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

416 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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

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

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

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

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

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

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 ...

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 ...

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 ...

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/60998/a-3d-spheroid-model-for-glioblastoma'>Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)</a>The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)The video demonstrates the generation of a three-dimensional ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

A Three-Dimensional Co-culture Model of Infiltrating T Cells in Tumor Spheroids

Source: Lin, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61392">Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture.</a> J. Vis. Exp. (2020).
This video demonstrates a method to establish a three-dimensional tumor spheroid and T cell co-culture. An agarose cast with microwells containing tumor spheroids is prepared, followed by the addition of T cells. After incubation, this setup allows studying the interaction of T cells with tumor spheroids in a controlled three-dimensional environment.

1. Generation of spheroids

	Prepare and autoclave 2% agarose in 1x phosphate-buffered saline (PBS) (e.g., 1 g agarose in 50 mL of 1x PBS) and autoclave ...