This research was supported by Cancer Research Foundation / Swiss Cancer League grant KFS-4125-02-2017 to RKD and National Institute of Health grant DK079307 to EKJ.

1. Cell culture
NOTE: Conduct cell handling under sterile conditions.
<ol>
	<li>Human Umbilical Vein Endothelial Cells (HUVECs) subculture
	<ol>
		<li>Coat 75 cm2 flasks with collagen (5 &#181;g/cm2) (rat-tail) overnight (ON) at room temperature (RT) or 2-3 h at 37 &#176;C, rinse with water, and allow the flask to dry.</li>
		<li>Grow HUVECs in growth medium (EBM-2, Endothelial Basal Medium-2) supplemented with glutamine (1x = 2 mM), antibiotic-antimycotic solution (AA; 100 &#181;g/mL of streptomycin, 100 &#181;g/mL of penicillin and 0.025 &#181;g/mL of amphotericin B), LSGS (2% v/v FCS, 1 &#181;g/mL of hydrocortisone, 10 ng/mL of human Epidermal Growth Factor, 3 ng/mL of human basic Fibroblast Growth Factor, 10 &#181;g/mL of heparin) and 10% FCS (Fetal Calf Serum) under standard tissue culture conditions (37 &#176;C, 5% CO2). Change the medium every 2 days until the cells reach 70%-80% confluence.</li>
		<li>Wash the sub-confluent cultures with 5 mL of HBSS (without Ca2+ and Mg2+) and add 3 mL of trypsin (0.25% diluted in HBSS (without Ca2+ and Mg2+)). 
		NOTE: HBSS can also be replaced with PBS.</li>
		<li>Incubate the cells at 37 &#176;C for 2 min (microscopically check whether the cells are detached and rounded up) and stop the detaching reaction by adding 6 mL of 10% FCS medium (DMEM-F12 or EBM-2, 10% FCS).</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min at RT and discard the supernatant.</li>
		<li>Suspend the cells in growth medium and seed in 75 cm2 flasks or culture dishes.</li>
	</ol>
	</li>
	<li>Michigan Cancer Foundation-7 (MCF-7, human breast adenocarcinoma cells) subculture
	<ol>
		<li>Grow human breast cancer cell lines in 75 cm2 flasks in growth medium (DMEM/F12 medium supplemented with a commercial glutamine (1x), antibiotic-antimycotic solution (AA; 100 &#181;g/mL of streptomycin, 100 &#181;g/mL of penicillin and 0.025 &#181;g/mL of amphotericin B), and 10% FCS&#160;under standard tissue culture conditions (37 &#176;C, 5% CO2). Change the medium every 2-3 days.</li>
		<li>Wash the subconfluent cell cultures (70%-80% confluence) with 5 mL of HBSS (without Ca2+ and Mg2+) and add 3 mL of trypsin (0.5% diluted in HBSS (without Ca2+ and Mg2+) at 37 &#176;C for 5 min (microscopically verify that the cells are detached). 
		NOTE: HBSS can also be replaced with PBS.</li>
		<li>Add 8 mL of 10% FCS medium to stop the detaching reaction, centrifuge at 250 x g for 5 min at RT and discard the supernatant.</li>
		<li>Suspend the pellet in growth medium and plate in 75 cm2 flasks or culture dishes.</li>
	</ol>
	</li>
</ol>
2. Spheroid formation
<ol>
	<li>Plate 5 x 105 cells/mL (HUVECs and MCF-7) in a 3.5 cm round dish and culture for 48 h.</li>
	<li>Treat or transfect the plated cells for 24 h or as desired.</li>
	<li>Optional step performed in 96-well U-bottom plate: Wash the cells with 1 mL of HBSS (Ca2+ and Mg2+) and stain HUVECs using a blue dye (0.5 &#181;g/mL) and MCF-7 cells using a green dye (0.5 &#181;M) diluted in the respective growth medium and place into a 37 &#176;C and 5% CO2 incubator for 30-40 min.</li>
	<li>Wash the cells with 1 mL of HBSS (without Ca2+ and Mg2+), add 1 mL of enzymatic detachment reagent (0.25% trypsin for HUVEC and 0.5% trypsin for MCF-7) to each round dish using a P1000 pipette, and then incubate for 2-5 min until the cells are detached.</li>
	<li>Collect each cell type separately in a 5 mL round-bottom Polystyrene tube and add 1 mL of 10% FCS medium using a P1000 pipette to stop the enzymatic reaction. Centrifuge the cell suspensions at 250 x g for 5 min.</li>
