The authors would like to acknowledge April Wiles and Dr. Roger Broderson for assistance with animal necropsy and gland isolation, respectively. Funding for this work was supported by the Philadelphia College of Osteopathic Medicine Centers for Chronic Disorders of Aging.

Procedures involving animal subjects in this protocol were reviewed and approved by the Institutional Animal Care and Use Committee of the Philadelphia College of Osteopathic Medicine and all techniques were conducted under strict ethical guidelines.
1. Sample Procurement and Processing
<ol>
	<li>Select appropriate animal subject and place into a CO2 chamber. For this experiment, use 8-10 week old female nulliparous B6.Cg-Cav1tmMIs/J and C57BI/6J.</li>
	<li>Turn on gas flow to 30-40%. Once the animal is visibly unconscious after about 2 min, open the gas valve to full pressure for an additional 5 min. 
	NOTE: Confirm animal death by observing visible signs of breathing (e.g. movement of the chest) for a period of 10 min after cessation of CO2 delivery. If the animal is still alive, it may be placed back in the CO2 chamber. Cervical dislocation may also be used although it is not recommended as blood may accumulate around the mammary glands interfering with the dissection and results.</li>
	<li>Following sacrifice, pin carcass in a supine position and saturate with 70% ethanol.</li>
	<li>Pinch the pelt just above the pubis using forceps and nick with small surgical scissors. Rotating the scissors, cut the pelt along the ventral midline moving caudal to cranial.</li>
	<li>With larger scissors or a hemostat, bluntly dissect the subcutaneous fascia bilaterally, using caution not to puncture the peritoneum. Cut along the horizontal margins at both distal ends of the incision.</li>
	<li>Pin the pelt flaps open and spray again with 70% ethanol. 
	NOTE: Although the use of 70% ethanol permitted better visual distinction between the gland and the surrounding subcutaneous tissue, be careful to avoid drying of the tissue as a result of use of this solution. To avoid drying, remove the gland in a time frame not to exceed 4 min. As an alternative to 70% ethanol, the investigator may also substitute a general isolate buffer, such as 1x PBS, to avoid tissue desiccation.</li>
	<li>Locating the mammary glands of interest, slide a #4 scalpel blade along the inside of the pelt flaps, cutting the mammary gland and associated cutaneous adipose free from the dermis. 
	NOTE: 3.5x surgical micro loupes may aid in easier visualization of the glands.</li>
	<li>Immediately submerge newly isolated gland in conical tube containing 10:1 solution-tissue volume of 10% neutral buffered formalin for 24-48 h. 
	NOTE: Depending on intent of study, many alternative fixatives may be used such as paraformaldehyde, ethanol for genomic studies, and commercially available RNA preserving buffers.</li>
	<li>Follow institutional protocols for paraffin embedding, submit samples to a vendor for processing, embedding, and slicing/slide mounting. Section tissues at 5 &#181;m. 
	NOTE: Due to excess adipose surrounding gland, consider removing 100-200 &#181;m of tissue before sectioning and staining.</li>
</ol>
2. Tissue Staining
<ol>
	<li>Immunohistochemistry
	<ol>
		<li>Place slides to be stained on heat block set at 58 &#730;C for 1 h to melt paraffin. 
		CAUTION: Melting paraffin wax may run off slide. Monitor closely or place wipe under slide to capture runoff wax. 
		NOTE: This step is not essential and may be skipped. If results are not as expected, adding this step back may improve results.</li>
		<li>Incubate slides in a slide jar containing 100% xylene for 30 min ensuring complete submersion. Repeat once. 
		NOTE: At this point, visual inspection should be performed to ensure that tissue sections are free of adipose tissue and paraffin. If additional adipose tissue or wax is evident, the slide should be re-submerged in xylene. Use caution to minimize added exposure to xylene as this can cause shrinkage of the tissue.</li>
		<li>Rehydrate tissues in a slide jar containing 100% ethanol for 10 min. Repeat once.</li>
		<li>Move slides to a slide jar containing 95% ethanol for 10 min. Repeat once.</li>
