We would like to thank Professor Chris Jones for contributing the KNS42 cell line. The Zeiss LSM880 confocal microscope with AiryScan used in this work is part of the Huddersfield Innovation and Incubation Project (HIIP) funded through the Leeds City Region Enterprise Partnership (LEP) Growth Deal. Credit for microscope image Figure 3: Carl Zeiss Microscopy GmbH, microscopy@zeiss.com.

1. Generation of Cell Spheroids
Day 1
<ol>
	<li>Prepare the standard culture medium as required by the cell line under investigation.</li>
	<li>Carry out all tissue culture-associated steps in a tissue culture hood using sterile handling techniques.</li>
	<li>Trypsinize and count cancer cells. Use 20 mL of cell suspension per plate. Keep the cell suspensions in clearly labeled sterile universal tubes.</li>
	<li>Add a predetermined number of cells to each well. Both the initial number of cells and ultimate spheroid size required depends upon the proliferation rate of the cell line being investigated. 
	NOTE: For established glioma cell lines such as U251 and KNS429,10, 5 x 103 cells/mL will produce a microscopically visible spheroid (200 or 800 &#181;m) after 4 days of incubation.</li>
	<li>Resuspend the cells in universal tubes by gentle inversion to avoid cell clumping. Pipette 200 &#181;L of the cell suspension into each well of a 96-well plate. If all wells are not required, it is advisable to add 200 &#181;L of 1x PBS to each empty well to avoid evaporation.</li>
	<li>Incubate the cells in an incubator as normal at 37 &#176;C. 
	NOTE: Cell lines such as glioma cancer cell lines will form spheroids within 24 h. Allow 3D cellular architecture to form by incubating the spheroid for 72 h.</li>
</ol>
Day 2
<ol start="7">
	<li>Check cells by bright-field microscopy after 24 h. Depending upon the cell line, cells may have formed a spheroid detectable in the bottom of the well. 
	NOTE: Established glioma cancer cell lines readily form spheroids within 24 h. Patient-derived glioma cancer cell lines may take up to 1-2 weeks.</li>
</ol>
2. Collagen Invasion Assay
Day 3
<ol>
	<li>Place collagen, 5x culture medium, 1 M NaOH, and one 20 mL tube on ice.</li>
	<li>Carefully and slowly add 10.4 mL of cold collagen into a chilled culture tube. Avoid bubbles. This quantity of collagen is enough for one 96-well plate. Upscaling is possible, but it is recommended that one 20 mL tube per plate is prepared at a time.</li>
	<li>Gently add 1.52 mL of cold sterile 5x culture medium. Avoid bubbles.</li>
	<li>Just before use, gently add 72 &#956;L of cold sterile 1 M NaOH. Keep solution on ice.</li>
	<li>Mix gently by pipetting. Avoid bubbles. Efficient mixing leads to a color change (from red to orange-red (pH 7.4) in the medium. Leave the mixture on ice until use.</li>
	<li>Crucial step: Remove 190 &#956;L of supernatant from the 96-well plate prepared on day 1. Be very careful not to disturb the spheroids that formed in the bottom of the well. Use the pipette at an angle towards the side, not the center, of the well.</li>
	<li>Gently add 100 &#956;L of the collagen mix to each well. To prevent any spheroid disturbance, pipette the mix down the side of the well. Avoid bubbles. Keep any remaining collagen mix in the 20 mL tube at room temperature to assess polymerization.</li>
	<li>Incubate plate in the incubator for at least 10 min to allow the collagen to polymerize. As a guideline, if the leftover collagen has set, becoming semisolid and sponge-like, the spheroids are ready to be treated with inhibitor.</li>
