We would like to thank members of Temple Bioengineering for valuable discussions. We thank David Ambrose at the flow cytometry core (Lewis Katz School of Medicine) for his assistance with cell sorting and Tony Boehm from the IDEAS Hub (College of Engineering, Temple University) for help with the 3D printing. We also thank our funding resources: American Cancer Society Research Scholar Grant 134415-RSG-20-034-01-CSM, Conquer Cancer Now / Young Investigator Award, National Institutes of Health, R00 CA172360 and R01 CA230777, all to BG.

1. Fabrication of a Spheroid Imaging Device (SID) to optimize spheroid embedding (Duration 1 day)
<ol>
	<li>Create the spacer using a 3D printer (Figure 1A, B&#160;and Supplementary File 1).</li>
	<li>Weigh out a 10:1 (wt/wt) ratio of base polymer:crosslinker in a plastic cup [e.g., 20 g of ethylbenzene base polymer and 2 g of silicone resin crosslinker to create polydimethylsiloxane (PDMS)].</li>
	<li>Thoroughly mix the PDMS solution in the plastic cup using a disposable pipette.</li>
	<li>Place the plastic cup into a vacuum chamber to remove the air bubbles from the mixture. Quickly release the vacuum pressure to remove the small amount of air trapped at the surface of the mixture and dissipate the remaining air bubbles.</li>
	<li>Incubate the 3D printed spacer at 100 &#176;C for 5 min to increase its flexibility.</li>
	<li>Clean the two glass plates that will be used to construct the PDMS mold thoroughly by wiping them with 100% isopropanol. If the glass plates were previously used to cast PDMS, be sure to remove any remaining old PDMS by gently scraping the glass plates with a razor blade and wiping them with 100% isopropanol.</li>
	<li>Construct the mold by placing the 3D printed spacer flush in-between the two clean glass plates.</li>
	<li>Seal the mold using large binder clips on the outside edges of the glass plates. Place two binder clips on the bottom edge and one on the top corner.</li>
	<li>Inspect the top part of the mold to ensure that the spacer is flush with the glass plates. This guarantees that no deformations will be present and that an even sheet of PDMS will be created. 
	NOTE: If the resulting sheet is not of uniform thickness, the clips need to be adjusted slightly.</li>
	<li>Cut the tip of a disposable pipette (~2 cm from the tip) and add the PDMS mixture at a slow and constant rate to the top left corner of the mold. Pour the mixture slowly to prevent the creation of large air pockets.</li>
	<li>Place the mold into a vacuum chamber to remove air bubbles that formed during the pour.</li>
	<li>Cure the PDMS by incubating the mold at 100 &#176;C for 1 h.</li>
	<li>Retrieve the mold from the incubator and allow it to cool to touch.</li>
	<li>Remove the binder clips and glass plates from the spacer containing the cured PDMS.</li>
	<li>Use a razor blade to cut through the seal that was created on all four sides of the mold between the spacer and the glass plate. With all four sides cut into, begin pulling apart the mold to reveal the PDMS sheet in the spacer.</li>
	<li>Carefully peel the new PDMS sheet off of the spacer using tweezers.</li>
	<li>On a cutting mat, punch out 17.5 mm diameter PDMS disks from the sheet, and then punch three evenly distributed 5.5 mm diameter holes, using different size biopsy punches. Here these 3-hole PDMS disks are referred as &#8220;inserts&#8221; (Figure 1C). 
	NOTE: Nonuniform cuts made into the PDMS, or a faulty bind via plasma treatment, may result in the future leaks.</li>
	<li>Clean each insert by gently removing any dust particles using tape.</li>
	<li>Stick the inserts onto a piece of double-sided tape and wrap the tape around the lid of a 10 cm Petri dish.</li>
	<li>Place the lid of the Petri dish in the plasma machine, along with the open 35 mm glass bottom dishes.</li>
	<li>Activate the surface of the inserts and of the glass via plasma treatment for 1 min at 300 mTorr. A hand-held plasma wand can be used.</li>
	<li>Using tweezers, quickly attach the upside, i.e., treated side, of one insert (Figure 1D) to the glass part of a glass bottom dish (Figure 1E). Repeat for all dishes.</li>
	<li>Use pointer finger and thumb to apply an even pressure while rotating the glass bottom dish. This will ensure stable securement of the insert to the glass bottom dish.</li>
	<li>Incubate the SIDs (Figure 1F) at 60 &#176;C for 20 min to strengthen the adhesion between glass and PDMS.</li>
	<li>Perform a second round of plasma treatment on the SIDs, using the same settings as in step 1.21 or with the hand-held wand. This will render the free PDMS surface adhere to the poly-L-lysine in the coating solution (see 1.26).</li>
	<li>Freshly prepare the following solutions.
	<ol>
		<li>Coating solution: 1x PBS containing 0.01% (vol/vol) poly-L-Lysine [e.g., add 10 &#181;L of 0.1% (vol/vol) poly-L-Lysine to 90 &#181;L of 1x PBS].</li>
		<li>Crosslinking solution: Distilled water containing 1x (vol/vol) glutaraldehyde [e.g., add 10 &#181;L of 10x glutaraldehyde to 90 &#181;L of distilled water].</li>
		<li>Storage solution: 1x PBS containing 10x (vol/vol) Penicillin-Streptomycin [e.g., add 1 mL of 100x Penicillin-Streptomycin to 9 mL of 1x PBS].</li>
	</ol>
	</li>
	<li>Add 35 &#181;L of coating solution per each hole and incubate for 1 h at room temperature.</li>
	<li>Aspirate the poly-L-Lysine and rinse the entire SID 3 times with distilled water.</li>
	<li>Add 35 &#181;L of crosslinking solution per each hole incubate for 30 min at room temperature.</li>
	<li>Aspirate the crosslinking solution and rinse the entire SID 3 times with distilled water.</li>
	<li>Add 70% ethanol into each SID and place under ultraviolet (UV) light for 30 min.</li>
	<li>Under the hood, aspirate ethanol and rinse the SID 3 times with distilled water.</li>
	<li>Add 2.5 mL of storage solution per each SID. 
	NOTE: At this stage, the SIDs can be stored at 4 &#176;C for a week. It was observed that the binding strength between PDMS and glass decreases over time. Beyond a week, users are recommended to test the SIDs for potential leakage before use. To do so, aspirate the storage solution and add 35 &#181;L of 1x PBS into each hole. After use, PDMS inserts can be peeled off the glass bottom dishes. To achieve optimal cleaning of the glass bottom dishes, several washes with isopropanol and hydrochloric acid should be used to remove PDMS residues.</li>
