Publications by authors named "Ilan Tsarfaty"

34 Publications

Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing.

Nat Commun 2021 03 12;12(1):1657. Epub 2021 Mar 12.

School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel.

Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure. Integration of these two features enables cutting-edge analysis of root development at single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms.
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http://dx.doi.org/10.1038/s41467-021-21802-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954861PMC
March 2021

Proteomic analysis of combined IGF1 receptor targeted therapy and chemotherapy identifies signatures associated with survival in breast cancer patients.

Oncotarget 2020 Apr 28;11(17):1515-1530. Epub 2020 Apr 28.

Institute of Oncology, Chaim Sheba Medical Center, Tel Hashomer 52620, Israel.

Clinical, epidemiological and experimental data identified the insulin-like growth factor-1 receptor (IGF1R) as a candidate therapeutic target in oncology. While this paradigm is based on well-established biological facts, including the potent anti-apoptotic and cell survival capabilities of the receptor, most Phase III clinical trials designed to target the IGF1R led to disappointing results. The present study was aimed at evaluating the hypothesis that combined treatment composed of selective IGF1R inhibitor along with classical chemotherapy might be more effective than individual monotherapies in breast cancer treatment. Analyses included comprehensive measurements of the synergism achieved by various combination regimens using the software. In addition, proteomic analyses were conducted to identify the proteins involved in the synergistic killing effect at a global level. Data presented here demonstrates that co-treatment of IGF1R inhibitor along with chemotherapeutic drugs markedly improves the therapeutic efficiency in breast cancer cells. Of clinical relevance, our analyses indicate that high IGF1R baseline expression may serve as a predictive biomarker for IGF1R targeted therapy. In addition, we identified a ten-genes signature with potential predictive value. In conclusion, the use of a series of bioinformatics tools shed light on some of the biological pathways that might be responsible for synergysm in cancer therapy.
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http://dx.doi.org/10.18632/oncotarget.27566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197451PMC
April 2020

The effect of maternal obstructive sleep apnea on the placenta.

Sleep 2019 06;42(6)

Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Study Objectives: Obstructive sleep apnea (OSA) during pregnancy has been associated with adverse maternal outcomes. However, the effect of maternal OSA on fetal growth is less clear. The placenta is a critical organ for fetal growth and development and the principal determinant of birthweight. We aimed to investigate the effect of maternal OSA on placental growth and function.

Methods: Placentas of women recruited to a prospective longitudinal study were consecutively obtained immediately after delivery. Each placenta was measured for length, width, and thickness. Total RNA was isolated for gene expression analysis of VEGF, VEGF receptor, PIGF, and leptin. Histological and morphometric evaluations of the placenta were performed.

Results: A total of 53 placentas were investigated. Ten women (19%) had OSA, and the weight of their placentas was significantly higher compared with the placentas of the controls (526.1 ± 83.9 vs. 425.7 ± 95.5 g, p = 0.004). There was a significant positive correlation between placental weight and the log apnea-hypopnea index even after controlling for maternal body mass index (BMI; r = 0.31, p = 0.04). The birthweight/placental weight ratio was significantly lower in women with OSA compared with controls (p = 0.03). Placental weight and newborn triceps adiposity thickness correlated positively after controlling for maternal BMI (r = 0.29, p = 0.04). Leptin expression was 1.8-fold higher in placentas of women with OSA compared with controls (p = 0.02). No histological differences were found between the groups.

Conclusions: Maternal OSA is associated with increased placental weight that correlated with OSA severity and neonatal adiposity independently of maternal BMI. Placental leptin overexpression may mediate/underlie the above findings.Trial Registration: Clinical Trials NCT00931099.
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http://dx.doi.org/10.1093/sleep/zsz072DOI Listing
June 2019

Mimp/Mtch2, an Obesity Susceptibility Gene, Induces Alteration of Fatty Acid Metabolism in Transgenic Mice.

PLoS One 2016 30;11(6):e0157850. Epub 2016 Jun 30.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Objective: Metabolic dysfunctions, such as fatty liver, obesity and insulin resistance, are among the most common contemporary diseases worldwide, and their prevalence is continuously rising. Mimp/Mtch2 is a mitochondrial carrier protein homologue, which localizes to the mitochondria and induces mitochondrial depolarization. Mimp/Mtch2 single-nucleotide polymorphism is associated with obesity in humans and its loss in mice muscle protects from obesity. Our aim was to study the effects of Mimp/Mtch2 overexpression in vivo.

