Name: a macrophage-tumor spheroid co-invasion assay
Uploaded: 2025-01-24T13:50:00.000+00:00
Duration: 9 min 1 s
Description: The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

The authors would like to thank Andrea Mordhorst for excellent technical support and cell culture and Martin Aepfelbacher for continuous support. Work on macrophage invasion in the SL lab is supported by Deutsche Forschungsgemeinschaft (CRC877/B13; LI925/13-1).

This protocol involves the use of primary human macrophages derived from donor blood samples. According to the ethical guidelines of the University Medical Center Hamburg-Eppendorf, blood samples were collected, and donors were compensated. Further processing of the samples was performed following given safety guidelines (e.g., treatment of untested samples). Work with primary human macrophages in this study has been judged unobjectionable by &#196;rztekammer Hamburg, Germany.
1. Generation of H1299 tumor spheroids in a scaffold-free approach (3 days in advance)
<ol>
	<li>Cultivate cancer cells in a 37 &#176;C incubator with 5% CO2 in 25 cm2 cell culture flasks (see Table of Materials) with cancer cell medium (Dulbecco&#39;s modified eagles&#39; medium [DMEM] + 1% penicillin-streptomycin [pen-strep] + 10% fetal bovine serum [FBS] + 0.1 mM nonessential amino-acids + 2 mM L-glutamine) until 80% confluence.</li>
	<li>Wash the cells once with Dulbecco&#39;s phosphate buffered saline (DPBS) and detach them by adding 1 mL of trypsin-ethylenediaminetetraacetic acid (EDTA) (0.25%, phenol-red) for 2 min. Stop the enzymatic reaction by adding 2 mL of cancer cell media and check the detachment under a light microscope. 
	NOTE: It is important that the cancer cells are separated and that no cancer cell clusters are present.</li>
	<li>Transfer the cell suspension into a 15 mL reaction tube and centrifuge it at 245 x g for 5 min. Afterward, remove the trypsin solution by pipetting or using a pump, wash the pellet subsequently by adding 5 mL of DPBS, and centrifuge them again under the same conditions.</li>
	<li>Resuspend the pellet in 5 mL of cancer cell medium and count the cells, e.g., with a Neubauer chamber. Transfer 8,000 H1299-GFP cells into a 96-well ultra-low adhesion plate (see Table of Materials) in a final volume of 25 &#181;L of cancer cell medium.</li>
	<li>Incubate the chamber for 3 days in the incubator at 37 &#176;C and 5% CO2. Check the spheroids under the microscope for uniformity. Do not consider spheroids with a lower density or unattached individual cancer cells for further experiments.</li>
</ol>
2. Preparation of primary human macrophages from donor blood samples
<ol>
	<li>If donor blood samples are not used for cell isolation immediately, store them at 4 &#176;C, with shaking.</li>
	<li>Transfer 20 mL of blood from a transfusion bag into a 50 mL reaction tube.</li>
	<li>Subsequently, prepare a new 50 mL tube and add 15 mL of lymphocyte separation medium (LSM).</li>
	<li>Carefully add the 20 mL of blood to each LSM-containing tube without mixing and centrifuge it at 450 x g for 30 min at 4 &#176;C.</li>
	<li>In the meantime, prepare a new 50 mL tube and add 10 mL of cold Roswell Park Memorial Institute (RPMI)-1640 medium.</li>
	<li>After centrifugation, transfer the dense, white phase (&#34;buffy coat&#34;) within the blood-containing tube into the prepared RPMI-containing tube and add up the volume to 50 mL with cold RPMI.</li>
	<li>Centrifuge the suspension at 450 x g for 10 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the cells in 10 mL of cold RPMI.</li>
	<li>Centrifuge the mix again at 450 x g for 10 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in 50 mL of RPMI.</li>
	<li>Centrifuge the sample again at 450 x g for 10 min at 4 &#176;C, discard the supernatant, and resuspend the cell pellet in 1.5 mL of cold monocyte buffer (0.5% human serum albumin + 5 mM EDTA in 25 mL of DPBS).</li>
