Name: analysis of cancer cell invasion and anti-metastatic drug screening using hydrogel micro-chamber array (hmca)-based plates
Uploaded: 2018-10-25T14:00:00.000+00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

This work is supported by the bequest of Moshe Shimon and Judith Weisbrodt.

1. HMCA Plate Embossing
NOTE: The complete process for the design and fabrication of polydimethylsiloxane (PDMS) stamp and HMCA imaging plate used in this protocol is described in detail in our previous articles15,16. The PDMS stamp (negative shape) is used to emboss the HMCA (positive shape) which consists of approximately 450 MCs per well (Figure 1A). As demonstrated in Figure 1B, each of the MCs has a shape of a truncated upside-down square-shaped pyramid (height: 190 &#181;m, small base area: 90 &#181;m x 90 &#181;m,&#160;and large base area:&#160;370 &#181;m x 370 &#181;m). The HMCA plate is used for the preparation and culturing of 3D tumor spheroids and thereafter, for invasion assay. Alternatively, a commercial stamp could be used for HMCA production.
<ol>
	<li>Prepare a solution of 6% low melting agarose (LMA). Insert the LMA powder and sterile phosphate buffered saline (PBS) (0.6 g of LMA per 10 mL of PBS) into a glass bottle with a magnetic stirrer. Place the bottle on a heating plate and stir the solution while heating to 80 &#176;C for a few hours until a uniform solution is achieved. Keep the solution at 70 &#176;C until use.</li>
	<li>Pre-heat the PDMS stamp to 70 &#176;C in the oven. Keep it warm until use. Alternatively, use a commercial stamp and follow the instruction.</li>
	<li>Place the commercial 6-well glass-bottom plates on a dry bath pre-heated to 75 &#176;C. Wait a few minutes until the plate is totally warm.</li>
	<li>Pour a drop (400 &#181;L) of pre-heated LMA onto the glass bottom of each one of the wells in the plate. Gently place the pre-heated PDMS stamp over the agarose drop. Incubate at room temperature (RT) for 5&#8211;10 min for pre-gelling and pre-cooling. Incubate at 4 &#176;C for 20 min to achieve full agarose gelation. Then, gently peel off the PDMS, leaving the agarose gel patterned with MCs. 
	NOTE: This step is done simultaneously in all the wells.</li>
	<li>Place the embossed plate in a light curing system equipped with ultraviolet (UV) lamp, and incubate for 3 min. 
	NOTE: Now the HMCA plate is UV sterilized.</li>
	<li>Fill the macro-wells with sterile PBS to ensure that they are kept humid, then cover the plate, wrap it with parafilm and store it at 4 &#176;C until use.</li>
	<li>Before use, empty PBS residuals from the HMCA plate. Place it in the biological hood.</li>
</ol>
2. Loading Cells and Spheroid Formation
<ol>
	<li>Collect the cells as follows: remove all medium from a 10 cm culture plate cell monolayer, wash twice with 10 mL of PBS to dispose of serum residuals, add 5 mL of trypsin (pre-warmed to 37 &#176;C) and incubate for 3 min in the incubator at 37 &#176;C. Then, shake the plate gently to ensure monolayer detachment, add 10 mL of complete medium (pre-warmed to 37 &#176;C) and pipette up and down until homogenous cell suspension is achieved. Centrifuge and wash the cell suspension with 15 mL of fresh medium, then, suspend at appropriate concentrations in fresh complete medium.</li>
	<li>Gently load the cell suspension (50 &#181;L, 150&#8211;360 x 103 cells/mL in medium, ~16&#8211;40 cells per MC) on top of the HMCA and allow the cells to settle by gravity for 15 min.</li>
	<li>Gently add 6&#8211;8 aliquots of 500 &#181;L fresh medium (total 3&#8211;4 mL) to the rim of the macro-well plastic bottom outside the ring and hydrogel array.</li>
	<li>Incubate HeLa cells for 72 h and MCF7 cells for 48 h at 37 &#176;C and 5% CO2, in a humidified atmosphere for the formation of spheroids.</li>
</ol>
3. Viability Detection by Propidium Iodide (PI) Staining
<ol>
	<li>Prepare a stock solution of PI (500 &#181;g of PI per 1 mL of PBS). Keep it at 4 &#176;C for about 6 months. Freshly prepare a dilution of 1:2,000 (0.25 &#181;g/mL), by the addition of 6 &#181;L of stock to 12 mL of complete medium without phenol red, for the 6-well HMCA plate (2 mL per well).</li>
	<li>Transfer the HMCA plate with 48-72 h spheroids, from the incubator to the biological hood. Remove all medium from the HMCA by leaning the tip end on the edge of the macro-well plastic bottom beside the hydrogel array, leaving the array tank filled with medium.</li>
	<li>Gently add 2 mL of PI dilution from Step 3.1 to each well, by leaning the tip end on the edge of the macro-well plastic bottom. Incubate the HMCA plate for 1 h at 37 &#176;C and 5% CO2, in a humidified atmosphere. Continue to Step 7. 
	NOTE: Other dyes could be used here as well, for example, Hoechst for nucleus staining, tetramethylrhodamine methyl ester (TMRM) for mitochondrial membrane potential staining, fluorescein diacetate (FDA) for live cells detection, Annexin V for apoptosis detection and others.</li>
</ol>
4. Collagen Mixture Preparation
<ol>
	<li>Prepare sterile double distilled water (DDW) by autoclave and a stock of 100 mL of 1 M NaOH filter-sterilized solution. Maintain the materials at RT, and the tips used for collagen mixture preparation frozen at -20 &#176;C, until use.</li>
	<li>10&#8211;20 min before use, place all intergrades including: sterile DDW, 1 M NaOH sterile solution, sterile 10x PBS, type I collagen from rat tail (stored at 4 &#176;C for 3&#8211; months) and a sterile tube into the ice bucket until totally cooled. Place the ice bucket inside the biological hood.</li>
	<li>Prepare 1,800 &#181;L of collagen mixture at a final concentration of 3 mg/mL, for 6-well HMCA invasion assay plate (300 &#181;L per well) as follows. Add the intergrades into the cooled tube in the following order: 515 &#181;L of DDW, 24.8 &#181;L of 1 M NaOH and 180 &#181;L of 10x PBS. Mix well by vortex and place back into the ice. Finally, add 1080 &#181;L of collagen to the mixture, vortex again and put back into ice.</li>
</ol>
5. HA and Collagen Mixture Preparation
<ol>
	<li>Dissolve 10 mg of HA in 2 mL of sterile DDW. Incubate the mixture at RT for a few minutes and vortex gently until the powder is totally dissolved. Store the stock solution at 4 &#176;C for up to two years.</li>
	<li>Right before use, place the HA stock solution in the ice bucket inside the biological hood.</li>
	<li>Prepare a 3 mg/mL collagen mixture as described in Step 4. Add 36 &#181;g (7.2 &#181;L) of HA into 1,800 &#956;L of ready to use collagen mixture, for a final concentration of 20 &#181;g/mL. Then, vortex again briefly and put back into ice.</li>
</ol>
6. ECM Mixture Addition
<ol>
