This work was supported by the S&#227;o Paulo Research Foundation (FAPESP 20/10544-1 and 20/10664-7). Some images were generated using BioRender.com.

The use of primary human cells was approved by the Institutional Human Research Ethics Committee (CAAE 57895822.0.0000.5416) of the School of Dentistry at Araraquara - UNESP. The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of cells for 3D cell culture
NOTE: All steps in this section must be performed within a laminar flow hood.
<ol>
	<li>Obtain single-cell suspensions of the cells that will form the heterospheroid. 
	NOTE: In this experiment, we generated spheroids (see step 2) consisting of OSCC cell line (SCC9), fibroblasts (primary cancer-associated fibroblast from tongue cancer biopsies), and monocytes (primary monocytes from healthy donors). Select the type of cells according to experimental interests.</li>
	<li>Check the cell number and viability using a hemocytometer and trypan blue staining. 
	NOTE: The cell ratio in this protocol is 5:3:2 (5,000 neoplastic: 3,000 fibroblasts: 2,000 monocytes), totaling 10,000 cells per spheroid. Optimize the ratio and total number of cells according to experimental interests.</li>
	<li>Separately dilute cells in 1.5 mL tubes with non-supplemented medium appropriate for each cell type (e.g., for neoplastic cells - DMEM/F12; for fibroblasts - DMEM; for monocytes - RPMI) to achieve an appropriate seeding density of 5,000 neoplastic cells per well, 3,000 fibroblasts per well, and 2,000 monocytes per well, or 500,000 cells per milliliter. 
	NOTE: To ensure an adequate number of cells for the experiment, use 20% more than the total required, considering potential losses during the staining and centrifugation steps.</li>
	<li>Label the different cell types separately using distinct fluorescent vital stains that remain in living cells through several generations. Add optimized concentrations of fluorescent dye to the medium containing cells (e.g., neoplastic: CFSE - green dye; fibroblast: CMTPX - red dye; monocyte, Violet BMQC - blue dye) and incubate at 37 &#176;C for 20 min. 
	NOTE: Select fluorescent probes with different excitation and emission spectra to avoid spectral overlap according to the specifications of the confocal microscope that will be used.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at room temperature to form a cell pellet. Aspirate the medium using a 200 &#181;L pipette and re-suspend in 1 mL of 1x PBS.</li>
	<li>Centrifuge at 400 x g for 5 min to form a cell pellet and re-suspend the total number of cells in 1 mL of supplemented culture medium according to the cell type/cell line used (e.g., neoplastic - 100 &#181;g/mL streptomycin, 100 IU/mL penicillin, 10% (vol) FBS, and 400 &#181;g/mL hydrocortisone; fibroblast and monocytes - 100 &#181;g/mL streptomycin, 100 IU/mL penicillin, 10% (vol) FBS). Check fluorescence intensity on the microscope and count the cells with trypan blue staining.</li>
</ol>
2. 3D cell culture
NOTE: This study employed the bioprinting magnetic field method11 to create heterospheroids. Here, cells are magnetized using biocompatible magnetic nanoparticles and subjected to gentle magnetic forces. Optimize the ratio of nanoparticles to cells according to the cell type. Alternative methods for establishing spheroid cultures may also be used. Conduct the steps in this section in a laminar flow hood.
<ol>
	<li>Add magnetic nanoparticles at a ratio of 1 &#181;L of nanoparticles to 20,000 cells, and thoroughly re-suspend by mixing 10 times.</li>
	<li>Centrifuge the cells at 400 x g, room temperature, for 5 min, and re-suspend by mixing 10 times. Repeat this cycle of centrifuging/resuspension two additional times. 
	NOTE: Magnetized cells will form a brownish-colored pellet.</li>
	<li>After completing the last centrifugation cycle, aspirate the medium and re-suspend the cells in 34 &#181;L per well (34 &#181;L/5,000 neoplastic cells; 34 &#181;L/3,000 fibroblast cells; 34 &#181;L/2,000 monocyte cells) of supplemented culture medium (DMEM/F12 containing 100 &#181;g/mL streptomycin, 100 IU/mL penicillin, and 5% (vol) FBS) for the neoplastic cells. 
	NOTE: Calculate the volume based on the well size. This protocol utilized 96-well plates, totaling 100 &#181;L of medium per well.</li>
	<li>Mix all the cells into a new tube for co-culturing, and then re-suspend.</li>
	<li>Transfer 100 &#181;L of co-cultured cells into each well of an ultra-low attachment plate.</li>
	<li>Insert the spheroid drive magnetic field under the plate to induce aggregation and spheroid formation, and place it in the incubator (5% CO2, 37 &#176;C) for 3 h. 
	NOTE: The time of exposure to the magnetic field can vary according to spheroid size or composition.</li>
	<li>Remove the magnetic field and place the plate in the incubator (5% CO2, 37 &#176;C) for at least 24 h to allow the production of extracellular matrix proteins and cell-to-cell/cell-to-matrix interactions.</li>
</ol>
3. Preparation of extracellular matrix-coated microporous membrane
NOTE: Conduct the steps in this section in a laminar flow hood.
<ol>
	<li>Add 50 &#181;L of the commercially available extracellular matrix to the center of a 24-well microporous membrane using a 200 &#181;L tip. 
