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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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+complex+3D+culture+models+to+unravel+novel+pathways+and+identify+accurate+biomarkers+in+breast+cancer.">The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer.</a> Advanced Drug Delivery Reviews. 69, 42-51 (2014).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> The Journal of Pharmacology and Experimental Therapeutics. 332 (3), 821-828 (2010).</li><li>Li, L., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+3D+Culture+System+to+Study+the+Interaction+between+Epithelial+Breast+Cancer+and+Its+Surrounding+Fibroblasts.">Optimizing a 3D Culture System to Study the Interaction between Epithelial Breast Cancer and Its Surrounding Fibroblasts.</a> Journal of Cancer. 2, 458-466 (2011).</li><li>Pickl, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+3D+and+2D+tumor+models+reveals+enhanced+HER2+activation+in+3D+associated+with+an+increased+response+to+trastuzumab.">Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab.</a> Oncogene. 28 (3), 461-468 (2009).</li><li>Nyga, A., Cheema, U., Loizidou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumour+models:+novel+in+vitro+approaches+to+cancer+studies.">3D tumour models: novel in vitro approaches to cancer studies.</a> Journal of Cell Communication and Signaling. 5 (3), 239-248 (2011).</li><li>Kimlin, L. C., Casagrande, G., Virador, V. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells.">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+SOX9+by+the+NF-kappaB+signaling+pathway+in+pancreatic+cancer+stem+cells.">Epigenetic regulation of SOX9 by the NF-kappaB signaling pathway in pancreatic cancer stem cells.</a> Stem Cells. 31 (8), 1454-1466 (2013).</li><li>Mathews, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">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>Mathews Griner, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+combinatorial+screening+identifies+drugs+that+cooperate+with+ibrutinib+to+kill+activated+B-cell-like+diffuse+large+B-cell+lymphoma+cells.">High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (6), 2349-2354 (2014).</li><li>Herter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three-dimensional+heterotypic+spheroid+model+for+the+assessment+of+the+activity+of+cancer+immunotherapy+agents.">A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.</a> Cancer Immunology, Immunotherapy. 66 (1), 129-140 (2017).</li></ol>

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 this protocol is that the spheroids are formed from traditional 2D tissue-cultured cells in 1536-plates using defined media referred to as SCM+KO+ITS. Once the cells are seeded in, they are sealed with breathable adhesive plate seals to prevent any evaporation over the duration of the 3 d spheroid formation. Spheroids can also be cultured for longer periods of time using this approach16. After 3 d of sphere formation, compounds are added for 5 d before assaying the changes in cell growth with a lytic-based proliferation reagent. After normalization to the control wells and calculation of IC50s between cells cultured in 2D vs. 3D, the changes in the compound-treated cells are compared to the control-treated cells.
Since the 1536-well plates are regular tissue-treated plates and not hydrogel-coated or cell-repellent plates, a limitation of this protocol is the use of the defined medium, which is a critical factor to the successful execution of this protocol. The KO serum replacement was initially tested for its ability to successfully aid in the growth of embryonic stem cells in 3D17 and in this protocol has been adapted for the growth of many tumor spheroid lines13,15,18,19. To note is that, although the media conditions are different for the growth of the 2D cells versus the 3D spheroids, we have previously demonstrated that the differential effects of drug sensitivity are mediated by the growth format, and not simply the differences in media composition9.
To ensure the success of this protocol, as a troubleshooting step, it is suggested that a series of assay development experiments are first carried out to optimize each cell line before embarking on a large-scale screen. In this example, all of the cell lines assayed were first evaluated to determine if they could be seeded at the same starting density of 500 cells per well. They were then assayed with DMSO-only plates to examine standard deviations (SD) and coefficients of variance (CVs) for the duration of the growth period. Even with this assay development in place, certain lines grew faster than others, and it would have been beneficial to further optimize the seeding densities and the duration of the experiment of these more challenging lines. Finally, the data presented here demonstrate that there is a tendency, rather than a universal phenomenon, for certain cell lines like the NSCLC line Calu-6 to be uniquely more sensitive to compounds targeting the MAPK pathway in 3D compared to those grown in 2D. Additional experiments are underway to determine what biological mechanisms might be contributing to regulating this striking difference in sensitivity.
The ability to screen spheroids in an ultra-HTS setting allows 3D cultures to be used early on in the drug discovery process and may enable the identification of novel chemical matter that is only active in a 3D setting. With so many clinical candidates never reaching the market due to a lack of efficacy in patients, albeit demonstrating growth inhibition in vitro and regression in vivo, teams have started questioning whether implementing 3D assays could bridge this gap earlier during the discovery process. A number of different plate types exist to culture cells in 3D when using 96-well or 384-well methods. Here, we have presented 1536-well methods which produce multiple foci of individual spheroids. In the future, these findings could be compared to similar screens using new technologies which are under development such as 1536-well plates which could grow cells as hanging drops, or plates which are coated with hydrogel/Matrigel and that are round-bottom, which would allow for the formation of one sphere per well. These conditions would introduce additional layers of complexity to the culture conditions used for screening, making them even more physiologically relevant, and could potentially represent an increased rate of translation from bench to bedside for novel therapies. These technologies would also enable scientific questions involving hypoxia or diffusion gradients, which require the use of larger spheroids.
The methods presented here focus on screening these spheroids against a small molecule library of compounds. In the future, these methods could expand to screen for novel biologics and biotherapeutics such as bi-specific antibodies or antibody-drug conjugates, where 3D cultures may provide more physiologically relevant antibody-antigen interactions. In addition, the use of 3D spheroids may be critical to developing disease-relevant assays for understanding targets and interventions in the emerging immuno-oncology space21.