Methods: Transgenic mice overexpressing Mimp/Mtch2-GFP were characterized and monitored for lipid accumulation, weight and blood glucose levels. Transgenic mice liver and kidneys were used for gene expression analysis.

Results: Mimp/Mtch2-GFP transgenic mice express high levels of fatty acid synthase and of β-oxidation genes and develop fatty livers and kidneys. Moreover, high-fat diet-fed Mimp/Mtch2 mice exhibit high blood glucose levels. Our results also show that Mimp/Mtch2 is involved in lipid accumulation and uptake in cells and perhaps in human obesity.

Conclusions: Mimp/Mtch2 alters lipid metabolism and may play a role in the onset of obesity and development of insulin resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157850PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928869PMC
July 2017

Modeling the Transitions between Collective and Solitary Migration Phenotypes in Cancer Metastasis.

Sci Rep 2015 Dec 2;5:17379. Epub 2015 Dec 2.

Center for Theoretical Biological Physics, Houston, TX 77005-1827, USA.

Cellular plasticity during cancer metastasis is a major clinical challenge. Two key cellular plasticity mechanisms -Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Amoeboid Transition (MAT) - have been carefully investigated individually, yet a comprehensive understanding of their interconnections remains elusive. Previously, we have modeled the dynamics of the core regulatory circuits for both EMT (miR-200/ZEB/miR-34/SNAIL) and MAT (Rac1/RhoA). We now extend our previous work to study the coupling between these two core circuits by considering the two microRNAs (miR-200 and miR-34) as external signals to the core MAT circuit. We show that this coupled circuit enables four different stable steady states (phenotypes) that correspond to hybrid epithelial/mesenchymal (E/M), mesenchymal (M), amoeboid (A) and hybrid amoeboid/mesenchymal (A/M) phenotypes. Our model recapitulates the metastasis-suppressing role of the microRNAs even in the presence of EMT-inducing signals like Hepatocyte Growth Factor (HGF). It also enables mapping the microRNA levels to the transitions among various cell migration phenotypes. Finally, it offers a mechanistic understanding for the observed phenotypic transitions among different cell migration phenotypes, specifically the Collective-to-Amoeboid Transition (CAT).
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http://dx.doi.org/10.1038/srep17379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667179PMC
December 2015

The motility-proliferation-metabolism interplay during metastatic invasion.

Sci Rep 2015 Sep 4;5:13538. Epub 2015 Sep 4.

School of Physics and Astronomy, The Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Metastasis is the major cause for cancer patients' death, and despite all the recent advances in cancer research it is still mostly incurable. Understanding the mechanisms that are involved in the migration of the cells in a complex environment is a key step towards successful anti-metastatic treatment. Using experimental data-based modeling, we focus on the fundamentals of metastatic invasion: motility, invasion, proliferation and metabolism, and study how they may be combined to maximize the cancer's ability to metastasize. The modeled cells' performance is measured by the number of cells that succeed in migration in a maze, which mimics the extracellular environment. We show that co-existence of different cell clones in the tumor, as often found in experiments, optimizes the invasive ability in a frequently-changing environment. We study the role of metabolism and stimulation by growth factors, and show that metabolism plays a crucial role in the metastatic process and should therefore be targeted for successful treatment.
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http://dx.doi.org/10.1038/srep13538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642550PMC
September 2015

Tumor invasion optimization by mesenchymal-amoeboid heterogeneity.

Sci Rep 2015 May 27;5:10622. Epub 2015 May 27.

School of Physics and Astronomy, The Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.

