This work was supported partially by (1) the Department of Defense Breast Cancer Research Program (US Army Medical Research Acquisition Activity) award #W81XVVH-10-1-0781 to LAL and VE, and (2) the Susan G. Komen Foundation grant IR122224446 to LAL and VE. Pathology support and tissue grossing was kindly provided by Inova Fairfax Pathology Department, Dr. Hassan Nayer, Dr. Geetha A. Menezes, and Dr. Charles Bechert. Patient consent and sample procurement was expertly guided by Inova Fairfax Hospital clinical research coordinators Holly Gallimore, Heather Huryk, and Emil Kamar.

Human breast tissue was collected from patients enrolled in a research study, with written informed consent, following Department of Defense, George Mason University, and Inova Health System Institutional Review Board&#8217;s approved protocols.
1. Prepare Nutrient Rich Medium with Growth Factors and Antibiotics
<ol>
	<li>Prepare stock solutions of insulin, epidermal growth factor (EGF), streptomycin sulfate and gentamicin sulfate.
	<ol>
		<li>Reconstitute insulin in sterile filtered water to a final concentration of 10 mg/ml. Aspirate 10 ml of Type 1 reagent grade water in a sterile 10 ml disposable syringe. Attach a 0.22 &#181;m polyethersulfone filter to the syringe. Dispense the sterile filtered water into a sterile 15 ml conical tube.</li>
		<li>Add 10 ml of sterile filtered water to a 100 mg vial of insulin. Mix the contents briefly on a vortex mixer. Keep the insulin vial on ice. Dispense 450 &#181;l of the insulin stock solution into labeled, sterile microcentrifuge tubes and store at -20 &#176;C. Insulin stock solution is stable until the expiration date on the vial.</li>
		<li>Prepare stock solution of epidermal growth factor (EGF): Add 500 &#181;l sterile water to 500 &#181;g EGF (stock 1 &#181;g/&#181;l). Mix the contents briefly on a vortex mixer. Keep the EGF vial on ice.
		<ol>
			<li>Prepare a working stock solution of EGF: Add 100 &#181;l of the stock EGF solution to 900 &#181;l nutrient medium (working stock will be 0.1 &#181;g/&#181;l). Mix the contents briefly on a vortex mixer. Dispense 50 &#181;l of the EGF working stock solution into labeled, sterile microcentrifuge tubes and store at -80 &#176;C. 
			NOTE: EGF stock solution (1 &#181;g/&#181;l) is stable for 1 year at -80 &#176;C; working stock solution (0.1 &#181;g/&#181;l) is stable for 2 months at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Weigh 40 mg of streptomycin sulfate on an analytical balance. Place the streptomycin sulfate into a sterile 15 ml conical tube. Add 4 ml of nutrient medium. Mix the contents briefly on a vortex mixer. The solution should be slightly pink. Store at 4 &#176;C protected from light. Streptomycin sulfate solution is stable for 7 days.</li>
		<li>Weigh 20 mg of gentamicin sulfate on an analytical balance. Place the gentamicin sulfate into a sterile 15 ml conical tube. Add 2 ml of nutrient medium. Mix the contents briefly on a vortex mixer. The solution should be yellow. Store at 4 &#176;C protected from light. Gentamicin sulfate solution is stable for 7 days.</li>
	</ol>
	</li>
	<li>Prepare two 200 ml batches of nutrient medium supplemented with human recombinant EGF (10 ng/ml final conc.), insulin (10 &#956;g/ml final conc.), streptomycin sulfate (100 &#956;g/ml final conc.) and gentamicin sulfate (20 &#956;g/ml final conc.).
	<ol>
		<li>Add 200 ml nutrient medium to a vacuum filter flask equipped with a 0.2 &#181;m polyethersulfone filter flask and a 250 ml receiving bottle. Add 20 &#181;l working stock EGF, 200 &#181;l stock insulin, 2 ml stock streptomycin sulfate, and 400 &#181;l stock gentamicin sulfate to the 200 ml of nutrient medium. Attach the filter flask to a vacuum. Filter the medium. Discard the filter and label the flask as &#8220;nutrient rich medium&#8221;. Store at 4 &#176;C for up to 14 days.</li>
	</ol>
	</li>
</ol>
2. Tissue Acquisition and Grossing
<ol>
	<li>In the operating suite, maintain sterile technique after procuring the breast tissue. Place the breast tissue in a sterile tray and cover the tray with sterile plastic wrap.
	<ol>
		<li>Transport the tissue in the covered tray to Radiology/Pathology as required by your institution. Do not open the tray. Transport times vary within institutions. Tissue can remain viable in this covered tray at RT for up to 45 min post excision. However, prompt processing of the tissue into nutrient rich medium provides optimal conditions for subsequent organoid culture.</li>
	</ol>
	</li>
	<li>Gross tissue dissection to identify areas of DCIS within the breast tissue: Use sterile gloves, blades, scalpels, tissue marking dyes, and vinegar during tissue dissection to maintain tissue sterility.
	<ol>
		<li>Clean the work area with 70% ethanol or 1% bleach. Open the sterile gloves and place the glove wrapper on the work surface. Place the sterile interior of the glove wrapper face up. Put on the sterile gloves.
		<ol>
			<li>Clean the surface of the specimen container with 70% ethanol. Remove the plastic wrap from the tissue specimen container and place the specimen on the glove wrapper.</li>
		</ol>
		</li>
		<li>Dip two cotton tipped swabs into the tissue marking dye. Apply the dye to the surface of the tissue by rolling the swabs across the tissue surface. 
		NOTE: Each pathology department has a standardized dye color/tissue orientation protocol. Tissue is labeled with dye to orient the tissue in relation to its position in the patient. The dye is blotted or painted onto the tissue. Do not pour the dye over the tissue. Pouring the dye can cause the dye to run/drip into tissue crevices thus confusing the orientation of surgical margins. Tissue orientation provides the surgeon and pathologist with anatomical landmarks to a) describe the tissue specimen, and b) determine the location of the surgical margins in relation to the tumor.</li>
