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Cancer-associated fibroblasts (CAF) have been reported to support tumor progression by a variety of mechanisms. However, their role in the progression of non–small cell lung cancer (NSCLC) remains poorly defined. In addition, the extent to which specific proteins secreted by CAFs contribute directly to tumor growth is unclear. To study the role of CAFs in NSCLCs, a cross-species functional characterization of mouse and human lung CAFs was conducted. CAFs supported the growth of lung cancer cells in vivo by secretion of soluble factors that directly stimulate the growth of tumor cells. Gene expression analysis comparing normal mouse lung fibroblasts and mouse lung CAFs identified multiple genes that correlate with the CAF phenotype. A gene signature of secreted genes upregulated in CAFs was an independent marker of poor survival in patients with NSCLC. This secreted gene signature was upregulated in normal lung fibroblasts after long-term exposure to tumor cells, showing that lung fibroblasts are "educated" by tumor cells to acquire a CAF-like phenotype. Functional studies identified important roles for CLCF1–CNTFR and interleukin (IL)-6–IL-6R signaling in promoting growth of NSCLCs. This study identifies novel soluble factors contributing to the CAF protumorigenic phenotype in NSCLCs and suggests new avenues for the development of therapeutic strategies. Cancer Res; 72(22); 5744–56. ©2012 AACR.
Cancer is initiated and progresses within a microenvironment that is itself altered as a consequence of the tumorigenic process. Stromal cells in contact with cancer cells secrete growth factors and cytokines that may act directly by signaling to tumor cells or indirectly by recruiting other stromal components to promote tumor progression (1–3). An important aspect of this process is the expansion of cancer-associated fibroblasts (CAF; refs. 4–6). CAFs are a diverse population of stromal cells with distinct characteristics in different tumors and tissues. To date, they have been best characterized in human breast and prostate cancer (7, 8) as well as in several mouse cancer models (9–11). However, the exact origins of these cells and how they acquire their tumor-promoting capacities remains poorly understood. Several studies have identified key CAF-secreted molecules that play a role in tumor progression. For example, in human breast cancer, CAFs recruit endothelial cells and signal directly to tumor cells via the cytokine SDF-1 (12). In mouse dermal, cervical and islet cell cancers, CAFs secrete a proinflammatory array of cytokines in response to NF-κB activation (11). CAFs have also been reported to modulate the self-renewal and proliferation of cancer stem cells in vitro and in vivo (13, 14). These observations link CAFs to the regulation of multiple processes that control tumor development. Thus, CAF–tumor cell interactions play an important role in cancer progression and represent a relatively unexplored target for cancer therapy. Consequently, understanding the precise molecular mechanisms that lead to tumor promotion by CAFs is an important goal in cancer biology.
Lung cancer is one of the leading causes of cancer death in the world. Non–small cell lung cancer (NSCLC) accounts for most lung cancer cases and the overall 5-year survival of patients with this disease remains approximately 15% (15). Most NSCLC tumors are characterized by a significant desmoplastic/fibrotic component at the time of diagnosis (16, 17). Several studies have suggested a role for lung fibroblasts in NSCLCs using in vitro models (18–20). Recently, CAF-secreted VEGF has been suggested to promote tumor invasion (21). Whether other secreted proteins also play a role in lung CAF-induced tumor proliferation has not been reported. Here, we report a systematic cross-species study of the molecular phenotype of normal lung fibroblasts and CAFs and identify novel mediators of tumor–stroma cross-talk in NSCLCs.
Oncogenic Kras-driven mouse models of lung cancer recapitulate many features of human NSCLCs (22), including oncogene-specific gene signatures (23). In this study, normal mouse lung fibroblasts and CAFs isolated from a Kras-driven lung adenocarcinoma model were used to identify individual genes and gene signatures relevant to the pathogenesis of lung cancer. While both normal mouse lung fibroblasts and CAFs contribute to tumor progression, lung CAFs fostered tumor growth in vivo more efficiently than normal mouse lung fibroblasts. A gene signature specific to lung CAFs including multiple secreted proteins and genes related to inflammatory processes was identified and shown to be a marker of poor survival in patients with NSCLCs. Mouse and human lung fibroblasts upregulated genes in this gene signature after exposure to tumor cells, strongly suggesting that CAFs derive from normal resident lung fibroblasts. Finally, a critical and nonredundant functional role for one member of the CAF-secreted gene signature, CLCF1, was confirmed using both knockdown and overexpression studies. While interleukin (IL)-6 also contributes to tumor growth, signaling through the IL-6 receptor (IL-6R) is dispensable for tumor growth. In contrast, blockade of CLCF1–CNTFR paracrine signaling significantly decreases the growth of tumor cells in vivo. Thus, these studies underscore a previously uncharacterized role for CLCF1 in paracrine signaling in NSCLCs.
Materials and Methods
Open reading frames for lacZ and clcf1 were obtained from Open Biosystems (Thermo Fisher). Il6 cDNA was synthesized from mouse CAF RNA with the DyNAmo cDNA Synthesis Kit (New England Biolabs). All open reading frames were PCR-amplified using reverse primers that lacked the stop codon for each gene, cloned into an entry D-TOPO vector, and transferred to a pL6/V5-DEST lentiviral vector using Gateway technology (Invitrogen). Mouse-raised antibodies to α-smooth muscle actin, vimentin, cytokeratin, β-actin (Sigma), and V5 (Invitrogen), and rabbit-raised antibodies to β-actin (LiCOR) were used.
Cell lines
LKR10 and LKR13 cells are mouse lung cancer cell lines derived by serial passage of minced lung adenocarcinoma tissues from 2 tumors isolated from separate lobes of the same KrasLA1 mouse (24). These cells were a gift of Dr. Julien Sage (Stanford University School of Medicine, Stanford, CA). A549 and H1299 cells were from American Type Culture Collection. LSZ2 cells were derived through xenograft passages from KrasLSLG12D mice and established as cell lines in vitro. All cell lines were grown in Dulbecco's' Modified Eagle's Media (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. MLE12 cells were purchased from American Type Culture Collection and grown according to vendor's specifications. These cells were originally isolated by Wikenheiser and colleagues (25).
Isolation of normal lung fibroblasts and CAFs
Mouse fibroblasts were isolated as described (26). Cells could be grown in vitro for 4 to 6 passages before replicative senescence became apparent under these culture conditions. Human normal lung fibroblasts and CAFs were isolated from normal lung or lung adenocarcinomas obtained with approval from the Stanford Institutional Review Board (Stanford, CA). Tissues were digested to obtain single cells and grown in 96-well culture plates before expansion for different analyses. For immortalization of normal mouse lung fibroblasts, cells were transfected using a 1:10 ratio of pEmpty Puro and pSV40Tag vectors using Lipofectamine (Invitrogen) and following manufacturer's specifications. The vectors were a gift of Dr. Steve Artandi (Stanford University School of Medicine, Stanford, CA).
Retro and lentiviral infections
Virus was produced by transfection into 293FT cells of a retroviral vector expressing eGFP (27) using FuGENE (Roche) according to manufacturer's recommendations. A pLKO.1 vector expressing mCherry was constructed by replacing the puromycin cassette with mCherry cDNA (Clontech). pLKO and pL6 vectors were used to produce lentivirus by transfection into 293FT cells as previously described (28). Viruses were filtered and applied directly to cells for infection at a multiplicity of infection (MOI) < 1.
Cell proliferation assay
Cells were plated into 96-well plates and treated with MTT. Experiments were read on the indicated days according to manufacturer's instructions (Cell proliferation Kit I, Roche). Data were normalized to day 0 of experiment. The 3T3 assays were as described (29) except for normal mouse lung fibroblasts and CAFs, 200,000 cells were plated and counted every 3 days.
Co-injection experiments
Xenograft experiments were carried out in accordance with Stanford Institutional Animal Care and Use Committee guidelines. For experiments with mouse lung cancer cells, 5 × 103 LKR10, LKR13, or LSZ2 were resuspended, alone or in combination with 5 × 104 normal mouse lung fibroblasts or CAFs (ratio 1:10), in 100 mL of serum-free DMEM and injected subcutaneously into the 2 lower flanks of athymic Balb/cnu/nu mice (Charles River). Beginning 1 week after injection, tumor dimensions were measured every 3 to 4 days, and tumor volume was calculated using volume = π/6 × (length) × (width)2. For human experiments, 5 × 105 A549 or H1299 cells were injected alone or in combination with 5 × 106 normal mouse lung fibroblasts or iCAFs.
Conditioned media and coculture experiments
Conditioned media from normal fibroblasts/CAFs: 1 × 106 lung fibroblasts and CAFs were plated into 10 cm and media were changed on the following day. Fresh media (DMEM, 10% FBS, and 1% penicillin/streptomycin) was added, and cells were grown for another 2 days. Media was then removed and concentrated using Amicon Ultra columns (Millipore). Total protein was determined using the BCA Kit (Pierce) and indicated concentrations were used to treat LKR10 or LKR13 cells. Conditioned media from LKR10: 2 × 106 cells were plated and grown in fresh media for 5 days, and conditioned media was harvested; 1:1 ratio of filtered conditioned media plus fresh media was used to treat normal mouse lung fibroblasts for 36 hours. For coculture, mCherry–normal fibroblasts and LKR10 cells were mixed in a 1:2 ratio for 5 or 15 days. Then, mCherry-positive cells were sorted out for gene expression analysis. Normal mouse lung fibroblasts were cocultured with CAFs for 5 or 15 days, and mCherry-negative cells sorted for mRNA analysis. Human normal lung fibroblasts were cocultured with GFP-expressing A549 cells at a 1:2 ratio for 12 and 24 days, followed by sorting of GFP-negative cells for gene expression analysis. G10 and G13 cells were cocultured with normal mouse lung fibroblasts overexpressing clcf1, il6, and control cells at a 1:5 ratio for 7, 14, and 21 days.
Western blotting
Cells were scraped, washed, and lysed in NP-40 buffer supplemented with protease inhibitor cocktail (Roche), 25 mmol/L sodium fluoride and 1 mmol/L sodium orthovanadate (Sigma). Protein samples were resolved by SDS-PAGE and transferred to Immobilon-FL membranes (Millipore) and incubated in blocking buffer (Li-COR Biosciences) for 1 hour before addition of primary antibodies. Antibody incubations and washing steps were done according to Li-COR protocols. Membranes were scanned using an Odyssey Imager (Li-COR Biosciences).
Immunofluorescence and trichrome staining
Cells were trypsinized and grown on glass slides overnight. Cells were fixed using 4% paraformaldehyde, processed, and stained with indicated primary antibodies. AlexaFluor secondary antibodies were from Invitrogen. Bioquant software was used to montage the entire xenograft sections and calculate the percentage of trichrome staining (Bioquant Inc.). Images were taken using an Eclipse E800 microscope (Nikon).
Quantitative reverse transcriptase-PCR
RNA was isolated using TRIzol reagent (Invitrogen) following manufacturer's specifications and cleaned-up using Qiagen miniRNA columns (Qiagen). cDNA was synthesized with a DyNAmo cDNA Synthesis Kit (New England Biolabs) and quantitative reverse transcriptase-PCR (qRT-PCR) was carried out using SYBRGreen (Applied Biosystems; see Supplementary Information for primer sequences).
Gene expression analysis
The Robust Multichip Average (RMA) Express 1.0.4 program (30) was used for background adjustment and quantile RMA normalization of the 45,101 probe sets encoding mouse genome transcripts. Linear Models for Microarray data (LIMMA; ref. 31) was used for comparison between the groups on RMA-normalized signal intensities. After preprocessing steps, a moderated Student t test was used to select differentially expressed genes between groups (CAFs and normal mouse lung fibroblasts). P values were adjusted for multiple testing using the Benjamini–Hochberg method. Fold change was calculated by comparing gene expression levels between CAFs and normal mouse lung fibroblasts samples and expressed as the ratio between the averages of normalized intensities of the 2 groups. Only genes with fold change threshold greater than 2 and adjusted P < 0.075 were considered as differentially expressed. The overexpressed genes identified from LIMMA were analyzed using Ingenuity Pathway Analysis (IPA) software (32). For network analysis, we chose to include only direct interactions in IPA. We used Onto-Express Ensemble (33, 34) for pathway analysis and Gene Ontology (GO) studies of the overexpressed genes from LIMMA. P values were computed using the hypergeometric distribution and adjusted for multiple hypotheses using Benjamini–Hochberg correction.
We conducted meta-analysis of 5 human NSCLC data sets obtained using DNA microarrays. Samples included tumor and stroma. Details of the meta-analysis procedure will be described elsewhere (Khatri and colleagues, manuscript in preparation). Briefly, we estimated the effect size for each gene in each data set as Hedges' adjusted g (35). The study-specific effect sizes for each gene were then combined into a single meta-effect size using a linear combination of study-specific effect sizes, fi, where each study-specific effect size was weighted by inverse of the variance in the corresponding study (Eq. A). After computing meta-effect size, significant genes were identified using Z-statistic, and p-values were corrected for multiple hypotheses testing using Benjamini–Hochberg false discovery rate (FDR) correction.
Survival analysis