	<li>Transfer the HMCA plate, with 48&#8211;72 h spheroids, from the incubator into the biological hood and place it on the ice. Incubate for 10 min until the plate is cooled. Pre-heat a solution of 1% LMA to 37 &#176;C in a water bath.</li>
	<li>Remove all medium from around the HMCA, by leaning the tip end on the edge of the macro-well plastic bottom beside the hydrogel array. Then, very carefully, remove all medium from the array tank with a fine tip/gel loading tip, by leaning the tip end on the array edge, gently and slowly sucking all medium out. 
	NOTE: After removing all medium from around the hydrogel array, the array tank is still filled with medium. This medium has to be removed very gently in order to avoid destruction of the hydrogel MC and dislocation of spheroids.</li>
	<li>Take 150 &#181;L of the collagen mixture or HA and collagen mixture (ECM mixture) with the pre-frozen fine tip and add into the array tank by attaching the tip to the array edge. Release the mixture slowly to avoid spheroid dislocation. Repeat this step with another aliquot of 150 &#181;L, for a total 300 &#181;L per well. Follow the same procedure for each well until all wells are filled. Then place the plate into the incubator for 1 h for the full gelation of the ECM.</li>
	<li>After the full gelation of ECM has been achieved, pipette 400 &#181;L of pre-warmed 1% LMA on top of the ECM gel. Cover the plate with its lid, incubate the plate at RT for 5&#8211;7 min and then for 2 min at 4 &#176;C, for agarose gelation. Finally, gently add 2 mL of complete medium by leaning the tip end on the edge of the macro-well plastic bottom. 
	NOTE: The agarose layer prevents the detachment of the collagen-gel matrix from the HMCA.</li>
</ol>
7. Invasion Assay Acquisition and Analysis
<ol>
	<li>Load the HMCA plate onto a motorized inverted microscope stage equipped with an incubator, pre-adjusted to 37 &#176;C, 5% CO2 and humidified atmosphere.</li>
	<li>Pre-determine the positions for image acquisition in each well so as to cover the whole array area, and use 10X or 4X objectives (this step is required for the image acquisition software used here, see the Materials Table below for details). Alternatively, use any image acquisition software to facilitate automatic scan of the entire required area, by choosing the&#160;&#34;Stich&#34; option.</li>
	<li>Acquire bright field (BF) images every 2&#8211;4 h for a total period of 24&#8211;72 h in order to follow the invasion of cells from the spheroid body into the surrounding ECM.</li>
	<li>Acquire fluorescent images for PI detection by using a dedicated fluorescent cube (excitation filter 530&#8211;550 nm, dichroic mirror 565 nm long pass and emission filter 600&#8211;660 nm).</li>
	<li>Export time-lapse BF/fluorescent images from the image acquisition software and save them in tagged image file format (TIFF) to a dedicated folder by using the &#34;Database&#34; tab in the toolbar and then the &#34;Export record files&#34; button. 
	NOTE: We developed a special in-house analysis code in MATLAB software that includes a designed graphic user interface (GUI) in which invasion analysis could be executed faster and easier. In this analysis code, the segmentation of the spheroids and invasion area is done semi-automatically using Sobel filter followed by the morphological operators Erosion and Dilation. After automatic segmentation of the areas, manual corrections were made where needed for better accuracy. After the segmentation completion, a set of parameters were automatically calculated by the software and exported to an excel file for further manipulation. The list of parameters are: basic spheroid sectional area at time = 0, invasion area of cells connected to spheroid body = main invasion area, area of cells separated from main invasion area, number of cells separated from main invasion area, average distance of invasion from the basic spheroid center of mass to the circumference of the main invasion area and separated cells and collective invasion distance parameter = d (d = &#8730; ((X2 - X1)2 + (Y2 - Y1)2) out of the X and Y spheroid center of mass coordinates, before (X1, Y1) and after (X2, Y2) collective invasion process). Alternatively, image analysis could be done with any other specialized software.</li>
	<li>Open the GUI and push the &#34;Load BF&#34; button. After the image is open, adjust the segmentation parameters (minimum size: &#62;1 and radius close: &#62;1) to achieve a precise segmentation of spheroid and its invasion area, then, press &#34;Region of interest (ROI) segmentation&#34; button for execution and a border will appear around each spheroid. If the automatic segmentation is incorrect, press the &#34;Delete&#34;&#160;or&#160;&#34;Add&#34; button and make the requested corrections manually. Repeat the same steps for subsequent images.</li>
	<li>Press the&#160;&#34;Tracking&#34; button to number the spheroids and the software will assign the same number for each spheroid in each of the time lapse images. Then, press the &#34;Measurement&#34; button, which will generate a spreadsheet with all parameters. Save this spreadsheet as an excel file for further use in results processing and statistics in excel software.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+concentration+within+a+3D+collagen+matrix+modulates+matrix+viscoelasticity,+but+not+fibroblast+response.">Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response.</a> Matrix Biology. 28 (6), 336-346 (2009).</li><li>Chanmee, T., Ontong, P., Itano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan:+A+modulator+of+the+tumor+microenvironment.">Hyaluronan: A modulator of the tumor microenvironment.</a> Cancer Letters. 375 (1), 20-30 (2016).</li><li>Zhao, Y., 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=Modulating+Three-Dimensional+Microenvironment+with+Hyaluronan+of+Different+Molecular+Weights+Alters+Breast+Cancer+Cell+Invasion+Behavior.">Modulating Three-Dimensional Microenvironment with Hyaluronan of Different Molecular Weights Alters Breast Cancer Cell Invasion Behavior.</a> ACS Applied Materials &amp; Interfaces. 9 (11), 9327-9338 (2017).</li><li>Wu, 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=A+novel+role+of+low+molecular+weight+hyaluronan+in+breast+cancer+metastasis.">A novel role of low molecular weight hyaluronan in breast cancer metastasis.</a> The FASEB Journal. 29 (4), 1290-1298 (2015).</li><li>Fisher, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+resistance,+high+molecular+weight+hyaluronic+acid,+and+longevity.">Cancer resistance, high molecular weight hyaluronic acid, and longevity.</a> Journal of cell communication and signaling. 9 (1), 91-92 (2015).</li></ol>