		<li>Pour distilled white vinegar (5% acetic acid) onto sterile gauze pads. Blot the dyed tissue with the vinegar soaked gauze. Discard the gauze. Vinegar is used to fix the tissue marking dyes.</li>
		<li>Cut the breast tissue into vertical slices approximately 5 mm thick. Do not cut all the way through the tissue (Figure 2). Observe/palpate the tissue for areas of calcification. Identify DCIS lesions by their characteristic firm, pale appearance surrounded by reddish, rubbery borders that feel gritty (due to calcium spicules). 
		NOTE: In some cases, comedo (pimple-like) areas can be seen as white dots, representing necrotic material from large diameter DCIS lesions.</li>
		<li>Cut out areas of DCIS/calcified breast tissue, including a small amount of surrounding breast tissue. Place the breast tissue into a sterile 50 ml tube containing 20-30 ml of nutrient rich medium prepared in step 1.
		<ol>
			<li>Mix the tissue and medium by gently inverting the tube several times. Discard the medium and add fresh medium. Place the tube containing the tissue and medium into an insulated container and transport the tissue to the tissue culture lab.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Tissue Culture
<ol>
	<li>Working in a biological safety cabinet, pour the tissue and a small amount of the nutrient medium into a sterile Petri dish. Using a sterile scalpel cut away and discard the fibrous tissue. Cut the breast tissue into pieces (organoids) approximately 3 mm2 (Figure 1C-D). Try to cut the tissue so that each organoid contains at least one discernable duct segment with surrounding stroma.</li>
	<li>Open the lid of the tissue culture flask. Using sterile tweezers or forceps, place the organoids into the flask. Close the lid. Discard the petri dish.</li>
	<li>Add 11 ml serum-free nutrient rich medium (prepared in step 1) to the tissue culture flask. Close the flask and swirl the flask so the organoids and medium are evenly distributed across the flask surface (Figure 1E-F).</li>
	<li>Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere for 2 days. Do not move the flask during this time.</li>
	<li>On day 2 post incubation, remove the flask from the incubator to check for potential bacterial/fungal contamination. Avoid sudden, sharp movements, or swirling of the flask. Place the flask on an inverted microscope stage.
	<ol>
		<li>Observe the medium for bacteria, yeast, and/or fungus. If no contamination is noted, return the flask to the incubator for an additional day. If contamination is noted, discard the flask and contents in an appropriate container.</li>
	</ol>
	</li>
	<li>On day 3 post incubation, replace the conditioned medium with fresh medium.
	<ol>
		<li>Place 11 ml of medium in a sterile tube at 37 &#176;C for 20-30 min. Remove the tissue culture flask from the incubator. Without disturbing the organoids, remove and discard the medium in the flask using a sterile serologic pipette.</li>
		<li>Using a new sterile serologic pipette, add 11 ml of the pre-warmed fresh medium. Very gently rotate the flask to distribute the medium across the flask surface.</li>
		<li>Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere.</li>
	</ol>
	</li>
</ol>
4. Maintenance of Established Organoid/Epithelial Cell Colonies
<ol>
	<li>Prepare fresh medium every 2 weeks and replace the media in the tissue culture flask 3 times per week.
	<ol>
		<li>Place 11 ml of medium in a sterile container at 37 &#176;C for 20-30 min. Remove the flask from the incubator. Using a sterile serologic pipette, remove and discard the conditioned medium from the flask, being careful not to disturb the organoids.</li>
		<li>Using a new sterile serologic pipette, add 11 ml of the pre-warmed fresh medium. Very gently rotate the flask to distribute the medium across the flask surface. Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>After 10 - 14 days in culture, remove any pieces of tissue in the culture flask that are not adherent.
	<ol>
		<li>Periodically harvest cells and/or organoids from the flask for propagation into new culture flasks, for xenograft transplantation, or for phenotypic and/or molecular analysis.</li>
		<li>Remove the tissue culture flask from the incubator. Spray the flask with 70% ethanol. Wipe off excess ethanol on the flask with a clean paper towel that has been sprayed with 70% ethanol.</li>
		<li>Organoid propagation:
		<ol>
			<li>Open the lid of the flask and place the lid face-up in the biological safety cabinet. Under microscopic visualization, locate the organoid(s) to be harvested. Use sterile tweezers or forceps to pick-up an organoid.</li>
			<li>To propagate the organoid in a new culture flask, place the organoid in the new flask. Add 11 ml fresh, warm medium. Incubate at 37 &#176;C, in a humidified 5% CO2 atmosphere as described in steps 3.3 &#8211; 3.6.3. Replace the cell culture medium in the tissue culture flask three times per week.</li>
		</ol>
		</li>
		<li>Harvesting cells:
		<ol>
			<li>Under direct microscopic vixualization, gently scrape and aspirate cells and mammospheres using a 1,000 &#181;l pipette with sterile disposable pipette tips. Aspirate the cells and surrounding medium. Dispense the cells/medium into a sterile microcentrifuge tube.</li>
		</ol>
		</li>
		<li>Spin the cells briefly in a mini-centrifuge at 12,100 x g for 5 sec. Remove and discard the medium.
		<ol>
			<li>For DNA analysis, immediately freeze the cell pellet on dry ice, in a small volume of medium (10 &#181;l), with long term storage at -80 &#176;C.</li>
			<li>For proteomic analysis, lyse the cells in 10 &#181;l of 8 M urea for mass spectrometry or 2x SDS tris-glycine buffer for western blotting/reverse phase protein microarrays. Alternatively, spin cells in a cytocentrifuge to make cell smears for immunohistochemical analysis.</li>
		</ol>
		</li>
		<li>After harvesting cells from a flask, remove and discard the remaining medium. Add 11 ml of warm, fresh nutrient rich medium. Incubate the flask at 37 &#176;C, in a humidified 5% CO2 atmosphere.</li>
	</ol>
	</li>
</ol>

