We would like to thank Samuel Zirbel for assistance in optimizing culture conditions and monitoring spheroid growth. We are grateful to Agilent technologies for providing the SeaHorse XFe96 analyzer, assay reagents and technical insights. We also thank Dr. Hemant Kocher at Barts Cancer Institute in United Kingdom for providing the PS1 cells. This work was supported in part by the Seena Magowitz Foundation for Pancreatic Cancer Research and a Stand Up To Cancer-Cancer Research UK-Lustgarten Foundation Pancreatic Cancer Dream Team Research Grant (Grant Number: SU2C-AACR-DT-20-16). Stand Up To Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C.

1. Culture of Pancreatic Cancer 3D Spheroids using Magnetic Bioprinting
<ol>
	<li>Using standard aseptic tissue culture technique, culture cells of interest in a T75 flask to a confluency of 70-80% in appropriate growth media. 
	NOTE: Typically, 5-7 &#215; 106 cells from a 70-80% confluent T75 flask were harvested. Two different cell types were used in this study - Patu8902 (pancreatic tumor cells) and PS1 cells (activated pancreatic stellate cells21). Both cell lines were cultured in Roswell Park Memorial Institute media 1640 (RPMI-1640) supplemented with 10% (v/v) fetal bovine serum(FBS), 1&#215; penicillin-streptomycin (p/s).</li>
	<li>Wash cells once with 5 mL of Dulbecco&#39;s phosphate buffered saline(DPBS).</li>
	<li>Detach cells from plastic surface by trypsinizing with 2 mL of 0.05% Trypsin-EDTA for 5-10 min at 37 &#176;C. Check for cell detachment under a microscope.</li>
	<li>Deactivate trypsin by addition of 8 mL growth media to detached cells. Avoid over trypsinization, as this can adversely affect the health/viability of cells.</li>
	<li>Pipet cells up and down to generate a single cell suspension and count cell density using an automated cell counter or a hemocytometer.</li>
	<li>In the meantime, equilibrate a vial of NS to ambient temperature.</li>
	<li>Calculate the amount of cells required for seeding the desired number of spheroids (wells) and aliquot to a new 15 mL conical tube. For example, to seed an entire 96 well plate with 5,000 cells/well, aliquot at least 5.5 &#215; 105 cells.</li>
	<li>Collect cells by centrifuging at 500 x g for 3 min. Carefully aspirate or decant the supernatant and resuspend the cells at a concentration of 1.0 x 106 cells/mL in growth media. Pipette gently using a wide bored pipet tip to avoid shearing. For example, resuspend 5.5 x 105 cells in 550 &#181;l of growth media.</li>
	<li>Magnetize the desired amount of cells using the equilibrated NS (step 1.6).
	<ol>
		<li>Directly add NS to the cell suspension in growth media and agitate gently. For efficient magnetic labeling of cells, use 10 &#181;L NS per 100 &#181;L cells (resuspended at 1 x 106 cells/mL). 
		NOTE: For a typical experiment, label 5.5&#215;105 Patu8902 cells in 550 &#181;L with 55 &#181;L NS and 1.1&#215;106 PS1 cells in 1100 &#181;L, (separately) with 110 &#181;L NS.</li>
		<li>Gently invert tube a few times to ensure cell suspension. Temporarily, transfer the cell-NS mix into a single well of a 24-well plate. Do this separately for each cell type to be magnetized. Cover plate and incubate cells at room temperature for 2 h with gentle shaking to allow NS binding to cell surface. 
		NOTE: Culture and assay of spheroids starting either from Patu8902 or PS1 alone cells, in addition to co-culture spheroids starting with both cell types premixed before printing on magnetic drives was successfully achieved using this method in independent experiments. A method for generating co-culture spheroids from Patu8902 and PS2 cells is described below in the subsequent steps.</li>
	</ol>
	</li>
	<li>Pipet up and down the magnetized cells gently to mix evenly and prepare a master mix containing the desired cell number. For seeding spheroids, use a total of 15,000 cells (5,000 Patu8902 + 10,000 PS1) and adjust to a total volume of 150 &#181;L per well of a 96-well plate. 
