Name: clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization
Uploaded: 2016-06-07T15:00:00
Description: Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

This study was approved by the Institutional Review Board of the Chonnam National University Hwasun Hospital and University of South Carolina. TMA samples composed of breast cancer and matched adjacent normal breast tissues were provided by the Biobank of Chonnam National University Hwasun Hospital, a member of the Korea Biobank Network.
1. Solution Preparation
<ol>
	<li>Prepare 1 L of DNase and RNase free water. Make all chemical solutions using diethylpyrocarbonate (DEPC) treated water. Tr...

<ol>
	<li>Shah, N. R., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+pathogenesis+of+breast+cancer:+Implications+in+diagnosis+and+treatment.">MicroRNAs in pathogenesis of breast cancer: Implications in diagnosis and treatment.</a> World J Clin Oncol. 5, 48-60 (2014).</li><li>Tang, H., 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=miR-185+suppresses+tumor+proliferation+by+directly+targeting+E2F6+and+DNMT1+and+indirectly+upregulating+BRCA1+in+triple-negative+breast+cancer.">miR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.</a> Mol Cancer Ther. 13, 3185-3197 (2014).</li><li>Ye, X. 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=Epigenetic+silencing+of+miR-375+induces+trastuzumab+resistance+in+HER2-positive+breast+cancer+by+targeting+IGF1R.">Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.</a> BMC Cancer. 14, 134 (2014).</li><li>Oneyama, C., 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=MicroRNA-mediated+downregulation+of+mTOR/FGFR3+controls+tumor+growth+induced+by+Src-related+oncogenic+pathways.">MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.</a> Oncogene. 30, 3489-3501 (2011).</li><li>McGuire, A., Brown, J. A., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+breast+cancer:+the+potential+of+miRNA+for+diagnosis+and+treatment+monitoring.">Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring.</a> Cancer Metastasis Rev. 34, 145-155 (2015).</li><li>Quesne, J. L., 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=Biological+and+prognostic+associations+of+miR-205+and+let-7b+in+breast+cancer+revealed+by+in+situ+hybridization+analysis+of+micro-RNA+expression+in+arrays+of+archival+tumour+tissue.">Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue.</a> J Pathol. 227, 306-314 (2012).</li><li>Fernandez, S., 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=miR-340+inhibits+tumor+cell+proliferation+and+induces+apoptosis+by+targeting+multiple+negative+regulators+of+p27+in+non-small+cell+lung+cancer.">miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.</a> Oncogene. 34, 3240-3250 (2015).</li><li>Yu, L., Zhang, J., Guo, X., Li, Z., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-224+upregulation+and+AKT+activation+synergistically+predict+poor+prognosis+in+patients+with+hepatocellular+carcinoma.">MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma.</a> Cancer Epidemiol. 38, 408-413 (2014).</li><li>Li, 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=microRNA-146+up-regulation+predicts+the+prognosis+of+non-small+cell+lung+cancer+by+miRNA+in+situ+hybridization.">microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.</a> Exp Mol Pathol. 96, 195-199 (2014).</li><li>Kriegel, A. J., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+in+situ+hybridization+for+formalin+fixed+kidney+tissues.">MicroRNA in situ hybridization for formalin fixed kidney tissues.</a> J Vis Exp. , e50785 (2013).</li><li>Ucar, 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=miR-212+and+miR-132+are+required+for+epithelial+stromal+interactions+necessary+for+mouse+mammary+gland+development.">miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.</a> Nat Genet. 42, 1101-1108 (2010).</li><li>Nuovo, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+microRNAs+in+paraffin+embedded,+formalin+fixed+tissues+and+the+co-localization+of+their+putative+targets.">In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.</a> Methods. 52, 307-315 (2010).</li><li>Kierzek, E., 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+influence+of+locked+nucleic+acid+residues+on+the+thermodynamic+properties+of+2'-O-methyl+RNA/RNA+heteroduplexes.">The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.</a> Nucleic Acids Res. 33, 5082-5093 (2005).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> CSH Protoc. 2008, (2008).</li><li>Li, L., 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=Sequential+expression+of+miR-182+and+miR-503+cooperatively+targets+FBXW7,+contributing+to+the+malignant+transformation+of+colon+adenoma+to+adenocarcinoma.">Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma.</a> J Pathol. 234, 488-501 (2014).</li></ol>

The goal of this study was to determine the level of miRNA expression in breast cancer tissues using miRNA in situ hybridization. It must be noted that in this experiment, all conditions were optimized for breast cancer tissue. Further optimization might be required for other tissue types. All solutions must be made with DEPC water and all containers to be used should be rinsed with DEPC treated water and subsequently autoclaved. Even slight RNase contamination can interfere with the final outcome of the experim...