The authors declare that they have no competing financial interests.

It is well documented that living organisms, characterized by their complex 3D multicellular organization are quite distinct from the commonly used 2D monolayer cultured cells, emphasizing the crucial need to use cellular models which better mimic the functions and processes of the living organism for drug screening. Recently, multicellular spheroids, organotypic cultures, organoids and organs-on-a-chip have been introduced8 for the use in standardized drug discovery. However, the 3D multicellular model&#39;s great complexity significantly jeopardizes its robustness, parallelization and data analysis which are crucial for assay efficiency.
Quantitative evaluation of invasion capacity typically measures the movement of cells through hydrogel materials. In order to measure the tumor invasion using traditional micro-well plates, large numbers of cancer cells are seeded in round bottom plates and forced to aggregate by centrifugation21,22. These plates restrict cell/spheroid interaction in adjoining wells, while the round bottom obstructs the optical quality and complicates image acquisition. In other methods, individual cells randomly encapsulated within the hydrogel, grow and function in a 3D environment but do not necessarily develop into mature spheroids23,24. Moreover, complicated procedures for cell positioning within hydrogel are required, like magnetic force-based cell patterning25 or two-photon laser irradiation of bioactive hydrogels26.
The HMCA-based technology presented herein proves to be advantageous over the above methods: cell positioning, spontaneous aggregation and creation of spheroids from few dispersed cells can be easily achieved, enabling the use of valuable limited cellular sample (e.g., cancer stem-like cells or primary cells derived from patient specimens). The system has control over both the nature of the cells that compose the spheroids as well as over their exact spatial location during long term experiments. In addition, the flat bottom of the MC facilitates the generation of numerous spheroids on the same focal plan, hence rapid illumination and image acquisition of large areas via a wide field microscope is possible, eliminating the need for complicated time-consuming confocal microscopy. The system is easy to handle and its practicality feasible. By applying simple operational procedures, we achieved high occupancy of spheroid populations in which approximately 50% of the spheroids have comparable size and a reliable analysis of cancer cell invasion capacity. In addition, the effect of drugs on invasion capacity can be analyzed simultaneously with their cytostatic and/or cytotoxic effects by using high-content analysis approach together with HMCA device cultured with numerous spheroids. Moreover, in the HMCA, all cellular elements share the same space and medium, making it possible to investigate spheroid-spheroid interaction which is usually mediated via cell-secreted cytokines, chemokines, hormones and the ECM27. This cross-talk facilitates the survival and the function of individual cells/small cell clusters in macro-well volume.
A critical step in the execution of the protocol is the addition of ECM mixture on top of the spheroids and validating its proper polymerization. The spheroids will not show migratory behavior even if they are embedded within ECM, unless assembly and cross-linking occur, and appropriate collagen fibers are formed. The collagen fibers can be observed by BF illumination, and we advise checking the plate under a microscope to confirm the creation of collagen fibers at the end of this phase. Since the number of spheroids is dependent on initial cell concentration and the percent of occupied MCs, the array should be checked after initial cell loading, and if cell occupancy is not optimal, re-seeding of the same array may be performed. Indeed, the size and geometry of the HMCA described herein were designed to accommodate relatively small cell clusters (up to 150 &#181;m in diameter). This could be considered a limitation since designing a new type of array will be necessary for analyzing cell migration from larger cell structures.
The current platform utilizes minimal cell number and small reagent volume, while at the same time providing a large sample size (thousands of spheroids per plate). In order to analyze 12 different compounds for anti-metastatic activity, 2 single 6-well HMCA based plates would be required, yielding the results from about 5,000 spheroids, while at least fifty 96-well plates would be needed to achieve the same outcomes. The ability to analyze the migration at individual-object resolution in the HMCA provides a high level of statistical robustness and accuracy, enabling the study of structural and functional invasion heterogeneity in numerous spheroids.
