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...

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 t...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.0K Views.  Bar-Ilan University. 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.

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

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Bioengineering

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

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 ...

Medicine

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 ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...