Team:Duke/Proof Of Concept


By the end of Phase 1 of our project, we have created proof-of-concepts of multiple aspects of our drug screening system:

  1. We have successfully introduced recombinant plasmids via non-viral delivery into patient-derived glioma cells, which is a cell line that is traditionally difficult to transfect. We demonstrated that foreign DNA can be introduced and expressed by the patient-derived glioma cells with high efficiency and expression. This proof-of-concept suggests that our plasmid reporter system has a high probability of being successfully transfected into the patient-derived glioma cells.
  2. We have successfully established a co-culture system of cancer cells and mini-brain organoids, which will allow us to perform accurate in vitro drug testing. We demonstrated that cancer cells can be grown alongside brain organoid and that several aspects of typical tumor-tissue interactions are recapitulated. This proof-of-concept suggests that a co-culture system has a high probability of being successful in our final reporter platform.
  3. We have successfully performed initial drug testing on our co-culture systems, which has followed expected trends. We demonstrated that our co-culture system can be used to easily and effectively determine the effects of applied treatments on tumor invasion and development. This proof-of-concepts suggests that our final co-culture system, with the integrated reporter, has a high probability of being able to meaningfully quantify drug effects in vitro.

Transfections of Glioma Cells

Although transfections are widely used to transfer plasmid DNA into common cell lines such as HEK 293T cells, plasmid transfections of glioma cells have not been well studied. Limited synthetic biology work has been performed on primary cells, including glioma cell lines and in organoid co-culture settings. Our goal is to optimize a protocol for transferring our plasmids into glioma cells that has high transfection efficiency and gene expression.

Low Lipofection Efficiency of Glioma Cells

We first used Lipofectamine 2000 and Lipofectamine LTX to introduce two established plasmids (pcDNA5-mCherry and FUGW-GFP) into the patient-derived glioma cells. However, we found that there was low transfection efficiency across all conditions, as evidenced by the sparse cells with fluorescence expression (Figure 1).

Figure 1. Lipofection of Patient-derived Glioma Cells using L2000 and LTX with pcDNA5-mCherry and FUGW-GFP

Successful Electroporation of Glioma Cells

Due to the low efficiency of the lipofection protocol, we switched to a different transfection strategy of introducing established plasmids into glioma cells. We used electroporation to introduce FUGW-GFP into the patient-derived glioma cells using the following electroporation parameters that are well-established for other cell lines (Rieger et al., 1998):

  1. 200V, 950 µF - time constant = 40.1 ms
  2. 250V, 950 µF - time constant = 38.1 ms
  3. 300V, 950 µF - time constant = 32.4 ms

Figure 2. Electroporation of Patient-derived Glioma Cells using different voltage conditions (200V, 250V, and 300V) with FUGW-GFP

From our electroporation of patient-derived glioma cells using the parameters described above, we concluded that the 250V condition had the best balance of transfection efficiency and fluorescence expression (Figure 2). The 300V condition had significantly fewer cells compared to the 200V and 250V conditions, suggesting that this condition caused high cell death. The 200V and 250V conditions had similar amounts of transfected cells, but the 250V condition had a higher fluorescence expression. Further refinement of our transfection protocol should investigate the most optimal amount of DNA added into the cells.

We successfully introduced recombinant plasmids into patient-derived glioma cells, which is a cell line that is traditionally difficult to transfect. We demonstrated that foreign DNA can be introduced and expressed by patient-derived glioma cells with high efficiency and expression. This proof-of-concept suggests that our plasmid reporter system has a high probability of being successfully transfected into the primary cells.

 In the next phase of our project, we will transfect all of our plasmid reporter candidates into patient-derived glioma cells to determine the most efficient plasmid reporter construct and continue to refine our transfection protocol to optimize gene expression. Should issues arise with non-viral methods of introducing our engineered plasmids into the primary glioma cells, we will investigate using lentiviral vectors, adeno-associated viruses (AAV), or CRISPR for transfection. After selecting our plasmid reporter and finalizing our transfection protocol, we will co-transfect the plasmid reporter with the DhdR plasmid into the patient-derived glioma cells to test the overall reporter system.      

Co-culture of Cancer Cells and Mini-Brain Organoids

As a proof of concept, a co-culture experiment was done using mature mini brains and a cancer cell line pre-labeled with green fluorescent protein, D425-GFP. This experiment allowed us to observe the interaction between and invasion of cancer cells and normal brain cells in the organoid (Figure 3).

