Name: systems biology of metabolic regulation by estrogen receptor signaling in breast cancer
Uploaded: 2016-03-17T14:00:00
Description: Watch this Scientific Journal Video about Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer at JoVE.com

This work was supported by grants from the National Institute of Food and Agriculture, U.S. Department of Agriculture, award ILLU-698-909 (ZME) and Pfizer, Inc (ZME). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the U.S. Department of Agriculture.

1. Preparation of RNA-Seq Samples
<ol>
	<li>Cell Culture and Treatment
	<ol>
		<li>Culture the MCF7 cells in Improved MEM (IMEM) plus phenol-red medium with 10% Heat inactive FBS, 1% penicillin/streptomycin at 37 &#176;C in humidified 5% CO2 atmosphere. 
		Note: Same cell line and cell culture condition is used throughout the study.</li>
		<li>Seed 100,000 MCF7 cells per well in 6-well plates. Have triplicates for each treatment. On day 3, remove the medium and add 2 ml fresh medium with or without 10<s...

<ol>
	<li>Hamilton, K. J., Arao, Y., Korach, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+hormone+physiology:+reproductive+findings+from+estrogen+receptor+mutant+mice.">Estrogen hormone physiology: reproductive findings from estrogen receptor mutant mice.</a> Reprod Biol. 14 (1), 3-8 (2014).</li><li>Madak-Erdogan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+genomics+of+gene+and+metabolic+regulation+by+estrogen+receptors+alpha+and+beta,+and+their+coregulators.">Integrative genomics of gene and metabolic regulation by estrogen receptors alpha and beta, and their coregulators.</a> Mol Syst Biol. 9, 676 (2013).</li><li>Gong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+analysis+identifies+gene+networks+regulated+by+estrogen+receptor+alpha+(ERalpha)+and+ERbeta+that+control+distinct+effects+of+different+botanical+estrogens.">Transcriptomic analysis identifies gene networks regulated by estrogen receptor alpha (ERalpha) and ERbeta that control distinct effects of different botanical estrogens.</a> Nucl Recept Signal. 12, e001 (2014).</li><li>Bergamaschi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forkhead+transcription+factor+FOXM1+promotes+endocrine+resistance+and+invasiveness+in+estrogen+receptor-positive+breast+cancer+by+expansion+of+stem-like+cancer+cells.">The forkhead transcription factor FOXM1 promotes endocrine resistance and invasiveness in estrogen receptor-positive breast cancer by expansion of stem-like cancer cells.</a> Breast Cancer Res. 16 (5), 436 (2014).</li><li>Madak-Erdogan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+extranuclear+pathway+inputs+in+the+regulation+of+global+gene+expression+by+estrogen+receptors.">Nuclear and extranuclear pathway inputs in the regulation of global gene expression by estrogen receptors.</a> Mol Endocrinol. 22 (9), 2116-2127 (2008).</li><li>Madak-Erdogan, Z., Lupien, M., Stossi, F., Brown, M., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+collaboration+of+estrogen+receptor+alpha+and+extracellular+signal-regulated+kinase+2+in+regulating+gene+and+proliferation+programs.">Genomic collaboration of estrogen receptor alpha and extracellular signal-regulated kinase 2 in regulating gene and proliferation programs.</a> Mol Cell Biol. 31, 226-236 (2011).</li><li>Madak-Erdogan, Z., Ventrella, R., Petry, L., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+roles+for+ERK5+and+cofilin+as+critical+mediators+linking+ERalpha-driven+transcription,+actin+reorganization,+and+invasiveness+in+breast+cancer.">Novel roles for ERK5 and cofilin as critical mediators linking ERalpha-driven transcription, actin reorganization, and invasiveness in breast cancer.</a> Mol Cancer Res. 12 (5), 714-727 (2014).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat Rev Genet. 10 (1), 57-63 (2009).</li><li>Invitrogen. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quant-iT&#8482; PicoGreen &#174; dsDNA Reagent and Kits. , (2008).</li><li>Lee, P. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+genetic+programming+optimised+Bowtie2+on+genome+comparison+and+analytic+testing+(GCAT)+benchmarks.">Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks.</a> BioData Min. 8, (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+analysis+of+ChIP-Seq(MACS).">Model-based analysis of ChIP-Seq(MACS).</a> Genome Biol. 9 (9), R137 (2008).</li><li>Gao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metscape:+a+Cytoscape+plug-in+for+visualizing+and+interpreting+metabolomic+data+in+the+context+of+human+metabolic+networks.">Metscape: a Cytoscape plug-in for visualizing and interpreting metabolomic data in the context of human metabolic networks.</a> Bioinformatics. 26, 971-973 (2010).</li><li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7 (3), (2006).</li><li>Mokry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+double+fragmentation+ChIP-seq+provides+nucleotide+resolution+protein-DNA+binding+profiles.">Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles.</a> PLoS One. 5 (11), e15092 (2010).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nat Rev Genet. 10, 669-680 (2009).</li><li>Landt, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22 (9), 1813-1831 (2012).</li><li>Hah, N., Kraus, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone-regulated+transcriptomes:+lessons+learned+from+estrogen+signaling+pathways+in+breast+cancer+cells.">Hormone-regulated transcriptomes: lessons learned from estrogen signaling pathways in breast cancer cells.</a> Mol Cell Endocrinol. 382, 652-664 (2014).</li></ol>

