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><tbody><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>214736</td><td></td></tr><tr><td>Permount</td><td>Thermo Fisher Scientific</td><td>SP15-100</td><td>mounting media for coverslipping whole mounts and H&amp;amp;E sections</td></tr><tr><td>Leica automated processor</td><td>Leica Biosystems</td><td>Model # ST5020</td><td></td></tr><tr><td>HM 355S Automatic Microtome</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>905200</td><td></td></tr><tr><td>Paraffin</td><td>Leica Biosystems</td><td>EM-400</td><td></td></tr><tr><td>Superfrost plus microscope slides</td><td>Thermo Fisher Scientific</td><td>4951PLUS-001</td><td>electrostatically charged</td></tr><tr><td>Fisher Finest 24x60x1</td><td>Thermo Fisher Scientific</td><td>12-548-5P</td><td>coverslips</td></tr><tr><td>Varistain Gemini ES Automatic slide stainer</td><td>Thermo Fisher Scientific</td><td>A78000014</td><td></td></tr><tr><td>Sta-On</td><td>Leica Biosystems</td><td>3803107</td><td>liquid adhesive; used in water bath at sectioning</td></tr><tr><td>Modified Harris Hematoxylin 72711</td><td>Richard-Allan Scientific (Part of Thermo Scientific)</td><td>72711</td><td></td></tr><tr><td>Eosin</td><td>Leica Biosystems</td><td>3801600</td><td></td></tr><tr><td>Lithium Carbonate</td><td>SkyTech Laboratories</td><td>LCQ500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Thermo Fisher Scientific</td><td>A38-500</td><td>needed in Carnoy&#039;s fixative</td></tr><tr><td>Chloroform</td><td>Macron Fine Chemicals</td><td>4440-04</td><td>needed in Carnoy&#039;s fixative</td></tr><tr><td>100% Ethanol</td><td>The Warner Graham Company</td><td>6505001050000</td><td>needed in Carnoy&#039;s fixative and washing slides</td></tr><tr><td>95% Ethanol</td><td>The Warner Graham Company</td><td>6505011137320190</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma-Aldrich</td><td>C1022-25G</td><td></td></tr><tr><td>Aluminum potassium sulfate dodecahydrate, Sigma Ultra</td><td>Sigma-Aldrich</td><td>A7210-500G</td><td></td></tr><tr><td>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 ...

sectioning-mammary-gland-whole-mounts-for-lesion-identification

Research

JoVE Journal

Cancer Research

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 ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Developmental Biology

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Medicine

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Biology

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Summary

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Chapters in this video

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

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

Mammary Epithelial Transplant Procedure

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Evaluation of Mammary Gland Development and Function in Mouse Models

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology