Scientific Publications

Ruthenium Counterstaining for Imaging Mass Cytometry
Journal of Pathology 2018-02

Imaging mass cytometry is a novel imaging modality that enables simultaneous antibody-based detection of more than 40 epitopes and molecules in tissue sections at subcellular resolution using isotopically pure metal tags. Essential for any imaging approach where antigen detection is performed is the so-called counterstaining that reveals the overall structure of the tissue. Counterstaining is necessary since antigens of interest are often present only in a small subset of cells while the rest of tissue structures are not visible. Since most biological tissues are nearly transparent or non-fluorescent, chromogenic reagents such as haematoxylin (for immunohistochemistry) or fluorescent dyes such as DAPI (that stains nuclei for epifluorescence and confocal microscopy) are utilized. Here, we describe a metal-based counterstain for imaging mass cytometry based on simple oxidation and subsequent covalent binding of the tissue components to ruthenium tetroxide (RuO4 ). RuO4 counterstaining reveals general tissue structure both in areas with high cell content and in stromal areas with low cellularity and fibrous or hyaline material in a manner analogous to haematoxylin in immunohistochemical counterstaining or eosin or other anionic dyes in conventional histology. Our new counterstain approach is applicable to any metal-based imaging technique and will facilitate the adaptation of imaging mass cytometry for routine applications in clinical and research laboratories.

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histoCAT: analysis of cell phenotypes and interactions in multiplex image cytometry data.
Nature Methods 2017-09

Single-cell, spatially resolved omics analysis of tissues is poised to transform biomedical research and clinical practice. We have developed an open-source, computational histology topography cytometry analysis toolbox (histoCAT) to enable interactive, quantitative, and comprehensive exploration of individual cell phenotypes, cell-cell interactions, microenvironments, and morphological structures within intact tissues. We highlight the unique abilities of histoCAT through analysis of highly multiplexed mass cytometry images of human breast cancer tissues.

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AirLab: a cloud-based platform to manage and share antibody-based single-cell research
Genome Biology 2016-06

Single-cell analysis technologies are essential tools in research and clinical diagnostics. These methods include flow cytometry, mass cytometry, and other microfluidics-based technologies. Most laboratories that employ these methods maintain large repositories of antibodies. These ever-growing collections of antibodies, their multiple conjugates, and the large amounts of data generated in assays using specific antibodies and conditions makes a dedicated software solution necessary. We have developed AirLab, a cloud-based tool with web and mobile interfaces, for the organization of these data. AirLab streamlines the processes of antibody purchase, organization, and storage, antibody panel creation, results logging, and antibody validation data sharing and distribution. Furthermore, AirLab enables inventory of other laboratory stocks, such as primers or clinical samples, through user-controlled customization. Thus, AirLab is a mobile-powered and flexible tool that harnesses the capabilities of mobile tools and cloud-based technology to facilitate inventory and sharing of antibody and sample collections and associated validation data.

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Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes
Development 2016-04

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro-assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene-regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo

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Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.
Cytometry A 2016-03

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows.

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Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1
PNAS 2015-12
Catena R

Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease?Tsp-1 axis as a potential antimetastatic therapeutic target.

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WormGUIDES: an interactive single cell developmental atlas and tool for collaborative multidimensional data exploration.
BMC Bioinformatics 2015-06

BACKGROUND:
Imaging and image analysis advances are yielding increasingly complete and complicated records of cellular events in tissues and whole embryos. The ability to follow hundreds to thousands of cells at the individual level demands a spatio-temporal data infrastructure: tools to assemble and collate knowledge about development spatially in a manner analogous to geographic information systems (GIS). Just as GIS indexes items or events based on their spatio-temporal or 4D location on the Earth these tools would organize knowledge based on location within the tissues or embryos. Developmental processes are highly context-specific, but the complexity of the 4D environment in which they unfold is a barrier to assembling an understanding of any particular process from diverse sources of information. In the same way that GIS aids the understanding and use of geo-located large data sets, software can, with a proper frame of reference, allow large biological data sets to be understood spatially. Intuitive tools are needed to navigate the spatial structure of complex tissue, collate large data sets and existing knowledge with this spatial structure and help users derive hypotheses about developmental mechanisms.
RESULTS:
Toward this goal we have developed WormGUIDES, a mobile application that presents a 4D developmental atlas for Caenorhabditis elegans. The WormGUIDES mobile app enables users to navigate a 3D model depicting the nuclear positions of all cells in the developing embryo. The identity of each cell can be queried with a tap, and community databases searched for available information about that cell. Information about ancestry, fate and gene expression can be used to label cells and craft customized visualizations that highlight cells as potential players in an event of interest. Scenes are easily saved, shared and published to other WormGUIDES users. The mobile app is available for Android and iOS platforms.
CONCLUSION:
WormGUIDES provides an important tool for examining developmental processes and developing mechanistic hypotheses about their control. Critically, it provides the typical end user with an intuitive interface for developing and sharing custom visualizations of developmental processes. Equally important, because users can select cells based on their position and search for information about them, the app also serves as a spatially organized index into the large body of knowledge available to the C. elegans community online. Moreover, the app can be used to create and publish the result of exploration: interactive content that brings other researchers and students directly to the spatio-temporal point of insight. Ultimately the app will incorporate a detailed time lapse record of cell shape, beginning with neurons. This will add the key ability to navigate and understand the developmental events that result in the coordinated and precise emergence of anatomy, particularly the wiring of the nervous system.