Survival analysis was conducted on both gene sets and individual genes. Four different datasets were used (36). Data were generated from RNA samples that included both tumor and stromal populations. Only samples at N0 and N1 stage and with time period less than 60 months for the study were analyzed. Each data set was normalized independently and standardization (mean centering), and scaling (dividing the centered value by the standard deviation) was conducted before uni- and multivariate analysis. High and low expressed groups were determined by taking the average of the standardized values across all samples for a specific gene. For gene sets, a summation of all the genes for a particular sample was calculated, and the average of the sum values was used to determine the high and low groups. Samples that had a value less than the calculated average were assigned as the low-risk group and the rest to high-risk group. Cox HRs were calculated for both uni- and multivariate analysis to estimate the relative risk along with confidence interval (CI) and log-rank tests.
Statistical analyses
A 2-tailed t test was used for comparisons of groups. Error bars correspond to either SD or SEM. Significant P values in the text correspond to <0.05 (*), <0.01 (**), or <0.001 (***).
CAFs promote lung tumor development in vivo
To compare the effect of normal mouse lung fibroblasts and lung CAFs on lung cancer progression, we used cells derived from the KrasLA1 mouse model of lung adenocarcinoma (24). KrasLA1 lung tumors contained a significant fibrotic component, suggesting a role for CAFs in the progression of these tumors (Fig. 1A). CAFs isolated from tumors in this model and normal lung fibroblasts from littermate wild-type mice were expanded in vitro for approximately 7 to 10 days before gene expression analysis or in vivo experiments (see Materials and Methods). Expression of fibroblast-specific protein-1 (fsp-1; ref. 37) was detected in both normal mouse lung fibroblasts and CAFs, whereas markers of other cells types were very low or undetectable (Supplementary Fig. S1A). Both normal mouse lung fibroblasts and CAFs showed expression of mesenchymal markers [α-smooth muscle actin (α-sma) and vimentin] by immunofluorescence (Fig. 1B) whereas they did not express cytokeratins (Supplementary Fig. S1B). As expected (12, 21), expression levels of α-sma were higher in CAFs than in normal mouse lung fibroblasts. It has been suggested that CAFs may be derived from epithelial cells that have undergone an epithelium-to-mesenchyme transition (38). However, KrasLA1 CAFs did not show recombination of the Kras locus, which occurs in epithelial tumors in this model (Supplementary Fig. S1C), showing that they are not of tumor origin. Finally, neither normal mouse lung fibroblasts nor CAFs formed subcutaneous tumors when injected alone (data not shown).
Next, mouse lung cancer cell lines derived from the same KrasLA1 model (LKR10 and LKR13) were grafted alone or co-injected with either normal mouse lung fibroblasts or CAFs (ratio 1:10) into immunodeficient mice. Tumors harboring CAFs were significantly larger than tumors harboring only tumor cells. Interestingly, tumors harboring normal mouse lung fibroblasts were also larger than tumors lacking fibroblasts, although significantly smaller than those harboring CAFs (Fig. 1C and D). Normal mouse lung fibroblasts and CAFs had similar proliferative capacity in vitro (Supplementary Fig. S1D). In addition, the fibrotic component was similar in tumors that incorporated normal mouse lung fibroblasts or CAFs (Fig. 1E) and closely recapitulated that of the KrasLA1 tumors (Fig. 1A). Thus, the differential contribution to tumor growth of CAFs and normal mouse lung fibroblasts is not likely to be due to a difference in their proliferative potential in vivo. The ability of normal fibroblasts and CAFs to influence tumor growth in vivo was maintained after 2 weeks in culture (Fig. 1F), showing that the tumor-promoting capacity of normal mouse lung fibroblasts and CAFs is stable over time and independent of ongoing contact with tumor cells, at least after 2 weeks in culture. Therefore, resident lung fibroblasts contribute to tumorigenesis in vivo and CAFs provide a further advantage to tumor growth.
CAFs have been proposed to promote tumor growth through direct interaction with tumor cells or by recruitment of other cells to the tumor microenvironment (11, 12). To determine whether CAFs from KrasLA1 mice directly stimulate the growth of tumor cells through secreted factors, LKR10 and LKR13 cells were treated in vitro with conditioned media from normal mouse lung fibroblasts or CAFs. Proliferation of both lung cancer cell lines was significantly increased by conditioned media from CAFs when compared with that of normal mouse lung fibroblasts or control medium (Fig. 1G and H). This suggests that CAFs act, at least in part, by secreting soluble ligands that directly stimulate the growth of tumor cells.
CAFs are characterized by a secretory and inflammatory expression profile
Gene expression microarrays were used to identify changes between normal mouse lung fibroblasts and CAFs that may contribute to their different tumor-enhancing capacity. Unsupervised hierarchical clustering separated normal mouse lung fibroblasts and CAF gene expression into 2 distinct clusters (Supplementary Fig. S2A). One hundred sixty-four genes were upregulated in CAFs compared with normal mouse lung fibroblasts (CAF-HIGH), whereas only 11 genes were downregulated in CAFs (CAF-LOW; fold change > 2 or <0.5 and FDR < 0.075; Supplementary Fig. S2B and Supplementary Table S1). Interrogation of the molecular pathways represented in CAF-HIGH genes using Onto-Express (34) identified cytokine–cytokine receptor interaction (P = 5.04 × 10−4), apoptosis (P = 0.0086), and Jak-STAT signaling (P = 0.0087) as pathways enriched in CAF-HIGH genes (Supplementary Table S2). In addition, genes coding for proteins secreted into the extracellular space were significantly enriched in the CAF-HIGH gene list (corrected P = 0.0026 and 0.0058, respectively) as were membrane proteins (corrected P = 5.8 × 10−4; Supplementary Table S3). Thus, extracellular proteins secreted by CAFs may represent a key component of their phenotype. A heat map for these 15 secretome genes is shown in Fig. 2A. Differential expression of most of these secreted genes in CAFs was validated by qRT-PCR (Fig. 2B).
To identify whether the gene signature of CAFs was centered around specific gene networks, we used a database of curated information about gene interactions (see Materials and Methods). This identified 4 major gene networks based on the human orthologues of the CAF-HIGH genes (Supplementary Table S4). These networks were enriched for molecular pathways of inflammatory response and disease, cell death, cellular growth and development, and cell-to-cell signaling and interaction (P < 0.0001; Supplementary Table S5). Interestingly, the inflammatory response pathway was represented in 2 networks, highlighting the biologic relevance of this cellular function in CAFs. The 15 secretome genes were particularly enriched in "network 1," which contained 10 of these secreted proteins (Fig. 2C). Thus, an inflammatory expression profile involving multiple secreted proteins is characteristic of lung CAFs. Collectively, these results identify secreted proteins that are part of an inflammatory phenotype in lung CAFs that might contribute to tumor development.
A CAF secreted-gene signature is a marker of poor survival in human NSCLCs
To probe the relevance of these findings to human NSCLC, we conducted cross-species gene expression analysis using publicly available human NSCLC gene expression data sets. As NSCLC samples often contain a significant percentage of tumor stroma, we reasoned that expression profile analyses from NSCLC biopsy samples could identify gene expression changes due to overexpression of stromal genes rather than genes expressed in tumor cells themselves. First, gene expression of the human orthologues of the mouse-defined secreted gene signature was assessed in 5 independent microarray data sets from normal human lung and NSCLC samples (36, 39–42). Several of these genes (CCL7, CCL13, CLCF1, EREG, IGFBP5, IL11, LIF, and NGF) were upregulated in NSCLCs compared with normal lung (Supplementary Fig. 3A and Supplementary Table S6). Next, the prognostic potential of the CAF gene signature was queried in a large expression profile data set of human NSCLC (36). In 2 independent data sets, univariate analysis revealed that high levels of the secreted CAF gene signature was associated with poor survival in patients with NSCLC (CAN/DF: log-rank test, P = 0.0002, HR = 3.16 ± 0.26; and UM: log-rank test, P = 0.002, HR = 2.93 ± 0.14; Fig. 3A and B). Survival prediction using this gene set was independent of stage and gender in a multivariate analysis (CAN/DF: log-rank test, P = 0.001, HR = 3.12 ± 0.26; and UM: log-rank test, P = 0.007, HR = 2.91 ± 0.14). These observations show that the secreted gene signature identified in mouse CAFs is highly relevant to human NSCLC and is a strong predictor of patient survival.
Tumor–stroma cross-talk directly alters the phenotype of resident lung fibroblasts to become CAFs
The mechanism by which normal lung fibroblasts are activated to become CAFs is poorly understood. For example, it is unclear whether tumor cells can directly activate normal mouse lung fibroblasts or whether other stromal components are first recruited into the tumor microenvironment leading to an indirect mechanism for normal mouse lung fibroblasts activation. Furthermore, it is unclear whether normal mouse lung fibroblasts are altered to become CAFs or whether CAFs perhaps arise from a rare subpopulation of either resident cells or cells mobilized from bone marrow–derived mesenchymal stem cells (37, 43–46). Therefore, we tested whether tumor cells could induce changes via secreted proteins that would directly lead to upregulation of CAF-secreted genes in normal mouse lung fibroblasts. Conditioned media from mouse tumor cells were used to treat immortalized normal fibroblasts (see Materials and Methods and Supplementary Fig. S4A), and the expression of the secretory gene signature was assessed by qRT-PCR 36 hours later. Normal mouse lung fibroblasts subjected to tumor cell conditioned media displayed increased expression (>1.5-fold) of mRNAs for 11 of 15 CAF genes identified by microarray analysis (Fig. 4A). Similar findings were observed in an independent normal mouse lung fibroblast line (Supplementary Fig. S4B). The upregulation of the secretory gene signature in normal mouse lung fibroblasts after a short exposure to tumor cell conditioned media suggests that CAFs may arise directly from reprogramming of resident normal mouse lung fibroblasts rather than requiring expansion of a rare subpopulation of cells within the stromal compartment. However, expression of these secreted proteins in normal mouse lung fibroblasts returned to basal levels after 72 hours of treatment (data not shown), suggesting that tumor cell conditioned media provide a transient effect that fails to permanently reprogram normal mouse lung fibroblasts toward a CAF phenotype.
To determine whether direct cell-to-cell contact or continuous stimulation by tumor cells would provide stable upregulation of the CAF-secretory gene signature, normal mouse lung fibroblasts and tumor cells were cocultured in vitro, and the expression profile of the secreted gene signature was studied in these "educated" normal mouse lung fibroblasts. Lung fibroblasts were modified to express a fluorescent reporter (mCherry) to allow isolation by fluorescence-activated cell sorter (FACS) after coculture (Supplementary Fig. S4C). An upregulation of 9 of 15 secreted proteins, similar to what was observed after conditioned media treatment, was observed after 5 days of coculture. A further increase in the expression level of the majority of secreted proteins (13 of 15 genes) was observed after 15 days (Fig. 4B). These results were confirmed using a coculture system incorporating human normal lung fibroblasts and GFP-positive NSCLC cell lines (A549), indicating the relevance of tumor–normal fibroblast interactions to human lung cancer. Exposure of human normal fibroblasts to NSCLCs for 12 days enhanced the expression of 9 genes of the secretory gene signature >1.5-fold (data not shown). Upregulation of 12 of 15 members of the secretory gene signature was further increased after 24 days of coculture in vitro (Fig. 4C). These studies show the existence of functional cross-talk between tumor cells and fibroblasts and suggest that (i) the CAF phenotype is the consequence of direct and chronic stimulation of normal lung fibroblasts by tumor cells and (ii) CAFs can be derived directly from resident lung fibroblasts and do not necessarily arise from a distinct population of mesenchymal cells recruited from a distant site.
While the above results suggest that tumor cells directly contribute to normal fibroblast–to-CAF transition, CAFs may also establish homotypic interactions with resident normal lung fibroblasts and contribute to further recruitment of fibroblasts toward a CAF phenotype. Treatment of normal mouse lung fibroblasts with CAF conditioned media did not upregulate expression of secretory genes (data not shown) indicating that, unlike tumor cell conditioned media, CAF conditioned media is not sufficient to induce gene expression changes in normal mouse lung fibroblasts. However, coculture of normal mouse lung fibroblasts and CAFs led to upregulation of expression of 8 of 15 CAF genes in normal mouse lung fibroblasts (Fig. 4D and Supplementary Fig. S4D). Taken together, these results suggest that tumor cells initiate a transition of normal mouse lung fibroblasts to a proinflammatory CAF state that is further propagated to other normal mouse lung fibroblasts by already activated CAFs.
