The authors would like to acknowledge Marc Ferrer at the National Center for Advancing Translational Sciences, National Institutes of Health for his support and guidance on the initial development of these assays. In addition, we would like to thank Alyson Freeman, Mariela Jaskelioff, Michael Acker, Jacob Haling, and Vesselina Cooke for their scientific input and discussion as project team members.

1. Culturing of 1536-well 3D Tumor Spheroids
<ol>
	<li>Prepare a fresh 3D tumor spheroid medium stem (cell medium + knockout serum replacement + insulin-transferrin-selenium) by adding the following reagents to 500 mL of Dulbecco&#39;s Modified Eagles Medium (DMEM/F12): 1x penicillin/streptomycin, 10 ng/mL of human basic fibroblast growth factor (bFGF), 20 ng/mL of human epidermal growth factor (EGF), 0.4% bovine serum albumin (BSA) (Fraction V), 1x insulin-transferrin-selenium, and 1% knockout (KO) serum replacement (do not freeze-thaw).</li>
	<li>Filter-sterilize the entire medium with the supplements through a 0.4 &#181;m bottle-top filter system.</li>
	<li>Detach the cancer cell lines, which have been growing according to recommended routine culture conditions, from the traditional cell-culture flasks by washing them 3x with 1x phosphate buffered saline (PBS) and adding an appropriate amount of 0.25% trypsin (1 mL per 5 cm2) for 5 min to create a thin layer over the cells. Neutralize the trypsin with 5x the amount of the medium containing the serum and proceed to count the cells. For example, use 25 mL of medium for 5 mL of trypsin.</li>
	<li>Adjust the concentration of the cell solution to 62.5 x 104 cells/mL in order to seed a total of 500 cells per well in 8 &#181;L of spheroid medium into the 1536-well tissue-culture-treated plates.</li>
	<li>In a biological safety cabinet, seed the cells using a sterilized, small stainless-steel-tipped cassette with a peristaltic-pump-based system. Between different cell lines, flush the tubing of the cassette with 1x PBS and 70% Ethanol for 10 s.</li>
	<li>Alternatively, seed the cells using a liquid handler located within a HEPA-filtered room using either a sterilized, small stainless-steel-tipped cassette and a peristaltic pump or an attached syringe pump, depending on the number of plates per cell line needed.</li>
	<li>Seal the plates using a breathable adhesive plate seal either manually or using a plate sealer.</li>
	<li>Place the plates in a spinning incubator set at 10 rpm and 37 &#176;C with 5% carbon dioxide (CO2) and 95% relative humidity. Allow the spheroids to form for 3 d.</li>
</ol>
2. Culturing of 1536-well 2D Cancer Lines
NOTE: The 1536-well 2D cancer lines should be cultured 24 h before the addition of the compound to the 3D plates.
<ol>
	<li>Prepare a 2D cancer cell medium by adding the following reagents to 500 mL of an appropriate growth medium for the selected lines: 1x penicillin/streptomycin and 10% fetal bovine serum (FBS).</li>
	<li>Filter-sterilize the entire medium with the supplements through a 0.4 &#181;m bottle-top filter system.</li>
	<li>Detach the cancer cell lines, which have been growing according to recommended routine culture conditions, from the traditional cell-culture flasks by washing them 3x with 1x PBS and adding an appropriate amount of 0.25% trypsin (1 mL per 5 cm2) for 5 min to create a thin layer over the cells. Neutralize the trypsin with 5x the amount of the medium containing the serum and proceed to count the cells. For example, use 25 mL of medium for 5 mL of trypsin.</li>
	<li>Adjust the concentration of the cell solution to 62.5 x 104 cells/mL in order to seed a total of 500 cells per well in 8 &#181;L of complete medium into 1536-well tissue-culture-treated plates.</li>
	<li>In a biological safety cabinet, seed the cells using a sterilized, small stainless-steel-tipped cassette with a peristaltic-pump-based system. Between the different cell lines, flush the tubing of the cassette with 1x sterile PBS and 70% Ethanol for 10 s.</li>
	<li>Alternatively, seed the cells using a liquid handler located within a HEPA-filtered robotics room using either a sterilized, small stainless-steel-tipped cassette and a peristaltic pump or the attached syringe pump, depending on the volume of the plates per cell line needed.</li>
	<li>Seal the plates using a breathable adhesive plate seal either manually or using a plate sealer.</li>
	<li>Place the plates in a spinning incubator set at 10 rpm and 37 &#176;C for 24 h prior to the compound addition, with 5% CO2 and 95% relative humidity.</li>
</ol>
3. Compound Addition
<ol>
	<li>Prepare 8-point, 3-fold serial dilutions of MAPK inhibitors in 100% dimethyl sulfoxide (DMSO) on a liquid-handling robot with a 10 mM top concentration. The proteasome inhibitor Bortezomib is used as a positive control for a complete cell killing to determine the dynamic range of the assay.</li>
	<li>Quick-spin all assay plates and compound plates at 100 x g to collect any condensation. Remove the adhesive seal from each plate and place a plate on an acoustic dispenser set-up to add a total of 8 nL of each compound/dilution representing a final concentration of 0.1% DMSO per well and a top compound dose of 10 &#181;M.</li>
	<li>After the addition of the compound is completed, place a custom-made, stainless-steel cell culture lid which prevents evaporation on the plate and place the plate into a spinning incubator at 37 &#176;C for 5 d, with 5% CO2 and 95% relative humidity.</li>
</ol>
4. Detection Reagent Addition and the Acquirement of Raw Data
<ol>
	<li>Pre-warm an adequate volume of a cell lysis reagent containing luciferin to detect changes in adenine triphosphate (ATP) and, thus, changes in the cell proliferation at room temperature or in a 37 &#176;C water bath. Add a total of 3 &#181;L of the detection reagent to each well using a peristaltic pump and incubate the plate at room temperature for at least 15 min.</li>
	<li>Capture the luminescence on a plate luminometer.</li>
</ol>
5. Data Processing
<ol>
	<li>Extract the raw data from the instrument and normalize all fields containing test compounds to the average of all wells containing DMSO alone as the neutral control. From this value, calculate the percent growth inhibition of each compound. This is completed using the formula function in Microsoft Excel where f(x)={(sample value relative light units (RLUs)/(average DMSO value RLUs)*100)}.</li>
	<li>Generate the dose-response curves and inhibitory IC50s by graphing the values of the normalized data from step 5.1 against the compound concentration using a graphing program. 
	NOTE: We analyzed the data using Helios, an internal NIBR software tool. To complete this function, we selected the nonparametric curve analysis tool.</li>
</ol>