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Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1.
Cancer Discov 2013-05

Metastatic tumors have been shown to establish permissive microenvironments formetastases via recruitment of bone marrow-derived cells. Here, we show thatmetastasis-incompetent tumors are also capable of generating suchmicroenvironments. However, in these situations, the otherwise prometastaticGr1(+) myeloid cells create a metastasis-refractory microenvironment via theinduction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. Bonemarrow-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis,which was restored by bone marrow transplant from Tsp-1(+) donors. We alsodeveloped a 5-amino acid peptide from prosaposin as a pharmacologic inducer ofTsp-1 in Gr1(+) bone marrow cells, which dramatically suppressed metastasis.These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1(+) myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1(+) cells, which,depending on the context, promote or inhibit metastasis, and suggest that thepeptide could be a potential therapeutic agent against metastaticcancer.SIGNIFICANCE: The mechanisms of metastasis suppression are poorlyunderstood. Here, we have identified a novel mechanism wherebymetastasis-incompetent tumors generate metastasis-suppressive microenvironmentsin distant organs by inducing Tsp-1 expression in the bone marrow?derivedGr1+myeloid cells. A 5-amino acid peptide with Tsp-1?inducing activity wasidentified as a therapeutic agent against metastatic cancer.

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Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration.
Cancer Cell 2013-01

The progression of cancer to metastatic disease is a major cause of death. Weidentified miR-708 being transcriptionally repressed by polycomb repressorcomplex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708targets the endoplasmic reticulum protein neuronatin to decrease intracellularcalcium level, resulting in reduction of activation of ERK and FAK, decreasedcell migration, and impaired metastases. Ectopic expression of neuronatinrefractory to suppression by miR-708 rescued cell migration and metastasisdefects. In patients with breast cancer, miR-708 expression was decreased inlymph node and distal metastases, suggesting a metastasis-suppressive role. Ourfindings uncover a mechanistic role for miR-708 in metastasis and provide arationale for developing miR-708 as a therapeutic agent against metastatic breastcancer.

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Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition.
Cancer Res 2012-03

Tumors systemically initiate metastatic niches in distant target metastaticorgans. These niches, composed of bone marrow-derived hematopoietic cells,provide permissive conditions for future metastases. However, the mechanisms bywhich these cells mediate outgrowth of metastatic tumor cells are not completely known. Using mouse models of spontaneous breast cancer, we show enhancedrecruitment of bone marrow-derived CD11b(+)Gr1(+) myeloid progenitor cells in thepremetastatic lungs. Gene expression profiling revealed that the myeloid cellsfrom metastatic lungs express versican, an extracellular matrix proteoglycan.Notably, versican in metastatic lungs was mainly contributed by theCD11b(+)Ly6C(high) monocytic fraction of the myeloid cells and not the tumorcells or other stromal cells. Versican knockdown in the bone marrow significantlyimpaired lung metastases in vivo, without impacting their recruitment to thelungs or altering the immune microenvironment. Versican stimulated mesenchymal toepithelial transition of metastatic tumor cells by attenuating phospho-Smad2levels, which resulted in elevated cell proliferation and accelerated metastases.Analysis of clinical specimens showed elevated versican expression within themetastatic lung of patients with breast cancer. Together, our findings suggestthat selectively targeting tumor-elicited myeloid cells or versican represents a potential therapeutic strategy for combating metastatic disease.