		<li>Move slides to a slide jar containing 75% ethanol for 5 min.</li>
		<li>Move slides to a slide jar containing 50% ethanol for 5 min.</li>
		<li>Boil 10 mM sodium citrate solution (pH 6.0) in a hot plate or a microwave and pour into Coplin jar. 
		CAUTION: Carefully monitor solution while heating and take care to avoid boil over. Container will be very hot. Use protection to avoid burns.</li>
		<li>Slowly place slides into Coplin jar, ensuring complete submersion, and incubate at 100 &#176;C for 10 min to retrieve epitopes. Remove and carefully dry slides.</li>
		<li>Draw a barrier around tissue with a hydrophobic marker.Add enough endogenous enzyme blocker (hydrogen peroxide and sodium azide, available commercially) to cover tissue and incubate for 10 min.</li>
		<li>Submerge slides in 1x phosphate buffered saline (PBS) for 10 min.</li>
		<li>Add enough 10% donkey serum in 1x PBS to cover tissue and incubate for 1 h at room temperature in a humidified chamber. 
		NOTE: The experiment can be paused here by storing the slides in the humidified chamber at 4 &#176;C overnight. While donkey serum was found to yield optimal staining results, the investigator may wish to test different sera such as bovine serum albumin (BSA), goat serum or horse serum to determine optimal results. If using BSA, the investigator should prepare this fresh before use.</li>
		<li>Decant excess blocker from slides and add properly diluted primary antibody in 1% serum directly to slides ensuring that the tissue is evenly covered in solution. Incubate for 30 min at room temperature in a humidified chamber. 
		NOTE: This step may be continued for longer time durations with humid chamber stored at 4 &#176;C. Make sure to include proper negative control slides (e.g. a slide with no primary antibody, known antigen-negative tissue, etc.) at this step.</li>
		<li>Carefully wash slides by flowing about 1 mL of diH2O over tissue and incubate in 1 change of 1x PBS for 5 min.</li>
		<li>Decant excess PBS and add enough horseradish peroxidase label to cover tissue and incubate for 30 min in a humidified chamber. 
		NOTE: At this step, the investigator may wish to proceed to fluorescent staining using fluorescently conjugated secondary antibodies. If this is desired, the next steps described should be modified accordingly.</li>
		<li>Carefully wash slides by flowing about 1 mL of diH2O over tissue and incubate in 1 change of 1x PBS for 5 min.</li>
		<li>Make diaminobenzidine (DAB) plus chromogen solution according to the manufacturer suggested ratio and mix by vortex. 
		NOTE: Many ready-made kits are commercially available aside from the one used in this protocol.</li>
		<li>Decant excess buffer from slides and add 2-3 drops (10-50 &#181;L) of chromogen stain directly to tissues and incubate for 5 min in humid chamber. Carefully wash slides by flowing about 1 mL diH2O over tissue. 
		CAUTION: DAB-chromogen is highly toxic. Avoid direct contact of stain with skin and collect flow off in hazardous waste.</li>
	</ol>
	</li>
	<li>Hematoxylin and Eosin (H&#38;E)
	<ol>
		<li>Following final rinse after chromogen incubation, submerge slides in Harris hematoxylin for 2-2.5 min. Rinse slides gently under tap water for 1-2 min. 
		NOTE: Take care to avoid direct jet of water onto tissues.</li>
		<li>Submerge slides for 2-3 s in differentiation solution (0.25 mL of hydrochloric acid in 100 mL of 70% ethanol). Rinse slides gently under tap water for about 1-2 min.</li>
		<li>Submerge slides in blue agent (4.5 mg calcium carbonate in 100 mL tap water, pH adjusted to 9.4) for 60 s. Rinse slides in 95% ethanol for 30 s.</li>
		<li>Submerge slides in alcoholic eosin Y for 2-3 min.</li>
		<li>Dehydrate tissue in 2 changes of 95% ethanol for 1 min each.</li>
		<li>Dehydrate tissue in 1 change of 100% ethanol for 1 min.</li>
		<li>Carefully dry slides with a lint-free wipe. Apply 1 drop (about 100-200 &#181;L) of synthetic, non-aqueous, resin-based mounting media to slide and apply coverslip. 
		NOTE: If a different staining method was elected, a different mounting media may be required. For instance, if the investigator chose a fluorescent-conjugated secondary antibody, an aqueous mount would be more ideal.</li>
