The Experimental Factor Ontology (EFO) is an application ontology driven by experimental variables including cell lines to organize and describe the diverse experimental variables and data resided in the EMBL-EBI resources. The Cell Line Ontology (CLO) is an OBO community-based ontology that contains information of immortalized cell lines and relevant experimental components. EFO integrates and extends ontologies from the bio-ontology community to drive a number of practical applications. It is desirable that the community shares design patterns and therefore that EFO reuses the cell line representation from the Cell Line Ontology (CLO). There are, however, challenges to be addressed when developing a common ontology design pattern for representing cell lines in both EFO and CLO.In this study, we developed a strategy to compare and map cell line terms between EFO and CLO. We examined Cellosaurus resources for EFO-CLO cross-references. Text labels of cell lines from both ontologies were verified by biological information axiomatized in each source. The study resulted in the identification 873 EFO-CLO aligned and 344 EFO unique immortalized permanent cell lines. All of these cell lines were updated to CLO and the cell line related information was merged. A design pattern that integrates EFO and CLO was also developed.Our study compared, aligned, and synchronized the cell line information between CLO and EFO. The final updated CLO will be examined as the candidate ontology to import and replace eligible EFO cell line classes thereby supporting the interoperability in the bio-ontology domain. Our mapping pipeline illustrates the use of ontology in aiding biological data standardization and integration through the biological and semantics content of cell lines.The online version of this article (10.1186/s12859-017-1979-z) contains supplementary material, which is available to authorized users. Tens of thousands of cell lines have been developed and used in experimental research, making the usage of cell lines a major tool for scientific discovery. A biomedical ontology is a set of human- and computer-interpretable terms and relations that represents various entities and the relations among these entities in a biomedical domain. Biomedical ontologies are critical to data and knowledge representation, standardization, integration, and computer-assisted reasoning. To better support cell line-based research, it is critical to have a standardized ontology that represents available cell lines, their associated cell types, tissues, and diseases, and how these entities connect to each other.The Experimental Factor Ontology (EFO) is a data-driven biomedical application ontology developed by the European Bioinformatics Institute (EMBL-EBI) to organize the diverse experimental variables needed to describe data resided in the EMBL-EBI resources, including cell line data . EFO aimCurrently, EFO cell line is loosely defined as a population of cell units and covers primary cell lines as well as permanent cell lines under ‘material entity’. On the other hand, CLO defines characteristics of cell line at a single-cell level and aims to cover the immortalized stable cell lines. With the different conceptual viewpoint of EFO and CLO cell lines, the population level definition of EFO cell line is suitable to model experiments where measurement is performed on cell line cultures. Even though cell line cultures were assumed homogeneous, recent studies reviewed the effects of heterogeneity of cells within the same culture –9. The aBesides the difference in the definition of individual-level and population-level of cell lines, EFO and CLO also have different coverages. EFO covers primary cell lines, stem cell derived cell lines, and immortalized permanent cell lines that can be cross-referenced with central cell line catalogs, while CLO only covers immortalized permanent cell lines. While the aim of this study is to align cell lines in CLO and EFO, cell lines that do not belong to the immortalized permanent cell lines mapping described above should remain in the scope of EFO-native primary cell lines.In this study, we have developed a mapping process that combines third-party cross referencing, lexical and biological content comparison, and semantic relation matching to compare, align and synchronize immortalized permanent cell lines available in EFO and CLO. This will allow for a better cell line knowledge integration within the scope of the OBO Foundry . AdditioThe input ontology OWL files of EFO version 2.85) and CLO (version 2.1.106) were downloaded from Ontobee  and the .85 and CIn order to achieve cell line mapping with high confidence and quality, a three-way mapping among EFO, CLO and Cellosaurus was first performed  in Fig. EFO cell lines that were not processed in the previous step were directly mapped to CLO cell lines  in Fig. SLD is shortest Levenshtein distance (SLD) among all the combinations of EFO and CLO cell line labels and synonyms [M is a function indicating whether the ith cell line related element in K was matched (+1) between EFO and CLO cell line, or unmatched (−1). K contained the cell line related information including diseases, cell type, anatomic location and species of origin extracted from the corresponding ontologies. The C-scores of each EFO cell line against all CLO cell lines were computed. For example, the mapping between EFO cell line (EFO_0002208) NIH3T3 and CLO cell line NIH-3 T3 (CLO_0004301) had SLD of 1  and synonyms . In brieSince there are biological and modeling differences between primary cell lines and immortalized permanent cell lines, it is necessary to check whether the remaining unmapped cell lines from the previous step were in fact immortalized permanent cell lines or not. There is not an explicit statement that distinguishes immortalized cell lines from primary cell lines in the EFO cell line classes. The identification of additional immortalized permanent cell lines from EFO was done by cross referencing to Cellosaurus since primary cell lines are not curated in the Cellosaurus  in Fig. http://www.ontobee.org/sparql).Alignment by cross-referencing between CLO and EFO was mined from Cellosaurus knowledgebase. The source files of EFO and CLO were then processed for ontology class property alignment. A set of regular expression rules were designed for normalization of special characters and comparison of cell line rdfs:label property. The semantics and other biological information enriched by CLO’s and EFO’s design patterns were retrieved and compared using the OWL API library . The chaCell lines in CLO and EFO have different naming strategies. In EFO, cell line naming convention is a mix of individual cell implication , or a cell line name with no indication of cell population . CLO focuses on the definition of individual ‘cell line cell’ and its laboratory-derived descendants. CLO:‘cell line’ is a population of CLO:‘cell line cells’. The definition of ‘cell line cell’ targets individual single cells, which offers the advantage of using CLO’s design pattern in single cell profiling data representation. Therefore, the aforementioned EFO’s cell line examples would be converted to ‘HEK-293 cell’ and ‘MCF 10A cell’ according to CLO’s naming convention to aid normalization between the two resources. The normalization also improves the readability and clarity of EFO cell line names when working in OBO space.has grain” in CLO. Even though cell line related biological information was mostly shared between EFO and CLO, two minor differences existed. First, EFO uses the object property “bearer of” to model disease while CLO uses “is model for”. Second, CLO “cell line cell” was “derives from” the CL class “cell” which can be “cell type” or another “cell line cell” , but in EFO “cell line” was “derives from” only “cell type”. Therefore, aligning EFO with CLO by importing CLO classes into EFO required adjustment within EFO so the differences in the two cell line design patterns were resolved.The design patterns of both EFO and CLO were similar with some minor variation, and CLO design pattern was used as the template to bridge EFO unique features Fig. . First, The results of the three intermediate steps, (i) EFO-Cellosaurus-CLO mapping, (ii) Direct EFO-CLO Mapping and (iii) Identify EFO Unique Cell Lines were illustrated in Fig. Using the 624 cell lines in the EFO-Cellosaurus-CLO mapping step as the gold standard, we compared and evaluated the improvement of cell line mapping when biological information and semantic relationship were incorporated into the mapping process. By using the cell line annotation alone , 439 out of 624 cell lines were mapped. An additional of 130 cell lines were recovered by integrating biological information and semantic relationship of disease definition. The percent mapping was improved from 70.4 to 91.2% when the biological and semantic content was added on top of the lexical and cross reference mapping criteria.To illustrate the similarities and differences between CLO and EFO, an example of the aligned cell line, “MCF 10A” is described to facilitate the comparison Fig. . MCF 10AWe have developed a mapping process that combines third-party cross referencing, lexical and biological content comparison, and semantic relation matching in order to compare, align and synchronize immortalized permanent cell lines available in EFO and CLO. In this study, 92.6% (1218 out of 1315) EFO native immortalized permanent cell lines were either aligned or added to CLO. The complementary information integrated from EFO and CLO provided a more comprehensive coverage of immortalized permanent cell lines and their related biological information. In addition, the imported CLO immortalized permanent cell lines defined as the biologically individual cell can help EFO to better represent and model single-cell experiments.Manual curation of the mapping between ontologies is time consuming, as illustrated here in cell line mapping between EFO and CLO. Our study also proposed an approach to discover new EFO-CLO cell line mapping exploiting the confidence score (C-score) that integrated cell line lexical and semantic information to aid the process of building semi-automated mapping pipeline. Thus, our study illustrated the advantage of utilizing biological content stored as semantic relations in ontology over just lexical comparison for cell line mapping. Using the EFO-Cellosaurus-CLO cross referenced cell lines as the gold standard. In this study, 91.2% of the EFO-CLO cell lines were mapped and validated. Our pipeline that integrated cell line-related biological information and its semantic relations showed improved mapping performance as to lexical matching only. Even though the design of this pipeline is specific for the cell line mapping from EFO to CLO, the usage of biological content and semantic relations defined in the ontologies could be generalized. For example, such information can be applied to probabilistic mapping tools using Bayesian network , 21 and In addition, biological information of a cell line can be examined by their ontology relations to improve the accuracy of the mapping. For example, cell line ‘SW684’ is defined in both EFO and CLO. In EFO, this cell line EFO_0002369) is derived from ‘connective tissue’. The same cell line in CLO (CLO_0009198) is derived from some ‘fibroblast’. In Cell Ontology (CL) [69 is derCell line naming in different laboratories remains an issue of inconsistency, and can lead to repeated usage of cell line names. In our study, we identified two occurrences of the cell line name “H9”. The CLO “H9” cell line is an immortalized human T-cell lymphoma cell line deposited with American Type Culture Collection (ATCC) . The EFOhttps://www.ebi.ac.uk/spot/oxo/). Establishing a framework for common disease ontology remains a practice requiring community agreement at large.Another issue throughout the mapping of EFO and CLO cell lines was the imprecise disease definition of a cell line from different resources. This will continue to be a major challenge as modeling of diseases is very difficult due to the dynamic of definition establishment that vary in different clinical communities and expertise. Though much needed, establishing a framework that consolidates the different aspects of disease semantics to be modeled with a common disease ontology is out of the scope of this study. Attempts to find a common ground that consolidates multiple disease vocabularies have been initiated in various projects such as the Monarch Initiative Disease Ontology , and theIn this study, a mapping process was developed order to compare, align and synchronize immortalized permanent cell lines available in EFO and CLO. The mapping pipeline combined third-party cross referencing, lexical and biological content comparison, and semantic relation matching in. A total of 92.6% EFO native immortalized permanent cell lines were either aligned or added to CLO, and the complementary information was merged. The imported CLO immortalized permanent cell lines defined as the biologically individual cell can help EFO to better represent and model single-cell dynamic under varied experimental conditions.Additional file 1: Table S1.EFO cell lines drawn from external sources. In the initial step of the EFO-CLO comparison and alignment process, there are 428 and 20 EFO cell lines which were imported from Cell Line Ontology and 20 in BRENDA Tissue and Enzyme Source Ontology respectively. These 448 EFO cell lines were excluded from the entire mapping process. File is stored in Microsoft Excel spreadsheet (xlsx) format. (XLSX 47 kb)Additional file 2: Table S2.Final EFO-CLO alignment result. The 874 EFO-CLO mapped cell lines aligned and merged into CLO (Tab. 1 in the excel file) and 344 EFO unique immortalized permanent cell lines added to CLO (Tab. 2 in the excel file). File is stored in Microsoft Excel spreadsheet (xlsx) format. (XLSX 54 kb)