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Overexpression of TMPRSS4 in non-small cell lung cancer is associated with poor prognosis in patients with squamous histology.
Br J Cancer 2011-11

BACKGROUND: Mortality rates in lung cancer patients have not decreasedsignificantly in recent years, even with the implementation of new therapeuticregimens. One of the main problems is that a large proportion of patients presentlocal or distant metastasis at the time of diagnosis. The need for identificationof novel biomarkers and therapeutic targets for a more effective management oflung cancer led us to investigate TMPRSS4, a protease reported to promote tumour growth and metastasis.
MATERIAL AND METHODS: In all, 34 lung cancer cell lines were used to evaluate theTMPRSS4 expression. Cell migration and clonogenic assays, and an in-vivo lungmetastasis model were used for functional analysis of the TMPRSS4 downregulation in H358, H441 and H2170 cell lines. The TMPRSS4 expression analysis in normal andmalignant lung tissue samples was performed by qPCR. Five differentmicroarray-based publicly available expression databases were used to validateour results and to study prognosis.
RESULTS: The TMPRSS4 knock down in H358, H441 and H2170 cells resulted in asignificant reduction in proliferation, clonogenic capacity and invasion. Asignificant (P<0.05) decrease in the lung colonisation and growth was found when mice were injected with TMPRSS4-depleated H358-derived clones, as compared withcontrols. Expression of TMPRSS4 showed a >30-fold increase (P<0.001) in tumoursin comparison with non-malignant samples. Levels in tumours with squamous cellcarcinoma (SCC) histology were found to be significantly higher (P<0.001) thanthose with adenocarcinoma (AC) histology, which was confirmed in data retrievedfrom the microarrays. Kaplan-Meier curves demonstrated that high levels ofTMPRSS4 were significantly associated (P=0.017) with reduced overall survival in the patients with SCC histology, whereas no correlation was found for the AC histology. CONCLUSION: Our results demonstrate that TMPRSS4 has a role in the lung cancerdevelopment. The potential use of TMPRSS4 as a biomarker for lung cancerdetection or as a predictor of patient's outcome warrants further investigation. Pubmed link


Optimization of 100 ?m alginate-poly-L-lysine-alginate capsules for intravitreous administration.
J Control Release 2012-03

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively itsapplicability. Producing reduced size microcapsules involves broadening thepossibilities to employ this technology in the treatment of many disorders.Nervous system diseases (NSD) represent a clear example of that. This workdescribes the feasibility of reducing the size of alginate-poly-L-lysine-alginate(APA) microcapsules up to 100 ?m in a highly monodisperse way using the novelFlow Focusing technique. C(2)C(12) myoblasts genetically engineered to expressthe triple reporter gene thymidine kinase-green fluorescent protein-luciferase(TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might beaffected due to the physicochemical changes arising from such dramatic sizereduction. Not only were negative effects at this level not noticed in terms ofcell viability, cell proliferation and KDR secretion, but once again thesuitability of APA microcapsules was also reinforced against other microcapsuledesigns. Furthermore, the fully viable and functional biosystems weresuccessfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks.

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Selenoprotein-P is down-regulated in prostate cancer, which results in lack of protection against oxidative damage.
Prostate 2011-06

BACKGROUND: Oxidative stress plays a role in prostate cancer (PrCa) initiationand development. Selenoprotein-P (SepP; a protein involved in antioxidantdefence) mRNA levels are down-regulated in PrCa. The main goal of our study wasto assess whether SepP protects prostate cells from reactive oxygen species (ROS)in prostate carcinogenesis.
METHODS: Modification of SepP levels and ROS conditions in C3(1)/Tag-derived celllines representing prostate epithelial neoplasia (PIN) lesions (Pr-111, with highSepP expression); and invasive tumors (Pr-14, with very low SepP expression).
RESULTS: Both Pr-111 and Pr-14 cells express ApoER2 (SepP receptor), whichsuggests that they may uptake SepP. Pr-14 cells had much higher ROS levels thanPr-111 cells and were highly sensitive to H(2)O(2)-mediated cytotoxicity. WhenSepP mRNA levels were knocked down with siRNAs in Pr-111 cells, a significantincrease in ROS and cell growth inhibition upon H(2)O(2) exposure was found.Subsequent administration of purified SepP in the culture medium of these cellswas able to rescue the original phenotype. Similarly, administration of SepP toPr-14 cells was able to reduce ROS concentrations. Administration of flutamidedecreased SepP mRNA levels whereas dihydrotestosterone or synthetic androgensinduced SepP expression, indicating the importance of androgens for SepPexpression. Immunohistochemical analysis using a PrCa tissue microarray furtherrevealed that SepP protein was reduced in 60.8% prostate tumors compared tobenign prostates.
CONCLUSIONS: Levels of SepP in prostate cells determine basal ROS levels andsensitivity to H(2)O(2)-induced cytotoxicity. Deregulation of SepP duringprostate carcinogenesis may increase free radicals, thus promoting tumordevelopment and de-differentiation.

