Results

2. Transformation and miniprep We first transformed our assembled plasmids into DH10b, but sequencing results show that DH10b performed not accurately on our shRNA fragments. We further chose the NEB stable for shRNA and got our plasmids for SIRPA KO tests. To increase the efficiency for the large plasmids(15kb+), we also troubleshooted and extended the recovery time during transformation to two hours and obtained desired results.

3. Plasmid Verifications After miniprep, we use PCR to amplify specific fragments to verify the accuracy of our plasmid. We used specific primers and NEB Q5 polymerase to make the selection master mix, this improved our verification efficiency. Figure 3 shows the PCR verification of CAR system and nsp system. We can observe that the plasmids from all the selected colonies have the correct size of the nsp fragments, and plasmids from 2 of the 12 colonies have the correct size of the CAR fragments. Figure 4 shows the PCR verification of the ribo-shRNA system, from which we can see that 4 of the 10 colonies have the correct size of the ribo-shRNA fragments.

We have also tried the colonies PCR to save the miniculture and miniprep resources. The defects of colony PCR is that we need to keep half of the colony on the plate for miniprep and this is hard to operate on small colonies.

However, verification PCR can only verify the size of the fragments and is not appropriate for small fragments. In order to make sure our plasmids are accurate enough, we further used sequencing to validate the plasmids performed well in PCR verification. As shown in Figure 5A and 5B, the nsP and nanoCAR system have identical sequences as expected. For ribo-shRNA system, some insertions were still observed after several attempts of troubleshooting, including changing bacterial strain from DH10b to Stbl.3, which is capable of cloning toxic and repeated sequences. Indeed, this is not the best case as these insertion mutations could bring uncertainty. Theoretically, however, a few extra bases upstream of hammerhead ribozyme and downstream of HDV ribozyme should not affect the functionality of the self-cleaving shRNA. The HH ribozymes, HDV ribozymes, and SIRPa shRNA are both intact, without any mutations. Due to time constraint and limited resources, we had made the difficult decision of continuing to use these ribo-shRNA systems.

2. Puromycin Killing Curve Our srRNA are designed to include a puromycin resistance gene, which gives transfected THP-1 an advantage in puromycin stress. In order to distinguish the cells with successful srRNA transfection, puromycin incubation was necessary to kill all THP-1 that do not confer puromycin resistance. The critical concentration of puromycin was determined by checking the percentage of viable cells after adding different concentrations (0-10 ug/mL) of the antibiotic to confluent THP-1 (1 to 5*105 cells/mL) for 7 days. The initial and final cell concentration were calculated via trypan blue cell counting at Day 1 and Day 7. Each concentration was done in triplicate, and the error bars indicate standard deviation. Referring to the Killing curve below, the critical concentration was found to be 5 ug/mL, which was chosen as the selection concentration for all future transfection. The figure below shows the microscopic images (100X) of THP-1 during the construction of the killing curve. The dead cells appear to be darkened, and smaller in size. An obvious trend of increasing proportion of dead cells can be observed when we move up to higher puromycin concentration. The images were taken before trypan blue cell counting at Day7.

3. srRNA Transfection Three types of srRNA were transfected into the wild-type THP1 by us; here are their main functional components.

• CAR srRNA: nsp 123 and nsp4 (which provide self-replication and TMP control function);
Chimeric Antigen Receptor (CAR) system; Puromycin resistance.
• nanoCAR srRNA: nsp 123 and nsp 4; nanobody Chimeric Antigen Receptor (nanoCAR) system; Puromycin resistance gene.
• SIRPA KO shRNA srRNA: nsp 123 and nsp 4; SIRPA KO shRNA; Puromycin resistance gene.

The detail function of each system has already been introduced in Engineering parts.

Following in vitro transcription, the srRNA was transfected into THP-1 via either electroporation (Neon™ Transfection System by Thermofisher Scientific) or lipofection (Lipofectamine™ 2000 Transfection Reagent by Thermofisher Scientific).

