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Method Article
We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.
Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.
Three dimensional (3D) culture models that more accurately mimic the tumor architecture and microenvironment in vivo are important for studies aimed to dissect the complex interactions between cells and their microenvironment and to test the efficacy of candidate therapies. Tumor dimensionality impacts oxygen and nutrient gradients, the uniformity of drug exposure, interstitial pressure/blood flow, and 3D architecture1-4. The presence of an appropriate stromal microenvironment contributes to tumor dimensionality and influences cell-ECM signaling and paracrine signaling between stromal cells and malignant epithelial cells. The effects of tumor dimensionality and the microenvironment on cellular function are well established, with both factors altering drug response1,3,5-8. Additionally, cellular growth kinetics, metabolic rates, and cell signaling differ between two dimensional (2D) culture and culture in 3D, with these factors affecting cellular response1,3,8-10.
In vitro, the tumor surrogate microenvironment can be modulated by including representative ECM constituents and stromal cell populations. Malignant epithelial cells are influenced by the ECM and cancer-associated stromal cells either in a synergistic/protective manner to promote tumor progression or in a suppressive manner to inhibit further tumor propagation5,6,10. In either context, the stroma can affect therapeutic response and drug delivery via paracrine signaling and/or by increasing interstitial pressure in the tumor resulting in decreased drug delivery1,6. Therefore, the addition of ECM and stromal cells into preclinical models will help recapitulate aspects of the tumor that cannot be modeled well in 2D culture.
Herein a method to establish breast cancer surrogates that incorporate a recapitulative microenvironment, including ECM constituents and stromal cells, in a 3D volume is described. In breast carcinoma, the stromal cell population is predominately comprised of cancer associated fibroblasts (CAF) and the stromal ECM is largely composed of collagen type I with a smaller proportion of matrix components that are found in the basement membrane, including laminin and collagen type IV1,4,11-13. Therefore, these components of the breast carcinoma microenvironment (i.e., CAF, collagen I, and basement membrane) have been incorporated into the surrogates. This method can be used to generate solid, un-perfused 3D surrogates (Figure 1A) or can be adapted to include perfusion of medium through the surrogate via a bioreactor system (Figure 1B). Both approaches are described here. This method could also be modified to include other stromal elements, such as tumor-associated macrophages, or to model other solid tumors by adjusting the cellular and ECM components, as appropriate.
For the breast carcinoma surrogate described here, we have utilized the MDA-MB-231 (231) breast cancer cell line, CAF previously isolated from human breast carcinoma14, and an ECM composed of 90% collagen I (6 mg/ml) and 10% growth factor reduced basement membrane material (BM). The surrogate is either grown in an 8-well chamber slide (solid surrogate) or a bioreactor system is utilized to provide continuous nutrient perfusion (perfused surrogate). Any perfusion bioreactor system that can accommodate a volume of ECM containing cells can be used15. As an example, we describe the preparation of the tissue surrogates in our bioreactor system. This system was developed in-house and is not commercially available. Because our focus here is on the preparation and analysis of the 3D tissue surrogates, we have not gone into extensive detail regarding the specifics of manufacture and assembly of our bioreactor system. However, a detailed description of this system and its development has been published16. In this bioreactor system, a polydimethylsiloxane (PDMS) flow channel is used to house the surrogate, which is supported by a PDMS foam (formed using methods similar to those described by Calcagnile et al.17). This volume is penetrated by 4 microchannels (each 400 µm in diameter) which are continuously perfused by medium via a microphysiologic pump to supply oxygen and nutrients to the surrogate.
Appropriate analysis of the surrogates is crucial to gain pertinent information regarding cellular function in response to treatment or other manipulations. Surrogates can be analyzed by various methods including direct imaging of intact surrogates using confocal microscopy or other means of non-invasive imaging, indirect cellular analysis by assaying the conditioned media, or perfusate, for secreted products, or analysis on histologic sections after fixation and processing to paraffin. One such parameter that can be evaluated on histologic sections is cell density. We present one approach to measure cell density (i.e., the number of nucleated cells per section area) using semi-automated image processing techniques applied to photomicrographs of surrogate histologic sections stained with hematoxylin and eosin (H&E). The cell density can be used as an indicator of the relative change in cell number over time or that results from varying growth conditions and treatments.
Figure 1. 3D volume and bioreactor system. A) Schematic of the process to generate solid 3D surrogates. Top: cartoon of solid 3D volume containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Bottom: top view of 8-well chamber slide containing surrogates. B) Schematic of the process to generate perfused 3D surrogates. Top: cartoon of 3D volume with channels to allow for medium perfusion and containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Middle: image of PDMS flow channel containing PDMS foam (black arrow) to be injected with cell+ECM and penetrated by polymer-coated stainless steel wires (pink arrow) measuring 400 µm in diameter; Bottom: image of the PDMS flow channel containing a surrogate and connected to the bioreactor system to allow for continuous medium perfusion (peristaltic pump and media reservoir not shown). C) Images of processing steps for both solid and perfused surrogates after culture. Left: image of the cryomold containing specimen processing gel and surrogate; Middle: image of a paraffin block containing a fixed and processed surrogate; Right: image of a glass slide with a H&E-stained histologic section of a surrogate. Please click here to view a larger version of this figure.
