The authors would like to acknowledge Pam Ovwigho, Tenette Jones, and Natasha Clayton, NIEHS, for their technical expertise and support with this project.

All animal use and procedures for this study were approved by the NIEHS Laboratory Animal
Care and Use Committee and conducted in an Association for Assessment and Accreditation of
Laboratory Animal Care-accredited facility.
1. Mammary Whole-mount Preparation2,3,6,7
<ol>
	<li>Remove the inguinal mammary glands from one side of a non-pregnant CD-1 female mouse, as described in reference3, and place them on an electrostatically charged slide.</li>
	<li>Cover the gland with non-stick paper or film and place another slide on top. Add pressure (i.e., water weights) to the slide to spread the gland out flat so that the gland will stick to the slide for fixation.</li>
	<li>Submerge the slides in fixative (e.g., 100% ethanol, chloroform, and glacial acetic acid in a 6:3:1 ratio) overnight at room temperature.</li>
	<li>Remove the glands from the fixative and wash in ethanol for 15-30 min. Following the 30-min wash, gradually change to water by pouring out 1/3 of the ethanol and adding water. Let the wash sit for about 5 min each time, and repeat with the addition of water 3 times.</li>
	<li>Stain (e.g., carmine alum solution) for 12-24 h. 
	NOTE: Thicker tissues will require longer staining times. See reference3 for staining details.</li>
	<li>Pour off the stain after the allotted time and rinse the slides in water for 30 s. Dehydrate the tissues by washing the slides in 70% ethanol for 15 min, followed by performing a 15&#160;min wash in 95% ethanol and a final wash in 100% ethanol for 20 min.</li>
	<li>Clear the fat from the tissues by placing them in xylene for a minimum of 24 h, or longer if the tissue is thick, so that any opaque or white areas are removed.</li>
	<li>Remove the tissue from the xylene and quickly add mounting medium to the slide to avoid tissue dehydration; place a coverslip on top. Allow the slide to dry for at least 48 h.</li>
	<li>Evaluate the whole-mounted tissue for abnormalities, as described in2. 
	NOTE: When lesions are detected in whole mounts and sectioning is necessary to establish their identity, the following steps should be followed.</li>
</ol>
2. Removal of the Mammary Whole Mount from a Glass Slide
<ol>
	<li>Remove the coverslip and mounting medium by submerging the mammary whole-mount slide in a glass staining jar filled with xylene&#160;overnight. 
	NOTE: Thicker tissues and slides with excessive mounting solution may require additional time for coverslip removal.</li>
	<li>Following the initial overnight soak, place the slides in fresh xylene and soak for 6 h. Perform one final soak in fresh xylene overnight. 
	NOTE: The coverslip will easily fall off the slide after the last soak.</li>
	<li>With a gloved hand, hold the slide and carefully remove the coverslip with a pair of forceps to ensure that it is removed in one piece.&#160; 
	NOTE: In the event that the coverslip is still attached to the glass slide, continue soaking until it can be removed with little effort.</li>
	<li>Once the coverslip is removed, hold the slide perpendicularly to a glass Petri dish containing xylene, carefully take a sharp, disposable razor blade and slide the blade in a single motion down the slide.&#160;</li>
	<li>Once the tissue is removed, quickly submerge the mammary pad into a xylene-filled glass Petri dish to ensure that the tissue does not air dry. 
	NOTE: Be careful with this step. The whole mount is thin (&#8804; 1 mm), and&#160;fragile from xylene processing. Any impressions or tears may change the morphology of the tissue and complicate later evaluations.</li>
</ol>
3. Tissue Processing
<ol>
	<li>Once the tissue is in the Petri dish, carefully transfer it to a labeled histology cassette. Grab the edge of the fat pad with blunt-nosed, serrated-tipped forceps to minimize tissue damage.
	<ol>
		<li>Using a razor, cut larger tissues (especially for rats) in half and place them in two separate labeled cassettes that will accommodate the tissue size. Place the tissue right-side up (use the lymph node as a reference). Ensure that the tissue without the lymph node is kept in the same, upright position when it is transferred to the cassette.</li>
	</ol>
	</li>
	<li>Place the cassette into a container of xylene for up to 2 h before placing it in the tissue processor. 
	NOTE: It is recommended to process the samples on the same day they are placed into the cassette. Extended soaking in xylene has not been tested.</li>
	<li>Load the maximum number of cassettes into the tissue processor by hand.</li>
	<li>Prior to beginning, program all the reagents that will be used for this process into the processor&#8217;s reagent list. To automate movement from one station to the next, use the menu options and create a new program. When all steps have been completed, select &#8216;OK&#8217; to proceed.&#160; 
	NOTE: The processor will allow the review of all station details before starting.
	<ol>
		<li>Automate the processor to soak the cassettes in xylene for 30 min at 37 &#176;C, followed by a second soak in fresh xylene under the same conditions. Automate the processor to remove the cassettes from the xylene solution and transfer the tissues to the next station containing a 1:1 xylene:molten paraffin mixture, set at 60 &#176;C, for 30 min. 
