination of NLR+PS increased prognostic accuracy to 0.72. The NLR was externally validated in a cohort of 126 subjects. Conclusions: We identified the NLR as a validated and objective index to improve patient selection for experimental therapies, with its normalization following treatment predicting for a survival benefit of 7 months. Prospective validation of the NLR is warranted. Copyright: \ua9 2014 Pinato et al