Although primary breast tumors are detected early in most cases, it is inevitable that many patients remain at risk for future recurrence and death due to micrometastases. We investigated interactions between the degradome and the adhesome that drive metastasis, and have focused on matrix metalloproteases (MMPs) within the degradome and integrins and E-cadherin within the adhesome.The aim of this study is to identify interaction networks between adhesion molecules and degradative enzymes in breast cancer metastasis.in vitro and in vivo.We compared non-metastatic  and metastatic  human breast cancer cell lines and xenografts, in which we measured growth rate, migration, invasion, colony formation, protein expression, and enzyme activity in vivo. These metastatic breast cancer models also displayed elevated heterophilic cell-extracellular matrix (ECM) and lower homophilic cell–cell adhesion compared with those of non-metastatic models. This was conferred by an increased expression of the heterophilic cell adhesion molecule integrin β1 (ITGB1) and a decreased expression of the homophilic cell adhesion molecule E-cadherin. Inhibition of MMPs in metastatic cells led to a reduced expression of ITGB1, and stimulation of ITGB1 resulted in higher MMP activities in metastatic cancer cells, demonstrating reciprocal dependencies between degradome and adhesome. Re-expression of E-cadherin (CDH1) led to an increased expression of the precursor form of ITGB1.The metastatic breast cancer lines and xenografts displayed higher expression and activity levels of MMPs, which was also confirmed by noninvasive imaging Our results point toward a concerted interdependence of MMPs, ITGB1, and CDH1 that is critical for breast cancer metastasis. In the first 10 years after diagnosis, up to 12% of Stage I/II breast cancer patients, whose cancers are locally contained within the breast, die. However, 60% of Stage III patients, whose cancers have invaded nearby lymph nodes, and over 90% of Stage IV patients, whose cancers have spread to distant organs, die within 10 years after diagnosis.2 Integrin β1 facilitates cell–ECM adhesion and movement of tumor cells into the stroma and thus assists in local invasion within the primary site and growth in metastatic sites.5 At least 24 different integrin complexes are capable of binding to distinct ECM ligands.6 Integrin can also form signaling complexes with oncogenic Her2, Met, and EGF receptors, and it can recruit MMPs to the cell surface for local degradation of ECM in human breast cancer.7 E-cadherin promotes homotypic tumor cell–cell adhesion and provides intercellular contacts that confine tumor cells to the primary tumor site.8 MMPs, integrins, and E-cadherin are all implicated in breast cancer metastasis.9Cancer cell to ECM and cell-to-cell adhesion is, to a large extent, conferred by integrins and E-cadherin.10 and that the cancer degradome contributes to tumor progression, invasion, cell–ECM communication, and the metabolome,12 the interaction between these two groups of molecular players and its role in metastasis is just emerging.13 Our hypothesis that cell adhesion molecules interact with the degradome is supported by (i) co-localized expression of adhesion molecules such as integrins and E-cadherin and degradative enzymes such as MMPs,14 (ii) regulation of MMP activities by integrins and E-cadherin,15 (iii) regulation of the integrin-mediated adhesome by MMPs,16 (iv) binding of key integrins as well as MMPs to the same extracellular matrix (ECM) components,17 and (v) interaction between integrins, E-cadherin, and membrane-bound MMPs in cell communication in several types of cancer.18 Recent findings also revealed that several types of cancer cells have associations between adhesion and degradome molecules.16 For example, in melanoma cells, integrin αVβ3 protein binds directly with MMP-2 and thereby localizes MMP-2 to cell surfaces, and hence assists in ECM, specifically collagen, degradation.13 In human melanoma cells, MMP-2 cleaves fibronectin into small fragments to enhance cell adhesion mediated by αvβ3 integrin.16 The co-localization of MMP-14 (MT1-MMP) and integrin β1 is necessary for local invasion by human adenocarcinoma, epidermoid carcinoma, and fibrosarcoma cells.14 It was also shown that MMP-14 and integrin form protein complexes that regulate fibronectin remodeling in murine myoblast cells.19 In human osteosarcoma cell lines, integrin α2β1 positively regulates the expression of MMP-1 and collagen-1α1.20Although previous studies have shown that integrin activation controls metastasis in human breast cancer21 They inhibit several MMPs, such as MMP-1, MMP-2, MMP-7 and MMP-9, by directly binding to Zn2+ ions in the active site.16 Clinical studies with these hydroxamate-based broad-spectrum MMP inhibitors were ineffective at doses associated with musculoskeletal toxicity in a phase III trial for metastatic breast cancers that were stable after chemotherapy.22 The second generation of hydroxamate-based MMP inhibitors was more selective for specific groups of MMPs, such as for example MMI-166, which is a selective inhibitor of MMP-2, MMP-9, and MMP-14.23 However, they still suffered from the general limitation of hydroxamate-based MMP inhibitors that drug metabolism leads to the loss of the hydroxamate Zn2+-binding group and thus were ineffective in human trials.22 The second generation of non-hydroxamate MMP inhibitors such as rebimastat, tanomastat, and SB-3CT were developed to avoid metabolic inactivation.24 However, their efficacies were inconsistent and opposing outcomes were obtained depending on the timing of administration. The third generation of MMP inhibitors, which are specific to only one target and possess no zinc-binding group, is currently being evaluated preclinically.25 MMPs are also involved in cytokine and chemokine activation and inactivation, cell-surface-receptor cleavage and release.26 The full potential of MMP inhibitors can only be explored once all MMP functions and all MMP interactions with other important molecules in cancer such as growth factors, apoptotic mediators, and adhesion molecules are fully understood.Over the past 40 years, several generations of MMP inhibitors have been investigated to potentially treat cancer development and metastasis. The first-generation of MMP inhibitors such as marimastat, ilomastat (GM6001) and batimastat were hydroxamate-based inhibitors based on the structure of collagen.in vivo tumor degradome activity and angiogenesis. The results provided novel insights into the molecular networks that comprise the cancer adhesome and degradome in breast cancers, and tested how their combined expression and activation drives cancer growth, invasion, ECM remodeling, and metastasis. Our results also suggest that, collectively, the adhesome and degradome molecules expressed in a given tumor can predict the metastatic risk of this primary tumor.In this paper, we have performed comparative studies with a set of non-metastatic  versus metastatic  human breast cancer cell lines and xenografts. Experiments were performed to determine growth rate, migration and invasion, colony formation, adhesion, aggregation, protein expression (western blot) and activity (zymography). Enzymatically activatable optical imaging probes were utilized to study 27 Human mammary fibroblasts were a kind gift from Dr Gary Luker at the University of Michigan. All cells were incubated at 37 °C with 5% CO2 in a humidified incubator.Human breast cancer cell lines, MDA-MB-231, SUM149, SUM159, BT-474, T-47D, and MCF-7, were obtained from the American Type Culture Collection . Cells were stably transfected with a construct containing cDNA of tdTomato as outlined in the 27Descriptions of cell-specific culture media are given in http://kmplot.com).28 Kaplan–Meier estimates of 10-year relapse-free survivals (RFS) were calculated with data collected from patients with systemic treatment. The median of gene expression was used to dichotomize data into high- and low-expression groups. Differences in survival curves were evaluated by log-rank test. Significantly different Kaplan–Meier estimators indicate longer RFS for patients with low expression of MMPs compared to the high-expressing group.The relationship of MMP gene expression and relapse-free survival was evaluated in an integrated multi-study breast cancer transcriptomic data set using Kmplotter . The heat map represents changes in relative content of adhesome and degradome gene expression levels in 17 metastatic breast cancer cell lines  and 11 non-metastatic breast cancer cell lines .A breast cancer microarray data set (GSE-16975) was analyzed where the breast cancer cell lines were grown to optimal cell densities for RNA extraction and hybridization on Affymetrix microarrays.http://string-db.org) computational tool and database with a high confidence interval of 0.7.30 The STRING network, composed of the proteins of interest, is constructed based on genomic context, high-throughput experiments, co-expression, and scientific reports.30 The network nodes are proteins and edges represent the predicted functional associations. A red line indicates the presence of fusion evidence; a green line neighborhood evidence; a blue line co-ocurrence evidence; a purple line experimental evidence; a yellow line text-mining evidence; a light blue line database evidence; a black line co-expression evidence.30 Clustering algorithms (Kmeans=2) were used to extract relevant modules.30 Inter-cluster edges are represented by dashed-lines.30Network analysis of potential protein interactions among adhesome and degradome proteins, whose expressions were different in metastatic versus non-metastatic cell lines in the Gene-e analysis, was carried out using the STRING-9.1 , immunoblotting protein assays, cell migration, and invasion assays. E-cadherin transfection was done using E-cadherin-GFP, which was a kind gift from Jennifer Stow (Addgene plasmid # 28009). Experimental details are provided in ex vivo fluorescence imaging. Four to six 2-mm thick fresh tissue sections were cut from the primary tumor using an adjustable tissue slicer . Tumor sections and whole lungs were imaged with IVIS camera settings at 1- and 2-s exposure time, binning factor of 8, field of view of 6.4 cm, and f number of 2. All experiments were carried out according to the approved guidelines of the Institutional Animal Care and Use Committees (IACUCs) of the Johns Hopkins University.Optical imaging was carried out using the Xenogen IVIS 200 Spectrum system. Enzymatically activatable optical imaging probes MMPSense-680 (NEV10126), and AngioSense-750 (NEV10011EX) from PerkinElmer  were injected into the tail veins of mice growing orthotopic tumor xenografts according to the manufacturer’s protocol. MMPSense is activated by MMP-2, -7, -9, -12, -13, and -14. AngioSense injection enables imaging of tumor blood vessels and was used for normalization of uneven delivery of the probes to the tumor. Each mouse was imaged at 24 h after the injection with the IVIS camera settings at 1 and 2 s exposure time, binning factor of 8, field of view of 18.8 cm, and f number of 2. Then, the animals were killed and tumor xenografts and lungs were excised for ex vivo imaging, tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin blocks. Serial sections of 5-μm thickness were cut from the formalin fixed, paraffin embedded tissue blocks and floated onto charged glass slides .32 A hemotoxylin and eosin stained section was obtained from each tissue block. Unstained sectioned were further processed for immunohistochemical detection of MMP-1, MMP-9, and ITGB-1 as detailed in Following t-test. P<0.05 was considered statistically significant. Box-and-Whisker