Name: characterization of the effects of migrastatic inhibitors on 3d tumor spheroid invasion by high-resolution confocal microscopy
Uploaded: 2019-09-16T13:45:00.000+00:00
Duration: 9 min 17 s
Description: 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

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>

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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><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 4 &amp;deg;C, 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.&amp;nbsp;&amp;nbsp;&amp;nbsp;</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;&amp;nbsp; 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 &amp;mu;m) 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&amp;nbsp;</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&amp;nbsp;</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, 3 mL)</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&amp;nbsp;&amp;nbsp;</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, 5 mL 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 &amp;mu;L) and single channel pipettes (10 &amp;mu;L, 50 &amp;mu;L, 200 &amp;mu;L 1 mL)</td><td>various</td><td>various</td><td>for cell and spheroid handling</td></tr><tr><td>Widefield microscopy</td><td>various&amp;nbsp;</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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

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

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

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

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 ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Bioengineering

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 ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

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

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Spheroid Assay to Measure TGF-β-induced Invasion

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

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

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

A 3D Spheroid Model for Glioblastoma

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model