Metastasizing tumor cells migrate through the surrounding tissue and extracellular matrix toward the blood vessels, in order to colonize distant organs. They typically move in a dense environment, filled with other cells. In this work we study cooperative effects between neighboring cells of different types, migrating in a maze-like environment with directional cue. Using a computerized model, we measure the percentage of cells that arrive to the defined target, for different mesenchymal/amoeboid ratios. Wall degradation of mesenchymal cells, as well as motility of both types of cells, are coupled to metabolic energy-like resource level. We find that indirect cooperation emerges in mid-level energy, as mesenchymal cells create paths that are used by amoeboids. Therefore, we expect to see a small population of mesenchymals kept in a mostly-amoeboid population. We also study different forms of direct interaction between the cells, and show that energy-dependent interaction strength is optimal for the migration of both mesenchymals and amoeboids. The obtained characteristics of cellular cluster size are in agreement with experimental results. We therefore predict that hybrid states, e.g. epithelial-mesenchymal, should be utilized as a stress-response mechanism.
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http://dx.doi.org/10.1038/srep10622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650638PMC
May 2015

Live time-lapse dataset of in vitro wound healing experiments.

Gigascience 2015 25;4. Epub 2015 Feb 25.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978 Israel.

Background: The wound healing assay is the common method to study collective cell migration in vitro. Computational analyses of live imaging exploit the rich temporal information and significantly improve understanding of complex phenomena that emerge during this mode of collective motility. Publicly available experimental data can allow application of new analyses to promote new discoveries, and assess algorithms' capabilities to distinguish between different experimental conditions.

Findings: A freely-available dataset of 31 time-lapse in vitro wound healing experiments of two cell lines is presented. It consists of six different experimental conditions with 4-6 replicates each, gathered to study the effects of a growth factor on collective cell migration. The raw data is available at 'The Cell: an Image Library' repository. This Data Note provides detailed description of the data, intermediately processed data, scripts and experimental validations that have not been reported before and are currently available at GigaDB. This is the first publicly available repository of live collective cell migration data that includes independent replicates for each set of conditions.

Conclusions: This dataset has the potential for extensive reuse. Some aspects in the data remain unexplored and can be exploited extensively to reveal new insight. The dataset could also be used to assess the performance of available and new quantification methods by demonstrating phenotypic discriminatory capabilities between the different experimental conditions. It may allow faster and more elaborated, reproducible and effective analyses, which will likely lead to new biological and biophysical discoveries.
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http://dx.doi.org/10.1186/s13742-015-0049-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4341232PMC
December 2016

MicroRNA miR-125a-3p modulates molecular pathway of motility and migration in prostate cancer cells.

Oncoscience 2014 30;1(4):250-261. Epub 2014 Apr 30.

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Israel.

Fyn kinase is implicated in prostate cancer. We illustrate the role of miR-125a-3p in cellular pathways accounted for motility and migration of prostate cancer cells, probably through its regulation on Fyn expression and Fyn-downstream proteins. Prostate cancer PC3 cells were transiently transfected with empty miR-Vec (control) or with miR-125a-3p. Overexpression of miR-125a-3p reduced migration of PC3 cells and increased apoptosis. Live cell confocal imaging indicated that overexpression of miR-125a-3p reduced the cells' track speed and length and impaired phenotype. Fyn, FAK and paxillin, displayed reduced activity following miR-125a-3p overexpression. Accordingly, actin rearrangement and cells' protrusion formation were impaired. An inverse correlation between miR-125a-3p and Gleason score was observed in human prostate cancer tissues. Our study demonstrated that miR-125a-3p may regulate migration of prostate cancer cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278297PMC
http://dx.doi.org/10.18632/oncoscience.30DOI Listing
January 2015

Interplay Between HGF/SF-Met-Ras Signaling, Tumor Metabolism and Blood Flow as a Potential Target for Breast Cancer Therapy.

Oncoscience 2014 11;1(1):30-38. Epub 2013 Dec 11.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University.

High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met-Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met-Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295761PMC
http://dx.doi.org/10.18632/oncoscience.6DOI Listing
January 2015

Chemotherapy-induced vascular toxicity--real-time in vivo imaging of vessel impairment.

J Vis Exp 2015 Jan 7(95):e51650. Epub 2015 Jan 7.

Sackler Faculty of Medicine, Tel Aviv University; Institute of Oncology, Davidoff Center and Rabin Medical Center;

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity. Yet, albeit the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform to evaluate the potential acute vascular toxicity of anti-cancer therapies. We have set up a platform of in vivo, high-resolution molecular imaging in mice, suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.
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http://dx.doi.org/10.3791/51650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354501PMC
January 2015

The three-way switch operation of Rac1/RhoA GTPase-based circuit controlling amoeboid-hybrid-mesenchymal transition.