	<li>Carefully aspirate the supernatant and suspend the cells in 1 mL of steroid-free medium (EBM-2, glutamine , antibiotic-antimycotic, 0.4% FCS sf (charcoal-stripped)). 
	NOTE: Steroid-free medium can be replaced with normal growth medium.</li>
	<li>Use 100 &#181;L of each cell suspension diluted with 10 mL of Diluent II (see Table of Materials) to determine the total cell number and calculate the amount of treatment medium (EBM-2, glutamine, antibiotic-antimycotic, 0.4% FCS sf, vehicle/Treatment) needed to obtain a cell suspension of 5 x 103 cells/mL or 3.4 x 105 cells/mL per cell type.
	<ol>
		<li>Cell count
		<ol>
			<li>Turn on the machine and flush by pressing the Function and Start buttons (set-up S3), with Diluent II solution in a cell counter vial.</li>
			<li>Use set up S4 and press Start to measure the blank with fresh Diluent II solution.</li>
			<li>Change the scintillation vial with 100 &#181;L of cell suspension in 10 mL of Diluent II solution and measure the samples: set-up S4 and press Start.</li>
			<li>Note the number of cells per mL on the digital display. 
			&#8203;NOTE: Cell counting can also be performed using other manual or automatic systems: Hemocytometer gridlines, light-scatter cell-counting technology, electrical impedance, imaging-based systems using cell staining, e.g., trypan blue.</li>
		</ol>
		</li>
		<li>96-well U-bottom plates
		<ol>
			<li>Prepare 5 mL of each cell suspension (5 x 103 cells/mL) and mix them to get a final volume of 10 mL in the ratio of 1:1.</li>
			<li>Use a manual repeating pipette to pipette out 100 &#181;L of the cell suspension into each well of the 96-well U-bottom plates.</li>
			<li>Place the plate into an incubator under standard tissue culture conditions and check for spheroids formation after 48 h.</li>
			<li>Optional step: Take pictures of the spheroids (40x) using a fluorescence stereo microscope. 
			NOTE: This method generates 96 spheroids (one spheroid/well) after 48 h (area 1-2 pixel2), and it is usually used for growth studies.</li>
		</ol>
		</li>
		<li>Hanging drop
		<ol>
			<li>Mix the cell suspensions (3.4 x 105 cells/mL) in a 1:1 ratio to get a final volume of 2 mL.</li>
			<li>Use a P20 pipette to seed the cell mixture in the form of 15 &#181;L drops on the inverted lid of a 10 cm Petri dish.</li>
			<li>Invert the lid with the drops and add 5 mL of base medium (EBM-2) at the bottom of the dish to avoid evaporation of the drops. 
			&#8203;NOTE: This method generates one spheroid/drop after 48 h. Ensure the drops are sufficiently apart from each other, so that they do not merge while switching the lid. Use more than one 10 cm Petri dish if necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Spheroid growth study
<ol>
	<li>Plate 100 &#181;L of HUVECs and MCF-7 cell mixture (5 x 102 cells/well) in 96-well U-bottom plates and place them in the incubator.</li>
	<li>48 h after plating, check for spheroids formation with an inverted microscope (Bright field). Take pictures (at least six pictures per treatment, 40x) at 96 h of culture.</li>
	<li>Open the pictures using an image processing software and process the image to 300 pixel/inch and save the image as a tiff file.</li>
	<li>Download the ImageJ software and open it. Click on the File option and go to Open.</li>
	<li>Convert the areas of interest to saturated black areas in a uniform manner to have a binary image (black and white). For this, select the Image option, choose Type | 8-bit. Now, the image is black and white.</li>
	<li>Use the Freehand Selection Tool to draw the border of the spheroids.</li>