In this paper, we described generation and integrative analysis of RNA-Seq, ChIP-Seq and metabolomics data from MCF-7 cells that are treated with E2. We developed a set of protocols strategizing to utilize the most efficient methods and user friendly soft wares which produce biologically relevant discovery. To our knowledge, ours is the first study to integrate -omics data from three different analysis and identified new metabolic pathways that ER&#945; directly regulated in breast cancer cells.
<p class="jove_conten...

Estrogens are important regulators of many physiological processes in both females and males including reproductive tissues, metabolic tissues, brain and bone1. In addition to beneficial effects in these tissues, estrogens also drive cancers that arise from mammary and reproductive tissues. Estrogens mainly work through ERs to induce cell type specific effects. Deep sequencing of transcripts regulated by ER&#945; using RNA-Seq and genome-wide ER&#945; DNA binding sites analysis using ChIP-Seq proved to be useful to understand how ER&#945; works in different tissues and cancers that arise from them. We and others have published gene expression profiles associated with different receptors (ER&#945; v.s. ER&#946;)2,3, different ligands3-5 and different coregulators2,4,6,7.
RNA-Seq is the main method to examine the transcriptome, offering higher precision and efficiency compared to microarray based gene expression analysis8. RNA obtained from cell lines2-4,7, tissues or tumor samples are sequenced, mapped to available genomic assemblies and differentially regulated genes are identified. Chromatin Immunoprecipitation (ChIP) is employed to dissect the transcription factor and coregulator chromatin binding to known regulatory binding sites. ChIP-Seq (ChIP followed by high throughput sequencing) provides unbiased detection of global binding sites. Metabolomics is another increasingly used system biology approach, which quantitatively measures, dynamic multiparametric response of living systems to various stimuli including chemicals and genetic perturbations.
By performing global metabolic profiling, a functional readout can be obtained from cells, tissues, and blood. In addition, information from transcriptome experiments do not always reflect actual changes in the level of enzymes that contribute to biochemical pathways. Combined analysis of transcriptome and metabolome data enables us to identify and correlate changes in gene expression with actual metabolite changes. Harnessing the information from all these large scale datasets provide the mechanistic details to understand the role of transcription factors regulating complex biological systems, especially ones that pertain to human development and diseases like cancer and diabetes.
The complex nature of the mammalian genome makes it challenging to integrate and fully interpret the data obtained from the transcriptome, cistrome and metabolome experiments. Identifying functional binding events that would lead to changes in expression of target genes is important because once functional binding sites are identified; ensuing analysis, including transcription binding (TF) motif analysis, could be performed with higher accuracy. This leads to the identification of biologically meaningful TF cascades and mechanisms. Also direct comparison of RNA-Seq and ChIP-Seq experiments are not always possible since the data from each experiment have differing scales and noises and in some cases meaningful signals are obscured by population noise. We are not aware of any study that integrated information from these three independent but related approaches to understand the direct metabolic regulation by ER&#945; in breast cancer. Therefore, our overall goal in this paper is to relate productive binding events to gene expression and metabolite changes. In order to achieve this goal, we integrated data from RNA-Seq, ChIP-Seq and metabolomics experiments and identified those estrogen induced ER&#945; binding events that would lead to gene expression changes in metabolic pathways. For the first time, we provide a complete set of protocols (Figure 1) for generating ChIP-Seq, RNA-Seq and metabolomics profiling and performing integrative analysis of the data to uncover novel gene circuits regulating metabolism of breast cancer cell lines.