1. Cell Culture
2. Preparation of Cells in ECM (6 mg/ml Bovine Collagen Type I + 10% BM)
Note: An ECM composed of 90% collagen I + 10% BM was chosen to model invasive breast carcinoma because the tumor stroma in this malignancy is composed primarily of collagen I with components of the BM, such as laminin, collagen IV, and entactin, comprising a smaller portion of the ECM12,13,18,19.
Preparation of cells in ECM (6 mg/ml Bovine Collagen Type I + 10% BM) | |
178.8 µl | Cell culture grade water containing the desired number of 231 cells (determined above) |
606 µl | Collagen I (10 mg/ml bovine), add drop by drop |
100 µl | Basement membrane, thawed |
100 µl | 10x DMEM (containing phenol red) with the desired number of CAF (determined above) |
15.2 µl | 7.5% (v/v) Sodium Bicarbonate, add drop by drop |
Table 1. Preparation of cells in ECM.
3. Surrogate Preparation
4. Surrogate Fixation and Processing (Figure 1C)
5. Sectioning and H&E Staining (Figure 1C, Right Panel)
H&E Staining | ||
Station | Solution | Time |
1 | Xylene | 5 min |
2 | Xylene | 5 min |
3 | Xylene | 5 min |
4 | 100% Ethanol | 5 min |
5 | 100% Ethanol | 5 min |
6 | 95% Ethanol | 5 min |
7 | 95% Ethanol | 5 min |
8 | Tap Water | 5 min |
9 | De-ionized Water | 5 min |
10 | Hematoxylin 7211 | 5 min |
11 | Tap Water | 5 min |
12 | Clarifier* | 10 dips |
*Richard Allan #7401 or 70% Ethanol + 0.5% HCl | ||
13 | Tap Water | 5 min |
14 | Bluing Reagent | 30 sec |
15 | Tap Water | 5 min |
16 | 95% Ethanol | 10 dips |
17 | Eosin-Y | 1 min |
18 | 95% Ethanol | 10 dips |
19 | 95% Ethanol | 10 dips |
20 | 100% Ethanol | 10 dips |
21 | 100% Ethanol | 10 dips |
22 | 100% Ethanol | 5 min |
23 | Xylene | 10 dips |
24 | Xylene | 5 min |
Table 2. H&E Staining.
6. Measuring Cell Density
Figure 2. ImageJ analysis. Screenshot of ImageJ processing. Please click here to view a larger version of this figure.
Figure 3. CellProfiler example pipeline. Screenshot of the pipeline designed to measure the number of nucleated cells in CellProfiler. Please click here to view a larger version of this figure.
Figure 4. CellProfiler pipeline: changing image to grayscale. Screenshot of "ColortoGray "module. Please click here to view a larger version of this figure.
Figure 5. CellProfiler pipeline: inverting image. Screenshot of "ImageMath" module. Please click here to view a larger version of this figure.
Figure 6. CellProfiler pipeline: identifying nuclei. Screenshot of "IdentifyPrimaryObjects" module. Please click here to view a larger version of this figure.
Figure 7. CellProfiler pipeline: identifying cells. Screenshot of "IdentifySecondaryObjects" module. Please click here to view a larger version of this figure.
Figure 8. CellProfiler pipeline: measuring objects. Screenshot of "MeasureObjectSizeShape" module. Please click here to view a larger version of this figure.
Figure 9. CellProfiler pipeline: filtering objects. Screenshot of "FilterObjects" module. Please click here to view a larger version of this figure.
Figure 10. CellProfiler pipeline: exporting data. Screenshot of "ExportToSpreadsheet" module. Please click here to view a larger version of this figure.
Figure 11. CellProfiler output following filtering. Screenshot of output screen in cell profiler following object filtering. Please click here to view a larger version of this figure.
Both solid and perfused 3D breast cancer surrogates were prepared as described above and grown for 7 days. Subsequently, surrogates were fixed, processed to paraffin, sectioned, and stained with hematoxylin and eosin, as described above. The number of nucleated cells per area (both 231 cells and CAF) of each surrogate was measured. As can be seen in Figure 12, representative photomicrographs of the H&E-stained sections demonstrate a higher concentration of cells prese...
