Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
Uploaded: 2016-05-09T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

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).

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The authors declare that they have no competing financial interests.

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.e., multiple cell types, ECM, and 3D volume) provide a more appropriated context to study biological processes because tissue architecture, signals from the microenvironment, and dimensionality are being taken into account 1,8. Other methods of 3D culture have been previously described including, the culture of cells on top of a 3D matrix29, the generation of spheroids30, hanging drop cutlures31, and the use of microfluidic devices32. While each of these systems has unique advantages, overall they fail to include either matrix components, stromal cell populations, or a representative dimensionality. The method of 3D culture described here includes each of these parameters and can be established using a bioreactor system to allow for longer growth periods that are difficult to achieve using solid 3D culture. In addition, perfusion of surrogates provides a greater degree of growth compared with solid surrogates.
Another feature of this approach for in vitro culture is the flexibility in the types of analyses that can be performed using the surrogate &#34;tissues&#34;. A variety of endpoints can be monitored indirectly during culture by evaluating the conditioned media, or perfusate, for soluble products (e.g., LDH as an indicator of cell death, specific secreted proteins or metabolites as an indicator of appropriate phenotypic function) or by utilizing non-invasive imaging of fluorescently or luminescently labeled cells to evaluate growth and surrogate architecture overtime. Following growth, the ECM can be digested using an ECM protease and cells can be isolated and used for additional analysis or further experiments, such as flow cytometric quantification33. Lysates of the cells removed from the ECM could also be evaluated genomically, at the mRNA transcript level, or for protein expression. Alternatively, endpoints can be measured directly from the fixed and processed surrogate &#34;tissues&#34; after the experiment has concluded. These include a variety of morphological parameters (e.g., nuclear/cell morphology and the degree of cell aggregation) on H&#38;E-stained histologic sections and other molecular analyses performed on histologic sections using immunohistochemistry (e.g., expression of Ki-67 as an indicator of cell proliferation) or in situ hybridization (e.g., RNA expression of specific gene products). It is also possible to perform additional molecular analyses, such as real-time, quantitative PCR or next generation sequencing, on these formalin-fixed, paraffin-embedded surrogates; however, due to molecular cross-linking induced by fixation and processing, snap-frozen surrogates are preferred for these types of analyses. Such molecular and morphologic evaluations can provide valuable information regarding cell growth, death, or response to pharmacological treatment throughout growth as well as following culture.
The main advantage of using the CellProfiler program for semi-automated quantification of the density of nucleated cells present in histologic sections of surrogates is the time savings that it provides. Although the total amount of time required per surrogate is variable and depends largely on the size of the surrogates and the number of images acquired, it is estimated that arriving at a cell density from the surrogate images requires 4-5 hours of hands-on time per surrogate when manually counting versus half an hour per surrogate when using the CellProfiler program. The majority of the hands-on-time using CellProfiler analysis is attributed to the measurement of the surrogate area in ImageJ. The CellProfiler program does require time to automatically process the images, but does not require hands-on time from the researcher other than importing the images.
