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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<li>Ivanov, D. P., Grabowska, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spheroid+arrays+for+high-throughput+single-cell+analysis+of+spatial+patterns+and+biomarker+expression+in+3D%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D.</a> Scientific Reports. 7, 41160(2017).</li>
<li>Spoerri, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+melanoma+heterogeneity+is+regulated+through+cell-matrix+interaction-dependent+changes+in+tumor+microarchitecture%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Phenotypic melanoma heterogeneity is regulated through cell-matrix interaction-dependent changes in tumor microarchitecture.</a> bioRxiv. , (2021).</li>
<li>Haass, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+cell+cycle+imaging+during+melanoma+growth%2C+invasion%2C+and+drug+response%5BTitle%5D)+AND+%22Pigment+Cell+%26+Melanoma+Research%22%5BJournal%5D)">Real-time cell cycle imaging during melanoma growth, invasion, and drug response.</a> Pigment Cell & Melanoma Research. 27 (5), 764-776 (2014).</li>
<li>Ahmad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clearing+spheroids+for+3D+fluorescent+microscopy%3A+combining+safe+and+soft+chemicals+with+deep+convolutional+neural+network%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Clearing spheroids for 3D fluorescent microscopy: combining safe and soft chemicals with deep convolutional neural network.</a> bioRxiv. , 428996(2021).</li>
<li>Villani, T., Rossi, A. E., Sherman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+characterization+of+3-D+cell+culture+models+grown+in+spheroid+microplates%5BTitle%5D)+AND+%22American+Laboratory%22%5BJournal%5D)">Image-based characterization of 3-D cell culture models grown in spheroid microplates.</a> American Laboratory. 50 (6), 12(2018).</li>
<li>Lloyd-Lewis, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+the+mammary+gland+and+mammary+tumours+in+3D%3A+optical+tissue+clearing+and+immunofluorescence+methods%5BTitle%5D)+AND+%22Breast+Cancer+Research%22%5BJournal%5D)">Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.</a> Breast Cancer Research. 18 (1), 127(2016).</li>
<li>Tainaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li>
<li>Spoerri, L., Beaumont, K. A., Anfosso, A., Haass, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+cell+cycle+imaging+in+a+3D+cell+culture+model+of+melanoma%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Real-time cell cycle imaging in a 3D cell culture model of melanoma.</a> Methods in Molecular Biology. 1612, 401-416 (2017).</li>
<li>Cold Spring Harbor. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+Dilution+Buffer+%28Abdil%29%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Antibody Dilution Buffer (Abdil).</a> Cold Spring Harbor Protocols. , (2018).</li>
<li>Pawley, J. B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPawley%2C+JB+%22Handbook+Of+Biological+Confocal+Microscopy%22">Handbook Of Biological Confocal Microscopy</a>. Pawley, J. B. , Springer. US. 20-42 (2006).</li>
<li>Sakaue-Sawano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Tissue+clearing+technique%3A+Recent+progress+and+biomedical+applications%5BTitle%5D)+AND+%22Journal+of+Anatomy%22%5BJournal%5D)">Tissue clearing technique: Recent progress and biomedical applications.</a> Journal of Anatomy. 238 (2), 489-507 (2021).</li>
<li>Browning, A. P., Murphy, R. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBrowning%2C+AP+%22Zenodo%22">Zenodo</a>. , (2021).</li>
<li>Browning, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+tumour+spheroid+structure%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Quantitative analysis of tumour spheroid structure.</a> eLife. 10, 73020(2021).</li>
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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><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 &amp;micro;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 &amp;micro;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&amp;prime;,N&amp;prime;-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>NaN&lt;sub&gt;3&lt;/sub&gt;</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

2.7K Views.  The University of Queensland. Mijn lab is gespecialiseerd in drie- en vierdimensionale tumormodellering. Tumor sferoïde modellen worden veel gebruikt in de biologie en de ontdekking van geneesmiddelen. Bestaande protocollen voor het verzamelen van gegevens uit deze experimenten zijn echter duur, moeilijk en beperkt.Dit protocol vereist gemakkelijk toegankelijke chemicaliën en apparatuur en is niet erg arbeidsintensief in vergelijking met de huidige methoden die vergelijkbare resultaten opleveren. Dit protocol is ...

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

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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

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

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

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

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

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

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

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

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

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

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

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

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

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

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

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

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Chemistry

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Bioengineering

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

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

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

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

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

Source: Dillard, P., et al. <a href="https://www.jove.com/video/58785/a-spheroid-killing-assay-by-car-t-cells">A Spheroid Killing Assay by CAR T Cells</a>. J. Vis. Exp. (2018).
Spheroids are a type of 3D cell culture model used to more accurately represent a cell&#8217;s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids&#8217; growth.

Source: Dillard, P., et al. A Spheroid Killing Assay by CAR T Cells. J. Vis. Exp. (2018).
Spheroids are a type of 3D cell culture model used to more ...

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

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