At this point in my research endeavors, I have been involved with several different techniques and experiments compared to when I began last semester. Recently, I have been making some crucial and important discoveries and findings while working with four different cell lines of MCF7 cancerous breast cells including the non-targeting sequence (NT), 59, 75, and 91. I have been utilizing the 3D hanging drop plate technique, which is where a plate of 12 columns and 8 rows are lined with tiny wells that contain no more than 50 microliters in volume each. The purpose of the hanging drop plate is to form spheroids at the bottom of each well that contains clumped spheres or cells. Growing cells in clusters or 3D mammospheres, being the proper term, mimics how cancerous tumors form in reality and thus is a considerably decent 3D tumor model. Being able to grow cells in 3D is a huge benefit to gathering more efficient results and puts us one step closer to essentially programming these cells to undergo apoptosis, just as we successfully did in 2D cells by knocking down PNUTS, the binding subunit to PP1.
Several difficulties I have encountered throughout this project has been shooting the spheroids through the hanging drop plate and into a conical tube for collection and extracting the protein from the mammospheres without breaking fully apart after being spun down. I have tried several methods of collecting the mammospheres from the hanging drop plates including pipetting off of the top or bottom of the plates to the more preferred shooting through method, which is where I pipette MCF7 media or PBS as well as air through each well to allow each mammosphere to drop through the top portion of the plate into a conical tube or 96 well plate. One downside to the hanging drop plate at the time of mammosphere collection is that some of the spheres may have already dropped through the top portion of the plate and settled on the bottom layer, thus reducing the amount of spheres I can essentially shoot through. Typically after collection and spinning down, the supernatant, which is the liquid on top of the pellet is dumped and carefully suspended up using Pasteur pipettes, however there are times where the pelleted spheres will follow with the uptake and sometimes break apart. This results in an even more challenging approach of lysing the mammospheres in order to collect their protein and actually wind up with a significant amount of it to continue on to a Western blot for further evaluation of the presence of Rb, Actin, or PNUTS. My protein assays have been yielding negative values for my protein concentrations, which means barely any protein was present in my 4 unknown samples.
However, I continued on with running a gel and eventually performing a Western blot to test for the presence of PNUTS and Actin. One idea Dr. Krucher had in mind was to find a gel that can hold a larger amount of volume in the wells compared to my limited amount of 50 microliters would could be effecting my results on the Western for protein presence. Just recently, Dr. Krucher ordered a new gel that we are currently practicing with now that can hold up to around 120 microliters in volume in each well, a significantly larger number that could positively alter my results and hopefully yield a more abundant amount of protein in my 4 samples. So far, the new gel seems to be significantly working, however I have not been able to run it fully through yet; I still need time to work with it and see if it is a better fit than the previous 7.5% smaller-welled gels I was using.
Even though I do not have definite results for this specific project yet, I am still eager and determined to continue my research and discover if whether or not this gel in fact is more significant and essentially if I can wind up causing cells to go through apoptosis, my ultimate, overall goal for all of my research. I have an accumulation of results from other projects that have helped me lead up to this point as well as findings from all of my more introductory work with cells growing in 2D environments. Dr. Krucher and I are still very engrossed in this project and how it will turn out; we are not going to give up hope or lose our motivation. Overall, my research projects have impacted me in such a positive and intriguing way, which I am very grateful for. I become excited when I achieve significant results or even over the smallest occurrences such as receiving pellets in all four of my samples after being spun down. Becoming involved in breast cancer research has sparked me to grow closer to my faculty advisor who has done so much for me up to this point in my undergraduate career as well as express my excitement and knowledge to my grandmother who is a two-time breast cancer survivor and in essence developing an even stronger connection with her. The field of research has opened my eyes to a very different and challenging world around me and I appreciate it and the individuals involved in it. There is so much more that this area of research can teach me, and I am definitely willing to explore it further and continue learning as much as I can as I resume my studies here at Pace.