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prevention+of+intraepithelial+neoplasia:+an+important+target+for+accelerated+new+agent+development.">Treatment and prevention of intraepithelial neoplasia: an important target for accelerated new agent development.</a> Clin Cancer Res. 8 (2), 314-346 (2002).</li><li>Lee, 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=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9 (5), 391-403 (2006).</li><li>Behbod, F., 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=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Res. 11 (5), (2009).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+malignant+nature+of+human+ductal+carcinoma+in+situ.">What is the malignant nature of human ductal carcinoma in situ.</a> Nat Rev Cancer. 11 (1), 68-75 (2011).</li><li>Gong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beclin+1+and+autophagy+are+required+for+the+tumorigenicity+of+breast+cancer+stem-like/progenitor+cells.">Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like/progenitor cells.</a> Oncogene. 32 (18), 2261-2272 (2012).</li><li>Simon, B., 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=Epithelial+glycoprotein+is+a+member+of+a+family+of+epithelial+cell+surface+antigens+homologous+to+nidogen,+a+matrix+adhesion+protein.">Epithelial glycoprotein is a member of a family of epithelial cell surface antigens homologous to nidogen, a matrix adhesion protein.</a> PNAS. 87 (7), 2755-2759 (1990).</li><li>Ma, X. 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=Gene+expression+profiles+of+human+breast+cancer+progression.">Gene expression profiles of human breast cancer progression.</a> Proc Natl Acad Sci U S A. 100 (10), 5974-5979 (2003).</li><li>Schnitt, S. J., Harris, J. R., Smith, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+prognostic+index+for+ductal+carcinoma+in+situ+of+the+breast.+Are+we+there+yet.">Developing a prognostic index for ductal carcinoma in situ of the breast. Are we there yet.</a> Cancer. 77 (11), 2189-2192 (1996).</li><li>Sgroi, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preinvasive+breast+cancer.">Preinvasive breast cancer.</a> Annu Rev Pathol. 5, 193-221 (2010).</li><li>Asch, H. L., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+keratin+expression+in+mouse+mammary+hyperplastic+alveolar+nodules+and+adenocarcinomas.">Heterogeneity of keratin expression in mouse mammary hyperplastic alveolar nodules and adenocarcinomas.</a> Cancer Res. 45 (6), 2760-2768 (1985).</li><li>LaBarge, M. A., Petersen, O. W., Bissell, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+picture+of+the+mouse+mammary+stem+cell.">The emerging picture of the mouse mammary stem cell.</a> Stem Cell Rev. 3 (2), 114-123 (2007).</li><li>Kim, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cap+associated+focal+adhesions+and+their+distinct+role+in+cellular+mechanosensing.">Actin cap associated focal adhesions and their distinct role in cellular mechanosensing.</a> Sci Rep. 2, 555 (2012).</li><li>Espina, V., Wysolmerski, J., Edmiston, K., Liotta, L. 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The authors have nothing to disclose.