	NOTE: The final volume dispensed into each well must be the same regardless of the number of cells used.</li>
	<li>Place a cell repellent 96-well plate atop the 96-well magnetic spheroid drive and dispense 150 &#181;L of cell mix into each well. Observe the cells slowly collect to the center of the well above the magnet. Cover and incubate the plate overnight in a 5% CO2 incubator set at 37 &#176;C with the magnetic spheroid drive still attached. Remove the magnetic drive and incubate for further growth for up to 7 days. 
	NOTE: It is critical to use cell repellent growth plates for spheroid printing using magnetic spheroid drives. Ensure that the spheroid drive with thinner smaller magnets is used, not the holding drive.</li>
	<li>Monitor the growth of spheroids every day. Replenish with fresh growth media every three days by placing the growth plate mounted on the magnetic holding drive at an angle and using a multichannel pipet to draw out spent media. 
	NOTE: Patu8902 and PS1 cells co-seeded at 15,000 cells/well will grow into 3D spheroids with a diameter of 400-600 &#181;m within 5-7 days. At this point spheroids are ready for metabolic characterization, drug evaluation, and other downstream applications.</li>
</ol>
2. Metabolic Assay of 3D Pancreatic Tumor Spheroids for Bioenergetics Pathways using the Extracellular Flux Analyzer
Note: In this section, the analysis of the metabolic functions of the spheroids using an extracellular flux analyzer with assay microplates specifically designed for 3D spheroids is described.
<ol>
	<li>Washing and Transfer of spheroids from growth plates to spheroid assay microplates. 
	NOTE: Spheroids may be used for metabolic assays when they reach a diameter of ~500 &#181;m.
	<ol>
		<li>The day prior to performing the assay, hydrate the probe or sensor cartridge with assay calibrant (200 &#181;L/well) in the provided utility plate and incubate overnight (4-18 h) in a humidified incubator (non-CO2) set at 37 &#176;C.</li>
		<li>Prepare the appropriate assay medium for the desired metabolic assay by supplementing the base media (see the Table of Materials) with either 2 mM L-glutamine for glycolysis stress test (GST) assay or with 1 mM pyruvate, 2 mM L-glutamine and 10 mM glucose for a mitochondrial stress test (MST) assay. 
		Note: The extracellular flux analyzer measures both mitochondrial respiration (OCR) and glycolysis (ECAR) of live cells in a 96-well plate format. These rates provide an overview of cellular metabolic function in samples under investigation. Please refer to the manual in the assay kits for detailed instructions.</li>
		<li>After adding assay specific supplements (refer Step 2.1.1), warm the supplemented assay medium in a 37 &#176;C water bath and adjust the pH to 7.4 &#177; 0.1 with 0.1N NaOH. Keep the medium warm until use.</li>
		<li>Reconstitute assay kit reagents in the calibrated assay medium to recommended stock concentrations. 
		NOTE: These are made at 10&#215; the final concentration desired in the wells. Stock concentrations for glucose, oligomycin and 2-deoxy-glucose (2-DG) are 100 mM, 100 &#181;M and 500 mM, respectively.</li>
		<li>Observe spheroids in the growth plate under a light microscope to check for spheroid morphology and overall uniformity; in terms of the shape and structure of the spheroids.</li>
		<li>Transfer the growth plate onto a magnetic holding drive and carefully aspirate ~120 &#181;L growth media. Gently wash spheroids with ~120 &#181;L of warmed and pre-calibrated assay media, aspirate and repeat the wash twice. Inspect the spheroids under the microscope again to ensure that spheroids are not washed away.</li>
		<li>Prepare the spheroid assay microplate by adding 180 &#181;L warm assay media to each well.</li>
		<li>Set a P20 micropipette to 10 &#181;L and fit a wide bored tip to it. If wide bored tips are not available, cut off the tips of regular pipette tips with a clean scissors or scalpel to widen the bore. 
		Note: This should reduce shearing and preserve the overall morphology of the spheroids while being transferred to assay plates.</li>
		<li>Use a warm (37 &#176;C) surface to carry out transfer of spheroids. Use an X-ray film viewer surface warmed with a white-light lamp to enhance contrast and aid visualization of spheroids during the transfer process.</li>
		<li>Carefully aspirate a single pre-washed spheroid from the cell repellent growth plate using a P20 micropipette fitted with a wide bore tip and gently transfer spheroid directly into the center of each well of a spheroid assay plate for carrying out the metabolic assay. Allow each spheroid to gently fall into the central micro-chamber of each well by pure gravity to populate the assay plate. 
		NOTE: It usually takes 5-8 s for each spheroid to fall into the center of the well by gravity. Do not pull out the pipette until the spheroids have settled down in the micro-chamber. Do not deposit spheroids in the 4 corner wells of the assay plate (A1, A12, H1, H12) since these will be used as background wells.</li>
		<li>Monitor the morphology and position of each spheroid to ensure that each spheroid is in the center of each well. If necessary, reposition to the center of well by aspirating out spheroid using a wide bore pipet tip, and subsequent deposition by gravity.</li>
		<li>Once all spheroids have been transferred, place the assay plate in a 37 &#176;C humidified air incubator (non-CO2) for one hour prior to assay.</li>
	</ol>
	</li>
	<li>Loading sensor cartridge with compounds and performing the assay
	<ol>
		<li>Prepare 10&#215; concentrated assay specific reagents in the assay specific media described in step 2.1.2. For example, to carry out GST, reconstitute glucose, oligomycin, and 2-DG in recommended volumes referred to in the assay manual. Both GST and MST assays were carried out using Patu8902-PS1 spheroids.</li>
		<li>Properly orient the sensor cartridge with rows labeled A-H on the left hand side and place a loading guide on top of the sensor cartridge. Ensure correct loading of desired port by orienting the loading guide so that the letter corresponding to the port to be loaded is at the upper left hand corner.</li>
		<li>Using a multichannel pipette, dispense reagents directly into injection ports. Hold loading guides in position using fingertips throughout the procedure. 
		Note: Each reagent will be injected in a recommended port referred in the assay manual. Avoid creating air bubbles but do not tap any part of the cartridge to remove air bubbles.</li>
		<li>Remove loading guide and position at eye level with cartridge to visually inspect injection ports for even loading.</li>
		<li>Create the experimental assay design/instrument protocol using the instrument controller Wave software (henceforth, analysis software) as described in section 2.3.</li>
	</ol>
	</li>
	<li>Assay design and execution using the analysis software
	<ol>
		<li>Create an assay template for the experiment
		<ol>
			<li>Open the analysis software and click &#34;Templates&#34; or choose from an existing assay template. Double click on &#34;Blank Template&#34;.</li>
			<li>Define experimental groups and conditions.
			<ol>
				<li>Define Injection strategies for ports A, B, and C. Leave port D empty. 
				NOTE: For GST assay, only these ports will be loaded with Glucose, Oligomycin and 2-DG. In case of MST assay, oligomycin, FCCP and rotenone will be loaded.</li>
				<li>Define pre-treatments if spheroids were treated with one or more test compounds and the control group. Define the assay media to include source, supplements, and other information.</li>
				<li>Define one or more cell type (e.g. Patu8902, PS1) seeing the density and passage number information.</li>
			</ol>
			</li>
			<li>Click &#34;Generate Groups&#34;.</li>
			<li>Click &#34;Plate Map&#34; tab and assign groups to the assay plate corresponding to the experimental design.</li>
			<li>Select the four corner wells as background wells. Ensure that there are no spheroids in these wells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Run assay on the analyzer
	<ol>
		<li>Click on the Instrument Protocol tab. Keep the default protocol commands by checking &#34;Calibrate&#34;, &#34;Equilibrate&#34; and &#34;Baseline Measurement Cycle&#34;.</li>
		<li>Click &#34;Injections&#34; and define compound for each port. Edit measurement cycles to 6 cycles from the default 3 cycles for spheroids. Keep the default 3 min mix, 0 min wait, and 3 min measure times. 
		Note: Default mix-wait-measure times are 3 min, 0 min, 3 min, respectively. Three basal rate measurements are typically taken prior to injection of first assay compound. These measurements can be calibrated and readjusted as per the experimental design.</li>
		<li>Review Protocol and Group Summary.</li>
		<li>Save assay design template.</li>
		<li>Proceed to pre-assay calibration once injection ports have been loaded with assay substrates. Transfer the cartridge with the utility plate to the extracellular flux analyzer. 
