{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block page_content %} Inspired by CAR-T cell therapy, we have developed a chimeric antigen receptor protein to treat cancer cachexia. We have chosen to target the cytokine IL-6 because it is a main immunoinflammatory cytokine that triggers signaling pathways that have been traced to the degradation of muscle and fat cells. We have successfully demonstrated expression of the CAR protein in HEK293 cells and binding to our 3xFLAG-IL6-mCherry protein via flow cytometry.
Representative photos of our first attempts are to the right.
Figure 1: First attempt at transfecting
HEK293 cells with 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR
After many iterations of plasmid construction, we ended up seeing substantial fluorescence from all three of our constructs! Representative photos from transfection of our finalized designs are to the right.
Figure 2: Final transfection of HEK293
cells with 3xFLAG-IL6-mCherry (left) and anti-IL6 Megf10 CAR
Figure 3: HEK293 cells transfected with our 3xFLAG-IL6-mCherry fusion protein
Figure 4: Incubating CAR HEK293 cells with 3xFLAG-IL6-mCherry freeze-thaw lysate
Figure 5: Incubating CAR HEK293 cells with IgG-eluted 3xFLAG-IL6-mCherry
This phase of our project was full of the most unknowns. We would not be following any other lab’s protocols nor be able to reference multiple papers that cited running similar experiments. Ultimately, we had three types of IL-6 protein solutions we wanted to try incubating with CAR-expressing HEK293 cells:
Figure 6: Incubating CAR HEK293 cells with 3xFLAG-IL6-mCherry freeze-thaw lysate
Ultimately, we have successfully achieved a proof-of-concept for the IL-6 binding component of our therapeutic
by demonstrating that IL-6 binds to CAR-expressing cells significantly more frequently than to control cells.
It took a few attempts to run everything smoothly, but our final protocol followed a precise timeline that
allowed us to see encouraging results!
Day 0: We transfected a 10cm dish of HEK293 cells with our 3xFLAG-IL6-mCherry fusion protein. mCherry
control wells were transfected too.
Day 1: We transfected a 12-well plate of HEK293 cells, half of the wells with our anti-IL6 Fcγ CAR and
half with our anti-IL6 Megf10 CAR. GFP control wells were transfected too.
Day 2: We extracted the cells of the IL6-transfected dish using a cell scraper and processed the cells
in 2 different ways.
Figure 7: Negative control backgating.
First, we used negative control cells to separate cell debris from actual HEK293 cells in our data. This was performed under the assumption that debris is significantly smaller in size than actual HEK293 cells. Thus, we strictly gated for the area around the hotspot and above, but discarded the area that tailed underneath. Retroactively, we applied our eGFP+ and mCherry+ gating to the negative control population to verify our gating stringency; as expected, neither eGFP+ nor mCherry+ cell populations were observed in the negative control HEK293 cells.
Figure 8: eGFP control backgating.
In Figure 8, we next used eGFP-expressing cells as a positive control. We intended to gate for green fluorescing cells (eGFP+) in this population, but due to the realization that we did not have a clear second peak representing the eGFP+ population, we decided to use this population to gate for mCherry+ instead. We knew these eGFP+ cells had no mCherry expression; therefore, our mCherry gate was decided where there was no fluorescent signal on the histogram. In the same manner, we determined our eGFP+ gate by using the mCherry-expressing population and selecting the portion with low to no mCherry signal. Retroactively, we verified that the majority of the eGFP-expressing population was correctly gated for eGFP+, and similarly for mCherry+.
Figure 9: Megf10 CAR with IgG-purified IL-6 backgating.
Figure 9 corresponds to cells transfected with our Megf10 CAR and incubated with IgG-purified IL-6. We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
Figure 10: Megf10 CAR with IL-6 freeze-thaw lysate backgating.
Figure 10 corresponds to cells transfected with our Megf10 CAR and co-cultured with IL-6 freeze-thaw lysate. We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
Figure 11: Fcγ CAR with IgG-purified IL-6 backgating.
Figure 11 corresponds to cells transfected with our Fcγ CAR and incubated with IgG-purified IL-6. We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
Figure 12: Fcγ CAR with IL-6 freeze-thaw lysate backgating.
Figure 12 corresponds to cells transfected with our Fcγ CAR and co-cultured with IL-6 freeze-thaw lysate. We first subsetted the entire population to only HEK293 cells, removing debris. Next, we gated for cells which were eGFP+, implying CAR expression. We then further subsetted this green population to cells which also fluoresced red (doubly positive), implying that the CAR bound to our fusion IL6-mCherry protein. As desired, a significant portion of the entire population fluoresces both green and red, indicating successful binding.
Finally, we aggregated the above data and concluded with exciting results: a significant percentage of cells expressed CARs and bound IL6-mCherry!!
Figure 13: Control and experimental cell populations positive for both green and red fluorescence. Being positive for both implies successful CAR expression and ability to bind IL-6.
From top to bottom, the populations are:
From the final chart comparing our different variables against each other, we can conclude that the Fcgamma CAR
incubated with IL-6 freeze-thaw lysate had the greatest double positive frequency of the parent population, but
the other three samples were significant as well and not too different from each other. We are pleased to find
that the incubations with freeze-thaw lysate were both higher than the incubations with IgG-eluted IL-6. This
supports our suspicion that the protein purification protocol had annihilated the concentration of IL-6 in those
solutions. We suspect that the manyfold greater concentration of IL-6 in the freeze-thaw lysate improved the
probability of CAR binding events.
Additionally, we were eager to compare the behavior of the Fcgamma vs. Megf10 intracellular component. Although
it is important to note that we were not able to compare its impact on the phagocytotic behavior of a
macrophage, we theorize that the intracellular component had an impact on the folding of the protein or even the
binding affinity of the protein. Due to the limited scope of the experiments we ran, we cannot conclude which is
more optimal than the other; we determine that they are both great candidates to move forward with and perform
macrophage experiments with. Based on phagocytic activity, it may become more apparent which CAR is preferable.
The precise protocols that we followed are detailed on this page and on
our experiments page and lab notebook. We have made every effort to thoroughly document our experiments
and ensure other researchers can reproduce our results, and we welcome questions or clarifications
addressed to igem-2023-students@mit.edu
For replication of our experiments, we recommend preparing all of the possible controls to account for all variables of the project. Namely, to test for binding specificity, we suggest incubating CAR+ cells with whole cell lysate (ideally with some sort of fluorescent reporter) for 6 hours and comparing the double positive data with the data we have achieved.
Moreover, it is unclear why we observed significant loss of protein yield during the two-step M-PER lysis protocol and anti-FLAG protein purification protocol. We urge future researchers to test anti-FLAG purification of the freeze-thaw lysate, with the goal of understanding where yield is lost. It is possible that the loss of protein lies in the M-PER lysis protocol. Additionally, if it is found that the FLAG tag is the issue, we urge the creation of a similar IL-6 protein tagged with an amino acid chain that can assist in a different method of non-denaturing purification.
We are extremely proud of having achieved a proof-of-concept that HEK293 cells expressing our CARs bind IL-6 significantly more strongly than cells lacking our CARs. Due to constraints on our time and resources, we were unable to progress further than this level of achievement. However, we have planned potential next steps that can be taken to achieve the loftier goal of developing a real, practical cachexia therapy.