	<li>Put the cell suspension on ice, add 250 &#181;L of anti-CD14 microbeads, and incubate for 15 min on ice.</li>
	<li>Prepare a separation column by addition of a filter at a magnetic separator rack and add 900 &#181;L of monocyte buffer for equilibration.</li>
	<li>Pour the cell suspension onto the filter and let it flow through into a waste tube by gravity.</li>
	<li>Wash the column, which contains the cells bound to the magnetic beads, by adding 1 mL of monocyte buffer.</li>
	<li>Prepare a fresh 50 mL tube containing 20 mL RPMI and replace the waste tube underneath the column.</li>
	<li>Add 3 mL of monocyte buffer to the column, remove it from the magnetic rack, and attach the stamp.</li>
	<li>Press the cells into the prepared 50 mL tube and fill it up to 30 mL of volume with RPMI.</li>
	<li>Count the cells (e.g., using a Neubauer counting chamber) under a light microscope and adjust the cell number by adding RPMI to 2 x 106 cells/mL.</li>
	<li>Seed 1 mL of the cell suspension into each well of a 6-well chamber and incubate the plate for 2-4 h at 37 &#176;C and 5% CO2.</li>
	<li>Check if the cells adhered properly and replace the RPMI with 1.5 mL monomedium (20% human serum + 1% pen-strep in RPMI).</li>
	<li>Change the medium after 24 h of incubation. Under these conditions, monocytes differentiate into macrophages within 6 days.</li>
</ol>
3. Macrophage preparation
<ol>
	<li>At the day of the experiment, detach the macrophages with an appropriate amount of accutase for the individual culture conditions. For example, use 500 &#181;L of accutase if the cells are seeded in a 6-well dish. Incubate the cells for about 40 min within the solution, wash them once with 2 mL of DPBS, and count them with a Neubauer chamber.</li>
	<li>For one spheroid, dilute the desired number of macrophages in 40 &#181;L of collagen I mix (2.5 mg/mL rat tail collagen I) and mix them by shortly vortexing the reaction tube. In the experiments conducted, 200,000 cells/mL have shown a detectable difference on the invasion of cancer cells. Keep the collagen-cell-mix on ice, as it will start to polymerize quickly at room temperature (RT).</li>
</ol>
4. Setup of the co-invasion assay
NOTE: Depending on the format of the imaging chamber, the purpose of the analysis, or the respective imaging setup, the tumor spheroid might need to be transferred from the ultra-low adhesion plate and the values for collagen and media adapted. The following steps describe the setup for one sample in a 15-well &#181;-slide.
<ol>
	<li>First, wash the spheroid by adding 300 &#181;L of DPBS. Cut the tip of a 1 mL blue pipette tip and take up the spheroid with DPBS. After aspiration of the spheroid settled into the tip, briefly push the pipette to the bottom of the imaging chamber to transfer the tumor-spheroid by surface tension. 
	NOTE: It is important to note that the friction at unmodified tips might disturb the integrity of the spheroid.</li>
	<li>Remove residual DPBS transferred with the spheroid as much as possible by pipetting.</li>
	<li>Swiftly add 40 &#181;L of collagen I/macrophage mix (see step 3.4) to the imaging chamber that contains the H1299-GFP spheroid.</li>
	<li>Transfer the plate into a cell incubator and incubate the plate for 30 min at 37 &#176;C and 5% CO2 under humid conditions to fully polymerize the collagen mix.</li>
	<li>After polymerization, carefully add 25 &#181;L of cancer-cell media to each well and incubate the plate for 3 days in the incubator under the previously described conditions. If needed, image the live sample with a laser scanning microscope at desired time points.</li>
</ol>
5. Additional fixation and staining
<ol>
	<li>In order to perform immunofluorescence staining, fix the macrophages and H1299-GFP spheroids within the collagen I matrix. First, carefully remove the supernatant from each sample with a pump without touching the matrix. 