Sci Rep 2014 Sep 23;4:6449. Epub 2014 Sep 23.

1] Center for Theoretical Biological Physics, Rice University, Houston, TX 77005-1827, USA [2] Department of Biosciences, Rice University, Houston, TX 77005-1827, USA [3] School of Physics and Astronomy and The Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv 69978, Israel.

Metastatic carcinoma cells exhibit at least two different phenotypes of motility and invasion - amoeboid and mesenchymal. This plasticity poses a major clinical challenge for treating metastasis, while its underlying mechanisms remain enigmatic. Transitions between these phenotypes are mediated by the Rac1/RhoA circuit that responds to external signals such as HGF/SF via c-MET pathway. Using detailed modeling of GTPase-based regulation to study the Rac1/RhoA circuit's dynamics, we found that it can operate as a three-way switch. We propose to associate the circuit's three possible states to the amoeboid, mesenchymal and amoeboid/mesenchymal hybrid phenotype. In particular, we investigated the range of existence of, and the transition between, the three states (phenotypes) in response to Grb2 and Gab1 - two downstream adaptors of c-MET. The results help to explain the regulation of metastatic cells by c-MET pathway and hence can contribute to the assessment of possible clinical interventions.
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http://dx.doi.org/10.1038/srep06449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171704PMC
September 2014

Functional molecular imaging of tumors by chemical exchange saturation transfer MRI of 3-O-Methyl-D-glucose.

Magn Reson Med 2014 Nov 18;72(5):1375-80. Epub 2014 Sep 18.

School of Chemistry, Tel Aviv University, Tel Aviv, Israel.

Purpose: To evaluate the feasibility to detect tumors and metastases by the chemical exchange saturation transfer (CEST) MRI technique using 3-O-Methyl-D-glucose (3OMG), a nonmetabolizable derivative of glucose that is taken up rapidly and preferentially by tumors and is entirely excreted by the kidneys.

Methods: In vivo CEST MRI experiments were performed on a Bruker 7 Tesla Biospec on implanted orthotopic mammary tumors of mice before and following i.p. injection of 3OMG. The CEST images were generated by a series of gradient-echo images collected from a single 1 mm coronal slice after a 1.2 s presaturation pulse, applied at offsets of ±1.2 ppm from the water and at B(1) power of 2.5 µT.

Results: Following 3OMG (1.5 g/kg) i.p. injection, an enhanced CEST effect of approximately 20% was visualized at the tumor within a few minutes. The signal slowly declined reaching half of its maximum at approximately 80 min.

Conclusion: Due to the large CEST effect of 3OMG and its low toxicity 3OMG-CEST may serve for the detection of tumors and metastases in the clinic.
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http://dx.doi.org/10.1002/mrm.25467DOI Listing
November 2014

Propagating waves of directionality and coordination orchestrate collective cell migration.

PLoS Comput Biol 2014 Jul 24;10(7):e1003747. Epub 2014 Jul 24.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.
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http://dx.doi.org/10.1371/journal.pcbi.1003747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109844PMC
July 2014

Benchmark for multi-cellular segmentation of bright field microscopy images.

BMC Bioinformatics 2013 Nov 7;14:319. Epub 2013 Nov 7.

Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, 69978, Israel.

Background: Multi-cellular segmentation of bright field microscopy images is an essential computational step when quantifying collective migration of cells in vitro. Despite the availability of various tools and algorithms, no publicly available benchmark has been proposed for evaluation and comparison between the different alternatives.

Description: A uniform framework is presented to benchmark algorithms for multi-cellular segmentation in bright field microscopy images. A freely available set of 171 manually segmented images from diverse origins was partitioned into 8 datasets and evaluated on three leading designated tools.

Conclusions: The presented benchmark resource for evaluating segmentation algorithms of bright field images is the first public annotated dataset for this purpose. This annotated dataset of diverse examples allows fair evaluations and comparisons of future segmentation methods. Scientists are encouraged to assess new algorithms on this benchmark, and to contribute additional annotated datasets.
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http://dx.doi.org/10.1186/1471-2105-14-319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826518PMC
November 2013

Molecular imaging of tumors and metastases using chemical exchange saturation transfer (CEST) MRI.