</ol>
2. Spheroid formation and embedding into collagen (Duration 4 days)
NOTE: For live imaging of spheroids, longitudinally or in time-lapse videos, use a cell line expressing a cytoplasmic and/or nuclear fluorescent protein. If such a cell line is available, follow the steps described in this section. Alternatively, in the section 3, a protocol is proposed to label cancer cells in spheroids using a cytoplasmic dye.
<ol>
	<li>Form spheroids of 4T1 and/or 67NR cells using the hanging drop technique, as previously described4,5,6,7, with 3,000 cells/40 &#181;L droplet and a 3-day incubation time. Add the bovine atelocollagen I solution last and maintain all solutions on ice, at all time.</li>
	<li>Identify the correctly formed spheroids using a bright-field microscope.</li>
	<li>Fill a 15 mL conical tube with 8 mL of pre-warmed complete medium.</li>
	<li>Collect spheroids with a P1000 pipette and transfer to the 15 mL conical tube. Wet the pipette tip by pipetting some complete medium in and out to prevent spheroids from sticking to the inside walls of the pipette tip and limit spheroid loss.</li>
	<li>Allow spheroids to sink to the bottom of the tube and carefully wash the spheroids by exchanging the medium. Repeat twice.</li>
	<li>Prepare a 5 mg/mL collagen I solution according to the manufacturer&#8217;s recommendations (alternate gelation procedure, see exemplary calculation below). Replace the distilled water with complete medium. When calculating the volume of medium to be used, take into account that spheroids will be added in 20 &#181;L of complete medium [e.g., for one SID, 3 x 30 + (3 x 30) x 20 % = 108 &#181;L of 5 mg/mL collagen I is needed (prepare 20 % extra to account for pipetting loss when handling viscous fluids); 22 &#181;L of complete medium + 10.8 &#181;L of 10x PBS + 1.2 &#181;L of 1 M NaOH + 54 &#181;L of 10 mg/mL collagen I stock].</li>
	<li>Collect all spheroids in 20 &#181;L of medium, using a P200 pipette, and add them to the collagen I solution prepared in step 2.6.</li>
	<li>Slowly mix the solution by pipetting up and down to avoid heterogeneity in the collagen I concentration, limiting bubble formation. Keep the solution on ice.</li>
	<li>Remove the storage solution from the SID(s) and wash 3 times with 3 mL of 1x PBS. After the final wash, leave the SID(s) dry so not to dilute the collagen I solution.</li>
	<li>Start a timer and dispense 30 &#181;L of the collagen I solution containing one spheroid into one of the three holes of the SID. Visually ensure that a single spheroid is contained in the 30 &#181;L.</li>
	<li>Repeat step 2.10 twice more to fill all the 3 holes of a SID.</li>
	<li>Use a 10 &#181;L pipette tip to re-center the spheroid if it is located close to the PDMS border. If two or three spheroids end up dispensed in one of the holes, same pipette tip can be used to separate the spheroids from each other. Stop the timer. 
	NOTE: Frequently inverting of the SID upside-down and upside-up, throughout the period of the collagen I polymerization and solidification, ensures that the spheroid is positioned at the vertical center of the ECM layer, and prevents invasion of cells in 2D. The frequency of inverting (flipping) should be maximized. As the flipping frequency is controlled by the spheroid &#8220;dispensing time&#8221; measured in 2.10-2.12, the dispensing time should be minimized. In our lab, dispensing time is 2 min on average.</li>
	<li>To vertically center the spheroid in the collagen layer, flip the SID upside-down and incubate at 37 &#176;C for dispensing time.</li>
	<li>Flip the SID upside-up and incubate at 37 &#176;C for dispensing time. 
	NOTE: The length of time that the spheroids spend in the upside-down orientation should be equal to the time spent in the upside-up orientation.</li>
	<li>Repeat steps 2.13 and 2.14 for 30 min, until the collagen I polymerizes.</li>
	<li>Add 2.5 mL of complete medium/SID and if needed, acquire an image of the spheroids for the initial timepoint.</li>
	<li>Repeat steps 2.10-2.16 if multiple SIDs are used.</li>
</ol>
3. Fluorescence labeling of spheroids
<ol>
	<li>Live imaging (Duration 6-7 days) 
	NOTE: If a cell line expressing a cytoplasmic and/or nuclear fluorescent protein is available, follow the steps described in the section 2. Alternatively, for cytoplasmic labeling, the following protocol is proposed.
	<ol>
		<li>Follow steps 2.1-2.5 of the protocol described in the section 2.</li>
		<li>Dilute the cytoplasmic dye to 25 &#181;M in 200 &#181;L of serum-free medium. 
		NOTE: While a red cytoplasmic dye is used in this experiment, any other available color should be suitable. Cancer cells were labeled inside spheroids using nuclear dyes diluted to 20 &#181;M in 200 &#181;L of serum-free medium (see Table of Materials).</li>
		<li>After the final wash, resuspend spheroids in the cytoplasmic or nuclear dye solution and incubate at room temperature for 20 min, protected from light. 
		NOTE: With this approach, labeling of the cells in the spheroid center will not be efficient. To achieve labeling of all cells, incubation can be extended overnight, by placing the dish on a rocker. Alternatives are described in Discussion.</li>
		<li>Wash spheroids 3 times in complete medium.</li>
		<li>Proceed to steps 2.6-2.17 of the protocol described in the section 2.</li>
		<li>Image spheroids via time-lapse imaging every 10 min for 24-72 h (Figure 2), or via longitudinal imaging, daily, for up to 7 days (Figure 3). Use a laser scanning confocal microscope, 10x air objective (0.4 numerical aperture and 3.1 mm working distance), 1024 x 1024 pixels, exposure time 8 &#181;s/pixel, pinhole 90 &#181;m, 6 x 15 &#181;m z-steps and 3 fields of view-each containing one spheroid. 
		NOTE: For time-lapse imaging, use minimal laser power and equip the microscope with an environmental chamber with temperature, humidity and gas control. Culture the embedded spheroids at 37 &#176;C for &#62; 8 h or overnight prior to imaging to minimize the time on the microscope.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining of spheroids (Duration 2 days) 