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systems biology of metabolic regulation by estrogen receptor signaling in breast cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Transcriptomics 
To analyze differentially expressed genes by E2 treatment, we chose to perform an RNA-Seq experiment. In addition to providing information about mRNA levels, RNA-Seq data can also be used to monitor changes in non-coding RNA (long non-coding RNAs, microRNAs) and alternative splicing events. We did not provide information on the analysis of non-coding RNAs or alternatively transcribed genes, since scope of our study is to identify protein coding genes that are importa...

Watch this Scientific Journal Video about Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer at JoVE.com

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

The overall goal of this experiment is to understand the direct metabolic regulation of ER Alpha in breast cancer, using genome-wide transcriptome, systrome, and metabolome data. This method can help answer key questions in the nucleoreceptor and cancer biology fields, such as the role of certain transcription factors in regulating metabolism to contribute to cell aggressiveness. The main advantage of this technique is that it gives a global picture of gene and resulting metabolic regulation nuance.Demonstrating the procedure will be Yiru Chen, a postdoctoral fellow from my laboratory. After culturing MCF7 cells according to the text protocol, harvest the cells by adding one milliliter of RNA isolation reagent to each well, and incubate at room temperature for five minutes. Use a pipette to collect the cell lysate and transfer it into 1.5 milliliter tubes.To isolate the RNA, add 200 microliters of chloroform to one milliliter of cell lysate, and vortex for 10 seconds. After incubating at room temperature for five minutes, centrifuge the samples at 16, 000 x g and four degrees Celsius for 15 minutes. Following centrifugation, carefully transfer the aqueous top phase to a new tube.Add 500 microliters of 100%isopropanol to the aqueous phase, and mix by inverting. Then, incubate at negative 20 degrees Celsius overnight. The following morning, centrifuge the samples at 1600 x g and four degrees Celsius for 10 minutes.Discard the supernatant, and add 750 microliters RNase-free 70%ethanol, and briefly vortex. Centrifuge the samples at 6, 000 x g and four degrees Celsius for five minutes. Then, remove the supernatant, and centrifuge the samples at 16, 000 x g and four degrees Celsius for one minute.With a gel loading tip, remove the remaining ethanol by pipetting. And set the tubes upside-down to air dry for 10 minutes. Then, add 20 to 50 microliters of DEPC water to dissolve the pellet, and purify and quantify the RNA according to the text protocol.Once MCF7 cells have been cultured according to the text protocol, add 250 microliters of 16%formaldehyde into five milliliters of medium to the cells to cross-link chromatin and incubate at room temperature for 15 minutes. Then use five milliliters of ice-cold 1X to wash the cells and stop the cross-linking reaction. Harvest the cells in 250 microliters of cell lysis buffer per plate.Then, to fragment the chromatin to the required sizes, sonicate the cells eight times for 30 seconds each at an amp of 30. Cool each sample on ice after each 30 second sonication. Next, after centrifuging the samples at 16, 000 x g and four degrees Celsius for 10 minutes, carefully pipette the supernatant without disturbing the pellet at the bottom.Apply a five microliter aliquot of the supernatant to a 1%agarose gel, and carry out electrophoresis to determine the DNA size. The ideal fragment length should be between 200 and 500 base pairs. To carry out immunoprecipitation of the chromatin, save 25 microliters of the cell lysate as input.Then, in a 50 milliliter tube, mix four milliliters of the cell lysate with 20 milliliters of IP buffer, ER Alpha antibody, and protein A and protein G-coded magnetic beads. Incubate the mixture by rotation at four degrees Celsius overnight, to allow the formation of chromatin-antibody complexes. After using wash buffers to wash the beads, according to the text protocol, add 500 microliters of elution buffer to the beads, and elute the DNA into four tubes.For the DNA input samples, elute into 125 microliters