This research was carried out at the Translational Research Institute (TRI), Woolloongabba, QLD. TRI is supported by a grant from the Australian Government.&#8239;We thank the staff in the microscopy core facility at TRI for their outstanding technical support. We thank Prof. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Prof. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, PA, for providing the cell lines. We thank Dr Loredana Spoerri for providing C8161 cryosection images.
This work was supported by project grants to N.K.H.: Australian Research Council (DP200100177) and Meehan Project Grant (021174 2017002565).

The protocol describes the preparation of a sufficient quantity of tumor spheroids to mount one 24-well plate (approximately 240-360 spheroids or 10-15 spheroids/well) at 200 &#181;L of agarose gel per well and 500 &#181;L of clearing solution per well. The complete procedure is illustrated in Figure 1.
1. 2% agarose-PBS gel preparation
<ol>
	<li>Weigh 0.5 g of low-melting agarose (see Table of Materials) in a glass bottle and add 25 mL of phosphate-buffered saline (PBS).</li>
	<li>Melt the agarose by boiling the solution in a microwave for 30-60 s with the lid closed but not sealed and with constant swirling. Ensure agarose is fully dissolved and the solution does not boil over. 
	NOTE: Gel can be stored at room temperature; check the volume before use to account for evaporation. Bring to the liquid state (microwave; 20-60 s with swirling) before using.</li>
</ol>
2. Preparation of the clearing solution
<ol>
	<li>Weigh 9 g of N,N,N&#8242;,N&#8242;-Tetrakis(2-Hydroxypropyl) ethylenediamine, 22 g of urea, 44 g of sucrose, 0.1 g of Triton X-100, and 24.9 g of deionized water (see Table of Materials). 
	NOTE: It is easier to weigh the approximate amount of N,N,N&#8242;,N&#8242;-Tetrakis(2-Hydroxypropyl)ethylenediamine and adjust the weight of other components to obtain the desired concentration.</li>
	<li>Heat the solution in a 56 &#176;C water bath with constant mixing and continue till all the crystals dissolve.</li>
	<li>Rest the solution at room temperature to allow the bubbles formed during mixing to rise to the surface. The solution is stable at room temperature for up to 2 months.</li>
</ol>
3. Spheroid preparation
<ol>
	<li>Generate spheroids using the agarose overlay method as previously described1,3,14. 
	NOTE: Spheroids generated by other methods are also compatible with this imaging protocol.</li>
	<li>Cut the tip of a 1 mL pipette tip using a scalpel to enlarge the orifice. 
	NOTE: All spheroids were handled with 1 mL or 200 &#181;L pipettes with cut tips.</li>
	<li>Using the pipette, aspirate the spheroids from the wells and transfer them to a clear 1.5 mL conical tube. Multiple spheroids from the same conditions can be combined in the same tube. 
	NOTE: Always allow the spheroids to settle to the bottom of the tube to aspirate the medium.</li>
	<li>Wash the spheroids in 1 mL PBS twice, add neutral 4% pre-warmed paraformaldehyde (PFA) solution, and incubate the spheroids at 37 &#176;C for 20 min.</li>
	<li>Remove the PFA solution and wash the spheroids twice with 1 mL of PBS.</li>
</ol>
4. Spheroid staining
<ol>
	<li>Using a pipette, transfer up to 15 spheroids into a 200 &#181;L PCR tube. 
	NOTE: For fragile spheroids, mount the spheroids in gel (steps 5.1-5.4) before proceeding.</li>
	<li>Permeabilize the cells using 200 &#181;L of 0.5% Triton X-100 in PBS. Place PCR tubes inside 50 mL screw-cap tubes and incubate the spheroids for 2 h at room temperature with mild agitation. 
	NOTE: All incubations are done with agitation on a rotor, roller, or shaker (see Table of Materials) unless specified otherwise. This is important to achieve uniform staining. Fixed spheroids in PBS can sometimes stick to the sides of the pipette tip. Rinsing the tip in 0.5% Triton X-100 minimizes the spheroids from sticking to the tips.</li>