	NOTE: The procedure described here is adapted and optimized from previously published protocols8,9. This method can be used after the protocol outlined in the sections 2 and 3.1.
	<ol>
		<li>Freshly prepare the following solutions.
		<ol>
			<li>Fixing solution: 1x PBS containing 4% PFA (vol/vol) [e.g., add 5 mL of 16% (vol/vol) PFA to 15 mL of 1x PBS].</li>
			<li>Fixing and permeabilizing solution: 1x PBS containing 4% PFA (vol/vol) and 0.5% Triton X-100 (vol/vol) [e.g., add 50 &#181;L of Triton X-100 to 10 mL of fixing solution].</li>
			<li>Blocking solution: 1x PBS containing 1% FBS (vol/vol) and 1% BSA (wt/vol) [e.g., dissolve 100 mg of BSA in 10 mL of 1x PBS, then add 100 &#181;L of FBS].</li>
			<li>Washing solution: 1x PBS containing 0.05% Tween 20 (vol/vol) [e.g., add 25 &#181;L of Tween 20 to 50 mL of 1x PBS].</li>
		</ol>
		</li>
		<li>Remove the culture medium from the SID(s) and wash once with warm 1x PBS.</li>
		<li>Add 2 mL of fixing and permeabilizing solution per SID and incubate at room temperature for 5 min.</li>
		<li>Remove the fixing and permeabilizing solution and add 2 mL of fixing solution per SID and incubate at room temperature for 20 min.</li>
		<li>Wash 3 times with the washing solution.</li>
		<li>Add 2 mL of blocking solution per SID and incubate at 4 &#176;C for 24 h with mild shaking. 
		NOTE: At this step, samples can be incubated at 4 &#176;C, over the weekend. Please note that a longer blocking time might interfere with the immunofluorescence labeling procedure.</li>
		<li>Dilute primary antibodies in blocking solution.</li>
		<li>Add 150 &#181;L of blocking solution with primary antibodies/well in a 48-well plate.</li>
		<li>Using fine tweezers, carefully detach the plug of collagen I containing a spheroid and transfer into a well. Repeat if multiple spheroids are labeled. Wipe any liquid that might remain on the tweezers when working with different antibodies, to prevent contamination.</li>
		<li>Incubate overnight at 4 &#176;C with mild shaking.</li>
		<li>Add 300 &#181;L of washing solution to empty and full wells.</li>
		<li>Carefully transfer the plug of collagen I containing a spheroid into a &#8220;wash&#8221; well.</li>
		<li>Wash 3 times with washing solution for 2 h, at room temperature and with mild shaking. 
		NOTE: Transferring the plugs of collagen I containing a spheroid, instead of aspirating the solutions, can help reducing sample loss and sample damage.</li>
		<li>Dilute secondary antibodies, 4&#8217;,6-diamidino-2-phenylindole (DAPI) and/or phalloidin in blocking solution.</li>
		<li>Add 150 &#181;L of blocking solution with secondary antibodies, DAPI and/or phalloidin per well.</li>
		<li>Using tweezers, carefully transfer the collagen plug containing the spheroids into the well. Repeat if multiple spheroids are labeled.</li>
		<li>Incubate at room temperature for 1 h with mild shaking.</li>
		<li>Repeat steps 3.2.11 and 3.2.12.</li>
		<li>Wash 3 times with washing solution for 30 min, at room temperature with mild shaking.</li>
		<li>Using a razor blade, cut a 3-hole PDMS insert into three parts so that each part contains a hole.</li>
		<li>Place two pieces of PDMS onto a microscope slide.</li>
		<li>Add a drop of mounting solution using a P200 tip with the end cut off. Prevent bubble formation.</li>
		<li>Carefully transfer a collagen I plug into each hole. 
		NOTE: If the spheroid was positioned at the top of the collagen plug, invert the collagen plug so that the spheroid ends up closer to the glass coverslip.</li>
		<li>Place a coverslip on top and seal using tape.</li>
		<li>Let the sample dry at room temperature for 10 min, protected from light.</li>
		<li>Store samples at 4 &#176;C, protected from light until imaging.</li>
	</ol>
	</li>
</ol>
4. Image processing to analyze cancer invasion over time
NOTE: The format required for this macro is a single-channel x,y,t image saved as a .tiff file.
<ol>
	<li>If multiple z-slices were acquired (i.e. x,y,z,t image), open the image in Fiji and select Image | Stacks | Z Project. It is recommended to use the Max Intensity option. Alternatively, a single z-slice can be used to run the macro. Save the image as a .tiff file.</li>
	<li>Create a separate &#8220;Processing&#8221; folder on the Desktop.</li>
	<li>Select File | Save As | Image Sequence. Use the TIFF format, update the digits number according to the number of frames, check the box to use slice label as file name and select Ok. Select the &#8220;Processing&#8221; folder created in step 2.</li>
	<li>Open one image from the &#8220;Processing&#8221; folder. It should be an x,y image, corresponding to a single timepoint.</li>
	<li>Select Image | Adjust | Auto Threshold. In the dropdown menu select the method Try all and check the box for white objects on black background.</li>
	<li>A montage image appears showing the result for each automated thresholding method.</li>
	<li>Identify the best automated thresholding method, for example RenyIEntropy.</li>
	<li>To confirm the choice for the automated thresholding method, open any other image from the &#8220;Processing&#8221; folder and test the chosen thresholding method using Image | Adjust | Auto Threshold.</li>
	<li>Close all images.</li>
	<li>Download the SpheroidAreaTime macro (Supplementary File 2).</li>
	<li>Open the Fiji macro by drag-and-drop.</li>
	<li>In line 58, update the automated thresholding method as needed.</li>
	<li>In line 62, update the size range according to the cell dimensions.</li>
	<li>Select Run.</li>
	<li>Select the &#8220;Processing&#8221; folder and type in &#8220;Processing&#8221; as the Parent folder, then select Ok.</li>
	<li>Once the run is over, save the table &#8220;Summary&#8221;. The first column indicates the name of the image; the third column indicates the spheroid area; the sixth, seventh and eight columns specify the parameters for an ellipse fitted onto the spheroid.</li>
	<li>The &#8220;Processing&#8221; folder now contains a processed image for each time point, with the extension _SpheroidArea. 
	NOTE: If the spheroid center is dim, fill it with white using the selection tools, for all timepoints, and then proceed to step 3. Similarly, the space around the spheroid can be filled with black to &#8220;clean&#8221; the image. If the image is clean, comment the line 61 to speed up the run.</li>
</ol>