	<li>To calculate the area of interest and to separate the object or the foreground pixels from the background pixels, use the threshold function: Select the Image option, choose Adjust and click on Threshold.</li>
	<li>Now a Threshold pop-up window will open. The top bar indicates the minimum threshold value: set the value at zero. The bottom bar indicates the maximum threshold value: move the bar until the area of interest becomes completely red. 
	NOTE: If the color is not red, select Red from the Threshold window.</li>
	<li>Go to Analyze | Set Measurements and select Area and Perimeter.</li>
	<li>To measure the area of interest, select the Analyze option and use the Analyze Particles tool. In the Analyze Particles window, set the Size to measure (0.00003-Infinity or 3-Infinity, depending on the size of the spheroids), select Summarize and Display Results. Then, click on OK.</li>
	<li>The results will show up in a chart. Copy the measurements into a spreadsheet to analyze the data. 
	&#8203;NOTE: The area is calculated as the number of total pixels; use the Total Area for calculation.</li>
</ol>
4. Spheroid immunohistochemistry study
<ol>
	<li>Sample preparation
	<ol>
		<li>Plate the cell mixture of HUVECs and MCF-7 (5 x 103 cells/well) in 96-well U-bottom plates in HUVEC growth medium for 96 h.</li>
		<li>Collect approximately 50 spheroids into a 1.5 mL tube with a cut tip of a P200 &#181;L pipette; allow them to settle to the bottom of the tube by gravity, and then discard the supernatant.</li>
		<li>Fix the spheroids with 500 &#181;L of 4% PFA for 1 h, RT.</li>
		<li>Boil 2% Nobel Agar solution in PBS usinga hot plate with a magnetic stirrer for 3-5 min to dissolve the agarose powder completely and cool down to around 60 &#176;C.</li>
		<li>Remove the PFA with a P1000 pipette, wash the spheroids with 500 &#181;L of PBS, and allow them to sediment. Discard the supernatant.</li>
		<li>Carefully pipette 600 &#181;L of agarose solution into the 1.5 mL tube with spheroids and place the tube immediately in a centrifuge with a horizontal rotor at 177 x g for 2 min.</li>
		<li>Add a short string in the middle of the agarose solution to easily remove the plug from the tube.</li>
		<li>Solidify agarose plug on ice or at 4 &#176;C. Add 500 &#181;L of PBS into the 1.5 mL tube to avoid drying out of the pellet. 
		&#8203;NOTE: The samples are ready for subsequent dehydration, paraffin embedding, sectioning, transfer onto the microscope slides, and IHC staining (performed by Sophistolab).</li>
	</ol>
	</li>
	<li>Image acquisition and processing
	<ol>
		<li>Acquire images of spheroid sections using a stereomicroscope.</li>
		<li>Save three images for each sample as .jpg files.</li>
		<li>Open the ImageJ software. Open the JPEG image, click on File and then on Open.</li>
		<li>Select Color and Color Deconvolution in the Image option.</li>
		<li>Select H DAB vectors in the Color Deconvolution 1.7 window, and the three images appear. 
		NOTE: The three images are: Color 1 represents only the Hematoxylin staining (blue/purple), and Color 2 represents only the DAB staining (brown). Color 3 is not needed for the image analysis with two stainings.</li>
		<li>Select the Color 1 window and set the Threshold: go to Image | Adjust and click on Threshold. The Threshold window appears.</li>
		<li>Leave the minimum threshold value set at zero and adjust the maximum threshold value to remove the background signal without influencing the true signal. Click on Apply.
		<ol>
			<li>Area measurement
			<ol>
				<li>Click on Analyze, choose Set Measurement. Select Area, Mean grey value, and Min and Max grey value and click on OK to confirm.</li>
				<li>Measure the size of the nucleus using the Analyze option, and then select Measure. 
