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.
Since I have returned to school after the conclusion of my winter break, I have been super excited to return to my research. Since I have returned, I have been conducting in a few new techniques and utilizing several different cell lines through experimentation. Dr. Krucher explained to me her hopes for her current work with 3-D’s. At the end of last semester, she informed me that another method of killing off cancerous cells in 3D cultures was needed and that she was looking into one that involved some drug. Currently, we are working with special plasmids of different cell lines and adding the drug called Dox (Doxycycline) to slowly diminish those cells. The plasmids are injected into the cell lines and must have a signal bind to the promoter region to initiate a response. I believe there is a fluorescence (Dharmafect) involved that will glow a green shade with the presence of protein or Rb in the breast cell lines. With PNUTS KD initiated in the cells lines again, there should be less of a green presence in the cells because they are undergoing apoptosis, or at least that is the hopes of this experiment. I am still new to this concept especially due to the fact that this technique is not set in stone with Dr. Krucher in regards to its functionality.
Currently, I am working with hanging-drop plates, which is probably one of the more tedious and challenging techniques I have been taught so far. Hanging-drop plates are difficult because they contain spheres of clumped cells that can easily drop through the wells and onto the bottom part of the plate. Eventually the spheres must be poked through each well, collected in a tube, run a protein assay to see how much protein I have in total, then run a western blot to see if Rb is present in my sample. However, I have retrieved some exciting results for Dr. Krucher through my most recent western blot. In essence, I feel I am finally contributing worthy results for my faculty advisor, even though she always does tell me I am doing a wonderful job and to not get discouraged when something doesn’t seem correct. Recently, Dr. Krucher has asked me to help out with her experiments as well while she is in lecture, which elates me and shows she is comfortable with me in the lab and the utilization of all materials on my own.
As I continue in my research, I have come to find that I enjoy it more and more each day. There is always something new to learn in research in general, and being that I am an avid learner, I am always willing to soak in new information in an exciting manner. Additionally, even though I am still learning new techniques here and there, I have becoming increasingly better in my performance in general which essentially makes me feel a bit more confident and have a more enjoyable experience. My coworker and I have even become closer and connect very well with each other. Even though there have been some challenges or set backs here and there such as the 3D’s not allowing enough cell death by PNUTS KD, this experience has been a positive, pleasing, and intellectual one so far.
Even though I have only been working about a semester in the research lab, I have learned an abundance of information and techniques in this short period of time. Personally, I feel I have made much progress already in the lab. Everything I partake in is on my own after I am shown what to do for the first time. This way I am able to learn more quickly and provide me the opportunity to learn for myself exactly how certain techniques are performed. More specifically, the techniques I have been taught and now mastered are Protein Extraction, Cell Lysis, Protein Concentration Assay, running of gels and the Western Blot, and 3-D culturing. 3-D culturing is difficult to master in the beginning due to the fact that you are working with Matrigel which is an extracellular-matrix material that changes between a more solid or liquefied substance based on the surrounding temperature. Therefore, culturing and feeding cells in such an environment must be taken with great care or then there will be no cells to work with for the experiment. Protein Extraction is a technique that dissolves Matrigel in order to release the cells and thus releasing the protein from them. Taking the samples from the Protein Extraction, a Cell Lysis procedure is performed to open up the cells to allow the proteins to be released followed by a Protein Assay, or a standard curve, to examine how much protein is truly in our sample as compared to the standards. After we determine how much protein is found in our sample, we can introduce the protein sample, a standard, and whatever other components are necessary into a gel and examine later in a Western Blot. Gel electrophoresis separates proteins by size and the Western Blot is used for protein analysis by identifying specific proteins with the use of monoclonal antibodies. Once the blot is completed, results of how much protein exhibited are apparent. These results allow me to conclude whether I received my expected results or if an error occurred due to my technical performance.
At this point in the semester, I feel I have obtained significant data even though my techniques and experiments have been mutually the same and repetitive each week. Repetition is not a bad thing though because through repetition I am perfecting my techniques and learning more and more with each recurring experiment. Especially with the research I am conducting in, I must make sure to keep a close eye on every detail and be very cautious and careful to not make a mistake due to the fact that these materials are super expensive. I have performed a few protein extraction, cell lysis, and protein assays, have cultured and fed MCF7 cells in 4 different slides over a span over several weeks, ran 2 gels and performed 2 western blots with varying times and examples per experiment. With each new experiment I run, my results become closer and closer to being more precise. At this point in time, I feel very comfortable with the proper techniques in order to carry out a full experiment with ease. I thoroughly enjoy everything I am working on and conducting in and each day I am more and more fascinated by my area of research. There is still so much to know and understand about breast cancer research and cancer in general therefore I strive to learn something new each day and continue to do so in the future.