Figure 3. Co-culture day 1-3 of cancer cells with a mini brain, with the glioma cells in green.

A starting density of 10000 cancer cells was seeded into a well with one mini brain in a 96-well plate. The system was then incubated in cerebral organoid media, and images were taken over the course of 5 days. On day 4 and 5, the mini brain had mostly disintegrated, as the cancer cells took over the main cell population (Figure 4).

Figure 4. Co-culture day 4-5. Notably, the mini brain population had been almost entirely taken over by the green cancer cells.

To further examine if the mini brains can provide a microenvironment to foster cancer cell growth and formulate cell-cell cross-talk, we co-cultured D425-GFP cells with mini brains and compared this to a system that had the same starting conditions but only cancer cells. The media condition does not support the growth of cancer cells due to its lack of zinc supplement, as the entire cell population died out after only 48 hours (Figure 4), but in the co-culture system the cancer cells were actively proliferating and showed invasion and development. We hypothesize that mini brains likely secreted some growth factors that supported cancer cell growth that was otherwise not present in the media. This system confirmed the establishment of a brain microenvironment in vitro and demonstrated that our co-culture system was interactive and supported cell communication.

Figure 5. The microenvironment in mini brains fostered growth of cancer cells in a 48 co-culture system. Green channel indicates alive cells; red channel indicates dead cells stained with a dead cell dye, ethidium homodimer-2. Note that the D425 population standing alone was entirely dead, while in the co-culture system there was obvious proliferation.

Moving forward, we will test the possibility to introduce drug titration in this system to see if there is a drug response initiated in the co-culture by examining the level of fluorescence.

Combining Co-culture with Drug Testing

To further demonstrate the robustness of the co-culture system, we set up a drug assay experiment that mimicked the process of testing drugs with patient-derived cell lines in a clinical setting. We used the same line as mentioned above, D425-GFP,  because it had green fluorescent reporters. This line is known to be a fast-growing cancer cell line that is resistant to temozolomide (TMZ) at high doses. The drug assay showed this trend as there were no significant differences in killing in two different TMZ doses (Figure 6).

Figure 6. TMZ drug assay over the course of 14 hours. There were no significant differences between the two doses.

We tested another drug, medroxyprogesterone acetate (MPA), which has been confirmed to have potent killing effects on D425-GFP, to show that the co-culture system conferred the same results as in previous studies. The drug was verified in patient-derived xenograft (PDX) models and pre-established cell lines and has been shown to cause strong and fast killing of the D425 line (He 2021) compared to MPA. Our co-culture system confirmed this trend and had results that agreed with previously published data (Figure 7).

Figure 7. The comparison between the killing effect of TMZ and MPA over the course of 26 hours. Note that the two higher concentrations of TMZ had lower efficiency in killing.  

The co-culture system also had the ability to demonstrate cell interactions and migrations visually. Cancer cell lines are usually known to be attracted to healthy cells in a co-culture system, which is the target interaction some drugs inhibit. Upon introduction of cancer cells in the co-culture system, they tend to migrate towards healthy cells and invade the interior of healthy organoids. A drug that inhibits such an effect should then cause the cancer cells to remain dispersed randomly in the system, preventing the typical congregation of cancer cells at the edges of the organoids. We tested this idea in the co-culture system and it was clear that one verified drug, tazemetostat (EPZ), demonstrated this effect (Figure 8).

Figure 8. Comparison of invasion distance between the cancer cells and the normal mini brains in untreated control and two different doses of EPZ groups. Note the different distances between the cancer cells and mini brains (arrows) increasing as the drug concentration increases, and the different total amount of cancer cells (green) in each condition. 

This demonstrated the functionality of our current co-culture platform, as it accurately recapitulated the differences between drug effects in varying doses and visually provided evidence that indicates inhibition of cancer cell migration. Moving forward, we expect to obtain glioma cell lines with implemented reporter systems for integration into co-culture, which will allow us to use these systems for drug screening purposes.


Rieger, J., Naumann, U., Glaser, T., Ashkenazi, A., & Weller, M. (1998). Apo2 ligand: A novel lethal weapon against malignant glioma? FEBS Letters, 427(1), 124–128.

Yang, R., Wang, W., Dong, M., Roso, K., Greer, P., Bao, X., Pirozzi, C. J., Bigner, D. D., Yan, H., Ashley, D. M., Zhabotynsky, V., Zou, F., & He, Y. (2021). Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells.