We thank all the members of our laboratories for their critical input and suggestions. This research was supported by the Key Project of Jiangsu Commission of Health (K2019030). Conceptualization was conducted by C.W. and Z.C., the methodology was performed by W.H. and M.L., the investigation was performed by W.H. and M.L., the data curation was performed by W.H., Z.Z., S.X., and M.L., the original draft preparation was performed by Z.Z., J.Z., S.X., W.H., and X.L., the review and editing was performed by Z.C., project administration was performed by C.W. and Z.C., and funding acquisition was conducted by C.W. All the authors have read and agreed to the published version of the manuscript.

1. Spheroid construction
<ol>
	<li>Anti-adhesion treatment of the culture plate
	<ol>
		<li>Pipette 100 &#181;L of anti-adhesion reagent into each well of a 48-well plate with a U-shape well bottom, and keep for 10 min. After 10 min, aspirate the coating reagent, and wash twice with sterilized PBS.</li>
		<li>Put the culture plate in an incubator (37 &#176;C in humidified air with 5% CO2) until use.</li>
	</ol>
	</li>
	<li>Cell preparation, collection, and counting
	<ol>
		<li>Use the culture medium specific to the cells to culture the cells in cell-culture flasks (Supplementary Table 2). For example, NCI-H23, CT-26 cells are cultured in RPMI 1640, and HT-29 cells are cultured in McCoy&#39;s 5A medium. These two media are supplemented with 10% heat-inactivated FBS and 1% P/S, respectively.</li>
		<li>Maintain all the cells in standard culture conditions (37 &#176;C in humidified air with 5% CO2) during proliferation. Here, the NCI-H23 cell line is used as an example in the following steps.</li>
		<li>Wash cells cultured in a T25 flask twice with 1x PBS to remove the culture medium (it is better to choose cells in the logarithmic phase and passage the cells at a confluency of 80%-90%).</li>
		<li>Treat the expanded cells with 1 mL of 0.25% trypsin/EDTA for 1- 2 min in an incubator at 37 &#176;C, 5% CO2. Confirm the cell shape (normally circular in this case) under the microscope, and then stop the trypsin treatment. To do this, aspirate the used trypsin/EDTA suspension in the T25 flask, and wash the cells with 4 mL of fresh medium.</li>
		<li>Transfer all of the suspension (5 mL) to a 15 mL tube. Use 1 mL of fresh medium to wash down the residual cells and add it to the tube. Centrifuge the cells at 186.48 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and add 10 mL fresh medium to the cell pellet, followed by gentle pipetting until the cells are in a homogenous suspension.</li>
		<li>Aspirate 0.1 mL of cell suspension into a new centrifuge tube, add 0.9 mL of fresh medium, and then pipette the suspension well.</li>
		<li>Extract 10 &#181;L of the cell suspension for cell counting. Carry out this process two or three times and take an average value.</li>
		<li>Dilute the suspension to reach a final seeding density of 50,000 cells/mL, according to the concentration obtained from the cell counting process in step 1.2.7.</li>
	</ol>
	</li>
	<li>Cell culture and spheroid formation
	<ol>
		<li>Add 200 &#181;L of the cell suspension to each well of a 48-well U-bottom plate.</li>
		<li>Wrap the sealing film around the plate and centrifuge it at 119.35 x g for 5 min at room temperature.</li>
		<li>Carefully take the plate out of the centrifuge and pull off the protective film. Then, add 5-8 mL of sterilized water into the water channel surrounding the wells (to prevent evaporation) and incubate at 37 &#176;C for 5 days. Do not change/supplement any water to the water channel during the period.</li>
		<li>Observe the cell aggregation during the following 5 days. 
		NOTE: In general, the cells start to clump as colonies within 5 days. However, the process of spheroid construction may be quicker or slower with different cell types and cell densities. Due to this, the cells must be observed every day using one of three possible methods. First, the cells can be observed through the bottom of the well plate. When the cells have yet not formed a spheroid, a single layer of cells can be seen at the bottom. When the cells form a spheroid, a dense 3D construct can be observed at the U-bottom of each well. Another method involves checking the cells under a microscope. When the cells become a tumor spheroid, the construction involves three layers (a proliferating layer, an inactive layer, and a necrotic core, from the outside to the inside of the spheroid), which have a transparency gradient. Finally, the color of the culture medium can also be used for observational purposes. This can be helpful when the three-layer structure cannot be clearly seen, even using a digital microscope. When the medium turns from purple-red to yellow, the process of embedding the spheroids in the gel can begin. The medium should not be replaced during the cell aggregation period.</li>
	</ol>
	</li>
	<li>Gel embedding 
	NOTE: The gels need to be stored at a temperature below &#8722;20 &#176;C. In particular, gels should be placed far away from the fridge door to avoid temperature fluctuations. Note that the gels are in a frozen state at this stage of the process.
	<ol>
		<li>Take the frozen gel from the &#8722;20 &#176;C fridge and place it on an ice box for the whole time during the experiment.</li>
		<li>Observe the cell spheroids under the microscope. Before the gel embedding begins, the status of the spheroids should again be checked with a digital microscope.</li>
		<li>Carefully remove 150 &#181;L of the medium. The plate should also be placed on the ice box.</li>