	<li>Replace the solution (step 4.2) with 200 &#181;L of Antibody dilution buffer (ABDIL)15 and incubate overnight at room temperature.</li>
	<li>Remove ABDIL, add 75 &#181;L of primary antibody in ABDIL, and incubate at 4 &#176;C for 2.5 days.</li>
	<li>Remove the supernatant and wash with 200 &#181;L of PBS with 0.1% Tween and 0.1% Triton X-100 (PBS-TT) twice&#160;and incubate with 200 &#181;L of PBS-TT for 4 h at room temperature.</li>
	<li>Remove PBS-TT, add 100 &#181;L of secondary antibody in ABDIL, and incubate at 4 &#176;C for 2.5 days. 
	NOTE: DAPI or other nuclear dye can be added at this stage. Most dyes require less incubation time than antibodies.</li>
	<li>Remove the supernatant and wash with 200 &#181;L of PBS-TT twice and incubate with 200 &#181;L of PBS-TT for 4 h. The spheroids are ready for mounting.</li>
</ol>
5. Mounting
<ol>
	<li>Using a 200 &#181;L pipette, transfer fixed and stained spheroids to 500 &#181;L PCR tubes at one tube per condition (approximately 10-15 spheroids).</li>
	<li>Replace the solution with 200 &#181;L of 2% (w/v) liquid agarose gel and centrifuge on the quick spin (at the fixed maximum speed, see Table of Materials) at room temperature for 30 s. 
	NOTE: Unless mounting immediately, place in a heating block at 50 &#176;C to avoid gel hardening.</li>
	<li>Aspirate the spheroids in ~50 &#181;L liquid agarose gel and dispense in well of a 24-well glass-bottom plate.</li>
	<li>Before the gel hardens, separate the spheroids using a pipette tip in the surrounding gel, and ensure the spheroids are covered with gel. Optionally, place the plate on ice to rapidly set the gel. 
	NOTE: The number of spheroids per well depends on the size of the spheroids. Spheroids are placed far away from each other so that only one spheroid enters the field of view while imaging. This is important for automated image processing.</li>
	<li>Add 500 &#181;L of clearing solution (step 2) per well, ensuring the gel is submerged. Incubate at RT for at least 24 h and image the spheroids in the clearing solution. Spheroids are stable in this solution for up to a month at room temperature and when protected from evaporation.</li>
</ol>
6. Imaging
<ol>
	<li>Choose an objective with a working distance long enough to encompass the entire spheroid, including the mounting height of the spheroid in the vessel. 
	NOTE: Objectives with a working distance of 3 mm or longer are required to image the equatorial plane of the spheroids. Higher magnification objectives with higher NA can allow for better resolution, but will limit the maximum depth at which the spheroids can be imaged.</li>
	<li>To identify the equatorial plane, adjust the focus till the largest surface area is reached in the XY plane and image with the required laser power percentage, detector voltage, gain, and offset settings. 
	NOTE: The laser power percentage, detector voltage, gain, and offset will vary greatly depending on the fluorophore, staining intensity, laser intensity, detector sensitivity, etc.</li>
	<li>For 3D images, set the start and end of the spheroids, and choose appropriate signal intensity at various z-depth using z intensity correction settings before imaging. 
	NOTE: For best image resolution, use the Nyquist sampling rate for x, y, and z (for details, see reference16).</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook Of Biological Confocal Microscopy. , 20-42 (2006).</li><li>Sakaue-Sawano, 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=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132 (3), 487-498 (2008).</li><li>Tian, T., Yang, Z., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+technique:+Recent+progress+and+biomedical+applications.">Tissue clearing technique: Recent progress and biomedical applications.</a> Journal of Anatomy. 238 (2), 489-507 (2021).</li><li>Browning, A. P., Murphy, R. 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Olympus contributed to the costs of publication.

