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WHY THIS TOPIC
Deciding on Triple Negative Breast Cancer (TNBC):
Triple Negative Breast Cancer (TNBC) is more difficult to treat because of its lack of estrogen, progesterone, and human epidermal growth factor 2 receptors (Jerusalem et al., 2016). These receptors are targeted in traditional treatments, making TNBC more important to study (Walter et al., 2020). Different stages of TNBC can behave differently and may react to treatment differently. Stages I and II occur when the cancer is still localized in the tissue, and in stages III and IV, the TNBC has metastasized and begun to spread to other parts of the body, typically via the bloodstream (National Breast Cancer, 2025). This research will specifically look into later stages of breast cancer where the cancer has metastasized, as it can be more difficult to treat and has a worse prognosis for patients. For localized TNBC, there is a 91% survival rate; however, TNBC that has metastasized has a 12% survival rate (American Cancer Society, 2025).
Deciding on Onametostat:
PRMT5 is a type II protein arginine methyltransferase, meaning it adds methyl groups to monomethyl arginine to create SDMA (U.S. National Library of Medicine, 2025). PRMT5 aids in mRNA splicing, DNA replication, cell signaling, and gene expression, which can promote cancer cell growth and cancer progression (Li & Pützer, 2008). PRMT5 is overexpressed in TNBC, causing more rapid growth and proliferation (Wang et al., 2023). When acted upon by Onametastat, a selective PRMT5 inhibitor, there is a decrease in methylation marks. This leads to changes in gene expression, improper splicing, and accumulation of faulty transcripts. In addition, there is increased DNA damage and less DNA repair, causing cell cycle arrest where the cell won't finish through the G1-S phase of mitosis (Hanard et al., 2018).
Deciding on E0771 cells:
E0771 cells are a cell line derived from spontaneous TNBC of C57BL/6 mice (Le Naour et al., 2020). E0771 cells are Lumina B cancer, which tend to grow faster, be more aggressive, and are more likely to recur, in addition to a poorer prognosis for patients (Cleveland Clinic, 2024). This makes E0771 cells are relevant model for TNBC. There are other types of cells that I could have used; however, my mentor from CU Anschutz was able to give me access to E0771 cells, making them the perfect choice for my project.
WHY WE CARE
On average, a women is diagnosed with Breast Cancer every two minutres in the United States (National Breast Cancer, 2025). It is predicted that in 2025, 316,950 women and 2,800 men will be diagnosed with breast cancer (National Breast Cancer, 2025). About 10-15% of all breast cancer diagnoses are TNBC (American Cancer Society, 2025). Because of its aggressive nature, prevalence and high mortality rates, there should be increase efforts in search of a better treatment. TNBC has a low five-year surival rate and a high recurrence rate (De Ruijter et al., 2010).
PREVIOUS RESEARCH
A study published in Nature News found that Onametostat can help to treat glioblastoma: an aggressive form of brain cancer (Lavogina et al., 2024). In addition, studies on lung cancer determined that PRMT5 is targetable for treatment specifically by utilizing Onametostat to target it (Brehmer et al., 2021). While Onametostat has shown promise in other cancer types, there is currently no research on the effectiveness of Onametostat in E0771 cells. This is important because all cancer types can respond differently, which can provide more insight into how other cancers will respond.
This is what a 96-well plate looks like
This is a hemocytometer, the cells are pipetted into the tiny slit in the middle at the bottom
This is what the grid of a hemocytometer looks like under the microscope
METHODOLOGY
The independent variable of this study is the 0.00025 μM concentration of Onametostat that will be added to the E0771 cells. The dependent variable is the growth of the cells after the 48-hour exposure period. The negative controls are the untreated cells where no Onametostat will be administered, leaving the cell growth uninterrupted. The constants of this experiment include: the 96-well plates where the drug will be administered, the cell culture protocols, the conditions in which the cells will be kept (5% CO2 and 37℃), and the non-culture-treated flasks (PHC, 2023).
Culturing Cells:
Cells will be stored in an incubator at 37℃ and 5% CO2 to mimic internal body conditions. Whenever I am working with that, all procedures will happen in a biological safety cabinet to help prevent contamination. Cells will be grown in suspension cultures. Suspension cultures are where cells are suspended in the media. This mimics later stages of breast cancer, such as stages 3 or 4, where the cancer has metastasized in the bloodstream. Cells will first be grown in a T25 flask, which means it is 25 cm squared. Once they reach 80% confluency, they will be transferred to a T75 flask, which is 75 cm square. This is because the cell growth is safer when there are more cells per square centimeter due to cell communication in growth. Once a T75 flask reaches 80% confluency, it will be split so the cells are in two separate flasks. This can be repeated until there are 4 flasks, for trials. While cells grow, media health will be monitored. The media will start out as a pinkish-red color, but as the cells suck up the nutrients to grow, it will start to turn a yellowish color. Once the media has changed color, it will be swapped with fresh media in order to give the cells enough nutrients to grow. Media will be composed of 450 mL RPMI, which is the main nutrient, 50mL fetal gro which provides proteins for the cells, and 5 mL penicillin, which is an antibiotic to help with contamination.
Trials:
For trials, one flask will go into one 96-well plate. Due to the edge effect, which is the idea that the wells on the outer edges of the flask will experience evaporation, only the inner 60 wells of each plate will be used. 30 of these wells will be controlled, and 30 wells will be experimental. Each well will receive 220 μL of cells with media, the only difference being the type of media. The control wells will be cells with the same media that they were grown in. The experimental cells will be in media that has a 0.00025uL concentration of Onametostat. The media will be pre-drugged with a 5-step dilution, starting with 1mg of pure Onametostat and the RPMI growth, and ending with 50 mL of RPMI media with a 0.00025μL concentration of Onametostat.
I will be conducting a cell growth assay, so cell counts will occur before and after the 48-hour exposure period. To collect cell counts, out of each well, 20 μL of media and cells will be extracted and placed into a microcentrifuge tube. Then, the microcentrifuge tube will receive 20 μL of trypan blue and be mixed. Then, 10ul of this cell and trypan blue mixture will be pipetted into a hemocytometer and placed under a microscope. This is what a hemocytometer looks like, and this is how it will look under the microscope. It allows the collection of cell counts to be analyzed over a certain amount of space. I will be counting cells on the upper and right lines. The trypan blue causes the dead cells to glow red, so those will be what is counted. This process will be repeated for all 60 wells. Then the 96-well plate will be placed in the incubator for 48 hours at 37℃ and 5%CO2. Then, after the 48-hour exposure time, the same process of trypan blue and hemocytometer will be repeated again for all 60 wells. I am conducting 3 trials, so this whole process will be repeated 3 times, with 3 separate plates, resulting in a total of 180 data points. 90 of which are control, and 90 that are experimental.
Data Analysis Plan:
These trials will consist of a primary cell count and then a secondary cell count after 48 hours, and the p-value of each trial will be recorded. The initial cell counts will be compared to the secondary cell counts of their respective wells. This data will be collected through three trials, with 180 data points being collected in each trial. These data points are composed of 90 control and 90 experimental. Statistical significance will be identified using a 2-sample left-tailed t-test between the control and experimental groups with a p-value of 0.05 or less. The equation: average # of cells per mL = (0.25T)10,000 will be used to calculate the number of viable cells in each sample, with T being the number of cells initially counted in the hemocytometer.
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