Sci Rep 2013 Oct 25;3:3045. Epub 2013 Oct 25.

School of Chemistry, Tel Aviv University, Tel Aviv, Israel.

The two glucose analogs 2-deoxy-D-glucose (2-DG) and 2-fluoro-2-deoxy-D-glucose (FDG) are preferentially taken up by cancer cells, undergo phosphorylation and accumulate in the cells. Owing to their exchangeable protons on their hydroxyl residues they exhibit significant chemical exchange saturation transfer (CEST) effect in MRI. Here we report CEST-MRI on mice bearing orthotopic mammary tumors injected with 2-DG or FDG. The tumor exhibited an enhanced CEST effect of up to 30% that persisted for over one hour. Thus 2-DG/FDG CEST MRI can replace PET/CT or PET/MRI for cancer research in laboratory animals, but also has the potential to be used in the clinic for the detection of tumors and metastases, distinguishing between malignant and benign tumors and monitoring tumor response to therapy as well as tumors metabolism noninvasively by using MRI, without the need for radio-labeled isotopes.
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http://dx.doi.org/10.1038/srep03045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365327PMC
October 2013

The structure of anthracycline derivatives determines their subcellular localization and cytotoxic activity.

ACS Med Chem Lett 2013 Mar 4;4(3):323-8. Epub 2013 Feb 4.

Department of Organic Chemistry and Department of Chemical Physics, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University , Ramat Aviv 69978, Tel Aviv, Israel.

The cytotoxic activities and subcellular localizations of clinically used and synthetic analogues of the anthracycline family of chemotherapeutic agents were studied. The structures of the anthracycline derivatives affected their cytotoxicity and the time required for these compounds to exert cytotoxic effects on tumor cells. Fluorescent DNA intercalator displacement experiments demonstrated that there was no correlation between the DNA intercalation properties and the cytotoxicity of the studied anthracycline derivatives. Confocal microscopy experiments indicated that structural differences led to differences in subcellular localization. All studied anthracycline derivatives were observed in lysosomes, suggesting that this organelle, which is involved in several processes leading to malignancy, may contain previously unidentified molecular targets for these antitumor agents.
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http://dx.doi.org/10.1021/ml3002852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027571PMC
March 2013

Met kinetic signature derived from the response to HGF/SF in a cellular model predicts breast cancer patient survival.

PLoS One 2012 25;7(9):e45969. Epub 2012 Sep 25.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

To determine the signaling pathways leading from Met activation to metastasis and poor prognosis, we measured the kinetic gene alterations in breast cancer cell lines in response to HGF/SF. Using a network inference tool we analyzed the putative protein-protein interaction pathways leading from Met to these genes and studied their specificity to Met and prognostic potential. We identified a Met kinetic signature consisting of 131 genes. The signature correlates with Met activation and with response to anti-Met therapy (p<0.005) in in-vitro models. It also identifies breast cancer patients who are at high risk to develop an aggressive disease in six large published breast cancer patient cohorts (p<0.01, N>1000). Moreover, we have identified novel putative Met pathways, which correlate with Met activity and patient prognosis. This signature may facilitate personalized therapy by identifying patients who will respond to anti-Met therapy. Moreover, this novel approach may be applied for other tyrosine kinases and other malignancies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457970PMC
May 2013

Emergence of HGF/SF-induced coordinated cellular motility.

PLoS One 2012 6;7(9):e44671. Epub 2012 Sep 6.

Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel.

Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation -resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up (resembling mesenchymal to epithelial transition (MET) phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation-based collective cell motility dynamics. Our quantitative phenotypes may guide future investigation on the molecular and cellular mechanisms of tyrosine kinase-induced coordinate cell motility and morphogenesis in metastasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044671PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435317PMC
March 2013

Secrets of tubule engineering by epithelial cells.

Proc Natl Acad Sci U S A 2012 May 23;109(18):6790-1. Epub 2012 Apr 23.

Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and School of Physics and Astronomy, The Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.

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http://dx.doi.org/10.1073/pnas.1202337109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345023PMC
May 2012

Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

PLoS One 2011 9;6(11):e27593. Epub 2011 Nov 9.

Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel.

Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027593PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212570PMC
April 2012

A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

Clin Cancer Res 2008 Jul;14(14):4612-21

Compugen Ltd., Tel Aviv, Israel.

Purpose: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway.

Experimental Design: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway.

Results: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF.

Conclusions: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.
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http://dx.doi.org/10.1158/1078-0432.CCR-08-0108DOI Listing
July 2008

Human endogenous retrovirus (HERV-K) reverse transcriptase as a breast cancer prognostic marker.

Neoplasia 2008 Jun;10(6):521-33

Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

A reverse transcriptase (RT) cDNA, designated HERV-K-T47D-RT, was isolated from a hormonally treated human breast cancer cell line. The protein product putative sequence is 97% identical to the human endogenous HERV-K retroviral sequences. Recombinant T47D-RT protein was used to generate polyclonal antibodies. The expression of HERV-K-T47D-RT protein increased in T47D cells after treatment with estrogen and progesterone. The RT-associated DNA polymerase activity was substantially increased after over-expressing a chimeric YFP-HERV-K-T47D-RT protein in cells. This RT-associated polymerase activity was significantly reduced by mutating the active site sequence YIDD to SIAA. Moreover, the endogenous RT activity observed in T47D cells was decreased by HERV-K-T47D-RT-specific siRNA, confirming the dependence of the endogenous enzymatic activity. To assess HERV-K-T47D-RT expression in human breast tumors, 110 paraffin sections of breast carcinoma biopsies were stained and subjected to confocal analysis. Twenty-six percent (28/110) of the tumor tissues and 18% (15/85) of the adjacent normal tissue, from the same patients, expressed the RT. HERV-K-T47D-RT expression significantly correlates with poor prognosis for disease-free patients and their overall survival. These results imply that HERV-K-T47D-RT might be expressed in early malignancy and might serve as a novel prognostic marker for breast cancer. Furthermore, these results provide evidence for the possible involvement of endogenous retrovirus in human breast carcinoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386537PMC
http://dx.doi.org/10.1593/neo.07986DOI Listing
June 2008

Diagnostic targeting of colon cancer using a novel fluorescent somatostatin conjugate in a mouse xenograft model.

Int J Cancer 2008 May;122(9):2044-9

Advanced Technology Center, Sheba Medical Center, Tel Hashomer, Israel.

Colorectal carcinoma is one of the more prevalent, highly malignant human tumors, occurring in about 7% of the population. However, if diagnosed and treated in its early stages, colon cancer is curable. In our study, we used a mouse xenograft model to investigate the capability of a fluorescent conjugate of a novel synthetic somatostatin (SST) analog to improve detection of human colorectal tumors that are characterized by over-expressed SST receptors. Human HT-29 colon carcinomas were induced in nude mice. After administration of the fluorescent SST conjugate, in vivo low- and high-magnification fluorescence microscopy, as well as high-resolution spectrally resolved imaging were performed, and the time-dependent biodistribution was determined quantitatively (using fiber-optic spectroscopy). Administration of the conjugate (at concentrations of 6 mg/kg body weight) enabled targeting small (1-5 mm diameter) tumors with high sensitivity and selectivity. Toxicity studies at dosages up to 1,000 mg/kg body weight did not reveal any drug related abnormalities. In conclusion, the SST conjugate significantly enhanced the detection of HT-29 colon tumors by fluorescence imaging because of a 5- to 8-fold increase in the contrast between malignant and normal tissues.
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http://dx.doi.org/10.1002/ijc.23353DOI Listing
May 2008

Preparing the "soil": the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells.

Cancer Res 2006 Nov 23;66(21):10365-76. Epub 2006 Oct 23.

Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.

Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel-enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-2977DOI Listing
November 2006

Mimp, a mitochondrial carrier homologue, inhibits Met-HGF/SF-induced scattering and tumorigenicity by altering Met-HGF/SF signaling pathways.

Cancer Res 2006 Sep;66(17):8687-97

Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.