This study uniquely demonstrates the collective invasion of breast cancer spheroid populations upon the exposure to DETA/NO. The cell clusters of MCF7 cells show amoeboid migratory phenotype, and quantitative changes in the morphology and location of each spheroid can be automatically attained. To the best of our knowledge, this is the first arrayed system that facilitates monitoring and analysis of collective invasion in numerous 3D structures. By analyzing the relative directions and the invasion distance for each spheroid, it is possible to study the mutual interaction between spheroids within the population. Additionally, the established influence of extracellular microenvironment factors on cancer cell behavior28 is illustrated here. Among these factors, the stiffness and composition of the microenvironment has been shown to strongly influence cell migration. HMCA technology provides a defined, biomimetic, in vitro platform, in which these properties of the ECM can be easily accessed and controlled. The stiffness of pure collagen type I hydrogel at the concentration of 3 mg/mL which was used in this study, is reported to be approximately 50-600 Pa29,30,31 and the addition of HA (20 &#181;g/mL; 0.4 wt %) did not change the stiffness of the hydrogel significantly32,33. Thus, the suppressive effect of medium molecular weight (MMW)-HA on the invasion of 3D HeLa spheroids which was demonstrated here is not due to stiffness changes. These results are in agreement with previous studies which showed a bimodal effect of HA on cancer cell invasion with high dependency on HA molecular weight34,35,36,37.
Future applications of the methods include side by side multi cell type culturing of tumor and stromal cells in different regions within the macro-well, and retrieval of specific spheroids for downstream molecular analysis.

Cancer death is attributed mainly to the dissemination of metastatic cells to distant locations. Many efforts in cancer treatment focus on targeting or preventing the formation of metastatic colonies and progression of systemic metastatic disease1. Cancer cell migration is a crucial step in the tumor metastasis process, thus, the research of the cancer invasion cascade is very important and a prerequisite to finding anti-metastatic therapeutics.
The use of animal models as tools for studying metastatic disease has been found to be very expensive and not always representative of the tumor in humans. Moreover, the extracellular microenvironment topology, mechanics and composition strongly affect cancer cell behavior2. Since in vivo models inherently lack the ability to separate and control such specific parameters which contribute to cancer invasion and metastasis, there is a need for controllable biomimetic in vitro models.
In order to metastasize to distant organs, cancer cells must exhibit migratory and invasive phenotypic traits which can be targeted for therapy. However, since most in vitro cancer models do not mimic the actual features of solid tumors3, it is very challenging to detect physiologically relevant phenotypes. In addition, the phenotypic heterogeneity that exists within the tumor, dictates the need for analyzing tumor migration at single-element resolution in order to discover phenotype-directed therapies, for instance, by targeting the metastasis-initiating cell population within heterogeneous tumors4.
The study of cell motility and collective migration is primarily conducted in monolayer cultures of epithelial cells on homogeneous planar surfaces. These conventional cellular models for cancer cell migration are based on the population analysis of individual cells invading through the membranes and ECM components5, but in such systems, the intrinsic differences between individual cells cannot be studied. Generating 3D spheroids either via scaffolds or in scaffold-free micro-structures is considered as a superior means to study the tumor cell growth and cancer invasion6,7,8. However, most 3D systems are not suitable to high throughput formats, and inter-spheroid interaction cannot usually be achieved since isolated individual spheroids are generated in each micro-well9. Recent studies involving the cancer migration are based on microfluidic devices3,10,11,12, however, these sophisticated microfluidic tools are difficult to produce and cannot be used for high throughput screening (HTS) of anti-invasive drugs.
Two main phenotypes, collective and individual cell migration, which play a role in tumor cells overcoming the ECM barrier and invading neighboring tissue, have been demonstrated13,14, each displaying distinct morphological characteristics, biochemical, molecular and genetic mechanisms. In addition, two forms of migrating tumor cells, fibroblast-like and amoeboid, are observed in each phenotype. Since both, invasion phenotypes and migration modes, are mainly defined by morphological properties, there is a need for cellular models that enable imaging-based detection and examination of all forms of tumor invasion and migrating cells.
The overall goal of the current method is to provide a simple and reproducible biomimetic 3D in vitro cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. Here, we introduce the HMCA-based 6-well imaging plate for the research of tumor invasion and anti-metastatic therapy. The technology enables the formation of large numbers of uniform 3D tumor spheroids (450 per well) in a hydrogel micro-chambers (MC) structure. Various ECM components are added to the spheroid array to enable the invasion of the cells into the surrounding environment. Invasion process is continuously monitored by short- and long-term observation of the same individual spheroids/invading cells and facilitates morphological characterization, fluorescent staining and retrieval of specific spheroids at any point. Since numerous spheroids share space and medium, interaction via soluble molecules between individual spheroids and their impact on one another is possible. Semi-automatic image analysis is performed by using in-house MATLAB code which enables faster and more efficient collection of large amount of data. The platform facilitates accurate, simultaneous measurement of numerous spheroids/invading cells in a time-efficient manner, allowing medium throughput screening of anti-invasion drugs.