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>Reagents</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>Consumables</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>Equipment</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 demonstrates that, when used for large-scale screening, differential compound sensitivity between cultures grown in 2D versus those in 3D can be observed. Each dot on the graph represents one compound&#39;s inhibitory concentration where there is a 50% cell killing or the compound&#39;s IC50 response in both settings. To demonstrate that the effect is not mediated solely by the different media, Calu-6 cells were cultured in the same media using either 2D tissue-cultured plates or 3D Scivax biofilm plates which prevent attachment. The increased sensitivity or lower IC50 for the 3D growth condition is seen in Supplemental Figure 1 for two compounds in the same media. Lines like Calu-6 demonstrate an increased sensitivity to the compounds when grown under 3D conditions, while lines like SK-MEL-30 are less sensitive in 3D compared to 2D. Table 1 includes additional background information about all the cell lines that were used in this screen.
Examples of representative dose-response curves are visualized in Figure 3. This figure demonstrates the differential efficacy of two compounds, RAF265 (Figure 3A) and RAF709 (Figure 3B), which inhibit the activity of RAF kinases. Both compounds are more potent in 3D conditions in Calu-6, which is a non-small cell lung cancer line harboring Q61K, an activating mutation in the protein KRAS. At the same time, both compounds are similarly potent in the 3D spheroids and 2D cultures from the HCT116 colon cancer line, which contains the G13D-activating mutation in KRAS. To demonstrate that the effect of the increased sensitivity is mediated by the growth condition of 3D versus 2D and not simply by the different media conditions, Calu-6 cells were grown using FBS under both conditions. As seen in Supplemental Figure 1, when both 2D and 3D conditions use FBS, the 3D culture still demonstrates an increased sensitivity to the compounds producing a lower IC50.
Finally, the 3D assay also permitted an observation of the phenomenon of paradoxical growth activation, which is known to occur when there is an incomplete inhibition of RAF dimers in highly activated RAS mutant lines, and not in the dimer-independent pathway activation found in BRAF V600E mutations11,12. This activation is seen in Figure 4, upon treatment with the BRAF inhibitor Dabrafenib under 3D conditions in KNS-62, an HRAS-mutated small cell lung cancer line, and also in the atypical-BRAF mutant pancreatic cell line BxPC-3. The growth is activated at low concentrations of the drug, and the growth is only repressed at high concentrations of the drug (Figure 4B). This activation was surprisingly not seen in the KRAS mutant line Calu-6 and is not to be expected in the V600E BRAF mutant line A375. We have observed a paradoxical activation of Calu-6 using other 3D growth models (data not shown).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57476/57476fig1.jpg" /> 
Figure 1: Representative images of cancer cells growing in 3D 1536-well plates. Cells were plated for a total of 3 d in 1536-well plates using a reagent dispenser and a small cassette. Bright-field images, acquired on an inverted microscope using a 4X lens, demonstrate the various 3D morphologies in the MAPK mutant cell lines Calu-6:KRAS, NCI-H1299:NRAS, SK-MEL-30:NRAS, and KNS-62:HRAS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig1large.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/57476/57476fig2.jpg" /> 