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VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A.
Mol Cancer 2010-12

BACKGROUND: Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189)have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms,designated as VEGF(xxx)b, generated through alternative splicing, have beendescribed. Previous studies have suggested that these isoforms may inhibitangiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpressthese isoforms and assess their angiogenic potential.
RESULTS: Recombinant VEGF121/165b proteins generated either in yeasts ormammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells withVEGF121/165b increased cell proliferation compared to untreated cells, althoughsuch stimulation was lower than that induced by VEGF165. Moreover, in vivoangiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1empty vector, as control) and xenotransplanted into nude mice showed increasedtumor volume and angiogenesis compared to controls. To assess whether theVEGF(xxx)b isoforms are differentially expressed in tumors compared to healthytissues, immunohistochemical analysis was conducted on a breast cancer tissuemicroarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-Aprotein expression in infiltrating ductal carcinomas compared to normal breastswas observed. A positive significant correlation (r = 0.404, p = 0.033) betweenVEGF(xxx)b and total VEGF-A was found. CONCLUSIONS: Our results demonstrate that VEGF121/165b are not anti-angiogenic,but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms areup-regulated in breast cancer in comparison with non malignant breast tissues.These results are to be taken into account when considering a possible use ofVEGF121/165b-based therapies in patients. Pubmed link


PDGFR signaling blockade in marrow stroma impairs lung cancer bone metastasis.
Cancer Res 2011-01

Bone microenvironment and cell-cell interactions are crucial for the initiationand development of metastasis. By means of a pharmacologic approach, using themultitargeted tyrosine kinase inhibitor sunitinib, we tested the relevance of theplatelet-derived growth factor receptor (PDGFR) axis in the bone marrow (BM)stromal compartment for the initiation and development of lung cancer metastasis to bone. PDGFR? was found to be the main tyrosine kinase target of sunitinibexpressed in BM stromal ST-2 and MC3T3-E1 preosteoblastic cells. In contrast, no expression of sunitinib-targeted receptors was found in A549M1 and low levels in H460M5 lung cancer metastatic cells. Incubation of ST-2 and human BM endothelial cells with sunitinib led to potent cell growth inhibition and induction ofapoptosis in a dose-dependent manner. Similarly, sunitinib induced a robustproapoptotic effect in vivo on BM stromal PDGFR?(+) cells and produced extensive disruption of tissue architecture and vessel leakage in the BM cavity.Pretreatment of ST-2 cells with sunitinib also hindered heterotypic adhesion tolung cancer cell lines. These effects were correlated with changes in cell-celland cell-matrix molecules in both stromal and tumor cells. Pretreatment of micewith sunitinib before intracardiac inoculation of A549M1 or H460M5 cells causedmarked inhibition of tumor cells homing to bone, whereas no effect was found whentumor cells were pretreated before inoculation. Treatment with sunitinibdramatically increased overall survival and prevented tumor colonization but not bone lesions, whereas combination with zoledronic acid resulted in markedreduction of osteolytic lesions and osseous tumor burden. Thus, disruption of thePDGFR axis in the BM stroma alters heterotypic tumor-stromal and tumor-matrixinteractions, thereby preventing efficient engagement required for bone homingand osseous colonization. These results support the notion that concomitanttargeting of the tumor and stromal compartment is a more effective approach forblocking bone metastasis.

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Using the transcription factor inhibitor of DNA binding 1 to selectively target endothelial progenitor cells offers novel strategies to inhibit tumor angiogenesis and growth.
Cancer Res 2010-09

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute toangiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth;however, the lack of a marker that can track EPCs from the BM to tumorneovasculature has impeded progress in understanding the molecular mechanismsunderlying EPC biology. Here, we report the use of transgenic mouse andlentiviral models to monitor the BM-derived compartment of the tumor stroma; thisapproach exploits the selectivity of the transcription factor inhibitor of DNAbinding 1 (Id1) for EPCs to track EPCs in the BM, blood, and tumor stroma, aswell as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directeddelivery of a suicide gene reduced circulating EPCs and yielded significantdefects in angiogenesis-mediated tumor growth. Additionally, use of the Id1proximal promoter to express microRNA-30-based short hairpin RNA inhibited theexpression of critical EPC-intrinsic factors, confirming that signaling throughvascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs, ourresults establish a strategy to track and target EPCs in vivo, clarifying thesignificant role that EPCs play in BM-mediated tumor angiogenesis.