There are limitations of the methods and the 3D surrogate cultures described here. As shown in Figure 12, the use of the perfusion bioreactor system provided a greater degree of growth than solid cultures, in which the accumulation of cells and the rate of proliferation is considerably slower. Although the perfusion bioreactor system used here provided a growth advantage, perfusion systems can also have their own limitations. For example, PDMS is often used in the manufacture of bioreactors due to its ease of use, inert properties, and reproducibility. However, this material is known to absorb hydrophobic molecules from cell culture media and may take up lipophilic drugs34-36. While other materials can be used instead of PDMS if these limitations are of concern, other materials available also come with their own drawbacks that must be considered. Because CellProfiler analysis is dependent on the parameters input for the diameter and circularity of the nuclei, optimization of these inputs for each surrogate/cell type may be necessary. It is also of note that the cell density measurement presented here is not a direct measurement of cell viability. It has previously been noted that a direct measurement of cell viability is challenging in 3D culture systems37. However, nuclear degradation is a part of both apoptosis and necrosis, the two predominate forms of cell death. In apoptosis, the nucleus fragments, a process called karyorrhexis, following chromatin condensation, or pyknosis38,39. This differs from necrosis, where the dying cell swells causing the cell membrane to rupture and release the contents of its cytoplasm. Histologically, necrosis is characterized by karyolysis (i.e., dissolution of a nucleus), followed by pyknosis and karyorrhexis40. Nuclei that have undergone these changes can be seen on H&#38;E-stained histologic sections but will not be recognized as intact nuclei by the CellProfiler program. These nuclear changes occur relatively late in the process of cell death however. Therefore, quantifying the number of nucleated cells per area may include cells that are in the earlier stages of cell death, but will nonetheless provide useful information regarding cell viability at the time of surrogate fixation. Other methods that could be used to indicate viability or growth include analyses of perfusates (e.g., Alamar Blue or LDH assays) or evaluation of proliferation in the surrogate &#34;tissues&#34; (e.g., via Ki-67 labeling by immunohistochemistry).
Critical steps in the protocol for surrogate setup include ECM polymerization, where temperature, pH, and time all must be monitored to ensure complete polymerization prior to bathing the surrogate in medium. In addition, keeping the cell+ECM mixture on ice prior to plating in an 8-well chamber slide or injection into a bioreactor system is crucial to preventing premature polymerization. Also to prevent polymerization during surrogate setup, the sodium bicarbonate used to increase the pH to 7 should be added to the mixture last. Finally, adequate incubation time at 37 &#176;C must be given to ensure complete ECM polymerization; 45 minutes has proven to be sufficient. Another important factor in surrogate preparation is the avoidance of bubble formation. In both the solid and perfused systems, it is best to avoid the formation of bubbles in the cell+ECM mixture as to not create a block to the flow of nutrients during culture. It is also important to avoid bubbles during medium introduction in the perfusion system where a bubble may function as an air embolism, blocking medium flow.
Proper fixation is important for downstream analysis; therefore, attention to standardized and consistent fixation (including the type of fixation and the amount of time in fixative) and processing is necessary to obtain replicative results. The duration and type of fixation is less of an issue for the H&#38;E staining described here, but these parameters may affect other types of molecular analysis of the surrogates. This is because formalin fixation, in particular, causes the cross-linking of molecules, including protein, DNA, and RNA in a somewhat time-dependent and molecule-specific manner41,42. This is of particular importance in immunohistochemical analysis, when antibody recognition may be hindered by cross-linking.