plots for cell migration, invasion, and adhesion were generated by BoxPlotR , where the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by R software , whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles, and outliers are represented by dots.33 A one-way analysis of variance was calculated from the number of migrated or invaded cells in all experimental groups. Post hoc comparisons using the Fisher least significant difference were explored to compare the mean of one group with the mean of another group. P<0.05 was considered statistically significant. In vivo MMP activity was measured for each pixel by calculating the ratio of MMPsense divided by Angiosense optical signal intensity using in-house software written in Matlab . Box plots for MMP activity were also generated using Matlab.Statistically significant differences between quantitative measurements were analyzed by unpaired Student’s 29 we have demonstrated that metastatic compared with non-metastatic breast cancer cells contain significantly (P<0.01) increased messenger RNA (mRNA) gene expression levels of integrin α-1, α-4, α-5, α-6, α-V, and β-1, a decreased level of E-cadherin, and increased levels of MMP-2, -3, -11, -14, -16, and -19 as shown in 34 It should be noted here that tail vein injected breast cancer cells represent a subpopulation of cancer cells with increased tendency to colonize a particular organ.35In an analysis of 1,144 genes from a panel of 28 human breast cancer cell lines given in the GEO data set GSE16795,To identify known and potential protein–protein interactions relevant to the adhesome and degradome, we used the STRING 9.1 software and database. STRING quantitatively integrates protein interaction data from multiple sources for a large number of organisms. Using STRING, we identified several members of the MMP family , integrin family  and the E-Cadherin molecule that have direct and indirect associations with each other, as well as associations with major ECM components such as collagen, laminin, and fibronectin . E-cadhe9 In our analysis of clinical data sets taken from the KM-Plotter database,36 we show that significantly increased expression levels of MMP-2 (P=1.5e−6), MMP-9 (P=0.027), and MMP-14 (P=0.00051) were predictive of a decreased RFS in chemotherapy treated ER- human breast cancer patients (n=211). High expression levels of MMP-1 (P=0.00012) and MMP-9 (P=0.00022) were predictive of a decreased RFS in endocrine treated ER+ human breast cancer patients (n=690) as shown in P=1.5e−6), MMP-9 (P=0.027), and MMP-14 (P=0.00051) is predictive of lower RFS in chemotherapy treated for ER− human breast cancer patients. High expression of MMP-1 (P=0.00012), and MMP-9 (P=0.00022) is predictive of lower RFS in endocrine treated ER+ human breast cancer patients. These Kaplan–Meier curves were generated using KM plotter from http://kmplot.com.Breast tumors with an overexpression of MMPs are associated with invasion and metastases.in vitro and in vivo/ex vivo experiments with human breast cancer cell lines and xenografts as outlined in in vivo and ex vivo (37). Metastatic tdTomato-expressing MDA-MB-231 and SUM159 cells grew a significantly (P<0.01) higher number of colonies compared with all three tdTomato-expressing non-metastatic cell lines on uncoated, collagen1 coated, and matrigel-coated plates. Metastatic tdTomato-expressing SUM149 cells formed fa significantly (P<0.01) increased number of colonies on uncoated surfaces, but not in collagen1, and matrigel-coated surfaces, compared with all three non-metastatic cell lines.We have performed comparative  ex vivo . Prolife ex vivo . Clonoge ex vivo 37. MetasWe also tested the migration and invasion capabilities of all tdTomato-expressing cell lines. In transwell migration and invasion assays, significantly fewer non-metastatic tdTomato-expressing cell lines  migrated or invaded through the 8 μm pores of a transwell chamber compared with metastatic tdTomato-expressing cell lines  with gelatin zymography. HMF alone are able to produce pro-forms and active forms of MMP-2 and MMP-9. The activity of active MMP-2 was enhanced when HMF were co-cultured with MDA-MB-231, and active MMP-9 activity was enhanced when HMF were co-cultured with SUM149 or SUM159 cancer cells , on the regulation of MMP secretion in cultured cells. When RGDS peptide was added to cell culture medium, the secreted gelatinases MMP-2 and MMP-9 increased in the metastatic MDA-MB-231 and SUM159 cell lines, as shown in P<0.01) when metastatic MDA-MB-231 and SUM159 cells were treated with the broad-spectrum MMP inhibitor marimastat or with the MMP-2 and MMP-9 inhibitor SB-3CT for 48 h  peptide, which is present in a variety of ligands, which are part of the ECM such as collagen, laminin, and fibronectin and thereby represents a major recognition system for cell adhesion.for 48 h . This su43 Although we were able to detect E-cadherin gene expression by qRT-PCR in the transfected cells  Increased expression and activity of MMP-2 and MMP-9 among others in metastatic breast cancer cell lines, xenografts, and lungs as compared with non-metastatic lines. (ii) Heterophilic adhesion, likely mediated by integrin β1, is increased in metastatic compared to non-metastatic lines. (iii) Homophilic adhesion, likely mediated by E-cadherin, is increased in non-metastatic compared to metastatic lines. (iv) Re-expression of E-cadherin reduced the expression of the mature form of integrin β1 in metastatic breast cancer cells. (v) A reciprocal interaction exists between integrin β1 and MMP-2/MMP-9 in metastatic breast cancer cells.9 Despite promising preclinical data, clinical trials using MMP inhibitors resulted in inconsistent outcomes. As evident from our data and the data of others,40 a key issue is that the types and levels of MMP expression and activity are quite variable across different breast tumors and the derived breast cancer cell lines, which is one of the main reason for the inconsistent outcomes in clinical trials using MMP inhibitors. However, systemic treatment with MMP inhibitors would be a good way of treating breast cancer metastasis, as MMP activity in metastatic sites is upregulated during or even prior to metastatic seeding,40 as also observed in the lungs of our metastatic tumor xenograft model.We observed that MMP-2 and MMP-9 activities, among other MMP activities, are increased in metastatic breast cancer cell lines, xenografts, and lungs as compared with non-metastatic lines. Our analysis of publicly available clinical and cellular expression data revealed that low levels of MMP-1, -2, -9, and -14 are important for breast cancer survival. However, a slew of other MMPs also confer breast cancer metastasis, depending on the particular cell lines studied. Since elevated MMPs are an important component of many aggressive tumors, it is a potential drug target for cancer therapy.46 In addition, β1 integrins initiate signaling cascades in the cell in response to extracellular chemokines (outside-in), and also transmit intracellular signals that change the way the cells interact with the ECM (inside-out).6 These signaling pathways regulate cell adhesiveness by changing the conformation of β1 integrin binding to the ECM.5 These dynamic adhesion processes are crucial for conferring the migration abilities of cancer cells, which degrade mammary basement membrane, the dense ECM surrounding the tumor, and the ECM of the walls of blood vessels.We showed that mature active integrin β1 expression was elevated in metastatic breast cancer cell lines that adhered to the ECM components collagen 1 and matrigel, while non-metastatic cell lines contained no integrin β1 and did not adhere well. We also observed that metastatic breast cancer cell lines displayed increased heterophilic adhesion. It is known that integrin heterodimers containing β1 subunits are receptors for various types of ECM molecules such as collagens, laminins, fibronectin, and tenascin, and thus have an essential role in cell–ECM adhesion.8 Loss of E-cadherin results in the weakening of cell–cell adhesion.8 On the other hand, increase in integrin-β1 subunits mediates cell–ECM interactions by linking signals from the environment to the actin cytoskeleton.6 Simultaneously, these two processes enhance the agility of metastatic breast cancer cells, which let them respond to external signals and execute successful migration and invasion.8In our study, E-cadherin was increased in non-metastatic breast cancer cell lines that adhere well to each other but not to fibroblasts, as compared with metastatic breast cancer cell lines. This finding is in good agreement with the suggestion that dynamic E-cadherin-mediated cell–cell adhesions and integrin-mediated cell–ECM adhesions govern the invasive and metastatic potential of tumors.47 The effect of re-expressing E-cadherin on integrin β1-subunits has, to the best of our knowledge, not been studied so far. Our data suggest that reducing the amount of active integrin β1 increases the expression of E-cadherin. Taken together, our data indicate that a reciprocal interaction exists between E-cadherin and integrin β1, which couples homophilic cell–cell and heterophilic cell–ECM adhesion in metastatic breast cancer cells. This will enable metastatic breast cancer cells to lose contact to other cells at a time when they start binding to the ECM while they migrate, and switch back to binding to cells instead of ECM when they arrest and colonize distant sites.We observed that E-cadherin gene re-expression in metastatic breast cancer cells reduced the amount of mature integrin β1 protein in metastatic breast cancer cells. In the clinical setting, it was shown that the secondary metastatic site can induce the re-expression of E-cadherin in cancer cells, which is a critical step in the survival of cancer cells in the new microenvironment.Vice versa, when we inhibited MMP activities in cell culture by either Marimastat or SB-3CT, the expression of integrin β1 was significantly reduced. Our experiments with the MMP inhibitors Marimastat and SB-3CT showed for the first time that inhibition of MMP activities can reduce the expression of active integrin β1, suggesting an association between active MMP-2/MMP-9 and integrin β1 protein expression in metastatic breast cancer cells. It has previously been shown in human umbilical vein endothelial cells that the physical associations of MMP-2 with integrin β1 can promote ECM degradation by these endothelial cells.48 In good agreement with these findings, our protein–protein interaction network analysis of human data also predicted an interaction between several MMPs and integrin β1.We found that RGD-stimulation of metastatic breast cancer cells upregulated the expression of MMP-2 and MMP-9. Metastatic breast cancer remains one of the most devastating cancers, and very few new treatments have revealed meaningful improvements in the survival of advanced stage breast cancer patients. Elevated MMP expression and activity within the degradome is characteristic of metastatic breast tumors and is associated with the development of distant metastases. Even though preclinical studies examining the effectiveness of MMP inhibition were encouraging, clinical studies turned out to be disappointing. We showed for the first time that inhibition of MMPs reduced the cell–ECM adhesion molecule integrin β1 expression. We also showed that re-expression of the cell–cell adhesion molecule E-cadherin reduced the active form of integrin β1 along with cell migration. We suggest that a treatment strategy that targets critical nodes in the adhesion-degradome network might be the most effective for clinical translation. Elevated MMP levels may be used to identify and monitor women at high risk of developing metastatic disease.11 Therefore, collagen degradation releases a significant amount of proline, which is in turn used in cellular metabolism as a source of energy. Proline can be metabolized by proline oxidase to generate reactive oxygen species as signaling molecules for epigenetic reprogramming, which regulates the redox homeostasis of cancer cells.49 It was recently shown that metabolic flux can cause changes in cancer cell adhesion and metastatic transformation.50 Future studies should focus on the relationship of the metabolome with the adhesome-degradome network in terms of driving breast cancer metastases.A quarter of the collagen residues in the tumor microenvironment are proline residues.In summary, our study shows for the first time that cancer cell adhesome and degradome interact in metastatic breast cancer cells, and are modulated during migration and invasion of these cancer cells. These results suggest that targeting nodes in the adhesome-degradome network of breast cancer may be an effective strategy for treating metastatic breast cancer.


Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of ‘LLO nanocarriers’ coupled with the stabilizing element of natural origin.The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated.The impact of LLO was evaluated in human leukemia cell lines The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes. Modern medicine undoubtedly can boast great successes in the control of tumor development and progression. Nevertheless, an increase in cancer-related mortality is still observed in developed countries –4. This Recently, novel anti-tumor agents with highly specific mechanism of actions have been discovered –11 and eIt should be noted that kernels of natural-origin attract special attention in the field of active agent delivery because of their certain properties, such as a cell-like size and high ligand binding ability. Furthermore, in combination with polyelectrolyte membranes, natural cores can build systems with high biocompatibility.Despite the numerous examined medicines and drug delivery systems, an optimal system that exhibits efficiency as an anti-tumor therapy with reduced side effects has not been yet obtained. An application of factors inducing tumor necrosis might introduce a major change in this area. However, in the majority of the proposed systems, drug molecules are released into the medium surrounding the tumor tissues and subsequently are adsorbed by healthy cells. A different approach to combat this issue involves the process of adsorption of the whole cytostatic carriers. The success of such a strategy is determined by the size of the carriers, which can be internalized via phagocytosis, and the efficiency of the anti-tumor factor immobilized within the carrier.in vitro in human peripheral blood mononuclear cells or Jurkat, WEHI-164 and IC-21 cell lines. The obtained results prove that our system allowed the cytostatic release in eukaryotic cells by exerting a lethal impact.We have described the stabilized nanosystems (SNS) based on elements of natural origin as the platform for antitumor factor delivery to the targeted cells. The main parts of the designed platform are 200-nm diameter preserved bacterial carriers that bear a cytostatic agent—listeriolysin O (LLO) –32. The Herein, we report the first platform for local LLO delivery in which bacterial nanocarriers are coupled with naturally derived stabilizing elements. The application of mostly natural elements is the unique feature of our platform. The membrane construction applied in the present system ensures the increase of the system avidity towards tumor cells. Thus, the SNS provides specific delivery of the cytostatic factor to the targeted cells and simultaneously reduces the number of potential side effects caused by the anti-tumor therapy.−1) at the beginning of the experiment. The examination was performed using FTS 3000MX spectrometer . Liquid samples were collected in a KBr pellet. Typically, thirty scans were performed at a resolution of 4 cm−1 and selectivity of 2 cm−1. Presented FTIR curves were analyzed using Essential FTIR software .The polyelectrolyte (PE) membrane on a substrate was analyzed by Fourier transform infrared spectroscopy (FTIR) . PeakForce Tappingeviously . Then, pFor surface forces acquisition, the silicon cantilever with a borosilicate glass colloidal particle of a 10 μm diameter were used . Spring constant value of a used cantilever was determined before experiment with ThermalTune method. The force-distant data were acquired in Nanoscope 8.15 software and analyzed in Origin 8.50 (OriginLab).The surface wettability angle of the applied polyelectrolyte membrane was analyzed using a surface energy analyzer  with dedicated software.hly gene sequence from Listeria monocytogenes 10403S chromosome and the gfpmut3b sequence were PCR amplified and fused to OE-PCR using specific oligonucleotides. The resulting hly, hly-gfp and gfp-hly genes were cloned into the pPSG-IBA series plasmids  using the StarGate Cloning System . Then, the recombinated pPSG-IBA plasmid was transformed into the E. coli BL21(DE3) production strain. The LLO, LLO-GFP and GFP-LLO proteins were purified from the bacterial cell lysates using Ni-NTA resin columns via affinity chromatography and concentrated with a centrifugal concentrator. Construction and purity was confirmed by SDS PAGE and western blot, and activity was assessed using the hemolytic test : 3375 exhibiting N-H stretching vibrations in PEI and HA; 2359, 2337 exhibiting N-H vibrations of hydrogen interactions in PEI and HA; 1366 exhibiting C-CHI and HA .To visualize the morphological structure of the polyelectrolyte shells, we applied the AFM technique. Both HA and PEI layers were deposited on mica substrate. The obtained data are presented in The AFM scratching method let us determine the thickness of membrane formed by PEI/HA system equal to 3 nm by analyzing the difference of height between the film and revealed surface of a solid substrate. The PEI/HA film show good homogeneity which is visible in large  and smalFad is adhesion force and z is the distance of sphere from a surface. The calculated average work of adhesion occurring between layers PEI and HA value was equal to 4.32±1.34×10−15 J (n = 4).The measure of interaction between the polyelectrolyte layers is the work of adhesion calculated by an integration of force—distance dependencies according to the following formula:The HA|PEI configuration is the most convenient for our platform due to its ability to sustain the bilayer structure because of relatively high work of adhesion between the layers as compared with interaction between PEI and weak polyelectrolytes .−12 [cm2×s-1].The cell cores (IC-21 cells) coated with PE membrane were used to estimate the applied membrane molecular weight cut-off value. Diffusive permeability was evaluated using a thermodynamic description of diffusive mass transport across a homogenous membrane (Fick’s law) and a two-compartment model . The dexTo evaluate the wettability of PEI|HA membrane on an alginic support, we used the surface energy analyzer. The value of the contact angle measured for the dedicated layer was 34.68±2.45 [°], indicating the relatively high hydrophilicity of the designed membrane system.To identify the most effective system for anti-tumor agent delivery, we designed and examined three different platforms. We studied the following systems: ‘LLO nanocarrier’, the basic platform consisting of the bacterial core nanocoated with a polyelectrolyte bilayer with active agent immobilized within; ‘ligand modified LLO nanocarrier’, ‘LLO nanocarrier’ modified by biotin-avidin-biotinylated transferrin complexes and ‘stabilized nanosystem’ (SNS) build with the stabilizing element bearing transferrin (‘cell core’) and ‘LLO nanocarriers’ coupled to the cell core via a biotin-avidin-biotin bridge.To prepare the ‘LLO nanocarrier’ , we coatThe construction of the third platform—the ‘stabilized nanosystem’ (SNS)–consisted of two main steps. First, transferrin ligands were anchored to the surface of the ‘cell core’. Second, the prepared ‘cell core’ was bound with the biotinylated ‘LLO nanocarriers’. The SNS were obtained as a result .Cells can absorb small elements by engulfing them in the process called phagocytosis. Accordingly, in the designed systems, we applied bacteria, the size of which allows adsorption by cells. Moreover, to prevent the destruction of the active agent during the experiment, the agent was immobilized within the PEI|HA bilayer covering the bacterial core.To boost the stability of the prepared LLO platform and intensify the effect towards targeted cells, we used a stabilizer, the eukaryotic cell core, to extend the system. Accordingly, previously studied ‘LLO nanocarriers’ were coupled to the ‘cell core’ via a biotin-avidin-biotin bridge. In addition, to facilitate cytostatic agent delivery to tumor cells, transferrin ligands were anchored to the surface of the cell core. The application of the stabilizer increases the final dose of delivered active substance and simultaneously reduces its unnecessary dissipation throughout body fluids. We selected a core of biological origin to minimize the extent of the platform and maintain non-immunogenicity of the system.Numerous tumor-associated receptors, including transferrin receptor, asialoglycoprotein receptor, and estrogen receptor, have been investigated using receptor-targeting approaches –44. We cAfter confirming nanocarriers coupling with the ‘cell core’, we applied confocal microscopy. To facilitate the analysis, we used FITC-labeled microbeads coated with a PEI|HA bilayer instead of non-fluorescent bacterial carriers. To detect transferrin, an anti-transferrin FITC antibody  was used. Microscopic observations are presented in The presence of the basic platform—LLO nanocarriers was confirmed using flow cytometry. Coating of the non-fluorescent bacterial core with polyelectrolyte layer with immobilized GFP-LLO resulted in obtaining meanly 86.9±5.3 FITC positive events.The presence of the individual elements of the designed systems, especially the polyelectrolyte layer, was confirmed indirectly by immunocytochemical reaction with an anti-transferrin antibody. Accordingly, we incorporated transferrin complexes within the HA layer of ‘ligand modified LLO nanocarrier’ or the ‘cell core’ of the SNS. After 24 hours of incubation in a culture medium, we assessed the formation of complexes using an anti-transferrin antibody (FITC-positive events) via flow cytometry. All experiments were performed in six repeatings. As shown in To examine the trace of nanocarrier interaction with the target cells, we also applied flow cytometry. To facilitate analysis, we substituted the bacterial cores of LLO nanocarriers with FITC-labeled microbeads (~200 nm diameter). Then, Jurkat cells were cultured in the presence of those ‘LLO nanocarrier’ platforms  for 24 hours for flow cytometry evaluation. The intensity of fluorescence in the FITC-A channel corresponding to the signals from the FITC emission wavelength was evaluated. In the experimental group, the fluorescence signal of FITC was relatively high (approximately 84%) compared with the control group (cells cultured without the “LLO nanocarrier”) at approximately 0.2% . The obtTo further assess whether the LLO molecules are adsorbed by cells via phagocytosis, we examined the impact of all constructed systems on cells exhibiting phagocytic functionality, namely the macrophage cell line IC-21 (mouse macrophage). No significant difference in cells viability was noted between the macrophage cells incubated in