	NOTE: Keep the extracellular matrix on wet ice. Do not touch the microporous membrane when placing the matrix on top.</li>
	<li>Remove bubbles using a sterile needle and ensure that the matrix covers the entire membrane surface.</li>
	<li>Place the membrane in the incubator (37 &#176;C) for 1 h to allow gel formation of the extracellular matrix.</li>
</ol>
4. Invasion assay
NOTE: Conduct the steps in this section in a laminar flow hood.
<ol>
	<li>Add 500 &#181;L of DMEM/F12 culture medium supplemented with 10% fetal bovine serum to the lower chamber of the microporous membrane as a chemoattractant.</li>
	<li>Carefully remove the medium from the formed spheroids and gently add 50 &#181;L/well of fresh non-supplemented DMEM/F12 medium over the heterospheroid. 
	NOTE: This step can be performed using a holding drive for the bioprinting magnetic field method.</li>
	<li>Cut the edge of a 200 &#181;L low retention tip with a sterile scissors. 
	NOTE: The size of the tip will depend on the size of the spheroid.</li>
	<li>Collect the spheroid and 50 &#181;L of medium with the low retention tip. 
	NOTE: Perform this step slowly and carefully to avoid disaggregation of the spheroid.</li>
	<li>Place the spheroid gently on top of the extracellular matrix. 
	NOTE: Do not touch the jellified extracellular matrix.</li>
	<li>Carefully add 150 &#181;L of non-supplemented DMEM/F12 medium drop by drop to reach a total volume of 200 &#181;L.</li>
	<li>Place the plate in the incubator (5% CO2, 37 &#176;C) for 48 h. 
	NOTE: The time of incubation can vary depending on the cell line or protocol.</li>
</ol>
5. Image acquisition
<ol>
	<li>Use a confocal microscope for imaging.</li>
	<li>Set up the microscope based on the excitation and emission characteristics of vital fluorescent stains used (e.g., CFSE dye, 488 nm laser, and 520 nm detection wavelength; fibroblast: CMTPX dye, 561 nm laser, and 610 nm detection wavelength; monocyte, Violet BMQC dye, 405 nm laser, and 516 nm detection wavelength).</li>
	<li>Load the plate onto the microscope stage.</li>
	<li>Locate spheroids/cells using the bright field at 10x magnification.</li>
	<li>Adjust the zoom to 40x magnification.</li>
	<li>Set laser power and detector gain for each fluorescent channel.</li>
	<li>Identify the Z-Stack starting point as the membrane focal plane by locating the membrane pores using the bright field.</li>
	<li>Capture three images below and five to eight images above the membrane along the z-axis for each channel, including the bright field. Ensure 10 &#181;m intervals between images, covering a total depth of 90-120 &#181;m in the z-axis.</li>
	<li>Save the project as a .czi extension file after generating the images.</li>
</ol>
6. Image extraction
NOTE: The usage of image software is explained below.
<ol>
	<li>Open the .czi file using the image editing software (see Table of Materials).</li>
	<li>Click on Processing in the top right corner, then select Single. Next, click on Method and choose Image Export.</li>
	<li>Select the file under Input.</li>
	<li>Click on the Parameters button, then select the following settings: Filetype (Tagged Image File Format - .TIFF), Compression (LZM), Resize (100%), Apply Display Curve And Channel Color, Individual Channel Images, Use Channel Names, Define Subsets (Channel, select all channels), Z-Position (extract all), Region (Full), Tiles (Export selected tiles) and Export to (select the file of destination). 
	NOTE: To extract merged channel images, select the following settings: Filetype (Tagged Image File Format - .TIFF), Compression (LZM), Resize (100%), Apply Display Curve And Channel Color, Merged Channels Image, Use Channel Names, Define Subsets (Channel, select the specific channels to overlay), Z-Position (extract all), Region (Full), Tiles (Export selected tiles) and Export to (select the file of destination).</li>
	<li>Click on the Apply icon located in the top right corner. 
	NOTE: This step will simultaneously export images from the different channels.</li>
</ol>
7. Image analysis
NOTE: The usage of free software is explained in this step.
<ol>
	<li>Click on File in the top left corner, select Open, choose the image, and then click on OK.</li>
	<li>Click on Analyze, then Set Measurements, and select Area, Integrated Density, and Display Label. Click on OK.</li>
	<li>Click on Analyze, and then Measure. 
	NOTE: Ensure the area of all images is the same. &#34;Integrated Density&#34; displays two values: (1) Raw Integrated Density (RawIntDen): Sum of all pixel values in the image; (2) Integrated Density (IntDen): Product of Mean Gray Value and Area. Since the image is RGB color and the area of all images is the same, consider using RawIntDen as the fluorescence measurement value. Differences in fluorescence values (intensity and quantity) among images may indicate variations in cell invasion and proliferation intensity. Another method to normalize the invasion pattern in the extracellular matrix toward the microporous membrane using co-cultures of different cell types is to calculate the ratio of RawIntDen values: 
	Ratio RawIntDen =&#160; &#160;<img alt="Equation 1" src="/files/ftp_upload/67114/67114eq01.jpg" style="margin: auto;" /> 
	A ratio of 1 represents the image/location along the z-axis with the highest invasion/localization of cells for that specific cell type.</li>
</ol>

