The overall goal of this procedure is to investigate and predict the efficacy of GnRH-based drug delivery systems in human cancer cell cultures. This method can help answer key questions in the targeted anti-cancer therapy field, such as how efficiently cells of different human cancer types take of GnRH targeted anti-cancer drugs. The main advantage of this method is that it uses new fluorescently labeled GnRH analogues that allow simultaneous use of confocal microscopy and flow cytometry analysis.
The implications of this technique extends toward anti-cancer therapy because GnRH receptors are highly expressed on several cancer cell types, enabling the selective targeting of them. Demonstrating the procedure will be Bianka Gurbi, a Ph.D.student from my laboratory. To begin, remove the cultured medium from each of the seven wells of a seeded microscopic slide.
Then, add 250 microliters of the preheated complete medium to the negative control well and 250 microliters of the preheated GnRH fit seed treating media to each of the remaining six wells. Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for five hours. After five hours of incubation, aspirate the medium from each well and wash the cells with 250 microliters of PBS.
Subsequently, add 250 microliters of fixing solution into each well and incubate the slide at room temperature for 10 minutes. Once the cells are fixed, discard the fixing solution and wash the cells with 250 microliters of PBS. Add 250 microliters of five micromolar fluorescent probe solution prepared in PBS into each well and incubate the slide at room temperature for 10 minutes.
After removing the fluorescent probe solution, carefully wash the cells twice with 250 microliters of PBS. Finally, add three to four drops of mounting medium into each well. Keep the slide in dark at room temperature until imaged under a microscope.
Remove the cultured medium from each of the five wells of a seeded microscopic slide. Next, add 250 microliters of the preheated complete medium to the negative control well and to the second well with the untreated cells. Pretreat the cells in the three remaining wells with the preheated treating media containing 10 micromolar GnRH FITC.
Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for one hour. Afterward remove the media only from the treated cells and wash the cells with 250 microliters of the preheated complete medium. Next, remove the wash medium.
Then, add 250 microliters of the preheated complete medium into each of the three wells. Incubate the slide in an incubator at 37 degrees Celsius, five percent carbon dioxide for one hour. After incubation, discard the media from all five wells and wash the cells with 250 microliters of PBS.
Then, remove the PBS and fix the cells with 250 microliters of fixing solution at room temperature for 10 minutes. Next, discard the fixing solution and wash the cells with 250 microliters of PBS. Add 250 microliters of blocking solution into each well and incubate the slide at room temperature for one hour.
Aspirate blocking solution from each well except the negative control. After washing the cells with 250 microliters of PBS, add 250 microliters of primary antibody solution to each well. Incubate the slide at room temperature for one hour in a dark place.
Remove solutions from each of the five wells and wash the cells with 250 microliters of PBS. Then, remove the PBS and add 250 microliters of secondary antibody solution. Incubate the slide at room temperature for one hour in the dark.
Aspirate the solution from each well and wash the cells with 250 microliters of PBS. Remove the wash PBS and then add 250 microliters of PBS containing five micromolar fluorescent probe into each well. Incubate the slide for 10 minutes at room temperature.
Remove the fluorescent probe solution from each well and carefully wash the cells twice with 250 microliters of PBS. Finally, add three to four drops of mounting medium into each well. Keep the slide in the dark at room temperature until imaged under a microscope.
Image the cells under an inverted confocal laser scanning microscope. Turn on the 488 nanometer and the 633 nanometer lasers. Check the laser amplification and the pinhole size.
Image the negative control sample. Optimize the signal observed for the nucleus and adjust the signal of FITC and Alexa 546 so that it is close to zero. Then, using the same parameters, image the treated samples.
Discard the complete medium from all seven wells of a seeded culture plate. Then, add one milliliter of preheated complete medium into the negative control well and one milliliter of the preheated treating media to the remaining six wells. Incubate the plate in an incubator at 37 degrees Celsius, five percent carbon dioxide for five hours.
After discarding the medium from all wells, carefully wash the cells twice with two milliliters of PBS. Remove the PBS and then add 500 microliters of Trypsin EDTA solution into each well. Incubate the plate at 37 degrees Celsius until cells detach, which will take approximately 10 minutes.
Using one milliliter of complete medium, neutralize the Trypsin in each well and shake the plate. Afterward suspend the cells by gentle pipetting. Transfer the suspensions into separate FACS tubes and centrifuge the samples at 150 times G at four degrees Celsius for four minutes.
Decant the supernatants carefully. Then, add 500 microliters of ice-cold PBS into each FACS tube and gently resuspend the cells. Keep the tubes on ice until analyzed by FACS.
Presented here is the analysis of GnRH uptake in pharynx carcinoma cells. Five hour treatment with one micromolar of fluorescent complex of GnRH resulted in slight, but distinguishable intake manifested as green fluorescent signal localized in pharynx cancer cells. Additional increase of complex dosage led to enhanced uptake, demonstrating dose dependency of GnRH uptake in pharynx carcinoma cells.
Receptor dependent uptake of GnRH and the cellular distribution of the ligand in its receptor in lung cancer cells were analyzed by simultaneous treatment with fluorescent GnRH complex and staining of the type one GnRH receptor and nuclei. The results show that the type one GnRH receptor is associated with the cell membrane while the GnRH protein is internalized in cells and remains in their cytoplasm. The GnRH uptake in pharynx carcinoma cells was also studied using FACS analysis.
The results show that the uptake of GnRH observed when cells were treated with one micromolar of GnRH conjugate was strongly enhanced when the dose applied to the cells was increased to 10 micromolar. Once mastered, this technique can be finished in eight hours if it is performed properly. While attempting this procedure, it's important to remember to protect the treated samples and fluorescent dyes from direct light.
Following this procedure, other methods like cell viability or apoptosis assays can be performed in order to answer additional questions like how efficiently the GnRH targeted drug can inhibit the growth of a given type of cancer. After this development, this technique paved the way for researchers in the field of cancer therapy to explore the efficacy of GnRH-based drug delivery systems in adhering cancer cells. After watching this video, you should have a good understanding of how to use these fluorescently labeled GnRH conjugates to explore the susceptibility of cancer cells for GnRH targeted therapies.