the presence of designed systems and negative control (population cultured without the system) during 24 hours of culture . These dAssessing the influence of the basic ‘LLO nanocarriers’ system or ‘modified LLO nanocarriers’ on human peripheral blood mononuclear cells there was no significant difference in the percentage of viable cells cultured in presence of ‘modified LLO nanocarriers’ compared with culture in presence of ‘LLO nanocarriers’ after two hours (p = 0.0516>0.05) or after 24 hours (p = 0.1637>0.05). However a significant difference in the percentage of viable cells in the group cultured with ‘LLO nanocarriers’ or ‘modified LLO nanocarriers’ compared with control (the cells without the addition of the systems) was observed after two hours (p = 0.0001<0.05) or after 24 hours (p = 0.0001<0.05) or after 24 hours (p = 0.0000<0.05) or after 24 hours (p = 0.0000<0.05) .It can be caused by lower transferrin receptors expression compared with actively growing cells of cell lines .Consequently, for further examinations the WEHI 164 cells were applied. All experiments were performed in six repeatings.Assessing the influence of the basic system (‘LLO nanocarriers’) on WEHI-164 cells, a significant difference in the percentage of viable cells in the experimental group compared with control I (population cultured in the presence of ‘LLO nanocarriers’ without incorporated GFP-LLO) was observed. Moreover, for ‘LLO nanocarriers’, the percentage of FITC-positive events in the experimental group after 24 hours of culture were reduced 3-fold compared with the culture examined after 2 hours of incubation, revealing GFP-LLO loss related to cell necrosis .To evaluate the influence of the ‘LLO nanocarriers’, ‘ligand modified LLO nanocarriers’ and SNS platforms on eukaryotic cells, we cultured WEHI-164 cell line in their presence. We observed a mean 35% decline in viable cells after 2 hours of incubation in the presence of ‘LLO nanocarriers’, a 48% decline in the presence of ‘ligand modified LLO nanocarriers’ and a 60% decline for the SNS. All examined systems exerted lethal effects on the evaluated cells. A significant difference in cell viability was noted between the negative control  and each one of the experimental groups after 2 hours of culture . NeverthAdditionally, the lethal impact of the ‘ligand modified LLO nanocarriers’ and the SNS was maintained up to 24 hours. Statistical differences in cell viability was noted between the cells cultured with the SNS compared with the ‘LLO nanocarriers’ (p = 0.0002<0.05) or the negative control (p = 0.0010<0.05) and between the cells cultured with the ‘ligand modified LLO nanocarriers’ compared with the ‘LLO nanocarriers’ (p = 0.0000<0.05) or the negative control (p = 0.0000<0.05). Importantly, there were the following two negative controls for each experiment: WEHI-164 cultured alone and the population cultured with the respective system without LLO incorporation.To estimate whether GFP-LLO delivered by designed platforms actually interacted with the cells, we evaluated the percent of FITC-positive events corresponding to signals from the GFP emission wavelength.The systems bearing transferrin (‘ligand modified LLO nanocarriers’ and SNS) demonstrated increased lethal effects on targeted cells after 2 hours of culture compared with the non-modified nanocarrier (‘LLO nanocarrier’). These results indicate that the presence of this protein supports the system’s effectiveness. As previously mentioned, proliferating tumor cells exhibit increased expression of transferrin receptors on their surface; thus, the platform with incorporated transferrin exhibits increased affinity towards tumor cells. Accordingly, a higher number of LLO nanocarriers reach the target, which increases the platform efficiency.After examining three different platforms, we conclude that the SNS is the most effective platform. Based on biologically derived elements, the unique design of the developed system supplies LLO to the targeted cells. The constructed system provides immediate cytotoxic effects on tumor cells while simultaneously ensuring the protection of the active agent from potential destruction during the experiment. Comparing the performance of constructed platforms in targeted cells, the increased lethal effects of systems bearing transferrin is observed, indicating that the ligand increases the platform affinity towards the tumor cells. Finally, the ‘cell core’ with transferrin ligands ensures system stability and enhances the titer of the LLO nanocarriers that are delivered to the targeted cells. Of note, the macromolecular substances involved because of phagocytosis induced visible intracellular effects after a few dozen minutes. This process cannot be observed in cases of macrophages because of their phagocytic function. The unique advantage of the system is its possible application in local listeriolysin O anti-tumor therapies. These experiments provided the basis for the further development of LLO delivery systems as an alternative to targeting chemotherapeutic drugs in local anti-tumor therapy.


In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the  Clostridium perfringens , a feature that enables a directed CLDN targeting for therapeutic approaches [The claudin (CLDN) protein family is a major structural and functional component of the tight junction complex in epithelial and endothelial cells ,2,3. Chaproaches ,17. Thisproaches ,17,18,19proaches ,21. Thisproaches . Thus, Cproaches ,24,25. Tproaches .Several studies indicated that cells derived from human and canine tumours reduce their CLDN expressions during cultivation ,7,18,27.This study reports comparative CLDN-1, -3, -4 and -7 expression analyses of non-neoplastic and neoplastic canine mammary gland tissue samples and thereof derived primary cultures and cell lines. The aim of the study was to identify stable CLDN expressing canine mammary gland derived cell lines, keeping the initial epithelial character.Six of the 17 primary cultures discontinued growth before reaching p.19: three of the five healthy tissues derived primary cultures , one of two lobular hyperplasia derived primary cultures (DT14/08R) and both simple carcinoma derived primary cultures . For details see For the evaluation of the epithelial character, early and late passages were immunocytochemically analysed for cytokeratin distribution. DT14/04R and DT14/05S2 did not exhibit any cytokeratins. DT14/07T partially kept the distribution of cytokeratins. DT14/08R and DT14/09T lost the cytokeratins. DT14/07R added on cytokeratins. For details see Eleven of the 17 primary cultures proliferated until p.19 and beyond and therefore resulted in cell lines: two of the five healthy tissue derived primary cultures , one of the two lobular hyperplasia derived primary cultures (T124), eight of the twelve neoplastic tissue derived primary cultures (simple adenoma derived (DT14/04T), complex adenoma derived primary cultures (T121), complex carcinoma derived primary cultures , carcinoma arising in a benign mixed tumour derived primary culture (T126), benign mixed mammary tumours derived primary cultures . For details see Seven cell lines partially kept the distribution of cytokeratins . Three cell lines lost the cytokeratins . One cell line added on cytokeratins (DT14/04T). For details see CLDN-1, -3, -4, -7, β-Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in RNAs of all examined passages of the primary cultures, cell lines and available original tissue samples via amplified fluorescence detection. Furthermore, all passages of the primary cultures, cell lines and the original tissue samples were analysed in one run. CLDN expressions were normalized to the reference genes ACTB and GAPDH revealing similar results. ACTB and GAPDH were chosen as they were already used for CLDN gene expression analyses in canine prostate tumour and mammary tissue and tumour derived cell lines [CLDN gene expression analyses of human tissues and intestinal carcinomas [CLDN expression was normalized only to ACTB or GAPDH it is clearly stated within the text.The QuantiGene Plex Assay allowed the simultaneous multiplexing of all genes of interest . Alterations of the CLDN-1, -3, -4 and -7 gene expressions decreased in relation to GAPDH, comparing the original tissue samples and the corresponding first passage. In five of six cell cultures the CLDN-1, -3, -4 and -7 gene expressions decreased in relation to ACTB. In one cell culture the CLDN-1, -4 and -7 gene expressions increased in relation to ACTB. However, in that cell culture CLDN-3 expression decreased in relation to ACTB. For details see In all three analysed primary cultures and all three analysed cell lines, the CLDN gene expression levels  in the latest analysed passage were displayed for CLDN-1 by four cell lines , for CLDN-3 by two cell lines (T124 and T120A) and for the CLDNs-4 and -7 by three cell lines . For details see High CLDN-1, -3, -4 and -7 gene expression levels decreased in the majority of the analysed primary cultures and cell lines in the beginning of the cultivation and remained low (lower than 0.1) during cultivation. The CLDN expression levels for the CLDN-1, -3, -4 and -7 genes. Cell line DT14/04T exhibited high CLDN expression levels for the CLDN-1, -4 and -7 genes. Therefore, the CLDN gene expression profiles were described for cell lines T120A, T124 and DT14/04T.Cell lines T120A and T124 showed high CLDN-1, -3 and -4 expressions were higher than the CLDN-7 expression. Regarding the expression profiles from p.10 to the latest analysed p.30, the CLDN-1, -3 and -7 gene expressions increased. The CLDN-4 expression increased from p.10 to p.20 but decreased slightly from p.20 to p.30.T120A: Cell line T120A was analysed in p.10, p.20 and p.30. Generally, the CLDN-1, -3, -4 and -7 genes. Regarding the expression profiles from p.02 to p.30, the CLDN gene expressions increased. Regarding the expression profiles from p.20 to p.30 the CLDN-4 gene expression increased, whereas the CLDN-1, -3 and -7 gene expressions decreased moderately.T124: Cell line T124 was analysed in p.02, p.10, p.20 and p.30. Passage 30 displayed comparable expression levels for the CLDN-1, -3, -4 and -7 expression levels of the latest analysed p.30 were at high levels. However, a decrease of expression was observed from p.10 for CLDN-3 and -7 and from p.20 for CLDN-1 and -4.DT14/04T: Cell line DT14/04T was analysed in p.03, p.10, p.20 and p.30. CLDN gene expressions during cultivation revealed highly correlating CLDN gene expression profiles in cell line T120A for the CLDN-1/-3, -1/-4 