To analyze the relevance of these findings in vivo and to determine whether normal lung fibroblasts could be stably reprogrammed to a CAF-like phenotype, fluorescently labeled normal mouse lung fibroblasts (mCherry) and tumor cells (GFP) were co-injected to form xenografts (Supplementary Fig. S4E). Tumors from these co-injections yielded similar volumes to those observed with primary lung fibroblasts (Supplementary Fig. S4F) showing that manipulation of lung fibroblasts and tumor cells with reporter constructs did not result in significantly phenotypic changes. Double staining with anti-mCherry and anti-vimentin antibodies showed the presence of exogenous, donor-derived normal mouse lung fibroblasts within expanding tumors (Fig. 4E). Thus, lung fibroblasts expand within xenografts and are an active and sustained component of developing tumors in vivo. The in vitro data above suggested that tumor cells can progressively drive the normal fibroblast–to-CAF transition and that this transition is further promoted by CAFs acting directly on normal mouse lung fibroblasts. To study whether lung fibroblasts could acquire a stable CAF-like phenotype after in vivo passage with tumor cells, normal mouse lung fibroblasts were sorted from tumors and expanded in vitro for approximately 7 to 10 days. Next, tumor cells were injected alone, with parental normal lung fibroblasts, or with fibroblasts isolated from tumor xenografts (here called induced CAFs or iCAFs). Secondary xenografts incorporating iCAFs were larger than those including "uneducated" primary normal mouse lung fibroblasts and the difference closely paralleled that between primary normal mouse lung fibroblasts and CAFs (Fig. 4F). These differences were not due to variations in the proliferation rate between iCAFs and normal mouse lung fibroblasts (Fig. 4G). iCAFs isolated from secondary xenografts (siCAF) also recapitulated the tumor-promoting abilities shown by iCAFs from primary xenografts (Supplementary Fig. S4G). These results indicate that normal mouse lung fibroblasts can be "educated" to display characteristics of CAFs in vivo and that the long-term interaction of these fibroblasts with tumor cells provides a point of no return from a CAF-like phenotype even after in vitro expansion for more than 1 week.
Next, we tested the relevance of the CAF phenotype established in mouse fibroblasts to human lung cancer oncogenesis. The normal mouse lung fibroblasts and iCAFs described above were mixed with the human NSCLC cell lines A549 or H1299. A549 and H1299 cells grew significantly better when mixed with iCAFs (Fig. 4H and Supplementary Fig. S4H). Thus, the acquisition of a CAF-like phenotype in resident lung fibroblasts leads to the promotion of oncogenesis in vivo via the direct stimulation of tumor cell proliferation by secreted proteins. These findings support the relevance of studies of mouse-derived lung CAFs to human lung cancer.
The cytokines clcf1 and il6 contribute to tumor progression in vivo
As normal mouse lung fibroblasts can transition to iCAFs that are phenotypically similar to CAFs isolated directly from tumors, the most functionally relevant secreted genes that drive the CAF phenotype should be common to both iCAFs and CAFs. To identify these genes, the expression profile of the CAF-secretory gene signature was queried in normal mouse lung fibroblasts compared with iCAFs. Six genes (clcf1, cxcl14, ereg, gdnf, il6, and ngf) in the original CAF signature were consistently upregulated by at least 1.5-fold in iCAFs (Fig. 5A and Supplementary Fig. S5A). Thus, this subset of the secreted gene signature may be the most critical for the CAF phenotype. As a further functional test, normal fibroblasts and CAFs were isolated from primary human lung adenocarcinomas (Supplementary Fig. S5B). Two pairs of matched primary human normal mouse lung fibroblasts and CAFs were analyzed for the expression of these 6 genes. Notably, only CLCF1 and IL-6 were upregulated in human CAFs compared with their matched normal mouse lung fibroblasts (Fig. 5B and Supplementary Fig. S5C), suggesting that these 2 genes may play a central role in driving the protumorigenic phenotype of CAFs.
To test the functional role of clcf1 and il6 in lung cancer in vivo, normal mouse lung fibroblasts were transduced with lentiviral vectors containing cDNAs coding for either of these 2 mRNAs (Fig. 5C and D). Fibroblasts overexpressing clcf1, il6, or control (lacZ) were co-injected with tumor cells. Co-injection with lung fibroblasts overexpressing clcf1 and il6 yielded significantly larger tumors (Fig. 5E and F). Similar results were obtained using an independent lung tumor cell line derived from the KrasLSLG12D mouse model (ref. 47; Supplementary Fig. S5D). Importantly, overexpression of clcf1 or il6 did not alter the proliferation rate of the corresponding fibroblasts (Fig. 5G). Thus, clcf1 and il6 secreted by CAFs play a direct role in stimulating tumor growth in vivo. This effect is likely due to direct stimulation of tumor cells by fibroblasts, as both clcf1 and il6 overexpressors increased the relative cell number of cancer cells when compared with control fibroblasts when cocultured in vitro (Supplementary Fig. S5E and S5F). No expression of either clcf1 or il6 was detected in tumor cells (Fig. 5H and I), suggesting that the primary source of both cytokines is derived from CAFs which establish a paracrine signaling axis to promote tumor cell growth.
Suppression of clcf1–cntfr paracrine signaling axis decreases lung cancer in vivo
Clcf1 and il6 act through a receptor complex containing specific α-subunit receptors, cntfr and il6r, which recognize and bind clcf1 and il6, respectively. These distinct α-subunits act together with the common β-subunit receptor gp130 to activate JAK-STAT signaling (48). Abrogating the ligand–receptor interaction via decreased expression of cntfr and il6r in tumor cells would thus be expected to halt tumor growth in vivo. To test this hypothesis, 2 independent short hairpin RNAs (shRNA) to cntfr and il6r were validated and used to infect LKR10 cells (Fig. 6A and Supplementary Fig. S6A). We co-injected CAFs along with cntfr-and il6r-deficient LKR10 cells or controls and assessed tumor volume. Tumor cells carrying single shRNAs directed against cntfr were significantly smaller than controls (Fig. 6B). A similar trend was observed with 2 independent il6r shRNAs; however the differences with regard to control cells were not statistically significant for both hairpins (Supplementary Fig. S6B). Importantly, loss of cntfr or il6r did not alter tumor cell growth in vitro, suggesting that the in vivo effect is likely due to an important role of this signaling pathway in mediating paracrine effects in vivo but not intrinsic tumor proliferation (Fig. 6C and Supplementary Fig. S6C). Taken together, the results of the overexpression and knockdown studies indicate that while both clcf1 and il6 stimulate tumor growth, il6–il6r signaling may be dispensable whereas loss of the clcf1–cntfr axis causes a consistent decrease in tumor formation, suggesting that the 2 signaling pathways are distinct and yet together constitute an important paracrine signaling network between CAFs and tumor cells in NSCLCs.
Accumulating evidence points to a critical role for CAFs in tumor progression (4–6). As increased stromal proportion is a marker of poor survival in patients with NSCLCs (16, 17, 49), CAFs are likely to play an important role in lung cancer progression. We used a cross-species approach to identify a secretory gene signature in CAFs that promotes lung cancer growth and to query individual components of this signature for relevance to the phenotype of CAFs and to human NSCLCs. These studies link secretion of CLCF1 and IL-6 from CAFs to tumor progression in vivo. CLCF1 and IL-6 are members of the IL-6 family (50). CLCF1 (cardiotrophin-like cytokine factor 1, also known as NNT-1 or BSF-3) is a cytokine that has been implicated in the pathogenesis of multiple myeloma (51) but has not been reported to play a role in NSCLCs. While autocrine secretion of IL-6 has been proposed to occur in lung cancer cell lines carrying EGF receptor (EGFR) mutations (52), our results suggest that IL-6 is produced primarily by CAFs and not tumor cells. Indeed, the functional analysis presented here suggests that CLCF1 and IL-6 secretion by CAFs play a significant role in the contribution of this component of the tumor microenvironment to tumor growth.
CLCF1 and IL6 are cytokines that signal through multimeric complexes that include the gp130 receptor/β-subunit and ultimately trigger a signaling cascade that is mediated by the Jak-STAT and mitogen-activated protein kinase (MAPK) pathways (50). The specificity of the cellular responses elicited by these cytokines is governed by the expression patterns of different α-subunit receptors, CNTFR for CLCF1, and IL-6R for IL-6, which can be activated by each cytokine independently (50). Interestingly, overexpression of another IL-6 family member upregulated in mouse CAFs, IL11, which signals through an alternative α-subunit receptor (IL11RA), does not induce tumor growth in vivo in our model system (data not shown), suggesting that distinct receptor complexes may be activated in specific tumor types. Our loss-of-function experiments show a more prominent role of the CLCF1–CNTFR axis in our model and suggest that CLCF1 may be particularly relevant to NSCLC progression. Thus, these results provide rationale for the development of new therapies targeting CAFs and suggest that inhibition of the CLCF1–CNTFR axis may be a novel strategy.
In vitro and in vivo experiments in models of breast and skin carcinoma have reported that tumor cells can induce a CAF-like phenotype in normal fibroblasts, suggesting that direct tumor cell–fibroblast cross-talk plays an important role in tumor development (11, 45). In this study, we show that transient upregulation of some CAF-specific genes in normal mouse lung fibroblasts can be induced by tumor-derived secreted cues. However, the ability of normal mouse lung fibroblasts to fully upregulate a secretory CAF gene signature is only achieved through long-term direct exposure to tumor cells. Our results suggest that while normal mouse lung fibroblasts are responsive to tumor-derived signals, the generation of normal mouse lung fibroblasts populations stably expressing a CAF-like gene signature (and thus, recapitulating the CAF tumor-promoting phenotype) likely involves genetic and/or epigenetic changes resulting from chronic tumor cell-to–normal fibroblast interactions (53, 54). The fact that normal mouse lung fibroblasts passaged through a xenograft (iCAFs) are phenotypically indistinguishable from primary CAFs after 3 to 4 weeks of in vivo growth (despite intermediate expansion in vitro for >1 week) supports the notion that long-term interaction between tumor cells and normal mouse lung fibroblasts leads to the acquisition of a stable CAF phenotype. In addition, in vitro experiments described here also show a role for human NSCLCs in the reprogramming of human normal fibroblasts. Thus, tumor cells are critical in regulating the reprogramming of lung fibroblasts to CAFs in both mouse and human lung cancer. Further work will be required to elucidate the molecular mechanisms that induce this transition.
Cross-species gene expression analysis has been used successfully to identify mouse gene signatures relevant to human NSCLCs (23). By analyzing mouse stromal cells, we identified a gene signature predictive of survival in human NSCLCs, supporting the relevance of studies of stromal compartments in mice to the pathogenesis of human cancer. Previous work identified an 11-gene signature of human CAFs in NSCLC samples with prognostic relevance (55). Surprisingly, we found no significant overlap between our mouse CAF-HIGH gene set and the upregulated CAF genes uncovered in the former study (data not shown). This discrepancy is likely due to the heterogeneous genetic background of the different human samples and highlights the ongoing value of cross-species comparison as a tool for functional genomics studies.
In summary, the data presented in this work describe a novel CAF gene signature with significant relevance in human NSCLCs. We show that CAF-derived CLCF1 and IL-6 exert specific paracrine effects to promote tumor growth in vivo and may be particularly important components of the CAF secretome. In addition, we establish that direct interaction between tumor cells and lung fibroblasts is sufficient to alter the phenotype of the later to become CAFs. These observations highlight a prominent role for CAFs in NSCLCs and provide a framework for further mechanistic studies to identify relevant therapeutic targets. Finally, these studies underscore the need to further elucidate the mechanisms that lead to the conversion of lung fibroblasts to CAFs as this may provide a way to inhibit the "education" of normal mouse lung fibroblasts to a protumorigenic CAF state.
Disclosure of Potential Conflicts of Interest
A.J. Butte is a consultant/advisory board member for NuMedii, Personalis, Ansh Labs, Roche, Johnson and Johnson, and Samsung Advanced Institute of Technology. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: S. Vicent, E.A. Sweet-Cordero
Development of methodology: S. Vicent, E.A. Sweet-Cordero
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Vicent, L.C. Sayles, Y. Zheng, A.K. Gillespie, N. Clarke, Y. Xu, J. Shrager, C.D. Hoang, E.A. Sweet-Cordero
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Vicent, L.C. Sayles, D. Vaka, P. Khatri, O. Gevaert, R. Chen, S. Plevritis, A.J. Butte, E.A. Sweet-Cordero
Writing, review, and/or revision of the manuscript: S. Vicent, J. Shrager, C.D. Hoang, A.J. Butte, E.A. Sweet-Cordero
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Vicent, L.C. Sayles, J. Shrager, E.A. Sweet-Cordero
Study supervision: S. Vicent, J. Shrager, A.J. Butte, E.A. Sweet-Cordero
Grant Support
This work was supported by the Alfonso Martín Escudero Foundation and the Tobacco-Related Disease Research Program (TRDRP) of California (UCAEF SPO#44443; to S. Vicent), the National Cancer Institute (1R01CA138256-01; to P. Khatri, A.J. Butte, and E.A. Sweet-Cordero), the Center for Cancer Systems Biology (CCSB) at Stanford [funded by a grant from the National Cancer Institute (U54 CA149145) to O. Gevaert and S. Plevritis], the Fund for Scientific Research Flanders (FWO-Vlaanderen to O. Gevaert), and an American Cancer Society Research Scholar Award (RSG-08-132-01-CDD; to E.A. Sweet-Cordero).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The authors thank Julien Sage, Laura Attardi, Steve Artandi, Monte Winslow, and all the members of the Sweet-Cordero laboratory for helpful discussions. They also thank Oihana Murillo for technical assistance with flow cytometric analyses.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received March 26, 2012.
- Revision received August 15, 2012.
- Accepted August 17, 2012.
- ©2012 American Association for Cancer Research.