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Use of a combination of biomarkers in serum and urine to improve detection of prostate cancer.
World J Urol 2010-12

OBJECTIVE: To measure a combination of novel molecular biomarkers in urine/blood samples of consecutive patients referring lower urinary tract symptoms (LUTS) notpreviously diagnosed, to improve prostate cancer diagnosis.
METHODS: Serum and urine samples from 113 men who went consecutively to theDepartment of Urology of our Institution. Biomarkers analyzed were AMACR andMMP-2 levels, and GSTP1/RASSF1A methylation status, in addition to PSA levels.Sensitivity, specificity, area under the ROC (AUROC) curves, and discriminantfunction analysis were assessed to determine the diagnostic potential of eachvariable alone or in combination.
RESULTS: Of the patients, 30.08% had PCa and the remaining ones were tumor free. Areas under the ROC (AUROC) curves were as follows: 0.476 for PSA, 0.532 forAMACR, and 0.706 for MMP-2. Sensitivity and specificity for methylation statuswere 53.3 and 45.9%, respectively. The combination of these biomarkers resultedin an AUROC curve of 0.788, which significantly outperformed AUROC curves for PSA(P = 0.0033) and AMACR (P = 0.0375). Sensitivity, specificity, positive andnegative predictive values for the combination of biomarkers were 57.1, 96.6,88.9, and 82.4%, respectively.
CONCLUSION: We conclude that analysis of this biomarker combination in bodyfluids improves very significantly the diagnosis of PCa compared to the PSA test.

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Improvement of the monitoring and biosafety of encapsulated cells using the SFGNESTGL triple reporter system.
J Control Release 2010-08

Cell microencapsulation may represent a breakthrough to overcome problemsassociated with cell therapy. Advances in material biocompatibility andproduction protocols have put this field close to its clinical application.However, issues such as the possibility of tracking cell-containingmicrocapsules, monitoring cell viability, and discontinuation of the therapeutic activity when necessary, still remain unsolved. We demonstrate here simultaneous monitoring and pharmacological control of myoblasts-containing alginatemicrocapsules, injected in immunocompetent mice after transduction with theSFG(NES)TGL triple reporter retroviral vector, which contains green fluorescence protein (GFP), firefly luciferase and herpes simplex virus type 1thymidine-kinase (HSV1-TK). Naked (as controls) or microencapsulated cells weresubcutaneously injected in C57BL/6J mice and followed up by luminometry. Signalfor naked cells disappeared 2 weeks after cell injection, whereas signal formicroencapsulated cells remained strong for 8 months, thus demonstrating thepresence of living cells. Treatment of mice with the thymidine-kinase substrateganciclovir caused death of microencapsulated myoblasts, as seen by a drasticdecay in the light emission and histological analysis. Hence, we conclude thatincorporation of the SFG(NES)TGL vector into microencapsulated cells representsan accurate tool for controlling cell location and viability in a non-invasiveway. Moreover, cell death can be induced by administration of ganciclovir, incase therapy needs to be interrupted. This system may represent a step forward inthe control and biosafety of cell- and gene- therapy-based microencapsulationprotocols.

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Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model.
BMC Cancer 2010-05

BACKGROUND: There is strong evidence demonstrating that activation of epidermalgrowth factor receptors (EGFRs) leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown tobe relevant drugs for lung cancer treatment. Recent studies demonstrate thatlapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, isclinically effective against HER-2-overexpressing metastatic breast cancer. Inthis report, we investigated the activity of lapatinib against non-small celllung cancer (NSCLC).
METHODS: We selected the lung cancer cell line A549, which harbors genomicamplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenicassays, and signaling cascade analyses (by western blot) were performed in vitro.In vivo experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy) were also carried out.
RESULTS: Lapatinib dramatically reduced cell proliferation (P < 0.0001), DNAsynthesis (P < 0.006), and colony formation capacity (P < 0.0001) in A549 cellsin vitro. Furthermore, lapatinib induced G1 cell cycle arrest (P < 0.0001) andapoptotic cell death (P < 0.0006) and reduced cyclin A and B1 levels, which areregulators of S and G2/M cell cycle stages, respectively. Stimulation ofapoptosis in lapatinib-treated A549 cells was correlated with increased cleavedPARP, active caspase-3, and proapoptotic Bak-1 levels, and reduction in theantiapoptotic IAP-2 and Bcl-xL protein levels. We also demonstrate that lapatinibaltered EGFR/HER-2 signaling pathways reducing p-EGFR, p-HER-2, p-ERK1/2, p-AKT, c-Myc and PCNA levels. In vivo experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PETanalysis) (P < 0.04) and smaller in size than controls. In addition, tumors from lapatinib-treated mice showed a dramatic reduction in angiogenesis (P < 0.0001). CONCLUSION: Overall, these data suggest that lapatinib may be a clinically usefulagent for the treatment of lung cancer. Pubmed link