<ol><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hallmarks+of+cancer%3A+The+next+generation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Hallmarks of cancer: The next generation.</a> Cell. 144, 646-674 (2011).</li>
<li>Noone, A., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNoone%2C+A+%22SEER+Cancer+statistics+review+1975-2015%2C+based+on+November+2017+SEER+data+submission%22">SEER Cancer statistics review 1975-2015, based on November 2017 SEER data submission</a>. , (2019).</li>
<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+tissue+morphogenesis+and+cancer+in+3D%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li>
<li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiment%22%5BJournal%5D)">A Simple hanging drop cell culture protocol for generation of 3D spheroids.</a> Journal of Visualized Experiment. , e2720(2011).</li>
<li>Berens, E. B., Holy, J. M., Riegel, A. T., Wellstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cancer+cell+spheroid+assay+to+assess+invasion+in+a+3D+setting%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A cancer cell spheroid assay to assess invasion in a 3D setting.</a> Journal of Visualized Experiments. 2015, (2015).</li>
<li>Tönisen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EP4+receptor+promotes+invadopodia+and+invasion+in+human+breast+cancer%5BTitle%5D)+AND+%22European+Journal+of+Cell+Biology%22%5BJournal%5D)">EP4 receptor promotes invadopodia and invasion in human breast cancer.</a> European Journal of Cell Biology. 96, 218-226 (2017).</li>
<li>Bayarmagnai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invadopodia-mediated+ECM+degradation+is+enhanced+in+the+G1+phase+of+the+cell+cycle%5BTitle%5D)+AND+%22Journal+of+Cell+Sciences%22%5BJournal%5D)">Invadopodia-mediated ECM degradation is enhanced in the G1 phase of the cell cycle.</a> Journal of Cell Sciences. 132, 227116(2019).</li>
<li>Dekkers, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+3D+imaging+of+fixed+and+cleared+organoids%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">High-resolution 3D imaging of fixed and cleared organoids.</a> Nature Protocols. 14, 1756-1771 (2019).</li>
<li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Taking+cell-matrix+adhesions+to+the+third+dimension%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Taking cell-matrix adhesions to the third dimension.</a> Science. 294, 1708-1712 (2001).</li>
<li>Boutin, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+imaging+and+nuclear+segmentation+analysis+protocol+for+cleared+3D+culture+models%5BTitle%5D)+AND+%22Science+Reports%22%5BJournal%5D)">A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models.</a> Science Reports. 8, 11135(2018).</li>
<li>Pampaloni, F., Richa, R., Ansari, N., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+spheroid+formation+recorded+with+light+sheet-based+fluorescence+microscopy%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Live spheroid formation recorded with light sheet-based fluorescence microscopy.</a> Methods in Molecular Biology. 1251, 43-57 (2015).</li>
<li>Marcello, M., Richards, R., Mason, D., Sée, V. Live imaging of cell invasion using a multicellular spheroid model and light-sheet microscopy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMarcello%2C+M+%22Advances+in+Experimental+Medicine+and%22">Advances in Experimental Medicine and</a>. Dmitriev, R. I. , Springer International Publishing. 155-161 (2017).</li>
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</ol>