		NOTE: The processor will then transfer the cassettes into a new chamber that contains molten paraffin for 1 h. After 1 h, the samples will automatically be transferred to a new chamber of fresh molten paraffin and will be processed for an additional 2 h. Both steps are carried out at 60 &#176;C.</li>
		<li>The processor then transfers the cassettes to the station containing molten paraffin for 1 h. Then, the samples are automatically transferred to the final station of fresh molten paraffin and processed for an additional 2 h. Both steps are carried out at 60 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Embedding the Processed Mammary Tissue
<ol>
	<li>Place the cassettes in the 58 &#176;C paraffin holding tank of the embedding station until ready to embed the tissue in the mold.&#160;&#160;</li>
	<li>Remove the cassette cover to determine the best mold size for the tissue. Make sure that the mold is big enough to accommodate the tissue, and leave enough room to have a tissue-free border of paraffin around the perimeter.</li>
	<li>Add 3-4 mm of molten paraffin to the mold and orient the &#160;mammary whole-mount surface, which was adjacent to the bottom of the glass slide and the bottom of cassette, facing up in the mold.&#160;</li>
	<li>Transfer the mold to a cooling plate and quickly adjust the tissue as needed so that it is parallel to the mold bottom. 
	NOTE: Once the paraffin hardens, the tissue will besecured in place.</li>
	<li>Using warm forceps, place the labeled, bottom half of the cassette on top of the mold; press firmly using the forceps.</li>
	<li>Add additional molten paraffin to the mold in a continuous motion to cover the entire cassette. Move the mold from the warm plate to a cold plate to complete hardening of the block.</li>
	<li>Remove the block from the mold when the paraffin completely solidifies to avoid cracks or air bubbles. 
	NOTE: Store the paraffin-embedded tissues at room temperature until ready to section.</li>
</ol>
5. Sectioning the Paraffin-embedded Mammary Tissue on the Microtome
<ol>
	<li>Prior to sectioning, incubate the paraffin blocks at -20 &#176;C for 1 h. 
	NOTE: There will be residual mounting medium in the block from the original coverslipped whole-mount tissue. Chilling the block improves block sectioning and ribboning.</li>
	<li>Prepare the microtome by turning on the water bath, containing fresh distilled water, and adjust the temperature to 42-45 &#176;C. Place a fresh, low-profile blade onto the microtome and set it at 4 &#181;m.&#160; 
	NOTE: Section quality is reduced with sections thicker than 4 &#181;m because of the presence of residual mounting medium in the tissue.</li>
	<li>Insert the block into the microtome with the wax facing the blade and aligned with the vertical plane.&#160; Then, moisten a section of gauze pad in cold water and place it onto the block for several minutes.</li>
	<li>Section the block by turning the large wheel in a clockwise motion, in combination with the coarse advanced wheel, until a full face or representative section of block is obtained. 
	NOTE: The tissue is thin because of xylene clearing during the whole-mount process. Align block properly with the blade to minimize the number of cuts in obtaining a representative section.&#160;&#160;&#160;&#160;</li>
	<li>Pick the ribbon section up by hand, carefully float the ribbon in the warm (45 &#176;C) water bath (prepared in advance), allow tissue wrinkles to dissipate and carefully float a section onto a clean glass slide.</li>
	<li>Place the slides upright on a drying rack to remove excess water. Incubate the slides in a slightly opened slide box overnight at 37 &#176;C.</li>
</ol>
6. Automated and Manual H&#38;E Staining of Sectioned Mammary Tissues
<ol>
	<li>Prior to staining, the slides are stored at room temperature.&#160;</li>
	<li>For automated staining, select H &#38; E from the stain options on the main menu.&#160; Deparaffinization, staining, and dehydration are all completed on the automated stainer. Use mounting medium and a coverslip to mount the sections.&#160;</li>
	<li>If manually staining the slides, deparaffinize the sections by immersing the slides in xylene for 5 min. Transfer to &#160;fresh xylene and repeat for 5 min.</li>
	<li>Rehydrate the slides by immersing them twice in 100% ethanol for 3 min each, followed by two repetitions in fresh 95% ethanol and two separate washes in 1X wash buffer at 5 min each.</li>
	<li>Rinse the slides in distilled water.&#160;</li>
	<li>Add the slides to the hematoxylin for 1-2 min. Remove the slides and rinse them with running tap water for 5 min.&#160; 
	NOTE: Filter hematoxylin prior to each use.&#160;&#160;</li>
	<li>Place the slides in 1% glacial acetic acid for 30 s to promote color differentiation, and then rinse in running tap water for 1 min.</li>
	<li>Add slides to 1X PBS for 30 s to 1 min to blue the tissue sections, followed by a 5 min rinse in tap water and then in 95% alcohol for 15 - 20 s.</li>
	<li>Counterstain with eosin solution for 30 s to 1 min. Following this, dehydrate the slides twice in fresh 95% ethanol, followed by two fresh repetitions in 100% ethanol. Perform each wash for 5 min.</li>
	<li>Clear the slides in xylene, with two changes in xylene for 5 min each.</li>
	<li>Coverslip the tissue section with mounting medium and dry flat overnight at room temperature.</li>
</ol>

