We would like to thank the Tulane Flow Cytometry and Cell Sorting Core as well as the Tulane Histology Core for their technical support. This work was supported by the Southeastern Society of Plastic &#38; Reconstructive Surgeons 2019 Research Grant and the National Science Foundation (EPSCoR Track 2 RII, OIA 1632854).

All human tissues were collected in accordance to protocol #9189 as approved by the Institutional Review Board Office of LSUHSC.
1. Seeding of Adipose-derived Stem Cells (ASCs) for cell sheets
<ol>
	<li>Purchase ASCs from commercial sources or isolate from primary human breast tissue by following established protocols8,9. Seed human breast ASCs at 70% density (~80,000 cells/cm2 surface area) onto 6-well standard tissue culture plates in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and 1% Penicillin/Streptomycin (BC-MPS Media). Ensure that the ASCs to be used for forming the cell sheets should be less than passage 10.
	<ol>
		<li>For each well of breast cancer microphysiological system (BC-MPS) that will be generated, seed cells in 1 standard tissue culture well and 1 well of similar size on poly(N-isopropylacrylamide) (pNIPAAm)-coated tissue culture plastic plate. One 6-well plate of BC-MPS will require one standard tissue culture 6 well plate (bottom) and one pNIPAAm-coated plate (top). The pNIPAAm plates can be purchased from commercial sources or can be coated in-lab10,11,12.</li>
	</ol>
	</li>
	<li>Culture ASCs in a tissue culture incubator at 37 &#176; C and 5% CO2. Change the media every 2-3 days in a biosafety cabinet.</li>
	<li>Culture the ASCs until they become 100% confluent and form a striated pattern. Depending on the confluence at which they were seeded, this will take 7-10 days. Confluent cell sheets are required to anchor the buoyant adipose breast tissue.</li>
</ol>
2. Preparation of supplies needed for BC-MPS
<ol>
	<li>To prepare gelatin for 6-well plates make a 12% gelatin solution in distilled H2O. Mix 19 g of gelatin type B (gel strength of ~225 Bloom) and 125 mL of H2O in a 250 mL glass media bottle. Add 1.6 mL of 1 M NaOH to the solution to neutralize the pH.</li>
	<li>Autoclave the solution under a liquid cycle for 25 min to sterilize the solution.</li>
	<li>Allow the solution to cool to 37 &#176;C. In a sterile biosafety cabinet add 16 mL of sterile 10x Hanks balanced salt solution (HBSS) to the solution to obtain a final volume of 160 mL. 
	&#8203;NOTE: The gelatin solution can be used right away or stored at room temperature for later use. The prepared gelatin is stable at room temperature for 1 month. If only a few plungers will be needed then a 10 mL solution can be made and sterile filtered on the same day as making BC-MPS as previously described7.
	<ol>
		<li>To prepare gelatin solution by filter sterilization, dilute 1 mL of 10x HBSS with 9 mL of H2O to make a 1x HBSS solution in a 15 mL conical tube. Add 100 &#181;L of 1 M NaOH to each 10 mL tube and then add 0.7 g of gelatin to the solution. Mix the solution vigorously to dissolve the gelatin.</li>
		<li>Incubate the tube in a 75 &#176;C water bath for 20 min shaking every 5 min. Use a 10 mL syringe and a 0.22 &#181;m syringe filter to filter the gelatin solution. Filter the gelatin solution directly onto the plungers in a biosafety cabinet.</li>
	</ol>
	</li>
	<li>3D print or use simple acrylic to make the plungers used for transferring the cell sheets. Ensure that the plungers fit loosely in the desired size of well. A standard 3D printed plunger is shown in Figure 1. The plungers can be designed using standard computer aided design software such as TinkerCad and printed using filament 3D printers that are compatible with polylactic acid (PLA)13,14.</li>
	<li>To prepare a rack for holding the plungers place a 15 mL tube rack in a biosafety cabinet. Place plungers upside down in the rack, so that the bottom of the plungers is facing up. Place a plastic box with a lid for transporting BC-MPS in the biosafety cabinet. It is recommended that the box be at least 35.5 cm length x 28 cm width x 8.25 cm height to fit 4 plates of BC-MPS. Remove the lid from the box and spray down the rack, plungers, and box with 70% EtOH.</li>
	<li>Prepare sterile razor blades and forceps by autoclaving or by placing them in a biosafety cabinet and washing with 70% EtOH.</li>
	<li>Spray the plungers and box with 70% EtOH and turn on the UV light in the biosafety cabinet for at least 30 min to sterilize the supplies.</li>
</ol>
3. Prepare gelatin plungers and apply to the upper cell sheets