The culture system described herein constitutes a new model for generating living pre-invasive neoplastic breast cells for basic and translational research studies. In the past, pre-malignant breast cancer progression has typically been studied using three different methods. The first method is histopathologic and genetic analysis of microdissected frozen or fixed human specimens12-14. The second method utilizes mouse models that contain hyperplastic alveolar nodules (HAN lesions) that are thought to be simila...

Proliferation of epithelial cells within the confines of breast ducts and alveoli (ductal carcinoma in situ) is recognized as an obligate precursor to invasive ductal and lobular breast carcinoma. Nevertheless, the molecular mechanisms and cell population dynamics that permit progression to invasive cancer are poorly understood. Elucidating the survival mechanisms used by pre-invasive breast carcinoma cells, or any pre-invasive tumor, may reveal therapeutic strategies for killing, or even preventing, pre-invasive neoplasms1. However, simple low-cost methods for functionally studying human pre-invasive lesions have been lacking. Although in vitro monolayer culture of transformed cell lines is an established laboratory method, the phenotype and genotype of these immortalized cell lines fails to recapitulate the molecular status of primary human tumor cells2. Furthermore, even the non-tumorigenic MCF-10A cell line, which recapitulates 3-D mammary gland architecture, fails to adequately represent the functional phenotype and molecular characteristics of an individual patient&#8217;s pre-invasive breast lesion3,4.
To determine if stem-like neoplastic cells capable of invasion existed within the ductal carcinoma in situ (DCIS) cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery (Figure 1)5. Our ex vivo breast organoid culture system does not rely on enzymatic tissue disruption, basement membrane extract matrix, or fibroblast depletion, for isolating and propagating mammosphere-forming cells from fresh human breast ductal carcinoma tissue6-8. Our new system is based on the principle of cell streaming/migration5. The discernible breast ducts, and surrounding stroma are submerged in a minimum volume of serum-free nutrient medium (just enough to cover the duct fragments) to maximize gas exchange, with the cut surface of the duct exposed to the culture medium, but in no specific orientation in the flask (Figure 1E-F). This culture system allows cells to migrate out of the duct and into/onto the autologous stroma and culture flask. The nutrient medium, supplemented only with Epidermal Growth Factor (EGF), insulin, and antibiotics, supports growth of mixed cell populations emanating from the organoid. The tissue culture flask has a removable, re-sealable lid that allows the organoids and/or cells to be harvested without disrupting the entire flask or neighboring organoids, while maintaining a sterile humidified environment.

