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 suspen...

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) ...

Our research group primarily focuses on the crosstalk between cancer and the immune cells. We aim to investigate the impact of various biological mechanisms on the evolution of head and neck cancer through the modulation of tumor immunity. The purpose of this protocol is to assess invasion within extracellular matrix and a spatial colocalization of different cell types in a multi-layered context.The advantage of this protocol is the use of heterospheroids incorporating two relevant non-neoplastic cell types alongside cancer cells to analyze invasion with extracellular matrix towards both a microporous membrane and chemotaxis stimuli. To begin add magnetic nanoparticles to the cell suspension and mix 10 times. Centrifuge the cells at 400G for five minutes at room temperature, and mix 10 times to resuspend the cells.Then transfer and mix all the cells in a new tube for co-culturing, and dispense 100 microliters of co-cultured cells into each well of an ultra-low attachment plate. Insert the spheroid drive magnetic field under the plate to induce aggregation and spheroid formation and place it in the incubator for three hours. Using a 200-microliter tip, add 50 microliters of ECM to the center of a 24-well microporous membrane.Remove bubbles with a sterile needle, ensuring that the matrix evenly covers the membrane surface. Incubate the membrane for one hour at 37 degrees Celsius for gel formation. To begin, prepare the 3D cell culture and the extracellular matrix-coated microporous membrane.Add 500 microliters of culture medium, supplemented with 10%fetal bovine serum to the lower chamber of the microporous membrane as a chemo attractant. Then carefully remove the medium from the formed heterospheroids, and gently add 50 microliters of fresh non-supplemented medium per well over the spheroid. Using sterile scissors, cut the edge of a 200 microliter low retention tip and collect the spheroid, along with 50 microliters of medium.Gently place the spheroid on top of the extracellular matrix. Next carefully add 150 microliters of non-supplemented medium drop by drop to reach a total volume of 200 microliters. Place the plate in the incubator for 48 hours.Click on file in the analysis software. Select open, then choose the image and click on okay. Then click on analyze, followed by set measurements to select area, integrated density, and display label.Click on okay. Finally click on analyze, and then measure. The bright field images represents spheroid or cell localization, identified by dark or black conglomerates.Images of the different cells exhibited variation in fluorescence intensity and quantity across different images along the Z axis, indicating different patterns of proliferation and invasion among cells. Using confocal microscopy, different channels were overlaid for visualizing colocalization patterns during invasion. Image analysis of the representative results presented indicated that monocytes invade first, followed by neoplastic cells, and fibroblasts are the last cells to invade the extracellular matrix.

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

Research

JoVE Journal

Cancer Research

227 Views.  Department of Diagnosis and Surgery - São Paulo State University (UNESP), School of Dentistry, Araraquara. Our research group primarily focuses on the crosstalk between cancer and the immune cells. We aim to investigate the impact of various biological mechanisms on the evolution of head and neck cancer through the modulation of tumor immunity. The purpose of this protocol is to assess invasion within extracellular matrix and a spatial colocalization of different cell types in a multi-layered context.The advantage of this protocol is the use of heterospheroids incorporating two relevant non...