<ol>
	<li>If the gelatin solution was previously prepared, heat it in a 37 &#176; C water bath to melt it.</li>
	<li>Pipette the gelatin solution onto the plungers in the biosafety cabinet using a 5- or 10-mL serological pipette. A plunger for 1 well of a 6-well plate will require ~2.5 mL of gelatin.</li>
	<li>Once the gelatin has solidified on the plungers (~30-45 min) move the pNIPAAm-coated ASC plates to the biosafety cabinet. Gently place the plungers into the wells of the pNIPAAm-coated ASC plates so that the gelatin contacts the ASCs. Place a metal washer (~7.5 g) on the plunger to weigh down the plunger so that gelatin is in direct contact with the ASC cell sheet. Leave the plungers on the ASC cell sheets at room temperature for 30 min.</li>
	<li>Gently move the plate with the plungers into the sterile box in the biosafety cabinet and place the lid on it. Move the box to a 4 &#176;C fridge for 30 min. If a 4 &#176;C fridge is not available place the plate on ice in an ice bucket in the biosafety cabinet for 30 min.</li>
</ol>
4. Preparation of cancer cell lines
<ol>
	<li>Seed the cancer cell lines in a standard T75 tissue culture dish and keep them in culture so that they are ~80% confluent on the day BC-MPS will be processed. (~200,000 cancer cells will be needed per BC-MPS). Grow the cancer cells in normal media. 
	NOTE: To identify and isolate the cancer cells it is recommended that the cells are transfected with a fluorescent protein to distinguish them from the ASCs and breast tissue. The number of cancer cells needed per BC-MPS has been optimized for MCF7 and MDA-MB-231 cells. Other cell lines may require further optimization.</li>
	<li>On the day that the BC-MPS is being prepared move the flask containing the cancer cells to the biosafety cabinet. Aspirate the media from the flask. Wash the flask with 5 mL of PBS.</li>
	<li>Add 1 mL of cell detachment solution to the flask containing the cancer cells and then incubate the flask in a 37 &#176;C incubator until the cells have detached (~2-5 min).</li>
	<li>In the biosafety cabinet add 9 mL of PBS to the flask, transfer the cells in PBS into a 15 mL conical tube and count the cell number using a hemocytometer.</li>
	<li>Centrifuge the cancer cells at 500 x g for 5 min at room temperature.</li>
	<li>Resuspend the cells in BC-MPS media so that there are 2 x 106 cells/mL.</li>
	<li>Place the cancer cells in a 37 &#176;C water bath until they are ready to be added to the human tissue.</li>
</ol>
5. Processing of human breast tissue
<ol>
	<li>In a biosafety cabinet wash the human breast tissue (HBT) 3x with 10 mL of sterile PBS.</li>
	<li>Use sterile forceps and a razor blade to coarsely mince the BC-MPS and try to remove as much fascia and connective tissue as possible. Failure to remove the connective tissue may result in the ASC upper layer to not properly anchor the HBT. 
	NOTE: The fascia and connective tissue can be identified by its white or clear appearance compared to the yellow color of the adipose tissue.</li>
	<li>Once the connective tissue has been removed use a sterile razor blade to finely mince the tissue until it has a homogenous liquid consistency. The tissue is finely minced when it can easily be pipetted using a 25 mL serological tip.</li>
	<li>Use a sterile razor blade to cut the tip off a p1000 pipette tip to assist in pipetting the minced HBT.</li>
	<li>In a 1.5 mL tube mix the minced HBT, cancer cell lines, and BC-MPS media (for 1 well of a 6 well plate this requires 200 &#181;L of minced HBT, 100 &#181;L of of BC-MPS media, and 100 &#181;L of cancer cells).</li>
	<li>Move the ASC plate that will be used for the bottom cell sheet from the incubator to the biosafety cabinet. Aspirate the media from the bottom ASC plate. Pipette the mixture onto the center of the well of the bottom ASC plate using a p1000 pipette tip that had the distal end cut off from step 5.4</li>
	<li>Move the box containing the upper ASC plate with the plungers on it to the biosafety cabinet. Gently remove the gelatin plungers from the pNIPAAm-coated plate and place on top of the HBT mixture. Add BC-MPS media to the well (for 1 well of a 6-well plate add 2 mL of media). Carefully move the bottom ASC plates with the BC-MPS mixture and plungers to the sterile plastic box and place the lid of the box on for transport. 
	NOTE: The 6-well plate lid will not fit back on the ASC plate while the plungers are on. Care must be taken to avoid contaminating the culture.</li>