Herein, a method of 3D culture has been described that incorporates components of the tissue microenvironment, including the extracellular matrix (ECM) and human stromal fibroblasts, in a volume that more closely models human breast cancer to allow for the development of a recapitulative 3D morphology. The 3D culture method described is more representative of human disease than traditional 2D cell culture in that multiple cell types are incorporated into a 3D volume of ECM. It has been noted that these parameters (i....
The authors declare that they have no competing financial interests.
The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).
Name | Company | Catalog Number | Comments |
Dulbecco's Modified Eagel Medium 1x (DMEM) | Corning CellGro | 10-014-CV | |
Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11150 | |
0.25% Trypsin + 2.21 mM EDTA 1x | Corning | 25-053-CI | |
Tissue Culture plates, 100 mm | CellTreat Scientific Products | 229620 | Sterile |
Tissue Culture plates, 35 mm | CellTreat Scientific Products | 229638 | For PDMS foam formation |
9" Glass pipette | Fisher | 13-678-20D | Sterile |
10 ml pipette | CellTreat Scientific Products | 229210B | Sterile |
1,000 µl piptette tips | FisherBrand | 02-717-166 | Sterile Filtered |
200 µl pipette tips | FisherBrand | 02-717-141 | Sterile Filtered |
10 µl pipette tips | FisherBrand | 02-717-158 | Sterile Filtered |
15 ml conical tubes | CellTreat Scientific Products | 229410 | Sterile |
50 ml conical tubes | CellTreat Scientific Products | 229422 | Sterile |
1.5 ml microcentrifuge tubes | FisherBrand | 05-408-129 | Sterile |
Trypan blue | Corning Cellgro | 25-900-CI | Sterile |
Sylgard 184 | Electron Microscopy Sciences | 24236-10 | PDMS elastomer and curing agent. Used for our in-house bioreactor. |
PDMS Foam | Made in-house for use in our in-house bioreactor. | ||
High Concentration Bovine Collagen Type I | Advanced Biomatrix | 5133-A | FibriCol ~10 mg/ml |
Growth Factor Reduced Matrigel (Basement Membrane) | Corning | 354230 | Basement membrane material |
Sodium Bicarbonate | Sigma | S8761 | |
Molecular Biology Grade Water | Fisher | BP2819-1 | |
DMEM 10x | Sigma-Aldrich | D2429 | |
Nunc Lab-Tek Chamber Slide System | Thermo Scientific | 177402 | 8-well |
Bioreactor | Made in-house. | ||
Spring-Back 304 Stainless Steel—Coated with PTFE polymer | McMaster-Carr | 1749T19 | Stainless steel wires to generate microchannels in our in-house bioreactor system. 0.016" Diameter |
BioPharm Plus platinum-cured silicone pump tubing, L/S 14 | Masterflex | EW-96440-14 | For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in. |
2-Stop Tubing Sets, non-flared PVC, 1.52 mm ID | Cole-Parmer | EW-74906-36 | For use in our in-house bioreactor system (with microperistalitic pump). |
Six Channel precision micro peristaltic pump | Cole-Parmer | EW-74906-04 | For use with our in-house bioreactor system |
Tuberculin Syringes | BD Medical | 309625 | 26 gauge 3/8 in. needle; Sterile |
Dissecting Tissue Forceps | FisherBrand | 13-812-36 | 5.5 inch |
Mini Tube Rotator | Boekel Scientific | 260750 | Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753) |
50 ml tube carousel | Boekel Scientific | 260753 | Used with mini tube rotator |
Bambino Hybridization Oven | Boekel Scientific | 230301 | Equipment option for surrogate rotation |
HistoGel Specimen Processing Gel | Thermo Scientific | HG-4000-012 | Specimen Processing Gel described in Step 5.2 |
Cryomold | Andwin Scientific | 4566 | 15 mm x 15 mm x 5 mm |
Tissue Marking Dye | Cancer Diagnostics, inc. | 03000P | Can be used to mark surrogates, allowing multiple samples to be included in one tissue cassette |
Hinged tissue cassettes | FisherBrand | 22-272-416 | |
Formalin | Fisher | 23-245-685 | |
GoldSeal Plain Glass Slides | Thermo Scientific | 3048-002 | |
Xylene | Fisher | X3P-1GAL | |
Ethanol, 200 proof (100%), USP | Decon Laboratories, Inc. | 2805M | |
Hematoxylin | Thermo Scientific Richard-Allan Scientific | 7211 | |
Clarifier | Thermo Scientific Richard-Allan Scientific | 7401 | |
Bluing Solution | Thermo Scientific Richard-Allan Scientific | 7301 | |
Eosin Y | Thermo Scientific Richard-Allan Scientific | 7111 | |
Cytoseal XYL mounting media | Thermo Scientific Richard-Allan Scientific | 83124 | |
Coverslips | Fisher Scientific | 12-548-5G |
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