				NOTE: The Results window gives the name of the image (Label), size of the image (Area), the average pixel intensity of the IHC image (Mean), and the minimum and maximum gray values (Min, Max). Use the Mean value for calculation.</li>
				<li>Repeat steps 4.2.6-4.7.1.2 for the Color 2 (and Color 3 if there is dual staining) window.</li>
			</ol>
			</li>
			<li>Cell quantification
			<ol>
				<li>Click on Process, choose the option Binary and select Watershed to separate cells.</li>
				<li>Go to Analyze and click on Analyze Particles. On the Analyze Particles pop-up window, set the size of the cells to exclude unspecific particles. Next, on the Show option, click on the drop-down box and select Outlines. Then, click on OK.</li>
				<li>Repeat steps 4.2.6-4.2.7 and cell quantification steps (section 4.2.7.2) for the Color 2 (and Color 3 if there is dual staining) window. 
				&#8203;NOTE: Now three windows will appear: 1) Summary results (number of cells counted, total area, average size, % of area and mean), 2) Results (respective to the cells that are counted), and 3) Drawing window (depicted image of the cells that are counted).</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Live/dead staining in Spheroids
<ol>
	<li>Prepare 4 mM stock solutions of Calcein AM in DMSO (stains live cells green) and 2 mM of Ethidium homodimer in DMSO (stains dead cells red) in 1.5 mL tubes.</li>
	<li>Calculate the amount of solution necessary to add 10 &#181;L/well of a mixture of Calcein AM and Ethidium homodimer diluted 1:50 in EBM-2.</li>
	<li>Add the staining mixture to the spheroids and place the plate in the incubator under standard tissue culture conditions for 30-60 min.</li>
	<li>Acquire pictures (40x) using the Fluorescence stereo microscope.</li>
</ol>
6. Spheroid&#39;s protein and RNA isolation
<ol>
	<li>Prepare cell suspensions at 3.4 x 105 cells/mL per cell type, mix them at a ratio of 1:1, and seed the cell mixture using a P20 pipette in the form of 15 &#181;L drops on the lid of a 10 cm Petri dish. Invert the lid and place it in the incubator under standard tissue culture conditions for 96 h.</li>
	<li>Use 5-6 mL of PBS to collect spheroids in a 15 mL round-bottom Polystyrene tube using 5 mL sterile polystyrene pipettes. 
	NOTE: It is also possible to use 15 mL conical tubes.</li>
	<li>Centrifuge the spheroids suspension at 250 x g&#160;for 5 min, and then carefully aspirate the supernatant.</li>
	<li>Add 500 &#181;L of trypsin (100%) and pipette up and down using a P1000 pipette for 30 s to disaggregate spheroids, achieving a cell suspension.</li>
	<li>Neutralize trypsin with 10% FCS medium, centrifuge the tubes at 250 x g for 5 min, and aspirate the supernatant.</li>
	<li>According to the manufacturer&#39;s protocol (specific kit for DNA-free RNA isolation), use 300 &#181;L of RNA lysis buffer to lyse the cell pellet. 
	NOTE: Lysis buffer can also be directly added to the intact spheroids (no trypsin step required) to extract RNA. Add 300 &#181;L of the lysis buffer to the pelleted spheroids, place tubes in ice, and triturate 10 times using a P200 pipette. Subsequently, isolate RNA as above. Spheroid dissociation may be needed if RNA recovery is low due to matrix interference.</li>
	<li>Alternatively, use 70 &#181;L of the Lysis buffer for protein isolation. Homogenize for 2-4 s by sonication and determine the protein concentration according to the manufacturer&#39;s protocol using a BCA Assay Kit. 
	NOTE: For protein isolation, pelleted spheroids can be lysed directly in lysis buffer followed by sonication. Use 25 &#181;g of total protein to perform western blot.</li>
</ol>