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<li>Mathews Griner, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+pharmacological+profiling+of+3D+tumor+models+of+cancer+cells%5BTitle%5D)+AND+%22Cell+Death+%26+Disease%22%5BJournal%5D)">Large-scale pharmacological profiling of 3D tumor models of cancer cells.</a> Cell Death & Disease. 7 (12), e2492(2016).</li>
<li>Polo, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responsiveness+to+PI3K+and+MEK+inhibitors+in+breast+cancer.+Use+of+a+3D+culture+system+to+study+pathways+related+to+hormone+independence+in+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.</a> PLoS One. 5 (5), e10786(2010).</li>
<li>Hatzivassiliou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Nature. 464 (7287), 431-435 (2010).</li>
<li>Bhargava, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Registered+report%3A+RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Elife. 5, e09976(2016).</li>
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<li>Mathews, L. A., Crea, F., Farrar, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+gene+regulation+in+stem+cells+and+correlation+to+cancer%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">Epigenetic gene regulation in stem cells and correlation to cancer.</a> Differentiation. 78 (1), 1-17 (2009).</li>
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<li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li>
<li>Horii, T., Nagao, Y., Tokunaga, T., Imai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li>
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<li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li>
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The authors are employees of Novartis, and some employees are also stockholders.

The methods presented here demonstrate a detailed protocol on how to produce tumor spheroids in 1536-well plates for large-scale compound screenings. These methods were initially adapted from work at the National Cancer Institute where tumor spheroids were grown in low- throughput assays in 6-well and 96-well plates to ask questions about genetic dependencies and compound sensitivity13,14,15. A critical and unique feature of thi...