The authors have nothing to disclose.

The 3D printed spacer was designed to create 1-mm thick sheets of PDMS that can then be used to easily create various shapes of PDMS, as required by the experimental applications. Due to the simplicity of its fabrication and the freedom to alter the design, this method of PDMS casting was chosen for the initial design of the SID. If high volume of SIDs is required, production can be made more efficient by creating a 3D-printed mold, which already contains PDMS disks with three equally spaced holes, and reducing the process to one step. This would eliminate the need to punch out each disk, along with the subsequent three holes, and decrease the overall preparation time.
While the 3-hole punch is developed for use with 35 mm glass bottom dishes, other sizes are available which allow for more holes and hence, more spheroids to be imaged in parallel. In addition, custom-size cover glass is also commercially available, which, in combination with 3D printed holders, can allow for high-throughput spheroid assays. With such an approach, limiting factor is the speed of data acquisition- for example, in our multicolor time-lapse confocal imaging, acquiring 3D stack of a single spheroid requires approximately 2.5 minutes. Therefore, acquiring 3D stacks for 3 spheroids in the SID requires approximately 8 minutes. As a result, to maintain our preferred frequency of imaging at 10 minutes per stack, we cannot increase the number of holes in the SIDs.
To record and quantify the invasion of living cancer cells in the 3D spheroid model, bright-field imaging can be used5. However, fluorescence microscopy is preferred, as it provides increased contrast, and ease and precision in the image processing. If the generation of a cell line expressing a cytoplasmic and/or nuclear fluorescent protein is not possible, we propose the use of the cytoplasmic dyes. As the retention time of cytoplasmic dyes inside cells is three days in our imaging conditions, cells should be labeled following the 3-day period in hanging drop, and immediately before the embedding. Labeling of cells post-embedding may non-specifically label the collagen, and reduce cell labeling. Imaging for longer than 3 days post-embedding requires the use of a different spheroid seeding protocol10 or cell lines stably expressing fluorescent protein(s).
We successfully formed and imaged spheroids containing anywhere from 60 to 5,000 cells/drop. Small spheroids are ideal for recording invasion over multiple days, as their entire invasion area can easily fit into a single field-of-view of higher magnification (20x-30x) objectives. In addition, they can easily be labeled throughout with cytoplasmic or nuclear dyes. Finally, due to the reduced scattering, each cell in the spheroid can be visualized and segmented. However, small spheroids are barely visible with naked eyes and may require additional labeling with tissue markers. In contrast, larger spheroids are easier to handle, but more susceptible to sinking to the bottom of the dish, due to their weight. Moreover, cells in the spheroid center are not always labeled when using dyes, or visible using confocal microscopy, which has penetration depth of approximately 100 micrometers. To visualize all cancer cells throughout the large spheroids using time-lapse imaging, multiphoton microscopy can be used, offering an extra advantage of collagen fibers visualization by second harmonic generation (SHG) without the need for labeling. Also, imaging can be done with light-sheet microscopy8,11,12, providing reduced image acquisition time and hence allowing for high-throughput spheroid assays, but also requiring more data storage and advanced image processing. If time-lapse videos are not required, our labeling procedure for fixed 3D spheroids can be further combined with optical clearing8,10. In addition, cryosectioning of the embedded spheroids can eliminate issues with penetration of dye or antibody during labeling, as well as penetration of light during imaging. In our hands, however, successful cryosectioning was limited to early stages of invasion and non-invasive cells, due to the technical challenges in preservation of long and fragile invasive strands.
Our protocol is also compatible with the use of nuclear dyes, to label cancer cells inside the spheroids, enabling single cell tracking of time-lapse data. The Fiji plugin TrackMate13 can be used to automate cell tracking and extract motility parameters of single cells, such as velocity, instantaneous speed and persistence.

During cancer progression, cancer cells can acquire a motile and invasive phenotype, enabling them to escape the tumor mass and invade into the surrounding tissues1. Eventually, these invasive cancer cells can reach and grow inside secondary organs, a process called cancer metastasis1. Metastasis causes more than 90% of cancer-related deaths2. One reason for this is that, while localized tumors are clinically manageable, no efficient methods exist for the elimination of invasive cancer cells once metastatic spreading has occurred. Therefore, the emergence of invasive cancer cells and the transition from a localized to an invasive disease is posing a major clinical challenge. Determining how cancer cells initiate and sustain an invasive behavior may lead to the development of novel potent therapies.
The 3D spheroid model is an ideal platform to investigate the motile behavior of cancer cells under controlled, yet physiologically relevant conditions3. Indeed, in this assay, spheroids of cancer cells are embedded inside extracellular matrix (ECM), for example collagen I, which mimics a simplified tumor. Then, imaging is used to visualize the invasion of cancer cells from the spheroid into the collagen matrix. However, multiple challenges limit this procedure.
The first challenge occurs at the embedding step, where the liquid collagen matrix can spread across the dish surface, causing the spheroid to touch the bottom of the dish. Consequently, cells from the spheroid spread on the two-dimensional (2D) surface, breaking the three-dimensional (3D) spheroid morphology. Increasing the volume of collagen is an efficient, but costly solution. To prevent cells from spreading on the 2D surface, while maintaining a minimal volume of collagen, we developed a spheroid imaging device (SID) by bounding a 1 mm-thick, 3-hole polydimethylsiloxane (PDMS) insert onto a glass bottom dish.
The second challenge of the spheroid assay is the labeling of cancer cells in spheroids, which is limited by the poor penetration of antibodies and fluorescent dyes, an effect that increases with the spheroid size. While the ideal solution for labeling cells is the establishment of cell lines stably expressing fluorescent protein(s), this option is mostly restricted to immortalized cell lines and is limited by the availability of fluorescent protein chimeras. Here, we describe an optimized protocol for immunofluorescence staining of fixed spheroids, as well as the efficient use of a cytoplasmic dye to label cells immediately before embedding the spheroid.
The third challenge of the spheroid assay is the lack of simple Fiji macros for semi-automated quantification of cell invasion over time. To address this challenge, we describe a simple methodology to analyze the spheroid area over time. We illustrate the advantages of this protocol using the 4T1 and 67NR cell lines as examples.