<table><tbody><tr><td>6 Micro-well Glass Bottom Plates with 14 mm micro-well #1.5 cover glass</td><td>Cellvis</td><td>P06-14-1.5-N</td><td>Commercial glass bottom plates which are used for HMCA embossing</td></tr><tr><td>UltraPure Low Melting Point Agarose</td><td>Invitrogen</td><td>16520100</td><td>A solution of 6% agarose is warmed up to 80 &amp;deg;C before use, a solution of 1% agarose is warmed to 37 &amp;deg;C</td></tr><tr><td>Trypsin EDTA solution B</td><td>Biological Industries</td><td>03-052-1A</td><td>Warmed to 37 &amp;deg;C before use</td></tr><tr><td>DMEM medium, high glucose</td><td>Biological Industries</td><td>01-055-1A</td><td>Warmed to 37 &amp;deg;C before use</td></tr><tr><td>Special Newborn Calf Serum (NBCS)</td><td>Biological Industries</td><td>04-122-1A</td><td>Heat Inactivated</td></tr><tr><td>DPBS (10x), no calcium, no magnesium</td><td>Biological Industries</td><td>02-023-5A</td><td>Kept on ice before use</td></tr><tr><td>NaOH, anhydrous</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td>Used for the preparation of 1 M NaOH solution</td></tr><tr><td>Cultrex Type I collagen from rat tail, 5 mg/mL</td><td>Trevigen</td><td>3440-100-01</td><td>Kept on ice before use</td></tr><tr><td>Hyaluronic acid sodium salt</td><td>Sigma-Aldrich</td><td>H5542-10MG</td><td>Kept on ice before use</td></tr><tr><td>Fisetin</td><td>Sigma-Aldrich</td><td>F505-100MG</td><td>Added to the culture medium, invasion inhibitor</td></tr><tr><td>DETA/NO</td><td>Enzo Life Sciences</td><td>alx-430-014-m005</td><td>Added to the culture medium, nitric oxide donor</td></tr><tr><td>PI</td><td>Sigma-Aldrich</td><td>P4170</td><td>Used at very low concenrtation without the need for washing</td></tr><tr><td>Dymax 5000-EC UV flood lamp complete system with light shield &amp;amp; Dymax 400 Watt EC power supply</td><td>Dymax Corporation</td><td>PN 39823</td><td>Used for HMCA plate sterilization by UV</td></tr><tr><td>Inverted IX81 microscope</td><td>Olympus</td><td></td><td>Used for automatic image acquisition</td></tr><tr><td>Incubator for microscope</td><td>Life Imaging Services</td><td></td><td>Essential for time lapse experiments with image acquisition, pre adjusted to 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; and keeping a humidified atmosphere</td></tr><tr><td>Sub-micron motorized stage type SCAN-IM</td><td>Marzhauser Wetzlar GmbH</td><td></td><td>Used to predetermine image acquisition areas, for automatic image acquisition</td></tr><tr><td>14-bit, ORCA II C4742-98 cooled camera</td><td>Hamamatsu Photonics</td><td></td><td>Highly sensitive, used for imaging</td></tr><tr><td>Fluorescent filter cube for PI detection</td><td>Chroma Technology Corporation</td><td></td><td>Filter cube specifications: excitation filter 530-550 nm, dichroic mirror 565 nm long pass and emission filter 600-660 nm</td></tr><tr><td>The Olympus Cell^P operating software</td><td>Olympus</td><td></td><td>Software used to control microscope, motorized stage, camera and image acquisition</td></tr><tr><td>Matlab R2014B analysis software</td><td>Mathworks</td><td></td><td>Used to develop in house graphic user interface for image analysis</td></tr><tr><td>Excel software</td><td>Microsoft</td><td></td><td>Used for data management, calculation, plot creation and statistics</td></tr></tbody></table>

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.

