Cancer Research
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Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates
Chapters
Summary October 25th, 2018
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.
Transcript
The goal of this simple reproducible biomimetic 3D cell-based system is to facilitate invasion capacity analysis in large population of tumor spheroid for anti-metastatic drug screening. The main advantage of our hydrogel micro-chamber array is that it allows the production of a large number of uniform spheroids that populate the same focal plane per well. The spheroid position is retained due to the hydrogel micro-chamber array structure even after extracellular matrix addition, facilitating the accurate continuous monitoring of the spheroids and the invasion process.
Further, the morphological characterization and fluorescent staining of numerous spheroids and invading cells can be performed in situ and analyzed at a single-element resolution in a time-efficient manner. Demonstrating the hydrogel micro-chamber array six-well imaging plate production procedure will be Maria Sobolev from our laboratory. To prepare the hydrogel micro-chamber array plates, first preheat six polydimethylsiloxane, or PDMS, stamps to 70 degrees Celsius, and place a six-well, glass-bottom plate on a dry bath pre-heated to 75 degrees Celsius for several minutes.
When the plate is warm, pour a 400-microliter drop of pre-heated low-melting agarose onto the bottom of each well of the six-well plate, and gently place one stamp over each drop. Allow the setup to cool for five to 10 minutes at room temperature, followed by 20 minutes at four degrees Celsius for full agarose gelation. When the agarose has solidified, gently peel off the PDMS from each well, leaving the agarose gels patterned with micro-chambers.
After allowing the UV lamp to reach its highest intensity, place the embossed plate in the light curing system for three minutes, and fill the UV-sterilized macro-wells with sterile PBS. Then, cover the plate, and wrap it with Parafilm for four-degree Celsius storage until use. To load the cells, remove the PBS from each well, and gently load 50 microliters of the cells of interest on top of each hydrogel array.
After allowing the cells to settle by gravity for 15 minutes, carefully add six to eight aliquots of 500 microliters of fresh medium to the rim of the macro-well plastic bottom outside of the ring and hydrogel array. Incubate the cells for the appropriate time period at 37 degrees Celsius and 5%carbon dioxide with humidity for the formation of spheroids. Then, transfer the spheroids into a biosafety hood on ice for 10 minutes.
When the plate has cooled, lean a pipette tip on the edge of the macro-well plastic bottom next to the hydrogel array, and remove all of the medium from around the array. Then, remove the medium from the array tank, lean a gel-loading pipette tip on the edge of the array tank very carefully, taking care not to disturb the micro-chambers and spheroids. Next, use a pre-frozen, fine pipette tip to add two aliquots of 150 microliters of the collagen mixture of interest at the edge of the array one at a time, releasing the mixture slowly to avoid spheroid dislocation.
After the second round of collagen has been added to each well, return the spheroids to the incubator for one hour for full gelation of the extracellular matrix. When the matrix has solidified, add 400 microliters of pre-warmed 1%low-melting agarose on top of the gel, and incubate the plate, covered at room temperature, for five to seven minutes, before cooling for two minutes at four degrees Celsius for agarose gelation. Then, leaning the pipette on the edge of the macro-well plastic bottom, gently add two milliliters of complete medium to each well.
To perform an invasion assay, load the hydrogel micro-chamber array plate onto a motorized inverted microscope stage equipped with a 37-degree Celsius and 5%carbon dioxide incubator with a humidified atmosphere, and use a four or 10x objective to pre-determine the positions for the image acquisition in each well so that the entire array area will be assessed. Then, acquire bright-field images every two to fours hours for a total of 24 to 72 hours to track the cell invasion from the spheroid body into the surrounding extracellular matrix. At the end of the image acquisition, export the time-lapse images from the image acquisition software for saving in a tagged image file format to a dedicated folder.
Next, open the graphic user interface, and click Load Bright Field. Adjust the segmentation parameters of the image to achieve a precise segmentation of the spheroids and their invasion areas, and click Region Of Interest Segmentation to create a border around each spheroid. If the automatic segmentation is incorrect, press Delete or Add, and make the appropriate corrections manually.
Click Tracking to number the spheroids in each of the time-lapse images. Then, click Measurement to generate a spreadsheet with all of the parameters, and save the spreadsheet in an appropriate format for data processing and statistical analysis. To examine the influence of hyaluronic acid on the spontaneous HeLa spheroid invasion process, in this experiment, two-day-matured spheroids were covered with collagen and hyaluronic acid mixture solution and compared to two-day-matured spheroids covered with collagen solution alone.
After 50 hours of incubation, the spheroids embedded in collagen and hyaluronic acid demonstrated a lower cell dispersion into the surrounding extracellular matrix compared to the spheroids embedded in collagen alone. Indeed, analysis of the invasion area kinetics over time for 99 spheroids at single-spheroid resolution clearly indicates that the addition of hyaluronic acid to the collagen inhibits cell dispersion out of the spheroid, resulting in smaller invasion areas. Treatment of the HeLa spheroid culture with a bioactive flavonoid with cyto-and migrastatic properties for 24 hours results in an inhibited cell dispersion around the spheroids compared to non-treated HeLa spheroids.
Indeed, analysis of the invasion areas of 488 spheroids at the end of the experiment revealed a significant inhibition after treatment with as little as 10 micromolar of the drug. Due to the low attachment characteristic of the hydrogel, this micro-chamber-based technology facilitate the formation of spheroid even in instances of very few cells. This is especially advantage in valuable limited cancer stem-like or patient-derived primary cell samples.
The hydrogel micro-chamber imaging plate allows analysis of the exact spatial location of the spheroids during long-term experiments for the evaluation of the invasion process at the single-element level and provides a high degree of statistical robustness and accuracy. Future applications of this methodology include side-by-side multi-cell culturing of tumor and stromal cells in different regions within the macro-well and the retrieval of individual spheroids for downstream molecular analysis.
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