	<li>Incubate the bottom plate in the box in an incubator at 37 &#176; C until the gelatin has melted, and the top ASC layer has begun to adhere to the bottom layer (~30 min).</li>
	<li>Move the box with the plates to the biosafety cabinet. Gently remove the plungers from the bottom plates. The tissue with the cancer cells will be anchored to the bottom of the well.</li>
	<li>Place the lid of the 6-well plate back onto the bottom plate and incubate at 37 &#176;C to completely melt the gelatin and allow the top layer to completely anchor to the bottom layer (~30-60 min).</li>
	<li>Gently move the plates to a biosafety cabinet and aspirate the media with a 10 mL serological pipette. Add 2 mL of fresh media to each well. Aspirate from the edge of the well and pipette onto the edge of the well to avoid dislodging the tissue.</li>
	<li>Maintain the BC-MPS at 37 &#176;C and 5% CO2 for the desired length of time and change media every 2-3 days.</li>
</ol>
6. Digestion of BC-MPS for analysis
<ol>
	<li>When the BC-MPS is ready to be analyzed, move the plate to the biosafety cabinet. Remove media with a serological pipette to avoid accidental dislodging of any tissue.</li>
	<li>Add 1 volume of PBS to each well.</li>
	<li>Remove the PBS with a serological pipette.</li>
	<li>Add 1 mL of cell disassociation solution to each well. Move the plate back to the incubator and incubate at 37 &#176;C for 5 min to allow the cells to detach. In a biosafety cabinet, use a cell scraper to completely detach the cells and the tissue from the culture plate.</li>
	<li>Transfer the solution with the tissue to a 15 mL conical tube using a serological pipette. Add 2 mL of PBS to each well to collect any remaining cells and transfer this solution to the conical tube. If the cells are fluorescent, wrap the tube in aluminum foil to protect it from light.</li>
	<li>Incubate the tube at 37 &#176;C under constant agitation in an orbital shaker at 1 x g for 10-20 min to completely dissociate cells from the tissue.</li>
	<li>In a biosafety cabinet use a serological pipette to disrupt any remaining clumps of cells in the tube and filter the sample through a 250 &#181;m tissue strainer into a new 15 mL tube. Pipette the solution slowly through the strainer so that it does not overflow.</li>
	<li>Rinse the strainer with 1 mL PBS to collect any cells still in the strainer.</li>
	<li>Centrifuge the samples at 500 x g at room temperature for 5 min. After centrifugation, the adipocytes will be floating on the top layer of the solution while the cancer cells and ASCs will be mixed together in a pellet at the bottom of the tube.</li>
	<li>To isolate the adipocytes gently pour the top layer into a new tube or alternatively cut the tip off a p1000 pipette tip and transfer the top layer to a new tube. Centrifuge the sample at 500 x g for 5 min again and use a syringe and needle to remove the solution from below the adipocytes so that only the adipocytes are remaining. The adipocytes are now ready for analysis</li>
	<li>After removing the adipocytes from the solution, separate the ASCs and cancer cells from each other for analysis if the cancer cells were previously transfected with a fluorescent protein. Aspirate the remaining solution from the tube containing the ASCs and cancer cells without disrupting the cell pellet. Resuspend the pellet in PBS or BC-MPS media and use flow cytometry to sort the cells based on the fluorescence.</li>
</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=Leptin+produced+by+obesity-altered+adipose+stem+cells+promotes+metastasis+but+not+tumorigenesis+of+triple-negative+breast+cancer+in+orthotopic+xenograft+and+patient-derived+xenograft+models.">Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models.</a> Breast Cancer Research: BCR. 21 (1), 67 (2019).</li><li>Cui, X. D., Gao, D. Y., Fink, B. F., Vasconez, H. C., Pu, L. L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+adipose+tissues.">Cryopreservation of human adipose tissues.</a> Cryobiology. 55 (3), 269-278 (2007).</li><li>Skardal, A., Shupe, T., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid-on-a-chip+and+body-on-a-chip+systems+for+drug+screening+and+disease+modeling.">Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling.</a> Drug Discovery Today. 21 (9), 1399-1411 (2016).</li><li>Clark, A. M., Allbritton, N. L., Wells, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+microphysiological+tissue+systems+of+cancer+metastasis+to+the+liver.">Integrative microphysiological tissue systems of cancer metastasis to the liver.</a> Seminars in Cancer Biology. , (2020).</li></ol>

