PROOF OF CONCEPT

Phase I Proof of Concept

During Phase 1 of our project, we created proof-of-concepts of multiple aspects of our drug screening system:

  1. We 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 (Figure 1).
    2. Figure 1. Lipofection of Patient-derived Glioma Cells using L2000 and LTX with pcDNA5-mCherry and FUGW-GFP, showing some successfully transfected cells

    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 successfully established a co-culture system of cancer cells and brain organoids, which will allow us to perform accurate in vitro drug testing. We demonstrated that cancer cells can be grown alongside brain organoids and that several aspects of typical tumor-tissue interactions are recapitulated, like cancer cell invasions (Figure 2).
    3. Figure 2. Co-culture days 1 and 5 of cancer cells with a brain organoid, with the glioma cells in green. Comparing the two shows the extent of glioma cell invasion of the minibrain over the course of the co-culturing experiment.

    This proof-of-concept suggests that a co-culture system has a high probability of being successful in our final reporter platform.

  3. We successfully performed initial drug testing on our co-culture systems, which followed the 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 (Figure 3).
    4. Figure 3. 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.

    This proof-of-concept suggests that our final co-culture system, with the integrated reporter, has a high probability of meaningfully quantifying drug effects in vitro.

Phase II Proof of Concept

Expression of DhdR in Mammalian System

We demonstrated that we could stably produce the bacteria-derived DhdR allosteric transcription factor in a mammalian cell line (HEK293T). To validate this, we performed a Western Blot on cells transfected with our DhdR construct. This construct included a FLAG-tag attached to the DhdR gene, allowing us to use the anti-FLAG antibody to detect the DhdR protein in our sample. We saw a protein band at the size consistent with the expected DhdR size (~25 kDa), suggesting that we were able to stably express this protein in vitro in mammalian cells (Figure 4).

Figure 4. Western blot results from SDS-PAGE of cell lysate, showing the presence of a band around 25 kDa in the 3’ DhdR and 5’ DhdR conditions and the lack of a band in the control condition

Fluorescence Changes in Response to D-2HG Levels

Ultimately, we want our reporter system to respond to varying D-2HG levels, given that this oncometabolite is linked to the activity of important oncogenic pathways and processes. Because fine-tuning robust responses to varying transcriptional states is a complicated process that requires rounds of iterations, we did not observe any significant changes in reporter expression due to differing D-2HG concentrations (Figure 5). Thus, we used modeling analyses to gain further insight into what might have been occurring. Through this work, we realized that N-terminal addition of the NLS and FLAG sequences may have interfered with transcription factor binding. In the following stages of our project, we hope to optimize the binding of the DhdR protein to the BS by replacing or removing the interfering parts.

Figure 5. Response of NODES reporter system to different doses of D-2HG. HEK293T cells were transfected with 2 vectors: one carrying 2 copies of DhdO binding site (BS5) with the reporter (mCherry) and the other containing DhdR transcriptional regulator or control GFP. mCherry expression levels were quantified in terms of median fluorescence intensity.