The authors have nothing to disclose.

Compared to 2D cell cultures, revolutionary 3D spheroid culture technology is a better and more powerful tool to reconstruct an organ&#39;s microenvironment, cell-cell interactions, and drug responses in vitro. This is the first protocol describing the formation of spheroids from multicellular (epithelial and endothelial) cell lines for breast cancer research. This protocol ensures spheroidal 3D growth of spheroids for up to 5 days, and spheroids can be examined after paraffin embedding, sectioning, and histolog...

The use of animals for experiments has limitations. Animal studies cannot accurately mimic disease progression in humans, and animals and humans do not have identical responses to pathogens. Additionally, restrictions in animal experimentation due to concerns for animal suffering and ethical problems1,2 increasingly constrain research programs. In vitro systems have been widely developed to circumvent the use of animals; moreover, the use of human cells has made in vitro models more relevant for the pathophysiological and therapeutic investigation. Conventional monolayer (2D) cell cultures are widely used because they mimic human tissues to some degree. However, 2D monocultures fail to mimic human organs, and 2D monocultures are unable to simulate the complex microenvironment of the original organs and mimic the in vivo situation3,4,5,6. Additionally, in monolayer cell cultures, drug treatments could easily destroy/damage all of the cells. Importantly, some of these limitations can be overcome by switching to multilayer three-dimensional (3D) cell cultures7,8. In fact, 3D culture models have been shown to better reflect the cellular structure, layout, and function of cells in primary tissues. These 3D cultures can be formed using a variety of cell lines, similar to a functional organ. Indeed, there are two models of 3D cultures. One model produces spheroids of aggregated cells that form clusters and reorganize them into spheres (scaffold-free models). The second yields organoids, which have a more complex structure and consist of combinations of multiple organ-specific cells, which are considered as a miniature version of organs9,10. Due to this, 3D culture systems represent an innovative technology with many biological and clinical applications. Thus, spheroids and organoids have numerous applications for disease modeling and studies related to regenerative medicine, drug screening, and toxicological studies6,11,12,13,14,15. Carcinogenic spheroids, derived from 3D technology, recreate the morphology and phenotype of relevant cell types, mimic the in vivo tumor microenvironment, and model cell communications and signaling pathways that are operational during tumor development16,17,18. In addition, to improve cancer biology understanding, tumor spheroids/organoids can also be used to identify a potential patient-specific anti-cancer therapy (personalized) and assess its efficacy, toxicity, and long-term effects19,20,21,22. Spheroids have opened prominent opportunities to investigate pathophysiology, disease modeling and drug screening because of their ability to preserve cellular and three-dimensional tissue architecture, the ability to mimic the in vivo situation, and the cell-cell interactions. However, one must also be aware of the limitations of this system, such as the lack of vascular/systemic component, functional immune or nervous system, and the system represents a reductionist approach as compared to animal models. Indeed, in contrast to animal models, 3D structures provide only an approximation of the biology within a human body. Understanding the limitations of the 3D method may help researchers to design more refined and valid processes for producing spheroids that better represent an organ at a larger scale23,24,25.
Cancer is the leading cause of death worldwide, and breast cancer is the most common cancer in women26,27,28. To mimic the complex microenvironment of breast cancer, breast cancer spheroids should be cultured using cells that play a prominent role in breast tumors, i.e., epithelial cells, endothelial cells, fibroblasts, and/or immune cells. Moreover, for a spheroid representing breast cancer, the expression of female hormone receptors (estrogen/progesterone receptors), ability to conserve the patient tumor histological status, and ability to mimic response to therapy should also be considered. Studies have shown that 3D co-culture systems have a cellular organization similar to that of the primary tissue in vivo, have the capability to react in real-time to stimuli, and have functional androgen receptors29,30,31,32. Hence, a similar approach could be useful to mimic a breast tumor in vitro. The purpose of the current protocol is to establish a new method of generating breast cancer spheroids. This method utilizes estrogen receptor-positive MCF-7 cells (an immortalized human cell line of epithelial cells) and vascular endothelial cells (HUVECs) or lymphatic endothelial cells (HMVEC-DNeo) to create a model that mimics or closely reflects the interactions between these cells within a tumor. Although MCF-7 (estrogen-responsive) and endothelial cells have been used to develop spheroids in the present study, other cells such as fibroblasts which represent ~80% of the breast tumor mass, could also be combined in the future to better represent and mimic breast tumor.
There are several methods to form spheroids, such as: 1) the hanging droplet method that employs gravity33,34; 2) the magnetic levitation method that uses magnetic nanoparticles exposed to an external magnet35, and 3) the spheroid microplate method that is performed by seeding cells on low-attachment plates36,37. On the basis of the existing methods, which use only one cell type, the present protocol has been optimized using epithelial and endothelial cells to better mimic the growth conditions of breast cancer tumors in vivo38,39,40,41. This method can be easily achieved in the laboratory at a low cost and with minimal equipment requirements. Based on the need/goals of the lab, different approaches were used to form spheroids and gain relevant cellular material from these spheroids. In this context, for DNA, RNA, or protein analysis, the 3D spheroids are produced by co-culturing endothelial and epithelial cells with the hanging drop method. However, for functional studies, for example, to monitor cell growth after short interfering (siRNA) transfection and/or hormone treatment, the spheroids are generated using U-bottom plates.
The purpose of this technical protocol is to provide a detailed step-by-step description for 1) forming breast cancer multicellular-type spheroids, 2) preparing samples for histological staining, and 3) collection of cells for extraction of RNA, DNA, and proteins. Both the inexpensive hanging drop method and the more expensive U-bottom plates are used to form spheroids. Here, a protocol for preparing (fixing) spheroids for sectioning and subsequent immunostaining with markers to assess cell proliferation, apoptosis, and distribution of epithelial and endothelial cells within a spheroid is provided. Additionally, this protocol shows a complete step-by-step analysis of histological data using ImageJ software. Interpretation of biological data varies depending on the type of experiment and the antibodies used. Sectioning of fixed spheroids and subsequent staining of the sections was performed by a routine pathology lab (Sophistolab: info@sophistolab.ch)