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 &#181;m 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&#38;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.
<img alt="Figure 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 
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 &#181;m 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&#38;E-stained histologic section of a surrogate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagel Medium 1x (DMEM)</td><td>Corning CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>0.25% Trypsin + 2.21&amp;nbsp;mM EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Tissue Culture plates, 100&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229620</td><td>Sterile</td></tr><tr><td>Tissue Culture plates, 35&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229638</td><td>For PDMS foam formation&amp;nbsp;</td></tr><tr><td>9&quot; Glass pipette</td><td>Fisher&amp;nbsp;</td><td>13-678-20D</td><td>Sterile</td></tr><tr><td>10 ml pipette</td><td>CellTreat Scientific Products</td><td>229210B</td><td>Sterile</td></tr><tr><td>1,000 &amp;micro;l piptette tips</td><td>FisherBrand</td><td>02-717-166</td><td>Sterile Filtered</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-141</td><td>Sterile Filtered</td></tr><tr><td>10 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-158</td><td>Sterile Filtered</td></tr><tr><td>15 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229410</td><td>Sterile</td></tr><tr><td>50 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229422</td><td>Sterile</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>FisherBrand</td><td>05-408-129</td><td>Sterile</td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td>Sterile</td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>PDMS elastomer and curing agent. Used for our in-house bioreactor.</td></tr><tr><td>PDMS Foam</td><td></td><td></td><td>Made in-house for use in our in-house bioreactor.</td></tr><tr><td>High Concentration Bovine Collagen Type I</td><td>Advanced Biomatrix</td><td>5133-A</td><td>FibriCol ~10 mg/ml</td></tr><tr><td>Growth Factor Reduced Matrigel (Basement Membrane)</td><td>Corning</td><td>354230</td><td>Basement membrane material</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S8761</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Fisher</td><td>BP2819-1</td><td></td></tr><tr><td>DMEM 10x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>Nunc Lab-Tek Chamber Slide System&amp;nbsp;</td><td>Thermo Scientific</td><td>177402</td><td>8-well</td></tr><tr><td>Bioreactor</td><td></td><td></td><td>Made in-house.</td></tr><tr><td>Spring-Back 304 Stainless Steel&amp;mdash;Coated with PTFE polymer</td><td>McMaster-Carr</td><td>1749T19</td><td>Stainless steel wires to generate microchannels in our in-house&amp;nbsp; bioreactor system. 0.016&quot; Diameter</td></tr><tr><td>BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td><td>Masterflex</td><td>EW-96440-14</td><td>For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td></tr><tr><td>2-Stop Tubing Sets, non-flared PVC, 1.52&amp;nbsp;mm ID</td><td>Cole-Parmer&amp;nbsp;</td><td>EW-74906-36</td><td>For use in our in-house bioreactor system (with microperistalitic pump).&amp;nbsp;</td></tr><tr><td>Six Channel precision micro peristaltic pump</td><td>Cole-Parmer</td><td>EW-74906-04</td><td>For use with our in-house bioreactor system</td></tr><tr><td>Tuberculin Syringes</td><td>BD Medical</td><td>309625</td><td>26 gauge 3/8 in. needle; Sterile</td></tr><tr><td>Dissecting Tissue Forceps</td><td>FisherBrand</td><td>13-812-36</td><td>5.5 inch</td></tr><tr><td>Mini Tube Rotator</td><td>Boekel Scientific</td><td>260750</td><td>Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td></tr><tr><td>50 ml tube carousel</td><td>Boekel Scientific</td><td>260753</td><td>Used with mini tube rotator</td></tr><tr><td>Bambino&amp;nbsp; Hybridization Oven</td><td>Boekel Scientific</td><td>230301</td><td>Equipment option for surrogate rotation</td></tr><tr><td>HistoGel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Specimen Processing Gel described in Step 5.2</td></tr><tr><td>Cryomold</td><td>Andwin Scientific</td><td>4566</td><td>15 mm x 15 mm x 5 mm</td></tr><tr><td>Tissue Marking Dye</td><td>Cancer Diagnostics, inc.&amp;nbsp;</td><td>03000P</td><td>Can be used to mark surrogates,&amp;nbsp; allowing multiple samples to be included in one tissue cassette&amp;nbsp;</td></tr><tr><td>Hinged tissue cassettes&amp;nbsp;</td><td>FisherBrand</td><td>22-272-416</td><td></td></tr><tr><td>Formalin</td><td>Fisher</td><td>23-245-685</td><td></td></tr><tr><td>GoldSeal Plain Glass Slides</td><td>Thermo Scientific</td><td>3048-002</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td></td></tr><tr><td>Ethanol, 200 proof (100%), USP</td><td>Decon Laboratories, Inc.</td><td>2805M</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7211</td><td></td></tr><tr><td>Clarifier</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7401</td><td></td></tr><tr><td>Bluing Solution</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7301</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal XYL mounting media</td><td>Thermo Scientific Richard-Allan Scientific</td><td>83124</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5G</td><td></td></tr></tbody></table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