Figure 2: A scatter plot comparing the 2D- versus the 3D-compound efficacy across cell lines. Spheroids, which were previously generated for 3 d, were then treated with compounds for an additional 5 d before the compound efficacy was determined. Each circle represents a unique compound response. Changes in the cell proliferation were detected using a lytic cell proliferation reagent, and IC50s were calculated using a four-parameter logistic regression analysis with the internal software called Helios. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig2large.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/57476/57476fig3.jpg" /> 
Figure 3: Dose-response curves comparing 2D- versus 3D-compound efficacy. The dose-response curves demonstrate the compound efficacy compared to the DMSO controls. The curves of the KRAS mutant Calu-6 and HCT116 cells treated with two RAF inhibitors, RAF265 and RAF709, are displayed here as a percentage in the change in cell growth (% growth inhibition) compared to the DMSO control wells. A population of the cells that were grown under 2D conditions appears in red, and those grown as 3D spheroids are in blue. The solid lines around the data indicate the 95% confidence intervals for each data set. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig3large.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/57476/57476fig4.jpg" /> 
Figure 4: Certain 3D cultures capture the phenomena of paradoxical growth activation. The dose-response curves demonstrate the compound efficacy (% growth inhibition) compared to the DMSO controls. The curves of the KRAS mutant Calu-6, BRAF V600E mutant A375, HRAS mutant KNS-62, and the atypical BRAF mutant BxPC-3 cells treated with a paradoxical activator Dabrafenib are displayed here. A population of the cells that were grown under 2D conditions appears in red, and those grown as 3D spheroids are in blue. The solid lines around the data indicate the 95% confidence intervals for each data set. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/57476/57476figS1.jpg" /> 
Supplemental Figure 1: Dose-response curves comparing the 2D- versus the 3D-compound efficacy in FBS containing media. The dose-response curves demonstrate the compound efficacy compared to the DMSO controls. The curves of the KRAS mutant Calu-6 cells treated with two RAF inhibitors, RAF265 and RAF709, are displayed here as a percentage in the change in cell growth (% growth inhibition) compared to the DMSO control wells. A population of the cells that were grown under 2D conditions appears in red, and those grown as 3D spheroids are in blue. Both conditions use FBS-containing media. The error bars represent standard deviations from 2 individual data sets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57476/57476figS1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Line Name</td>
			<td>Lineage</td>
			<td>BRAF</td>
			<td>RAS</td>
		</tr>
		<tr>
			<td>A-375</td>
			<td>Melanoma</td>
			<td>V600E</td>
			<td>WT</td>
		</tr>
		<tr>
			<td>BxPC-3</td>
			<td>Pancreatic</td>
			<td>V487-P492&#62;A</td>
			<td>WT</td>
		</tr>
		<tr>
			<td>Calu6</td>
			<td>Non-Small Cell Lung Cancer</td>
			<td>WT</td>
			<td>KRAS Q61K</td>
		</tr>
		<tr>
			<td>HCT 116</td>
			<td>Colorectal</td>
			<td>WT</td>
			<td>KRAS G13C</td>
		</tr>
		<tr>
			<td>IPC-298</td>
			<td>Melanoma</td>
			<td>WT</td>
			<td>NRAS Q61L</td>
		</tr>
		<tr>
			<td>KNS-62</td>
			<td>Squamous Lung</td>
			<td>WT</td>
			<td>HRAS Q61L</td>
		</tr>
		<tr>
			<td>NCI-H1299</td>
			<td>Lung</td>
			<td>WT</td>
			<td>NRAS G12D</td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>Pancreatic</td>
			<td>WT</td>
			<td>KRAS G12D</td>
		</tr>
		<tr>
			<td>SKMEL-30</td>
			<td>Melanoma</td>
			<td>WT</td>
			<td>NRAS Q61K</td>
		</tr>
	</tbody>
</table>
Table 1: Cell line information.

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.5K 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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

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 ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

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 ...