<table><tbody><tr><td>100 mm &amp;times; 20 mm tissue-culture treated culture dishes</td><td>Corning</td><td>CLS430167</td><td></td></tr><tr><td>149MULTI0C1</td><td></td><td></td><td></td></tr><tr><td>35 x 10 mm Tissue Culture Dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>5 mL Serological Pipet, Polystyrene, Individually Packed, Sterile</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>Adobe Photoshop</td><td></td><td></td><td>Version: 13.0.1 (64-bit)</td></tr><tr><td>Antibiotic Antimycotic Solution (100&amp;times;)</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma-Aldrich</td><td>17783</td><td></td></tr><tr><td>CD31 (cluster of differentiation 31)</td><td>Cell Marque</td><td>131M-95</td><td>monoclonal mouse ab clone JC70</td></tr><tr><td>CellTracker Green CMFDA (5-chloromethylfluorescein diacetate)</td><td>Invitrogen</td><td>C7025</td><td></td></tr><tr><td>CK8/18 (cytokeratins 8 and 18)</td><td>DBS</td><td>Mob189-05</td><td>monoclonal mouse ab, clone 5D3</td></tr><tr><td>CKX41 Inverted Microscope</td><td>Olympus Life Science</td><td></td><td>Olympus DP27 digital camera</td></tr><tr><td>Cleaved Caspase 3</td><td>Cell Signaling</td><td>9661L</td><td>polyclonal rabbit ab</td></tr><tr><td>Collagen (rat tail)</td><td>Roche</td><td>11 179 179 001</td><td></td></tr><tr><td>Coulter ISOTON II Diluent</td><td>Beckman Coulter</td><td>844 80 11</td><td>Diluent II</td></tr><tr><td>Coulter Z1, Cell Counter</td><td>Coulter Electronics, Luton, UK</td><td></td><td></td></tr><tr><td>Dehydrated Culture Media: Noble Agar</td><td>BD Difco</td><td>BD 214220</td><td></td></tr><tr><td>Dermal Microvascular Endothelial Neonatal, Pooled Donors (HMVEC-DNeo)</td><td>Lonza</td><td>CC-2516</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich</td><td>D6434</td><td></td></tr><tr><td>EBM-2 Endothelial Cell Growth Basal Medium-2</td><td>Lonza</td><td>190860</td><td></td></tr><tr><td>Ethidium homodimer</td><td>Sigma-Aldrich</td><td>46043</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Thermo Fisher Scientific</td><td>SH30070</td><td></td></tr><tr><td>Fetal Calf Serum Charcoal Stripped (FCS sf)</td><td>Thermo Fisher Scientific</td><td>SH3006803</td><td></td></tr><tr><td>Fluorescence stereo microscopes Leica M205 FA</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>GlutaMAX Supplement (100x)</td><td>Gibco</td><td>35050038</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Gibco</td><td>14175053</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>HUVEC &amp;ndash; Human Umbilical Vein Endothelial Cells</td><td>Lonza</td><td>CC-2517</td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health, USA</td><td></td><td>Wayne Rasband&lt;br/&gt; Version: 1.52a (64-bit)</td></tr><tr><td>Ki67</td><td>Cell Marque</td><td>275R-16</td><td>monoclonal rabbit ab, clone SP6</td></tr><tr><td>Leica fully motorized fluorescence stereo microscope</td><td>Leica Microsystems</td><td>M205 FA</td><td></td></tr><tr><td>Leica Histocore Multicut Rotary Microtome</td><td></td><td>149MULTI0C1</td><td></td></tr><tr><td>Low