	<li>Add the drugs or inhibitors at 2x concentration to the culture medium. Add the medium gently to each well (100 &#181;L per well). Again, pipette the medium down the side of the well to avoid spheroid disturbance.</li>
	<li>Observe and image each spheroid by bright-field microscopy at times T = 0 h, 24 h, 48 h, and 72 h to assess drug activity. Then return the plate to the incubator. 
	NOTE: Depending on the invasive behavior of the cell line, migration away from the original spheroid core may be observed from 24 h onwards.</li>
</ol>
3. Preparation of Collagen Embedded Spheroids and Migratory Cells for Confocal Microscopy
<ol>
	<li>Place the plate in a tissue culture hood and gently remove the supernatant (200 &#181;L). Again, take care not to disturb the spheroid and avoid touching the collagen, as this may interfere with the collagen plug.</li>
	<li>Replace the supernatant with 100 &#181;L of 1x PBS. Repeat this wash step 3x.</li>
	<li>Remove the final wash and replace with 4% formaldehyde in 1x PBS (100 &#181;L per well). 
	CAUTION: Formaldehyde is a potential carcinogen. Handle with care in accordance with health and safety guidelines.</li>
	<li>Place the 96-well plate on a lab bench, cover with foil, and leave for 24 h at room temperature.</li>
	<li>Carefully remove the formaldehyde and replace with 1x PBS. Repeat this 1x PBS wash 3x.</li>
	<li>Prepare 0.1% Triton X-100 in 1x PBS. Remove the 1x PBS wash and replace with 100 &#181;L of the Triton X-100 solution. Incubate for 30 min at room temperature. In the meantime, prepare the blocking solution with 1x PBS and 0.05% skimmed milk powder and mix thoroughly.</li>
	<li>Remove Triton X-100 and wash 3x with 1x PBS. Add 100 &#181;L of blocking solution to each well and incubate for 15 min.</li>
	<li>Dilute the required primary antibody in blocking buffer at the predetermined concentration. Here, use anti-mouse IgG acetylated tubulin antibody (1:100).</li>
	<li>Centrifuge the primary antibody-blocking buffer mix for 5 min at 15,682 x g. Carefully remove the blocking solution and add the supernatant (25&#8722;50 &#181;L) to each well. Incubate in the dark at room temperature for 1 h.</li>
	<li>Remove the antibody solution and wash 3x with 1x PBS (100 &#181;L per well).</li>
	<li>Dilute the secondary antibody in the blocking buffer at the recommended or predetermined concentration in addition to any additional fluorescent stains. Here, use 1:500 anti-mouse fluorophore-488 conjugated antibody, phalloidin-594 (1:500) for actin staining, and the DNA stain (DAPI).</li>
	<li>Again, centrifuge the secondary antibody solution for 5 min at 13,000 rpm.</li>
	<li>Remove the blocking solution from each well and add 25&#8722;50 &#181;L of the secondary antibody/phalloidin/DAPI mix. Incubate in the dark for 1.5 h at room temperature.</li>
	<li>Remove secondary antibody-dye solution and wash 3x with 1x PBS (100 &#181;L per well).</li>
	<li>Carefully lift individual collagen plugs by suction with a plastic pipette (200 &#181;L) onto the center of a high-quality plain glass slide.</li>
	<li>Add one drop of a suitable mountant to the collagen plug, ensuring the plug is completely covered. Avoid bubbles.</li>
	<li>Apply coverslip of the optimal thickness for the microscope objective that will be used for imaging and allow to set overnight. Store the slides at room temperature in the dark.</li>
</ol>
4. Fluorescence Microscopy
<ol>
	<li>Capture fluorescent images using a suitable confocal microscope.</li>
</ol>