and -3/-4 genes, in cell line T124 for the CLDN-1/-3 and -4/-7 genes and in cell line DT14/04T for the CLDN-1/-4 and -3/-7 genes. For details see Analyses of the correlation of CLDN-1, -3, -4 and -7 gene expressions were analysed using conventional PCR in cDNA of early and late passages of cell cultures T120A, T121, T124, DT14/04R, DT14/04T, DT14/06T and DT14/10 allowing a qualitative statement. Previous to the conventional PCR, contamination with genomic DNA were excluded for all cDNAs using a primer assay for an intro sequence of C-X-C chemokine receptor type 4 gene.CLDN-1 gene expression was equal in four cell lines  and one primary culture (DT14/04R). In two cell lines , CLDN-1 expression differed from negative in the early to positive in the late passage. CLDN-3 gene expression was equal in three cell lines  and lost in another cell line (DT14/06T). In one primary culture (DT14/04R) CLDN-3 expression differed from negative in the early to positive in the late passage and in two cell lines  the other way round. CLDN-4 gene expression was equal in three cell lines  and lost in three cell lines . In one primary culture (DT14/04R) CLDN-4 expression differed from negative in the early to positive in the late passage. CLDN-7 gene expression was equal in three cell lines  and lost in two cell lines . In one primary culture CLDN-7 gene expression differed from negative in the early to positive in the late passage (DT14/04R) and the other way round in one cell line (T121). For details see Immunocytochemistry detected CLDN-1, -3, -4 and -7 proteins in cell cultures T120A, T124, DT14/04T, DT14/04R, DT14/10, DT14/06T and T121. The late passages of cell cultures DT14/04T and DT14/10 lacked CLDN-4 protein.Immunofluorescence revealed a cell specific expression of the CLDN-1, -3, -4, and -7 proteins. While cell line DT14/06T p.44–49 did not show an expression of the CLDNs, cell lines T120A, T124 and DT14/04T did. The expression of CLDN-1, -3 and -4 proteins was found in cell line T120A p.37–42 at the cell membrane, but there was no signal for CLDN-7. All four analysed CLDN-1, -3, -4 and -7 proteins were found in cell line T124. In cell line T124 p.15 the CLDN-3 and -7 proteins were localized at the cell membranes, CLDN-1 and -4 were punctually localized in the cytoplasm. In cell line DT14/04T p.16–19 CLDN-1 and -7 proteins were strongly distributed in the cell-to cell contact regions, CLDN-4 was weakly expressed in p.5 and CLDN-3 was not detected in p.16–19. For details see Clostridium perfringens (CPE) in order to kill cancer cells by directed CLDN binding. In a xenografical mouse model, the intratumourally injected CPE reduced the size of CLDN expressing tumours initiated by a human breast cancer cell line [Mammary tumours commonly occur in female dogs . Treatmeell line . A recomell line . Human oell line . Furtherell line . Additioell line ,16,22,24CLDN-1, -3, -4 and -7 gene expressions decreased after transfer to culture conditions when compared to the original tissue samples. Furthermore, CLDN expressions remained low during longitudinal cultivation over 30 passages. These results match our previously published study, showing low to missing CLDN expression in long term established canine mammary non-neoplastic and neoplastic tissue derived cell lines ZMTH3, MTH53A and MTH52C [CLDN-3 and -4 gene expression patterns was reported for cultured human ovarian cancer cell lines that show a lower CLDN expression than primary ovarian tumours [In order to develop specific CLDN targeting therapeutic approaches for canine mammary gland tumours, the availability of CLDN expressing cell lines is a precondition. In the present study, comparative CLDN gene and protein expression analyses on eleven cell lines and six primary cultures derived from canine mammary gland non-neoplastic and neoplastic tissues were performed. In the majority of the herein analysed cell lines and primary cultures (fourteen of seventeen) the d MTH52C . Furtherd MTH52C . Human p tumours . The rea tumours . Metasta tumours . Epithel tumours  and it i tumours ,29. HoweCLDN genes increased comparing an early passage and p.30, but did not reach constant expression levels before p.30. Further, these cell lines kept the epithelial character throughout the observed cultivation period. To the best of our knowledge, this is the first study to longitudinally analyse the CLDN expressions up to passage 30. Comparisons to other studies are difficult as information about the analysed passages are missing.However, in three of the herein evaluated new established canine mammary cell lines  expression of the Correlation coefficient analyses of the three CLDN expressing cell lines identified in the present study indicated that culturing conditions do not affect all CLDNs in the same manner. These findings might be associated to the functional ability of different CLDNs to specifically heterophilically interact with each other. CLDN-3 is able to interact heterophilically with CLDN-1 or CLDN-2, whereas the latter are not able to interact with each other . ImmunofIn summary, the data reported here show that cultivation of canine mammary cells has negative effects on the expression of different CLDNs. However, three epithelial canine mammary lobular hyperplasia, adenoma and carcinoma derived cell lines kept the expression of the analysed CLDN genes and proteins during cultivation until p.30. These three canine mammary gland tissue derived CLDN expressing epithelial cell lines represent valuable tools for further studies regarding CLDN targeted tumour therapeutic approaches.Five normal canine mammary gland tissue samples, two lobular hyperplasias, one simple adenoma, one complex adenoma, two simple carcinomas, three complex carcinomas, one carcinoma arising in a benign mixed tumour and two benign mixed mammary tumours were provided by the Small Animal Clinic, University of Veterinary Medicine Hannover, Germany, the Institute of Veterinary Medicine, Georg-August-University Göttingen, Germany, and the veterinary practice of Dr. M. Schilling, Bielefeld, Germany; in total 17 canine mammary gland tissue samples. ® Mini Kit . For details see Samples for diagnostic procedures were stored in formalin. Histological classification was performed in the Department of Pathology of the University of Veterinary Medicine Hannover, Germany, according to the proposed classification of canine mammary tumours . DependiTM, Darmstadt, Germany) containing 20% fetal calf serum  and 200 IU/mL penicillin and 200 mg/mL streptomycin . Afterwards, cells were transferred into sterile 25 cm2 flasks containing 5 mL medium 199 (20%). The cultures were incubated in 5% CO2/air at 37 °C. During the first days, the cells were monitored on a daily basis observing cell growth, depending on respective characteristics. The culture medium was changed at least twice per week. Reaching confluency, the cells were passaged by adding 1 mL TrypLETM Express  and then the cell suspension was split and transferred to sterile cell culture flasks, containing 5 mL medium 199 (20%) per flask.Non-neoplastic and neoplastic tissue samples were stored in Hanks Medium , 2% penicillin and streptomycin  until being processed. A small portion of the initial tissue sample was chopped into small pieces followed by collagenase treatment  at 37 °C until cell connections dissolved, for a maximum of 4 h. The dissociated cells were washed with medium 199  00 (p.00). After first splitting, the cells were termed p.01, higher numbers indicate the number of the certain passage. The term “primary culture” referred to cell cultures between p.00 and p.19. Reaching p.20 and above, the certain “primary culture” was furthermore designated as “cell line”.Primary cultures were analysed in p.00 and additionally in another early passage . In case cell growth discontinued before the tenth passage, the latest viable passage was analysed. In case cells grew beyond p.10 but discontinued growth before p.19, then p.10, and additionally the latest viable passage, were analysed. Cell lines were analysed in p.00, in an early passage (p.01–p.03) and then in p.10, p.20 and p.30.Depending on the cell number of a passage, pellets were generated according to TM Express  to the flasks. The cells were counted using Cellometer SD100 cell counting chambers  and CellometerTM Auto T4 (Nexcelom Bioscience), pelleted at 1000 g for 10 min and stored adequately until further usage.The cells were detached by adding 1 mL TrypLEFor histological analyses, pellets were stored in 4% paraformaldehyde. For gene expression analyses, using either QuantiGene Plex Assay  or conventional PCR, cell pellets were stored at −80 °C.Cells were lysed at a final concentration of 400 cells/µL Lysis Mixture using the QuantiGene Sample Processing Kit (Affymetrix), according to the manufacturer’s instructions. Lysates were stored at −80 °C until usage.® Mini Kit (Qiagen) including a digestion step for genomic DNA using the RNase-Free DNase Set (Qiagen) according to the manufacturer´s instructions. An additional step for digestion of genomic DNA was performed using RQ1-DNAse  according to the manufacturer’s instructions. The total amount of RNA was quantified using Synergy 2  controlled by Gen5™ Reader Control and Data Analysis Software. RNAs were stored at −80 °C until usage.5–30 mg of frozen tissue samples were homogenized using the Tissue Lyser II  and 5 mm Stainless Steel Beads (Qiagen) according to the manufacturer’s instructions (Qiagen). RNA was isolated using the RNeasyTM columns (Qiagen). RNA was isolated using the RNeasy® Mini Kit (Qiagen), RNase-Free DNase Set (Qiagen) and RQ1-DNAse (Promega) as described in Cells were homogenised using QIAshredderCLDN-1, -3, -4, -7, β-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expressions were simultaneously analysed in isolated RNA (12.5 ng/µL) of canine mammary non-neoplastic and neoplastic tissue samples and cell lysates (400 cells/µL) of different passages of thereof derived primary cultures and cell lines using QuantiGene Plex Assay (Affymetrix) according to the manufacturer’s instructions, Vortemp 56  and Luminex 100/200 Systems . Luminex 100/200 Systems detected the amplified fluorescence signals generated by the QuantiGene Plex Assay (Affymetrix). For each run, background controls and process controls were performed as duplicates. As process controls, samples that were to be analysed were chosen. Samples were run as unicates. Normalization was performed towards two reference genes, ACTB [GAPDH [es, ACTB  and GAPDB [GAPDH . AvailabCLDN-1: XM_845155; CLDN-3: NM_001003088; CLDN-4: XM_005620962; CLDN-7: XM_546584; ACTB: XM_536888; GAPDH: NM_001003142.Probes for the QuantiGene Plex Assay (Affymetrix) were ordered based on the mRNA sequences provided by the National Center for Biotechnology Information (NCBI) and custom designed by Affymetrix . The accession numbers for the mRNA sequences were as follows: CLDN expression alterations from original tissue samples to p.00 were analysed in cultures DT14/06R, DT14/06T, DT14/06TS, DT14/07T, DT14/08R and DT14/09T, as RNA from tissue samples and lysates from the first cultivated cells were available.CLDN expression was declared as “high”, if the expression level in the latest analysed passage was higher than cut off 0.1, as “low”, if the expression level was lower than cut off 0.1. Normalization was performed towards the reference genes ACTB and GAPDH, all values refer to the reference gene value 1.x and y are passage series for two different CLDNs of the same cell line. “xm” and “ym” are the mean values of the CLDN gene expression data of a passage series in a certain cell line. For a better visibility of correlations, each value of each CLDN gene expression profile was scaled with a factor. The value of the factor is equal to the mean value of the expression time series such that all scaled expression profiles have the same mean value.The correlation coefficient according to Pearson is an empirical correlation coefficient and can be applied to calculate linear correlations between two time-dependent gene expression profiles, which were obtained from the cultivation of cell lines T120A, T124 and DT14/04T. The correlation coefficient is defined by the following equation:C-X-C chemokine receptor type 4 (cxcr4) which can only be found on genomic DNA, but not on cDNA. Integrity of the RNA and thereby cDNA quality of all samples was tested using a PCR assay for the reference gene ACTB (ACTB up/ACTB lo). cDNA was further analysed if contaminations were excluded in the cDNA and negative controls and if integrity of RNA was verified. Primer sequences have been published previously [cDNA was synthesised using M-MLV-Reverse Transcriptase (Promega), 500 ng of total RNA and AP2-Primer according to the manufacturer´s instructions. Two negative process controls were included. To check reagents for contamination, a negative control was performed once for each run that included the reverse transcriptase but no RNA, so that no cDNA was synthesisable from sample RNA. To check the RNA for contamination, an additional negative control was performed for each sample and included RNA but no reverse transcriptase, so that cDNA could not be synthesised from the RNA. Genomic DNA contamination was excluded in cDNA and negative controls using a PCR assay (CXCR4 upVIIIa/CXCR4 lo VIII) for an intron sequence of the reference gene eviously .CLDN-1, -3, -4 and -7 gene expressions were analysed in 1 µL cDNA of an early and late passage of cell cultures T120A, T121, T124, DT14/04R, DT14/04T, DT14/06T and DT14/10, using PCR assays  and real-time PCR primer assays  for conventional PCR, as published previously [eviously . The staTM Express Enzyme  followed by mechanical scraping  if enzymatic detachment was incomplete. Detached cells were centrifuged at 1000× g for 10 min, fixed in 4% paraformaldehyde and embedded in paraffin wax. Pellets of all primary cultures and cell lines were immunocytochemically stained with a panel of antibodies detecting various cytokeratins (CK), for details see 2O2 in 70% ethanol for 30 min, and blocking of non-specific binding with inactivated goat serum . Primary antibodies were applied for 1 h at ambient temperature (for anti-CLDN antibodies) or overnight at 4 °C (for anti-CK antibodies) followed by incubation with biotinylated goat anti-mouse or goat anti-rabbit secondary antibodies , respectively. Subsequently, avidin-biotin-peroxidase reagent (Vector Laboratories) was used according to the manufacturer’s instructions for 30 min and for anti-CLDN antibodies amplification was achieved using biotinylated tyramine as described elsewhere [2O2, 3,3′-diaminobenzidine  was applied to induce a brown colour reaction, and sections were counterstained with Mayer’s haematoxylin.For immunocytochemical characterisation of the cytokeratin distribution in the cell cultures, cell pellets from early and late passages of the cultured cells were detached with TrypLElsewhere . SupplemImmunofluorescence was performed for cell cultures T120A, p.37–42; T124, p.15; DT14/04T, p.4 and p.16–19; and DT14/06T, p.44–49. The cells were cultivated on collagen coated cover slips. After achieving a confluency of about 75%, the cells were fixed with 4% formaldehyde for 10 min at room temperature and permeabilized with 0.3% Triton X-100 in PBS for 15 min at 37 °C. BSA  in PBS was used for 30 min at 37 °C to block and saturate the non-specific binding positions. Then the cells were stained over night at 4 °C for CLDN-1, -3, -4 and -7 proteins with the respective primary antibodies according to


ID3 increases the melanoma sensitivity to vemurafenib short-term treatment. In addition, we observe an ID3-mediated regulation of cell migration and of the expression of resistance-associated genes such as SOX10 and MITF. In sum, these data suggest ID3 as a new key actor of melanoma adaptive resistance to vemurafenib and as a potential drug target.Adaptive resistance to targeted therapy such as BRAF inhibitors represents in melanoma a major drawback to this otherwise powerful treatment. Some of the underlying molecular mechanisms have recently been described: hyperactivation of the BRAF-MAPK pathway, of the AKT pathway, of the TGFβ/EGFR/PDGFRB pathway, or the low MITF/AXL ratio. Nevertheless, the phenomenon of early resistance is still not clearly understood. In this report, we show that knockdown of neural crest-associated gene  BRAF mutations in melanoma patients (50-60%), numerous small molecule inhibitors have been tested on melanoma [V600BRAF mutations [BRAF-mutated metastatic melanoma [BRAF mutations do not respond to vemurafenib treatment at all, probably due to intrinsic resistance mechanisms such as amplification of tumor promoter genes or loss of tumor suppressor genes [Most skin cancer related deaths are attributed to melanoma. It is responsible for more than 75% of death while it represents 4% of all skin cancers only . The genmelanoma . BRAF inients 50-0%, numermelanoma . The 3-yor genes .Major investigations are currently ongoing to understand the resistance mechanisms acquired during BRAF inhibitor monotherapy and BRAF/ MEK inhibitor combination therapy. Several models such as primary human melanoma xenograft models or vemurafenib resistant melanoma cell lines have been established and extensively studied . While aID3) is part of the ID gene family of helix-loop-helix (HLH) transcription factors which are considered as negative regulators of transcription [ID3 is involved in cell cycle progression and survival of neural crest progenitors [ID3 as a new molecule involved in melanoma adaptive resistance to vemurafenib and in the regulation of melanoma migration.Inhibitor of differentiation protein 3 (cription . ID3 is genitors . This gegenitors . In thisBRAF-mutated melanoma tumors derived from 21 patients either at the beginning of their treatment with BRAF inhibitors or at the time of disease progression. The resistance mechanisms developed by the tumors in the course of the treatment were investigated and most were involved in MAPK pathway activation [ID3 significantly upregulated in the resistant tumors compared to before treatment (p = 0.0077) and this upregulation (fold change > 2) was observed in 38% of the patients , DMSO-treated cell lines, differentiated cells such as normal human melanocytes (NHM) and less differentiated cells such as pluripotent stem cell-derived neural crest cells (D1NC) generated in our laboratory [ID3 upregulation, we also observed in this analysis an upregulation (log2-fold change ranging from 1 to 2) of pluripotency markers  and of AXL in the vemurafenib-treated cell lines compared to DMSO-treated control cells. Conversely, we observed a downregulation (log2-fold change ranging from -1.4 to -3.8) of differentiation markers   Figure . Moreove) Figure . Thus, tID3 in the response of melanoma cells to drug treatment, we treated several melanoma cell lines with vemurafenib or in combination with trametinib. The design of this experiment was the following: four BRAF-mutated melanoma cell lines  and one BRAF-WT cell line (SKmel23) were treated with increasing doses of vemurafenib for 96h  and trametinib (3 μM) for 96 h). We first confirmed by qPCR that not only the expression of t Figure .ID3 knockdown cell lines  and one ID3 overexpressing cell line (WM266-4). We verified by western blot or qPCR either silencing or overexpression of ID3 in all genetically modified cell lines. Indeed, ID3 expression was greatly impaired in A375 and HT144 cell lines and it was silenced more than 50% in SKmel28   became significantly more resistant to the drug treatment than the control cell line (WM266-4 EV)  Figure . Convers) Figure . Of note266-4 EV .ID3 expression and that, changes in ID3 expression level can regulate early resistance of melanoma cells, suggesting a role for ID3 in adaptive resistance to these drugs.Together, these data show that vemurafenib treatment or combination treatment with trametinib upregulate ID3 function in melanoma progression, we further characterized ID3 knockdown and ID3 overexpressing cell lines with respect to cell proliferation, cell migration and cell cycle states. Indeed, by using a scratch-like assay, we observed a delay in the migration rate of all three ID3 knockdown cell lines  compared to the control cell lines, ranging from 10% to 25% reduction. Conversely, the ID3 overexpressing cell line (WM266-4 ID3) migrated significantly faster than the control line (WM266-4 EV), enhancing from 40% to almost 60% after 4h  compared to the control lines   . Among tSOX10 and MITF. We observed a 2 to 4-fold upregulation in ID3 knockdown cell lines compared to their control lines   .In sum, these results show that ID3 controls melanoma cell migration and regulates mesenchymal markers, without affecting cell proliferation or cell cycle states. They also show that ID3 expression inversely correlates with that of SOX10 and MITF in melanoma cell lines and in melanoma patient samples.ID3 expression  in response to vemurafenib (or to the combination vemurafenib + trametinib) in vitro and in patients’ tumors. We also showed that modulation of ID3 expression regulates the