		<li>Embed each spheroid in the gel by adding the liquid gel slowly from the wall side of the well while moving the pre-cooled pipette tip around and inside the well. Wait for 5 min and if the gel does not spread evenly, gently pipette the gel with a 10 &#181;L pipette tip. Each well contains a tumor spheroid, 25 &#181;L of 3.5 mg/mL gel, and 50 &#181;L of complete culture medium. Add 75 &#181;L of medium to the controls as well. 
		NOTE: Each well contains one spheroid.</li>
		<li>Incubate the plate at 37 &#176;C for 30 min until hydrogelation is fully completed. Confirm the gelation status under the microscope.</li>
		<li>Overlay 125 &#181;L of the fresh medium on each sample.</li>
		<li>Culture the spheroids for another 7-10 days. Prepare groups of spheroids with four to six wells each and choose at least three of them for analysis. 
		&#8203;NOTE: If performing a drug test, prepare two groups for one sample. One group is used for viability testing, while the other is used for image capturing and analysis.</li>
	</ol>
	</li>
</ol>
2. Drug treatment
<ol>
	<li>Dissolve the drug according to the manufacturer&#39;s instructions. Prepare 100x working solutions with DMSO. Prepare at least five doses of the drug in serial dilution. Here, the lung cancer therapeutical drug, AMG 510, is used as an example. The composition is shown in Supplementary Table 1.</li>
	<li>Use 0.1% DMSO as a positive control.</li>
	<li>Add 125 &#181;L of drug-treated medium to each well and place the plate back into the incubator (37 &#176;C in humidified air with 5% CO2). At this stage, each well contains a tumor spheroid, 25 &#181;L of 3.5 mg/mL gel, and 175 &#181;L of the medium. The controls contain 200 &#181;L of the medium.</li>
</ol>
3. Spheroid viability
<ol>
	<li>Measure the spheroid viability using an Alamar Blue assay kit according to the manufacturer&#39;s guidelines. Measure the viability using a microplate photometer (absorbance at 570 nm and 600 nm) after Alamar Blue treatment is carried out.</li>
	<li>Measure the viability at day 1, day 4, day 7, and day 10 after embedding the spheroids in the gel, respectively, or as indicated. 
	NOTE: When using an Alamar Blue assay, at least 16 h of reacting time is required. Therefore, add 20 &#181;L of Alamar Blue in the afternoons of day 0, day 3, day 6, and day 9.</li>
	<li>Aspirate 100 &#181;L of the supernatant medium from each well to a new test plate and add 80 &#181;L of fresh medium to each well of the culture plate. Then, replace another 100 &#181;L of the drug-treated medium. Ensure there are no remains of Alamar Blue in the well. 
	&#8203;NOTE: Medium replacement is carried out on each occasion that the viability is tested, and the drug-treated medium of the imaging-analysis groups needs to be replaced on the same day.</li>
</ol>
4. Spheroid observation and deep learning analysis through images in the drug test
<ol>
	<li>Imaging
	<ol>
		<li>Aspirate 100 &#181;L of the medium out before imaging.</li>
		<li>Place the plate on the stage. Obtain digital images of the spheroids using an automated microscope with a 10x objective (2x objective first). The microscope can focus and centralize these spheroids automatically. 
		NOTE: The autofocus function and the algorithm used have been previously reported by Yazdanfar et al.17.</li>
		<li>Wait for the automatic imaging. Four images are acquired for each spheroid. An integrated image is formed and processed with the software connected to the high-content imaging system.</li>
		<li>Click on the &#34;Image patch process&#34; button and choose the integrated images in the software.</li>
		<li>Choose &#34;U-NET model&#34;, and type in the conversion rate (10x objective images have a conversion rate of 3.966). Click at the bottom of the screen below, to start the image processing. Then, save the diameter, perimeter, and roughness data in spreadsheet software.</li>
		<li>Calculate the excess perimeter index (EPI). The EPI is the ratio between the spheroid perimeter (P0) and the equivalent perimeter (Pe), as calculated by Eq. 1. Measure the area of the spheroid at the focal plane (S) using Image J. Then, calculate the equivalent perimeter of the spheroid using Eq. 2. 
		EPI = (Po&#8722; Pe)/Pe&#160; &#160; &#160; &#160;(Eq. 1) 
		<img alt="Equation 1" src="/files/ftp_upload/65125/65125eq01.jpg" style="margin: auto;" />&#160; &#160; &#160; (Eq. 2) 
		NOTE: The spheroid perimeter is generated by the software with deep-learning algorithms based on the developed U-NET model.</li>
		<li>Add 100 &#181;L of fresh medium with the drug and place the plate back into the incubator (37 &#176;C in humidified air with 5% CO2).</li>
	</ol>
	</li>
	<li>Spheroid inhibition
	<ol>
		<li>Calculate the tumor growth inhibition (TGI) using Eq. 3. The relative spheroid volume (RTV) is the terminal volume over the original volume (Eq. 4). The spheroid volume (V) is calculated automatically by the software using Eq. 5 according to the spheroid diameter output. 
		TGI = (RTVcontrol&#8722; RTVtreatment)/RTVcontrol &#215; 100%&#160; &#160; &#160; (Eq. 3) 
		RTV = Vterminal/Voriginal&#160; &#160; &#160; &#160;(Eq. 4) 
		V = 4/3&#960;(d/2)3&#160; &#160; &#160; (Eq. 5) 
		NOTE: The TGI is obtained with reference to in vivo growth inhibition, and the tumor weights are replaced with RTV. RTVcontrol is the relative spheroid volume of the control group, RTVtreatment is the relative spheroid volume of the drug tested group, Vterminal is the volume of the spheroid on the last day, Voriginal is the volume of the spheroid on the first culture day, and d represents the spheroid diameter.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