	NOTE: To avoid sample destruction, the supernatant can also be removed by manual pipetting without using a pump.</li>
	<li>Add 50 &#181;L of 3.7% formaldehyde in DPBS to each well and incubate the samples for 5 min on ice. Afterward, remove the fixative solution and add 50 &#181;L of fresh fixing solution again to it. This step is important to achieve a more uniform formaldehyde level within the well. Incubate the samples overnight at 4 &#176;C. 
	NOTE: Depending on the amount of collagen I mix, the incubation times might extend to several hours.</li>
	<li>Remove the fixative on the next day and wash the samples by repeated removal of at least two times DPBS depending on the volume of the imaging chamber. Briefly incubate them for 5 min on ice on a shaker before removing the DPBS by pipetting. The samples are now ready for staining.</li>
	<li>Stain the samples by adding a 1:100 mix of 4&#39;,6-diamidino-2-phenylindole (DAPI) (1 mg/mL, to stain nuclei), and phalloidin 568 (to stain F-actin) in DPBS for at least 30 min and wash them twice with DPBS before imaging.</li>
</ol>
6. Imaging and analysis of different parameters of tumor growth
NOTE: The following steps can be performed for either fixed live-cell imaging samples.
<ol>
	<li>For the observation of the tumor-spheroid, use an inverted laser scanning microscope equipped with a 10x objective that allows imaging of a z-stack of roughly the lower third of the spheroid. 
	NOTE: Due to the thickness of the sample, a 2-photon microscope might also be advantageous. For optimal identification of individual cells, higher laser power and exposure time might be needed.</li>
	<li>After the acquisition, combine the stacks by z-projection using Fiji, which is ImageJ software (Fiji). Further, process the resulting channel of the H1299-GFP signal by applying an auto threshold (e.g., Huang auto-threshold) and remove residual noise by despeckling the image depending on the amount of background and signal intensity.</li>
	<li>Measure the spheroid parameters using Fiji.
	<ol>
		<li>Area: Measure the spheroid size with the Wand tool.</li>
		<li>Perimeter: Include the Perimeter into the measurement settings.</li>
		<li>Diameter: Include the Feret&#39;s Diameter into the measurement settings.</li>
		<li>Circularity: Include the Shape Descriptors into the measurement settings.</li>
	</ol>
	</li>
	<li>Measure the individual cancer cell parameters using Fiji.
	<ol>
		<li>Measure the number of invading cells.
		<ol>
			<li>Choose the central spheroid with the Wand tool and delete the signal. This will remove the spheroid as well as all connected signals of cells, which are still attached to the spheroid, from the analysis.</li>
			<li>Afterwards, use the Analyze Particle tool or quantify all residual individual signals. 
			NOTE: The result will be the number of invading cancer cells. In addition, the result could also show the representative area covered by the cancer cells and be interpreted as collective invasion or single-cell invasion, depending on the size of the area. To further validate the number of invading cancer cells, a mask of the analyzed particles could be laid above the DAPI channel, and nuclei counted.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Further, evaluate the results using a statistical program such as GraphPad Prism or Microsoft Excel. 
	NOTE: This protocol has been shown to be both robust and flexible. It has been used in our lab more than 50 times, always with primary human macrophages from at least 3 donors, to account for donor variability. In its current form, the protocol has been shown to lead to a success rate of 90-95%. However, as the setup is complex, varying results might also be an outcome, as the interaction of macrophages for every spheroid is individual. Thus, performing the experiment at least 3 times with 5 spheroids is suggested to get a statistically significant result.</li>
</ol>