Molecular characterization of the Ggamma-globin-Tag transgenic mouse model of hormone refractory prostate cancer: comparison to human prostate cancer.
Prostate 2010-05

BACKGROUND: Prostate cancer (PrCa) has a high incidence in Western countries and at present, there is no cure for hormone refractory prostate cancer. Transgenicmouse models have proven useful for understanding mechanisms of prostatecarcinogenesis. The characterization of genetically modified mouse PrCa modelsusing high-throughput genomic analyses provides important information to guideappropriate experiment applications for such model.
METHODS: We have analyzed the transcriptome of the hormone refractory and highly metastatic Fetal Globin-SV40/T-antigen (Ggamma-globin-Tag) transgenic mouse modelfor PrCa compared to normal mouse prostate tissue. Gene expression patterns foundin Ggamma-globin-Tag mouse prostate tumors were compared with publicly available human localized and metastatic prostate tumors (GEO accession # GSE3325) through hierarchical cluster analysis, Pearson’s rank correlation coefficient, and SelfOrganizing Feature Maps (SOM) analyses.
RESULTS: Ggamma-globin-Tag tumors clustered closely with human metastatic tumors and gene expression patterns had a significant correlation (P < 0.01), unlikehuman localized primary tumors (P > 0.6). Bioinformatic analyses identifiedderegulated genetic pathways and networks in Ggamma-globin-Tag tumors, whichdisplayed similarities to alterations in human PrCa. Changes in the expression ofgenes involved in DNA replication and repair (Rb1, p53, Myc, PCNA, DNMT3A) andgrowth factor signaling pathways (TGFbeta2, ERK1/2, NRas, and Notch1) arederegulated in the Ggamma-globin-Tag tumors, suggesting their key role in theoncogenic process. Identification of an enrichment of putative binding sites for transcription factors revealed eight transcription factors that may be important in Ggamma-globin-Tag carcinogenesis, including SP1, NF-Y, CREB, Elk1, and E2F.Novel genes related to microtubule regulation were also identified inGgamma-globin-Tag tumors as potentially important candidate targets for PrCa.Overexpression of stathmin-1, whose expression was increased in human metastatic prostate tumors, was validated in Ggamma-globin-Tag tumors byimmunohistochemistry. This protein belongs to the SV40/T-antigen cancer signatureidentified in previous studies in prostate, breast, and lung cancer mouse models.
CONCLUSIONS: Our results show that the Ggamma-globin-Tag model for hormonerefractory PrCa shares important features with aggressive, metastatic human PrCa.Given the role of stathmin-1 in the destabilization of microtubles and taxaneresistance, the Ggamma-globin-Tag model and other SV40/T-antigen driventransgenic models may be useful for testing potential therapies directed atstathmin-1 in human prostate tumors.

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VEGF elicits epithelial-mesenchymal transition (EMT) in prostate intraepithelial neoplasia (PIN)-like cells via an autocrine loop.
Exp Cell Res 2010-02

Vascular endothelial growth factor (VEGF) is overexpressed during the transition from prostate intraepithelial neoplasia (PIN) to invasive carcinoma. We havemimicked such a process in vitro using the PIN-like C3(1)/Tag-derived Pr-111 cellline, which expresses low levels of VEGF and exhibits very low tumorigenicity in vivo. Elevated expression of VEGF164 in Pr-111 cells led to a significantincrease in tumorigenicity, invasiveness, proliferation rates and angiogenesis.Moreover, VEGF164 induced strong changes in cell morphology and celltranscriptome through an autocrine mechanism, with changes in TGF-beta1- andcytoskeleton-related pathways, among others. Further analysis ofVEGF-overexpressing Pr-111 cells or following exogenous addition of recombinantVEGF shows acquisition of epithelial-mesenchymal transition (EMT) features, with an increased expression of mesenchymal markers, such as N-cadherin, Snail1,Snail2 (Slug) and vimentin, and a decrease in E-cadherin. Administration of VEGF led to changes in TGF-beta1 signaling, including reduction of Smad7 (TGF-betainhibitory Smad), increase in TGF-betaR-II, and translocation of phospho-Smad3 tothe nucleus. Our results suggest that increased expression of VEGF in malignantcells during the transition from PIN to invasive carcinoma leads to EMT throughan autocrine loop, which would promote tumor cell invasion and motility.Therapeutic blockade of VEGF/TGF-beta1 in PIN lesions might impair not only tumorangiogenesis, but also the early dissemination of malignant cells outside theepithelial layer.