The microenvironment plays an important role in tumor growth. It may affect the provision of extracellular matrices, oxygen gradients, nutrition, and mechanical interaction and, thus, affect gene expression, signal pathways, and many functions of tumor cells19,20,21. In many cases, 2D cells do not produce such effects or even produce opposite effects, thus affecting the evaluation of drug treatments. However, the emergence of 3D...

Cancer is one of the diseases most feared by human beings, not least because of its high mortality rate1. In recent years, the possibility of treating cancer has increased as new therapies have been introduced2,3,4,5. Two-dimensional (2D) and three-dimensional (3D) in vitro models are used to study cancer in a laboratory setting. However, 2D models cannot immediately and accurately assess all of the important parameters that indicate antitumor sensitivity; therefore, they fail to fully represent in vivo interactions in drug therapy testing6.
Since 2020, the global three-dimensional (3D) culture market has been greatly boosted. According to one report from NASDAQ OMX, the global value of the 3D cell culture market will exceed USD 2.7 billion by the end of 2025. Compared with 2D culture methods, 3D cell culturing exhibits advantageous properties, which can be optimized not only for proliferation and differentiation but also for long-term survival7,8. By such means, in vivo cellular microenvironments can be simulated to obtain more accurate tumor characterization, as well as metabolic profiling, so that genomic and protein alterations can be better understood. Due to this, 3D test systems should now be included in mainstream drug development operations, especially those with a focus on screening and evaluating novel antitumor drugs. Three-dimensional growths of immortalized established cell lines or primary cell cultures in spheroid structures possess in vivo features of tumors such as hypoxia and drug penetration, as well as cell interaction, response, and resistance, and can be regarded as a stringent and representative model for performing in vitro drug screening9,10,11.
However, these 3D culture models also suffer from several problems that may take some time to solve. Cell spheroids can be formed using these protocols, but they differ in certain details, such as culture time or embedding gels12, so these constructed cell spheroids cannot be well controlled under a restricted size range. The size of the spheroids may influence the consistency of the viability test and imaging analysis. The growth microenvironments and growth factors also vary, which may lead to different morphologies due to differences in the differentiation among cells13. There is now an obvious need for a standard, simple, and cost-effective method for constructing all types of tumors with controlled sizes.
From another perspective, although homogeneous assays and high-content imaging approaches have been developed to evaluate morphology, viability, and growth rate, the high-throughput screening of 3D models remains a challenge for various reasons reported in the literature, such as the lack of uniformity in the position, size, and morphology of tumor spheroids14,15,16.
In the protocol presented here, we list each step in the construction of 3D tumor spheroids and describe a method for spheroid observation and analysis using a high-throughput, high-content imaging system that involves auto-focus, auto-imaging, and analysis, among other advantageous characteristics. We show how this method can produce 3D tumor spheroids of uniform size that are suitable for high-throughput imaging. These spheroids also demonstrate a high sensitivity to cancer drug treatment, and morphological changes in the spheroids can be monitored using high-content imaging. In summary, we demonstrate the robustness of this methodology as a means to generate 3D tumor constructs for drug evaluation purposes.