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

<ol>
	<li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+Mechanistic+insights+and+clinical+challenges.">Tumor metastasis: Mechanistic insights and clinical challenges.</a> Nat Med. 12 (8), 895-904 (2006).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Friedl, P., Alexander, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+invasion+and+the+microenvironment:+Plasticity+and+reciprocity.">Cancer invasion and the microenvironment: Plasticity and reciprocity.</a> Cell. 147 (5), 992-1009 (2011).</li><li>Wu, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cancer+cell+invasion:+Patterns+and+mechanisms.">Plasticity of cancer cell invasion: Patterns and mechanisms.</a> Transl Oncol. 14 (1), 100899 (2021).</li><li>Pijuan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration,+invasion,+and+adhesion+assays:+From+cell+imaging+to+data+analysis.">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Front Cell Dev Biol. 7, 1-16 (2019).</li><li>Nagy, &. #. 1. 9. 3. ;. G., Sz&#233;k&#225;cs, I., Bony&#225;r, A., Horvath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+automatic+monitoring+of+cancer+cell+invasion+into+an+epithelial+monolayer+using+label-free+holographic+microscopy.">Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy.</a> Sci Rep. 12 (1), 1-13 (2022).</li><li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutat Res Rev Mutat Res. 752 (1), 10-24 (2013).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> J Vis Exp. 88, e51046 (2014).</li><li>Kapa&#322;czy&#324;ska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+and+3D+cell+cultures+-+A+comparison+of+different.">2D and 3D cell cultures - A comparison of different.</a> Arch Med Sci. 14 (4), 910-919 (2016).</li><li>Cacciamali, A., Villa, R., Dotti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+cultures:+Evolution+of+an+ancient+tool+for+new+applications.">3D cell cultures: Evolution of an ancient tool for new applications.</a> Front Physiol. 13, 1-15 (2022).</li><li>Trindade, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+3D+cell+culture+State+of+the+art+and+current+advances.">Magnetic 3D cell culture State of the art and current advances.</a> Life Sci. 286, 1-8 (2021).</li><li>Han, S. J., Kwon, S., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+applying+multicellular+tumor+spheroids+in+preclinical+phase.">Challenges of applying multicellular tumor spheroids in preclinical phase.</a> Cancer Cell Int. 21 (1), 1-19 (2021).</li><li>Lin, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cancer+cell+invasion+and+T-cell+cytotoxicity+in+3D+culture.">Monitoring cancer cell invasion and T-cell cytotoxicity in 3D culture.</a> J Vis Exp. 160, e61392 (2021).</li></ol>

The authors declare that they have no conflicts of interest.

Three-dimensional spheroid cultures are a powerful approach to investigate various aspects of cell biology. In this study, we report a protocol for evaluating neoplastic cell invasion using heterospheroids that mimic the tumor microenvironment in a multilayered and more dynamic approach. This method allows for the assessment of co-localization and patterns of invasion in the presence of non-neoplastic stromal cells.
There are some critical steps that warrant consideration. First, it is importa...