<ol>
	<li>Gandalovi&#269;ov&#225;, A., 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=Migrastatics-anti-metastatic+and+anti-invasion+drugs:+promises+and+challenges.">Migrastatics-anti-metastatic and anti-invasion drugs: promises and challenges.</a> Trends in Cancer. 3 (6), 391-406 (2017).</li><li>Delgado-L&#243;pez, P. D., Corrales-Garc&#237;a, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+in+glioblastoma:+a+review+on+the+impact+of+treatment+modalities.">Survival in glioblastoma: a review on the impact of treatment modalities.</a> Clinical and Translational Oncology. 18, 1062 (2016).</li><li>Foreman, P. 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=Oncolytic+virotherapy+for+the+treatment+of+malignant+glioma.">Oncolytic virotherapy for the treatment of malignant glioma.</a> Neurotherapeutics. 14, 333 (2017).</li><li>Rodrigues, T., 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=Emerging+tumor+spheroids+technologies+for+3D+in+vitro+cancer+modelling.">Emerging tumor spheroids technologies for 3D in vitro cancer modelling.</a> Pharmacology and Therapeutics Technologies. 184, 201-211 (2018).</li><li>Sirenko, O., 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=High-content+assays+for+characterizing+the+viability+and+morphology+of+3D+cancer+spheroid+cultures.">High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.</a> Assay and Drug Development Technologies. 13 (7), (2015).</li><li>Wu, Q., 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=Bionic+3D+spheroids+biosensor+chips+for+high-throughput+and+dynamic+drug+screening.">Bionic 3D spheroids biosensor chips for high-throughput and dynamic drug screening.</a> Biomedical Microdevices. 20, 82 (2018).</li><li>Mosaad, E., Chambers, K. F., Futrega, K., Clements, J. A., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microwell-mesh:+a+high-throughput+3D+prostate+cancer+spheroid+and+drug-testing+platform.">The microwell-mesh: a high-throughput 3D prostate cancer spheroid and drug-testing platform.</a> Scientific Reports. 8, 253 (2018).</li><li>Jonkman, J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+way+you+slice+it-a+comparison+of+confocal+microscopy+techniques.">Any way you slice it-a comparison of confocal microscopy techniques.</a> Journal of Biomolecular Techniques. 26 (2), 54-65 (2015).</li><li>Pont&#233;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+human+glia+cells+in+culture.">Neoplastic human glia cells in culture.</a> Human Tumor Cells in Vitro. , 175-206 (1975).</li><li>Takeshita, I., 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=Characteristics+of+an+established+human+glioma+cell+line,+KNS-42.">Characteristics of an established human glioma cell line, KNS-42.</a> Neurologica Medico Chirurgica. 27 (7), 581-587 (1987).</li><li>Bance, B., Seetharaman, S., Leduc, C., Bo&#235;da, B., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+acetylation+but+not+detyrosination+promotes+focal+adhesion+dynamics+and+cell+migration.">Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and cell migration.</a> Journal of Cell Science. 132, (2019).</li><li>Bacon, T., 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=Histone+deacetylase+3+indirectly+modulates+tubulin+acetylation.">Histone deacetylase 3 indirectly modulates tubulin acetylation.</a> Biochemical Journal. 472, 367-377 (2015).</li><li>Jackman, L., 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=Tackling+infiltration+in+paediatric+glioma+using+histone+deacetylase+inhibitors,+a+promising+approach.">Tackling infiltration in paediatric glioma using histone deacetylase inhibitors, a promising approach.</a> Neuro-Oncology. 20, i19-i19 (2018).</li><li>Bhandal, K., 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=Targeting+glioma+migration+with+the+histone+deacetylase+inhibitor+MI192.">Targeting glioma migration with the histone deacetylase inhibitor MI192.</a> Neuro-Oncology. 19, i12-i12 (2017).</li><li>Del Duca, D., Werbowetski-Ogilvie, T. E., Del Maestro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+preparation+from+hanging+drops:+characterization+of+a+model+of+brain+tumor+invasion.">Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion.</a> Journal of Neuro-oncology. 67 (3), 295-303 (2004).</li><li>Bender, B. F., Aijian, A. P., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+spheroid-based+invasion+assays.">Digital microfluidics for spheroid-based invasion assays.</a> Lab on a Chip. 16 (8), 1505-1513 (2016).</li><li>Vinci, 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=Advances+in+establishment+and+analysis+of+three+dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biology. 10, 29 (2012).</li><li>H&#228;rm&#228;, V., 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=Quantification+of+dynamic+morphological+drug+responses+in+3D+organotypic+cell+cultures+by+automated+image+analysis.">Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis.</a> PLOS ONE. 9 (5), e96426 (2014).</li></ol>