A protocol for obtaining high-quality two- and three-dimensional images of tumor spheroids is presented here. Existing methods, such as CLARITY, See deep brain (SeeDB), and ScaleS, often cause size distortion by up to 30%, while techniques such as Benzyl Alcohol/ Benzyl Benzoate (BABB) and 3D imaging of solvent-cleared organs (3DISCO) can quench fluorescent protein18. Many of these methods are designed for clearing tissue with structural integrity and distort the size and structure when applied to spheroids18. In contrast to other protocols that use commercially available expensive clearing solutions, this protocol uses readily available consumables while maintaining optical clarity and endogenous fluorescence and minimizing size distortion. Embedding spheroids in agarose-PBS gel provides structural support for spheroids and minimizes osmotic shock when the clearing solution is added. This is crucial when imaging fragile spheroids post drug treatment. It is assumed that this optical clearing method is suitable for spheroids formed by any method since this protocol is adapted from whole tissue clearing. The assumption is based on the similarity in the spheroids obtained by different spheroid formation methods. The choice of fixative can affect the spheroid size as well as endogenous fluorescence. This clearing method is suitable for spheroids fixed with neutral 4% PFA solution. Further testing is required to check its compatibility with other fixatives.
Given that this technique allows multiple spheroids to be mounted simultaneously in a multi-well plate, it is well suited to quantitative analysis pipelines that require spheroid structure information from up to 360 spheroids per 24-well plate. Microscopes with automated stage and plate mapping functions can make imaging less manual. Even though this method is quicker and easier than sectioning, it is currently unsuitable for complete automation. However, images obtained by this method are suitable for automated image processing4,19, and the rate at which spheroids can be mounted using this protocol lends to quantitative analysis of spheroid inner structure20,21,22.
For staining whole spheroids, the antibody concentration, volume, and incubation time need to be optimized for every antibody. As a guide, use 2.5x of the recommended 2D immunofluorescence antibody concentration and 100-200 &#181;L of antibody, depending on the number of spheroids per tube. Ensure that all spheroids are covered in staining solution when on the rotor. The incubation time depends on many factors, including the size and density of spheroids and the antibody, and can range from 16-72 h. Despite the method allowing for signal detection deeper within the spheroid, fluorophores excited by UV cause significant light scattering, leading to a low signal-to-noise ratio. Care must be taken when choosing fluorophores to visualize the target protein. For example, staining less abundant and structural proteins with longer wavelength fluorophores and more abundant protein or nuclear stains with shorter wavelength fluorophores will achieve the best result. Finally, cleared spheroids still show light loss due to scattering in the necrotic core, evident in the y/z-dimension of images obtained of 600 &#181;m spheroids (Figure 2C).
Higher magnification imaging with higher NA objectives is possible with spheroids mounted and cleared using this protocol, but the objective&#39;s working distance limits the imaging depth. For an oil immersion lens, it is important to use oil that has an RI of 1.51 for the best result.
To summarize, the agarose-PBS gel embedded clearing method allows for visualization of cells deep inside spheroids using commonly available consumables. Spheroids mounted and cleared using this method undergo minimal size distortion and maintain their structural integrity, allowing the collection of high-quality data relating to the inner spheroid structure and subsequent automated quantification.