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Through a variety of programs - for high school and college leaders to first-time political candidates and influential business and community leaders - the Sorensen Institute has established itself as a powerful and effective force for restoring public confidence in our political system. At the heart of every Sorensen program are three central themes: ethics in public service, the power of bipartisanship, and a concentrated study of public policy issues.
Meet the esteemed alumni who completed our program and continued on to improve their communities.
Our leadership development programs focus on Virginia residents interested in becoming active in public service.
Know someone who would be a good candidate for a Sorensen program? Tell us about them!
This award will be presented annually to the individuals who model the spirit and mission of Sorensen through the trust, civility and respect they have consistently shown toward others. They create sustainable efforts toward the betterment of a more decent and tolerant civic life and culture for the people of the Commonwealth.
HSLP 2017: Day 14
Reported by Ava Liberty (McLean High School)
HSLP 2017: Day 13
Reported by Carolyn Lemens (Annandale High School)
After a rather alarming early morning fire drill, the class headed back to the College of Nursing for the budget game, not yet aware of the difficulties that were to come.
We walked into the room confident that a budget would be easy to pass; we would simply need to cut unnecessary spending and raise needed revenue. Once we were divided into a house and a senate group, we quickly realized that things were not so simple.
HSLP 2017: Day 12
Reported by Neil Kothari (Thomas Jefferson High School for Science and Technology)
HSLP 2017: Day 11
Reported by: Pari Kapila (Douglas S. Freeman High School)
Compared to other days at the HSLP, this day definitely felt like a slight break, as we were able to spend the morning working in our policy groups. Even though we're starting to feel the weight of our policy projects on our shoulders, we had a calm, productive start to our day. Everyone was working hard on their policy proposals in order to reach the 10 p.m. deadline for the proposals' first draft. It was amazing to really see all of our work finally come together.
HSLP 2017: Day 10
Reported by Julianna Jones (New Kent High School)
After a restful Sunday we headed back into the classroom and kicked our culminating project work into high gear. We started off the day with three fascinating speakers. Samantha Sedivy works as a judicial law clerk for the sixth circuit of Virginia. She described the law school admissions process, her law school experience, and getting involved in politics in high school and college. She also told us of many interesting cases that she's been involved with, including one about a man who faked his own death.
HSLP 2017: Day 9
Reported by Avery Gagne (Maggie L. Walker Governor's School) and Megan McEvoy (Roanoke Catholic School)
Check out the Sorensen political links page to discover new insights into politics in Virginia. We seek out new informational resources and frequently update the page with new links so bookmark it for later (or set it as your homepage if you like).