of elution buffer. Next, place the tubes in a heating block and cover them with foil, and place a weight on top to prevent them from opening. Incubate the tubes at 65 degrees Celsius overnight.To isolate the DNA from the pulldown samples using a ChIP DNA isolation kit, cool the tubes at room temperature for five minutes. In the meantime, warm up the elution buffer to 65 degrees Celsius. Add 625 microliters of binding buffer to each tube.If a precipitant forms, add another 250 microliters of binding buffer until the solution is clear. Load the sample onto the column and centrifuge at 16, 000 x g for 30 seconds. Then use 250 microliters of wash buffer to wash the column twice.With 15 microliters of elution buffer, elute the DNA. Combine the DNA from the four tubes of the same pulldown to arrive at 55 microliters of DNA. Measure DNA concentration and carry out QPCR according to the text protocol.Using MCF7 cells cultured according to the text protocol, add five milliliters of ice-cold 1X PBS to the plate. Then tilt the plate several times before removing the PBS. Repeat two more times, removing as much PBS as possible after the last wash.Add 750 microliters of pre-chilled acetone to each plate, and scrape the cells. Then, combine the cells from two plates into a two milliliter tube on ice. To count the cells for normalization, for each treatment, use two extra plates for cell quantification.Then, to detach the cells from the plates, add two milliliters of HE buffer and incubate for five minutes. Mix the cells thoroughly by pipetting in the HE buffer. Then, use 20 microliters of the cell solution in a hemocytometer to count the cell number under a microscope.Store the samples at negative 80 degrees Celsius before using gas chromatography mass spectrometry analysis to identify and quantify the metabolites. Perform integrative analysis according to the text protocol. This figure shows the integrity and overall quality of RNA samples purified for an RNA-Seq experiment.The analysis shows all samples have sharp and clean bands of 18S and 28S RRNAs, indicating the integrity and purity of the samples. An RNA Integrity Number, or RIN, of 10, indicates intact RNA. Six represents partially degraded RNA, and a three indicates a sample that is strongly degraded.In these experiments, all RNA samples received RIN values between 9.6 and 9.9. Carrying out ultra high throughput sequencing resulted in approximately 18 million high-quality sequencing reads per sample. A representative fast QC report, which analyzes the overall quality of the raw data, is shown here.For each read, the quality score was 32 to 34 for the first five base pairs, and 36 to 38 from six to 100 base pairs. Among 17, 990, 863 reads generated from this sample, most of them had a quality score higher than 34. This figure shows an overview of about 100 metabolites represented by peaks that show changes in MCF7 cells due to estradiol treatment.The top 10 metabolites with significant changes from E2 treatment are presented in this table. And this table lists the pathways that were up-and down-regulated upon E2 treatment. Once mastered, this technique can be done in a week if it is performed properly.While attempting this procedure, it's important to remember to pay attention to the health of the cells, and quality of the samples that will be analyzed. Following this procedure, other methods like exome sequencing or GRO-Seq that rely on sequencing data can be performed in order to answer additional questions like the identity of additional MRNAs, enhancer MRNAs, or long non-coding RNAs whose transcription are induced by treatments. After its development, this technique paved the way for researchers in the field of nuclear receptors to explore metabolic regulation by nuclear receptors in cell culture systems or in mice.After watching this video, you should have a good understanding of how to analyze and integrate data from multiple Omics approaches. Don't forget that working with Trizol, formaldehyde and methanol can be extremely hazardous and precautions such as wearing personal protection equipment such as gloves and lab coats should always be worn while performing this procedure.

systems-biology-metabolic-regulation-estrogen-receptor-signaling

Research

JoVE Journal

Medicine

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...