Three-dimensional (3D) cell cultures, such as spheroids, provide biologically realistic and reproducible models of aggregate cell growth1,2. These models are fast becoming commonplace in both basic research and drug development, where differences in spheroid size and structure are examined between treatments to ascertain drug efficacy3,4. In these contexts, the ability to collect detailed information from a large number of spheroids is highly advantageous, both from a statistical power perspective and to allow rapid assessment of cell behavior across several treatments.
Commonly used techniques for obtaining detailed microscopy images of spheroid structure are either time-consuming, expensive, or produce poor quality images that do not retain key quantitative features such as spheroid size4,5. For example, histological techniques based upon cryosectioning can provide high-quality images but are often time-consuming, require skilled labor, and often create sectioning artefacts6,7, while elegant technologies, such as single plane illumination microscopy (SPIM)8 and multiphoton microscopy9 require specialized microscopes that are not readily available. Modern microscopy technologies have recently enabled the so-called optical sectioning, where spheroids are placed within a refractive index matched clearing solution and images are obtained using confocal microscopy4,5. While these techniques have the potential to produce a high yield, common problems include spheroid movement while imaging, size distortion while clearing, and the high expense of proprietary clearing solutions. Furthermore, many existing protocols apply only to relatively small spheroids of less than 300 &#181;m in diameter or depth up to 100 &#181;m, limiting the technology to the early stages of tumor growth5,10,11.
The present protocol allows semi-high-throughput, high-yield collection of detailed spheroid images using a low-cost refractive index-matched clearing solution derived from whole organ clearing procedures12,13. To prevent spheroid movement during imaging and provide structural support to reduce size distortion, the spheroids are mounted in agarose-PBS gel in a 24-well #1.5 glass bottom plate. Since this technique allows for multiple spheroids to be mounted in each well in a 24-well plate, up to 360 spheroids (15 spheroids/well) can be rapidly mounted and imaged across various experimental conditions. A refractive-index-matched clearing solution constructed from readily available consumables is used to clear mounted spheroids and the surrounding gel optically. After a settling period of 24 h, this protocol provides high-quality 2D and 3D images of spheroid structure, even for relatively large spheroids (approximately 700 &#181;m in diameter), with less than 2% size distortion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#1.5 glass bottom 24-well plate</td>
			<td>Celvis</td>
			<td>P24-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;L clear PCR tubes</td>
			<td>Sigma</td>
			<td>HS4422</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson Immuno research</td>
			<td>715-605-151</td>
			<td>Dilution used 1:500</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>Final concentration 2% w/v</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-10MG</td>
			<td>Final concentration 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>MILLI Q</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Thermofisher</td>
			<td>A-31573</td>
			<td>Dilution used 1:500</td>
		</tr>
		<tr>
			<td>DRAQ7</td>
			<td>Thermofisher</td>
			<td>D15106</td>
			<td>Dilution used 1:250</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Ratek</td>
			<td>DBH10</td>
			<td>or similar equipment</td>
		</tr>
		<tr>
			<td>Hypoxyprobe Kits</td>
			<td>Hypoxyprobe</td>
			<td>HP1-1000Kit</td>
			<td>Antibody dilution used 1:500</td>
		</tr>
		<tr>
			<td>Low-melting agarose powder</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td>Final concentration 2% w/v</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Sharp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetrakis(2-Hydroxypropyl)ethylenediamine</td>
			<td>Sigma</td>
			<td>122262</td>
			<td>Final concentration 9% w/w</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>Final concentration 150 mM</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Final concentration 0.10% w/v</td>
		</tr>
		<tr>
			<td>p27 Kip1 (D69C12) XP</td>
			<td>Cell Signalling technology</td>
			<td>3686S</td>
			<td>Dilution used 1:500</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution</td>
			<td>Proscitech</td>
			<td>C004</td>
			<td>Final concentration 4% w/v</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermofisher</td>
			<td>18912014</td>
			<td>Final concentration 1x</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quickspin minifuge</td>
			<td></td>
			<td></td>
			<td>or similar equipment</td>
		</tr>
		<tr>
			<td>Roller</td>
			<td>Ratek</td>
			<td>BTR10-12V</td>
			<td>or similar equipment</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Ratek</td>
			<td>RSM7DC</td>
			<td>or similar equipment</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Ratek</td>
			<td>EOM5</td>
			<td>or similar equipment</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>Final concentration 44% w/w</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 7.4</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>Final concentration 20 mM</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Final concentration 0.10% v/v</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Final concentration 0.1% v/v</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>Final concentration 0.1% v/v</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5379</td>
