The overall goal of this procedure is to study the phenotypic consequences of mesenchymal-epithelial transitions in sarcomas. This method can help answer key questions about why epithelial-like sarcomas have superior outcomes relative to sarcomas that are more mesenchymal-like. The main advantage of this technique is that it provides an easy, reproducible means to study downstream gene regulatory networks and phenotypes associated with mesenchymal-epithelial transitions in sarcomas.
By better understanding phenotypic plasticity in sarcomas, we hope to identify therapeutic interventions that will enable us to shift sarcomas toward a more indolent epithelial-like state. Demonstrating the procedure will be two undergraduates in our laboratory, Jackson Xu and Shivee Gilja. On the first day of the transduction, plate three times 10 the fifth HEK293T cells in each of two wells of a six well plate in two milliliters of supplemented DMEM.
Incubate the plate at 37 degrees Celsius with 5%carbon dioxide. After culturing overnight, examine the cells under a microscope. They should be 40 to 60%confluent.
Next, in separate tubes containing 200 microliters of serum free medium with 1.8 micrograms of p delta 8.9 and 0.2 micrograms of pCMV-VSV-G helper plasmids, dilute two micrograms of the empty vector and two micrograms of the vector containing GRHL2. In another tube, dilute four microliters of a lipid-based transfection reagent in 200 microliters of serum free medium per transfection. This will be eight microliters and 400 microliters for two samples.
Add 200 microliters of transfection reagent mixture to each plasmid solution, and then incubate the samples for 20 minutes at room temperature. Meanwhile, use a vacuum aspirator to remove the medium from the HEK293T cells. Rinse the cells once with one milliliter of PBS and then replace the PBS with 800 microliters of serum free medium.
After 20 minutes have passed, add 400 microliters of transfection reagent plasmid mixture dropwise to each well of the plated cells. Incubate the dish for two hours at 37 degrees Celsius in 5%carbon dioxide. Following the incubation, carefully remove the transfection medium via vacuum aspiration.
HEK293T cells are loosely adherent, so when aspirating the medium, be careful not to remove the cells from the bottom of the dish or flask. Replace the aspirated medium with two milliliters of supplemented DMEM. Return the cells to the incubator for overnight culture.
The next day, refresh the medium on the transfected HEK293T cells, then plate three times 10 to the fifth RD human rhabdomyosarcoma cells in each well of a six well plate containing two milliliters per well of supplemented DMEM. Culture both plates overnight. On the fourth day, use a pipette to collect the viral medium from HEK293T cells and transfer it into two new 15 milliliter conical tubes, one for lentivirus containing an empty vector and another for lentivirus containing the GRHL2 plasmid.
Carefully replace the medium with new DMEM and place the HEK293T cells back in the incubator overnight. Next, add two microliters of 10 milligrams per milliliter polybrene per milliliter of viral medium into two new 50 milliliter conical tubes, one for the empty vector transfection and another for the GRHL2 transfection. Remove the plunger from a three milliliter syringe and attach a 0.45 micrometer polyethersulfone filter to the tip of the syringe.
Add the viral medium collected earlier into the barrel of the syringe and plunge it into one of the new 50 milliliter conical tubes containing polybrene. Repeat this step with the other sample. Then remove medium from RD cells by vacuum aspiration and add the two milliliters of each of the filtered viral medium samples to separate wells of the RD plate.
Incubate the RD cells at 37 degrees Celsius in a humidified incubator. The next day, repeat the viral medium collection and add the infection medium to the same wells containing the previously infected RD cells. The HEK293T cells can be discarded after the viral medium has been collected.
Incubate the cells for two more days. On the seventh day, wash the RD cells with one milliliter of PBS, then add 200 microliters of 0.05%trypsin to each well. Incubate at 37 degrees Celsius for five minutes.
Add two milliliters of supplemented medium and then transfer the cell suspension to new 15 milliliter conical tubes. Centrifuge the cells at 250 g's for five minutes at room temperature. After the spin, aspirate the medium and re-suspend the cells in one milliliter of DMEM supplemented with 5%FBS and 1%penicillin streptomycin.
To filter the cells in preparation for flow cytometry, use a pipette to apply the one milliliter RD cell suspension through a sterile 30 micrometer tube top filter into flow cytometry tubes. Place the tubes on ice. Using a flow cytometer, sort the EGFP positive cells into 1.5 milliliter microcentrifuge tubes containing 0.5 milliliters of DMEM supplemented with 10%FBS and 1%penicillin streptomycin.
Place the sorted cells back on ice. Plate the EGFP positive cells from both empty vector and GRHL2 transductions in a total of one milliliter of supplemented DMEM in separated wells of a 12 well plate. Then place the cells in the incubator for culture.
The yield of EGFP positive cells after flow cytometry is usually pretty low, in the range of 50, 000 to 100, 000 cells. Because of this, the cells may need to be cultured for an extra 10 to 14 days before proceeding to the next step. In a microcentrifuge tube, add three microliters of each 50 micromolar miR-200 mimic to 300 microliters of serum free medium.
In a separate tube, add nine microliters of 50 micromolar negative control micro RNA to 300 microliters of serum free medium. Next, in another microcentrifuge tube, add six microliters of siRNA specific transfection reagent to 600 microliters of serum free medium. Divide 300 microliters of this mixture into two tubes, one for each of the two miR mixtures.
Combine the 300 microliters of each miR mixture with a mix of 300 microliters of transfection reagent. Incubate for 20 minutes at room temperature. While incubating, prepare a cell suspension of 600, 000 EGFP positive RD cells expressing the MD vector or GRHL2 in 2.4 milliliters with 250 cells per microliter of serum free medium per treatment.
In a 24 well plate, add 100 microliters per well of the miR-200 or the negative control mix into six wells. Then add 400 microliters of each cell suspension into three wells of each miR mix. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide overnight.
The next day, change the medium to fully supplemented DMEM. Collect cells two days later for analysis using the appropriate buffer. Image each well every two hours with a 10X objective using an automated live cell imager.
These time lapse images show mesenchymal-epithelial transition, or MET, in RD cells expressing GRHL2, EGFP and miR-200 miRNAs. The cells change from spindle shaped to cobblestone in appearance, with increased cell to cell adhesions and a more rounded morphology. MET is not observed in other treatment conditions.
Instead, cells exhibit an elongated, spindle shaped morphology with few cell to cell attachments. During MET induction, the morphological change in RD cells upon GRHL2 and miR-200 overexpression was accompanied by up regulation of the epithelial marker, E-cadherin, as measured by qPCR and western blotting. Addition of miR-200s up regulated E-cadherin alone, but combined miR-200s and GRHL2 overexpression synergistically enhanced E-cadherin expression.
As demonstrated by immunofluorescence microscopy, there was an increase at cell to cell junctions of epithelial adhesion molecules, EpCAM and TJP-1, indicated by the white arrows. Up regulation of epithelial proteins was accompanied by down regulation of mesenchymal genes ZEB1 and Notch1 by QRT-PCR. Induction of MET reduced the anchorage independent growth of RD cells, as measured by soft agar growth assays.
The growth inhibition in soft agar was driven by miR-200s alone, as overexpression of GRHL2 led to an increase in anchorage independent growth. This is consistent with previous reports showing GRHL2 expression induces anchorage independent growth. Following this procedure, other methods, including assays to measure changes in cell migration, invasion, metastasis and response to therapy can be performed in order to elucidate the phenotypic consequences of MET in sarcomas.