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

<ol>
	<li>Lendeckel, U., Venz, S., Wolke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages:+shapes+and+functions.">Macrophages: shapes and functions.</a> ChemTexts. 8 (2), 12 (2022).</li><li>Dallavalasa, 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=The+role+of+tumor+associated+macrophages+(TAMs)+in+cancer+progression,+chemoresistance,+angiogenesis+and+metastasis+-+Current+status.">The role of tumor associated macrophages (TAMs) in cancer progression, chemoresistance, angiogenesis and metastasis - Current status.</a> Curr Med Chem. 28 (39), 8203-8236 (2021).</li><li>Pan, Y., Yu, Y., Wang, X., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+macrophages+in+tumor+immunity.">Tumor-associated macrophages in tumor immunity.</a> Front Immunol. 11, 583084 (2020).</li><li>Boutilier, A. J., Elsawa, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Polarization+States+in+the+Tumor+Microenvironment.">Macrophage Polarization States in the Tumor Microenvironment.</a> Int J Mol Sci. 22 (13), 6995 (2021).</li><li>Wenzel, E. 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=Intercellular+transfer+of+cancer+cell+invasiveness+via+endosome-mediated+protease+shedding.">Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding.</a> Nat Commun. 15 (1), 1277 (2024).</li><li>Anfray, C., Ummarino, A., Calvo, A., Allavena, P., Torres Andon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+tumor-associated+macrophages+in+the+tumor+microenvironment.">In vivo analysis of tumor-associated macrophages in the tumor microenvironment.</a> Methods Mol Biol. 2614, 93-108 (2023).</li><li>Hey, S., Wiesner, C., Barcelona, B., Linder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIF16B+drives+MT1-MMP+recycling+in+macrophages+and+promotes+co-invasion+of+cancer+cells.">KIF16B drives MT1-MMP recycling in macrophages and promotes co-invasion of cancer cells.</a> Life Sci Alliance. 6 (11), e202302158 (2023).</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>Shi, X., 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=Mechanism+insights+and+therapeutic+intervention+of+tumor+metastasis:+latest+developments+and+perspectives.">Mechanism insights and therapeutic intervention of tumor metastasis: latest developments and perspectives.</a> Signal Transduct Target Ther. 9 (1), 192 (2024).</li><li>Li, G. N., Livi, L. L., Gourd, C. M., Deweerd, E. S., Hoffman-Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+morphological+changes+of+neuroblastoma+cells+in+response+to+three-dimensional+matrices.">Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices.</a> Tissue Eng. 13 (5), 1035-1047 (2007).</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 (2020).</li></ol>

The authors declare that no competing interests exist.