sensitivity of the cells to vemurafenib. Interestingly, ID3 expression has been involved in non-small cell lung cancer resistance to chemotherapy [ID3 expression is also higher in a model of doxorubicin-resistant melanoma cell line compared to its control cell line [In this report, we show that melanoma cells upregulate otherapy . ID3 expell line .SOX10 was described to activate the TGFβ-EGFR-PDGFRB signaling and to lead to BRAF inhibitor resistance [Although the mechanisms by which vemurafenib regulates ID3 are not yet known, suppression of sistance . Our gensistance , 29. Morsistance . Althougsistance , 32.PTEN and p53 in addition to BRAF. Mutations in these important tumor suppressor genes seem not to influence the sensitivity to vemurafenib in comparison to A375 or WM266-4  or in melanoma cell lines treated or not with vemurafenib (flow cytometry) could be performed.Finally, two hypotheses can be formulated to explain ID3 sensitizes melanoma cells to vemurafenib treatment, suggesting that ID3 or ID3 downstream targets’ pharmacological inhibition may help improving current treatment targeting resistant melanoma.Ultimately, we found that the absence of Human melanoma cell lines  were cultured in DMEM  with 10% FBS (Biochrom), 0.1mM β-mercapthoethanol , 1% non-essential amino acids (NEAA) and 1% Penicilin/Streptomycin . Normal human melanocytes (NHM) were isolated from donor foreskins according to the ethical regulation  and were cultivated in medium 254  supplemented with 100x human melanocyte growth supplement (HMGS) . Human neural crest cells were derived from hiPSC following a previously published protocol [ID3 shRNA or a non-targeting shRNA (Sigma-Aldrich). WM266-4 cell line was transduced with either an empty lentiviral vector (pLX304) or the vector encoding for human ID3 (Addgene).A375, HT144 and SKmel28 cell lines were transduced with a lentiviral expression vector (pLKO.1) encoding for a human ID3 knockdown/ NT cell lines, and purified with RNeasy kit (Qiagen). Labeled RNA was hybridized to whole-genome BeadChip Sentrix arrays HumanHT-12 v4 from ILLUMINA  following the manufacturer's indications. Microarray scanning was carried out using an iScan array scanner.Total RNA from three independent experiments was isolated from primary melanocytes, human neural crest cells, melanoma cell lines A375, SKmel28 and WM266-4 (+/- treated with vemurafenib) and A375 P-values were inferior to 0.05, log2-expression values of the differentially expressed genes were represented.As test for significance, a Bayes test was used on the bead expression values of the two groups of interest. The average expression value is calculated as mean of the measured expressions of beads together with the standard deviation of the beads. After selecting the genes, which ID3 overexpressing cells (compared to control vector) were then uploaded to IPA software  to evaluate the most regulated signaling pathways.Regulated genes in vemurafenib-treated cells (compared to DMSO) or in ® solution. The mixture was incubated for 30 min at room temperature and then added to HEK293T producer cells. The collected supernatant was concentrated by ultracentrifugation and the virus was used to infect the cells. The virus production was done in a Biosafety level II laboratory, according the safety instruction. After 48h of transduction the cells were washed with PBS and normal fresh medium was added to the cells.Lentiviral particles were produced in HEK293T cells. HEK293T cells were approximately 60% confluent on the day of transfection. The plasmid with the gene of interest along with packaging plasmids VSV-G and Δ8.9 was mixed in the DMEM and X-treme GENEID3 shRNA were selected by using puromycin (0.5-0.8 μg/ml) and cell lines with ID3 overexpressing vector were selected using blasticidin (5-8 μg/ml) for 4-6 days.Cell lines with Total RNA isolation from melanoma cell lines , neural crest cells (D1NC) and normal human melanocytes (NHM) was done using RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. The RNA was treated with DNase I on the column. RNA concentration and quality were measured by NanoDrop ND1000 spectrophotometer. cDNA was synthesized using the Revert Aid First Strand cDNA synthesis kit (Thermo scientific) according to the manufacturer's protocol.ID3-engineered melanoma cell lines . In all experiments, rRNA 18s was used as the housekeeping gene and the values were normalized to it. Relative gene expressions were quantified by calculating (ΔΔCt). Primers used are as follow: 18S F: GAGGATGAGGTGGAACGTGT, 18S R: TCTTCAGTCGCTCCAGGTCT, ID3 F: GGA GCTTTTGCCACTGACTC, ID3 R: TTCAGGCC ACAAGTTCACAG, SOX10 F: GGCTTTCTGTCTGG CTCACT, SOX10 R: TAGAGGGTCATTCCTGGGGG, MITF F: GCTCACAGCGTGTATTTTTCC, MITF R: TC TCTTTGGCCAGTGCTCTT, FN1 F: GGTGAC ACTTATGAGCGTCCTAAA, FN1 R: AACATGTA ACCACCAGTCTCATGTG, SLUG F: TGGTCAA GAAACATTTCAACGCC, SLUG R: GGTGAGGATC TCTGGTTTTGGTA, SNAIL F: GAGGCGGTGGC AGACTAG, SNAIL R: GACACATCGGTCAGACCAG.Quantitative real-time PCR (qPCR) was performed using SYBR Green  on a 7500 real-time PCR system . RNA samples were isolated from ID3-engineered melanoma cell lines  by using 1% Triton-X-100 lysis buffer containing a protease inhibitor cocktail . The protein concentration was determined using the Pierce BCA protein assay kit (Thermo scientific). Proteins were resolved on SDS-PAGE and transferred to PVDF membranes (Merck Millipore). Later the membrane was probed with primary antibodies and then with HRP conjugated secondary antibody. The bands were visualized using Luminata Forte western HRP substrate (Merck Millipore) according to manufacturer's protocol. The band intensities were quantified using ImageJ software (Fiji). The primary antibodies used are as follow: ID3 , SOX10 (Abcam), MITF (Abcam), GAPDH (CST).Protein samples were extracted from human melanoma cell lines  and from ID3-ingeneered melanoma cell lines  via two methods. Culture silicone inserts from Ibidi (scratch-like assay), 70 000 cells were seeded for 24 h and were serum-starved overnight. The inserts were removed and cell migration was observed every 4 hours up to 28 hours. TScratch software was used for the quantitative analysis of the data. Alternatively, Boyden chamber system was used according to manufacturer's protocol (Trevigen).Cell migration was studied on all ID3-engineered melanoma cell lines  using Alamar blue (Invitrogen) for up to 6 days. 2500 or 5000 cells were seeded in triplicates in a 96-well plate. After 24 hours Alamar blue (10% of the medium) was added and the plates were incubated for 4 hours at 37°C. Fluorescence was measured with excitation wavelength at 530-560 nm and emission wavelength at 590 nm using the Tecan Infinite 200 Pro plate reader. For cell cycle analysis, A375, SKmel28, and WM266-4 ID3-engineered cells were seeded (4–6 × 105) in 6-well plates in triplicates and incubated at 37°C for 18-24 hours. After cell collection and centrifugation, cell pellets were resuspended in ice-cold PBS and fixed with pre-cooled 70% ethanol. Cells were then washed with ice-cold PBS and treated with RNase for 30 min at 37°C. Propidium iodide (50 μg/ml) was used to stain the cellular DNA. Cell cycle stages were analysed by flow cytometry .Cell proliferation was measured in all ID3-engineered cell lines  were seeded in 96-well plates (2500 cells per well). After 16-18 hours, increasing concentrations (0.001-10 μM) of Vemurafenib (PLX4032) or DMSO were added to the cells. Combination treatement was performed with Vemurafenib and Trametinib (GSK1120212) at ratio 1:1. Cell viability was measured every 24 hours up to 4 days using Alamar blue as described above.Human melanoma cell lines  and


Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression , but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117. Nucleoside analogs are synthetic, chemically modified nucleosides that due to their resemblance can be incorporated into RNA and DNA to inhibit their synthesis and subsequently inhibit cell division . This haA cytidine analog, fluorocyclopentenylcytosine (RX-3117) , has shode novo DNA methylation patterns in DNA, which is important during embryogenesis -RX-3117 and [2-14C]-uridine as described earlier -RX-3117 (90.5 mCi/mmol) for 120 minutes as described earlier -uridine (r = 0.828 for the cell lines and r = 0.878 for the xenograft cells) and [3H]-RX-3117 (r = 0.803 for the cell lines and r = 0.915 for the xenograft cells), while this was not the case for UCK1   and in a panel derived from seven xenograft cells . The UCK activity data of the 6 cell lines were previously obtained [t cells) .We also investigated the role of UCK2 in sensitivity to RX-3117 in the above mentioned cell lines. We observed a non-linear correlation of UCK2 mRNA expression and sensitivity to RX-3117 . Cells wIn these cell lines we also investigated the formation of activated RX-3117 nucleotides . The forNext to the mRNA expression we also measured protein expression with two newly developed antibodies . InteresEarlier we showed that an excess of the UCK2 substrates uridine and cytidine would protect cells from cytotoxicity of RX-3117 by preventing that RX-3117 is activated by UCK2. As a read-out for a mechanistic effect we analyzed the cell cycle distribution of A549 and SW1573 cells after exposure to 33.3 μM RX-3117 for 24 hrs or RX-3117 with 100 μM uridine or 100 μM cytidine. PI stained cells showed an increase in the S-phase when treated with RX-3117  in A549 This study demonstrates that the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) is activated by UCK2 to its monophosphate. UCK1 does not play a role in this process. Moreover, UCK2 expression is high in a number of target tumor tissues.UCKs catalyze the phosphorylation of uridine and cytidine and their respective synthetic analogs . UCK actin vivo activity against various tumor types, including gemcitabine resistant tumors [Our findings provide novel perspectives for RX-3117, since it can circumvent resistance to other widely used drugs such as gemcitabine, commonly used for treatment of several solid tumors including lung  and panct tumors , 6. AddiIn addition to the pathway by which RX-3117 is metabolized, the mechanism of action by which it exerts its cytotoxic activity is equally important. Earlier it was shown that RX-3117 downregulates DNMT1 .To summarize, this study showed that UCK2, but not UCK1, is solely responsible for the phosphorylation of RX-3117 into its monophosphate metabolite. This distinctive metabolism by tumor cells compared to e.g. gemcitabine offers a potential for clinical application due to its lack of cross resistance.