The parameters of electroporation were chosen after thorough literature reviews, which is 1700V, 20ms, 2 pulses based on Laoharawee et al. (2022). Not to our surprises, the cell viability after electroporation was low, as shown in the photo below (100X), taken 18 hours after transfection. It is obvious that the morphology of most cells do not look spherical and regular, many of them appeared to be smaller and darker than usual. Extra 2-3 days of recovery was given to the cells for returning to a healthy state. Regarding the high level of apparent cell damage from electroporation, we also performed lipofection to transfect srRNA during the extra recovery time. The cells appeared to be in normal condition, with regular shape as shown in photo below (100X). As the transfection efficiency is not 100%, some of the cells died after addition of puromycin, we centrifuged at lower speed for a longer time to remove the dead cells. After one or two passages, most of the cells were able to survive in the puromycin added medium. This proves that our srRNA was successfully transfected into the cells and kept expressing in the cells. Whether the rest systems work and can enhance the immunoreaction need further co-culture and flow cytometry tests to validate.

4. M0-macrophage Differentiation Following recovery and puromycin selection, the trasnfected THP-1 underwent M0-macrophage differentiation by incubating with PMA for 24 hours, and then switching to PMA-free medium for another 48-72 hours. As shown in the microscopic images above (100X), THP-1 displayed similar morphology to M0-macrophage after 24 hours of PMA incubation, which is commonly characterized as circular and slightly elongated (Hourani et al., 2023). Also, the migration from suspension to adherent nature further proves the successful differentiation. M1-macrophages are classically activated by IFN-γ and LPS, which have a dendritic-like, elongated morphology with protruded pseudopods (Hourani et al., 2023). Referring to the photos above, the macrophages showed a distinct difference to M0-macrophage after 72 hours in PMA-free medium. Under light microscope, the macrophages show similar shape and features with M1-macrophages. This could be a very initial and rough indication that our srRNA system continuously secretes IFN-γ to polarize M0- into M1-macrophages, since IFN-γ or LPS were not present in PMA-free medium. However, it should be noted that some M2-macrophages, like M2a and M2b, also shared similar elongated morphologies (Hourani et al., 2023).

Therefore, light microscope inspection of macrophage morphology can only serve as a rough and brief hint of M1-macrophage polarization. Nevertheless, these CARma macrophages were subjected to co-culture testing with HepG2-GFP. The promising result of co-culture, detailed in the next section, also served as an indirect, rough indication of phagocytic, anti-tumour M1-macrophages.

5. Co-culture 5.1 Phagocytosis observation Before quantifying co-culture, we firstly observed the phagocytosis under the fluorescence microscope. The HepG2 we chose for co-culture contain green fluorescent protein(GFP) gene, they will produce green fluorescent signal under blue light(about 488 nm). We observed the cells under normal light and UV light to identify the cell type.As shown in Figure 5.1, 5.2 and 5.3, we can observe that THP1 attaches with HepG2-GFP and this can be considered as phagocytosis. Some ongoing phagocytosis can also be observed (Figure 5.3), however, we can only observe the cells at 25 degree environment so that THP1 performs low activities and we cannot observe the whole phagocytosis process.


5.2 Quantifying phagocytic effect analysis In order to test if our CAR system can enhance the phagocytosis of cancer cells by macrophages and whether our TMP system works, CAR/nanoCAR transfected THP1 and HepG2-GFP are grown in a Direct Co-Culture manner, GFP signal was regularly measured as well(excited at 488 nm). As table 1 shows, we co-culture wild-type THP1 (WT), CAR srRNA lipofection transfected THP1 (lipofection CAR group), nanoCAR srRNA electroporated THP1 (electroporation nanoCAR group), and nanoCAR srRNA lipofection transfected THP1 (lipofection nanoCAR group) with HepG2-GFP in wells contain 180uL of the culture medium of the same formulation (M10:RPMI=1:2), and added 14.4 ug of the trimethoprim (TMP) to half of the wells.

To improve the confidence of our data, we did a triplicate for each group.

A. Validation of the phagocytosis of THP1 Figure 5.4 shows the changes in fluorescence intensity in each well over time. The control group is HepG2-GFP seeding in the same medium as the experimental groups. We chose lipofection CAR in TMP free medium as the representative to see the phagocytosis condition. We found that all of the wells in this group have lower fluorescence intensity than the control all the time, which means the amount of HepG2-GFP in experimental groups is lower than the control. This indicates that the cells after transfection have a good phagocytic effect.

Moreover, we want to test whether our CAR system enhances the phagocytic effect of THP1 compared to the wild-type THP1.