		NOTE: This usually completes within 15-20 min and calibrates the sensors to conduct OCR and ECAR measurements.</li>
		<li>After calibration of the cartridge, replace the utility plate with the pre-warmed assay plate containing 3D spheroids and initiate the assay. After the measurements are completed, export the data to spreadsheet or graphing and statistical software to analyze the data.</li>
	</ol>
	</li>
</ol>

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The authors George W. Rogers and Andrew Neilson are employees/shareholders of Agilent Technologies that produces reagents and instruments used in this Article. All other authors declare that they have no competing financial interests.

Using pancreatic cancer, the 3rd leading cause of cancer related deaths in the United States24, as an example and model, a rapid and consistent method was developed to grow and assay 3D spheroids using the co-culture of pancreatic cancer cells and activated pancreatic fibroblasts. Using magnetic bioprinting, spheroids ranging in size from 400-600 &#181;m in diameter were obtained. These were subjected to metabolic assays to successfully test the functionality of the cultured 3D spheroids. This method is simple, consistent and can be easily adapted to other cell types.
While this methodology will accelerate and add to the translational value of the assays performed with 3D spheroids, it can be improved by developing techniques that allow multiplexing of spheroid manipulation and handling. This should further reduce the overall time, cost and labor required to carry out the assay. Additionally, spheroid volumes may be used to normalize OCR signals as reported by others6. Spheroid volume normalized by average volume of a cell could be used to determine OCR per cell, but since growth of spheroids will not be identical, calculating an absolute OCR per cell might be challenging. Currently, this methodology is limited by the lack of an efficient tool to multiplex spheroid handling and transfer from growth plates to assay environment.
The method described is modified and adapted from the magnetic bioprinting technology, and further validated to show the functional relevance by applying the cultured spheroids to a downstream metabolic assay. The method provides an important tool to generate robust, viable and functional spheroids that contain the two major cell types found in most tumor tissues, cancer cells and cancer associated fibroblasts, which can be employed for other investigational assays, such as drug screens. The relative ease and efficiency of the NS methodology is a major improvement over other methods25, such as the hanging drop method2,26 of spheroid generation.
Robust spheroids generated as such provide an in vitro tumor model that includes stromal cells, which are often missing from many 3D culture studies. The possibilities for application of 3D spheroid models generated as such are vast. For example, such models can be employed to investigate cell-cell interactions, bioenergetics shifts and to test genetic susceptibility and dependence of various therapeutic regimens. 3D spheroids that recapitulate the in vivo tumor microenvironment serve as highly relevant and useful tool for drug screening studies and those investigating the mechanisms of action of current and novel therapeutics. Metabolic assays probing energy pathways using spheroids cultures with mutant cells may help shed new light into the role of those genes and related pathways in the pathobiology in a relevant 3D model of cancer, like the spheroids described in this study.
The successful application of this method relies foremost on the health of cells required for magnetic printing, which is why cell growing in logarithmic phase should be harvested and magnetized. It is critical to use the magnetic spheroid drive to print magnetized cells and not the holding drive, which should be utilized only during the spheroid washing and media replenishing steps of the described method. The other most critical aspect for successful readout of metabolic assays is to maintain the morphology of the spheroids during the transfer process from the growth plate to the assay plate.
Overall, this protocol has been established for generation of 3D spheroids that contain both cancer and stromal cells, following the subsequent metabolic assay of the spheroids using the extracellular flux analyzer. The key features of this method are that it is quick, easily adaptable and consistent, relevant to and mimics the in vivo tumor microenvironment, is comparatively inexpensive and may serve as a new tool for studying tumor biology and developing anticancer agents.