		<li>Allow slides to set overnight at room temperature.</li>
	</ol>
	</li>
	<li>Picrosirius Red (PSR) Staining
	<ol>
		<li>Select slides to be stained and follow immunohistochemistry protocol through step 6 (50% ethanol soak).</li>
		<li>Submerge slides in PSR stain for 1 h.</li>
		<li>Submerge slides in 0.5% acetic acid for 1-2 s, twice to differentiate stain.</li>
		<li>Dehydrate tissues in 2 changes of 95% ethanol for 1 min each.</li>
		<li>Dehydrate tissues in 1 change of 100% ethanol for 1 min.</li>
		<li>Clear slides by briefly submerging in 100% xylene for about 3-4 s.</li>
		<li>Carefully dry slides with a lint-free wipe. Apply 1 drop (about 100-200 &#181;L) of synthetic, non-aqueous, resin-based mounting media to slide and apply coverslip.</li>
	</ol>
	</li>
</ol>
3. Sample Analysis
<ol>
	<li>Ductal Analysis
	<ol>
		<li>Select slides stained for &#945;-SMA. Using a light microscope fitted with a camera-mounted objective, collect representative images at 20X magnification.</li>
		<li>Using ImageJ (NIH), distinguish and count ducts in each image. These can be enumerated manually or one may assign a numerical score to individual ducts. To assign a numerical score, open ImageJ and select Plugins. Next select Analyze and choose Cell Counter from the drop down menu. Click Initialize then highlight Type 1 to begin labeling ducts. When finished, select Results to view the ductal count. 
		NOTE: Take care to verify structures are ductal and not vascular.</li>
		<li>In &#39;Set Measurement&#39; options, check &#39;Perimeter&#39; and &#39;Area&#34;.</li>
		<li>Using the freehand polygon tool, draw a line around each duct at the myoepithelial compartment (visibility aided by &#945;-SMA stain). Select &#39;Measure&#39; and record the perimeter and area.</li>
		<li>Again using the freehand polygon tool, draw a line along the apical side of the ductal epithelium within the interior of the lumen. Select &#39;Measure&#39; and record the perimeter and area.</li>
		<li>Subtract the perimeter of the luminal compartment from the perimeter of the myoepithelial compartment in order to obtain the circumference of the ductal epithelium. Subtract the area of the luminal compartment from the area of the myoepithelial compartment to obtain the area of the ductal epithelium.</li>
	</ol>
	</li>
	<li>Immunohistochemistry Analysis
	<ol>
		<li>Upload brightfield images to an analytical software, such as ImageJ or FIJI Suite. 
		NOTE: This protocol outlines the steps for staining analysis using ImageJ and the IHC Toolbox plugin.</li>
		<li>Open the IHC Toolbox from the Plugin menu. In the &#34;Select Model&#34; combo box that opens, select appropriate stain (i.e. H-DAB, PSR, etc.). Select the &#34;Color&#34; option to isolate the stain. This will open a result window. 
		NOTE: This method is appropriate if a protein of interest is located in the extracellular or cytosolic spaces, or is bound to the plasma membrane. If the protein of interest is nuclear, selecting &#34;Nuclei&#34; will prompt the plugin to analyze the image for positively stained nuclei.</li>
		<li>Use the Color Chooser Slide to ensure proper isolation of the stain without background inclusion or excessive stain exclusion. 
		NOTE: If the automatic mode is unable to detect the stain or does not result in acceptable images, draw a square Region of Interest (ROI) over an identified stained region. On the IHC Toolbox window, select &#34;Train&#34; to direct the plugin to the appropriate target.</li>
		<li>Convert the image to 16-bit. Go to Image &#8594; Type &#8594;&#160;16 bit.</li>
		<li>Threshold the image. Go to Image&#160;&#8594;&#160;Adjust &#8594;&#160;Auto Threshold.</li>
		<li>Set the image measurement scale by drawing a line ROI directly over the scale bar in the image then assigning the length. To set the scale bar, go to Analyze &#8594;&#160;Set Scale. Input the number of pixels per the desired unit of length (e.g. 120 pixels per 50 &#181;m).</li>
		<li>Measure the resultant area and mean grey value. Go to Analyze &#8594;&#160;Set Measurements and collect the measurement by clicking Analyze &#8594;&#160;Measure.</li>
	</ol>
	</li>
</ol>