The authors declare no conflict of interest.

A novel way to create cancer cell spheroids for identification of migrastatic drug activity using high-resolution confocal microscopy is described. The use of low adherent plates over other techniques, such as hanging drops15, has facilitated a means of generating reproducible and uniform spheroids for use in the collagen migration and invasion assays. The critical points in this protocol are the removal of growth medium from the 96-well plate prior to the cell spheroid embedding in a collagen mat...

Three dimensional spheroid technologies for preclinical drug discovery and the development of potential cancer drugs are increasingly being favored over conventional drug screens; thus, there is more development of migrastatic &#8212; migration and invasion preventing &#8212; drugs. The rationale behind these developments in cancer treatment are clear: 3D spheroid assays represent a more realistic approach for screening potential anti-cancer drugs as they mimic 3D tumor architecture more faithfully than cell monolayer cultures, recapitulate drug-tumor interactions (kinetics) more accurately, and allow the characterization of drug activity in a tumor-related setting. In addition, the rise of resistance to chemotoxic drugs in many cancer types and high death rates among cancer patients due to metastasis potentiated by the ability of cancer cells to migrate to distant tumor sites supports the inclusion of chemotherapeutic agents targeting the migratory potential of cancer cells as adjuvant treatment in future clinical cancer trials1. This is particularly the case in highly invasive cancers, such as high-grade glioblastomas (GBM). GBM management includes surgery, radiotherapy, and chemotherapy. However, even with combination treatment, most patients relapse within 1 year of initial diagnosis with a median survival of 11-15 months2,3. Huge advances in the field of 3D technology have been made over the last few years: rotative systems, microfabricated structures and 3D scaffolds, and other individual assays are being continually improved to allow routine testing on a large scale4,5,6,7. However, results obtained from these assays must be analyzed in a meaningful manner because data interpretation is often hindered by attempts to analyze 3D-generated data with 2D image analysis systems.
Despite being preferable in terms of image acquisition speed and reduced photo-toxicity, most wide-field systems remain limited by resolution8. Thus, apart from data read-outs relating to drug efficacy, detailed effects of drug action on 3D cellular structures of migrating cells are inevitably lost if imaged using a wide-field system. Conversely, confocal laser scanning microscopy (CLSM) captures high quality, optically sectioned images that can be reconstructed and rendered in 3D post-acquisition using computer software. Thus, CLSM is readily applicable to imaging complex 3D cellular structures, thereby enabling interrogation of the effects of anti-migrastatic inhibitors on 3D structures and in-depth analyses of cell migration mechanisms. This will undoubtedly guide future migrastatic drug development. Here, a combined workflow of spheroid generation, drug treatment, staining protocol, and characterization by high-resolution confocal microscopy is described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>for glioma invasion assay; this is offered by many distributors/manufacturers and will need to be determined for both the type of assay intended and cell lines used. For glioma cancer cell lines Collagen rat tail type 1 ( e.g. Corning) is the preferred choice. Collagen should be stored at 4oC, in the dark, until required. It is not advisable to mix collagen from different batches as this may affect the consistency of the polymerized collagen.&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopy imaging</td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>needed at 5X concentration for collagen solution for glioma invasion assay;&#160; this may be purchased in powdered form, made up in double distilled water and, depending upon final composition of the growth medium, completed with any additives required. The complete 5X solution should be filtered through a syringe filter system (0.22 microns) before use.</td>
		</tr>
		<tr>
			<td>Foetal calf serum</td>
			<td>Sigma</td>
			<td>F7524-500ML</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopy imaging</td>
		</tr>
		<tr>
			<td>High glucose DMEM</td>
			<td>Gibco</td>
			<td>41965062</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Inhibitor</td>
			<td>Tocris</td>
			<td>various</td>
			<td>various - according to experimental design; inhibitors can be purchased from manufacturers such as Selleckchem and Tocris. These manufacturers offer detailed description of inhibitor characteristics, links to associated references and suggestion of working concentrations. As with all inhibitors, they may be potentially toxic and should be handled according to health and safety guidelines. Inhibitors are prepared as stock solutions as recommended by the manufacturer. As an example we used the migrastatic inhibitor MI-192 to demonstrate the use of such inhibitors. We have tested a range of migrastatic inhibitors in this way with comparable results.</td>
		</tr>
		<tr>
			<td>Mountant</td>
			<td>various</td>
			<td>various</td>
			<td>for microscopic imaging</td>
		</tr>
		<tr>
			<td>NaOH (1 M)</td>
			<td>various&#160;</td>
			<td>various</td>