			<td>Final concentration 22% w/w</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the ability of this clearing method to provide high-quality two- and three-dimensional images, spheroids with diameters of 300-600 &#181;m were grown from Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) transduced melanoma cell lines FUCCI-WM164 and FUCCI-WM983b9,17, which express monomeric Kusabira Orange2 (mKO2) and monomeric Azami Green (mAG) protein when in Gap1, and early S-phase/Gap2/mitosis phase of the cell cycle, respectively according to procedures provided in step 31,3,14. The spheroids were then fixed with 4% formaldehyde solution at 37 &#176;C, permeabilized, and stained with anti-p27kip1/anti-rabbit Alexa Fluor 647 anti-pimonidazole/anti-mouse Alexa Fluor 647, DAPI, or DRAQ7 (see Table of Materials) (Figure 2). All microscopy files are uploaded to the GitHub repository (https://github.com/ap-browning/SpheroidMounting). Compared with PBS-mounted spheroids, the clearing solution provides high-clarity images with minimal size distortion (Figure 2A). The protocol allows for high-resolution imaging of cellular-level details deeper into the spheroids without histological sectioning, and the cross-sectional image was obtained from a 20x air objective (0.7 NA) at a resolution of 4096 x 4096 px without stitching (Figure 2B). Using a lower magnification and lower numerical aperture objective with a longer working distance, 3D confocal images that provide cellular-level detail at a depth of at least 200 &#181;m can be obtained (Figure 2C). Spheroids were also cryosectioned and stained as per the protocol by Spoerri et al.4 and compared to whole spheroid staining (Figure 2D,E). Figure 2D shows the hypoxic region of the spheroid stained by pimonidazole, and Figure 2E shows p27kip1 staining that marks cell cycle arrest (yellow) and DAPI nuclear stain (gray). Protein localization and staining pattern are similar between cryosectioning and clearing and hence are unaffected by this clearing method.
Signal intensity correction in z allows the imaging of the whole spheroid. However, light-scattering due to the necrotic core limits the ability to image the far side of the spheroid. Deeper penetration and less scattering of a far-red fluorophore, such as DRAQ7 nuclear stain, allows for even further improved 3D spheroid structure representation (Figure 3). Movie 1 shows the 3D rendering of the FUCCI spheroids stained with DRAQ7. A thinner z-slice may allow for better z-resolution, but this significantly increases the imaging time and photobleaching of the fluorophores.
To determine whether the clearing solution causes size distortion, twelve spheroids in 2% agarose-PBS gel were imaged at 6 h, 12 h, 24 h, 72 h, and 168 h following the introduction of the clearing solution. The images were summarized by determining the diameter of the spheroid, defined based on a sphere with the same cross-sectional area as the spheroid (Figure 4A). While the spheroids are observed to slightly increase in size over the first 6 h, indicated by a diameter fold-change of between 2% and 6% (Figure 4B), after 24 h to 72 h, the spheroids return to a size approximately equal to the corresponding size in PBS post PFA fixation (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64103/64103fig01.jpg" /> 
Figure 1: Illustration of the spheroid mounting and clearing protocol. Fixed and stained spheroids are transferred to a 500 &#181;L tube; excess fluid is replaced by 200 &#181;L agarose-PBS gel and centrifuged. Spheroids are then transferred to a glass-bottom well in a 24-well plate. After the gel is allowed to solidify, 500 &#181;L clearing solution is added, and spheroids are allowed to equilibrate. <a href="https://www.jove.com/files/ftp_upload/64103/64103fig01large.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/64103/64103fig02.jpg" /> 
Figure 2: Comparison of cleared and uncleared FUCCI human melanoma spheroids. Coloring indicates cell nuclei positive for mKO2 (red), which indicates cells in gap 1; and cell nuclei positive for mAG (green), which indicates cells in gap 2.&#8239;(A)&#8239;Spheroids grown from 5000 FUCCI-WM983b cells, harvested at day 10 and imaged in agarose-PBS gel and 24 h after clearing solution is added. Comparing brightfield and confocal images before and after clearing solution shows minimal size distortion and a large gain in clarity. Images are obtained using a 10x objective.&#8239;(B)&#8239;Spheroids grown from FUCCI-WM164 cells permeabilized using Triton X-100 and stained with DRAQ7, staining all cell nuclei. Image is obtained using a 20x objective (0.75 NA), demonstrating the clearing solution allows high-resolution imaging of cell-level details.&#8239;(C) 3D images (10x, 0.4 NA) were obtained of FUCCI-WM164 spheroids in PBS, and 24 h after the clearing solution was added. Adjusting laser power, voltage, and offset at different z-plane allows imaging deeper inside the spheroid. (D,E) Comparison between cryosection and cleared whole spheroid stained for pimonidazole and p27kip1. (D) Pimonidazole staining in magenta shows the hypoxic region in the spheroids. Red and green indicate FUCCI. (E) Cryosections and cleared spheroid showing DAPI (gray) and p27kip1 (yellow). <a href="https://www.jove.com/files/ftp_upload/64103/64103fig02large.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/64103/64103fig03.jpg" /> 
Figure 3: Clearing allows for imaging deeper into the spheroid with minimal light loss. Confocal microscopy images of a FUCCI human melanoma spheroid at 10x magnification and lower NA (0.4), allowing imaging at higher z-depth with minimal signal loss.&#8239;(A)&#8239;3.88 &#181;m slices of spheroid nuclei stained with DRAQ7.&#8239;(B-D)&#8239;y/z-resolution in the 488 (mAG), 568 (mKO2), and 647 nm (DRAQ7) channels, respectively. <a href="https://www.jove.com/files/ftp_upload/64103/64103fig03large.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/64103/64103fig04.jpg" /> 
Figure 4: Clearing solution has minimal impact on spheroid size.&#160;(A) Distribution of initial spheroid size (equivalent diameter) in PBS gel (n = 12 spheroids).&#8239;(B)&#8239;Diameter fold-change over time since addition of clearing solution. At 0 h, spheroids are in PBS gel only.&#8239;(C)&#8239;Distribution of diameter fold-changes at 12 h, 24 h, and 72 h. <a href="https://www.jove.com/files/ftp_upload/64103/64103fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: 3D rendering of a FUCCI spheroid stained with DRAQ7. <a href="https://www.jove.com/files/ftp_upload/64103/Movie S1.zip" target="_blank">Please click here to download this Movie.</a>

Watch this Scientific Journal Video about Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids at JoVE.com

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

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 ...

rapid-optical-clearing-for-semi-high-throughput-analysis-tumor

Research

JoVE Journal

Cancer Research

2.6K Views.  The University of Queensland. 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.

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

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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, ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

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 ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...