B. Validation of the effect of CAR system To identify whether the CAR system improves the effect of phagocytic, we compared the change rate of the growth of GFP signal between 0h-19h and 19h-44h. As shown in Figure 5.5, the growth of the GFP signal decreased in the nanoCAR groups and the WT group, while nanoCAR transfected THP1 reduced the growth of the GFP signal more significantly. We can infer that the CAR system did improve the effect of phagocytic. At the 43rd hour of co-culture, we refresh the medium (keep the TMP concentration) to ensure the cells can stay in good condition and avoid the influence of cellular metabolites. We measured the GFP signal 23 hours later and compared the change in the growth of the GFP signal between 66h-43h (after refreshing the medium) and 43h-19h to further prove our previous conclusion.
Figure 5.6 indicates that there is a certain degree of recovery of the growth of HepG2. The increased extent of the growth of the GFP signal in the two nanoCAR groups is less than in the WT group, which means both the lipofection nanoCAR group and electroporation nanoCAR group restricted the growth of HepG2 better than the WT group. We also did the two-sample t-test and calculated the p-value. From the result, we can infer that the CAR system can enhance the phagocytosis of THP1 significantly. (95% confidence interval for lipofection nanoCAR, 97.5% for electroporation nanoCAR)

C. Validation of the TMP system We want to further explore the influence of TMP on the cells. We compared the growth of the GFP signal after 43 hours of co-culture(Figure 5.7a). Among all three experimental groups, the growth of the GFP signal slowed down when adding 0.08 mg/mL of TMP into the co-culture medium. From previous results, we have concluded that THP1 with the CAR system can depress the growth of HepG2. Combining this conclusion with the data we observed in Figure 5.6, we can infer that TMP can depress the expression of the srRNA transfected into the cells. As we mentioned before, we refreshed the medium at the 43rd hour of co-culture. To validate our conclusions, we did the comparison again after 88 hours of the co-culture(Figure 5.8). The results remain in line with our expectations. The two-sample t-tests indicate that the difference between TMP-free and adding 0.08 mg/mL of TMP is significant. (97.5% confidence interval for lipofection nanoCAR, 95% for electroporation nanoCAR)

D. Validation of the effect of nanobody In addition, the impact of nanobody on the CAR system was analyzed (Figure 5.7b and Figure 5.7c). The growth of the GFP signal in both nanoCAR groups is less than in the lipofection CAR group, and we can observe the same effect after the addition of TMP. However, the results of the two-sample t-test were relatively non uniform. The significant difference can only be ensured between the two lipofection groups. These data partially indicate that the introduction of nanobody can enhance the function of the CAR system. As we mentioned before, we refreshed the medium at the 43rd hour of co-culture. To validate our conclusions, we did the comparison again after 88 hours of the co-culture(Figure 5.8). The results remain in line with our expectations. The two-sample t-tests indicate that the difference between TMP-free and adding 0.08 mg/mL of TMP is significant. (97.5% confidence interval for lipofection nanoCAR, 95% for electroporation nanoCAR)

E. Limitation and further improvement We use DMSO to dissolve TMP and add it to our culture medium as trimethoprim(TMP) is an organic which is hard to dissolve in the culture medium. Initially we plan to further detect whether there is a quantitative relationship between the concentration of TMP and the expression of srRNA. However, higher concentrations of TMP need a higher amount of DMSO to dissolve. Due to the previous test, 1.5% of DMSO showed a 10% inhibition of cell growth (Tunçer et al., 2018). We only analyze the data from the well with 0 mg/mL and 0.08 mg/mL TMP concerning the toxicity of TMP to the cells. In the future we will try to find another more capable solvent and further test the quantitative relationship, this will also help the next step in animal testing. In addition, the results only indicate that there is a significant effect of applying nanobody when we compare the lipofection nanoCAR group with the lipofection CAR group. There will be a higher degree of credibility in the conclusions if we supplement the data of the electroporation CAR group and compare it with the electroporation CAR group.

F. Conclusion

1. The THP1 that we transfected CAR system srRNA in can have phagocytosis with HepG2 and perform better than WT THP1.
2. After the introduction of the K-turn system into srRNA, we can depress the expression of srRNA by addition of TMP.
3. Nanobody can improve the phagocytic effect.

Remarks: the two-sample t-test we did in our analysis. In order to have a better approximation of p-value, we used the following linear interpolation: Where p1, p2 and t1, t2 are the value in the p-value table, p, t are the value of the sample.