			<td>NaOH can be either purchased at the required molarity or prepared from solid form. The prepared solution should be filter sterilized using a syringe filter system. One M NaOH is corrosive and care should be taken during solution preparation.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>various</td>
			<td>various</td>
			<td>for fixing spheroids and cells; make up at 4%, caution health hazard, ensure that health and safety regulations are adhered to for collagen solution for invasion assay</td>
		</tr>
		<tr>
			<td>Pastettes (graduated pipette, 3ml)</td>
			<td>various</td>
			<td>various</td>
			<td>for invasion assay, solution removal</td>
		</tr>
		<tr>
			<td>PBS, sterile for tissue culture</td>
			<td>Sigma</td>
			<td>D1408-500ML&#160;&#160;</td>
			<td>needed for cell culture of glioma cell lines and washes for staining</td>
		</tr>
		<tr>
			<td>Pen/strep (antibiotics)</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>needed for cell culture of glioma cell lines</td>
		</tr>
		<tr>
			<td>Primary antibody, secondary antibody, DAPI, Phalloidin</td>
			<td>various</td>
			<td>various</td>
			<td>there are many manufacturers for these reagents, for secondary labelled antibodies we recommend Alexa Fluor (Molecular Probes). Here we used for primary antibodies mouse anti-acetylated tubulin antibody (1/100, Abcam). For secondary antibodies we used 1/500 anti-mouse Alexa Fluor 488 conjugated antibody, Molecular Probes. For nuclear stain we used DAPI (many manufacturers) and the actin stain Phalloidin (many manufacturers) both used at recommended dilution of 1/500.</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>needed for collagen solution for glioma invasion assay at 5X concentration</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td>needed for collagen solution for glioma invasion assay at 5X concentration</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td>for trypsinisation</td>
		</tr>
		<tr>
			<td>Ultra low attachment plates</td>
			<td>Sigma/Nunc</td>
			<td>CLS7007-24EA</td>
			<td>for glioma invasion assay; a low adherent plate is required, with 96-well plates preferred to allow for large-scale screening of compounds under investigation. There are several low adherence plates commercially available; it is advisable to test a variety of plates for optimum spheroid generation. In our experience Costar Ultra Low Cluster with lid, round bottom, works best for the generation of spheroids from glioma cancer cells in terms of 100% spheroid formation and reproducibility. These plates were also successfully used for the generation of glioma spheroids from patient-derived material, bladder and ovarian cancer cells in our laboratory. In addition, stem or progenitor neurospheres can be used in these plates to facilitate the generation of standardized neurosphere-spheroids</td>
		</tr>
		<tr>
			<td>Stripettes (serological pipettes, sterile, 5ml and 10 ml)</td>
			<td>various e.g. Costar</td>
			<td>CLS4488-50; CLS4487-50</td>
			<td>for tissue culture and collagen preparation</td>
		</tr>
		<tr>
			<td>various multichannel (50 - 250 uL) and single channel pipettes (10 uL, 50 uL, 200 uL 1 mL)</td>
			<td>various</td>
			<td>various</td>
			<td>for cell and spheroid handling</td>
		</tr>
		<tr>
			<td>Widefield microscopy</td>
			<td>various&#160;</td>
			<td>various</td>
			<td>for observation of spheroid generation and spheroid imaging; here wide-field fluorescence images were captured using an EVOS FL cell imaging system (Thermo Fisher Scientific)</td>
		</tr>
		<tr>
			<td>Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective</td>
			<td>Zeiss</td>
			<td>quote from manufacturer</td>
			<td>Confocal images were captured using a Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective. Diode 405nm, 458/488/514 nm argon multiline and HeNe 594nm lasers were used to excite Phalloidin 594, Alexa Fluor 488, and DAPI respectively. For each image a single representative optical section were captured, with all settings, both pre- and post-image capture, maintained for comparative purposes. All images were subsequently processed using the associated Zen imaging software and Adobe Photoshop.</td>
		</tr>
	</tbody>
</table>

characterization of the effects of migrastatic inhibitors on 3d tumor spheroid invasion by high-resolution confocal microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

Three-dimensional spheroid technology is advancing the understanding of drug-tumor interactions because it is more representative of the cancer-specific environment. The generation of spheroids can be achieved in several ways; low adherence 96-well plates were used in this protocol. After testing several products from different manufacturers, the plates used here were chosen because they consistently performed best in terms of successful spheroid production and uniformity. The replacement...

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Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

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6.8K Views.  University of Huddersfield. The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Video: Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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 ...

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

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

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

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

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

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 ...

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 ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...