Breast cancer is among the most common malignancies affecting the female population across the globe. Over 1.3 million cases of breast cancer are reported every year worldwide1,2. Although tumor cells have been traditionally regarded as biologically homogenous and highly proliferative, it has become evident that breast cancer is genetically and clinically heterogeneous. Resistance to prevalent anticancer drugs is a hallmark of advanced breast cancers, leading to the mortality in the majority of patients through its facilitation of cancer progression and distance metastasis3.
MicroRNAs (miRNAs) are a class of small RNA molecules approximately 22 nucleotides long that regulate gene expression by blocking mRNA translation or mediating mRNA degradation4. More than 2,000 different miRNAs have so far been identified in humans, which are linked to human diseases5. An increasing array of studies have demonstrated that miRNAs can function as oncogenes and tumor suppressors and are often dysregulated in tumors6. miRNAs strongly impact all of the primary tumorigenesis by controlling the major regulators of cell cycle progression, senescence, apoptosis and autophagy, along with those of tumor cell motility, invasion and metastasis7.
Aberrant miRNA expression has been also associated with clinical implications such as stage, differentiation, prognosis and response to adjuvant therapy8. In particular, increased miR-146 expression is correlated with poor prognosis in lung cancer patients9. Another study has demonstrated that lost expression of miRNA-let7b is linked to malignant phenotypes and, consequently, poor survival6. Understanding the types of cells and structures that express miRNAs is an important part of understanding the mechanics through which alterations in miRNA expression influence cell and tissue phenotypes10. Certain miRNAs found to be secreted in stromal cells are taken in by epithelial cells and specifically act upon their targets. In the same vein, miR-212 and miR-132 are secreted by stroma and regulate the epithelial-stromal interactions required for ductal outgrowth during mammary gland development11. The overall objective of this paper is to explain a detailed protocol to detect miRNA expression in breast cancer tissues through miRNA in situ hybridization. We have optimized all conditions to assay the levels of miRNA-489 expression in breast cancer patient samples. To this end, we used a DIG labelled locked nucleic acid (LNA) probe in our experiment. Some of its nucleotides had an LNA modification, that is, a methylene bridge connecting the 2&#39; oxygen atom and 4&#39; carbon atom of the ribose backbone which fixes the nucleotide such that it stays &#34;locked in place&#34; after hybridization. As a result, it is much more difficult for the hybridized complex to denature12,13.

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ELF-97</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td style="width:119px;">E6604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td align="right" style="width:119px;">750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">t-RNA</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">109541</td>
			<td>Reconstitute in DEPC water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
			<td style="width:119px;">D1815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RVC</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:119px;">50-812-650</td>
			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miR-489 probe&#160;</td>
			<td style="width:159px;">Exiqon</td>
			<td style="width:119px;">38599-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease free BSA</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">711454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody-anti DIG</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td align="right" style="width:119px;">1609478</td>
			<td></td>
		</tr>
	</tbody>
</table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Human breast cancer tissue was used to determine miRNA-489 expression. Mammary gland duct and epithelial cells were found to express significantly higher miRNA-489 levels than adjacent tumor tissue in two patients (Figure 1A and 1B). This clearly demonstrates that miRNA-489 expression has been lost in tumor tissue, suggesting tumor suppressive activity of miRNA-489. To further confirm these results, tumor tissue and adjacent normal tissue from the same pa...

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

The overall goal of this Micro RNA In Situ Hybridization is to detect the localization of the Micro RNA of interest in breast cancer tissue. So the message can answer the key question in breast cancer field. Such as the was the micro RNA of interest is unclear or tumor suppressant.The major advantage of this technique is that it can detect cellular nuclearization of of interest in breast cancer tissue. Begin tissue preparation by removing paraffin from previously prepared breast tissue sections. Immerse the slides into a Coplin jar in fresh Xyline two times for ten minutes each.Re-hydrate the tissue section with incubations in decreasing concentrations of ethanol in double distilled water for five minutes each. Next, immerse the tissue in diethylpyrocarbonate or DEPC treated water for five minutes. While the tissue is immersed make boundaries around the tissue section with a hydrophobic barrier pen.After the DEPC immersion, fix the tissue with 4%PFA for 15 minutes. Follow the fixation with a PBS wash. Then, immerse the tissue in 0.3%Triton X-100 in PBS for ten minutes.Incubate the sample with 50 micrograms per microliter of Proteinase K solution for 15 minutes at 37 degrees Celsius. Then incubate the tissue sections with triethanolamine and acetic anhydride for five minutes at room temperature. And follow with a PBS wash.Wash the tissue thoroughly. And incubate the tissue section with 120 microliters of hybridization buffer for two hours at room temperature. Next, dilute the five prime DIG labeled NLA probe to a final concentration of 3 picamolar in 120 microliters of hybridization solution.Then add three microliters of 40 nanograms per microliter stock to the 120 microliters of hybridization buffer. And incubate the mixture at 95 degrees Celsius for five minutes. Insert the tissue section into a ten by eight inch container and cover the tissue with 120 microliters of the probe so that it is completely covered.Wet two paper towels and cover the container to create a humidity chamber. Incubate the tissue section overnight at 54 degrees Celsius. After hybridization, wash the tissue with five X saline sodium citrate buffer or SSC for seven minutes at 57 degrees Celsius.Next, wash the tissue section with one X SSC twice. And wash the tissue section with 0.2 X SSC. Then, wash the tissue section with 0.2 X SSC for seven minutes at room temperature.Incubate the tissue section in PBS for ten minutes. Incubate the tissue section with blocking buffer at room temperature for one hour in a humid chamber. Dilute the anti DIG AP antibody one to 300 in blocking buffer.And incubate the tissue section with the antibody at 4 degrees Celsius overnight. The next day, wash the sections with wash buffer three times for five minutes each. Follow the ELF 97 commercial kit instructions to develop the tissue section with alkaline phosphatase that produces size three fluorescent light.Next, wash the section with wash buffer for five minutes. Stain the section with 100 microliters of 2.5 nanograms per microliter Hoeshct dye for five minutes. And follow the stain with a five minute wash with wash buffer.Use 15 microliters of mounting buffer per tissue section. And wipe excessive mounting solution with a tissue wipe. Then, seal the slide with clear nail polish to prevent leaking.Finally observe the section with a fluorescent microscope at a total magnification of 100 X.Human breast cancer tissue from two patients was used to determine Micro RNA 489 expression. Memory gland duct and epithelial cells were found to express significantly higher Micro RNA 489 levels than adjacent tumor tissue. After watching this video, you should have a good understanding of how to perform Micro RNA In Sito hybridization by using breast cancer patient samples.

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Research

JoVE Journal

Medicine

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

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 ...

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 ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

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.

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 ...