In the last decade, numerous in vitro 3D models have been developed to recapitulate and investigate the in vivo tumor biology, microenvironment and growth conditions of cancer cells1,2. Two-dimensional (2D) monolayer cell culture systems with uniform exposure to biochemical factors and investigational compounds fail to replicate the native 3D tumor-stromal interactions exposed to a gradient of compounds diffusing through the extracellular matrix proteins (ECM)3,4. Thus, in comparison to 2D tissue culture models, 3D cancer models have emerged to show better potential at simulating the tumor microenvironment and served as important tools to better understand in vivo tumor characteristics, such as hypoxia, desmoplasia, dormancy, drug penetrance, toxicity and therapeutic resistance5,6. To this end, 3D models have potential to bridge the gap between 2D cell culture and whole animal models by mimicking in vivo tumor features, while being relatively inexpensive and optimized for rapid generation and consistency. These advantages are being exploited to accelerate translational research in many areas including cancer biology, morphogenesis and tissue engineering7,8.
In a surge of evolving 3D tissue culture methods, magnetic levitation techniques have recently been developed and described for growth, assaying and imaging of spheroids derived from various cell types9,10,11,12. Magnetic 3D bioprinting exploits the use of magnetic forces to engineer tissues by magnetizing cells with biocompatible nanoparticles and printing them in multi-well formats. This allows rapid production of consistent, near identical 3D spheroids, which can be harnessed and employed for a plethora of downstream applications for biochemical and biophysical investigation10. Here we have adapted the magnetic bioprinting technique using a biocompatible material called Nanoshuttle (NS) composed of iron oxide, poly L-lysine and gold nano particles to label pancreatic cancer cells and fibroblasts. NS attaches electrostatically to the plasma membrane, is not known to bind to any specific receptors, and releases off the cell surface within a week. It requires very low magnetic forces (30pN), enough to aggregate but not harm cells and does not affect cell viability, metabolism or proliferation to make it extremely biocompatible for 3D cultures10,13,14,15.
In this study, using pancreatic cancer as an example and model, we describe the generation and metabolic assay of 3D cancer cell-fibroblast spheroids. Starting from cells cultured in 2D vessels, we illustrate the culture and growth conditions of pancreatic tumor-fibroblast co-culture spheroids using magnetic bioprinting. Cultured spheroids were then used in functional metabolic assays using an extracellular flux analyzer, a technology demonstrated to simultaneously measure the two major energy producing pathways, glycolysis and mitochondrial respiration, in a variety of live cells and tissues16,17,18,19,20. Glycolysis was measured as a change in the extracellular acidification rate (ECAR), while mitochondrial respiration or oxidative phosphorylation was measured as oxygen consumption rate (OCR). We propose that this method developed for pancreatic tumor spheroids can serve as a backbone for optimizing and translating 3D tumor spheroid generation and assay to other cell/tissue types.

<table style="border-collapse:collapse;width:646pt" width="861">
	<colgroup>
		<col style="width:186pt" width="248" />
		<col style="width:140pt" width="187" />
		<col style="width:89pt" width="119" />
		<col style="width:230pt" width="307" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:186pt;height:15.75pt" width="248">0.05% Trypsin EDTA</td>
			<td class="xl22" style="width:140pt" width="187">Sigma</td>
			<td class="xl34" style="width:89pt" width="119">25300-054</td>
			<td class="xl35" style="width:230pt" width="307"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl19" height="42" style="width:186pt;height:31.5pt" width="248">96 well cell repellant plate</td>
			<td class="xl19" style="width:140pt" width="187">USA Scientific/ Griener Bio-One</td>
			<td class="xl26" style="width:89pt" width="119">655970</td>
			<td class="xl21">spheroid growth plate</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">Cellometry Cell counting slides</td>
			<td class="xl19" style="width:140pt" width="187">Nexcelom&#160;</td>
			<td class="xl26" style="width:89pt" width="119">CHT4-SD100-002</td>
			<td class="xl21"></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl19" height="20" style="width:186pt;height:15.0pt" width="248">D-Glucose</td>
			<td class="xl19" style="width:140pt" width="187">Sigma</td>
			<td class="xl26" style="width:89pt" width="119">D9434</td>
			<td class="xl21"></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl19" height="20" style="width:186pt;height:15.0pt" width="248">DPBS</td>
			<td class="xl19" style="width:140pt" width="187">Gibco</td>