The unique HMCA imaging plate is used for the invasion assay of 3D tumor spheroids. The entire assay, beginning with the spheroid formation and ending with the invasion process and additional manipulations, is performed within the same plate. For the spheroid formation, HeLa cells are loaded into the array basin and settle in the hydrogel MCs by gravity. The hydrogel MCs, which have non-adherent/low attachment characteristics, facilitate the cell-cell interaction and the formation of 3D tumor spheroids. Figure 2A illustrates the cells settled in the MCs, following the use of 150 x 103 cells/mL loading solution (i.e., ~16 cells per MC calculated value). It is clear that the occupancy of cells per MC is not evenly distributed through the array, and the average obtained statistic distribution is 14.78 &#177; 3.60 cells per MC. The table in Figure 2B summarizes the cell distribution per MC for the whole HMCA plate. The average coefficient of variation (CV) of the cell distribution within a macro-well is 43.33%, while between the macro-wells it is 24.37%. The differences in cell occupancy are crucial and directly dictate the size of spheroids formed and their homogeneity throughout the plate. In order to reduce/minimize the variability created by the number of cells per MC, the analysis of the results is performed at single-element resolution. The plots in Figure 2C-D illustrate the size (sectional area) distribution of 3-day spheroids. It is clearly exemplified in Figure 2D that calculating the growth ratio of each spheroid (sectional area at day 3/day 2) has a more homogeneous distribution than the absolute size distribution (Figure 2C), yielding a CV of 15.48% and 52.80%, respectively. Spheroid diameter distribution of 3-day spheroids yields a CV of 24.52% and the growth ratio (diameter at day3/day2) is 7.45%. These CV values are similar to17 or higher than18 other reports attaining cell loading by gravity. In order to eliminate the measured parameter variance resulting from the variability in the initial seeded cell number per MC, it is preferable to normalize the selected parameters to the initial cell number or to any other measured parameter of the same object, such that each spheroid has its internal control. This approach could be easily applied by tracking the object on the HMCA imaging plate.
The process of spheroid formation and growth could be easily followed on the HMCA imaging plate.&#160;Figure 3A-B&#160;shows a BF image of one region on the HMCA imaging plate containing 24 h HeLa cell aggregates and the respective image of mature, 5-day 3D multicellular objects, which are becoming more spherical and denser. It is clearly shown that most objects retain their location during the 5-day incubation period. The analysis of shape (sphericity) and size (sectional area) during the spheroid formation is presented in Figure 3C. The scatter plot demonstrates the increases in both parameters from day 1 to day 5; 0.544 &#177;0.020 a.u. to 0.684&#177; 0.016 a.u. for sphericity (p &#60; 0.05) and 0.00356&#177; 0.00013 mm2 to 0.00538 &#177; 0.00015 mm2 for sectional area (p &#60; 0.05), respectively. While 24-h aggregates are small and have non-regular shape, the respective 5-day spheroids are bigger and acquire spherical form.
The detection of spheroid cell viability is executed by using low concentration PI for staining19 which circumvents the need for dye washing. PI penetrates the cell membrane and then intercalates into the DNA of non-viable cells. Figure 4A shows 5-day HeLa spheroids stained with PI (in red). Analysis of the percentage of red area (representing dead cells) out of each spheroid sectional area is summarized in Figure 4B. The histogram shows that 85% of spheroid population has a maximal value of 5% red stained area and the average is 3.01 &#177; 2.34 % (left histogram). Sectional area of spheroids (right histogram) shows a normal distribution with average of 0.02447 &#177; 0.00487 mm2.
After producing 3-day mature HeLa spheroids, the invasion assay is performed within the HMCA imaging plate. The effect of variables, such as ECM composition, inhibitors and activators, on the 3D tumor spheroid invasion process could be examined. For invasion assay performance, the medium is gently removed and replaced with ECM solution pipette over the spheroids into the MC array basin. After the full gelation of the ECM solution, complete medium is added into the macro-well. (1) In order to examine the influence of HA on the HeLa spheroid spontaneous invasion process, the spheroids were covered with collagen and HA mixture solution (Figure 5A) and compared to those covered with collagen only solution (Figure 5C), and BF images were acquired right after ECM gelation (t = 0 h). The kinetic of invasion process was followed by imaging the same regions every 5 h for a period of 63 h. After 50 h of incubation, the spheroids embedded in collagen and HA (Figure 5B) showed lower cell dispersion into the surrounding ECM as compared to the spheroids embedded in collagen only (Figure 5D). The analysis of the invasion area kinetic over time, for 99 spheroids at single-spheroid resolution is summarized in Figure 5E. Figure 5E plots the mean/average invasion area over time of the two groups, and clearly demonstrates that the addition of HA to collagen inhibits the cell dispersion out of the spheroid, resulting in smaller invasion areas. The average of the slopes resulting from linear regression adjustment curves for each of the spheroids embedded in collagen (0.001973 &#177; 0.000894 mm2/h) as compared to collagen and HA solution (0.001410 &#177; 