Name: time-resolved fluorescence imaging and analysis of cancer cell invasion in the 3d spheroid model
Uploaded: 2021-01-30T13:00:00
Description: Watch this Scientific Journal Video about Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model at JoVE.com

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 ...

<ol>
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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 proce...

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&#8217;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&#8217;s phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,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</stron...

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 ...

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