			<td class="xl26" style="width:89pt" width="119">14190-144</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">Glycolysis Stress Test Kit</td>
			<td class="xl19" style="width:140pt" width="187">Agilent Technologies</td>
			<td class="xl26" style="width:89pt" width="119">103020-100</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">L-Glutamine</td>
			<td class="xl19" style="width:140pt" width="187">Sigma</td>
			<td class="xl26" style="width:89pt" width="119">59202C</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">Mitochondrial Stress Test Kit</td>
			<td class="xl19" style="width:140pt" width="187">Agilent Technologies</td>
			<td class="xl26" style="width:89pt" width="119">103015-100</td>
			<td class="xl21"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">n3D Magnetic Spheroid drive</td>
			<td class="xl19" style="width:140pt" width="187">Nano3D Bioscience</td>
			<td class="xl26" style="width:89pt" width="119">655841</td>
			<td class="xl21">Part of Spheroid Bioprinting Kit</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl19" height="42" style="width:186pt;height:31.5pt" width="248">n3D Magnetic Spheroid holding drive</td>
			<td class="xl19" style="width:140pt" width="187">Nano3D Bioscience</td>
			<td class="xl26" style="width:89pt" width="119">655841</td>
			<td class="xl21">Part of Spheroid Bioprinting Kit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">n3D NanoShuttle-PL&#160;</td>
			<td class="xl19" style="width:140pt" width="187">Nano3D Bioscience</td>
			<td class="xl26" style="width:89pt" width="119">655841</td>
			<td class="xl21">Part of Spheroid Bioprinting Kit:&#160; store at 4&#176;C</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">Patu8902 cells</td>
			<td class="xl19" style="width:140pt" width="187">Laboratory Stock</td>
			<td class="xl19" style="width:89pt" width="119"></td>
			<td class="xl21">pancreatic ductal adenocarcinoma cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">PS1 cells</td>
			<td class="xl19" style="width:140pt" width="187">Laboratory Stock</td>
			<td class="xl19" style="width:89pt" width="119"></td>
			<td class="xl21">activated pancreatic stellate cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">RPMI1640</td>
			<td class="xl19" style="width:140pt" width="187">Gibco</td>
			<td class="xl26" style="width:89pt" width="119">11875-093</td>
			<td class="xl21">growth media for cells, spheroids</td>
		</tr>
		<tr height="45" style="height:33.75pt">
			<td class="xl19" height="45" style="width:186pt;height:33.75pt" width="248">SeaHorse XFe96 extracellular flux analyzer</td>
			<td class="xl19" style="width:140pt" width="187">Agilent Technologies</td>
			<td class="xl26" style="width:89pt" width="119"></td>
			<td class="xl21">extracellular flux analyzer</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:186pt;height:15.75pt" width="248">Sodium Pyruvate</td>
			<td class="xl19" style="width:140pt" width="187">Sigma</td>
			<td class="xl26" style="width:89pt" width="119">S8636</td>
			<td class="xl21"></td>
		</tr>
		<tr height="24" style="height:18.0pt">
			<td class="xl19" height="24" style="width:186pt;height:18.0pt" width="248">XF Base media</td>
			<td class="xl19" style="width:140pt" width="187">Agilent Technologies</td>
			<td class="xl26" style="width:89pt" width="119">102365-100</td>
			<td class="xl21">base media for metabolic assays on XFe96</td>
		</tr>
	</tbody>
</table>

preparation and metabolic assay of 3-dimensional spheroid co-cultures of pancreatic cancer cells and fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

The general workflow for magnetic bioprinting and extracellular flux analysis of 3D spheroids 
Figure 1&#160;depicts an overview of the entire process described in this protocol. Pancreatic cancer cells (Patu8902) and activated stellate cells (PS1) were cultured in tissue culture flasks containing appropriate growth media to a confluency of 70-80%. Cells were detached from the 2D culture vessel and washed, cell density was determined and cells finally resuspended in growth media at 1&#215;106 cells/mL. NS, a nanoparticle assembly consisting of gold, iron oxide, and poly-L-lysine was used to magnetize cells. Individually magnetized Patu8902 and PS1 cells were then mixed and printed onto 96-well cell repellent growth plates mounted on a magnetic spheroid drive. We have optimized the 15,000 to be the total number of cells for seeding a single well (5,000 Patu8902 cells mixed with 10,000 PS1 cells to generate a single spheroid). After incubation under appropriate tissue culture conditions for 5-7 days, 3-dimensional spheroids were obtained, during which time the growth of these spheroids was monitored and recorded. After washing with assay media, spheroids were physically transferred onto a spheroid microplate to carry out metabolic assays using an extracellular flux analyzer for simultaneous measurement of glycolysis and mitochondrial respiration.