subset: commoncrawl


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Help us with our dream of inspiring people and get involved in the motivation of the people around you. Get used to saying kind things to others who need it and you'll be doing your fellow humans a great service.
What does getting involved in inspiring others mean?
It means you speak and act in ways that motivate people through love and respect. It's very hard to help someone work harder if their hard work is not noticed and appreciated. Be that person that keeps the chins of the people around them up by always being their emotionally to encourage and inspire.
We promise that if you suffer from lack of motivation and want of initiative that by helping others to maintain their inspirations it will redound and rebound into you threefold so that not only are you inspiring others but inspiring yourself as well.
So get involved in the motivation of your community, whatever it may be!


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I have a SanDisk 16GB MicroSD HC card in a SanDisk adapter. I use it in my digital camera.
I spent the day taking important pictures that will not be able to be re-taken. When I got home, I ejected the card from my camera and inserted it into my laptop. I was ready to open it and therefore accidentally clicked Enter when it said I needed to format the card. A pop-up came up saying the card could not be formatted. After ejecting and reinserting the card, it says that the card needs formatted before it can be used.
I asked my more computer savvy friend to see if they knew what was wrong. They said they would try using a recovery program, but would need to format it first. They inserted it into their smart phone and tried formatting it that way, with no success.
I think the images are probably lost, but just wanted to get a second opinion. It would be very awesome if anyone knew of a way to copy the deleted files or anything, as it will be quite troublesome if they cannot be recovered. Thank you in advance for your help and suggestions.
EDIT: When trying to open the card in recovery program a pop-up says
D:\ is not accessible.
The volume does not contain a recognized file system.
Please make sure that all file system drivers are loaded and that the volume is not corrupted.
message edited by Mmai
You could try this program. Use the free version. Have you tried putting the card back in the camera and seeing if they show up. You can format it in your camera. If you format it, but will loose everything on it. In the future, use a wire that plugs into your camera and the computer and upload them from the camera. When you eject a card from a device under the wrong conditions, you can screw up the data. Good Luck.
"Accidentally Formatted SD Card"
No you didn't...you weren't able to. Most likely the file table is corrupt or the card simply died. See if this helps:
There are a couple of utilities that may be able to restore/repair the partition. Testdisk and Easeus Partition recovery. Links to both below.
Whatever you do, DO NOT write anything to the card.
Look for the card in Windows Disk Management. If it shows there and the file type is RAW, then riider is correct and the file system is corrupted. At any rate, it can be fixed. Recuva may not be able to recover anything from an external storage device.
I suggest you make a copy of the SD card somewhere.
message edited by OtheHill
If you still have the camera, does it read the card? If so is there an option to connect the whole camera and transfer?


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Transform Your Outfit With...In Accessories - 03/14/13
Over recent years designer handbags have come a long way to transform the women look. They are a-la-mode and not without reason! They reflect a woman's personality and attitude towards life, style and fashion.
Patriotic Designer Shoes Set the...In Shoes - 04/17/12
With the Olympics just around the corner, designer shoes for summer are showing a trend toward the patriotic. The latest deck shoes, created by one of Britain's longest-standing manufacturers of boat shoe designs, come in three-toned leather, red...
The Best Clarks Boots for Men...In Shoes - 03/31/12
Clarks boots for men and women are a favorite among shoe lovers. These boots come in different styles to match every fashion preference. Check out this article to learn some of the nicest pairs available.
Police WatchesIn Accessories - 03/30/12
With a great reputation in the clothing accessories niche, Police watches are thought to be some of the most affordable and sought-after watches around. Being available in a myriad of designs and styles with keen prices, they are the choice of...
How To Make A Leather Tri-Fold...In How To - 12/13/11
If you'd like to save a little cash this holiday season and still give someone a gift that they will really enjoy, then you might consider how to make a leather wallet on your own. If you're handy with a thread and needle, or a sewing machine if...
Information About Pleaser ShoesIn Shoes - 12/06/11
Pleaser is a powerhouse that specializes in specialty footwear and caters exclusively to the needs of different alternative lifestyles. When they first started however, they only imported and distributed footwear for urban fashion. Throughout the...
Must Have Beach AccessoriesIn Accessories - 11/26/11
Next time you go to the beach, make your beach experience fun, exciting and stylish by packing the right accessories. Whether you're going on a holiday cruise or the Jersey shore, you can dress for the beach in style just like you would for any...
Get Your Meal Prep Area in Order...In Ideas - 11/06/11
No one enjoys preparing an evening meal in a cluttered kitchen, especially at the end of a tiring day. So you should take the time to put your meal preparation area in order with the right kitchen organizers. Doing so will make life much easier.
Rain Boots - All AboutIn Shoes - 11/01/11
An essential accessory to have during the rainy season is a pair of rain boots. This is something that everyone needs to stay dry and protected from the harsh weather conditions. In the past, these kinds of boots were quite basic and available in...
Online Apparel Shopping Made Interesting With CustomizationPosted in Ideas - Monday, October 24, 2011 @ 02:25 AM
You come across various occasions in your life such as birthdays, family get-together, office parties and events, your child's first birthday, world cup cricket matches, football matches, any political issue in the country that requires your participation and alike. So, in all such occasions, custom t-shirts teamed with a denim or any other apparel from your wardrobe, would be the apt outfit to express your support or presence in that particular occasion.


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Shock as Juventus agree £250k-a-week with Arsenal striker
Unfortunately, Arsenal cannot win the Premier League title that has eluded them for over 10years and the club now stands the risk of losing some of their key players.
Juventus are reportedly confident that they can put together a package that will entice Alexis Sanchez to Italy and fight off the competition, Paris Saint Germain and Atletico Madrid, although probably not China.
With 18 months left on his existing Arsenal contract, it's thought that talks between the club and Alexis have stalled because we won't give him the £250,000-a-week he wants.
Juve, however, are convinced that they can, while the rich Chinese clubs are reportedly ready to offer him £400,000-a-week.
Until now, PSG have been the favourites to land him since they are more likely to afford it.
I'm not confident that Alexis will stay in north London and as much as I rate him for obvious reasons, we can't force him to stay. If he goes, he goes and we rebuild.


