Results and Data
April 12, 2025
All the data, pictures, graphs, and other results compiled in this project.
Verification of IL-22 Knock out
To begin, the presence of MDSCs around 4T1 cells was verified by staining the MDSCs and inputting the culture into the cell sorter. This established that MDSCs did, in fact, congregate around cancer cells, specifically 4T1s. In mice without a tumor, there was a median of about 2.5% MDSCs in the spleen, around 0% in the blood, and 10% in the bone marrow. In mice with 4T1 tumors, there was 9% MDSCs in the spleen, 38% in the blood, 30% in the tumor, and 38% in the bone marrow. This shows a substantial increase in the amount of MDSCs in healthy mice versus cancerous mice. The next step was to verify and quantify the IL-22 expression of 4T1 cells (Figure 5). A batch of unstained cells acted as a control and showed 0.487% IL-22 expression. On the other hand, 62.509% of the stained culture represented IL-22, indicating that the stain did work and MDSCs were present. Since the control had a near 0% in the stain range, the cells are not naturally that color, meaning that 62.5% of stained cells are all MDSCs: the stain specifically targeted MDSCs.
After the cells were transfected with the modified plasmid, the presence of the plasmid was verified through studying the fluorescence of the GFP gene addition using a confocal microscope. The green “blurs” and “spots” are cells that have been successfully modified, as shown in Figure 6. In order to determine the exact percentage of cells that fluoresce, the cell sorter was the primary device used. The percentage of normal 4T1s that were green was 0.24%, which acted as a control to prove that the cells do not fluoresce on their own. The modified 4T1 cells were 5.05% green, representing the amount of cells that incorporated the modified plasmid.
The cells were also modified with TdTomato, a genome addition that fluoresces red, in order to track the progress of cancer cells once replaced in a mouse. If the 4T1 cells were taken out again, the modified cells could be verified through their red color. It would facilitate research and the collection of results of the tumor once it became metastatic. The red cells seen through the confocal microscope indicate that the stain did work, and the cells would be identifiable for future application and research.
Discussion
The presence of MDSCs near 4T1 cells was confirmed, and supported established research. The significant increase in healthy mice versus mice with cancer was distinct. This data was expected, as previous studies have already shown the correlation between cancer and MDSCs. The correlation between cancer cells and IL-22 production was also obvious. The unstained cells, which acted as a control, had a less than 1% fluorescence, which means that the cells don’t naturally glow in that color range; however the stained cells were 62.5% which is a clear indication that MDSCs do congregate around 4T1s.
Once the modified 4T1s were examined in the cell sorter, only 5% displayed the GFP addition. This number is higher than the control fluorescence of 0.24%, but not significantly enough. The low percentage of green fluorescence means that the IL-22 knockout, i.e. the plasmid transfection was not sufficiently positive. The low percentage of transfection is not usable for further research or to test the hypothesis. In order to examine and conduct a proper and complete gene knockout, transfected cells must not grow to confluency. Unfortunately, our 4T1 cells grew to near 100% confluency before transfection, which meant that even if the cells integrated the plasmid with their genome, the modified cell colonies would not be able to grow. Thus the knockout was not positive enough for further research. After successfully transfecting the cells, we would have waited for the individual cell lines to grow to confluency which would allow spare cells in case something went wrong; however, the time frame of this project did not allow for growth. Thus, in-depth research and results on the outcome of the IL-22 knockout was not possible. Primary data and research did not proceed far enough to support or deny the initial hypothesis.