0.000941 mm2/h) are significantly different (p = 0.003, t-test: two-sample assuming equal variances). (2) In order to examine the influence of Fisetin (a bioactive flavonoid found in several fruits and vegetables which shows cytostatic and migrastatic activities by acting on several molecular targets including phosphatidylinositol 3-kinase (PI3K)/Akt/c-Jun N-terminal kinases (JNK) signaling and Wnt/&#946;-catenin signaling downregulates matrix metalloproteinases (MMPs) and others20) on HeLa spheroid spontaneous invasion process, increasing concentrations of the drug were added to the complete medium and BF images were acquired at t = 0 (Figure 6A) and 24 h later (Figure 6B-C). As demonstrated, 10 &#181;M of Fisetin (Figure 6C) inhibits the dispersion of cells around the spheroids as compared to non-treated HeLa spheroids (Figure 6B). The invasion area analysis of a dose response experiment (Figure 6D) exhibits significant inhibition at 10-40 &#181;M of Fisetin (p &#60; 0.003), attaining saturation at the concentration of 10 &#181;M and higher. (3) It has been shown that NO, at a nano-molar level, contributes to a more aggressive breast cancer phenotype by stimulating tumor expansion and migration16. In order to examine the influence of NO on the collective invasion process of MCF7 spheroids embedded in collagen, 1 &#181;M of diethylenetriamine/nitric oxide donor (DETA/NO) was added to the complete culture medium and BF images were acquired at t = 0 and t = 20 h. Dramatic changes in the morphology and locations of 3D spheroids during exposure to NO donor are evident (Figure 7A-B). The spheroids lose their sphericity and become more elongated acquiring amoeboid migratory phenotype. Concomitantly, enhanced translocation of the spheroids within the surrounding collagen matrix was observed (Figure 7C). The average elongation value significantly increased from 1.305 &#177; 0.193 a.u. to 1.477 &#177; 0.298 a.u., (p &#60; 0.0006). The average migration distance measured for the same spheroids was significantly extended, 0.0788 &#177; 0.0575 mm and 0.3164 &#177; 0.3365 mm in the absence and in the presence of DETA/NO, respectively (p &#60; 4 x 10-6).
Image analysis of the spheroid invasion was achieved by semi-automatic in-house MATLAB software. The acquired time lapse images of spheroid formation and invasion within the HMCA consists of numerous spheroids in one image (4-6 spheroids at 10X magnification and 25-30 spheroids at 4X magnification). The analysis of spheroid formation, growth and invasion was concomitantly performed on all spheroids in the image. The key parameters extracted for spheroid invasion are presented in Figure 8A, which shows a representative image segmentation of 4 spheroids at a single time point (t = 16 h). The main invasion area defined by a red border, as well as the separated cells which disengaged and dispersed around it (indicated by black arrows), were tracked and numbered for each spheroid over time. Spheroid size before invasion at t = 0 is defined by a blue border. The mean/average invasion distance from spheroid center of mass to the invasion area circumference including separated cells is calculated from the encircled red segmentation and indicated by yellow arrows. Data extracted from this time-lapse experiment is summarized in Figure 8B-D. Figure 8B exhibits the elevation of the mean invasion distance from each spheroid center over time. Figure 8C exhibits the elevation of the total invasion area in each of the spheroids over time (including the main invasion area and separated/disengaged-cell area). Figure 8D1-4 shows the magnitude of separated-cell area compared to the total invasion area of each spheroid (over time). The cumulative number of disengaged cells is 33, 7, 4 and 5 for spheroids #1, #2, #3 and #4, respectively. The analysis of cumulative numbers of disengaged cells over 20 h of invasion obtained from 126 HeLa spheroids embedded in collagen is shown in Figure 8E. It is demonstrated here that there is a vast distribution in the number of disengaged cells in the examined population and the average value is 12.3 &#177; 12.8 cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58359/58359fig1.jpg" /> 
Figure 1: The HMCA imaging plate. (A) Image of one macro-well embossed with HMCA. (B) A cross section of one MC within the HMCA imaging plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58359/58359fig2.jpg" /> 
Figure 2: Homogeneity of the cell distribution and spheroid growth ratio in the HMCA imaging plate. (A) An overlay of BF and fluorescent images of the HMCA loaded with HeLa cells pre-stained with Hoechst 33342 1 &#181;g/mL (loading concentration 150 x 103 cells per mL). Images were acquired right after the cell settlement in the MC. Image magnification 4X, Scale bar = 500 &#181;m. (B) The table summarizes the distribution of HeLa cells within the HMCA-6-well imaging plate. The cells were counted from up to 90 MC/macro-well. The results are presented as mean &#177; standard deviation (SD), CV (SD/mean100) is calculated within and between the macro-wells. (C) Distribution histogram of the sectional area of 3 day spheroids, n = 418, determined from BF images. (D) Distribution histogram of the spheroid growth ratio (sectional area at day 3/day 2). Growth ratio is calculated at single-element resolution for each of the 418 spheroids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58359/58359fig3.jpg" /> 