<ol>
	<li>Rudel, R. A., Fenton, S. E., Ackerman, J. M., Euling, S. Y., Makris, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+exposures+and+mammary+gland+development:+State+of+the+science,+public+health,+implications,+and+research+recommendations.">Environmental exposures and mammary gland development: State of the science, public health, implications, and research recommendations.</a> Environ Health Perspect. 119 (8), 1053-1061 (2011).</li><li>Keenan, K. P., Wallig, M. A., Hascheck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+via+Nurture:+Effect+of+Diet+on+Health,+Obesity,+and+Safety+Assessment.">Nature via Nurture: Effect of Diet on Health, Obesity, and Safety Assessment.</a> Toxicol Pathol. 41 (2), 190-209 (2013).</li><li>Stanko, J. P., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+branching+density+in+rat+mammary+gland+whole+mounts+using+the+Sholl+analysis+method.">Quantifying branching density in rat mammary gland whole mounts using the Sholl analysis method.</a> J Vis Exp. , (2017).</li><li>Filgo, A. 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=Mammary+gland+evaluation+in+juvenile+toxicity+studies:+Temporal+developmental+patterns+in+the+male+and+female+Harlan+Sprague+Dawley+Rat.">Mammary gland evaluation in juvenile toxicity studies: Temporal developmental patterns in the male and female Harlan Sprague Dawley Rat.</a> Toxicol Pathol. 44 (7), 1034-1058 (2016).</li><li>Tucker, D. K., Foley, J. K., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+high-quality+hematoxylin+and+eosin-stained+sections+from+rodent+mammary+gland+whole+mounts+for+histopathologic+review.">Preparation of high-quality hematoxylin and eosin-stained sections from rodent mammary gland whole mounts for histopathologic review.</a> Toxicol Pathol. 44 (7), 1059-1064 (2016).</li><li>Hvid, H., Thorup, O., Oleksiewicz, M. B., Sjogren, I., Jensen, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+preparation+of+tissue+sections+from+the+rat+mammary+gland.">An alternative method for preparation of tissue sections from the rat mammary gland.</a> Exp Toxicol Path. 63 (4), 317-324 (2011).</li><li>Munoz-de-Toro, 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=Perinatal+exposure+to+bisphenol-A+alters+peripubertal+mammary+gland+development+in+mice.">Perinatal exposure to bisphenol-A alters peripubertal mammary gland development in mice.</a> Endocrinology. 146 (9), 4138-4147 (2005).</li><li>Padilla-Banks, E., Jefferson, W. N., Newbold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+the+phytoestrogen+genistein+alters+mammary+gland+growth+and+developmental+programming+of+hormone+receptor+levels.">Neonatal exposure to the phytoestrogen genistein alters mammary gland growth and developmental programming of hormone receptor levels.</a> Endocrinology. 147 (10), 4871-4882 (2006).</li><li>Nikaido, 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=Effects+of+maternal+xenotestrogen+exposure+on+development+of+the+reproductive+tract+and+mammary+gland+in+female+CD-1+mouse+offspring.">Effects of maternal xenotestrogen exposure on development of the reproductive tract and mammary gland in female CD-1 mouse offspring.</a> Reprod Toxicol. 18 (6), 803-811 (2004).</li></ol>

The authors have no conflicting interests to declare in relation to the research, authorship, and/or publication of this article.

The mammary gland whole mount is a powerful tool that may be used to illustrate the normal mammary development and morphological alterations that may arise and persist following exposure to chemicals, including endocrine disruptors. When a whole mount and H&#38;E section from the same animal are assessed together, they can provide an accurate visual snapshot of early alterations, which can progress into a more diseased state.
The ability to obtain this useful information lies in ensuring that ...