Culture and growth of pancreatic tumor-fibroblast spheroids 
To obtain functional and robust spheroids, the epithelial cancer cell line Patu8902 and activated stellate cells PS1 were cultured in RPMI-1640 supplemented with fetal bovine serum. Pat8902 and PS1 cells magnetized with NS were then mixed together in a ratio of 1:2, so as to seed a total of 15,000 cells per well in a growth plate. Within 24 h of printing the cells onto the magnetic drives, cells focalized to the center of the well exactly on the top of each magnetic pin under each well. Growth of Patu8902-PS1 spheroids was monitored by imaging on days 2, 4, 7 and 9 after seeding cells. Figure 2 shows quantification of the average diameter of representative spheroids at various time points above. The diameter of spheroids increases from 414 &#181;m on day 2 to 596 &#181;m on day 7 post printing. There was no significant difference between growth on days 7 and 9, and thus all further experimentation was limited to 7 days of spheroid growth. We noticed that the spheroids acquire a sharper morphology especially around the outer edges and the NS is progressively more evenly diffused throughout the volume of the spheroid with more days in culture.We also estimated the sphericity of 10 spheroids based on the length of their major and minor axes. The average sphericity is 0.92 &#177; 0.04 for Patu8902 alone (tumor), 0.95 &#177; 0.04 for PS1 (stromal) alone and 0.94 &#177; 0.02 for co-culture spheroids.
Functional metabolic assay of pancreatic 3D spheroids using the extracellular flux analyzer 
To test the functionality of the spheroids, we employed metabolic and bioenergetics analysis to probe the energy pathways in spheroids. We carried out a glycolysis stress test on spheroids cultured as described above. To this end, spheroids generated from either Patu8902 or PS1 cells in addition to those resulting from a co-culture of the two cell types were subjected to the assay. Interestingly, all three types of spheroids, whether originating from distinct or mixed cells, exhibited a biologically functional response to the GST assay. Upon injecting a saturating concentration of glucose, the spheroids types tested show increases in the glycolytic pathway as evidenced by an increase in ECAR (Figure 3). When, mitochondrial ATP production was inhibited using oligomycin, energy production shifts to glycolysis and pushed cells to maximal glycolytic capacity, seen as further increase in ECAR. Inhibition of glycolysis using 2-DG, a glucose analog that competitively binds glucose hexokinase, resulted in a decrease in ECAR, confirming that the prior increase in ECAR was due to glycolytic activity. We noticed that the spheroids tested in this study responded in this fashion, although the PS1 alone spheroids (average diameter 250 &#181;m) had a relatively lower signal, possibly due to their smaller size and lower glycolytic capacity compared to the Patu8902 cancer cells (average diameter 600 &#181;m). In general, a higher ECAR signal from tumor cell derived spheroids compared to those from relatively smaller PS1 fibroblast derived spheroids, could possibly be attributed to the cancer cells&#39; inherent metabolic inclination towards glycolysis22,23. All three types of spheroids were derived from seeding the same number of cells, although we noticed spheroid size variation among each type, with PS1 alone spheroids being the smallest, followed by co-culture spheroids, and Patu8902 alone spheroids being the largest.