The invasion of cancer cells remains an important yet understudied topic in the context of co-invading immune cells like macrophages. Collective and individual cancer cell invasion are critical processes during metastasis and have been shown to lower the survival rate of cancer patients due to the multiplicity of infestations of different organs8,9. In vivo studies are complex and are restricted to laboratories with access to animal housing facilities. Moreover, it is also difficult to control in vivo conditions or to specifically manipulate individual aspects. Therefore, the need for a more accessible system remains essential to answer basic questions in the first line of research.
With the method presented here, we developed a way in which i) immune cell and ii) cancer cell behavior, iii) growth and development of a solid spheroid, and iv) the impact of cells in the surrounding tumor microenvironment (TME) on ECM components can be further analyzed. It can be used to compare the impact of modified (e.g., depleted for specific regulators by siRNA treatment) macrophages on the invasiveness of individual cancer cells from a solid spheroid. Whether the physical rearrangement of the TME or secreted factors represents the main cause of the observed cancer cell behavior is currently unclear and needs to be analyzed in more detail.
In addition, also the cancer cells themselves could be manipulated, for example by siRNA treatment or knockout of specific regulators. Moreover, the analysis can be improved by comparing the number of counted nuclei within the identified cell profile to allow for a more precise determination of invading cancer cells.
We have used this assay to determine the impact of macrophages in the TME on the invasion of tumor cells. However, it should also be noted that tumor cells are likely to influence the activity of macrophages, possibly through secreted factors within the media. It would thus also be a worthwhile endeavor to identify changes in the macrophages, such as altered polarization status (M1 vs. M2) by immunofluorescence staining using respective antibodies. In the past, the comparison between cells growing in monolayers and those cultured in a 3D environment has shown significant differences in their expression profiles10.
In addition, fluorescence-activated cell sorting (FACS) of macrophage subpopulations and their integration into the experimental procedure could be instructive. Last but not least, more detailed imaging of the contact areas where macrophages interact with cancer cells or the tumor spheroid surface could lead to the identification of further mechanisms relevant to 3D invasion and interaction.
Of note that can not be controlled fully is the precise positioning of the spheroid in the center of the well after the addition of the collagen-mix. The amount of collagen underneath the spheroid is especially hard to regulate. Here, other methods have been established to allow for precise positioning of the spheroid, e.g., on top of agarose molds with higher sample numbers11. However, as the release of cytokines is a common mechanism, all spheroids within this multi-spheroid assay are exposed to secreted factors, and the number of immune cells acting on a single spheroid is hard to control.
One basic limitation of this protocol is the ability of cancer cell lines to form uniform spheroids, thus being only applicable to a subset of cell lines. For example, MeWo melanoma cells form uneven, sheet like 3D structures but no uniform spheroids.
It should also be noted that the assay is highly adaptable, as many of its features can be modified, such as the ECM material, the cell number or by adding specific factors such as cytokines to the supernatant. It should, therefore, be highly suitable for initial in vitro studies of the cancer cell/immune cell interaction and can be tailored to the specific research question that is currently addressed.

Macrophages are a major part of the innate immune system and represent the first line of defense in many pathological conditions, such as infections or the clearing of cell debris after injuries1. During the last decades, the impact of immune cells on the progression of cancer has been an aspect of many studies. Accordingly, it has been shown that macrophages can facilitate metastasis by associating with primary tumors and becoming tumor-associated macrophages (TAMs)2. Macrophages can change their expression profile when exposed to cancer cells, favoring the escape of metastasis from the immune system3. In addition, it has been shown that cancer cells can utilize defects in the extracellular matrix (ECM) generated by macrophages to escape from the primary tumor and that their behavior is also manipulated by the uptake of secreted factors, including extracellular vesicles (EVs)4,5. This interplay of physical and chemical aspects requires the development of new methods to characterize the impact of immune cells on tumor spreading and the surrounding microenvironment.
Different approaches have been developed to study immune cell behavior in the context of metastasis in vivo6. However, as for all experiments&#160;including animals, a license for animal testing and an animal facility is needed; these approaches already demand a lot of development and preparation. Furthermore, the analysis is often complicated, especially in regard to live cell imaging, as not all specimens are accessible for most microscopical setups. The development of new methods to test hypotheses first in qualified in vitro conditions is also necessary and timely, as science and society aim for animal-free research to reduce the number of sacrificed animals.
In a recent publication7, we investigated the impact of primary human macrophages on the invasiveness of invading cancer cells from a tumor spheroid. For this purpose, we established a collagen I-based macrophage-tumor spheroid co-invasion assay. In this context, we aimed to elucidate the role of fast recycling pathways of the membrane-type 1 matrix metalloproteinase (MT1-MMP) in macrophages. We tested macrophages that were depleted for the super processive kinesin KIF16B, which we identified as&#160;a major driver of MT1-MMP recycling, and their impact on invasive capabilities of H1299- Green Fluorescent Protein (GFP) cells from a solid spheroid. KIF16B-depleted macrophages show reduced levels of the membrane-bound MT1-MMP on their surface, while the H1299 cells themselves were untreated.
To our knowledge, no similar method has been described that allows for full imaging and analysis of macrophage-tumor-spheroid co-invasion. Although we focused in our recent publication7 on the analysis of individually migrating cells distant to the spheroid, the assay allows for multiple further investigations such as the number of invading strands, proteins involved in cancer cell-macrophage interaction, amount of collagen I degradation or other spheroid properties like the length of its perimeter.