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.

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&#38;E-stained sections demonstrate a higher concentration of cells present in the perfused surrogates (Figure 12A) compared to the solid surrogates (Figure 12B), even though the density of cells initially incorporated into the surrogates was the same in both solid and perfused conditions. This visual representation of cell growth is supported by a higher mean number of cells per area (density) in the perfused surrogates (n=3) compared to the solid surrogates (n=3), as determined by the CellProfiler analysis (Figure 12C). To obtain the data in Figure 12C, a complete histologic cross-section of each surrogate was imaged, requiring multiple images for each surrogate, and the images were analyzed as described above. Then, the total number of nuclei for each surrogate was divided by the total area of the surrogate (expressed as the number of nuclei/1 x 106 pixels2), providing a cell density for each surrogate. To validate the use of CellProfiler for nucleated cell quantification, the results using the CellProfiler program were compared with those obtained by simply manually counting the number of cells present per area in each of the 6 surrogates (Table 3). The cell density of each surrogate was calculated as described above. No significant difference was found in the cell densities obtained by manual counting and CellProfiler analysis for either perfused surrogates (p=0.855) or solid surrogates (p=0.553). To support that the difference in cell density between the solid and perfused surrogates correlates with a difference in cell growth, cell proliferation of each surrogate was evaluated via immunohistochemical analysis of Ki-67 labeling (Figure 12D)27. The same trend was found, with a significantly higher percentage of proliferating cells in the perfused surrogates (n=3) compared to the solid surrogates (n=3), indicating a higher growth rate in the perfused surrogates and correlating with the increased cell density of these surrogates. While the cell type (i.e., breast cancer epithelial or CAF) was not evaluated in these measurements, immunostaining for cell type-specific markers could be used to better understand the growth or distribution of a specific cell population. Protocols detailing this process have been previously published 25,28.
<img alt="Figure 12" src="/files/ftp_upload/54004/54004fig12.jpg" /> 
Figure 12. Representative Results. A) Representative image of a H&#38;E-stained section from a perfused surrogate grown for 7 days (400X original magnification). B) Representative image of a H&#38;E-stained section from a solid surrogate grown for 7 days with medium changed every 2 days (400X original magnification). C) Comparison of the mean number of nucleated cells per area, or cell density, following 7 days growth of perfused (no change of media) and solid surrogates (media changed every 2 days). D) Comparison of Ki-67 labeling index (i.e., the percentage of cells in a population that express Ki-67), a measure of cell proliferation, following 7 days growth of perfused and solid surrogates. Data represent the mean &#177; SEM, n=3 per condition, ** indicates p&#8804;0.01 and *** indicates p&#8804;0.001 (Student&#39;s t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54004/54004fig12highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Experiment</td>
			<td>CellProfiler: nucleated cells per area</td>
			<td>Manual: nucleated cells per area</td>
			<td>Average CellProfiler</td>
			<td>Average Manual</td>
			<td>P value (Unpaired t Test)</td>
		</tr>
		<tr>
			<td>Perfused Surrogate 1</td>
			<td>88.178</td>
			<td>75.532</td>
			<td>97.225</td>
			<td>99.901</td>
			<td>0.855</td>
		</tr>
		<tr>
			<td>Perfused Surrogate 2</td>
			<td>107.528</td>
			<td>117.812</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfused Surrogate 3</td>
			<td>95.967</td>
			<td>106.359</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solid Surrogate 1</td>
			<td>19.797</td>
			<td>17.480</td>
			<td>16.491</td>
			<td>12.991</td>
			<td>0.553</td>
		</tr>
		<tr>
			<td>Solid Surrogate 2</td>
			<td>8.612</td>
			<td>5.650</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solid Surrogate 3</td>
			<td>21.065</td>
			<td>15.844</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3. Cell densities obtained by manual or CellProfiler analysis for 3 solid and 3 perfused surrogates.

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

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.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.6K Views.  University of Alabama at Birmingham. 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.

Video: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

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 ...

Cancer Research

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

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

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

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

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

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

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Medicine

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

Biology

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

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Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

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

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks