Name: alternative strategy to analyze in vitro cell invasion of 3d cultures
Uploaded: 2024-08-09T14:30:00.000+00:00
Duration: 4 min 35 s
Description: Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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

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	<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><tbody><tr><td>96 Well Bioprinting Kit</td><td>Greiner Bio-one</td><td>&amp;nbsp;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&amp;nbsp;</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 &amp;micro;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 &amp;micro;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

368 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

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. ...

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 ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Biology

Tumor Cell Invasion Assay: A 3D In Vitro Native Matrix-based Method to Assess Epithelial-mesenchymal Cell Interactions

Source: Ng, Y. Z. et al. <a href="https://www.jove.com/v/51321"> Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion</a>. J. Vis. Exp. (2014)
This video describes the technique of utilizing a native matrix to study tumor cell invasion. The technique used in this assay provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix.

Test inwazji komórek nowotworowych: natywna metoda 3D oparta na macierzy in vitro do oceny interakcji komórek nabłonkowo-mezenchymalnych

Source: Ng, Y. Z. et al.  Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion. J. Vis. Exp. (2014)
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Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

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

Spheroid Assay to Measure TGF-β-induced Invasion

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

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

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

In vitro Cell Migration and Invasion Assays

Test inwazji komórek nowotworowych: natywna metoda 3D oparta na macierzy in vitro do oceny interakcji komórek nabłonkowo-mezenchymalnych