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The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer.
Br J Cancer 2009-03

Radiotherapy (RT) is a common treatment for localised prostate cancer, but cancause important side effects. The therapeutic efficacy of RT can be enhanced bypharmacological compounds that target specific pathways involved in cellsurvival. This would elicit a similar therapeutic response using lower doses ofRT and, in turn, reducing side effects. This study describes the antitumouractivity of the novel Akt inhibitor8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one(Palomid 529 or P529) as well as its ability to decrease radiation-activatedphospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potentantiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory50 (GI50) <35 microM. In addition, P529 significantly enhanced theantiproliferative effect of radiation in prostate cancer cells (PC-3). Analysisof signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF,MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg(-1)P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreasedtumour growth was due to reduced proliferation and increased apoptosis (asassessed by PCNA and caspase-3 immunostaining). Our results show the antitumourefficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer. Pubmed link


Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis
Oncogene 2008-09

Metastasis is the primary cause of death in patients with breast cancer.Overexpression of c-myc in humans correlates with metastases, but transgenic miceonly show low rates of micrometastases. We have generated transgenic mice thatoverexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF)in the mammary gland, which develop high rates of pulmonary macrometastases. Geneexpression profiling revealed a set of deregulated genes in Myc/VEGF tumorscompared to Myc tumors associated with the increased metastatic phenotype.Cross-comparisons between this set of genes with a human breast cancer lungmetastasis gene signature identified five common targets: tenascin-C(TNC), matrixmetalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signalingblockade or knockdown of TNC in MDA-MB-435 cells resulted in a significantimpairment of cell migration and anchorage-independent cell proliferation. Miceinjected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrateda significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapseafter surgical removal of the primary tumor and (3) incidence of lung metastasis.Our results demonstrate that VEGF induces complex alterations in tissuearchitecture and gene expression. The TNC signaling pathway plays an importantrole in mammary tumor growth and metastases, suggesting that TNC may be arelevant target for therapy against metastatic breast cancer. Pubmed link


Phenotypic and genetic characterization of circulating tumor cells by combining immunomagnetic selection and FICTION techniques.
J Histochem Cytochem 2008-07

The presence of circulating tumor cells (CTCs) in breast cancer patients has beenproven to have clinical relevance. Cytogenetic characterization of these cellscould have crucial relevance for targeted cancer therapies. We developed a methodthat combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool forinvestigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancercells. Nucleated cells were separated by double density gradient centrifugationof blood samples. Tumor cells (TCs) were immunomagnetically isolated with ananti-cytokeratin antibody and placed onto slides for FICTION analysis. Forimmunophenotyping and genetic characterization of TCs, a mixture of primarymonoclonal anti-pancytokeratin antibodies was used, followed by fluorescentsecondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheralblood and characterized by FICTION. Because genetic amplification of TOP2A andErbB2 (HER-2) in breast cancer correlates with response to anthracyclines andherceptin therapies, respectively, this novel methodology could be useful for abetter classification of patients according to the genetic alterations of CTCsand for the application of targeted therapies.

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Alternative splicing: an emerging topic in molecular and clinical oncology.
Lancet Oncol 2007-04

Alternative pre-mRNA splicing is a key molecular event that allows for proteindiversity. Through this process, a single gene increases its coding capacity byexpressing several related proteins with diverse and even antagonistic functions.Aberrant splicing has been found to be associated with various diseases,including cancer. Mutations in splicing regulatory elements within the nucleotidesequence and alterations in the cellular-splicing-regulatory machinery bothresult in changes in the splicing pattern of many cancer-related genes. Theanalysis of cancer-specific alternative splicing and its molecular consequencesis promising. In this review we summarise the current knowledge on the mechanismsgoverning abnormal alternative splicing in cancer and the biological consequencesassociated with the alteration of splicing in some relevant cancer-related genes.The use of alternative splicing as a potential source for new diagnostic,prognostic, predictive, and therapeutic tools is also discussed.

