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

Summary

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.

Abstract

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.

Introduction

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 invasion^6,7,8. 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 devices^3,10,11,12, 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 demonstrated^13,14, 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.

Protocol

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 articles^15,16. 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 µm, small base area: 90 µm x 90 µm, and large base area: 370 µm x 370 µm). 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.

1. 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 °C for a few hours until a uniform solution is achieved. Keep the solution at 70 °C until use.
2. Pre-heat the PDMS stamp to 70 °C in the oven. Keep it warm until use. Alternatively, use a commercial stamp and follow the instruction.
3. Place the commercial 6-well glass-bottom plates on a dry bath pre-heated to 75 °C. Wait a few minutes until the plate is totally warm.
4. Pour a drop (400 µL) 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 5–10 min for pre-gelling and pre-cooling. Incubate at 4 °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.
5. 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.
6. 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 °C until use.
7. Before use, empty PBS residuals from the HMCA plate. Place it in the biological hood.

2. Loading Cells and Spheroid Formation

1. 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 °C) and incubate for 3 min in the incubator at 37 °C. Then, shake the plate gently to ensure monolayer detachment, add 10 mL of complete medium (pre-warmed to 37 °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.
2. Gently load the cell suspension (50 µL, 150–360 x 10³ cells/mL in medium, ~16–40 cells per MC) on top of the HMCA and allow the cells to settle by gravity for 15 min.
3. Gently add 6–8 aliquots of 500 µL fresh medium (total 3–4 mL) to the rim of the macro-well plastic bottom outside the ring and hydrogel array.
4. Incubate HeLa cells for 72 h and MCF7 cells for 48 h at 37 °C and 5% CO_2, in a humidified atmosphere for the formation of spheroids.

3. Viability Detection by Propidium Iodide (PI) Staining

1. Prepare a stock solution of PI (500 µg of PI per 1 mL of PBS). Keep it at 4 °C for about 6 months. Freshly prepare a dilution of 1:2,000 (0.25 µg/mL), by the addition of 6 µL of stock to 12 mL of complete medium without phenol red, for the 6-well HMCA plate (2 mL per well).
2. 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.
3. 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 °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.

4. Collagen Mixture Preparation

1. 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 °C, until use.
2. 10–20 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 °C for 3– months) and a sterile tube into the ice bucket until totally cooled. Place the ice bucket inside the biological hood.
3. Prepare 1,800 µL of collagen mixture at a final concentration of 3 mg/mL, for 6-well HMCA invasion assay plate (300 µL per well) as follows. Add the intergrades into the cooled tube in the following order: 515 µL of DDW, 24.8 µL of 1 M NaOH and 180 µL of 10x PBS. Mix well by vortex and place back into the ice. Finally, add 1080 µL of collagen to the mixture, vortex again and put back into ice.

5. HA and Collagen Mixture Preparation

1. 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 °C for up to two years.
2. Right before use, place the HA stock solution in the ice bucket inside the biological hood.
3. Prepare a 3 mg/mL collagen mixture as described in Step 4. Add 36 µg (7.2 µL) of HA into 1,800 μL of ready to use collagen mixture, for a final concentration of 20 µg/mL. Then, vortex again briefly and put back into ice.

6. ECM Mixture Addition

1. Transfer the HMCA plate, with 48–72 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 °C in a water bath.
2. 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.
3. Take 150 µL 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 µL, for a total 300 µL 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.
4. After the full gelation of ECM has been achieved, pipette 400 µL of pre-warmed 1% LMA on top of the ECM gel. Cover the plate with its lid, incubate the plate at RT for 5–7 min and then for 2 min at 4 °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.

7. Invasion Assay Acquisition and Analysis

1. Load the HMCA plate onto a motorized inverted microscope stage equipped with an incubator, pre-adjusted to 37 °C, 5% CO₂ and humidified atmosphere.
2. 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 "Stich" option.
3. Acquire bright field (BF) images every 2–4 h for a total period of 24–72 h in order to follow the invasion of cells from the spheroid body into the surrounding ECM.
4. Acquire fluorescent images for PI detection by using a dedicated fluorescent cube (excitation filter 530–550 nm, dichroic mirror 565 nm long pass and emission filter 600–660 nm).
5. 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 "Database" tab in the toolbar and then the "Export record files" 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 = √ ((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.
6. Open the GUI and push the "Load BF" button. After the image is open, adjust the segmentation parameters (minimum size: >1 and radius close: >1) to achieve a precise segmentation of spheroid and its invasion area, then, press "Region of interest (ROI) segmentation" button for execution and a border will appear around each spheroid. If the automatic segmentation is incorrect, press the "Delete" or "Add" button and make the requested corrections manually. Repeat the same steps for subsequent images.
7. Press the "Tracking" 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 "Measurement" 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 Micro-well Glass Bottom Plates with 14 mm micro-well #1.5 cover glass	Cellvis	P06-14-1.5-N	Commercial glass bottom plates which are used for HMCA embossing
UltraPure Low Melting Point Agarose	Invitrogen	16520100	A solution of 6% agarose is warmed up to 80 °C before use, a solution of 1% agarose is warmed to 37 °C
Trypsin EDTA solution B	Biological Industries	03-052-1A	Warmed to 37 °C before use
DMEM medium, high glucose	Biological Industries	01-055-1A	Warmed to 37 °C before use
Special Newborn Calf Serum (NBCS)	Biological Industries	04-122-1A	Heat Inactivated
DPBS (10x), no calcium, no magnesium	Biological Industries	02-023-5A	Kept on ice before use
NaOH, anhydrous	Sigma-Aldrich	S5881-500G	Used for the preparation of 1 M NaOH solution
Cultrex Type I collagen from rat tail, 5 mg/mL	Trevigen	3440-100-01	Kept on ice before use
Hyaluronic acid sodium salt	Sigma-Aldrich	H5542-10MG	Kept on ice before use
Fisetin	Sigma-Aldrich	F505-100MG	Added to the culture medium, invasion inhibitor
DETA/NO	Enzo Life Sciences	alx-430-014-m005	Added to the culture medium, nitric oxide donor
PI	Sigma-Aldrich	P4170	Used at very low concenrtation without the need for washing
Dymax 5000-EC UV flood lamp complete system with light shield & Dymax 400 Watt EC power supply	Dymax Corporation	PN 39823	Used for HMCA plate sterilization by UV
Inverted IX81 microscope	Olympus		Used for automatic image acquisition
Incubator for microscope	Life Imaging Services		Essential for time lapse experiments with image acquisition, pre adjusted to 37 °C, 5% CO2 and keeping a humidified atmosphere
Sub-micron motorized stage type SCAN-IM	Marzhauser Wetzlar GmbH		Used to predetermine image acquisition areas, for automatic image acquisition
14-bit, ORCA II C4742-98 cooled camera	Hamamatsu Photonics		Highly sensitive, used for imaging
Fluorescent filter cube for PI detection	Chroma Technology Corporation		Filter cube specifications: excitation filter 530-550 nm, dichroic mirror 565 nm long pass and emission filter 600-660 nm
The Olympus Cell^P operating software	Olympus		Software used to control microscope, motorized stage, camera and image acquisition
Matlab R2014B analysis software	Mathworks		Used to develop in house graphic user interface for image analysis
Excel software	Microsoft		Used for data management, calculation, plot creation and statistics