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 initi...

<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>

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...

Watch this Scientific Journal Video about Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy at JoVE.com

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 ...

This protocol is demonstrating that we can now determine the effect of migrastatic inhibitors in detail in 3D cancer models. The main advantage is the ease and all-in-one approach with which this technique can be carried out. This technique allows the assessment of migrastatic drugs in cancer research in a 3D setting, allowing the testing of drugs in a more tumor environment-relevant setting.This method may be applied to testing of cytotoxic drugs in cancer or, for example, to assess the effect of radiation on cancer proliferation and cell migration. It is useful to practice the medium removal step and collagen replacement with ordinary 96-well plates to become confident. Also, training on a confocal microscope is essential.Visual demonstration of this technique is critical for researchers to understand the required delicate handling of the spheroids and their use in confocal microscopy. On the first day of experimentation, have the standard culture medium, in this case, U251, ready for the appropriate cell line. In a tissue culture hood, add 0.5 milliliters of trypsin into the culture to trypsinize the cancer cells.Count the cancer cells on a hemocytometer, and dilute to a concentration of five times 10 to the third cells per milliliter. Keep the cell suspensions in clearly labeled, sterile universal tubes. Resuspend the cells by gentle inversion to avoid cell clumping.Use a pipette to add 200 microliters of the cell culture to each well in a 96-well plate. Add 200 microliters of 1X PBS to each empty well to avoid evaporation. Incubate the cells in an incubator at 37 degrees Celsius.After 24 hours, check the cells by bright-field microscopy. Cell lines such as glioma cancer cell lines form spheroids in the bottom of the well within 24 hours. Incubate the spheroids for another 48 hours to allow a 3D cellular architecture to form.On the third day, place collagen, 5X culture medium, one-molar sodium hydroxide, and one 20-milliliter tube on ice. Carefully and slowly add 10.4 milliliters of cold collagen into the chilled culture tube. Avoid bubbles.Then, gently add 1.52 milliliters of cold sterile 5X culture medium. Avoid bubbles. Just before use, gently add 72 microliters of cold sterile one-molar sodium hydroxide, and keep the solution on ice.Mix gently by pipetting and avoiding bubbles. Efficient mixing leads to a color change from red to orange-red in the medium. Leave the mixture on ice until use.Next, to remove 190 microliters of supernatant from the 96-well plate prepared on day one, use a pipette at an angle towards the side of the well. Be very careful not to disturb the spheroids that formed in the bottom of the well. Gently add 100 microliters of the collagen mix to each well, pipetting down the side of the well.Avoid bubbles and any spheroid disturbance. Keep any remaining collagen mix in the 20-milliliter tube at room temperature to assess polymerization. Incubate the plate in the incubator for at least 10 minutes to allow the collagen to polymerize.When the leftover collagen becomes semisolid and sponge-like, the spheroids are ready to be treated with inhibitor. Add the inhibitors at 2X concentration to five milliliters of culture medium. Pipette 100 microliters of the medium gently down the side of each well to avoid spheroid disturbance.Observe and image each spheroid by bright-field microscopy at times of zero hour, 24 hours, 48 hours, and 72 hours to assess drug activity. Then, return the plate to the incubator. Now, place the plate in a tissue culture hood, and gently replace the supernatant with 100 microliters of 1X PBS.Take care not to disturb the spheroid, and avoid touching the collagen. Repeat this wash step three more times. Remove the final wash in each well, and replace with 100 microliters of 4%formaldehyde in 1X PBS.Place the 96-well plate on a lab bench, cover with foil, and leave for 24 hours at room temperature. The next day, carefully remove the formaldehyde, and replace with 1X PBS. Repeat this wash three more times.Then, replace the 1X PBS wash with 100 microliters of freshly prepared Triton X-100 solution. Incubate for 30 minutes at room temperature. In the meantime, prepare the blocking solution with 50 milliliters of 1X PBS and 05%skimmed milk powder in a 50-milliliter tube, and mix thoroughly.After 30 minutes, remove the Triton X-100, and wash three times with 1X PBS. Add 100 microliters of the blocking solution to each well, and incubate for 15 minutes. Next, dilute anti-mouse IgG acetylated tubulin antibody in the blocking buffer at a ratio of one to 100.Centrifuge the primary antibody-blocking buffer mix for five minutes at 15, 682 times g. Carefully remove the blocking solution in each well, and add 25 to 50 microliters of the antibody blocking buffer supernatant from the tube. Incubate in the dark at room temperature for one hour.Then, remove the antibody solution from each well, and wash three times with 100 microliters of 1X PBS. Dilute secondary antibody in the blocking buffer at the recommended concentration in addition to any additional fluorescent stains. Again, centrifuge the secondary antibody solution for five minutes at 15, 682 times g.Remove the blocking solution from each well, and add 25 to 50 microliters of the secondary antibody supernatant. Incubate in the dark for 1 1/2 hours at room temperature. Remove the secondary antibody dye solution, and wash three times with 1X PBS, 100 microliters per well.Carefully lift individual collagen plugs by suction with a plastic 200-microliter pipette onto the center of a glass slide. Add one drop of a suitable mountant to cover the collagen plug. Position a coverslip on top of the sample, and store the slide at room temperature in the dark.Three-dimensional spheroid technology is advancing the understanding of drug-tumor interactions because it is more representative of the cancer-specific environment. In this study, potential anti-or pro-migratory effects were detected. This is especially noticeable in KNS42 with seemingly no migration in either the control or treated spheroids.Cell death was observed at the highest inhibitor concentration of 10 micromolar. Nuclear fragmentation and migrating cells were evident in U251 cells. U251 cell spheroids had spikes radiating away from the original spheroid, with increasing cell rounding in cells apparent with increasing inhibitor concentration.Collapsed microtubules and nuclear fragmentation were observed in KNS42. Sheetlike protrusions with single cell spikes indicate KNS42 migration. At the lowest inhibitor concentration, this phenotype was pronounced but was lost with increasing inhibitor concentration.It is crucial to leave all the reagents on ice until ready. Otherwise, the collagen starts polymerizing before it has all been added to the spheroids. There is scope to quantify the images generated by confocal microscopy to assess the effects of, for example, migrastatic drugs on cell morphology.It allowed us, for the first time, to confirm the effect of the migrastatic inhibitor MI-192 on microtubule acetylation levels and to explore this further in terms of cell migration. Extra care should be taken when handling formaldehyde for the fixation step. The usual health and safety guidelines apply.

characterization-effects-migrastatic-inhibitors-on-3d-tumor-spheroid

Research

JoVE Journal

Cancer Research

6.7K Views.  University of Huddersfield. Questo protocollo sta dimostrando che ora possiamo determinare l'effetto degli inibitori emicrastatici in dettaglio nei modelli di cancro 3D. Il vantaggio principale è la facilità e l'approccio all-in-one con cui questa tecnica può essere eseguita. Questa tecnica consente la valutazione dei farmaci emicrastatici nella ricerca sul cancro in un ambiente 3D, consentendo il test di farmaci in un ambiente più rilevante per l'ambiente tumorale.Questo metodo può essere applicato alla spe...