<table><tbody><tr><td>Ethanol</td><td>Fisher</td><td>A405-P</td><td>prepare a 70% solution in Type 1 reagent grade water</td></tr><tr><td>18 M&amp;Omega;-cm water, sterile filtered</td><td></td><td></td><td>sterile filtered, Type 1 reagent grade water</td></tr><tr><td>10 cc plastic disposable syringe, sterile</td><td>BD</td><td>305482</td><td></td></tr><tr><td>0.2 &amp;micro;m polyethersulfone (PES) syringe filter, sterile</td><td>Thermo Scientific</td><td>194-2520</td><td></td></tr><tr><td>15 ml polypropylene conical tubes, sterile</td><td>Fisher</td><td>14-959-49B</td><td></td></tr><tr><td>50 ml polypropylene conical tubes, sterile</td><td>Fisher</td><td>05-539-6</td><td></td></tr><tr><td>1.5 ml low retention microcentrifuge tubes, sterile</td><td>Fisher</td><td>02-681-331</td><td></td></tr><tr><td>nutrient medium, DMEM-F12/HEPES</td><td>Invitrogen</td><td>11330-032</td><td>with L-glutamine</td></tr><tr><td>Insulin, human recombinant</td><td>Roche</td><td>11376497001</td><td>10 mg/ml stock</td></tr><tr><td>Epidermal Growth Factor (EGF), human recombinant</td><td>Millipore</td><td>GF144</td><td>100 &amp;micro;g/ml stock</td></tr><tr><td>Streptomycin sulfate</td><td>Sigma-Aldrich</td><td>S1567</td><td>10 mg/ml stock</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma-Aldrich</td><td>G19114</td><td>10 mg/ml stock</td></tr><tr><td>Filtration flask and filter top, sterile</td><td>Millipore</td><td>SCGPU02RE</td><td>0.22 &amp;micro;m PES membrane</td></tr><tr><td>25 ml sterile, disposable pipettes</td><td>Fisher</td><td>4489</td><td>paper-plastic wrapped</td></tr><tr><td>10 ml sterile, disposable pipettes</td><td>Fisher</td><td>4488</td><td>paper-plastic wrapped</td></tr><tr><td>Tissue marking dyes (black, blue, red, green, yellow and orange)</td><td>CDI</td><td>MD2000</td><td>after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td></tr><tr><td>Cotton tipped applicators, sterile</td><td>Fisher</td><td>23-400-115</td><td>single use only</td></tr><tr><td>Gauze pads, 10 x 10 cm, sterile</td><td>Fisher</td><td>2187</td><td></td></tr><tr><td>Plastic transfer pipettes, sterile, disposable</td><td>Samco</td><td>202-20S</td><td></td></tr><tr><td>Vinegar, white distilled</td><td>household use</td><td></td><td>5% acetic acid; after opening use only with sterile pipettes</td></tr><tr><td>#10 scalpels, sterile, disposable</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Petri dish, sterile</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>TPP 115 cm&lt;sup&gt;2&lt;/sup&gt; flask, with removable lid</td><td>MidSci</td><td>90652</td><td>screw cap with filter</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Fisher</td><td>13-998-074</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C, humidified chamber</td></tr><tr><td>inverted light microscope</td><td>Olypmus</td><td>IX51</td><td></td></tr><tr><td>8 M urea</td><td>Fisher</td><td>BP169-500</td><td>optional, for mass spectrophotometric analysis of cultured cells</td></tr><tr><td>2X SDS tris-glycine buffer</td><td>Life Technologies</td><td>LC2676</td><td>optional, for proteomic analysis of cultured cells</td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>optional, for preparing cell smears</td></tr></tbody></table>

non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Workflow for procuring and culturing sterile breast ductal carcinoma in situ tissue
Breast tissue sterility is maintained from the operating room to the cell culture lab via minor changes in typical hospital pathology workflow (Figure 1). Tissue is transported in a sterile container with a plastic film cover which allows radiologic assessment while maintaining tissue sterility. Gross tissue processing of a breast lumpectomy or mastectomy sample is performed with sterile ...

Watch this Scientific Journal Video about Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres at JoVE.com

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

non-enzymatic-serum-free-tissue-culture-pre-invasive-breast-lesions

Research

JoVE Journal

Biology

12.6K Views.  George Mason University. Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for >6 months.

Video: Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

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

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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 ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Cancer Research

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

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

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

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

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

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

Developmental Biology

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 ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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

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

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

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient ...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

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Przeglądaj więcej filmów

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

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

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

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

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

Quantification of Self-renewal in Murine Mammosphere Cultures

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Isolation and Culture of Primary Human Mammary Epithelial Cells