Even though everything may seem controlled and understood with my research, many questions still arise here and there as I finish each experiment and move onto the next. A few weeks ago, my mentor, Nancy Krucher, mentioned to me that she is working on finding a new drug or procedure that can be introduced into 3-D cultures in order to induce apoptosis within the cells. PNUTS KD was recently found to not work in the 3-D cultures as it did in the 2-D cultures. Therefore, she must find a replacement product in order to continue on with our experiments with 3-D’s. In the meantime, I have been working on perfecting my techniques which I have successfully done. If I recall correctly, I do remember Dr. Krucher mentioning that she has an idea of something that might work but it does take several weeks to initiate. If her sample experiment seems to provide her with the expected results, then she will inform me of such product and allow me to begin working with it in conjunction with my own experiments. Overall, my general questions would be centered on what else I am able to learn at my stage in research. I am ecstatic to see what else is in store for me, what new product will indeed induce apoptosis in my breast cells, and to be able to continue working with Dr. Krucher.
The proposed title of my research is as follows: “Induction of PNUTS Knockdown in 3-D cultures of acinar-structured breast cells leads to dephosphorylated Rb.” The purpose of my project is to observe the growth of breast cells in a 3-D environment as well as introduce a protein called PNUTS into the culture to examine if the expected phosphorylation of Rb and apoptosis occurs. I have some research providing support for apoptosis through induction of PNUTS siRNA into two different breast cell lines (MCF7’s & Hs578T’s) causing activation of Rb-directed PP1 activity thus leading to dephosphorylated Rb and finally apoptosis all in a 2-D environment. Therefore, I am expanding on these results and shifting from a 2-D environment to a 3-D one and focusing on if Rb can indeed be dephosphorylated and if apoptosis will continue to occur through these similar procedures.
Due to the fact that conducting in research takes a large sum of time to develop skills for and to gain an abundance of knowledge, my goals are to become completely familiar and comfortable with all of the necessary techniques for my project as well as develop a larger collection of facts and information on breast cancer research in general; hopefully I am able to accomplish these two goals by the end of this semester. Additionally, I hope to obtain a lot of useful and credible results not only for myself but also for my mentor, Dr. Nancy Krucher, to utilize for her own work. A few objectives I have for my project are to maintain a collection of healthy MCF7 cells as they grow in their cultures, to completely understand the purpose and technique of the western blot within the next week or two, and to oversee and carry out the full experimental design which roughly includes culturing, addition of PNUTS, and the western blot, all within the next few weeks. Furthermore, I hope to gain a positive experience and stronger skill set in the methods of research in addition to growing a deeper passion and acceptance for research and its many unique factors and procedures. I hope to become widely knowledgeable on my topic in addition to be able to effectively decipher other researchers’ results, journals, articles, etc to help me make use and create a better understanding of my own compiled work. Finally, I hope to achieve very substantial and successful results from my project in order to progress further and continue to conduct in research with Dr. Krucher after the conclusion of this specific project.
A few of the experimental methods I will be engaging in are cell culturing, plating and feeding cells, protein extraction in 3-D’s, cell lysis, and protein assays, administration of the PNUTS siRNA protein sequence, and the western blot. I must practice the art of cell culturing in a 3-D environment which is new not only to myself but to many other researchers as well due to the fact that working in 3-D cultures and utilizing 3-D models is a somewhat brand new concept being expanded on in the field of research. The MCF7 cells I am working with are plated in small, plastic, 8 chamber slides fixated with a laminin and collagen matrix called Matrigel. Cells must be fed every four days in order to grow and form clusters that resemble acini like structures which are the true arrangements that are found in the ducts in breasts. Protein extraction will take place in order remove the cells from the Matrigel by dissolving the matrix and forming a pellet of the cells through centrifugation in a microcentrifuge tube. Through cell lysis procedures and protein assays, I will be able to determine the amount of protein in my sample. Once I introduce PNUTS into my experiment, I will be able to perform gel electrophoresis and western blotting techniques in order to examine the protein sequences present in my sample, and more importantly, to center my attention on the activity of the Rb protein.