Serum Growth Supplement Kit (LSGS Kit)</td><td>Gibco</td><td>S003K</td><td></td></tr><tr><td>MCF-7 cells &amp;ndash; human breast adenocarcinoma cell line</td><td>Clinic for Gynecology, University Hospital Zurich</td><td></td><td>Provived from Dr Andr&amp;egrave; Fedier obtained from ATCC</td></tr><tr><td>Nunclon Sphera 96U-well plate</td><td>Thermo Fisher Scientific</td><td>174925</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) 1x</td><td>Gibco</td><td>10010015</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Protein Lysis Buffer</td><td>Cell Signaling, Danvers, USA</td><td>9803</td><td></td></tr><tr><td>Quick-RNA Miniprep Kit</td><td>Zymo Research</td><td>R1055</td><td></td></tr><tr><td>RNA Lysis Buffer</td><td>Zymo Research</td><td>R1060-1-100</td><td>Contents in Quick-RNA Miniprep Kit</td></tr><tr><td>Rotina 46R Centrifuge</td><td>Hettich</td><td></td><td></td></tr><tr><td>Round-Bottom Polystyrene Tubes, 5 mL</td><td>Falcon</td><td>352003</td><td></td></tr><tr><td>Sonicator</td><td>Bandelin electronics, Berlin, DE</td><td></td><td></td></tr><tr><td>Tecan Infinite series M200 microplate reader</td><td>Tecan, Salzburg, AU</td><td></td><td></td></tr><tr><td>Tissue Culture Flasks 75</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T3924</td><td></td></tr></tbody></table>

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.

The spheroids model using epithelial and endothelial co-cultures is required to closely mimic in vivo conditions of breast tumors for in vitro experiments. The scheme in Figure 1 depicts the protocol to form spheroids with breast cancer epithelial cells and vascular or lymphatic endothelial cells (Figure 1). Each cell type is seeded separately in a 3.5 cm round dish and treated with growth stimulators/inhibitors or transfected with oligonucleot...

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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.

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 ...

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Research

JoVE Journal

Cancer Research

4.5K Views.  University of Zurich and University Hospital Zurich. De hierin beschreven sferoïden van borstkankercellen zullen onderzoekers in staat stellen moleculaire mechanismen van endotheliale celinteracties van kankercellen te onderzoeken die betrokken zijn bij proliferatie en metastase van kankercellen. Het belangrijkste voordeel van dit 3D-model is dat het een meer realistische cellulaire opstelling biedt die de betrouwbaarheid van gevolgtrekkingen met betrekking tot de pathofysiologie en behandeling van borstkanker verbetert. De procedure wordt ged...

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicine

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Bioengineering

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Biology

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.

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

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