<table><tbody><tr><td>1 N NaOH</td><td>Honeywell Fluka</td><td>60-014-44</td><td></td></tr><tr><td>10X Dulbecco&amp;rsquo;s phosphate-buffered saline (PBS)</td><td>Gibco</td><td>SH30028.LS</td><td></td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>Alfa Aesar</td><td>43368-9M</td><td></td></tr><tr><td>1X Dulbecco&amp;rsquo;s phosphate-buffered saline (PBS)</td><td>Gibco</td><td>20012027</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>48-well plate</td><td>Falcon</td><td>T1048</td><td></td></tr><tr><td>Alexa Fluor 647 phalloidin</td><td>Life Technologies</td><td>A20006</td><td></td></tr><tr><td>Anti cortactin antibody</td><td>Abcam</td><td>ab33333</td><td>1 to 200 dilution</td></tr><tr><td>Anti E-cadherin antibody</td><td>Invitrogen</td><td>13-1900</td><td>1 to 100 dilution</td></tr><tr><td>Bovine atelocollagen I solution (Nutragen)</td><td>Advanced Biomatrix</td><td>501050ML</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A4503-50G</td><td></td></tr><tr><td>CellTracker Red CMTPX Dye</td><td>Invitrogen</td><td>C34552</td><td></td></tr><tr><td>Conical tubes</td><td>Falcon</td><td>352095</td><td></td></tr><tr><td>Coverslips</td><td>FisherBrand</td><td>12-548-5E</td><td></td></tr><tr><td>Disposable container</td><td>Staples</td><td></td><td>Plastic cups</td></tr><tr><td>Disposable transfer pipette</td><td>Thermo Scientific</td><td>202</td><td></td></tr><tr><td>DMEM</td><td>Fisher Scientific</td><td>11965118</td><td></td></tr><tr><td>Double-faced tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E7023-500ML</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Bio-Techne</td><td>S11550</td><td></td></tr><tr><td>Fluoromount-G</td><td>eBioscience</td><td>00-4958-02</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882-100mL</td><td></td></tr><tr><td>Hoescht nuclear stain</td><td>Thermo Fischer</td><td>62249</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fischer</td><td>S25371A</td><td></td></tr><tr><td>MatTek dish (glass bottom dish)</td><td>MatTek Corporation</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Methyl cellulose</td><td>Sigma Aldrich</td><td>M6385-100G</td><td></td></tr><tr><td>MilliQ water</td><td></td><td></td><td></td></tr><tr><td>Penicilin/streptomycin solution</td><td>Thermo Fischer</td><td>15140122</td><td></td></tr><tr><td>Petri dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Pipet tips</td><td>Fisherbrand</td><td>02-707</td><td></td></tr><tr><td>Pipets</td><td>Gilson</td><td>F167300</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>Primary antibodies, user specific</td><td></td><td></td><td></td></tr><tr><td>Rat Tail Collagen I</td><td>Corning</td><td>47747-218</td><td></td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td>Secondary antibodies, user specific</td><td></td><td></td><td></td></tr><tr><td>Slides</td><td>Globe Scientific</td><td>1354W-72</td><td></td></tr><tr><td>Sylgard 184 Silicone</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>Tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Triton X100</td><td>Sigma Aldrich</td><td>10789704001</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>655204-100ML</td><td></td></tr></tbody></table>