We have recently shown that Mimp, a mitochondrial carrier protein homologue, is induced by Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling and decreases the mitochondrial membrane potential in DA3 mammary adenocarcinoma cells. We show here that induction of Mimp leads to growth arrest in response to HGF/SF by arresting cells at the S phase of the cell cycle. Induction of Mimp or its transient expression does not lead to apoptosis. Mimp also attenuates HGF/SF-induced cellular scattering in vitro and tumor growth in vivo. The exogenous induction of Mimp at levels similar to its endogenous induction by HGF/SF increases the level of the Met protein and its phosphorylation by HGF/SF but reduces the levels of Shc and prevents the HGF/SF-induced tyrosine phosphorylation of Grb2 and Shc. In contrast, the level of phosphatidylinositol 3-kinase (PI3K) increases following Mimp induction and the level of phosphorylated PI3K in response to HGF/SF is unaffected by the exogenous induction of Mimp. Moreover, exogenous Mimp prevents the HGF/SF-induced transcription of the serum response element-luciferase reporter gene. Our results show that Mimp expression reduces Met-HGF/SF-induced proliferation and scattering by attenuating and altering the downstream signaling of Met. These data show a new link between a tyrosine kinase growth factor receptor and a mitochondrial carrier homologue that regulates cellular growth, motility, and tumorigenicity.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2294DOI Listing
September 2006

In vivo direct molecular imaging of early tumorigenesis and malignant progression induced by transgenic expression of GFP-Met.

Neoplasia 2006 May;8(5):353-63

Van Andel Research Institute, Grand Rapids, MI 49503, USA.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.
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http://dx.doi.org/10.1593/neo.05634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592452PMC
May 2006

HGF/SF increases tumor blood volume: a novel tool for the in vivo functional molecular imaging of Met.

Neoplasia 2006 May;8(5):344-52

Van Andel Research Institute, Grand Rapids, MI 49503, USA.

Molecular functional and metabolic imaging allows visualization of disease-causing processes in living organisms. Here we present a new approach for the functional molecular imaging (FMI) of endogenous tyrosine kinase receptor activity using Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), as a model. HGF/SF and Met play significant roles in the biology and pathogenesis of a wide variety of cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have previously shown that Met activation by HGF/SF increases oxygen consumption in vitro and results in substantial alteration of blood oxygenation levels in vivo, as measured by blood oxygenation level-dependent magnetic resonance imaging. Using contrast medium (CM) ultrasound imaging, we demonstrate here that HGF/SF induces an increase in tumor blood volume. This increase is evident in small vessels, including vessels that were not detected before HGF/SF treatment. The specificity of the effect was validated by its inhibition using anti-HGF/SF antibodies. This change in tumor hemodynamics, induced by HGF/SF and measured by CM ultrasound, is further used as a tool for Met FMI in tumors. This novel noninvasive molecular imaging technique may be applied for the in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.
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http://dx.doi.org/10.1593/neo.05685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592450PMC
May 2006

In search of drug treatment for genetic defects in the DNA damage response: the example of ataxia-telangiectasia.

Semin Cancer Biol 2004 Aug;14(4):295-305

Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

Most genetic disorders are severe diseases that cannot be treated. In the majority of them, enzyme and gene therapy can be significantly curtailed by technical difficulties and the nature of the physiological defects and affected tissues. A rational search for drug treatment for such diseases must be based on understanding the corresponding molecular defects. For example, in a disease stemming from a defective signaling pathway, a drug that can activate redundant pathways could be useful. Screening for such a drug would then depend on the availability of a laboratory assay that faithfully reflects the molecular defect in the corresponding disease, and a technology for applying the assay in a high throughput setup. Deficiencies in various components of the DNA damage response lead to genomic instability syndromes characterized by tissue degeneration, sensitivity to DNA damaging agents, and cancer predisposition. A typical example is ataxia-telangiectasia (A-T), caused by deficiency of the nuclear protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. ATM phosphorylates a multitude of substrates, each of which in turn modulates a branch of the damage response network. A certain redundancy among ATM and related proteins gives hope that activation of ATM-redundant activities might form a basis for drug treatment of A-T. This article describes a high throughput strategy for drug screening for A-T that is based on the above principles. A similar strategy can potentially be applied to drug screening for other genetic disorders.
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http://dx.doi.org/10.1016/j.semcancer.2004.04.009DOI Listing
August 2004