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Bespoke Presentation Skills Training
We work with businesses, schools and individuals to help people present with confidence, and improve their personal impact in presentations and meetings.
What makes us different?
At Love Public Speaking, we believe that a person's ability to communicate is the biggest determiner of the results they get. Our training programmes use techniques from cognitive behavioural therapy and neuro-linguistic programming (plus a few other secrets) to break through people's limiting beliefs and create a permanent shift in their presentation style. We believe in learning by doing, so all our training programmes are highly practical, with rigorous speaking exercises that relate to real life scenarios. As our clients tell us, the impact of our training goes far beyond better presentations, because when people gain speaking confidence they become stronger team players, more resilient workers, and powerful future leaders.
How do we do it?
We have a team of experienced trainers and a consistent training methodology that delivers lasting results. We go into businesses and schools to train teams of staff, as well as working on a 1:1 basis with individuals. Click the buttons below to view our training programmes.
"I can't rate Love Public Speaking highly enough. The training here was well beyond your normal technique training… Utterly transformative for my career."
C. Forshew, CEO, Seed
In-house training to develop your team's presentation skills, pitching skills, and gravitas when dealing with difficult people. Let us know where your team needs help, and we'll design a bespoke programme packed with practical speaking exercises.
Client Testimonials
"If you're looking for the perfect coach to up-skill your staff and turn them into accomplished speakers; Love Public Speaking is certainly the number one option."
Daniel Magill, TSB Bank
"The best training I have experienced in this role. Led by a warm, positive, engaging and intelligent trainer."
Tony Muirhead, Head of Philosophy, St. Dominics College
""At the beginning of the day I rated my public speaking confidence level a 2 out of 10, and by the end I was a 7. The trainer was excellent, I think she is born to do it. She's got a very positive vibe and this increased the energy levels in the room."
Rithma Fonseka, IT Manager
We're proud to say that of our last evaluation survey in November 2018, delegates rated the quality of our workshops an average of 4.9 out of 5. Take a look at what they said…
Blog & Tips on presenting
Check out our blog to read our expert trainers' top tips and tricks on presentation skills, confidence and overcoming imposter syndrome.


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In this article, we will show you, How to Format Power BI Card with example. This includes changing the category Label Colors, enabling Card Title, background colors etc.
To demonstrate these formatting options, we are going to use the Card that we created earlier. Please refer Card in Power BI article to understand the steps involved in creating a Card.
How to Format Power BI Card
Please click on the Format button to see the list of formatting options that are available for this Card.
Format Data Label of a Card in Power BI
Data Label is the numeric value (Sales Amount, Total Product Cost etc) that is displayed by the card. As you can see from the below screenshot, we changed the Color to Green, Display Units to Thousands, text Size to 40, and Font Family to Arial Black.
Format Category Label of a Power BI Card
Category Label means Columns Name that we used in the Card. From the below screenshot you can see, we change the Color to Brick Red, Font Family to Georgia, and Text Size to 35.
Format Power BI Card Title
By toggling the Title option from Off to On you can enable the title of a Card.
From the below screenshot you can see, we change the Title Text to Total Sum of Sales, Font Color to Golden, Title Alignment to center, Font Family to Georgia, and Text Size to 30.
Format Power BI Card Background Color
You can add Background color to a Card by toggling Background option to On. For the demonstration purpose, we added some random color with 57% transparency.
Format Power BI Card Borders and General Settings
You can add Borders to a Card by toggling Border option from Off to On.
Use General Section to Change the X, Y position, Width and height of a Card
Thank You for Visiting Our Blog


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4.3 - 22 Reviews
Hansard Scholars Programmehttps://www.hansardscholars.uk/
Hansard Scholars Programme is a program provider offering 1 abroad program. Programs offered include Study Abroad and Internship abroad programs in 1 country such as The United Kingdom.
For over 20 years, the Study & Scholars Programme has run a series of courses aimed at improving accessibility and understanding of the British political system. Hundreds of undergraduate and postgraduate students from around the world have participated in our prestigious Hansard Scholars Programme, and we have successfully delivered political education and training programmes to dozens of organisations from both the public and private sectors.
Study Abroad with Hansard Scholars Programme
December 7, 2019
Hansard Scholars Programme: London - Hansard Society
Are you an undergraduate interested in politics and current affairs? Would you enjoy studying and internin...
Featured Programs


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RV, marine and auto supplier Hopkins Manufacturing Corp. continues to diversify with the acquisition of F3 Brands, the leading provider of oil drains, drain pans, lifting aids and automotive ramps. F3 is the non-fuel containment business of Blitz USA.
"This acquisition improves our ability to provide a complete range of products for both our customers and do-it-yourself consumers across North America," said Bradley T. Kraft, president and CEO of Hopkins. "Our FloTool brand of innovative, task-specific funnels makes fluid transfer easier for all of the different automotive, farm, marine, and power-sport applications. Adding F3's product offering creates a complete line for the DIY'er."
In addition, the Emporia-based firm announced it had made a "significant strategic investment" in Paradise Road LLC, a Surf City Garage spin-off.
While Surf City Garage is the recognized leader in super-premium Enthusiast Grade brand detailing products, Paradise Road, based in Huntington Beach, Calif., is "an entirely separate company and brand exclusively focused on providing high-quality, premium-level appearance products for the typical car owner at mid-market prices," according to Hopkins.
As part of their joint venture, Hopkins will assist Paradise Road by leveraging its global distribution network. The entire line of products will be rolled out to retail stores over the next year.


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AoC- August Birthday Card Collection
I just have one… Happy Birthday Cahya or we calls him (BayMax)
I just have one… Happy Birthday Cahya or we calls him (BayMax)
Maybe its already too late for this because i broke my handphone before so I have to find another way to get my picture data from it. Anyway these are the July's birthday card collection The theme for these are … Continue reading
Hello for the Coins (Cindy On Interesting Followers) Happy Earth Day lets save our earth guys by planting trees, do not littering, save energies, walking and many other things so my friend and I made this DIY Earth Day Card … Continue reading
Happy 1st Anniversary for both of you hope you will have a long life love (3L). These card present just for you
Happy Birthday Ocep… at 16th March I made this card and gave it to him secretly by asking our lecture to do that for us…


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Main Street is taking shape and now we have a good idea how great the middle school is going to look; not only aesthetically, but also great place for learning for the students, staff and community. It will be another gem in Soledad. This could not have been accomplished without our great community. Thank you for supporting the Soledad Unified School District! It is Happening in Soledad!!
October 4, 2018
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22 Aug Praise God for His faithfulness
I would like to thank and praise God for his grace and his hand in all that we do. I am particularly glad for the opportunity to be a part of NPEVAC (National Partnership to End Violence Against Children in Sri Lanka).
NPEVAC is a new initiative to end violence against children by 2030; recently launched as part of a bold, new collaborative partnership between the government, UN agencies, international organisations, civil society, faith groups, the private sector, the media, children, and other key stakeholders. The launch was held on June 1 with the participation of many government officials including the Minister for Women and Children. I was able to contribute through providing input and support although LEADS is a relatively small organisation among many other partners.
I also thank God for giving me a chance to be a part of the staff retreat. The focus of the retreat was to promote collaborative engagement among staff and I returned with new ideas and perspectives.
Finally, I thank God that in spite of a busy schedule, I was able to organise the certificate ceremony and book launch of "Lighting a Lamp." These workshops were held last year and we were able to evaluate the benefit of organising such workshops. We came to know that wardens were implementing what they gained in the workshops and came back with queries and inquiries for further help.
Please pray for God's wisdom to lead the team and provide the needed support for the staff. Also pray for favour in the eyes of government officers.
— Nevedita Jeevabalan, Sri Lanka, Co-MP with AsiaCMS


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Refine search
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Number of results: 257
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Hotel accommodationÁgua Hotels Nelas ParQ
Telephone: +351 232 941 470
Fax: +351 232 944 914
E-mail: email@example.com
Website: http://www.aguahotels.pt
Hotel accommodationAlbergaria o Gato
Telephone: 284760000
Fax: 284760001
E-mail: email@example.com
Website: http://www.hotelogato.com
Hotel accommodationAlmedina Coimbra HotelAlmedina Hotel is located in a city known for its narrow streets, "patios", stairs and typical arches of the medieval age. The city of Coimbra stands over the River Mondego, and it's considered a world heritage. Our hotel's philosophy is to (...)
Telephone: +351 239 855 500
Fax: +351 239 829 906
E-mail: email@example.com; firstname.lastname@example.com
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Hotel accommodationAmazónia Lisboa HotelThe Amazónia Lisboa Hotel it's situated in the very heart of the city, near Praça Marquês de Pombal, and offers 192 modern rooms (including Jr. Suite and 2 rooms for handicapped) – half of them have their own sunny balcony.The swimming pool, (...)
Telephone: +351 213 877 006
Fax: +351 213 879 090
E-mail: email@example.com
Website: http://www.amazoniahoteis.com
Hotel accommodationAmérica Diamond's HotelThe America Diamond´s in Lisbon, Located in the heart of the city, next to Marquês de Pombal and Eduardo VII Park, the offers a confortable accommodation in historical building beautifully restored in the heart of Lisbon contrasting with its modern (...)
Telephone: +351 21 352 11 77
Fax: +351 21 353 14 76
E-mail: firstname.lastname@example.com
Website: http://www.americadiamondshotel.com
Hotel accommodationAqua HotelThe Aquahotel is a unique hotel, where guest are presented with unique, readiness and the elegance of a personalized service, which makes the Aquahotel the best place to stay.Fully equipped with services and facilities to accommodate the customer (...)
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Fax: +351 256 575107
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Hotel accommodationAuramar Beach Resort
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Hotel accommodationAxis Ponte de Lima Golf Resort HotelAxis Ponte de Lima Golf Resort is a building of contemporary architecture, located on the golf course is only 1.5 km from the historic center of Ponte de Lima.
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Website: http://www.axishoteisegolfe.com
Hotel accommodationBenavente Vila HotelIn the heart of the Ribatejo, the Benavente Vila Hotel integrates a concept of modernity combined with the traditions from the Ribatejo region. Located in the historic center of the village Benavente and 30 minutes from Lisbon, visitors are embraced (...)
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Fax: +351 263 518 215
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Hotel accommodationBraga Flag HotelBraga Flag Hotel (formerly Comfort Inn Braga) offers air-conditioned rooms with a TV. It is located near the A3 motorway exit, 400 meters from Ferreiros Train Station and 40 km from Francisco Sá Carneiro Airport.All rooms have simple furnishings and (...)
Telephone: +351 253 000 600
Fax: +351 253 000 600
E-mail: firstname.lastname@example.com
Website: http://www.bragaflaghotel.com


