Method Article

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

DOI:

10.3791/62009

⸱

April 23rd, 2021

 ,  ,  ,  ,  ,  , 
1Department of Surgery, Louisiana State University Health Sciences Center, 2Department of Bioinnovation, Tulane University, 3Tulane University School of Medicine, 4Department of Biological and Agricultural Engineering, Louisiana State University, 5Department of Medicine, Tulane University School of Medicine

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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'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'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).

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Protocol

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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

  1. 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'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.
    1. 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.
  2. Culture ASCs in a tissue culture incubator at 37 ° C and 5% CO2. Change the media every 2-3 days in a biosafety cabinet.
  3. 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.

2. Preparation of supplies needed for BC-MPS

  1. 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.
  2. Autoclave the solution under a liquid cycle for 25 min to sterilize the solution.
  3. Allow the solution to cool to 37 °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.
    ​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.
    1. 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 µL 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.
    2. Incubate the tube in a 75 °C water bath for 20 min shaking every 5 min. Use a 10 mL syringe and a 0.22 µm syringe filter to filter the gelatin solution. Filter the gelatin solution directly onto the plungers in a biosafety cabinet.
  4. 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.
  5. 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.
  6. Prepare sterile razor blades and forceps by autoclaving or by placing them in a biosafety cabinet and washing with 70% EtOH.
  7. 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.

3. Prepare gelatin plungers and apply to the upper cell sheets

  1. If the gelatin solution was previously prepared, heat it in a 37 ° C water bath to melt it.
  2. 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.
  3. 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.
  4. 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 °C fridge for 30 min. If a 4 °C fridge is not available place the plate on ice in an ice bucket in the biosafety cabinet for 30 min.

4. Preparation of cancer cell lines

  1. 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.
  2. 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.
  3. Add 1 mL of cell detachment solution to the flask containing the cancer cells and then incubate the flask in a 37 °C incubator until the cells have detached (~2-5 min).
  4. 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.
  5. Centrifuge the cancer cells at 500 x g for 5 min at room temperature.
  6. Resuspend the cells in BC-MPS media so that there are 2 x 106 cells/mL.
  7. Place the cancer cells in a 37 °C water bath until they are ready to be added to the human tissue.

5. Processing of human breast tissue

  1. In a biosafety cabinet wash the human breast tissue (HBT) 3x with 10 mL of sterile PBS.
  2. 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.
  3. 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.
  4. Use a sterile razor blade to cut the tip off a p1000 pipette tip to assist in pipetting the minced HBT.
  5. 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 µL of minced HBT, 100 µL of of BC-MPS media, and 100 µL of cancer cells).
  6. 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
  7. 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.
  8. Incubate the bottom plate in the box in an incubator at 37 ° C until the gelatin has melted, and the top ASC layer has begun to adhere to the bottom layer (~30 min).
  9. 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.
  10. Place the lid of the 6-well plate back onto the bottom plate and incubate at 37 °C to completely melt the gelatin and allow the top layer to completely anchor to the bottom layer (~30-60 min).
  11. 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.
  12. Maintain the BC-MPS at 37 °C and 5% CO2 for the desired length of time and change media every 2-3 days.

6. Digestion of BC-MPS for analysis

  1. 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.
  2. Add 1 volume of PBS to each well.
  3. Remove the PBS with a serological pipette.
  4. Add 1 mL of cell disassociation solution to each well. Move the plate back to the incubator and incubate at 37 °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.
  5. 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.
  6. Incubate the tube at 37 °C under constant agitation in an orbital shaker at 1 x g for 10-20 min to completely dissociate cells from the tissue.
  7. 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 µm tissue strainer into a new 15 mL tube. Pipette the solution slowly through the strainer so that it does not overflow.
  8. Rinse the strainer with 1 mL PBS to collect any cells still in the strainer.
  9. 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.
  10. 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
  11. 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.

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Results

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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 th...

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Discussion

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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...

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Disclosures

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The authors declare that they have no competing interests.

Acknowledgements

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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 & Reconstructive Surgeons 2019 Research Grant and the National Science Foundation (EPSCoR Track 2 RII, OIA 1632854).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AccumaxInnovative Cell Technologies1333Cell disassoication solution for separation of BC-MPS
AccutaseCorning25-058-CICell detachment solution for passaging of cells
BioStor Container 16ozNational Scientific Supply CoMPCE-T016For Transport of sterile tissue
Cell Culture 75 cm flasksCorning430641UFor culturing ASCs
Conical Tubes 15mL ThermoScientific339650
Curved ForcepsThermoScientific1631T5For maneuvering tissue while mincing 
DMEM low glucose, w/ GlutamaxGibco10567-014For culturing ASCs and BC-MPS
FBS QualifiedGibco26140-079
GelatinSigmaG9391
HBSS 10xGibco14185-052
NaOHSigma221465
Nunc UpCell 6 well platesThermoScientific174901Top ASC cell sheet
PBSGibco10010-023
Pen/Strep 5,000UGibco15070-063
Petri Dish 150 cmFisherBrandFB0875714For holding tissue while mincing 
Razor BladesVWR55411-055Single Edge for mincing tissue
Strainer 250um ThermoScientific87791For separation of BC-MPS
Tissue Culture 6 well platesCorning3506Bottom ASC cell Sheet
Weights/WashersBCP FastenersBCP672For weighing plungers down 1/2" inner diameter

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Tags

Breast Cancer ModelingAdipose Stem CellsExtracellular MatrixCancer Cell MotilityMetabolic CrosstalkFlow CytometryFluorescence MicroscopyTissue Dissociation

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