The mammary whole mountis a useful and inexpensive method implemented in many rat and mouse studies to understand both normal and chemically-induced, abnormal development. In general, a mammary gland collected at various stages during rodent development (i.e., adolescence, puberty, mid- to late-gestation, and the involution phase) will show morphological changes in tissue and cellular architecture influenced by paracrine, endocrine, and autocrine factors1. In the aging rat, the epithelial and stromal portions of the gland become increasingly dense, which makes measuring morphological parameters difficult in a whole mount. Thus, it is common practice to collect a gland for histological evaluation to identify changes on a microscopic level. Both processes are especially useful in studies involving chemical exposure (i.e., environmental, or pharmaceutical) or to examine the morphological changes accompanied by genetic alterations.
Techniques and capabilities for using the mammary gland in risk evaluation continue to evolve. While whole-mount preparation is routine and standardized, modifications to sectioning continue2,3. Many research groups from various backgrounds (i.e., academia, government, and industry) have adapted coronal/longitudinal sections of mammary tissue as a preferred method, while some labs still use cross-cut sections through the skin4. The latter method results in an inordinate representation of epidermis (skin), rather than the tissue of interest: the mammary gland epithelium2. Coronal or longitudinal sections are more useful for characterizing normal tissue5 and greatly improve the detection of abnormalities and lesions due to the increased surface area and number of structures present. Overall, this makes the whole mount a suitable method for the comparative histopathological assessment of the mammary gland1,2. Whether using a mouse or a rat, retrieval, and preservation of the 4th and 5th inguinal glands is highly recommended for the comparison of the whole mount to a contralateral H&#38;E section.
When used in combination, the H&#38;E section and whole mount provide an accurate account of the cellular and morphological changes induced by environmental exposure. This is especially true in certain rodent strains in which the model possesses a low susceptibility to tumor formation or a tumor is not grossly visible. In some circumstances, obtaining the required tissue may not always be possible using both techniques (i.e., insufficient tissue quantity, resources, or unexpected experimental results). In our case, abnormalities were observed in the mammary whole mount, whereas histopathological findings in the contralateral H&#38;E gland were mostly normal. The overwhelming discrepancy between the contralateral glands led us to develop an economical and efficient procedure to identify the abnormalities in the whole mounts. Using a modified processing and embedding procedure, high-quality H&#38;E-stained sections were prepared from paraffin-embedded whole mounts. We envision this protocol becoming a powerful detection tool for chemical exposure effects, enhancing inter-lab comparisons.

<table border="0" cellpadding="0" cellspacing="0" width="1013">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Xylene</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:152px;">214736</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Permount</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP15-100</td>
			<td>mounting media for coverslipping whole mounts and H&#38;E sections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Leica automated processor</td>
			<td>Leica Biosystems</td>
			<td>Model # ST5020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">HM 355S Automatic Microtome</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>905200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Paraffin</td>
			<td>Leica Biosystems</td>
			<td style="width:152px;">EM-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Superfrost plus microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">4951PLUS-001</td>
			<td>electrostatically charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fisher Finest 24x60x1</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">12-548-5P</td>
			<td>coverslips</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Varistain Gemini ES Automatic slide stainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>A78000014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Sta-On</td>
			<td>Leica Biosystems</td>
			<td>3803107</td>
			<td>liquid adhesive; used in water bath at sectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Harris Hematoxylin 72711</td>
			<td>Richard-Allan Scientific (Part of Thermo Scientific)</td>
			<td>72711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Eosin</td>
			<td>Leica Biosystems</td>
			<td>3801600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Lithium Carbonate</td>
			<td>SkyTech Laboratories</td>
			<td style="width:152px;">LCQ500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Glacial Acetic Acid</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">A38-500</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Chloroform</td>
			<td>Macron Fine Chemicals</td>
			<td style="width:152px;">4440-04</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505001050000</td>
			<td>needed in Carnoy&#39;s fixative and washing slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">95% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505011137320190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Carmine Alum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">C1022-25G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:385px;">Aluminum potassium sulfate dodecahydrate, Sigma Ultra</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">A7210-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Automation wash buffer, 20X</td>
			<td>Biocare Medical</td>
			<td>TWB945</td>
			<td></td>
		</tr>
	</tbody>
</table>

sectioning mammary gland whole mounts for lesion identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

This method is effective at assisting with diagnoses that may have otherwise been missed if the original contralateral H&#38;E section of the mammary pad did not show any histological changes. However, the outcome will only be useful if care is taken during the initial whole-mount preparation, as well as during the preparation of the tissue for the histological evaluation. Paraffin embedding will provide protection and will help to preserve the tissue for future sectioning.
<p class="...

Watch this Scientific Journal Video about Sectioning Mammary Gland Whole Mounts for Lesion Identification at JoVE.com

Sectioning Mammary Gland Whole Mounts for Lesion Identification

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

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10.3K Views.  University of North Carolina at Chapel Hill. We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Video: Sectioning Mammary Gland Whole Mounts for Lesion Identification

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...