<table><tbody><tr><td>0.5-10 &amp;mu;L Pipette tips</td><td>AXYGEN</td><td>T-300</td><td></td></tr><tr><td>1.5&amp;nbsp;mL Boil proof microtubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>100-1000&amp;mu;L Pipette tips</td><td>KIRGEN</td><td>KG1313</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Nest</td><td>601052</td><td></td></tr><tr><td>200 &amp;mu;L Pipette tips</td><td>AXYGEN</td><td>T-200-Y</td><td></td></tr><tr><td>3D gel</td><td>Avatarget</td><td>MA02</td><td></td></tr><tr><td>48-well U bottom Plate</td><td>Avatarget</td><td>P02-48UWP</td><td></td></tr><tr><td>50 mL Centrifuge Tube</td><td>Nest</td><td>602052</td><td></td></tr><tr><td>Alamar Blue</td><td>Thermo</td><td>&amp;nbsp;DAL1100</td><td></td></tr><tr><td>Anti-Adherence Rinsing Solution</td><td>STEMCELL</td><td>#07010</td><td></td></tr><tr><td>Certified FBS</td><td>BI</td><td>04-001-1ACS</td><td></td></tr><tr><td>Deionized water</td><td>aladdin</td><td>W433884-500ml</td><td></td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>DMSO</td><td>sigma</td><td>D2650-100ML</td><td></td></tr><tr><td>Excel sofware&amp;nbsp;</td><td>Microsoft office</td><td></td><td></td></tr><tr><td>Graphpad prism sofware&amp;nbsp;</td><td>GraphPad software&amp;nbsp;</td><td></td><td></td></tr><tr><td>High Content Imager and SMART system</td><td>Avatarget</td><td>1-I01</td><td></td></tr><tr><td>Image J software</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Insulin-Transferrin-Selenium-A Supplement (100X)</td><td>Gibco</td><td>51300-044</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>PBS</td><td>Solarbio</td><td>P1020</td><td></td></tr><tr><td>Penicillin/streptomycin Sol</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Scientific Fluoroskan Ascent</td><td>Thermo</td><td>Fluoroskan Ascent</td><td></td></tr><tr><td>T25 Flask</td><td>JET Biofil</td><td>TCF012050</td><td></td></tr><tr><td>Trypsin, 0.25% (1X)</td><td>Hyclone</td><td>SH30042.01</td><td></td></tr></tbody></table>

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.

Figure 1A,B shows the process used for constructing tumor spheroids in this study. We first seeded the cells in a 48-well U-bottom plate. This step is almost the same as that used in 2D cell culture. We kept the plate in a common incubator with water surrounding the wells so that the deposited cells started to form spheroids in a self-assembly process. Under normal operational conditions, most types of tumor spheroids were completely formed after 5 days when a targeted mediu...

Watch this Scientific Journal Video about Generation of 3D Tumor Spheroids for Drug Evaluation Studies at JoVE.com

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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 ...

generation-of-3d-tumor-spheroids-for-drug-evaluation-studies

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

2.0K Views.  Southeast University. Dit protocol demonstreerde een gestandaardiseerde methode voor het construeren van driedimensionale tumoren van sferoïden. En deze methodologie bood een hoog circuit en een hoge inhoudsanalyse van 3D-tumorconstructies. Door gebruik te maken van een standaardmethode van tumorachtige sferoïde constructie en een beeldvormings- en analysesysteem met hoge doorvoer, kunnen de effectiviteit en nauwkeurigheid van medicijntests gevormd op driedimensionale sferoïden drastisch worden verhoogd....

Video: Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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.

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 ...

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, ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Bioengineering

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 ...

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

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

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

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

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

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

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

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

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

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Biology

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/60998/a-3d-spheroid-model-for-glioblastoma'>Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)</a>The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)The video demonstrates the generation of a three-dimensional ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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