Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Transcript

Our modified 3D assay cultures can be used to investigate the role of various components in a cancer microenvironment, leading to the identification of novel biomarkers and drug targets. This method enables one to study changes in the cellular phenotype and molecular signaling that closely resemble the immune system. It can also be modified to meet specific requirements of a study.

This method can help identify novel biomarkers that can aid in early detection of breast cancer. Also, molecular insights can be utilized for identifying novel drug targets. This method can be used to study both tumorigenesis and morphogenesis after perturbation of gene or genes that can provide insight into the role played by the gene or genes.

To begin, trypsinize the cells by aspirating the medium and washing with 2 milliliters of PBS. Add 900 microliters of 0.05%Trypsin-EDTA and incubate at 37 degrees Celsius for around 15 minutes or till the cells are completely trypsinized. Prepare a bed of lrECM in eight wells of a chambered cover glass slide by coating each well with 60 microliters of lrECM.

Place it in a 37 degree Celsius carbon dioxide incubator for a maximum of 15 minutes. To prepare the cell suspension, add 5 milliliters of re-suspension medium to quench the trypsin activity and spin the cells at 112 times g for 10 minutes at 25 degrees Celsius. Aspirate the spent medium and re-suspend the cells in 2 milliliters of assay medium.

Mix the suspension well to ensure the formation of a single cell suspension. Count the cells using a hemocytometer and calculate the volume of cell suspension needed to seed 6, 000 cells in each well. According to the number of wells required, dilute the cell suspension in the overlay medium.

Add 400 microliters of the diluted cell suspension to the prepared lrECM beds, carefully ensuring not to disturb the lrECM bed. Incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator. After 3 hours, add 200 nanomoles of PAF to each well.

Add the same concentration of PAF during every media change, and replenish the cells with fresh medium every 4 days. After 20 days of culturing, carefully pipette out the medium from each well and wash the wells with 400 microliters of pre-warmed PBS. Fix the acinar structures by adding 400 microliters of freshly prepared 4%paraformaldehyde and incubating for 20 min at room temperature.

Rinse the wells once with ice-cold PBS and permeabilize with PBS containing 0.5%Triton X-100 for 10 minutes at 4 degrees Celsius. After 10 minutes, pipette out the Triton X-100 solution carefully and rinse with 400 microliters of freshly prepared PBS glycine. Add 400 microliters of the primary blocking solution comprising 10%goat serum in immunofluorescence buffer and incubate at room temperature for 60 minutes.

Remove the primary blocking solution, add 200 microliters of 2%secondary blocking antibody prepared in the primary blocking solution, and leave it at room temperature for 45 to 60 minutes. Prepare the primary antibody in 2%secondary blocking antibody solution in a 1:100 dilution. Remove the secondary blocking solution, add the freshly prepared antibody, and incubate overnight at 4 degrees Celsius.

Carefully pipette the primary antibody solution and wash it thrice with 400 microliters of immunofluorescence buffer. Next, prepare a 1:200 dilution of the fluorophore-conjugated secondary antibody in the primary blocking solution and incubate the slides in the secondary antibody solution for 40 to 60 minutes at room temperature. Rinse the slides with 400 microliters of immunofluorescence buffer for 20 minutes, followed by two washes with PBS for 10 minutes each.

Counter-stain the nuclei with PBS containing 0.5 nanograms per milliliter of nuclear stain for 5 to 6 minutes at room temperature. Wash the slides thrice with 400 microliters of PBS to remove the excess stain. Carefully pipette out the entire PBS and ensure removal of the residual solution.

Then add one drop of mounting reagent into each well and allow it to stand at room temperature overnight. Finally, image the slides under 40x or 63x magnification on a confocal microscope, taking optical Z-sections of 0.6 millimeter step size. The images represent the nuclei of cells in acini stained with a nuclear stain.

The 3D construction of the acini and the phase contrast image of MCF10A acini grown on lrECM for 20 days are shown here. The centermost section shows the presence of a hollow lumen. PAF disrupts the polarity and induces EMT-like changes in MCF10A breast acini.

MCF10A cells were grown as 3D on top'cultures in lrECM. The cultures were treated with PAF on day 0, 4, 8, 12, and 16. The cultures were maintained for 20 days and then immunostained for alpha6-integrin and nuclear stain, GM130, a marker for apical polarity, and Vimentin, an EMT marker.

The centermost stack of the respective acini has been shown here. The representative data is for 40-50 acini from three biologically independent experiments. The lrECM bed needs to be made carefully such that it is evenly spread without any air bubbles.

Also, the cell suspension should be mixed evenly and added slowly to avoid clumping. One can obtain protein and RNA from the 3D cultures that can then be used to investigate the different molecular pathways.

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Journal (Cancer Research)

Journal (Cancer Research)

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

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