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I am a science fiction and fantasy writer who supports inclusivity in publishing and writing. I also draw mice.
Welcome to my Ko-fi Page. If you enjoy my content, please consider supporting what I do. Thank you.
Ko-fi helps creators get support from people who love what they do.Get Your Free Page


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dmartora: Hi all, help and guidance appreciated.
I want to buy a couple of the self balancing electric scooters (mini segway, hoverboards... Etc.) for the kids (ish). I've decided to buy their affection through a couple of really unnecessary purchases that will make the march to Christmas simpler.
I don't usually have reservations about buying on-line and having stuff shipped over, but on this occasion I was trying to find local suppliers in NZ.
My thinking is that these are still relatively new products, just about entering generic brand and mass production, meaning you can probably pick up lots of alternative unbranded products across the standard sellers like eBay, AliExpress and also through Amazon etc.
My challenges are... reliability, especially after they've been chucked about by kids which they inevitably will be, and so the ability to have an easily accessible support and repair point, rather than the whole parcelling it up, trying to find out how to do it etc. etc. There's also the GST on international purchases I need to consider as I doubt I'll slip through any net with those things.
So, does anyone know of a local reseller that also offers ownership of after sales, or am I just being a bit of a scaredy cat whiner, and the potential savings of buying from an eBay reseller in China would off set the costs elsewhere.
Even better, if anyone actually a product recommendation, irrespective of source, then it would be useful.
I decided to buy from Glideboar
helping others at evgenyk.nz
Finch: Just noticed Harvey Norman is now selling the boards...
richms:Finch: Just noticed Harvey Norman is now selling the boards...
$998! WTF, Guess they have to allow for the inevitable 40% off of whatever, but that is really taking the .... for pricing.


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Özyeğin University, Çekmeköy Campus Nişantepe District, Orman Street, 34794 Çekmeköy - İSTANBUL
Phone : +90 (216) 564 90 00
Fax : +90 (216) 564 99 99
E-mail: firstname.lastname@example.com
Message from the Director
Message from the Director
Message from the Director
The world is changing at an increasing speed given the developments in the information and communication technologies. One fundamental impact is on the definition of value in the society. Value increasingly means not only finding the right knowledge and creating insights but also facilitating the real-time delivery mechanisms to act on the knowledge and insights. Ozyeğin University with its entrepreneurial focus and the PhD program provides a unique proposition for the next generation academic leaders.
The PhD program's mission is to educate the leaders who will be instrumental in charting the next generation businesses in the connected economy. This mission is achieved by a combination of guided, self-paced research opportunities followed after completing a rigorous foundational two-year program.
The essential tools and techniques as well as necessary skills and technical knowledge are acquired during the first two years. The first year provides a comprehensive orientation to the academic discipline. The PhD candidate is expected to acquire a broad perspective in the key study areas through introductory survey lectures that cover finance, marketing, operations and management. A holistic understanding with the right academic rigour provides abundant opportunities to be creative both from a methodology and an implementation point of view.
During the last two years, the PhD candidates work with their advisors of their choice, identify their topics of interest and strive to complete their dissertations in their chosen fields. This is where the ideas find plenty of space to flourish and turn into meticolusly prepared academic papers.
The business world is increasingly under pressure to keep-up with the changing nature of value. The stake holders demand increasingly innovative and technology-driven solutions. Once respected higher-education degrees become an expected venture for any individual who is serious to make significant contribution. Such ventures turn out to provide fertile grounds for data scientists and for acquiring deep-area expertise, key traits for the next generation leaders. I hope to see your application to the program if this path excites you.
Assoc. Dr. Bige Saatçoğlu
PhD Program Academic Director


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Theft of Swords by Michael J. Sullivan
Orbit: November 23, 2011
Genre: Fantasy
Source: Free From Library
Beach Vacation
Theft of Swords is a pretty traditional fantasy read. There's adventures and sword fights and a dash of magic thrown in for good measure. If you enjoy more descriptive works in the fantasy genre Theft of Swords is likely to appeal. For those who appreciate a greater depth of characterization, you may want to approach with caution.
Royce Melborn, a mysterious master thief, and his equally enigmatic mercenary partner, Hadrian Blackwater, have been contracted to steal a sword. Unfortunately the theft does not go as planned and the duo are implicated in the murder of the king. So, naturally, they kidnap the new king, with the help of the new king's sister. Politics, they just got more exciting. The plot of Theft of Swords kicks off early and the author maintains the pace with interconnected events. After doing some digging about this series I realized that Theft of Swords was originally self-published in installments. For me, the original publication schedule was evident in the disjointed separations of the book making for an overly lengthy reading experience. There is also a lot of emphasis on the description of the surroundings and the actions of characters – again, this isn't my thing so I wasn't always engrossed in reading to the point that I couldn't walk away from it for a few days.
What originally drew me to Theft of Swords was the characters. A thief and a mercenary go on adventures? Um, yes please. Of course, my expectation was that I would get to delve into the minds of these characters. But after reading 600 pages, I have to confess that I don't particularly feel like I "know" the characters. Both Royce and Hadrian remain as mysterious and unknowable as they did at the beginning of the book. While tidbits about each character are sprinkled throughout, I never really escaped the feeling that both characters were kind of empty. Yes, there were some flashes of humour from Royce and Hadrian but by and large they remain a mystery to me. Instead, the secondary characters of the princess, new king and other characters our heroes meet along the way seem to be more fleshed out than the supposed main characters. That said, I also recognize that this is the first in a trilogy and there are also prequel novels that focus on the characters, so this could explain some of the distance I felt when it came to the main characters. Take my thoughts with a grain of salt.
Theft of Swords certainly wasn't my favourite fantasy read, but it was one that I felt had some potential if the characters are further explored. The world and the political plot that goes along with it are also intriguing enough to make me want to read the second book. So, if you're looking for a more descriptive fantasy read, Theft of Swords just might be for you.
Similar Reads
Django Wexler's The Thousand Names
is a fantasy that I highly recommend and I think it creates a common ground in the descriptive emphasis without compromising on it's characters. Each book in the series has only become better than the first.
Swords and Scoundrels would be my second recommendation as a follow-up to Theft of Swords. Like Theft of Swords, Julia Knight keeps her focus on the action and adventure of her characters rather than their emotional complexity. If you were a fan of the descriptive nature of Theft of Swords, Knight's fantasy will appeal to you.


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I am a PhD student in Economics at the University of Chicago. Before beginning my PhD, I worked at the Center for the Economics of Human Development with Professor James Heckman. I study how education, knowledge, and skills affect quality of life, both directly and through expanding the budget set through increased earnings. But I am also interested in how people's expectations, beliefs, and preferences shape decisions that affect the accumulation of human capital.
I take inspiration from striking historical and contemporary economic trends: black-white gaps in college attendance, high school graduation, and incarceration rates; the polarization of wealth; human capital and economic growth; and the public finance of educational institutions.


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Sunday, November 21, 2010
カムイ外伝 "a.k.a" Kamui The Lone Ninja (2009)
Once a powerful ninja, Kamui walks away from his violent and murderous ways. His travels bring him to a seashore village where he meets Hanbei, a fisherman who shares the former ninja's sense of honor. Though Hanbei's wife is wary of the stranger, the fisherman and Kamui become good friends. Life at the seaside seems idyllic but Kamui doesn't get to enjoy the peace for very long when, one day, a band of pirates arrive... It seems that his past life is catching up on him, and everything and everyone is not as it seems. Now he must draw upon his shadowy arts if he hopes to escape with his life.
Country: Japan
Language: Japanese
Download links:
Posted by dasblach at 3:26 AM


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Undergraduate Honors Theses
With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors
Thesis Defended
Spring 2016
Document Type
Type of Thesis
Departmental Honors
Molecular, Cellular, & Developmental Biology
First Advisor
Dr. James Orth
Second Advisor
Dr. Jennifer Martin
Third Advisor
Dr. Xiang Wang
Exportin-1 (XPO-1) is responsible for the movement of cargo proteins out of the nucleus and into the cytoplasm. Selective inhibitors of nuclear export (SINE) bind XPO-1 at cysteine-528, which results in the sequestration of cargo proteins in the nucleus. SINE drugs are currently being developed and tested in the treatment of many types of cancers. One of the cargos, p53 may play an important role in the efficacy of SINE. To test the necessity of p53 in the action of SINE drugs, matched pairs of cell lines with wildtype or functionally disrupted p53 were analyzed for differences in their cell fates after treatment. The drug Nutlin targets the p53 degradation pathway and was used in comparison to SINE. SINE-induced cell death and arrest can occur independently of p53. Nutlin induced arrest in G-1 phase independent of p53, but cell death is largely dependent on p53. The two drugs in combination result in a strong death response in cells with and without p53.
Recommended Citation
Doak, Andrea E., "With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors" (2016). Undergraduate Honors Theses. 1188.
Included in
Cancer Biology Commons, Cell Biology Commons, Medicinal Chemistry and Pharmaceutics Commons, Molecular Biology Commons


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ETL Testing - Tasks
ETL testing is done before data is moved into a production data warehouse system. It is sometimes also called as table balancing or production reconciliation. It is different from database testing in terms of its scope and the steps to be taken to complete this.
The main objective of ETL testing is to identify and mitigate data defects and general errors that occur prior to processing of data for analytical reporting.
ETL Testing – Tasks to be Performed
Here is a list of the common tasks involved in ETL Testing −
- Understand the data to be used for reporting
- Review the Data Model
- Source to target mapping
- Data checks on source data
- Packages and schema validation
- Data verification in the target system
- Verification of data transformation calculations and aggregation rules
- Sample data comparison between the source and the target system
- Data integrity and quality checks in the target system
- Performance testing on data


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Further Detail
Suntime Antigua Patio set
This metal garden furniture has four coloured aluminium recliner chairs, a 1.2m round table, and colour coordinated parasol.
Suave and sophisticated, the textilene fabric on this stylish collection is enhanced by the shiny rust free aluminium framework.
Extremely comfortable and hard wearing, this furniture will be the focal point of your patio or lawn.
1 x 1.2m Round Table
1 x 2.2m Parasol
4 x 110cm x 58cm x 66cm Recliner
All glass table tops are tempered safety glass and comply with British Standards.