time-resolved fluorescence imaging and analysis of cancer cell invasion in the 3d spheroid model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Due to its biocompatibility, PDMS is widely used for microfabrication of confining wells, stamps and molds, which revolutionized micropatterning and microfluidic devices. In the method described here, it is used to create SIDs, customizable wells that optimize spheroid embedding and imaging procedure. Figure 1 illustrates the major components used in the fabrication of the SIDs. To cast the PDMS mold, a 1-mm thick spacer is 3D printed (Figure 1A,B), placed between the two glass plates, sealed with large clips. Pouring and baking PDMS in the space between the plates forms an 1-mm thick sheet of PDMS. The SID schematic (Figure 1C) indicates the dimensions of the optimal device, however slight variation occurs in the hole distances, due to manual punching of holes using biopsy punches (Figure 1D). Figure 1D,F indicate clean circular cuts with evenly spaced holes within the PDMS insert.
Using the SIDs facilitates efficient embedding, and hence recording of the invasion of cancer cells inside collagen I using time-lapse (Figure 2, Video 1) or longitudinal (Figure 3) imaging. Despite using the same inversion frequencies for all the SIDs during the collagen I polymerization, spheroid positions inside the collagen plug will slightly vary. Therefore, it is important to use an objective with a long working distance (&#62;1 mm). Otherwise, focusing and imaging may be difficult for spheroids positioned close to the top of the collagen plug. In contrast, spheroids positioned close to the bottom of the collagen plug, will have cells which move to the glass surface and migrate on the glass, instead of invading into the collagen I matrix. Videos of such spheroids need to be discarded. The thickness of the collagen plug, here approximately 800 &#181;m, is controlled by the volume dispensed in each hole of the SID and adjusted to invasion distances spheroids exhibit in this protocol. Thickness of the collagen plug can be lowered by dispensing a lower volume of collagen I in each hole of the SID, when using smaller or less invasive spheroids.
For proper analysis of the invasion using the Fiji macro, it is critical for cancer cells to be properly labeled over the course of the imaging session. As noted in the step 4.17., the image processing step allows for image correction if the labeling is sub-optimal. While we illustrate longitudinal imaging over the course of 6 days (Figure 3), which requires the stable expression of cytoplasmic and/or nuclear fluorescent protein, similar longitudinal imaging could be performed over a shorter time using labeling with dyes.
Following live imaging, we present some results from the immunolabeling procedure for the epithelial cadherin (E-cadherin), cortactin and F-actin (Figure 4). In these examples, we used the 4T1 and 67NR cell lines. Figure 5 shows step-by-step illustration of the image processing procedure using the Fiji macro to measure the area of the spheroid over time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61902/61902fig1v2.jpg" /> 
Figure 1: Fabrication of the SIDs.&#160;(A) Top view schematic representation of the spacer. (B) 3D printed spacer. (C) Top view schematic representation of the SID. A 3-hole PDMS inserts (D) is bound to a glass bottom dish (E), creating the final SID (F). Dimensions are in millimeters. <a href="https://www.jove.com/files/ftp_upload/61902/61902fig1v2large.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/61902/61902fig2v2.jpg" /> 
Figure 2: Live imaging of spheroids.&#160;Representative 20 h and 40 h timepoints from Video 1, maximum projection of a 4T1 spheroid. 4T1 cells were labeled using a cytoplasmic dye. Scale bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61902/61902fig2v2large.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/61902/61902fig3v2.jpg" /> 
Figure 3: Longitudinal imaging of spheroids.&#160;Representative micrographs (maximum projection) of a mixed 4T1/67NR spheroid imaged daily. 4T1 and 67NR cells stably express the cytoplasmic mScarlet and green fluorescent protein (GFP), respectively. Scale bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61902/61902fig3v2large.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/61902/61902fig4v2.jpg" /> 
Figure 4: Immunofluorescence imaging of spheroids.&#160;Representative micrographs (maximum projection) of a 4T1 spheroid fixed 2 days after embedding in collagen I. E-cadherin (cyan, A), cortactin (yellow, B) and F-actin (magenta, A and B) were labeled. Scale bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61902/61902fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61902/61902fig5v2.jpg" /> 
Figure 5: Image processing analysis.&#160;Step-by-step illustration of the image processing procedure using the Fiji macro (A) to measure the area of the spheroid over time (B). <a href="https://www.jove.com/files/ftp_upload/61902/61902fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Representative video of a 4T1 spheroid imaged every 10 min for 46 h and 10 min. 4T1 cells were labeled using a cytoplasmic dye. Scale bar, 100 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/61902/Video_1.avi" target="_blank">Please click here to download this video.</a>
Supplemental file 1: 3D model of the spacer (STL file). <a href="https://cloudflare.jove.com/files/ftp_upload/61902/JoVE_3D_Spacer.SLDPRT" target="_blank">Please click here to download this file.</a>
Supplemental file 2: SpheroidAreaTime macro. <a href="https://cloudflare.jove.com/files/ftp_upload/61902/SpheroidAreaTime.ijm" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model at JoVE.com

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

time-resolved-fluorescence-imaging-analysis-cancer-cell-invasion-3d

Universidad Científica del Sur

Research

JoVE Journal

Biology

6.4K Views.  Temple University. Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

Video: Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicine

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Cancer Research

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...