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<li>Barkan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metastatic+growth+from+dormant+cells+induced+by+a+col-I-enriched+fibrotic+environment%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Metastatic growth from dormant cells induced by a col-I-enriched fibrotic environment.</a> Cancer Res. 70 (14), 5706-5716 (2010).</li>
<li>Levental, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li>
<li>Wang, J. P., Hielscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibronectin%3A+How+Its+Aberrant+Expression+in+Tumors+May+Improve+Therapeutic+Targeting%5BTitle%5D)+AND+%22J+Cancer%22%5BJournal%5D)">Fibronectin: How Its Aberrant Expression in Tumors May Improve Therapeutic Targeting.</a> J Cancer. 8 (4), 674-682 (2017).</li>
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The authors have nothing to disclose.

In the paper, we have described a technique to isolate intact mouse mammary glands for downstream histological analyses of ECM expression and ductal morphology. With respect to analyses of ductal morphology, this methodology enables the rapid investigation of ductal architecture based off of stained histological sections. Other methods of ductal analyses rely on injections of dyes to enable visualization of the ductal tree, methods which may be technically challenging and time consuming.
In br...

Breast cancer is characterized by a substantial degree of tissue fibrosis1,2,3,4. Referred to as the ECM, this non-cellular entity is found in varying degrees in all tissues and is primarily comprised of a complex meshwork of fibrillar and non-fibrillar collagens, elastin, and glycoproteins in addition to various signaling molecules that are sequestered in this matrix. Under homeostatic conditions, the deposition and degradation of the ECM is tightly controlled.5 During breast tumorigenesis, the balance of ECM deposition and degradation is disrupted. As such, breast tumors have been reported to express abundant ECM proteins such as collagens, fibronectin and tenascin-C amongst others.6 The abnormal expression of these proteins in addition to increased patterns of matrix crosslinking has been documented to promote breast tumor progression, metastasis and therapy resistance1,3,4,7,8,9.
To assess ECM composition and ductal morphology, isolation of intact mammary glands was performed. Here, we used female nulliparous mice deficient for caveolin-1, an integral membrane protein which has been linked to an aggressive breast tumor signature10,11,12, and control female nulliparous B6 mice. Histological processing and staining of these tissues permitted the identification of several ECM proteins along with characterization of ductal morphology.
Overall, the isolation of whole, intact mammary glands gives researchers the opportunity to investigate tissue-wide morphological or cellular changes occurring in response to exogenous or endogenous factors. Drawbacks of the technique are associated with analyses of 2 dimension (2D) tissue sections as opposed to a 3D perspective, which would yield a more complete picture of the complex morphology of the ductal tree. Given the complexity of cell-cell and cell-ECM interactions that take place in the mammary gland, the isolation of whole, intact glands is advantageous for efficiently analyzing ductal morphology and protein expression in various regions of the murine mammary gland.

<table><tbody><tr><td>Light Microscope</td><td>Olympus</td><td>BX43</td><td></td></tr><tr><td>Microscope Camera</td><td>Olympus</td><td>DP73</td><td></td></tr><tr><td>Image Analysis Software</td><td>Olympus</td><td>cellSens Entry software</td><td></td></tr><tr><td></td><td>NIH</td><td>ImageJ</td><td></td></tr><tr><td>3.5x-R Surgical Micro Loupes</td><td>Rose Micro Solutions</td><td></td><td>Magnification at researcher&#039;s preference</td></tr><tr><td>Mayo Scissors</td><td>Medline</td><td>DYND04035</td><td></td></tr><tr><td>Staining Rack</td><td>Fisher Scientific</td><td>121</td><td></td></tr><tr><td>Staining Dish</td><td>Fisher Scientific</td><td>112</td><td></td></tr><tr><td>Coplin Jars</td><td>Fisher Scientific</td><td>19-4</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>12-550-15</td><td>Size appropriate for tissue</td></tr><tr><td>IHC EnVision+ Kit (HRP, Mouse, DAB+)</td><td>Dako</td><td>K400611-2</td><td></td></tr><tr><td>Picrosirius Red Kit</td><td>Abcam</td><td>AB150681</td><td></td></tr><tr><td>Eosin Y, alcoholic</td><td>Sigma-Aldrich</td><td>HT110132</td><td></td></tr><tr><td>Harris Hematoxylin</td><td>Sigma-Aldrich</td><td>HHS16</td><td></td></tr><tr><td>Donkey Serum</td><td>EMD Millipore</td><td>S30</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Xylenes, Reagent Grade</td><td>Sigma-Aldrich</td><td>214736</td><td></td></tr><tr><td>Ethanol, 200 proof</td><td>Sigma-Aldrich</td><td>792780</td><td>suitable for molecular biology</td></tr><tr><td>Phosphate Buffered Saline, 1x</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Sodium Citrate</td><td>Fisher Scientific</td><td>S279-500</td><td></td></tr><tr><td>Calcium Carbonate</td><td>Sigma-Aldrich</td><td>202932</td><td></td></tr><tr><td>Permanent Mounting Medium</td><td>Dako</td><td>S1964</td><td></td></tr><tr><td>Eukitt&#039;s Mounting Medium</td><td>Sigma-Aldrich</td><td>3989</td><td></td></tr><tr><td>Fibronectin antibody</td><td>Abcam</td><td>AB23750</td><td></td></tr><tr><td>Tenascin-C antibody</td><td>Abcam</td><td>AB108930</td><td></td></tr><tr><td>Alpha Smooth Muscle Actin antibody</td><td>Abcam</td><td>AB124964</td><td></td></tr><tr><td>Dako Envision Dual Link System HRP</td><td>Dako</td><td>K4065</td><td></td></tr></tbody></table>

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.

Female mice have 5 pairs of mammary glands. Specifically, there is one pair of cervical glands (#1), two pairs of thoracic glands (#2 and #3), one pair of abdominal glands (#4), and 1 pair of inguinal glands (#5) (Figure 1A). Here, we isolated the #4 glands as they are readily identifiable. In some circumstances, both #4 and #5 glands were isolated together as distinction between the two was difficult. To isolate intact #4 abdominal mammary g...

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

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 ...

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16.5K Views.  University of Nebraska Medical Center. 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. 

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

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.

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 ...

Cancer Research

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Medicine

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

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

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 ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

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 ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...