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Increased expression of VEGF121/VEGF165-189 ratio results in a significant enhancement of human prostate tumor angiogenesis.
Int J Cancer 2007-05
Catena R | Muniz-Medina V | Moralejo B | Javierre B | Best CJ | Emmert-Buck MR | Green JE | Baker CC | Calvo A

Vascular endothelial growth factor (VEGF) is a proangiogenic factor upregulatedin many tumors. The alternative splicing of VEGF mRNA renders 3 major isoforms of121, 165 and 189 amino-acids in humans (1 less amino-acid for each mouse VEGFisoform). We have designed isoform specific real time QRT-PCR assays toquantitate VEGF transcripts in mouse and human normal and malignant prostates. Inthe human normal prostate, VEGF(165) was the predominant isoform (62.8% +/-5.2%), followed by VEGF(121) (22.5% +/- 6.3%) and VEGF(189) (p < 0.001) (14.6%+/- 2.1%). Prostate tumors showed a significant increase in the percentage ofVEGF(121) and decreases in VEGF(165) (p < 0.01) and VEGF(189) (p < 0.05).However, the amount of total VEGF mRNA was similar between normal and malignantprostates. VEGF(164) was the transcript with the highest expression in the mouse normal prostate. Unlike human prostate cancer, tumors from TRAMP micedemonstrated a significant increase in total VEGF mRNA levels and in each of the VEGF isoforms, without changes in the relative isoform ratios. Morpholinophosphorodiamide antisense oligonucleotide technology was used to increase therelative amount of VEGF(121) while proportionally decreasing VEGF(165) andVEGF(189) levels in human prostate cell lines, through the modification ofalternative splicing, without changing transcription levels and total amount ofVEGF. The increase in the VEGF(121)/VEGF(165-189) ratio in PC3 cells resulted in a dramatic increase in prostate tumor angiogenesis in vivo. Our resultsunderscore the importance of VEGF(121) in human prostate carcinoma anddemonstrate that the relative expression of the different VEGF isoforms has animpact on prostate carcinogenesis. Pubmed link


2-methoxyestradiol induces mammary gland differentiation through amphiregulin-epithelial growth factor receptor-mediated signaling: molecular distinctions from the mammary gland of pregnant mice.
Endocrinology 2007-03
Huh JI | Qiu TH | Chandramouli GV | Charles R | Wiench M | Hayer GL | Catena R | Calvo A | Lavalle TM | Desprez PY | Green JE

Levels of 2-methoxyestradiol (2ME(2)), an endogenous metabolite of estradiol, arehighly elevated during late stages of pregnancy when mammary glands havedifferentiated with the formation of alveolar structures producing milk proteins.Based upon our previous demonstration that 2ME(2) induces mammary ductal dilationassociated with expression of mammary differentiation markers when administeredto transgenic mice that spontaneously develop mammary cancer, we studied theeffects of 2ME(2) on normal mammary gland development. The results of this study demonstrate that 2ME(2) can induce a partial differentiation of normal mammaryglands in virgin mice, as evidenced by the appearance of limited numbers ofalveolar cells and significantly increased expression of the differentiationmarkers beta-casein and whey acidic protein. 2ME(2)-induced differentiation isassociated with inhibition of expression of inhibitor of differentiation 1 (Id-1)in normal mammary epithelial cells through elements in the 5′-flanking region of the Id-1 gene. Microarray analysis revealed that 2ME(2)-induced differentiationof the mammary gland shares some significant similarities in gene expression withthat of mammary glands from late-stage pregnancy, including elevated expressionof many milk protein differentiation markers. However, several genes aredifferentially regulated between 2ME(2)-treated mammary glands and differentiatedmammary glands through pregnancy. Significantly, amphiregulin, ATF3, serpine2,and SOX6 were up-regulated in 2ME(2)-treated mammary glands but not in mammaryglands from pregnant mice. Using the SCp2 differentiation cell line system, wedemonstrate that 2ME(2) induces differentiation through the down-regulation ofId-1 and up-regulation of amphiregulin. Administration of amphiregulin to SCp2cells induced differentiation, whereas inhibition of 2ME(2)-induced expression ofamphiregulin by small interfering RNA blocked differentiation. Estrogenreceptor-negative SCp2 cells differentiate in response to 2ME(2), but notestradiol, suggesting that 2ME(2) operates through an estrogenreceptor-independent mechanism. These data demonstrate that 2ME(2) can induce apartial differentiation of the mammary gland through mechanisms that differ from those normally used during pregnancy.

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