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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-10 &#956;L Pipette tips</td>
			<td>AXYGEN</td>
			<td>T-300</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5&#160;mL Boil proof microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000&#956;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 &#956;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>&#160;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&#39;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&#160;</td>
			<td>Microsoft office</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism sofware&#160;</td>
			<td>GraphPad software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Content Microscope 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

Research

JoVE Journal

Cancer Research

1.9K Views.  Southeast University. 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. 

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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 ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

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 ...

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

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

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

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

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 &#181;m zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 &#181;m of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

The present protocol develops an image-based technique for rapid, non-destructive, and label-free regional cell density and viability measurement within 3D tumor aggregates. Findings revealed a cell-density gradient, with higher cell densities in core regions than outer layers in developing aggregates and predominantly peripheral cell death in HER2+ aggregates treated with Trastuzumab.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

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

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

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

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