The authors declare that they have no competing interests.

New systems for modeling human breast cancer are needed to develop a better understanding of the disease. Development of human microphysiological systems to model disease settings that include native ECM and stromal cells will increase the predictive power of pre-clinical studies. The BC-MPS model presented here is a newly developed system that overcomes the limitations of previous models allows for the evaluation of BC in its native HBT environment. This system can be used with cancer cell lines or with tumor explants a...

Each year, more than 40,000 US women die of breast cancer (BC)1. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials2,3. New models are needed to improve the drug development pipeline and our understanding of BC. The NIH Microphysiological (MPS) Program delineated the features required for breakthrough models for improving translating basic science into clinical success4. These included the use of primary human cells or tissues, stable in culture for 4 weeks, and inclusion of native tissue architecture and physiological response.
Current in vitro BC models, such as two-dimensional culture of BC cell lines, membrane insert co-culture, and three-dimensional spheroids and organoids, do not meet the NIH&#39;s MPS criteria because none of these recapitulate native breast tissue architecture. When extracellular matrix (ECM) is added to these systems, breast ECM is not used; instead, collagen gels and basement membrane matrices are used.
Current in vivo systems, such as patient derived xenografts (PDX), similarly do not meet the NIH&#39;s MPS criteria because murine mammary tissues vastly differ from human breasts. Moreover, immune system-BC interactions are increasingly recognized as key in tumor development, but the immunocompromised murine models used for generating PDX tumors lack mature T cells, B cells, and natural killer cells. Furthermore, while PDX allows for primary breast tumors to be maintained and expanded, the resulting PDX tumors are infiltrated with primary murine stromal cells and ECM5.
To overcome these challenges, we have developed a novel, ex vivo, three-dimensional human breast MPS that meets the NIH MPS criteria. The foundation of our breast MPS is made by sandwiching primary human breast tissue (HBT) between two sheets of adipose-derived stem cells (ASCs), also isolated from HBT (Figure 1). Plungers for transferring the cell sheets to sandwich the HBT can be 3D printed or made from simple acrylic plastics (Figure 1H,I). This technique adapts our previously described approach for culturing primary human white adipocyte tissue6,7. The breast MPS can then be seeded by a BC model of choice, ranging from standard BC cell lines to primary human breast tumors. Here, we show that these BC-MPS are stable in culture for multiple weeks (Figure 2); include native elements of HBT such as mammary adipocytes, ECM, endothelium, immune cells (Figure 3); and recapitulate the physiological interactions between BC and HBT such as metabolic crosstalk (Figure 4). Lastly, we show that BC-MPS allows for the study of amoeboid movement of BC cells throughout HBT (Figure 5).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>1333</td>
			<td>Cell disassoication solution for separation of BC-MPS</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Corning</td>
			<td>25-058-CI</td>
			<td>Cell detachment solution for passaging of cells</td>
		</tr>
		<tr>
			<td>BioStor Container 16oz</td>
			<td>National Scientific Supply Co</td>
			<td>MPCE-T016</td>
			<td>For Transport of sterile tissue</td>
		</tr>
		<tr>
			<td>Cell Culture 75 cm flasks</td>
			<td>Corning</td>
			<td>430641U</td>
			<td>For culturing ASCs</td>
		</tr>
		<tr>
			<td>Conical Tubes 15mL&#160;</td>
			<td>ThermoScientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>ThermoScientific</td>
			<td>1631T5</td>
			<td>For maneuvering tissue while mincing&#160;</td>
		</tr>
		<tr>
			<td>DMEM low glucose, w/ Glutamax</td>
			<td>Gibco</td>
			<td>10567-014</td>
			<td>For culturing ASCs and BC-MPS</td>
		</tr>
		<tr>
			<td>FBS Qualified</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS 10x</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc UpCell 6 well plates</td>
			<td>ThermoScientific</td>
			<td>174901</td>
			<td>Top ASC cell sheet</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep 5,000U</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 150 cm</td>
			<td>FisherBrand</td>
			<td>FB0875714</td>
			<td>For holding tissue while mincing&#160;</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>Single Edge for mincing tissue</td>
		</tr>
		<tr>
			<td>Strainer 250um&#160;</td>
			<td>ThermoScientific</td>
			<td>87791</td>
			<td>For separation of BC-MPS</td>
		</tr>
		<tr>
			<td>Tissue Culture 6 well plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Bottom ASC cell Sheet</td>
		</tr>
		<tr>
			<td>Weights/Washers</td>
			<td>BCP Fasteners</td>
			<td>BCP672</td>
			<td>For weighing plungers down 1/2&#34; inner diameter</td>
		</tr>
	</tbody>
</table>