To test mitochondrial function in 3D spheroids, we subjected co-culture spheroids to a MST assay. The MST measures key parameters of mitochondrial function by directly measuring OCR of cells (Figure 4A and 4B). A serial injection with compounds (oligomycin, FCCP, and a mix of rotenone and antimycin A) was used to measure key mitochondrial parameters such as, ATP production, maximal respiration, and non-mitochondrial respiration. These parameters and basal respiration rates were further used to calculate proton leak and spare respiratory capacity (Figure 4C). A decrease in OCR in response to oligomycin, an inhibitor of ATP synthase (complex V) correlates to the mitochondrial respiration associated with cellular ATP production. Spheroids were incubated with oligomycin for a longer duration to allow maximal penetration and efficient response time. A second injection with the uncoupler FCCP stimulates maximal oxygen consumption and a corresponding increase in OCR. The FCCP-stimulated OCR was used to calculate spare respiratory capacity, a measure of the ability of the cell to respond to increased energy demand. The third injection; a mix of rotenone, (a Complex I inhibitor), and antimycin A (a Complex III inhibitor) blocks mitochondrial respiration, seen as a sharp decrease in OCR (Figure 4B).
Overall, our observations confirm the functionality of cultured spheroids using an extracellular flux analyzer as a measure of their metabolic footprint/activity both in terms of the glycolytic and mitochondrial potential as described above.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56081/56081fig1.jpg" /> 
Figure 1. Schematic representation of the methodology for the culture and assay of pancreatic cancer cells/fibroblast spheroids. Patu8902 and PS1cells were cultured, trypsinized, washed and counted. For seeding each spheroid, Patu8902 (5,000 cells/well) and PS1 fibroblasts (10,000 cells/well) were magnetized using NS and printed on a cell repellent growth plate on day 1. After seeding, the growth plate was mounted on a magnetic spheroid drive (with one magnet under each well of the 96 well format growth plate) and allowed to culture for 2-7 days to obtain 3D spheroids. The resulting spheroids were washed and transferred to the spheroid assay microplates to carry out the metabolic assays on the extracellular flux instrument. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56081/56081fig2.jpg" /> 
Figure 2. Growth of pancreatic tumor-fibroblast spheroids. (A) A representative image of Patu8902-PS1 co-culture spheroid corresponding to the day in culture is shown on the top and the size in diameter of the spheroid (&#181;m) is quantified below. Progressive increase in spheroid size and diffusion of NS particles (dark dots) throughout its volume is evident between days 2 and 7. Spheroids were kept in culture for an additional 2 days after this, which did not result in a significant increase in spheroid diameter. (B) Representative pictures of spheroids derived from tumor cells (Patu8902), stroma cells (PS1) and co-cultured cells (Patu8902 + PS1) is shown. (C) Sphericity data is depicted from 10 individual spheroids from each group. Error bars represent standard deviation (SD). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56081/56081fig3.jpg" /> 
Figure 3. Glycolysis Stress Test (GST) with pancreatic spheroids. (A) Schematic representation of a typical glycolytic function assay with various assay parameters depicted. (B) An example of a GST test performed on the pancreatic spheroids cultured using the methodology described. Glucose injection ramps up glycolysis seen as an increase in ECAR, with further increase on addition of oligomycin to shut off mitochondrial respiration. ECAR falls in response to 2-DG, a competitive analog of glucose, by blocking glycolysis. All spheroids are noted to exhibit a functional response to the GST assay. (C) An output of the GST assay using the manufacturer&#39;s report generator depicting the three major parameters of the GST assay. Error bars represent standard error of mean (SEM). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56081/56081fig4.jpg" /> 
Figure 4.&#160;Mitochondrial Stress Test (MST) with Patu8902-PS1 spheroids. (A) Schematic representation of a typical mitochondrial function assay with various assay parameters is depicted. (B) An example of a MST on the pancreatic tumor-fibroblast spheroids is shown. (C) Co-culture 3D spheroids exhibit a functional response to the MST assay. As expected, mitochondrial function measured as a decline in OCR was noticed when spheroids were challenged with oligomycin, an ATP synthase inhibitor. Using FCCP to ramp up electron flow resulted in maximal OCR, which immediately collapsed upon inhibition of mitochondrial respiration using a -cocktail of mitochondrial complex inhibitors. Error bars represent standard error of mean (SEM).&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig4large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts at JoVE.com

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

preparation-metabolic-assay-3-dimensional-spheroid-co-cultures

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.1K Views.  Translational Genomics Research Institute. Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Video: Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

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 ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...