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Coaching App
At JourneyPure, we know all-too-well just how delicate the first year of recovery after treatment can be. There is no straight line to success, rather the road to recovery is usually filled with unpredictable twists and turns. Knowing this, we have developed JourneyPure Coaching, an app that is in a class of its own and designed to help provide an extra layer of support and guidance for that first year after completing one of our programs.
Our Commitment to Your Sobriety
The JourneyPure Coaching App
Before our patients finish their treatment program and transition back home, we work with them to develop an aftercare plan that meets their recovery needs. Part of that aftercare plan is setting the patient up with a recovery coach who will work with him or her through the JourneyPure Coaching App.
The app, which is to help the patient remain connected to our facility and their recovery, offers a number of different features that can aid in the patient continuing to live a healthy lifestyle free from substance abuse. Some of the most notable features of the JourneyPure Coaching app include:
- HIPAA-compliant secure messaging between the patient and his or her personal recovery coach
- Nutritional guidance, including a place to log meals
- Daily recovery log where patients can share their thoughts, feelings, and emotions
- Connections to local healthcare providers who can offer additional services as needed
- Information regarding alumni events and gatherings
At JourneyPure, we focus on providing our clients with a holistic treatment approach so that they can heal their minds, bodies, and spirits. Part of that holistic approach includes implementing good sleep habits, a nutritional diet, and regular exercise. Through the JourneyPure Coaching app, patients can track their sleep, meals, and exercise. Their recovery coach can view all logs, allowing for a sense of accountability to develop between the patient and the coach. Additionally, patients will have one recovery coaching session each week, where they can discuss their progress, setbacks, and continued goals.
Benefits of the JourneyPure Coaching App
Any medium geared towards providing those in recovery with support can be beneficial, including the JourneyPure Coaching app. The most obvious benefit of our state-of-the-art app is that it provides former patients with a sense of security in their new-found recovery, as they have a built-in line of communication with professionals who they know and respect. Attempting to go through recovery alone can be extremely difficult, which is why each individual using our app can contact their recovery coach at any time, as well as benefit from their weekly session with him or her.
Those who have a past of substance abuse or alcohol abuse can also have a past of disorganization and chaos throughout their days. But, through the JourneyPure Coaching app, individuals will start to develop a strong sense of structure by being able to log their sleep, meals, and exercise. When in treatment, patients learn that eating well, getting enough rest, and exercising can all help improve upon things such as stress, depression, and anxiety (to name a few). Having an easy, electronic place to log rest, meals, and exercise can not only help individuals structure out their days, but also help keep them accountable in doing these things.
Just because a patient leaves JourneyPure after his or her program has been completed does not mean that he or she no longer requires further care. Through the JourneyPure Coaching app, individuals will be connected to healthcare providers in their communities that can offer therapeutic and medical services to meet their recovery needs. Continuity of care is extremely important when in recovery because if an individual stops doing the things that keep him or her sober, it can be simple to relapse.
The JourneyPure Coaching app also keeps individuals in the know about events and gatherings, such as meetings held at the facility. Keeping connected to a treatment program can allow for an individual to utilize his or her alumnus to not only continue to recover but also to help support others who are either currently at JourneyPure or those who have also graduated from the program.
The JourneyPure Difference
At JourneyPure, we recognize the skill and determination it takes in order to get sober and maintain recovery. While patients are enrolled in our programs, we will work to treat the mental, emotional, physical, and spiritual impacts of their substance use disorder and restore those parts of themselves. We also pride ourselves in seeing our patients through their recovery by sticking with them after they leave the program, such as through the JourneyPure Coaching app. When someone attends JourneyPure, they become part of the JourneyPure family, and we treat them as such.
If you or a loved one need professional addiction treatment, do not wait any longer. Contact us at JourneyPure right now.


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Search Results
Gyokuto is the Guardian Spirit of Okatsu.
Guardian Spirits can be chosen to provide the player with certain benefits.
Activate Living Weapon by pressing △ + O (B + Y on Xbox/Steam controller) when the Amrita Gauge is full.
Pronounced "Gyokuto" in Japanese, Japan is home to many legends about the moon-hares, said to be capable of great speed. Some say that when lightning flashes across the sky, it is actually a moon-hare traveling to Earth and back. Legend has it that they spend their time on the moon making mochi ricecakes, or medicine; either way, these stories show them to have kind and caring spirits. always working hard to make something useful. In time, the moon-hare came to be seen as the guardian of travelers, who would often venerate them to help ensure safe passage.
How to Obtain
- Tokai Region, Main Mission "Memories of Death-Lilies" - Mission Level 78
Strategies and Notes
- Gyokuto's 100 Ninjutsu Power equates to roughtly a 15% increase in Ninjutsu Damage.
- The Guardian Spirit Talisman effect involves Gyokuto laying Lightning traps in front of the player. Gyokuto lays more traps than Hi-Nezumi and Gyokuto's traps don't vanish on contact with the enemy. Gyokuto's traps deal very little damage but inflict Lightning debuff very easily . Also, it does not stagger or knock down.
- Gyokuto is a speed oriented Guardian Spirit, and improves the quality of Low Stance, Ninjutsu, Dashing and Elixirs.
Table Key
|Rate at which the Living Weapon gauge decreases if you are hit while using the Living Weapon.
|Rate at which the Living Weapon gauge is reduced when you consume Ki while using the Living Weapon. The higher it is, the more you can use it.
|Rate at which the Living Weapon gauge recovers when you absorb Amrita while using a Living Weapon.
|The effectiveness of a Living Weapon that is active when your Life is under a certain level.
|Amrita Gauge Rate
|The rate at which you gain Amrita. The higher it is, the qicker the gauge fills. When the Amrita gauge hits maximum, you can invoke a Living Weapon.
16 Feb 2017 11:30
Gyokuto is acquired by completing the main mission Memories of Death-LiliesReplyReplies (0)


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8 Steps to manage your web presence that are free
- Google your business.. Add the words "best," & "review". What does it say? (Based on what it says will somebody buy your product, can they even find you? This is the new digital shelf)
- Claim and customize your map listings ( Google Places, Bing & Yahoo business listings, Manta, CitySearch, Merchant Circle, any associations). Find out what you need to fix here.
- Claim dedicated review sites. Monitor reviews. How does your brand appear? (Yelp, Angie's List, Epinions, Consumer Search, BBB, Trust Link)
- Set up content channels & social pages ( Youtube, Picasa, Metacafe, Vimeo, SlideShare, Facebook, Twitter, Linkedin)
- Consistently Create content to answer questions. (ask yourself why would someone follow my page? Original & Interesting, Unique discounts, offers & special, tag content to maximize search discovery, don't forget video)
- Determine what your Zero Moments are.. ( use google tools to determine search volumes, keywords etc)
- Integrate social into your business & ask for the invite ( b.cards, marketing materials, website, storefront, pos, QR codes, receipts, existing advertising)
- Speed beats perfection. So jump in now.
Peerless Digital Marketing creates integrated web presence strategies. Multi-screen strategies that help businesses engage with potential customers at the right time and place in the online world. Call Ryan Today 916-450-1335.
Specialties: Internet Marketing & Digital Media Strategy | Google Adwords Certified | PPC | Google Analytics | Merchant Accounts | Programmatic DSP Display | Ad Trafficking | Social Ads| Video Ads | Email Marketing | Web Site Conversion Strategies | Adobe Creative Suite | Word Press | Yelp | Data- Aggregators & Listings | Phone Tracking| Mobile App | Geo-Fence | IP Targeting | Live Chat Technologies | YouTube Video | Internet Radio- Pandora & Spotify |


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Pesa will supply 14 additonal Atribo diesel trains to Trenitalia. This extends the total Trenitalia 'Swing' fleet to 58 units.
Italian national passenger operator Trenitalia exercises an option to buy 14 Atribo 3-car diesel trains from Polish rolling stock manufacturer Pesa. The contract is worth PLN 200 million (€ 47 million). Deliveries of the 'Swing' designated trains are due in September 2019.
The order is part of a framework contract between Pesa and Trenitalia signed in December 2013. The original 2013 order was for 40 diesel trains and an option for 20 units. Trenitalia already lifted an option for 4 units earlier.
Order & delivery scheme:
- 2013 order: 40 units, delivery 2014/2015, numbered ATR 220 Tr 001-040;
- 2016 order: 4 units, delivery 2016/2017, numbered ATR 220 Tr 041-044;
- 2018 order: 14 units, planned delivery 2019
Pesa Atribo (ATR 220)
The 3-car Atribo DMU design for regional non-electrified lines (a.k.a 219M), is derived from Pesa's 218M type built for several Polish regional operators. Besides Trenitalia, several Italian regional railways are operating Atribo DMUs: Ferrovie del Sud Est, Ferrovie Nord Milano and Trasporto Passeggeri Emilia-Romagna.
The Atribo for Trenitalia has 21 First Class seats and 129 fixed and 4 tip-up seats in the Second Class. The trains with a Voith prime mover, can be operated in multiple with up to three sets. One unit features two toilets and a visual and acoustic passenger information system.