Figure 3: Tracking spheroid formation in the HMCA imaging plate. BF image of HeLa cells (loading concentration 120 x 103 cells per mL) incubated in the HMCA for 1 day (A) and 5 days (B). Image magnification 4X, Scale bar = 500 &#956;m. (C) Scatter plot of 3D object sphericity as a function of its sectional area, after 1 day (blue diamonds) and 5 days (brown squares) of incubation post cell loading. Each marker represents the analyzed data from a single spheroid, n = 83. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58359/58359fig4.jpg" /> 
Figure 4: Detection of cell viability in mature spheroids. An overlay of BF and fluorescent images of 5 day spheroids (loading concentration 360 x 103 cells per mL) stained with PI 0.25 &#181;g/mL (A). Image magnification 4X, Scale bar = 500 &#181;m. (B) Distribution histogram of the percent of PI area relative to the sectional area of the spheroid (left panel) and distribution histogram of sectional area (right panel), n = 84. Percent of PI area is calculated at single-element resolution for each of the 84 spheroids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58359/58359fig5.jpg" /> 
Figure 5: Influence of ECM composition on spheroid invasion process. BF images of 3 day HeLa spheroids embedded in a mixture of 3 mg/mL collagen and 20 &#181;g/mL HA (A) and 3 mg/mL collagen (C), at t = 0 h and after 50 h of incubation (B) and (D), respectively. Image magnification 10X, Scale bar = 200 &#181;m. The HMCA imaging plate was loaded onto the stage of the inverted microscope equipped with incubator. Images were acquired every 5 h and the kinetic analysis is presented in plot (E), n = 99. Each curve represents the mean &#177; SD of invasion area increase over time, for collagen and HA (blue diamonds) and for collagen only (brown squares). Average linear regression curve and its equation and R-squared value are indicated above the curves. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58359/58359fig6.jpg" /> 
Figure 6: Fisetin inhibits spontaneous invasion of HeLa spheroids. BF images of 3 day HeLa spheroids, embedded in a mixture of 3 mg/mL collagen (A) at t = 0 and then, 24 h after addition of 0 &#181;M (B) and 10 &#181;M Fisetin (C) to the complete culture medium. Image magnification 4X, Scale bar = 500 &#181;m. Analysis of invasion area for each spheroid at end point (t = 24 h) is presented in plot (D), n = 488. The curve represents mean &#177; SD of invasion area as a function of 0-40 &#181;M Fisetin. Fisetin treatment significantly inhibits the invasion at 10 &#181;M (P = 4.65 x 10-6), 20 &#181;M (P = 0.0021) and 40 &#181;M (P = 4.86 x 10-8), using t-test: two-sample assuming equal variances. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58359/58359fig7.jpg" /> 
Figure 7: NO induces collective invasion in MCF-7 breast cancer spheroids. BF images of 2 day MCF-7 breast cancer spheroids, embedded in collagen (A) at (t = 0) and (B) after 20 h exposure to 1 &#181;M DETA/NO. Spheroid ROI segmentation is defined by red border, overlaid and numbered in red on BF images A and B. ROIs defined by blue border, overlaid on BF image B represent spheroid size and location at t = 0. Magnification 4X, Scale bar = 500 &#181;m. (C) A scatter plot of the correlation between elongation factor and migration distance of individual breast cancer spheroids upon the exposure to 1 &#181;M DETA/NO (brown squares) as compared to control (blue diamonds) at t = 20 h, n = 54. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58359/58359fig8.jpg" /> 
Figure 8: HeLa cell spontaneous invasion image analysis. BF images of 3 day HeLa spheroids, 16 h after embedding in collagen (A). Magnification 10X. Spheroid invasion area and disengaged cells are defined by red border, spheroid size at t = 0 is defined by blue border, mean invasion distance from spheroid center of mass is indicated by yellow arrows and disengaged cells are indicated by black arrows. Plot the increase over time of the mean invasion distance from spheroid center of mass (B) and total invasion area (C). The curves represent spheroid #1 (blue diamonds), spheroid #2 (brown squares), spheroid #3 (green triangles) and spheroid #4 (purple Xs). (D) Bar chart of the main (blue) and separated cell (brown) invasion area increase over time. (E) Distribution histogram of the cumulative number of separated cells per spheroid during 20 h of invasion process, n = 126. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58359/58359fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

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.

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 ...

analysis-cancer-cell-invasion-anti-metastatic-drug-screening-using

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.0K Views.  Bar-Ilan University. Het doel van dit eenvoudige reproduceerbare biomimetische 3D-celgebaseerde systeem is om invasiecapaciteitsanalyse te vergemakkelijken bij grote populatie tumorsferoïde voor anti-gemetastaseerde geneesmiddelenscreening. Het belangrijkste voordeel van onze hydrogel micro-kamer array is dat het mogelijk maakt de productie van een groot aantal uniforme sferoïden die hetzelfde brandpuntsvlak per goed bevolken. De sferoïde positie blijft behouden als gevolg van de hydrogel micro-kamer array str...

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Medicine

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetics

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.

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 ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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 ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

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

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