modeling breast cancer in human breast tissue using a microphysiological system

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

Stability in culture 
BC-MPS is a stable microphysiological system that can be cultured in vitro for up to at least 14 days. A brightfield image of the ASC cell sheets was taken at 100x magnification to display the striated pattern of the confluent sheet (Figure 2A). The ASC cell sheets are stable in culture for at least 4 weeks. BC-MPS at 14 days in culture in one well of a 6 well plate was imaged with a color camera demonstrating that ...

Watch this Scientific Journal Video about Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System at JoVE.com

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

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Research

JoVE Journal

Cancer Research

4.0K Views.  Louisiana State University Health Sciences Center. This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Video: Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating investigators to analyze and interpret genes, samples and clinical data across different types of cancer. Herein, we describe a practical operation procedure, taking ID1 (Inhibitor of DNA binding proteins 1) as an example, to characterize the expression patterns of biomarker and survival predictors of breast cancer based on pooled clinical datasets derived from online accessible databases, including ONCOMINE, bcGenExMiner v4.0 (Breast cancer gene-expression miner v4.0), GOBO (Gene expression-based Outcome for Breast cancer Online), HPA (The human protein atlas), and Kaplan-Meier plotter. The analysis began with querying the expression pattern of the gene of interest (e.g., ID1) in cancerous samples vs. normal samples. Then, the correlation analysis between ID1 and clinicopathological characteristics in breast cancer was performed. Next, the expression profiles of ID1 was stratified according to different subgroups. Finally, the association between ID1 expression and survival outcome was analyzed. The operation procedure simplifies the concept to integrate multidimensional data types at the gene level from different databases and test hypotheses regarding recurrence and genomic context of gene alteration events in breast cancer. This method can improve the credibility and representativeness of the conclusions, thereby, present informative perspective on a gene of interest.

Here, we present a protocol to explore the biomarker and survival predictor of breast cancer based on the comprehensive analysis of pooled clinical datasets derived from a variety of publicly accessible databases, using the strategy of expression, correlation and survival analysis step by step.

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating ...

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

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

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

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

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...