Invasion is a process in which neoplastic cells migrate through the extracellular matrix into the surrounding tissue1,2. As the tumor progresses, neoplastic cells are exposed to increasingly complex microenvironments. The tumor microenvironment includes various components of the extracellular matrix and non-neoplastic cell types. Stromal cells, including fibroblasts and resident macrophages, remodel the microenvironment by secreting extracellular matrix components and growth factors, and cytokines, which in turn influence neoplastic cell functions3. Stromal and infiltrating leukocyte cells also directly interact with neoplastic cells, with each other, and with the extracellular matrix in a three-dimensional microenvironment. Altogether, the TME composition influences the efficacy and pattern of neoplastic cell invasion4.
The intricate nature of the tumor microenvironment requires the implementation of efficacious methodologies for comprehending intercellular interactions within tumor tissue. Most in vitro assays used to assess invasion have primarily been conducted in traditional 2D models and/or single cell-type&#160;cultures5,6,7,8, which&#160;oversimplifies the complex process of invasion and has multiple limitations, such as the lack of tissue architecture, stromal components, and in vivo reproducibility. Spheroid cultures represent one model of in vitro 3D cultures&#160;that mimics tumor mass morphology, allowing for cell-cell and cell-matrix interactions involving physical and biochemical features9. The use of 3D culture models has grown in the last decades, and there is evidence that various biological outcomes, such as response to treatment, cell morphology, and gene expression, are different in comparison with the 2D model9,10.
This protocol demonstrates a method for assessing in vitro invasion in a human extracellular matrix using 3D spheroid cell culture. Moreover, we employed heterospheroids comprised of neoplastic cells, fibroblasts, and monocytes to mimic the tumor microenvironment with two relevant non-neoplastic cell types. The imaging of cells by confocal microscopy along different planes of invasion towards a microporous membrane enables the visualization of this process in a more dynamic and multilayered context.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well Bioprinting Kit</td>
			<td>Greiner Bio-one</td>
			<td>&#160;655840</td>
			<td>Spheroid drive magnetic field. Holding drive. 96 well microplate, cell-repellent surface</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Invitrogen, ThermoFisher Scientific</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX</td>
			<td>Invitrogen, ThermoFisher Scientific</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Violet BMQC Dye</td>
			<td>Invitrogen, Thermo Fisher</td>
			<td>C10094</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>31600-034</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix with Myogel</td>
			<td>doi: 10.1186/s12885-015-1944-z</td>
			<td></td>
			<td>Mix composed of myogel (2.4 mg/mL), collagen (0.8 mg/mL) and non-supplemented medium of neoplastic cell&#160;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>12657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Nanoshuttle-PL</td>
			<td>Greiner Bio-one</td>
			<td>657841</td>
			<td>Magnetic nanoparticles</td>
		</tr>
		<tr>
			<td>Needle 23 G</td>
			<td>Medix</td>
			<td>AHMD004</td>
			<td>25 x 0.60 mm (23 G x 1)</td>
		</tr>
		<tr>
			<td>PBS pH 7.2 (1x)</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>2012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5,000 U/mL)</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips</td>
			<td>Axygen, Corning Inc.</td>
			<td>TF-200-L-R-S</td>
			<td>Pipet tip with barrier (filter), 200 &#181;L Low Retention Filter, short, maximum recovery</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>TransWell 24 well</td>
			<td>Costar, Corning Inc.</td>
			<td>3422</td>
			<td>Transwell Permeable Supports 6.5 mm Insert, 24 well plate; 0.8 &#181;m Polycarbonate Membrane</td>
		</tr>
		<tr>
			<td>Zen 3.4 (blue edition)</td>
			<td>Zeiss Group</td>
			<td></td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

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.

This study presents an alternative method for assessing cell invasion in vitro using spheroids composed of different cell types. It enables the analysis of cell invasion patterns in a multilayered context and the examination of cell co-localization dynamics during invasion (Figure 1).
The bright-field images represent spheroid/cell localization, identified by dark/black agglomerates (Figure 2A). The plane of the membrane is d...

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

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) ...

alternative-strategy-to-analyze-in-vitro-cell-invasion-of-3d-cultures

Research

JoVE Journal

Cancer Research

297 Views.  Department of Diagnosis and Surgery - São Paulo State University (UNESP), School of Dentistry, Araraquara. 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.

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

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 ...