<table><tbody><tr><td>15 &amp;micro;-Slide Angiogenesis&amp;nbsp;</td><td>ibidi</td><td>81506</td><td></td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>00-4555-56</td><td></td></tr><tr><td>Alexa Fluor 568 Phalloidin&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A12380</td><td></td></tr><tr><td>CD14 MicroBeads</td><td>Miltenyi Biotec</td><td>130-050-201</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Binder</td><td></td><td></td></tr><tr><td>Collagen I Rat Tail&amp;nbsp;</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>DAPI</td><td>AppliChem</td><td></td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>DPBS</td><td>Anprotec</td><td>MS01Y71003</td><td></td></tr><tr><td>FBS</td><td>Bio&amp;amp;Cell</td><td>FBS. S 0613</td><td></td></tr><tr><td>Fiji&amp;nbsp;</td><td>NIH</td><td>ImageJ 2 Version: 2.3.0/1.53s</td><td></td></tr><tr><td>Formaldehyde 37%&amp;nbsp;</td><td>252549-500ml</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>H1299-GFP cell line</td><td></td><td></td><td></td></tr><tr><td>Human serum albumin</td><td>Sigma-Aldrich</td><td>A5843</td><td></td></tr><tr><td>Leica TCS SP8 X</td><td>Leica</td><td></td><td></td></tr><tr><td>l-glutamine&amp;nbsp;</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Lymphocyte Seperation Medium (LSM) 1077</td><td>PromoCell</td><td>C-44010</td><td></td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>nonessential amino-acids&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11140050</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Gibco</td><td>81275-034</td><td></td></tr><tr><td>TC-Platte 96 Well, BIOFLOAT, R&amp;nbsp;</td><td>SARSTEDT</td><td>83,39,25,400</td><td></td></tr><tr><td>VORTEX-GENE 2&amp;nbsp;</td><td>Scientific Industries</td><td></td><td></td></tr></tbody></table>

a macrophage-tumor spheroid co-invasion assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Figure 1&#160;shows an H1299-GFP spheroid imaged at the respective days of incubation in a collagen I matrix. A respective brightfield image, taken at day 0, also shows the primary human macrophages cocultured with the spheroid. The representative image recorded on day 3 of the experiment is enlarged after processing. Different parameters that can be analyzed are indicated, including the number of invading cells, sites of collective invasion, and spheroid perimeter. The table included shows the results gained from this image. The number of detected signals matches the visual impression. No disruptions of the central spheroid are visible, indicating actual individual cancer cell invasion rather than cells derived from spheroid debris. The results of different spheroids and conditions can now be compared via statistical analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67374/67374fig01.jpg" /> 
Figure 1: Representative results. (Top panel) H1299-GFP spheroid imaged at day 0, day 1, day 2, and day 3 of incubation in a collagen I matrix. (Middle left panel) A brightfield image of the spheroid showing the cocultured primary human macrophages. (Middle right panel) A representative image acquired at day 3 enlarged after processing, showing the different parameters that can be analyzed (spheroid perimeter in red; traced manually for better visualization). (Bottom panel) The results obtained from the image on the middle right panel. Note: Macrophages are not visible, as only the GFP channel was recorded to elucidate the cancer cell behavior. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67374/67374fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

A Macrophage-Tumor Spheroid Co-Invasion Assay

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

a-macrophage-tumor-spheroid-co-invasion-assay

Nanyang Technological University

Research

JoVE Journal

Cancer Research

449 Views.  University Medical Center Hamburg-Eppendorf. Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion. 

Video: A Macrophage-Tumor Spheroid Co-Invasion Assay

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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

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

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

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

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 ...

A Macrophage-Tumor Spheroid Co-Invasion Assay

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Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A 3D Organotypic Melanoma Spheroid Skin Model

A Spheroid Killing Assay by CAR T Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

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

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

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