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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<li>Robado de Lope, L., Alcíbar, O. L., Amor López, A., Hergueta-Redondo, M., Peinado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour-adipose+tissue+crosstalk%3A+fuelling+tumour+metastasis+by+extracellular+vesicles%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">Tumour-adipose tissue crosstalk: fuelling tumour metastasis by extracellular vesicles.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 373 (1737), 20160485(2018).</li>
<li>Insua-Rodríguez, J., Oskarsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extracellular+matrix+in+breast+cancer%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">The extracellular matrix in breast cancer.</a> Advanced Drug Delivery Reviews. 97, 41-55 (2016).</li>
<li>Sabol, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Breast+Cancer+Research%3A+BCR%22%5BJournal%5D)">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/pubmed?term=((Cryopreservation+of+human+adipose+tissues%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Organoid-on-a-chip+and+body-on-a-chip+systems+for+drug+screening+and+disease+modeling%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">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/pubmed?term=((Integrative+microphysiological+tissue+systems+of+cancer+metastasis+to+the+liver%5BTitle%5D)+AND+%22Seminars+in+Cancer+Biology%22%5BJournal%5D)">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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&quot; 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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Cancer Research

4.2K 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

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

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

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

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

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

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

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

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

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 ...

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 ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Medicine

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Bioengineering

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Summary

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