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The following information contains the conclusion to my Pre-Trials, the current progress of my Experimental Trials, and my plans for the conclusion of my experiment.
To Recap...
I am investigating how the addition of the antibiotic Novobiocin Sodium Salt to a PARP inhibitor treatment of RIN-m Rat Beta Cells with Insulinoma impacts the health of the cell culture. PARP inhibitors causing fatal oxidative stress in a subset of tumor cells with deficiencies in DNA repair. PARP inhibitor resistance in tumor cells is an emerging problem, and the inhibition of the DNA repair by Novobiocin Sodium Salt is a potential pathway for reversing resistance. This research will contribute to the academic conversation around increasing PARP inhibitor efficacy in tumorigenic cells, in order to improve their cancer treatment potential.
Pre-Trials
When I left off in my last blog post, I was waiting on a batch of the RIN-m cells to continue multiplying to 80% confluency (80% of the surface area of the flask covered in cells), the milestone for transferring the cells to my dosage testing. RIN-m cells grow slowly, taking anywhere from 7-14 days to reach confluency (Picture 1). I said in my last blog post that I was predicting the cells would be ready to split by 01/31/2022. They were about 70% confluent on the 31st, but my school was canceled for two days in a row (Wednesday, 02/02, and Thursday, 02/03). This prevented my dosage testing until 02/09/2022, stalling my research for one week (Picture 2). The results of my dosage testing are in the table in this document. From this data, I found two conclusions:
Trial Concentrations: 230 uM Olaparib, and 300 uM Novobiocin. This is the concentration that yielded the first noticeable effect at the least concentration, meaning it is the most likely to have the least effect on the NIH/3T3s while having an effect on the RIN-m cells.
Data Collection Timeline Update: Seven days, not 24 hours. I needed more cells to increase the reliability of my hemocytometer assay.
That was the conclusion of my Pre-Trials, ending on 02/13/2022. Healthy NIH/3T3 stock cultures (frozen batches of cells that can be transferred between labs, allowing researchers to obtain multiple cell lines in one laboratory) were still being obtained, so I re-focused on collecting my data for my RIN-m cells. Experimental trials were to begin effective immediately.
Picture 1: This picture illustrates the pattern of cell coverage that is observed when a culture is at confluency. This specific plate took 19 days to reach confluency, due to incorrect temperature calibrations and the characteristic slow growth of cells plated directly from frozen stock.
Picture 2: This picture shows the 96-well plate that I seeded with cells for my dosage testing. The darker colored cells (6 X 5) in the middle contain my cell cultures, while the rest of the lighter colored cells are media blanks that prevent evaporation.
In The Thick of Research
My partially completed plan for experimental trials goes as follows. I am running six trials on 8 different experimental groups, whose treatments and concentrations can be observed on the following table (Table 1) (Picture 3). Each 96-well plate constitutes a trial, with 40 data points possible for each trial. A data point is defined as the experimental results (calculated as the number of surviving cells) per well of the 96-well plate. The data is collected by gathering the cells from each well in a process called passaging, and running the cells through a hemocytometer assay to assess the total number of cells in each well (Picture 4). I can get 40 hemocytometer assays done in about 3 1/2 hours, limiting my ability to run more than two trials in one week.
Currently, I am in the midst of my second and third experimental trial. The lab received new NIH/3T3 vials during the week of 02/20 - 02/26, a culture of which has been established by other Biotechnology researchers (Sophia McHenry, Allie Floyd, Kailey Sprengel, and Kyla Lauretti; check out their research HERE!). While waiting for those cells, I completed my 1st experimental trial on groups 1-4. I also started my 2nd and 3rd experimental trials on the same groups, and am planning on collecting 120 data points by the end of next week (Picture 5). After Rock Canyon's Spring Break, I will be running the remaining experimental trials on the NIH/3T3 control line (groups 5-8). At the end of my research, which should be 04/09/2022, I will have collected 240 data points (30 for each experimental group).
Table 1: This table shows the 8 treatment groups in my experimental trials, and what compounds they are exposed to.
Picture 3: This picture shows me creating experimental treatment solutions in a Biological Safety Cabinet, meant to keep a sterile area through a shield of positive air pressure.
Picture 4: This picture shows cells lying on a hemocytometer grid, with the white circles being live cells and the blue circles being dead cells. This example came from a confluent flask, hence the exorbitant amount of cells on the grid.
Picture 5: This picture shows my second experimental trial, labeled by experimental group.
Experimental Conclusion
Following the acquisition of my 240 data points, the data will be visually represented by a bar graph depicting the average number of viable cells for each group. Because the data is a mean, the data will be analyzed through an independent t-test to determine statistically significant differences between the RIN-m treatment groups (1-3) and the negative control (4). The RIN-m treatment groups and control will be analyzed in comparison to each other, as well as the NIH/3T3 cells (which serve as an indicator of general cell non-cytotoxicity). A statistically significant difference in the viable cell count of the treatment groups would indicate Olaparib and Novobiocin efficacy. The value from this statistical test, called a t-value, will be used to determine a p-value, deducing the chance that the difference between the groups is resultant of chance. A p-value of less than .05 will be considered statistically significant. The possible conclusions from the results of my experiment are as follows:
No statistically significant difference between the RIN-m negative control group and RIN-m treatment groups is found:
This result would indicate that the Olaparib and Novobiocin treatments were ineffective at limiting the cell division in the RIN-m tumor cells. This could be from a total lack of efficacy, an ineffective dosage, or a lack of drug uptake in the cells. All possibilities would be equally likely, and further research would have to be done to figure out the true cause.
A statistically significant difference between the treatment and control groups is found with the RIN-m cells, and not the NIH/3T3 cells:
This result would indicate that the Olaparib and Novobiocin treatments effectively targeted the tumor cells, while leaving the non-tumor cells unharmed. This result would show promise for an Olaparib + Novobiocin tumor treatment, and lead to a replication study with other types of cells.
A statistically significant difference between the treatment and control groups is found with both the RIN-m cells and the NIH/3T3 cells:
This result would indicate that the Olaparib and Novobiocin treatments are simply cytotoxic to all cells, and should not be used in developing a targeted cancer therapy.
In The Spirit Of Research
Research holds a special place in my heart—it is the way to take things that we do not like about the world, and change them for the better. That being said, research is often frowned upon for its very nature. Research is a process of systematic failure, a way to exhaust all possible conclusions until you find one that could be correct. Then, you run with that conclusion until it fails as well, and go back to the board to draw a clearer-than-ever picture of our universe. It is difficult for supporters of research to keep up their support, as things inevitably fall behind schedule, don't work, and go over budget. Thank you for taking the time to read these blogs, it does for for the continuation of research than you know.
Sources
All factual information in this post not directly obtained through my own pre-trials was taken directly from my AP Research Proposal, which can be accessed here.
The research that Sophia McHenry, Allie Floyd, Kailey Sprengel, and Kyla Lauretti are conducting can be viewed here.
All visuals were taken/created by the researcher.
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