In the past decade, more and more studies have implemented the use of 3D cell culture models to understand concepts that are not fully recapitulated by growing cells in 2D on plastic. Examples of these concepts include the alternations in normal epithelial cell polarity1 where the spatial orientation of apical and basal layers of cells are lost, as well as the role of the extracellular matrix in regulating survival and cell fate. Oncology research, in particular, has used 3D models to understand the basic biology of cancer cells and the differences between 2D and 3D cell culture systems3,4. The development of more sophisticated cell culture techniques and their further adaption to multi-well formats has enabled the search for new drugs in 3D settings. In contrast to cells grown under 2D conditions, 3D models of tumors range in complexity from layered cellular systems5 to single-cell-type spheres of different sizes, to complex multi-cell-type spheres6,7,8. The discovery of novel compounds or biologics that potently induce cell death in these 3D model systems is, therefore, of high interest in drug development campaigns. Endpoints of these assays are often identical to those performed in 2D cultures to assess changes in cell proliferation, but when conducted in a more physiologically relevant setting, they may reveal the true level of dependency of the target gene or pathway being interrogated.
As introduced above, a variety of model systems have been developed to study drug responses in 3D culture systems, but the majority use either 96 or 384-well microtiter plates and are not easily adaptable to the high-throughput screening (HTS) formats often used in drug discovery screening campaigns. Such systems include the use of hanging droplet technologies, spheroid cultures, pulsating cells with magnetic particles to induce levitation, and cultures incorporating natural or synthetic gels such as collagen, Matrigel, or polyethylene glycol (PEG)2. Here, we present the detailed methods of a previously developed technique to produce 3D spheroid cultures from established cancer cell lines in a 1536-well plate format. In this protocol, a highly defined medium is used which prevents the attachment of normally adherent cell lines9. This system has limitations (i.e., it cannot fully recapitulate a complex model system of cancer), but nevertheless, these assays enable a high-throughput screening of large collections of small molecules and crosswise comparisons of drug response between 2D and 3D cultures against a variety of cell lines and compounds.
The cell lines selected to demonstrate the methods in this article all harbor mutations in genes related to the MAPK signaling pathway, a pathway which is highly dysregulated in cancer, and for which many therapeutics are available. Many of the lines have activating oncogenic mutations in the Kirsten Rat Sarcoma virus also referred to as KRAS, the Neuroblastoma RAS or NRAS, the Harvey Rat Sarcoma virus oncogene or HRAS, and the associated kinases Rapidly Accelerated Fibrosarcomas, also known as RAFs. Recent literature suggests that the inhibitors of different nodes of this pathway are uniquely more efficacious in a subset of the cell lines when grown under 3D conditions9,10. One study found that when cancer cells with active RAS were cultured in 3D, they demonstrated an increased sensitivity to MAPK inhibitors, and further, that this approach could identify pathways and targeted inhibitors that might be missed in the traditional 2D setting. The goal of this study is to present the methods used to culture these cell lines, and further, to demonstrate the differential responses to these inhibitors which can be observed only when using 3D cell culture systems.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>ATCC</td><td>30-2006</td><td>Purchased as a prepared solution. This reciepe was found to be preferred compared to other vendors.</td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>Lonza</td><td>12-604F</td><td>Purchased as a prepared solution.</td></tr><tr><td>Roswell Park Memorial Institute (RPMI)</td><td>Lonza</td><td>12-115Q</td><td>Purchased as a prepared solution.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-500</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x 0.25% Trypsin with Ethylenediaminetetraacetic acid (EDTA)</td><td>Hyclone</td><td>SH30042.01</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>Purchased as a prepared solution.</td></tr><tr><td>10 ng/mL Human basic Fibrobast Growth Factor (bFGF)</td><td>Sigma</td><td>F0291</td><td>Resuspend in sterile water.</td></tr><tr><td>20 ng/mL Human Epidermal Growth Factor (EGF)</td><td>Sigma</td><td>E9644</td><td>Resuspend in sterile water.</td></tr><tr><td>0.4% Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A9418</td><td>Resuspend in sterile PBS and filter.</td></tr><tr><td>1x Insulin Transferrin Selenium (ITS)</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>1% KnockOut (KO) Serum Replacement</td><td>Gibco</td><td>10828-028</td><td>Add fresh right before use.</td></tr><tr><td>100% Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>276855-100ML</td><td></td></tr><tr><td>CellTiter-Glo</td><td>Promega</td><td>G7573</td><td>Purchased as a prepared solution.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Small Combi Cassette</td><td>ThermoFisher</td><td>24073295</td><td></td></tr><tr><td>Small Multiflow Cassette</td><td>BioTek</td><td>294085</td><td></td></tr><tr><td>1536-well sterile TC plates (white)</td><td>Corning</td><td>3727</td><td></td></tr><tr><td>1536-well sterile TC plates (black)</td><td>Corning</td><td>3893</td><td></td></tr><tr><td>Scivax Plates</td><td>MBL International</td><td>NCP-LH384-10</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adhesives seals</td><td>ThermoFisher</td><td>AB0718</td><td></td></tr><tr><td>Spinning Incubator</td><td>LiCONiC</td><td></td><td></td></tr><tr><td>Stainless Steel Lids</td><td>The Genomics Institute of Novartis Research Foundation (GNF)</td><td></td><td></td></tr><tr><td>ATS Acoustic Dispensor</td><td>EDS Biosystems</td><td></td><td></td></tr><tr><td>Echo Acoustic Dispensor</td><td>Labcyte</td><td></td><td></td></tr><tr><td>Luminometer</td><td>Envision-Perkin Elmer</td><td></td><td></td></tr><tr><td>Peristaltic Pump- Multidrop Combi</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Liquid Handler-Multiflow FX</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

generation of high-throughput three-dimensional tumor spheroids for drug screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

A variety of established cancer cell lines known to grow well under 2D culture conditions were tested using the methods outlined here. Representative images from a variety of MAPK mutant cancer cell lines (Calu-6:KRAS, NCI-H1299:NRAS, SK-MEL-30:NRAS, and KNS-62:HRAS) are seen in Figure 1. These images demonstrate that although the cell lines have various morphologies, each formed 3D structures in the 1536-well assay plates. Figure 2</stro...

Watch this Scientific Journal Video about Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening at JoVE.com

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

generation-high-throughput-three-dimensional-tumor-spheroids-for-drug

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Cancer Research

8.6K Views.  Novartis Institutes for Biomedical Research. Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Video: Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Bioengineering

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 ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

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 ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

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

Medicine

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...